Reinert 1977

Chapter n Haploids
J. Reinert et al. (eds.), Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture
© Springer-Verlag Berlin Heidelberg 1977
1. Anther Culture: Haploid Production and Its Significance
J. REINERT and Y. P. S. BAJAJ
1. Introduction
Haploid plants have the gametophytic number of chromosomes, that is a single

set of chromosomes in the sporophyte. Their significance for plant improvement,
and as a tool in various disciplines of plant science has been stressed (see KASHA,
1974a). Since haploids are of great importance, especially in studies on the induc-
tion of mutations and also for the production of homozygous plants, they are
needed in large numbers. However, the conventional methods (KIMBER and RI-
LEY, 1963; MAGOON and KHANNA, 1963), employed by plant breeders for their
production are cumbersome, laborious and not very efficient. With the introduc-
tion of techniques for the induction of androgenesis by the culture of excised
anthers (GuHA and MAHESHWARI, 1964), isolated pollen (NITSCH, 1974a) and by
chromosome elimination through hybrid embryo culture (KAsHA and KAo, 1970)
it has become increasingly evident that tissue culture methods could considerably
speed up the production of haploids for breeding programs. These topics are dealt
with in detail in the subsequent articles in this chapter.
In 1953, TULECKE, for the first time, observed that mature pollen grams of a
gymnosperm Ginkgo bi/oba can be induced to proliferate in culture to form hap-
loid callus. This and subsequent work (TULECKE, 1959; TULECKE and SEHGAL,
1963) established that pollen grains, under in vitro conditions, can be manipu-
lated to bypass their primary role of forming sperms, and instead can be induced
to form haploid tissue. It was not until 1964 that GUHA and MAHESHW ARI re-
ported the direct development of embryos from microspores of Datura innoxia by
the culture of excised anthers. Later BOURGIN and NITSCH (1967), obtained com-
plete haploid plants of N icotiana tabacum. Since then, there has been a steadily
increasing stream of information. In this article, which is intended to serve as a
general introduction to the chapter on haploids, the methods of anther culture
and the significance of haploids for plant improvement are discussed.
2. Techniques
The technique for the excision and culture of anthers is relatively simple and
efficient. Closed flower buds, which have anthers (Fig. 1 A) containing uninucleate
micros pores (Fig.2A), ar$! most suitable for the induction of androgenesis. The
flower buds are excised, surface-sterilized in batches of 25 with a 5% solution of
commercially available bleach or a 1% calcium hypochlorite solution for 10 min,
252 1. REINERT and Y. P. S. BAJAJ
Anther Culture: Haploid Production and Its Significance 253
and then washed twice with sterile, distilled water. However, the flower buds
obtained from plants grown in green houses, if dissected carefully, need no steri-
lization. First an incision is made on one side of the flower bud and the stamens
are gently taken out with a pair of fine forceps and collected in a sterile petri dish.
The filament from the stamen is then carefully removed and five anthers are
normally transferred to each culture vessel. During excision of anthers special
care should be taken to ensure that they are not injured in any way. Damaged
anthers should be discarded, as they often tend to produce callus from parts other
than pollen. The anthers are cultured on agar-solidified medium in glass tubes or
small petri dishes. They can also be grown in liquid media in Erlenmeyer flasks
and kept on a slow rotary shaker. The cultures are incubated at 24-27° C and
exposed to light of about 2000 lux for a 14 h day. Although unopened flowers with
their sepals and petals touching each other have been observed to be suitable for
androgenesis by excised anther culture, this is only true of young plants. However,
older plants, especially towards the end of flowering, frequently produce small
buds which, although appearing to be at a suitable stage, contain anthers with a
heterogeneous mixture of microspores and young pollen. In such anthers the
numher of abnormal pollen also increases and androgenesis is not only delayed
but considerably reduced. Therefore, it is important that anthers should be taken
from relatively young plants which have been grown under good light conditions
(DUNWELL and PERRY, 1973).
Depending upon the plant species it takes about 3-8 weeks for pollen plantlets
to emerge from the anthers (Fig. 1 B). The plantlets (Fig. 1 D) when approxiIllately
5 cm tall can be removed from the culture and freed from agar by gently washing
with running tap water. The plantlets are then transferred to small pots contain-
ing autoc1aved soil. To reduce sudden shock and to prevent dessication, it is
advisable to cover these plantlets with glass beakers (Fig. IE), and to keep them in
a well-lit, humid green-house. Mter one week the beakers are removed, and after a
further two weeks, when the potted plantlets have grown to a sufficient size
(Fig. 1F) they are again transferred into larger pots (Fig. 1G) where they even-
tually develop to maturity and flower.
Haploids can be diploidized to produce homozygous plants by two methods;
(1) Colchicine treatment: the plantlets while still attached to the anther are
treated for 24-48 h with 0.5% colchicine solution (KASPERBAUER and COLLINS,
1972; BURK et ai., 1972), washed thoroughly and replanted. However, if mature
haploid plants are available then colchicine-lanolin paste (0.4%) may be applied
to the axils of the leaves (TANAKA and NAKATA, 1969). (2) Stem-segment culture: it
is known that haploid callus cultures frequently undergo endomitosis to form
Fig. 1 A-H. Culture of excised anthers and the regeneration of haploid plants (A) Tobacco
anther at the time of inoculation (containing uninucleate microspores). (B, D) Anthers 4 and 6
weeks after culture; note the emergence of embryos and plantiets from the bursting anther.
(C) 5-Week-old potato (Solanum tuberosum) anther undergoing proliferation to form callus.
(E)6-week-old haploid tobacco plants transferred from culture tube to pots. To prevent dessica-
tion, the plants are kept in high humidity under glass beakers for a couple of days. (F) Same,
2 weeks after trasnfer to pots; note the unfolding of the leaves. (G) A haploid plant showing
flowering 8 weeks after transfer to soil (total time after culture of anther is 14 weeks). (H) A
chromosome squash from root tip ofa tobacco plant showing haploid number (n=24)
254 J. REINERT and Y. P. S. BAJAJ
(A) (8) (C)
Fig.2A-1. In vitro induction of androgenesis from microspores of Nicotiana tabacum. (A)

Uninucleate microspore in an anther at the time of inoculation. (B) Binucleate microspore
showing two similar nuclei taken from an anther grown for 4 days, after cold treatment.
(C) Four nucleate pollen grain taken from IO-day-old culture. (D- G) 2 and 3-weeks old mi-
crospores in cultures showing early stages of androgenesis. (H, I) Heart and torpedo-shaped
haploid embryos from a 4-week-old culture. (From REINERT et al., 1975)
diploid cells and this property can be exploited to obtain homozygous diploids
(NITSCH, 1969; KAMEYA and HINATA, 1970; KOCHHAR et ai., 1971). In such experi-
ments a small segment of stem is grown on an auxin-cytokinin medium to induce
callus formation (NITSCH, 1972). During callus growth diploid homozygous cells
are produced by endomitosis from which a large number of isogenic diploid
plants can be differentiated.
3. Culture Media and Nutritional Requirements
Basal media (see Table 1) of WHITE (1943), MURASHIGE and SKOOG (1962), and
NITSCH and NITSCH (1969), with slight modifications and the addition of growth
regulators, have been used for the culture of excised anthers. The normal level of
sucrose is 2-4%, however, barley (CLAPHAM, 1971), tomato (SHARP et aI., 1971 a),
and wheat (OUYANG et aI., 1973) anthers have been observed to grow better on
media with 6-12% sucrose; this seems to be an osmotic effect rather than a need
for a higher carbohydrate level.
The iron in the medium plays a very important role and is indispensable.
Although androgenesis can be initiated in tobacco without any iron, the proem-
bryos do not develop beyond the globular stage. Chelating agents such as FeED-
TA (NITSCH, 1969) and FeEDDHA (RASHID and STREET, 1973) are more effi-
cient than ferric citrate as the source of iron.
The nutritional requirements of the .excised anthers are much simpler than
those of isolated microspores. In the isolated microspores it is obvious that cer-
tain factors responsible for the induction of androgenesis, which might have been
provided by the anther, are missing, and these have to be provided through the
medium. This is certainly the case in tobacco, where excised anthers can be
cultured successfully on a simple basal medium, while the isolated microspores
require higher amounts of nitrogen in the form of amino acids (NITSCH, 1974a;
REINERT et ai., 1975).
In plants such as tobacco (NITSCH, 1969) and Atropa (RASHID and STREET,
1973), complete androgenesis can be realized on simple media, but in most cases
(see Table 2) a certain balance of auxin/cytokinins or complex additives such as
casein hydrolysate, yeast extract, coconut milk and other extracts have to be incor-
porated into. the medium. These observations support the assumption that the
requirement for auxin and cytokinin depends on their endogenous level in the
anther.
Media rich in growth regulators encourage the proliferation of tissues other
than microspores (i.e. anther wall, connective and filament) and should be avoid-
ed, because in such cases mixed calli with cells of different ploidy levels are
obtained (NISHI and MITSUOKA, 1969; DEVREUX et aI., 1971; NARAYANASWAMY
and CHANDY, 1971; ENGVILD et ai., 1972; IYER and RAINA, 1972; SUNDERLAND et
ai., 1974; WAGNER and HESS, 1974; Chap. III. 1 of this Vol.). This would necessitate
a laborious screening program for the selection of haploid cells and plants.
Incorporation of activated charcoal into the medium has stimulated the in-
duction of androgenesis in tobacco anthers (ANAGNOSTAKIS, 1974). Our work
256 J.REINERT and Y.P.S.BAJAJ
Table 1. Composition of some of the basal media commonly used for the culture of excised
anthers
WHITE MURASHIGE NITSCH and NITSCH

(1943) and SKOOG (1969)
(1962)
M acronutrients mgll
Ca(N03h ·4H zO 288
KN0 3 80 1900 950
NH 4N0 3 1650 720
KG 65
KH zP04 170 68
NaH zP04 .4H zO 19
CaCl z ·2H zO 440 166
MgS0 4 ·7H zO 737 370 185
Na ZS04 200
M icronutrients
Fez(S04h 2.5
FeS04· 7HzO 27.8 27.8
Na2-EDTA 37.3 37.3
MnS0 4 ·4H zO 6.7 22.3 25
H 3B0 3 1.5 6.2 10
ZnS04 ·4H zO 2.2 8.6 10
KI 0.75 0.83
Na zMo04 ·2H zO 0.25 0.25
CuS04· 5HzO 0.025 0.025
CoCl z ·6H 2O 0.025
Organic
Biotin 0.05
Glycine 3.0 2.0 2
Inositol 100 100
Nicotinic acid 0.5 0.5 5
PyridoxilJe-HG 0.1 0.5 0.5
Thiamine-HCl 0.1 0.1 0.5
Folic acid 5
Sucrose 20000 30000 20000
(BAJA] et ai., 1976) with Nicotiana tabacum cv. Badischer-Burley has shown that
the percentage of androgenic anthers can be raised from 41 to 91 by the addition
of activated charcoal (2%) to the medium ~ig. 3), however, it seems that charcoal
can also cause small ill(;reases in the induction of diploid plants (Table 3). Al-
though no satisfactory explanation can be given at present for the stimulatory
effect of charcoal, it seems likely that it might be responsible for removal of
inhibitory substances from the medium. In this connection FRIDBORG and ERIKS-
SON (1975) have observed that charcoal enhances the induction of embryogenesis
in cell suspensions of carrot, where embryo formation cannot normally be in-
duced by the omission of auxin from the medium (see REINFRT, 1968). Likewise
they observed abundant root formation in old cultures of Allium cepa when
transferred to charcoal medium; such cultures do not normally produce roots.
This led them to conclude that charcoal might be removing auxin from the
~
"c
;: 100
<t
o eo
c
"em 60
"0
~ ... 0
~
o
Fig. 3. Effect of various concentrations of charcoal

on androgenesis in tobacco anthers grown for o
12 weeks. (After BAJAJ et al., 1976) Charcoal %
medium. Although charcoal has highly increased the efficiency of adrogenesis in

tobacco anthers it does not seem to be so much effective for the anthers of Atropa
belladonna and Petunia hybrida (BAJAJ, unpublished). It is possible that in plants
like tobacco charcoal absorbs inhibitory substances and thus reduces the number
of potential pollen embryos which would normally have aborted. However, it
seems more likely that the level of growth regulators, both endogenous and
exogenous, is regulated by absorption onto charcoal.
4. Induction of Androgenesis
In culture, microspores undergo various modes of androgenesis (Fig. 4) which

lead to the formation of haploids either directly by embryogenesis, or indirectly
via callus formation (Fig. 5). From the geneticist's point of view the direct forma-
tion of embryos from pollen is preferable to the indirect formation of embryos via
callus. Haploid plants or tissues have been obtained from species of a number of
families (for literature see Chap. 111.1). These are Aegilops caudata, Arabidopsis
thaliana, Asparagus officinalis, Atropa belladonna, Brassica oleracea, Capsicum
annuum, Coffea arabica, Datura innoxia, D. metel, D. meteloides, D. muricata,
D. wrightii, Fragaria virginiana, Hordeum vulgare, Lolium multiflorum, Lotus corni-
culatus, Lycium halimifolium, Lycopersicon esculentum, Nicotiana alata, N. attenu-
ata, N. glutinosa, N. knightiana, N. otophora, N. raimondii, N. rustica, N. sylvestris,
N. tabacum, Oryza sativa, Pelargonium hortorum, Petunia auxillaris, P. hybrida,
Secale cereale, Setaria italica, Solanum dulcamara, S. melongena, S. nigrum, S. tu-
berosum, S. verrucosum, Triticale and Triticum aestivum.
In addition, in this dynamic field androgenesis has recently been induced in
Agropyron repens (ZENKTELER et al., 1975), Brassica napus (THOMAS and WEN-
ZEL, 1975a), Bromis inermis-(ZENKTELER et al., 1975), Digitalis purpurea (COR-
DUAN and SPIX, 1975), Fest,!ca pratensis (ZENKTE~ER et al., 1975), Freesia (BAJAJ
and PIERIK, 1974), Hyoscyamus niger (CORDUAN, 1975; RAGHAVAN, 1975), Paeonia
hybrida(SuNDERLAND, 1974), P.lutea (ZENKTELER et aI., 1975) Pharbitis nil (SANG-
Table 2. Some plant species a of economic and horticultural importance in which haploid plants/calli have been obtained by the culture of excised tv
VI
00
anther/pollen
Plant species Family Inoculum Medium b Growth response Referencec
(mgfl)
Asparagus officinalis Liliaceae anther mod. MS+NAA (O.I)+BA (0.2) Callusing and plants PELLETIER et al. (1972), RAQUIN
(1973), HONDELMANN and WILBERG
(1973)
Brassica oleracea Cruciferae pollen NM+CM(lO%) Callusing KAMEYA and HINATA (1970)
x B. alboglabra
B. napus anther LS+2,4-D (O.l)+CM (10%) Callusing and embryos THOMAS and WENZEL (197Sa)
Capsicum annum Solanaceae anther MS + kin(l)+ 2,4-D(I) Callusing and embryos GEORGE and NARaYANASWAMY
+ RNA-nucleotide (40) (1973), WANG et al. (1973a)
Coffea arabica Rubiaceae anther mod.L.S. + 2,4-D(0.1) + kin (0.1) Callusing and embryos SHARP et al. (1973)
Fragaria virginiana Rosaceae anther mod. MS+NAA(2)+kin(S) Callusing and plants ROSATI et al. (197S)
Freesia sps Iridaceae anther MS + NAA(O.S) + kin(l) Callusing and plants BAJAJ and PIERIK (1974)
+ PBA(2)+ CH(SOO)
Hordeum vulgare Gramineae anther WM+NAA(I)+CM(IS%) Callusing and plants CLAPHAM (1971), MALEPSZY and
GRUNEWALD (1974)
Lilium longiljlorum Liliac~e anther mod MS+thiamin HCI(4) Callusing SHARP et al. (1971 b)
Lotus corniculatus anther mod LS + IAA(O.I) + kin(O.I) Callusing NIIZEKI and GRANT (1971)
Lycopersicon esculentumSolanaceae anther mod.MS + NAA(2) + kin(1) Callusing and plants GRESSHOFF and Doy (1972)
L. esculentum pollen WM+2,4-D(6) Callusing SHARP et al. (1971a)
L. esculentum pollen NandN+IAA(O.I)+antherextract Callus, embryos DEBERGH and NITSCH (1973)
N icotiana tabacum anther Nand N Embryos and plants BOURGIN and NITSCH (1967), !-<
NAKATA and TANAKA (1968), :;g
NITSCH and NITSCH (1969), ~
SUNDERLAND and WICKS (1969,
1971) ~
N. tabacum pollen Nand N + glutamine (800) Embryos and plants NITSCH (1974a, b), REINERT et al.
+serine (100)+ IAA(O.I) (197S), BAJAJ et al. (197S)
8-
+ zeatin (0.01) + inositol (SOO)
!-<
;c
Oryza sativa Gramineae anther MS+NAA(I) Callus, embryos and NIIZEKI and DONO (1968, 1971), ~
plants NISHI and MITSUOKA (1969), HARN al
(1969, 1970), GUHA et al. (1970), i!:
GUHA-MuKHERJEE (1973)
t
Table 2. (continued) >
::s
::r
Plant species Family Inoculum Medium b Growth response Referencec ~
(mg/l)
-
()
a
c...
(1)
Paeonia hybrida Ranunculaceae anther MS+NOA(O.l) Embryos and plants SUNDERLAND (1974)
-
P.lutea anther MS + IAA(l) + kin(l) + CH(500) Embryos ZENKTELER et al. (1975) ::r:
po
Pelargonium hortorum Geraniaceae anther mod MS+NAA(2)+ Callus and plants ABO-EL-NIL and 'E.
kin(2.5)+CM (15%) HILDEBRANDT (1971) §;
Petunia hybrida Solanaceae anther mod MS + BA(0.2)+ NAA(O.1) Callus, embryos BERNHARD (1971), RAQUIN and
and plants PILET (1972) ~
0
Q..
PopullJs sp. Salicaceae anther Callusing SATO (1974) g
Primula obconica Primulaceae anther MS + 2,4-D(0.5) + Zeatin (2) Callusing and plants BAJAJ (1976) g.
Saintpaulia ionantha Gesneriaceae anther Blaydes m~dium (1966) Plants HUGHES et al. (1975) ::s
Secale cereale Gramineae anther MS + 2,4-D(0.25) Callus, embryos and THOMAS and WENZEL (1975b) po
::s
Q..
plants
anther RAJNA and IYER (1973) :::;'
Solanum melongena Solanaceae Band N +CM(15%) Callusing and plants til
S. tuberosum anther MS+NAA(O.Ol) Embryos and plants DUNWELL and SUNDERLAND (1973) CIl
dQ.
7riticale Gramineae anther MS + 2,4-D (2) Callusing WANG et al. (1973b) ::s
7riticum aestivum anther MS +2,4-D (2) Callus, embryos and OUYANG et al. (1973), PICARD S
0
po
plants (1973), PICARD and DE BUYSER ::s
0
(1973), WANQ et al. (1973a), BAJAJ (1)
(1976)
Vitis vinifera Vitaceae anther mod. MS+NAA (O.l)+kin (0.3) Callusing GRESSHOFF and Doy (1974)
Zeamays Gramineae anther WM+2,4-D(2) Callusing and roots MURAKAMI et al. (1972)
Z. mays pollen NM + serine(loo) + glutamine Embryos NITSCH (Chap. II. 2)
(800)
For ploidy level of the plants regenerated by the anther culture refer to Chap. I1I.l.
b Different media have been used by various workers for the same plant species, however, in the table only the simple medium is given.
For detailed references see Chap. III.1 and page 331.
Abbreviations: Basal media: Band N: BOURGIN and NITSCH (1967), MS: MURASHIGE and SKOOG (1962), LS: LINSMAIR and SKOOG (1965),
Nand N: NITSCH and NITSCH (1969), NM: NITSCH (1974 a), WM:WHITE(1943).
Supplements: IAA: Indoleacetic acid, NAA: Naphthyleneacetic acid, NOA: Naphthoxyacetic acid, CH: Casein hydrolysate, CM: Coconut milk, N
kin: kinetin, BA: Benzylamino purine, PBA: 6-(benzylamino)-9-(2-Tetrahydropyranyl-9H-purine, 2,4-D: Dichlorophenoxyacetic acid. ~
260 J.REINERTand Y.P.S.BAJAJ
Table 3. The effect of charcoal (2%) on the percentage of androgenic tobacco anthers and
on the ploidy of regenerated plants (The results are based on 500 plants)
Control Charcoal medium
Percentage
Androgenic anthers 41 93
Haploids 96.5 95
Diploids 3.5 5
WAN and NORREEL, 1975), Populus (SATO, 1974), Primula obconica (BAJAJ, 1976),
Saintpaulia ionantha (HUGHES et al., 1975) and Vitis vinifera (GRESSHOFF and
Doy, 1974).
5. Ontogeny of Androgenesis
In vivo, ,as a res~lt of meiosis in pollen mother .cells, pollen tetrads are formed,
which are eventually released in the form of microspol'es. The newly formed
microsspore is highly cytoplasmic with a central nucleus. With the increase in
volume of the microspore and vacuolation, the nucleus is pushed towards the
periphery. By the ftrst mitosis, a large and diffuse vegetative cell and a small dense
generative cell are foqned. The former remains quiescent while the latter divides
to form sperms (Fig.4A).
In culture, although androgenesis can be induced in anthers at the tetrad stage
(GRESSHOFF and Doy 1972 b), or at the binucleate pollen stage (KAMEYA and
HINATA, 1970), however, microsporesjust before, or at the time offirst mitosis, are
most suitable for the induction of androgenesis (NITSCH and NITSCH, 1969). Mi-
crospores in cultured anthers exhibit various modes of development (SUNDER-
LAND, 1974) leading to androgenesis. Some notable ones represented in Figure 4,
are; 1. After ftrst mitosis the vegetative cell divides repeatedly to form a haploid
embryo (Fig.4B) as is the case in Nicotiana (SUNDERLAND and WICKS, 1971),
Datura (hER and RAINA, 1972), and Hordeum (CLAPHAM, 1971).2. The genera~
tive nucleus usually remains quiescent or aborts after a few divisions, but, occa-
sionally it (Fig.4C) does take part in androgenesis (DEVREUX et al., 1971).
3. The micros pore nucleus instead of dividing to form a generative and a
vegetative nucleus, gives rise directly to two similar nuclei (NITSCH, 1974a)
or there is a direct segmentation (RASHID and STREET, 1973, 1974) wHere both the
daughter cells are involved in androgenesis (Fig.4D). 4. In sQme cases, as in
Datura (SUNDERLAND et al., 1974) two similar nuclei (Fig.2B).formed as a result
of direct division of the microspore nucleus or of the vegetative one, fuse with one
another (Fig.4E) and this results in the formation ofhom@zygous diploids.
Irrespective of the early events in the division of the microspore nucleus there
are two modes of androgenesis, the direct and the indirect! wh.ich are diagrammat-
ically represented in Figure 5. 1. Direct androgenesis: in thIS type the microspore
behaves like a zygote and undergoes various stages of embryogeny simulating
$,
M IO,.ospore Moth r Coif
$,
M lcr'Ol!!Ipore T.trad
OlplOldlzcnlon
t
~
"
VI
O iplold P,.a.rnbryO
!
"' "
<;::"
Hopfoid EmbryO Diploid Er"I"'tbryo
Fig. 4A-E. Diagrammatic illustration of microsporogenesis, and various modes of develop-

ment of pollen under in vivo and in vitro conditions. (A) Normal second mitosis of pollen in
vivo forming two sperms and the germination of pollen to form a pollen tube. (B-E) In vitro
behavior of pollen. (8) Repeated division of the vegetative nucleus and the abortion of the
generative nucleus. (C) Formation of a haploid embryo as a result of repeated division of the
generative nucleus, while the vegetative nucleus aborts. (D) Haploid embryo formation as a
result of repeated division of two similar nuclei of a pollen. (E) Homozygous diploid embryo
formed by fusion of two similar nuclei of the pollen after first mitosis. (Modified from
DEVREUX, 1970)
those in vivo, as in Atropa, Datura and Nicotiana. The embryos, mostly at the
globular stage, are released from the exine (Fig. 2G) and develop further
(Fig. 2 H, I). Finally the cotyledons unfod and the plantlets.emerge from the anthers
(Fig. 1B, D) in 4-8 weeks. 2. Indirect androgenesis: in contrast to the direct andro-
genesis, the micros pores instead of undergoing embryogenesis, divide a few times
to form a callus which bursts through the anther wall (Fig. 2D-F). This mode of
development is quite common (see Table 2) and is usually caused by complex
media, and in 'cases where the polarity seems to be disturbed. The callus either
differentiates to form embryos, or roots and shoots on the same medium, or it has
to be transferred to another medium. The callus-derived plants are mostly unde-
sirable as they exhibit genetic variations and polysomy. For details on the ploidy
status of anther-derived plants see Chapter III.l.
262 J. REINERT and Y. P.S. BAJAJ
F"tO\Ner" Sud
Excls
aJrn
d Anthers
+
III
III
CII
C
m
Proer"'nbryo
t
Antne,.
Culture
\lJ
Callusing
\
0
CO
0
III
~
CII
~
0
Ol
CO
0
~
Androgenes i s
>- E"'~ryO
I ~
~ a1
~
.0
VI
E
UJ HOplOi(l VI
Callus
I
Regeneration
~
HaplOiO Haplo i d
Plontlet Plontlet
HOplOld Plont
Fig. 5. Schematic representation of the culture of excised anthers and the development of
haploid plants directly by embryo formation, or through haploid callus
6. Stability of Haploid Cell Cultures
One of the main problems with tissue cultures, especially those of haploid origin,
is the maintenance of their genetic stability in long term cultures (D'AMATO 1975;
Chap. 111.1). For their use in somatic cell genetics, it is essential that the cultures
remain haploid during the early stages when the mutants are being selected. The
possibility of using para-fluorophenylalanine (PFP) to stabilize or even enhance
the growth of haploid cultures has been explored by various workers, however,
there is considerable disparity in the results. GUPTA and CARLSON (1972) observed
that PFP (9 mg/l) inhibited the growth of diploid tobacco (Nicotiana tabacum cv.
Havana Wisconsin 38) callus of stem origin, whereas the growth of haploid cells
remained uneffected. Likewise, DIX and STREET (1974) observed growth inhibition
20
i CONTROL
ii
:J 10
Fig. 6. Effect of 10 mgjl of para fluoro- Z
phenylalanine (PFP) on frequency distri-
bution of values for the nuclear DNA
o
II
contents of cell suspension of halploid 01
20
Nicotiana tabacum cv. Badischer-Burley
grown for 4 weeks. DNA content was
~II PFP
estimated by microspectrophotometty of e 10
100 Feulgen-stained nuclei. The PFP ~
treated cultures show an increase in the
number of haploid cells with correspond-
ing decrease in the diploid cells. (From 5 10 20 30 40 so
BAJAJ and GROBLER, unpublished) DNA Content - Arbitrary Units
Table 4. Effect of various concentrations of PFP on the growth (percentage of control) of

haploid stem-explants of Nicotiana tabacum cv. Badischer Burley cultured for 6 weeks.
(From BAJAJ and GROBLER, unpublished)
Weeks PFP (mgjl)

after
culture 5 10 15
Hap!. Dip!. Hap!. Dip!. Hap!. Dip!.
1 140 130 150 110 160 130

3 280 130 250 70 346 110
6 150 140 210 140 190 100
of diploid cultures of Nicotiana sylvestris by PFP (37.5 mg/I), but did not observe
any preferential growth of haploid cells in mixed .cultures, and stated that it is not
the ploidy level, but the genotype, that determines the sensitivity to PFP. Recent-
ly, MATTHEWS and VASIL (1976) reported that PFP inhibits the growth of both
haploid and diploid pith-explants of Nicotiana tabacum, and suggested that PFP
acts as a selective inhibitor which reduces the growth rate of haploid cells less
than that of the cells of higher ploidy.
In our experiments (BAJAJ and GROBLER, unpublished) with N icotiana tabacum
cy. Badischer-Burley stem explants, the optimal effect ofPFP was observed 3 weeks
after the initiation ofthe cultures (Table 4), and 15 mg/l was found to be more effec-
tive than 5 or 10 mg/l both for the haploid, as well as for diploid cultures. Whereas
15 mg/l of PFP was stimulatory for the haploid tissues, it slightly inhibited the
growth of diploid explants. Almost similar results are obtained with Atropa bella-
donna.
In cell suspensions obtained from androgenic plants, initially about 70% of
the cells were haploids, but, within 4-6 weeks the level decreased to about 30%.
After eight months of repeated subculturing, only 5% of the cells showed the
haploid DNA level. PFP enhanced the maintenance of such cultures for up to four
264 J.REINBitT and Y.P.S.BAJAJ
weeks (Fig. 6). The behavior of such cells in long-term PFP cultures is under
investigation.
The disparity in the results obtained by various workers could be due to the
differential sensitivity of various genotypes to PFP. Like DIX and STREET (1974),
we also observed that results varied a great deal with different plants. The physio-
logical state and the age of the plant appear to be of utmost importance, and in
order to critically evaluate the effect of PFP on growth, especially of the stem-
explants, it might be necessary to grow the haploid and diploid plants under the
same conditions. In addition our microspectrophotometric data clearly reveal
that PFP at 10 mgfl helps to maintain the haploidy in short-term cell suspension
cultures. This is an area which needs to be further explored as it might prove to be
helpful in preventing polysomy in haploid cultures, or in understanding the mech-
anism underlying the increase in ploidy.
7. Significance and Uses of Haploids
Haploids are required for two main reasons, (1) The presence of one set of chro-
mosomes facilitat~s the isolation of mutants, and (2) isogenic diploids can be
obtained by chromosome diploidization. Their significance for plant improve-
ment has been appreciated (see KIMBER and RILEY, 1963; MAGOON and KHAN-
NA, 1963). Although, by conventional inbreeding and backcrossing, it is possible
to obtain pure lines, this is a time-consuming process. With the availability of the
in vitro methods there has been a renewed interest in the production of haploids
(see DEVREUX, 1970; SUNDERLAND, 1970; MELCHERS, 1972; NITSCH, 1972; PAN-
DEY, 1973; KASHA, 1974a; BAJAJ, 1976). By anther culture, haploids can be ob-
tained in a matter of weeks, and by doubling their chromosome number homozy-
gous diploids can be procured in a single generation. These fertile homozygous
plants can be used for producing the inbred lines required to utilize hybrid vigor.
Such pollen-derived homozygous diploid plants offer the advantages that they
show normal meiotic segregation (COLLINS and SADASAVAIAH, 1972) and that
they do not lose desirable characters by segregation.
With a view to inducing mutations, NITSCH et al. (1969) SUbjected yonng
tobacco plants (while still emerging from the anthers) to 1500 and 3000 rads of
gamma irradiations and obtained a high proportion of abnormalities in their size,
shape and color. In a later study NITSCH (1972) reported that white-flowered
tobacco mutants developed when anthers were grown on a medium supple-
mented with N-3-nitrophenyl-N-phenylurea. Likewise, X-ray-subjected anthers
produced 40% haploid plants with aberrent phenotypes, and about 6% with
chromosomal aberration (DEVREUX and SACCARDO, 1971).
Haploid cell cultures are also useful material for the study of somatic cell
genetics (SMITH, 1974), especially for mutation and cell modification (see Chap.
IV.3). By employing agar-plating techniques and suspension cultures, isolated
protoplasts, pollen and cells (Fig. 7) can be handled like microbes, and can be
treated with various mutagenic chemicals and irradiations. The advantage of the
haploid cells is that the mutants can be easily diploidized to form homozygous
An~her lcul~ure
/icrospor~
1
ErT1bryoge"esis
~
Haploid Callus
(cells and lrmOPlas~s)
Haploid ~ Mutagenesis
Plorts .. (radiatianJ and crernicalS)
j -i-_oo
Chromosome Doubling Selection
(colchicine 'trea'trnent and
endomitosis)
1
Homozygous Diploid Haploid Mutant
Plants Plants
Fig. 7. Schematic representation for the induction of homozygous diploids and mutants from
haploid plants, cells and protoplasts obtained through anther culture
diploids. In this connection isolated pollen grains (NITSCH, 1974a; REINERT et al.,
1975) and possibly their protoplasts (COCKING, 1973; BAJAJ, 1974, 1975) would be
better suited for this purpose.
TULECKE (1960) isolated arginine-requiring strains of cells from Ginkgo pollen
by substituting auxin in the medium with L-arginine. Later, CARLSON (1970)
obtained auxotrophic mutant cell lines by treating haploid tobacco cells with
ethyl methane sulfonate, and regenerated plants from such cells. Since then, mu-
tant cell lines have been obtained by other workers by treating N icotiana taba-
cum and Petunia hybrida cells with 5-bromodeoxyuridine (MALIGA et al., 1973a),
streptomycin (BINDING et al., 1970; MALIGA et ai., 1973 b) and DNA base analogs
(LESCURE, 1973). By subjecting haploid Nicotiana tabacum cells to methionine
sulfoximine CARLSON (1973) also claimed to have regenerated mutant plants
which showed a considerably lower level of infection when inoculated with wild-
fire pathogen, Pseudomonas tabaci.
Since haploid plants can be regenerated from the isolated mesophyll proto-
plasts of tobacco (OHYAMA and NITSCH, 1972; BAJAJ, 1972), Petunia (BINDING,
1974) and Datura (SCHIEDER, 1975) they should form an additional tool for study-
ing the effect of various mutagens. Mesophyll protoplasts have the advantages of
being relatively homogenous, divide more synchronously and are genetically
more stable than the callus cultures.
Employing haploid tomato (Lycopersicon esculentum) cell cultures Doy et al.
(1972, 1973 a, b) reported their transformation by the incorporation of phage
lambda (Bacterial virus). However, the observation that these cells grow on a
medium containing galactose and lactose for some time, is not sufficient proof for
their transformation. This work has obvious shortcomings but could be extended
to other systems in order to find out the feasibility of employing haploid cells for
transformation studies. More conclusive results could be expected using proto-
266 J.REINERT and Y.P.S.BAJAJ
plasts instead of callus cells because of the possibility of a better uptake of the
transforming factor.
The importance of haploids in cereal breeding has been dealt with in detail in
article 3 and 4 of this chapter. To summarize, in addition to other advan~
tages, haploids can be used to obtain homozygosity for genes in cases where it is
normally difficult to achieve, such as self-incompatible alleles in rye. In barley,
monoploids provide a means of gamete selection, and if they are produced from
F 1 hybrids then the number of generations of self pollination, that are normally
required to produce uniform lines, are eliminated. This saving of the number of
generations ·is very important in winter barley because of the time required for
vernalization. A second advantage of doubled monoploids is the reliability of the
selection provided. When screening for yield, quality or disease resistance one can
be sure that due to homozygosity the desirable characters will not be lost because
of segregation from heterozygous loci in later generations (see JENSEN, 1974a). Of
the various other uses of cereal haploids, the most remarkable one is the one-step
transfer of cytoplasm from one line to another (CHASE, 1952). Using Zea mays
monoploids GOODSELL (1961) and KERMICLE (1973) successfully transferred the
genotypes of inbred lines into cytoplasm that caused male sterility. In addition
they could be used in (1) recurrent selection, (2) fixing quantitative characters so
that they can be analyzed and selected and (3) the identification of superior crosses
for more extensive exploitations (Chap. 11.4).
Potato (Solanum tuberosum) haploids have been sought after for years
(LAMM, 1938) because of their potential in breeding programs (HOUGAS and PELO-
QUIN, 1958). However, the methods employed for their production are laborious
and involve wide crosses. By anther culture (DUNWELL and SUNDERLAND, 1973)
haploids and subsequently the homozygous diploids can be obtained in a rela-
tively short time. The fertile dihaploids obtained from tetraploids can be used for
breeding at the diploid level. .
Haploids will be of immense importance for the improvement of some crop
plants. For instance, the haploids of complex hybrids of Coffea arabica (see Chap.
1.6) are of special interest in relation to 'coffee rust disease' because such hybrids
result from the incorporation of resistance genes of wild coffee after artificial
pollination. The objective of work with these crosses is to combine multiple
desirable genes in a single genotype. Six known dominant genes confer rust
resistance, and 1/64 of the microspores in F 1 from a hexahybrid cross will carry
all such genes, providing there are no linkages. Furthermore, the resulting totally
homozygous diploids will have a double dose of the genes for resistance and will
have an advantage over their parent lines because of the phenomenon of gene
dosage. When a haploid line of a disease-resistant heterozygous wild species,
having one or more genes for rust r~istance is used, the product following diploi-
dization should have a higher potential in field utilization, if there is a gene dosage
effect. .
In some plants of horticultural importance, such as Freesia (BAJAJ and
PIERIK, 1974) which are normally propagated vegetatively by means of corms, it
takes 8-10 years to produce a clone which is large enough for commercial pur-
poses. In these cases anther culture has obvious advantages. Specific desirable
gene combinations may be linked in a haploid form and then converted to the
fertile homozygous diploid or polyploid forms in a relatively short time.
Another use of haploids is in relation to self-incompatible plants. The haploids
from self-incompatible plants would be highly desirable as they would have sev-
eral applications, some of which outlined by de NETIANCOURT and DEVREUX
(Chap. 111.6), are (1) the use of completely homozygous self-incompatible diploid
lines as parental material (or the production of F 1 hybrid seed, and (2) for the
production of haploid plants from interspecific hybrids between self-compatible
and self-incompatible species. Such plants would theoretically be genetically dif-
ferent from one another and would carry different combinations of paternal and
maternal chromosomes. After diploidization they should constitute an extensive
collection of substitution lines.
In addition they could be used in (1) recurrent selection, (2) fixing quantitative
characters so that they can be analyzed and selected, (3) the identification of
superior crosses for more extensive exploitations (Chap. 11.4).
Amongst other uses, homozygous plants, obtained through anther culture,
have also been employed for the selection of breeding lines of N. tabacum with
high alkaloid content (COLLINS et at., 1974). Haploid protoplasts can also be used
as a means of clonal propagation of rare haploids, and in cases where the fre-
quency of haploid induction by anther culture is low.
However, it must be pointed out that there are some drawbacks in the tech-
nique of anther culture. One is that the plants may not originate from pollen only
but also from various other parts of the anther. This results in plants with various
levels of ploidy (NISHI and MITSUOKA, 1969; DEVREUX et at., 1971; NARAYAN AS-
wAMyand CHANDY, 1971; ENGVILD et at., 1972; IYER and RAINA, 1972; SUNDER-
LAND et at., 1974; WAGNER and HESS, 1974). The ploidy status of these plants has
been dealt with in detail in Chapter III.1, and it is evident that an extensive
screening and selection of such anther-derived plants is required. However, this
can be partially overcome by the culture of isolated pollen (Chap. 11.2). There is,
of course, the possibility of fusion of pollen nuclei, but this would be an advan-
tage, because homozygous cells would be directly obtained without any applica-
tion of colchicine.
References see page 331.

2. Culture of Isolated Microspores
C.NITSCH
1. Introduction
In 1953 Tulecke succeeded in growing a callus from the pollen of Ginkgo biloba
when it was cultured in vitro. The callus had the haploid number of chromo-
somes, however it has not been possible to regenerate plants from such callus.
After the discovery by GUHA and MAHESHWARI (1964) that Datura anthers placed
in culture stimulated the pollen to produce embryos, and the production of haploid
plants from anther culture of Nicotiana by BOURGIN and NITSCH (1967) opened a
new line of research which is especially attractive for geneticists and plant breed-
ers. This discovery would be even more significant if the knowledge already
available for single cells in vitro could be applied. In 1970 KAMEYA and HINATA
using more or less the same culture medium as that used in anther culture ob-
served the formation of cell clusters in mature pollen grains of Brassica. These
pollen grains were released from pre-sterilized anthers and cultured in drops of
between 50 and 80 in slide glass holes. These authors however did not show that
the cell clusters observed were derived from single grains, so 'the possibility of
agglomerate of pollen grains is not excluded. Later, SHARP et al. (1972) with
Lycopersicon escuientum, and PELLETIER (1973) with Nicotiana tabacum stimulated
colony formation by the nurse-culture technique, however it is only in the case of
Nicotiana that whole plant could be obtained using Petunia callus tissue for
nursing. The following pages are therefore devoted to a description of the isola-
tion of the pollen and its cultural conditions (NITSCH, 1974a) a technique which
has been successfully applied to N. tabacum cv. Badischer Burley (REINERT et ai.,
1975; BAJAJ et al., 1976). As will be shown, this involves two different steps, each
of equal importance. First, the induction of the microspore to change its normal
sexual evolution and follow a vegetative pathway; second, the development of the
induced micros pore towards embryogenesis.
2. Induction towards aNon-Sexual Pathway
From the work done on anther culture, successful for a fairly large number of
species (see REINERT and BAJAJ, Chap. ILl), the authors are unanimous in saying
that the state of the pollen at the initiation of culture is critical. In order to
produce haploid tissue, anthers should be planted at the time of the first pollen
mitosis or very soon after.
Culture of Isolated Microspores 269
0-------°. . . . . ,
o I ............ ,
~ 10 "',
,
Ir) /
.....
" " 0 pro - E
.'" :::'
::,'
01
-<:: 1 no cold
~.
I ~pro-E
OL-~----L-----~ ______- L_ _ _ __
o 3 7 12
Days in culture
Fig. 1. Effect of cold shock on the pollen nuclei. Flower buds of N. tabacum placed at SO C
for 3 days before taking out the anthers (upper curve), and anther planted just after excising
the bud from the plant (lower curve). In both cases, microspores isolated from the anther
3 days after placing the anther in culture. 1000 microspores stained by Feulgen's method are
observed for each point. Pro-embryos at day 12 have 4 identical nuclei
By modifying their normal development, we hope to trigger isolated micros-

pores into behaving as somatic cells which would give rise to plantlets. The idea
was therefore to prevent the microspore from forming a generative nucleus, think-
ing that, liberated from the influence of the generative cell, the vegetative cell
could grow as any somatic cell. Any treatment which can modify the mitosis has
therefore been studied.
1. Following the work of SAX in 1937 on Tradescantia, NITSCH and NORREEL
in 1972 carried out some experiments in which plants underwent a cold shock at
the time of the first pollen mitosis. A sudden change in temperature i.e. from 20° C
down to 3 or 5° C respectively for Datura or Nicotiana was given either to the
whole plant, to the flower-bud, to the dissected anther or to the isolated micros-
pores either in the light or in the dark.
The effect on the microspore nuclei was analyzed by observing grains which had been
fixed for 2-3 days in 1: 3 vIv acetic acid: ethanol and stained in Schitrs reagent after 10 min
hydrolysis in 60° C 1 N HC!.
The greatest effect is obtained when the cold treatment is given to the flower
bud detached from the plant and kept in the dark in a humid environment. The
percentage of pollen in which the mitosis has been modified (i.e. containing two
identical nuclei instead of one vegetative and one generative) is higher after a cold
shock, as is shown in Figure 1.
2. It is known from the literature that low temperature breaks down micro-
tubules. As was suggested by JENSEN (1974 b) this effect could be either replaced
or increased by gravity. Indeed, for Nicotiana sylvestris where it is difficult to trigger
the development of the micros pore in culture, gravity increases the number of
haploid plants produced by pollen culture.
The flower buds are centrifuged down at 500 g in a 5° C refrigerated centrifuge for 1 h
before anthers are dissected out.
The same type of response has been obtained with Zea mays although these
results are still preliminary and unpublished.
270 C.NrrscH
100 0 Colchicine 10- 3 M. Colchicine
'"co
'cv
a. 65
"C
~
"C
co 50
:>
J:
36.5 39.5
.... 25.5
u.'"
0
o
Fig. 2. Effect of colchicine on the first pollen mitosis of N. tabacum. Nuclei of 1000 grains
stained with Feulgen are observed 3 days after the colchicine treatment. 1 n grains with one
nucleus, V+ G: grains with one vegetative and one generative nucleus, id: grains with
2 identical nuclei, D: dead grains
3. Certain chemicals are also known to have an effect on mitosis, namely the
growth substances. As s.hown by DEBERGH and NITSCH in 1973 for tomato, the
first divisions towards embryogenesis seem to be auxin-dependent. However, the
monocotyledons . appear to be more generally auxin-dependent. NnZEKI and
OONO (1968) with rice, PICARD and DE BUYSER (1973), and OUYANG et al. (1973)
with wheat, indicate the absolute requirement for auxin in the medium for the
production of haploid plants by anther culture of these species. As we shall see
later in the discussion on the nutritional requirements for microspore culture, we
believe that growth substances have to be used with great care in order to avoid
dedifferentiation of the tissue.
4. Another way to induce microspores to grow as somatic cells is by altering
their mitoses with the use of colchicine. This chemical is known (JENSEN, 1974 b)
to stop mitosis at metaphase by inactivation of the spindle mechanism : weak
doses of colchicine are used for doubling the chromosome number of a cell. The
action of colchicine on the micros pore could therefore be particularly advanta-
geous, since it would change the microspore into a homozygous diploid cell. The
plants originating from such cells would have the great advantage of being fertile
unlike the normal haploid plants. Experiments done with N. tabacum cy. Red
Flowered and Coulo produced 15-20% fertile diploid plants. This percentage is
even higher with N.sylvestris.
Experimental procedure: anthers of N. tabacum or N. sylvestris are floated under vacuum
in basal medium containing 0.05% colchicine with 2% dimethylsulfoxide, used as a carrier,
for 4--12 h in the dark. The anthers are then taken out of the colchicine medium, washed, and
placed in fresh liquid medium for three days (inductive period) in a culture chamber at 25-
27° Cand a light intensity of 3000 lux.
The micros pores are then isolated from the anthers and placed in culture as
described below. The observations made on the nuclei at the start of the culture
are shown in Figure 2. After the colchicine treatment, the number of uninucleate
grains increased from 4.5-19%; it is also interesting to note that these uninucleate
grains have a much larger nucleus then the normal microspore nucleus. As might
be expected the number of dead grains has also increased. Another advantage of
Fig. 3. Induced microspores of N. tabacum after 5days in culture (x 1200). m.' grains in which
the mitosis has been modified: 2 identical nuclei, n .' grains with normal mitosis :.v .' vegetative
nucleus, g.' generative nucleus, d .' dead grain id.' grains with 2 identical nuclei
doubling the chromosome number of the micros pore might be the production of
homozygous diploid in species in which the haploid plants could not survive.
The important factor in obtaining the maximum number of micros pores
which develop along the vegetative pathway, is the use of anthers which contain
the maximum possible number of grains which are in mitosis to start the induc-
tion.
The inductive period is rather short (from two to five days) but nevertheless
essential, and has to be adapted to each species. It is by staining the micros pores
following Feulgen's method and observing the presence of grains with two identi-
cal nuclei that one can be sure of a high ind'uction frequency (Fig. 3). Thus having
observed grains with two identical nuclei, one can go on to culture the isolated
microspores by placing them on an appropriate medium which will be described
later.
3. Technique for the Isolation of Microspores
The unopened buds are sterilized by a 2 min immersion in a freshly prepared

filtered aqueous solution 7% of calcium hypochlorite followed by rinsing twice
with sterile water.
When the species requires only an induction by physical shock given to the
whole bud, as in Datura, the anthers are dissected and pollen extracted immedi-
272 C.NrrSCH
Fig. 4. Technique for microspore culture (a) anther or small flower buds are squeezed against
the side of the beaker to push out the pollen into the basal medium. (b) nylon sieve held onto
pyrex cylinder by a rubber band. (c) filtering anther mixture through the sieve. The arrow
shows the pollen suspension obtained. (d) pollen, centrifuged down, is washed twice with
fresh medium. (e) clean pollen suspension distributed into 5 cm pyrex dish: 2.5 ml per dish at
10 3 grains per ml. (I) 14 petri dishes sealed and placed together in a larger dish. Plantlets are
seen in the dish after 3 weeks
ately. On the other hand if the species requires an extra inductive period in the
presence of chemicals, the anthers are floated on the inductive medium in liquid
culture for two or three days. This is so in N icotiana as well as cereals. In short the
micros pores are extracted from the anthers at the end of the inductive period
when microspores with two identical nuclei can be seen (Fig. 3).
In the case of large anthers, as in Nicotiana, about 50 anthers are placed in a
small beaker containing 20 ml of basal medium (see Fig. 4a). The pollen grains are
then squeezed out of the anthers by pressing them against the side of the beaker
with the piston of a syringe. Anther tissue debris is removed by filtering the
suspension thus obtained through a nylon sieve (Fig. 4 b) having a pore diameter
which is slightly wider than the diameter of the pollen (e.g. 40 microns for Nico-
tiana or Datura, 25 microns for tomato, 100 microns for maize etc.). This pollen
suspension (Fig. 4c) is then centrifuged down at low speed (500-800 revolution min
for 5 min) the supernatant containing fine debris is discarded and the pellet of
pollen resuspended in fresh medium and washed twice (Fig. 4d). The rinsings are
particularly important if the anther tissue contains inhibitory· substances which
would prevent any growth of the microspores. At this point the pollen is mixed
with an appropriate culture medium at a density of 10 3 to 10 4 grains per ml. The
final suspension is pi petted into 5 cm wide pyrex petri dishes at 2.5 ml per dish
(Fig. 4e). To ensure good aeration, the layer of liquid in the dish should be as thin
as possible. Each dish is then sealed with parafilm to avoid dehydration, and 14
dishes are placed together in a 20 cm-wide dish (Fig. 4f). These are incubated at
27-30° C for Nicotiana. In species in which the flower buds are very small, as for
example with fruit trees or cereals, it is difficult to remove the anthers. In these
cases we separated each floret from the main axis, cutting off the bud at the level
of the receptacle and treated the whole bud in the same manner as for the anthers
of N icotiana. A pollen suspension can be just as easily prepared using these whole
buds as by using only anthers.
The micros pores are best grown in liquid medium, but if necessary can also be
grown by plating in a very soft agar medium (0.6% agar) Here again the layer of
medium in the dish should be very thin.
4. Requirements for the Growth of the Embryo
4.1 Nutrient Requirement
After numerous experiments in anther culture, a few authors came to the conclu-
sion that some species need only a rather simple medium to produce haploid
plants (l.P.NITSCH" 1969 for Nicotiana, DUNWELL and SUNDERLAND, 1973 for
potato). In Nicotiana and Datura NITSCH showed that pro-embryos can be pro-
duced on 2% sucrose in distilled water. That is to say that mineral salts, minor
elements, vitamins and growth substances, all constituents generally used in tissue
culture, are not necessary for the first phase of development of the micros pores.
To grow these pro-embryos to complete plants, the same author showed that only
iron plus sucrose were necessary. This work gave the first indication that the
274 C.NrrSCH
.J::;
<II
'0200
.... Fig. 5. Effect ofmyo-inositol on the
CI>
c.. growth of androgenetic embryos of
N. tabacum. Induced microspores
-
<II
:§ 100
are placed on basal medium
c:
supplemented with myo-inositol at
a:'" , different concentrations. Average
0
-ex> 10- 3 3'10- 3 10- 2 3.10- 2 10-1 number of plantlets from 6 dishes
Log. Cone. Mol. Inositol after one month in culture
production of haploid plants from anther culture was a two-step procedure,

namely induction and nutrition, and led us to believe that part of the nutritional
requirement came from the surrounding tissue.
4.1.1 Effect of Sugar

In anther culture, sucrose was shown to be absolutely necessary for most species
and was not successfully replaced by other sugars. The influence of glucose shown
by HOMFS (1967) on the development of somatic embryos of carrot was found to
be negative for induced micros pores of Solanaceae. On the other hand the positive
effect of a high concentration of myo-inositol shown by NORREEL and NITSCH
(1968) on the production of somatic embryos in carrot tissue could be reproduced
for Nicotiana micros pores. Figure 5 indicates the effect of different concentrations
of myo-inositol added to the basal medium. A concentration of 0.5% allows the
development of an average of 58 plants per dish, that is around 0.5% of the total
number of pollen in culture.
4.1.2 Effect of Amino Acids

Amino acids have long been known to be necessary for the development of
zygotic embryos. In 1953 PARIS et al. studied the effect of aspartic acid and
glutamic acid and their amides on the development of zygotic embryos of Datura.
Glutamine was found to increase the growth of these embryos. In our experi-
ments, glutamine added to the basal medium allowed the development of micros-
pores isolated from the anther as shown in Figure 6. A concentration of 3 x 10 - 3
M on an average produces plants from around 2% of the grains per dish. Aspara-
gine, lysine and threonine did not give any growth. Hydroxyproline, on the other
hand, in preliminary experiments produces plantlets at a concentration of
3 x 10- 4 M. The partial success of nurse culture technique for the production of
haploid plants from pollen by SHARP et al. in 1972 with tomato, and PELLETIER,
1973 for Nicotiana suggest that the microspore benefits for its development from
nutrients originating from the anther tissue. This hypothesis was confirmed when
in 1972 we succeeded in growing microspores of Datura innoxia isolated from the
anther on medium conditioned by an aqueous extract of embryogenic anther
tissue. Further experiments on the analysis of such extracts have shown the
importance of another amino acid, namely serine, for the development of the pro-
embryo (NITSCH, 1974a, b). Thus a completely defined medium has been proposed
.r:.
en
400
'i5
~
a. 300
en
:§
1\
aC 200
Fig. 6. Effect of L-glutamine of! the
growth of androgenetic embryos of
N. tabacUm. Induced microspores
-.
o
Q)
~ 100
are placed on basal medium :l
o~~~--~,----~,~,~~,~
supplemented with L-giutamine at Z
different concentrations. Average
number of plantiets from 6 dishes - 00 10. 4 10.3 3· 10.3 10.2
after one month in culture Log. Cone. Mol. Glutamine
for the production of haploid plants by in vitro culture of isolated microspores.

The basal medium is the classical medium used for anther culture (in mg/l):
Mineral salts: KN0 3 (950), NH 4N0 3 (725), Mg S04, 7 H 20 (185), CaCl 2 (166),
KH 2P0 4 (68); Iron: 5 mlfl of a solution obtained by dissolving in 1 liter of distilled water
5.57gof FeS04, 7 H 2 0 and 7.45 g of Na2 EDTA; and: 2% sucrose to which is added myo-
inositol at 2.5 x 10- 2 M, glutamine at 5 x 10- 3 M and serine at 10- 3 M. The pH is adjusted to
5.8 with NaOH and the sterilization done by filtration.
An average of 5% of the pollen of Nicotiana tabacum placed in culture devel-
oped to plantlets in this medium. Figure 7 shows the development of N icotiana
and corn microspores grown on such complete medium after having been induced
towards embryogenesis. For Nicotiana, the induction requires two to three days
at 5° C for the intact buds followed by three days in the anther on the basal
medium in liquid culture prior to the isolation of the microspores. For maize the
induction requires low temperature followed by colchicine. The results with Zea
mays L. are still very preliminary and unpublished.
4.2 Effect of the Environment
4.2.1 Temperature
As for any culture, the temperature at which the cells are grown varies for each
species, for example, we have shown that N. tabacum gives more plants at 27° C
than at 22° C. As a rule, one can say that the optimal temperature for pollen
development for a species is a few degrees higher than the temperature under
which undifferentiated callus tissue grows best.
4.2.2 Light
Plantlets can develop from anther cultures kept in the dark. However, it has been
shown that placed in the light, the anthers will develop many more and stronger
plants. A series of preliminary experiments done on the -effect of light on the
growth of plantlets from isolated micros pore cufture is summarized in Figure 8.
As one might expect, microspores lacking protection from the anther tissue are
276 C.NrrSCH
Nicotiana
3 days 10days 21 days

Maize
10 days 21 days 35days

Fig. 7. In vitro behavior of isolated micros pores of tobacco and maize. Upper N icotiana taba-
cum, 3, 10, and 21 days in culture Lower Zea mays L. 10, 21, and 35 days IJ:l culture (both in
the same complete medium described in the text)
more sensitive to light than when they are grown inside an anther. Red fluorescent
light or low intensity white light (500 lux) gave the best results. From these
experiments we could also see that the sensitivity of the micros pore to light is
more important during the first ten days of the culture. Moreover the microspores
grown under red fluorescent light developed faster than those under white light.
.r:.300
<II
'ij
......
a.
~200
C
a
.
!!
Fig. 8. Effect of various types of

light on the growth of N. tabacum o 100
microspores. Average number of ~
O~ ~ ~LL--
E
plantIets from 5 dishes after one
month in culture. D total darkness,
Z'"
B blue intensity (500 lux) same as o B H L R
H, R red fluorescent light Effect of light
In the red-light-grown culture, the small embryos were already visible with the
naked eye after ten days while 15 days were necessary for cultures kept under low-
intensity white light.
5. Advantages of Isolated Microspore Culture

over Anther Culture
5.1 The Influence of the Anther Tissue Can Be Detrimental
1. Although anther culture of about two dozen species as different as N icotiana

and Oryza or Triticum has been successful in producing haploid plants, the num-
ber of plants produced is still very limited and many attempts were unsuccessful. -
In most cases however, the first divisions of the microspore were observed in
culture. This interruption in the development of the micros pore may be due to
growth-inhibiting substances leaking out of the degenerating anther tissue.
2. In other instances when no inhibition is observed, the diploid tissue, more
precisely the connective tissue, is growing very actively. The growth of this diploid
tissue is competitive with the growth of the haploid micros pore which is very
soon submerged by a profuse diploid callus. One commonly reads in the literature
that various workers culture anthers on media containing growth substances and
leave them for several weeks or even months. In such cases the haploid tissue
competes with the other tissue but the presence of growth substances in the
medium inhibits the developments of the embryo. The cells originating from the
microspore, because of the presence of hormones in the medium, become undiffer-
entiated and grow as a callus. Taking the microspores out of such an environment
and placing them on a medium with sugars and amino acids rather than auxins
will preserve the embryogenic capacity of the micros pore and produce haploid
plantlets in large number. On the other hand if one lets the haploid callus grow
and then induce it to regenerate plants, it is well known that the ploidy of the plants
thus obtained will be very variable and numerous chromosomal alterations and
anomalies are generated during culture of undifferentiated tissue (see Chap. 111.1).
278 C.NrrsCH
It is therefore suggested that micros pores should be liberated from the anther
tissue just after the first divisions are observed. However more work is needed to
be able to induce this first division in the isolated cells.
5.2 A Homogeneous Population Will Allow Better Experimentation
1. Unlimited numbers of cells which develop freely into embryos in a liquid

medium provide an ideal system for morphological and biochemical analysis.
This technique might help us to understand more about the difTerentation of root
and shoot from an isolated cell.
2. Besides the fundamental research which is possible with this new technique
of isolated microspore culture, one important field of research is that of genetic and
plant breeding. The production of large numbers of haploid plants and homozy-
gous diploids is a big step forward for geneticists.
The isolated microspores mixed at random will give a population more homo-
geneous than anther culture, moreover the efficiency of mutagenic treatment is
greater for single cells than for cells surrounded by another tissue. The techniques
used in microbiology for plating as well as for selection of a given character are
directly applicablt; to the microspore culture.
To conclude, it is shown that isolated micros pore culture present all the
advantages of anther culture plus many more. The number of plants obtained is
higher, and the time required to produce them is shorter. It is now possible, at
least for Nicotiana and Datura, to start from a microspore and by doubling the
chromosome number of the microspore, collect seeds from the homozygous di-
ploid five months later. This technique, when applied to the cereals and other
plants of economic importance, will be an excellent tool for the plant breeders.

3. Haploid Induction in Cereals
D.H.CLAPHAM
1. Introduction
The first confirmed haploid cereal arose parthenogenetically from 1riticum com-
pactum after a cross with Aegilops cylindrica (GAINES and AASE, 1926). By 1940, the
sporadic occurrence of haploid plants, spontaneously or following experimental
treatment, had also been described for Hordeum, Oryza, Secale and Zea (KIMBER
and RILEY, 1963). Haploids were used by cytologists to reveal the tendencies for
chromosomes to pair non-homologously (see DARLINGTON, 1965).
Two encouraging recent developments are the discovery that the cross be-
twen Hordeum vulgare and H. bulbosum yields haploid H. vulgare progeny
(KASHA and KAO, 1970), and that anther culture can yield haploid plants
(GUHA and MAHESHWARI, 1964, 1966). Of the many methods that have been
proposed for raising haploids of higher plants, anther culture has given most
promise of being both effective and of general application. This simple technique
was soon extended to rice with some success (NIIZEKI and OONO, 1968, 1971);
immature anthers containing uninucleate microspores were cultured on ,agar cul-
ture medium in test tubes. After about two months, calli appeared from up to 9%
of the anthers and sometimes could be differentiated to give haploid plants.
Further studies confirmed that the calli developed from the microspores rather
than from anther somatic tissue. Barley, wheat, Tritica~e and rye are other major
cereals from which haploids have been obtained through anther culture (see
Table 1). However at present yields are not high enough.,
This article reviews progress in anther culture of cereals, considers its merits
and shortcomings in comparison with other methods of raising haploids, and
discusses the use of haploids in cereal breeding.
2. Methods of Haploid Induction in Cereals

2;1 Anther Culture
2.1.1 Procedures with Cereal Anther Culture
How the parent plants are grown, from which anthers for culture are to be taken,
can strongly 'influence the yield of plantlets. For example, barley gives better
results with plants grown out of doors than in the greenhouse; there are seasonal
drifts in yield (DALE and I-lUMPHREYS, 1973); and results are poor with material
grown out of season under lights (see also 2.1.6). The genotype of the parent
Table 1. Summarising the results obtained with anther culture of various cereals N
00
0
Species An- Induction medium Initial % of b Embryo Regeneration Nature of Reference
thera conditions productive or medium plantlets
stage Minerals" Growth of anthers callus
(and % sucrose) co~onent incubation
(m )
Aegilops caudata x 2-3 Miller (3%) 2,4-D2 25° C, C Omit 2,4-D Albino, haploid KIMATO and
umbellulata 100 lux SAKAMOTO
(No success with (1972)
6 other Aegilops
sp.)
Hordeum vulgare 2-3 MS (12% or e.g. TIBA 0.02 22-27° C, up to 30 C No change Majority CLAPHAM
cv Sabarlis 3%) or 16 h light or albino, up to (1971, 1973)
IAA 1, BAP 1, 15W/m 2 11 40% green
CM 10% ploidy x,2x
alanine 400 or4x
H. vulgare MS (12%) e.g. IAA 1.5, 26° C, up to 6 C e.g.MS Green and MALEPSZY
cv Amsel and BAP 1.5, 2000 lux (sucrose 3%) albino, x, 2x, and
others 2,4-DO.5 +IAAO.2, and GRUNEWALDT
BAPO.4, polyploid (1974)
CMIO%
Oryza sativa 2-3 Miller (3%) NAAI 28° C, dark up to 9 C No change Green or NnZEKI and
japonica or or or albino, OONO (1968,
various cvs, also MS(3%) 2,4-D 2-10 basal + IAA 2, ploidy x-5x 1971);
japonica x indica K4 NISHI and
hybrids MrrsUOKA
(1969)
!='
Oryza sativa indica 2-3 Miller (3%) CM 15%, 25° C, dark up to 26% E Omit auxins Green, haploid GUHA et al. ;:t:
Of 18 cvs, YE 1000, IAA 2, (1970), l:l
t"'
2 primitive ones 2,4-D2, K 1 GUH,A- >
."
from Assam best or CM 15% MUKHERJEE
(1973)
=
~
Table 1. (continued) ::x::
II>
"e.
Species An- Induction medium Initial %ofb Embryo Regeneration Nature of Reference
thera conditions productive or medium plantlets ~
stage Minerals· Growth of anthers callus Q.
-=
(and % component incubation c:(')
sucrose) (mgjI) -o=·
Oryza sativa 2-3 Miller (6%) e.g. YE 1000, 18-26° C, up to 50 C Basal+IAA 6-87% of 2nd Division S·
(")
ssp Hsien, 2,4-D2 9-11 h light 0.2-2, plantlets albino, (1974) t1>
Ken~ and F1 1500 lux KO.2-2 rest green.
...
t1>
hybnds Ploidy x-4x, ~

nearly 50%
diploid
Secale cereale 1-5 Nitsch (3%) or 2,4-D 0.1-0.5 or 25° C, dark 0,14 EorC No change Green or WENZEL and
F 1 hybrid winter MS (3%) 2,4-D 0.25 albino, THOMAS (1974),
types including ploidy x-4x THOMAS and
genes for short WENZEL
culm and self- (1975b),
fertility from THOMAS et al.
S. vavilovii (1975)
S. montanum 2-3 MS (12%) e.g. IAA 0.2, 24-26° C, 1 EorC Plantlets not ZENKTELER and
CH200 dark or light, obtained MISIURA (1974)
1500 lux
Setaria italica 2-3 Miller (3%) YE 3000, 28°C, C Change Green, haploid BAN et al.
2,4-D 1, dark hormones or diploid (1971)
IAA 1, K 1, or tolAA2,
2,4-D 1, K2 K2-4
Triticale 2-3 MSJ6%) e.g. 2,4-D 2, 25-30°C, up to 17 C Basal + NAA 29 albino, WANG et al.
CMI5% light O.2,CM 15% 12 green, (1973b)
1 albino-green,
all haploid
Triticum aestivum 1-3 MS(3-11%; 2,4-D 0.7-2, 15-26° C up to 3 CorE MS (sucrose One third OUYANG et al.
Winter and better at higher LH 200-300 or 9-11 h light 3%), IAA 0.2, albino, rest (1973)
spring cvs and concns; (for embryos) 1500 lux KO.2 green, haploid N
F1 hybrids usually 6%) CM30% or occasionally 00
diploid -
Table 1. (continued) N
~
Species An- Induction medium Initial % of b Embryo Regeneration Nature of Reference
ther a conditions productive or me<lium plantiets
stage Minerals c Growth of anthers callus
(and % co~onent incubation
sucrose) (m )
T. aestivum 2-3 MS (6%) 2,4-D 2, K 3 or 17-28° C, 1 CorE Basal + 9 green, WANGet al.
Spring wheat (for embryos) dark NAA 0.2, K 1 7 albino, (1973a)
hybrids CM20% haploid
T. aestivum 4 Miller (12%) e.g: 2,4-D 0.4 21-23° C, 0.5 C Miller Green and PICARD and
Spring and winter 16 h light (sucrose 2%) albino, DEBUYSER
cvs; male-sterile IAAO.2 haploid (1973)
line; F 1 hybrid
T. aegi/opoides White (3%) 2,4-D20 25°C, dark 3 C Callus only, FUJII (1970)
haploid
T. dicoccoides White (3%) 2,4-D20 25° C, dark 18 C Callus only,
(No success with haploid FUJII (1970)
T. monococcum,
durum, aestivum
and speltum)
Zeamays 2-3 White (2 %) or e.g. 2,4-D 3-5 0.4 C e.g. basal Roots only, MURAKAMI
Miller (3%) +2,4-D2 haploid? et al. (1972)
a
Anther stage, see Table 1.
b
'% of productive anthers' is a rough guide only. Sometimes the figures apply to short-term experiments with specific varieties, sometimes P
to average results over a whole season. p::
c (')
MS=minerals after MURASHIGEand SKOOG (1962); Miller = minerals after MILLER (1963); Nitsch = minerals after BOURGIN and NITSCH (1967); t'"
White = minerals after WHITE (1943).
CH = casein hydrolysate, CM = coconut milk, LH = lactalbumin hydrolysate, YE = yeast extract, BAP= 6-benzylarninopurine, TIBA = triiodobenzoic ~
acid, K = kinetin. ~
Haploid Induction in Cereals 283
Table 2. Suggested standard nomenclature for anther staging. (After SUNDERLAND, 1974)
Stage 1: Tetrads or young microspores just released from callose wall. G 1 of the cell cycle.
Stage 2: Midphase microspores. Exine well-developed. Vacuole present but nucleus still in
G1.
Stage 3: Latephase microspores. Vacuole present. Nucleus in S phase or G2.
Stage 4: First pollen grain mitosis.
Stage 5: Generative and vegetative nuclei present. Generative nucleus cut otT by a wall.
Microspore vacuole still present.
Table 3. Influence of genotype on the growth response of wheat anthers cultured at

uninucleate pollen stage. (After BAlAl, 1976)
Cultivar Number of % of anthers

anthers cultured forming callus
1. Maris Ensign 180

2. Rothwell Sprite 120 1.6
3. Janus 310 1.6
4. Chinese Spring 250 1.6
5. Kolibri 800 1.7
6. Tilli 421 1.6
7. Cardinal 329 1.5
8. Maris Freeman 180 1.1
9. Maris Widgeon 75
10. Maris Huntsman 129 0.8
11. Maris Templar 111
12. Maris Dove 85
13. Maris Nimrod 167
14. Luna 129
15. Jubilar 3 151
16. Golden Valley 86
17. Champlein 141 1.7
18. Bersee 175 0.6
19. Rampton Rivett 172
20. 1877 209
21. Benno 780 114
plants, as well as their physiological status, affects success with anther culture,
some varieties being much more responsive than others (see GUHA-MuKHERJEE,
1973).
Inflorescences are removed and the stage of development established by
crushing an anther in acetic orcein or carmine and examining the pollen under the
microscope. Anther:s from stage 1 to stage 5 (Table 2), are recommended for differ-
ent cereals. The inflorescence is usually surface-sterilized with 5% hypochlorite
solution, but one can often omit this and simply remove the anthers aseptically
from the florets in a sterile cabinet. The anthers are then placed on an agar culture
284 D.H.CLAPHAM
medium in tubes or petri dishes, the composition of successful media being out-
lined in Table 2. With a few exceptions, the concentrated mineral solutions of
MURASHIGE and SKOOG (1962), MILLER (1963), and BOURGIN and NITSCH (1967)
are preferred to the weaker one of WHITE (1943); an auxin such as 2,4-D, NAA or
IAA is almost always included; and sucrose concentrations in the range 6-12%
often give higher rates of pollen callus formation than the 2-3% normally recom-
mended for plant tissue culture. Coconut milk, yeast extract, lactalbumin hydro-
lysate, and various cytokinins and amino acids sometimes help, but no generaliza-
tion is possible. The requirements for vitamins have not been critically examined.
The cultures are incubated, usually at 25-28° C and initially in darkness. Some
but not all rice strains gave higher rates of callus formation at 30° C than at 23-
26° C, but the higher temperature was less favorable for subsequent differentia-
tion (2nd Division, 1974). After 3-8 weeks the cultures are checked for anthers
forming callus or plantlets. These are put under lights directly or after transfer-
ence to fresh culture medium to encourage differentiation and further growth.
The media for differentiation or regeneration vary considerably (Table 1).
High concentrations of auxins such as 2,4-D are avoided, since they promote
callusing rather than differentiation; NAA or IAA and a cytokinin are often
added over the range 0.2-2 mg/l; the sucrose concentration is usually 2-3%;
natural extracts are sometimes included. Rates of differentiation of calli to give
green or albino shoots vary greatly/ with species and variety, as well as with the
composition of the culture mediurh, from a high of 90% (1riticale, WANG et al.,
1973b) to zero. Plantlets of rice and barley sometimes regenerate directly from
callus on the initial medium in the absence of strong auxins (NIIZEKI and OONO,
1971; CLAPHAM, 1973). Sometimes a plantlet grows directly out of an anther on
the initial medium without visible callus formation e.g. with rice (GUHA et aI.,
1970),wheat (OUYANG et al., 1973; WANG et al., 1973a) and rye (THOMAS and
WENZEL, 1975) although it may start callusing later if left on a medium with
2,4-D.
Green plantlets with a developed root system are transferred to compost and
grown further, preferably in a mist propagator at first. Then root-tips are taken
for chromosome counts. Plantlets of pollen origin from haploid to pentaploid
have been observed (Table 2).
It is often desirable to obtain homozygous diploids from the haploids, if
doubling has not occurred "spontaneously" in vitro. Colchicine has been used
with varying degrees of success (see Chap. 11.4; CHASE, 1969 for the doubling of
barley and maize haploids respectively). T~eeapping technique of BELL (1950) is of
wide application and neady 100% success with its use has been reported for
barley haploids (ISLAM and SPARROW, 1974). The use of the mitotic synchronizer
5-aminouracil before colchicine treatment has been recommended for grass spe-
cies (see CLARKE, 1969). The origin and significance of diploid plantlets direct
from anther culture is discussed in Sections 2.1.2. and 2.1.4. (See also Chapter 11.4.).
Haploids set seed in the field after spontaneous doubling at low frequency. In
maize about 10% give viable diploid progeny, in barley about 3% (CHASE, 1969;
SUBRAHMANYAM and KASHA, 1973). The rate is under genetic control. In rice it
can be increased by chronic gamma radiation, which is of value in mutation
breeding (TANAKA, 1970).
2.1.2 Pathways to Multicellular Pollen Grains
Anthers can be removed from culture at intervals, fIxed, and squashed in chroma-
tin stains and examined (see SUNDERLAND, 1974 for general review; CLAPHAM,
1971 for Lolium and barley; IYER and RAINA, 1972 for rice; OUYANG et al., 1973,
WANG et al., 1973a; PICARD, 1973 for wheat; SUN et aI., 1974a for Triticale;
WENZEL and THOMAS, 1974, THOMAS and Wenzel, 1975b, for rye. Also relevant,
8 C 0 E F
QG)~~-®
~
G® Q®
J L N P
(j)
~
~
Fig. 1 (\-R. Some stages in cereal pollen development, in vivo and in vitro. (A-F) normal
development in vivo. Stage numbers after SUNDERLAND'S (1974) standard nomenclature.
(A) Young microspore, vacuole undeveloped. Stage 1. (B) Mid- to latephase microspore, vac-
uole present. Stage 2-3. (C) The normal polarized first pollen mitosis. Stage 4. (D) Young
bicellular grain. Generative nucleus against the wall, usually on the opposite side from the
germ~pore. Vacuole (not shown) begins to regress. Stage 5. (E) After the second pollen mito-
sis. Starch grains begin to form. Vacuole (not shown) much reduced. (F) Mature pollen grain
packed with starch. Two sperm and a vegetative nucleus, no vacuole. (G) Bicellular grain of
wheat in vitro. (After OUY ANG et al., 1973). (H, I) Triticale, in: vitro. See text. (After SUN et
al., 1974a). (J, K, L, M) Bi- and multinuclear grains in vitro. Various possible origins, future
obscure. In some cases, thin cell wall is perhaps present. (J) Often seen in cereal anther
culture. (K) Rye (after WENZEL and THOMAS, 1974). (L,M) Lolium multiflorum (after CLA-
PHAM, 1971). (L) Generative- and vegetative-type nuclei in mitosis. (M) Two generative- and
two vegetative-type nuclei. (N, 0, P) Sorghum purpureo-sericeum (n = 5) with B chromosomes,
in vivo. Nucleoli shown, B chromosomes in black (after DARLINGTON and THOMAS, 1941), see
also text. (N) 6-celled pollen grain. (0) Vegetative cell and 5 generative cells derived from it;
strongly polarized arrangement. (P) Diploid nucleus in anaphase, after nuclear fusion. Lag-
ging B chromosomes. (Q) Rye, in vitro. Two triploid vegetative-type nuclei in mitosis and a
generative-type nucleus in interphase. (After THOMAS and WENZEL, 1975b). (R) Multicellular
pollen grain of barley. (After CLAPHAM, 1971)
286 D.H.CLAPHAM
though concerned mainly with anthers in vivo, are DARLINGTON and THOMAS,
1941, on B chromosomes in Sorghum and BENNETT and HUGHES, 1972, for effects
of ethrel on wheat). It is then seen that some of the microspores develop more or
less normally into starch-filled tricellular pollen; some die at one- or two-celled
stages; and some form multicellular or multinuclear grains (see Fig. 1 and
Table 2).
In barley, eight-celled pollen grains are formed from uninucleate micros pores
after six days of culture (Fig. 1). Some of them have nuclei in mitosis and are alive
and healthy when taken from culture; others were dead or dying. The
causes of embryo abortion are not well understood. The obvious possibilities
are: inadequate diffusion of nutrients; pr~sence of toxic products from the dying
anther wall; chromosome errors arising from various causes; in hybrid material,
action of deleterious recessives segregating after meiosis. The high death-rate at
each stage makes it difficult to establish the exact pathway(s) of development of
microspores that grow further into embryos or callus.
On the basis of work with Datura and N icotiana species, where androgenesis is
under good experimental control, SUNDERLAND (1974) distinguishes three routes
to embryogenesis. In route A, the microspore passes through the normal polar-
ized first pollen mitosis to form a vegetative cell with a large diffuse nucleus and a
generative cell with a small compact nucleus. Then the vegetative cell, and after-
ward~ its derivatives, divide repeatedly till an enlarged multicellular pollen grain is
formed. These cells have nuclei resembling that of the original vegetative cell. The
generative cell can also divide but only to a limited extent and the derivatives
eventually abort. In route B, the microspore enters a non-polarized mitosis to give
two cells of roughly equal size with diffuse nuclei. Then one or both continue to
divide to give a multicellular grain. Route C begins with a normal polarized first
pollen mitosis, as in route A. It is distinguished from route A by the participation
of the generative cell in embryogenesis. The wall between the cells breaks down,
the generative nucleus enters mitosis simultaneously with a vegetative nucleus,
and they divide on a common spindle to give two equal diploid cells. It is sup-
posed that these multiply to give a diploid pollen embryo. Variations of route C
can lead to triploid or tetraploid embryos. At present route C has been estab-
lished.only for Datura innoxia.
Nuclear fusion is not confined to pollen grains on route C. In Datura, haploid
nuclei on route B sometimes fuse to give diploid nuclei and it is at least possible
that vegetative nuclei on route A can fuse, although this has not been demon-
strated. Diploid and tetraploid nuclei can also arise without fusion, by endomito-
sis or endoreduplication. These events are common in callus cultures (see SUN-
DERLAND, 1973 a) and are believed to account for some of the non-haploid plant-
lets from rice anther culture (NnZEKI and OONO, 1971).
Route A as well as route B seem to occur in wheat (OUYANG et al., 1973) and
probably in Lolium and barley (CLAPHAM, 1971), and route A occurs in rice (IYER
and RAINA, 1972). In Triticale, SUN et al. (1974a) have described a pathway that
can be regarded as a form of route B. The cells or more often free nuclei arising
from the first pollen mitosis are physiologically different even when the nuclei are
of the same size. One nucleus, showing properties of the normal generative cell,
divides 1-4 times to form up to 16 fra: nuclei that stain intensely with Feulgen.
The second nucleus, surrounded by cytoplasm that stains intensely with pyronine,
after some delay divides repeatedly with wall formation to give the multicellular
pollen grain. The free nuclei are pushed 'against the pollen wall and lost when the
exine and intine split (Fig. 1). The same auth<}rs report that route A also occurs in
Triticale; the development is of the same type except that nuclei after the first
pollen mitosis are unequal in size.
In species such as barley and rye, where diploid and higher ploidy mitoses are
seen at early stages, it is likely that a variation of route C is involved at least
sometimes, but this has not yet been demonstrated. THOMAS and WENZEL (1975b)
have found a three-celled grain in rye anther culture containing two triploid
nuclei of vegetative type in mitosis and a generative-type nucleus in interphase
(Fig. 1). This stage could be reached either by endomitosis and/or fusion of vege-
tative-type nuclei in route A, or, assuming two generative-type nuclei were origi-
nally formed, by a variation of route C (less probable). Triploid plantlets were in
fact obtained in addition to the diploid and tetraploid ones.,
The origin and future of the multinuclear grains often seen in cereal anther
culture (Fig. 1) are not well understood. Some may arise in vivo from irregular
cleavage at meiosis (SUNDERLAND, 1974) but others probably arise in vitro from
normal microspores by free nuclear division. In barley, pollen grains with up to 30
free nuclei are formed but they degenerate after 15 days of culture (DELANNAY,
personal communication).
Figure 1 shows in "ivo pollen grains and extra divisions induced by B chromo-
somes in Sorghum purpureo-sericeum (DARLINGTON and THOMAS, 1941). After a
normal but delayed first pollen mitosis, the vegetative cell could divide repeatedly
till 3-5 generative cells were piled up against the wall opposite the germ-pore.
Deviations from this pattern, ascribed by the authors to shifts of polarization,
could give multicellular grains resembling those seen in cereal anther culture (e.g.
Fig. 1 N). Nuclear fusion was also reported (Fig. 1 P). These authors speculate on
the causation of what they call "encapsulated tumors".
2.1.3 Pollen Embryos, Pollen Calli, and Somatic Calli

Some of the unaborted multicellular pollen continue to grow and organize into
structures that can reasonably be called "pollen embryos", particularly as the
early development of the true cereal embryo is quite flexible (MAHESHWARI, 1950).
Others, after breaking through the pollen exine and intine, form a less organized
"pollen callus" (see Fig. 2).
It is highly desirable if pollen embryos rather than callus are formed, since it
reduces the opportunity for chimaeras and also for the chromosome aberrations.
The ideal situation for the breeder is the production of doubled haploid embryos,
as in Datura innoxia (SUNDERLAND, 1974), and perhaps infrequently in rye
(THOMAS and WENZEL, 1975b).
Anther somatic tissue, e.g. the filament, also forms callus in some species (such
as wheat, OUYANG et al., 1973; BAJAJ, 1976; rye, WENZEL and THOMAS, 1974). It is
usually, but not always, obvious that this is happening.
288 D. H. CLAPHAM
Fig. 2 (A) Multicellular pollen grain in rye (WENZEL and THOMAS, 1974). (B) Rye pollen
embryo, with scutellum and ·coleoptile, after bursting through the anther wall. (THOMAS and
WENZEL,1975b)
Fig. 3 (A) Barley plantlet from anther culture. (B) Meta-anaphase of meiosis in haploid
barley from anther culture, showing five univalents and a bivalent. (CLAPHAM, 1973)
2.1.4 Testing for Homozygosity of Diploid Plantlets from Anther Culture

The origin of the diploid plants is important because of the varying genetic
consequences. Diploid plants from the anther somatic tissue of a hybrid plant
have the same hybrid genotype, and are of no interest. Diploid plants doubled
from normal haploid pollen are presumably homozygous and are certainly of
value. There is, however, another possible category of diploid plants, those de-
rived from unreduced pollen following total or partial suppression of meiosis
(SUNDERLAND et al., 1974). Methods of suppressing meiosis include failure of first
or second anaphase, or reunion of two daughter nuclei at second telophase.
Provided that pairing and crossing-over take place at the first meiotic division,
the eventual diploid micros pores are not only heterozygous but are of genotypes
differing from each other and from the parent plant (DARLINGTON, 1965). To
distinguish the desirable homozygous diploids from the undesirable heterozygous
ones is easy for self-pollinated species. It is only necessary that the parent plants
were heterozygous for genetic markers. Then the plants derived from anther
culture are selfed and the progenies examined for segregation. Such experiments
have been done with rice, barley and wheat.
In progenies of 16 diploid pollen plants from F 1 hybrid rice (2nd Division,
1974) 15 were strictly uniform and were presumably homozygous. The 16th segre-
gated for plant height and panicle form, but the segregation was far simpler than
in the progeny raised from seed from the F 1. Probably mutation(s) occurred in
diploid cells in vitro.
Woo et al. (1973) obtained six diploid plants from cultivars Indica x Japonica
rice hybrids. Tpe phenotypes differed slightly among themselves and at first the
plants were thought to be of pollen origin (Woo and TUNG, 1972). But five of
them set seed, and the progenies were non-uniform, showing segregation for four
characters. Therefore, it was interpreted that these five diploid plants were in fact
heterozygous and derived from somatic tissue, and the original slight phenotypic
differences were ascribed to cytoplasmic effects. (An alternative possibility is that
the plants were derived from unreduced diploid pollen, see above.) The sixth
diploid plant was sterile, but had properties making it plausible that it was
derive<i from normal pollen (see also NnZEKI and OONO, 1971).
In progenies from 18 diploid pollen plants obtained from hybrid barley and
examined for segregation (JONES and PICKERING, 1973), all but one gave every
indication of being completely homozygous. The one exceptional progeny segre-
gated for a low temperature chlorina mutation but was otherwise highly uniform
(see Fig. 4).
OUYANG et al. (1973) studied a fertile plant derived by anther culture from a
hybrid wheat, and demonstrated exhaustively that the progeny was sufficiently
uniform to be considered homozygous. The authors implied that chromosome
doubling occurred during the callus phase in vitro rather than later in the field.
To conclude: diploid plants from cereal anther culture, unless clearly derived
from the filament, are on present evidence usually doubled haploids and homozy-
gous, but caution is needed. The issue is important, since nearly half of the rice
plants (2nd Division, 1974) and more than half of the barley plants (CLAPHAM, 1973)
are diploid.
2.1.5 The Albinos

Why do so many albinos, i.e. chlorophyll-deficient shoots, appear in cereal anther
culture? Table 2 shows that they are frequent in Aegilops, Hordeum, Oryza, Secale,
290 D.H.CLAPHAM
Fig. 4. Photograph showing the origin of diploid plantlets from normal haploid microspores
of barley. The diploid plantles were derived from anthers taken from hybrid barley; their
progenies, shown here, are each highly uniform but differ from each other with respect to
segregating marker genes. (Courtesy of the Welsh Plant Breeding Station)
Triticale, and Triticum. The albinos of barley and rice can be white, yellow, light
green and striped or chimerical in various ways. In barley (CLAPHAM, 1973) and
rice (SUN et aI., 1974 b) they have been studied electron microscopically. Some
sections through an albina and a xantha barley plant unexpectedly showed vir-
tually normal chloroplasts, with well-developed grana; these were nevertheless
either inactive photosynthetically, or lay in patches of normal tissue. More in
accordance with expectation, other sections showed only proplastids, with globuli
and sometimes starch grains and concentric lamellar systems, but without grana.
With the rice albinos, nothing more developed than proplastids with concentric
lamellar systems, the authors commenting particularly on the absence of ribo-
somes from the plastids; the rest of the cell was normal. A first point is that the
albinos do contain plastids, so that they have not originated from a cell com-
pletely lacking in plastids, as may be true of the generative cell.
Following are some ofthe possible suggestions for the origin of the albinos:
1. Pollen carrying chlorophyll mutations preferentially form plantlets in vi-
tro. In barley, the spontaneous rate of chlorophyll mutation was found to be
about six per 9000 spike progenies (NYBOM et al., 1956). This indicates, on certain
assumptions, an upper limit of about 3.3 chlorophyll mutations per 10000 ga-
metes. Such a rate is just high enough to make it possible (though unlikely) that
pollen carrying chlorophyll mutations form the albino plantlets in barley anther
culture. However, such an hypothesis cannot account for the albinos from wheat
anther culture, since wheat, being a hexaploid, displays spontaneous chlorophyll
mutations at an enormously lower rate-too low to measure accurately. It is
therefore reasonable to exclude "mutation" in the ordinary sense of the word as

the main cause of albinism in cereal anther culture.
2. The appearance of the proplastids in barley and rice albinos recalls the
regression of the plastids during meiosis, described for Lilium longiflorum by
DICKINSON and HESLOP-HARRISON (1970). The plastids regress between leptotene
and pachytene, losing the greater part of the lamellar systems and most of the
ribosomes. The mitochondria disorganize and at the same time the ribosomes are
lost from the ground cytoplasm of the pollen mother cell. Plastid lamellar systems
and ribosomes reappear at late tetrad and early micros pore stages. It is possible
that this re-organization is complete at a later stage of pollen development in
cereals than in Lilium, at least for a fraction of the pollen, and is inhibited in vitro.
SUNDERLAND (1974) has noticed that in barley cv Sabarlis grown at 20° C, 15-
20% of the pollen grains just before anthesis (in vivo) are small, with little staina-
ble cytoplasm, and were retarded in development. Similar pollen dimorphism
occurs in Petunia, and in rye (WENZEL and THOMAS, 1974)
3. Another possibility is that the plastids disorganize in vitro. Ultrastructural
studies on tobacco pollen show that during the initial inductive period of anther
culture the plastids in the vegetative cell undergo a fundamental re-organization;
it is suggested (SUNDERLAND, 1973 b) that errors could occur at this stage that are
not corrected. However such a process does not occur in Datura (DUNWELL and
SUNDERLAND, 1974) and has not been studied in cereals.
The peculiarities of pollen should not be overstressed, since cultured Grami-
naceous cells in general are liable to albinism. Thus GAMBORG et al. (1970)
obtained exclusively albino plants from a somatic cell culture of Bromus inermis;
and WENZEL and THOMAS (1974) obtained 50% albinos from the anther filament
of rye. In other families too it is often difficult to obtain green somatic callus
cultures (see EL HINNAWY, 1974). This would direct attention more to the condi-
tions of culture. Unfortunately attempts to raise the frequency of green plants by
alterations of the culture medium have not so far' been very successful with cereal
anther culture (CLAPHAM, 1973; DALE and HUMPHRIES, 1973).
2.1.6 Approaches to Cereal Anther Culture

Success with anther culture is strongly dependent on the genotype of the parent
plant (GUHA-MuKHERJEE, 1973 on rice; BAJAJ, 1976 on wheat). It may be possible
to exploit a genotype that responds favorably to anther culture by using it as one
of the parents in a cross with the hope that the haploids are easily obtained from
the hybrids as well.
There have been various proposals for adjusting the physiological status of the
parents before anther culture. PICARD (1973), who emphasizes the importance of
route B development in which the first pollen mitosis is non-polarized and results
in two equal cells, recommends cold shocks to the wheat spike before anther
culture. This is believed to promote the non-polarized mitosis and formation of
pollen callus. An alternative approach is to spray plant growth substances on the
plants before anther culture (PANDEY, 1973). Ethrel (2-chlorethylphosphonic
acid), a source of ethylene and used as a male-sterilizer in several crop plants, can
292 D. H. CLAPHAM
induce additional mitoses in wheat (BENNETT and HUGHES, 1972), rice (WANG et
al., 1974) and Petunia pollen in vivo (BAJAJ, 1975).
As regards modifications of the culture medium, the inclusion of activated
charcoal has recently been recommended for anther culture (NAKAMURA and
ITAGAKI, 1973; ANAGNOSTAKIS, 1974; BAJAJ et al., 1976). Eventually anther cul-
ture can be expe<;ted to give way increasingly to pollen culture (NITSCH, 1974a);
the recent work of WENZEL et al. (1975), in which isolated rye microspores devel-
oped into multicellular structures, is the °first success of this kind with cereals.
2.2 Other Means of Haploid Induction in Cereals

The following brief account is an attempt to put aather culture in perspective as a
means of raising haploids in cereals. It is mainly concerned with work appearing
since the reviews of KIMBER and RILEY (1963) and MAGOON and KHANNA (1963).
2.2.1 Use of the Cross H. vulgare x H. bulbosum

This valuable technique is reviewed by JENSEN (Chap. 11.4 of this vol.). BARCLAY
(1975) has recently obtained haploid wheat plants at high frequency after crossing
Triticum aestivum cultivar Chinese Spring with H. bulbosum.
2.2.2 Use of Maize "Stock 6" and Its Derivatives in Conjunction with Genetic
Markers
Work on maize haploids is well developed (CHASE, 1969). The spontaneous rate of
haploidy in maize normally varies from 0.1 to 1 per thousand depending on the
material examined (RANDOLPH, 1940); however, COE (1959) discovered a genetic
strain, "stock 6", that on selfing gave the very high rate of 343 haploids per 10,616
plants (3.2%). An important feature of this strain is that it can be used as pollen
parent for inducing haploids in other genetic material. For example SARKAR et aI.,
(1972) developed a derivative of "stock 6" suitable for certain conditions that
induced a haploid frequency of 1.65% on four diverse female lines. The haploids
were recognized by screening for an appropriate genetic marker. An effective
method applicable when the female line is homozygous for factors determining
colored aleurone and colored scutellum was to cross with the "stock 6" derivative
(homozygous for the color inhibitor factor C 1) and then screen for kernels with
coloreq scutellum and colorless aleurone. These are the haploids, in which the
embryo has developed parthenogenetically and the endosperm after fertilization
(COE and SARKAR, 1964). This method does not require the seed to be germinated
in order to identify the haploids, so that thousands of kernels can easily be
screened. Even if only one in 200 are picked out as haploids, the method is
thoroughly practical. An effective means of doubling maize haploids has, how-
ever, yet to be found (CHASE, 1974).
2.2.3 Androgenesis and the "Indeterminate Gametophyte" Mutation in Maize

KERMICLE (1969) has discovered an interesting mutation ig "indeterminate game-
tophyte" in a highly inbred strain, Wisconsin-23. About 3% of the embryos in ig
embryo-sacs are paternal haploids. Such androgenetic haploids differ from those
obtainable from anther culture in that the cytoplasm comes from the embryo-sac.
Their special use is discussed in Section 3.4. The rate of spontaneous androgenesis
in maize is otherwise very low, about one in 80000 (CHASE, 1969).
The general value of the ig mutation is unknown. The rate of androgenesis is
strongly dependent on the genotype of the pollen parent, although the ig muta-
tion is strictly sex-limitea to the female parent and acts only in the embryo sac.
Other "effects of the ig mutation are: (a) half the kernels on ig ig plants and one
quarter on ig ig have higher than normal endosperm ploidy level, (b) about 6% of
the kernels with normal endosperm have more than one embryo.
2.2.4 Effect of Aegilops Cytoplasm on Cultivar Salmon of Triticum

KIHARA and his associates (see TSUNEW AKI et al., 1974) have found that the
cytoplasm of Aegilops caudata and five other Aegilops species induce at very
high frequency haploids (11-56%) and twins (0.5-15%) in the progeny of a bread
wheat variety, Salmon. The large majority of the twins were haploid-diploid. The
special feature of Salmon wheat is that it is a Triticale derivative, overwhelmingly
Triticum, but with a small part of chromosome lB including the nucleolar orga-
nizer replaced by a piece of rye chromosome lB, and two other minor changes.
Single and twin haploids arise from the parthenogenetic development of the egg-
cell; the diploid partner of a haploid-diploid twin arises by fertilization of a
synergid. The plants with Aegilops cytoplasm and Salmon nucleus were produced
by successive backcrossing of the initial hybrid to Salmon as pollen parent. They
are male-sterile but female-fertile. The frequency of haploids in the progeny de-
pends on the pollen parent used, as so often in haploid parthenogenesis, but is
always unusually high. Unfortunately it is not easy to exploit the special features
of this system.
2.2.5 Twins
A favorite old method of finding haploids is by searching among twin seedlings.
In cereals this is not very effective. Even the case of Aegilops-Salmon wheat
described above is not an exception, since the haploids are more frequent as
singles than as twins. KAWAKAMI (1967), in a large survey of cereal twins, quotes
frequencies of twins from his own results as 0.034% in Triticum (Einkorn, Emmer
and common wheat), 0.032% in Hordeum, 0.227% in Secale, 0.059% in Avena,
and 0.019% in Oryza. These were nearly always of the diploid-diploid or diploid-
triploid type. The highest frequency of haploid-diploid twins occurred in Triticum
(1 in 25) compared with 1 in 200 for Avena and none for Secale.
2.2.6 Colchicine-Induced Somatic Reduction

FRANZKE and Ross (1952) reported that "Experimental 3", a partially inbred
selection from an interspecific Sorghum cross~ gave a number of true:breeding
plants after treatment with colchicine. These proved to be doubled haploids (see
KASHA, 1974b). It was also possible under special conditions to raise undoubled
294 D.H.CLAPHAM
haploids. For example, CHEN and Ross (1963) treated 20 diploid derivatives of
"Experimental 3" with colchicine under red light at 20° C and high humidity.
Three out of the four survivors were haploid. Similar colchicine effects have been
reported for the barley cultivar, Decatur (GILBERT, 1963). Since special colchicine-
reactive cultivars are required, the phenomenon seems to be of limited applica-
tion.
2.2.7 Fluorophenylalanine-Induced Reduction

Para-fluorophenylalanine is used to induce haploid sectors in diploid colonies of
certain fungi, such as Aspergillus niger (LHOAS, 1961) and Ustilago violacea (DAY
and JONES, 1971). Its effects have also been studied in higher plants (see Chap.
II. 1). NITSCHE (1973) treated hybrids of Festuca pratensis x Lolium multiflorum
(2n = 4x = 28) with 3-fluorophenylalanine and examined root tips for chromosome
counts. Cells most frequently contained 21 chromosomes, but sometimes as few as
seven. Cells with anomalous counts were still present four months after treatment.
Fluorophenylalanine is worth further study with cereals.
3. Uses of Haploids in Cereal Breeding
3.1 Production of Homozygous Varieties in Self-Pollinated Cereals
The obvious application of haploid techniques to cereal breeding is as a quick

route from heterozygotes to homozygotes (EAST, 1930). In self-pollinated crops,
this is usually done by five to seven generations of self-fertilization. The alterna-
tive is to raise haploids from the heterozygous material and then double the
chromosome number by colchicine or otherwise. The resulting diploid plants are
strictly homozygous except in as much as colchicine may promote mutation. The
conventional method takes three years, even if two generations can be grown each
year, whereas the homozygotes are available in one year if haploids are used.
Whenever breeding program begins with a cross to give hybrid material from
which pure or nearly pure lines must be derived, as is usually the case, the haploid
method appears to offer advantages. Yet plant breeders are not always convinced.
It is best to compare briefly the merits and shortcomings of established methods
with those of haploid techniques. The established approach of the pedigree
method is discussed here.
In the pedigree method, selected parents are crossed, the F 1 seed grown, and
then succeeding generations raised by self-pollination till a high degree of homo-
zygosity is attained in about the F 7 • The plants are spaced to permit strong
selection from the F 2 onwards, at first on a single plant basis to eliminate geno-
types with undesirable major genes, later on a family basis to improve complex
characters such as yield. The important feature of the pedigree method is the ~ay
in which selection, assessment and to some extent multiplication of the new
potential variety is carried out while at the same time the material becomes more
homozygous. An unpromising cross can be abandoned completely at the F 2 stage.
Drawbacks of the method are that it is time- and land-consuming, and th~ breeder
tends to be confused by heterozygous advantage at the early stages 6f selection.
The drawbacks can be reduced by using the compromise method outlined by
MAC KEY (1962), ,in which selection is postponed till the F 4' followed by reselec-
tion at a later stage.
The procedure with haploid techniques might be as follows. The initial cross is
made, the F 1 plants grown, haploids are derived from the F 1 and doubled to give
homozygotes. At this stage some selection for simple characters may be possible,
but normally the doubled haploids must be allowed to set seed and the progenies
examined; otherwise the effects of colchicine or the tissue culture origin of the
plants will interfere with assessment, which in any case is very difficult on a single
plant basis. In fact, if seed on the doubled plants is set in the greenhouse, it may be
best to grow another generation in the field before comparing with control varie-
ties, raised from seed set in the field (WALSH et al., 1973). If this is done, the time-
saving potential of the haploid method can be reduced but not eliminated.
What do the different approaches offer? The haploid technique probably
saves two or more years as compared with the pedigree method. In terms of labor,
the advantage is less obvious, although quantitative comparisons of the methods
are hard to make. NEI (1963) in his interesting analysis begins by supposing that
the initial cross differs with respect to n genes, presumed unlinked. He then points
out, in effect, that a minimum of ~" plants must theoretically be grown for all
homozygous genotypes to be represented using haploid techniques, whereas 4" is
the corresponding figure using conventional breeding methods. This does not
really do justice to the pedigree method, the essence of which is that the unprom-
ising plants or segregating families are rejected in the early generations of selfing,
thus removing the need for growing all genotypes.
If, for example, the parents in the initial cross differed by as few as ten genes,
presumed unlinked, a minimum of 410 = 1024 doubled haploids would have to be
grown for the chance occurrence of all segregants, including the optimal one.
KASHA (1974b) estimates that with the Hordeum vulgare x H. bulbosum tech-
nique, 100 doubled haploids of barley can be raised per :worker per week, so that
one person would have to work for 2112 months. Far more plants need to be
grown using the pedigree method, but handling them is for the most part easy and
rapid. In practice, a breeder wishes each year to test many crosses, in each of
which a hundred or more genes are segregating. There is then no chance of
securing optimal combinations of genes; it is rather a matter of rejecting most of
the material with a minimum of effort. In such circumstances haploid techniques
are too laborious. They have the further disadvantage, as compared with the
pedigree method, of restricting the number of opportunities for tight linkages to
be broken by crossing-over. For a fuller analysis arriving at broadly similar
conclusions see WALSH (1974).
Haploid techniques have' perhaps most potential when selecting for characters
that must be assessed by a complicated test, such as a chemical analysis for
quality, and' when the character is under the control of a number of genes with
dominant effect (MELCHERS, 1972). These circumstances are, however, ratheli spe-
cialized. Haploid techniques might also conceivably be used in the breeding of
homozygous parents for hybrid varieties of wheat and barley (MELCHERS, 1972).
296 D. H. CLAPHAM
The newly-developed doubled haploid stocks would initially be highly uniform, a

possible attraction; but in the course of seed multiplication, owing to mutation
they are likely to become as variable as ordinary inbred lines.
3.2 Production of Hybrid Varieties in Cross-Pollinated Cereals
The ftrst step in the breeding of cereals such as maize and sorghum is usually the
derivation from heterozygous material of pure lines for use as parents of the
intended single-cross or double-cross hybrids. The haploid method can be used to
produce these pure lines with considerable saving of time. In fact, with maize
there is much practical experience of using the haploid method, described by
CHASE (1969). It has been employed successfully (CHASE, 1974) in a commercial
plant breeding venture, even without the beneftt of a high-frequency haploid-
inducing line such as "stock 6". It can be expected to become more popular with
maize breeders now that such lines are available.
Criticisms sometimes made of haploid techniques are:
1. With haploid techniques, there is only one opportunity for intrachromoso-
mal gene recombination to occur, whereas with conventional inbreeding it can
occur at each generation ofselfing. CHASE (1969) writes'however: "It is quite clear
from theoretical considerations that if intrachromosomal gene recombination is
required, it is more efficient to provide opportunity for recombination to occur
before inbreeding is initiated than during the inbreeding process." This drawback
of rapid inbreeding is recognized by maize breeders, who frequently adopt the
procedure known as "recurrent selection". This is intended to allow maximum
opportunity for recombination and to concentrate favorable genes. Cycles of
crossing among selected parents and reselecting among the progeny are inter-
posed between successive generations of self-pollination (see ALLARD, 1964). This
makes the production ofinbred lines into a lengthy thoughtful process, to which
the rapid haploid technique may be preferred.
2. Cross-pollinators tend to resist rapid approaches to homozygosity, owing
to the segregation of deleterious recessives and to the other causes of inbreeding
depression. Probably for this reason, it initially proved very difficult to raise
haploids of rye unless inbred lines were used as starting material (NORDEN-
SKIOLD, 1939). Similarly with maize, haploids are easier to induce in inbred than
in heterozygous material (CHASE, 1969). It is therefore sometimes claimed that the
haploid method is too extreme for use with cross-pollinators. Alternatively one
might suppose that a technique such as anther culture is very effective at picking
out the few viable combinations of genes that can be ftxed in homozygous diploid
form. Indeed recently, doubled haploids have been obtained from. hybrid rye by
anther culture (THOMAS and WENZEL, 1975 b).
3. SPRAGUE (1967) depreciated haploid techniques by claiming that the prob-
lem in maize breeding is the recognition and testing, not the production, of inbred
lines suitable as parents of hybrid varieties. However CHASE (1969) thinks that
breeders too often laboriously accumulate unmanageable numbers of unremarka-
ble inbreds when they would be better advised to try for excellent ones rapidly via
haploid techniques.
3.3 Use of Haploids in Mutation Breeding

The use of haploid cells and protoplasts for mutation experiments is discussed in
Chapter V of this volume. Alternative approaches to haploid mutation breeding
are: (1) to induce mutants by treating florets with acute doses of X- or y-radia-
tion, or with chemical mutagens before anther culture, (2) to incorporate a chemi-
cal mutagen into the culture medium, (3) to treat haploid plantlets with an acute
dose of a mutagen (DEVREUX and DE NEITANCOURT, 1974) or (4) to grow haploid
plants in a gamma field and expose to chronic gamma radiation (TANAKA, 1970).
Approach (1) has the advantage of avoiding the formation of chimeras that will
arise if whole plants are treated with a mutagen.
The obvious advantage of haploids is that they display mutations with reces-
sive effects in single dose. This is a very important point if mutagenesis and
selection is being applied to cell cultures. Otherwise the advantages of haploid
techniques in cereal mutation breeding seem to lie particularly with the ease of
selecting micromutants from the genetically homogeneous M2 progenies ob-
tained from doubled, mutated haploids, as compared with the genetically hetero-
geneous M2 progenies if the mutagen is applied to diploids.
To illustrate, TANAKA (1970) has experimented with haploid rice plants multi-
plied vegetatively from a single genotype, and irradiated in a gamma field at
various doses for a hundred days. Tillers propagated from buds that had been
irradiated at an early stage of development frequently gave diploid panicles with
fertile seed: 152 panicles with at least five fertile seed were obtained from 9315
plants derived from 440 irradiated plants. More than half of the radiation-induced
diploid panicles gave pure lines, about 40% being mutant with small but signifi-
cant changes of character. The conclusion is that irradiating haploid plants in a
gamma field is an effective way of producing pure lines with micromutations.
The reason why nearly half of the radiation-induced diploid panicles gave
genetically mixed lines, often segregating for gross mutations, is presumably be-
cause mutations were sometimes induced after diploidization. This clearly re-
duces the efficiency of the method, which remains, nevertheless, still considerable.
3.4 Miscellaneous Uses
CHASE (1952) suggested the use of androgenesis in maize as a technique for the
one-step transfer of cytoplasm from one line to another. The idea is that an
androgenetic haploid, originating from a male gamete (virtually devoid of cyto-
plasm) and developing without fertilization in the embryo sac, should have the
pollen parent's nuclear genotype and the seed-bearer's cytoplasm (see CHASE,
1969). If the seed-bearer has cytoplasmically determined male sterility, the (dou-
bled) androgenetic haploid should have the same. This has been confirmed experi-
mentally by GOODSELL (1961) and KERMICLE (1973). On occasions, however, an-
drogenetic plants are unexpectedly male-fertile instead of male-sterile, the reason
for which is obscure (CHASE, 1969). Of course, androgenetic plants from anther
culture are of no use here.
Of the many other uses of haploids in cereal botany, following are some of the
examples. (1) A cytological study of haploids led to the discovery of the control of
298 D. H. CLAPHAM
disomic inheritance in wheat by chromosome 5B (see KIMBER and RILEY, 1963).

(2) Haploid maize has been used in the study of the synaptonemal complex (GIL-
LIES, 1974). (3) Further applications to cereal breeding are conceivable (e.g. 4x
derivatives from 8x Triticale). (4) Doubled haploids from self-incompatible
plants such as rye should be homozygous at the incompatibility loci-a condition
hard to obtain by other means.
4. Conclusions
Progress is being made with cereal anther and pollen culture and a degree of
success is reported for a number of species; however, the techniques need consi-
derable further development. The yield of plantlets is usually low and unreliable;
the pollen rarely organizes directly into an embryo, but passes through a callus
phase, with the result that chromosome aberrations may arise; albinos rather
than green plants too frequently differentiate from ,the callus. It is likely that in the
near future some of these problems will be overcome. If doubled haploid embryos
and plantlets can be obtained in reasonable yield direct from culture, as with
Datura innoxia (SUNDERLAND et al., 1974), the method would be particularly
attractive as avoiding the need for later colchicine treatment.
Another in vitro method, very effective, is available for barley and wheat, and
depends on chromosome elimination in the embryo resulting from a cross with
H. bulbosum. In maize, parthenogenesis and androgenesis in vivo can be exploited
as routes to haploidy.
Cereal haploids will probably make their greatest impact on plant breeding
indirectly via cell and protoplast culture and advances in biochemical genetics. In
the nearer future, haploids can be used effectively in the production of parents for
hybrid varieties, particularly of cross-pollinated cereals. They also show promise
in connection with mutation breeding and research.
Acknowledgments. I am grateful to the Editors, to Dr. T. Eriksson, and to my colleagues
at the Dept. of Genetics and Plant Breeding for help with the manuscript, and to Dr.
K. Deiannay, Dr. N. Sunderland and Dr. E. Thomas for sending copies of their unpub-
lished papers.

4. Monoploid Production by Chromosome Elimination
C.J.JENSEN
1. Introduction
The production of monoploids in relatively large frequencies is of great value to
breeders and geneticists. Where monoploids can be induced in crop plants they
may be used to simplify patterns of inheritance and accelerate breeding programs
as the quickest possible way to homozygosity. Monoploids constitute moreover
an ideal material on which to base advanced genetics in higher plants (analogous
to fungal or microbial genetics) and through cell culture faciliatate an understand-
ing of the biochemistry, developmental processes and evolutionary aspects of
higher plants.
The purpose of this account is to give the status on monoploid production by
the chromosome elimination method in barley and the impact of in vitro culture
on this achievement.
Agricultural barley (Hordeum vulgare L., sensu lata) is a diploid species
(2n = 2x = 14). It is one of the oldest cereals known to man and ranks fourth in the
world's cereal production (only surpassed by wheat, rice and maize). Barley is self-
pollinating, annual and found as either two- or six-rowed, spring or winter types.
Traditionally the barley grain has been used in human diet, animal feed, and in
producing alcoholic beverages. Its conspicuous chromosomes, morphological
characters, and ease of handling have made barley a valuable object for mutation
studies. Genetically it is among the best-known higher plants (for a general review
see FR0IER et al., 1959; BELL and LUPTON, 1962; NILAN, 1964).
Monoploids are here defined as sporophytes with the basic chromosome num-
ber (i.e. the gametic chromosome number (DE FOSSARD, 1974). They have a single
genome or chromosome set (2n = x = 7). Monoploids are generally sterile but by
doubling the chromosomes, homozygous diploid fertile plants are produced.
They have been reported to occur spontaneously in barley (see JENSEN, 1974a, for
references).
Monoploids have been found in a number of species (for a review see KIMBER
and RILEY, 1963; CHASE, 1%9,1974; KASHA, 1974a, b). Although m.onoploid spo-
rophytes are smaller in size than the corresponding diploid plants they need not
be feeble plants. Their vegetative growth may be just as vigorous as the corre-
sponding diploids. The may also have a long life-span as exemplified by the
monoploid sporophyte of 1huja plicata (2n =x = 11) (T. gigantia var. gracilis, Pohl-
heim, which has been a sporophyte for over 100 years). Like almost all mono-
ploids it is completely sterile. A monoploid of horticultural interest is th~ Pelar-
gonium cultivar "Kleine Liebling" (2n=x=9) which has been reproduced vegeta-
tively over a number of years. Maize was the first crop where monoploids have
been produced systematically and employed as doubled monoploids in breeding
programs (CHASE, 1969, 1974).
300 C. J. JENSEN
Monoploids are an ideal tool in cell culture where they form a simple system
(only one allele) in biochemistry and cell genetic analysis (ZENK, 1974). Their
usefulness in cytogenetics has already been exemplified by SADASIVAIAH and
KASHA (1971), and GILLIES (1974). Perhaps their use in in vitro studies (e.g. auxo-
trophic mutants) will become more important than their application in breeding
programs and conventional genetics of higher plants. The use of monoploids in
various disciplines will depend, of course, on the ease and reliability of their
production. The present account deals with current practices of producing mono-
ploids in barley.
2. Scope
Monoploids as sporophytes in barley (2n = x = 7) may be induced by altering the

normal development of microspores (potential male gametophytes) or by
utilizing the female gametophyte. They can best be produced by interspecific
hybridization followed by chromosome (genome) elimination. The hybridization
method consists of crossing cultivated barley, Hordeum vulgare L. (2n = 2x = 14),
with the wild, diploid cross-pollinating and perennial H. bulbosum L. (2n = 2x = 14).
For practical purposes, monoploids have to be produced in relatively high
frequencies from all possible genotypes of barley. The interspecific hybridization
of barley with H. bulbosum allows for a production of relatively large numbers of
monoploids. This technique, the Bulbosum method, consists of the following
steps: the female gamete of barley is fertilized by the H. bulbosum gamete. Zygote
and embryo induction is high, but during this process the chromosomes of H. bul-
bosum are eliminated, leaving the barley genome in the embryos. The latter are
cultured in vitro as immature embryos. Plantlets from these monoploid embryos
can be made to give fertile flowers bearing homozygous offspring following an
efficient chromosome doubling technique.
Emphasis is placed here on an outline of current techniques in the production
of monoploids, and on indicating that the Bulbosum method can be made attrac-
tive to serve breeders and geneticists by improving its stability and efficiency.
Recent results on anther culture of barley are also given and discussed in relation
to pollen or microspore culture m~thods.
The frequencies of spontaneous monoploids are far too low for utilization in
breeding or genetic studies. If monoploids should become a tool in the various
disciplines of plant sciences, applied as well as in basic research, methods must be
available to allow a high frequency production from all possible genotypes with-
out changing the genetic character of the original material.
3. Usage and Need for Monoploids

The advantages of monoploids as tools in plant breeding or genetics become
more apparent when their direct applicatIon is visualized. Some of the advantages
have already been listed (JENSEN, 1974a) as: (1) they provide the quickest possible
Monoploid Production by Chromosome Elimination 301
way towards complete homozygosis, (2) they may serve to recover recessives,
(3) by sampling gametes as monoploids linkage data can be obtained directly,
(4) doubled monoploids give an immediate product of stable recombinants from
species crosses, (5) monoploids can be used to determine homology within a
genome and between genomes, (6) they are ideal objects on which to study
mutation frequencies and spectra, (7) in cell and protoplast culture they provide
an ideal system for fundamental cell biological problems (i.e. biosynthesis),
BREEDING METHODS
MONOPLOIDS VS. CONVENTIONAL
GAMETES
tlETlROZYGOTE
GENOTYPE
PHENOTYPE c=>
1:1:1:1
------1
9:
t - --
EffiCIENCY [
i Of SHrEO HETEROlYGOTE
-
Of GAMETfS
• OESIREO GENOTYPE •
OR n " n2 "
Fig. 1. Advantages of monoploids and doubled monoploids in breeding methods and in

genetic analyses. By turning female gametes (from F 1 plants) directly into sporophytes and
doubling their chromosome number, the segregation product is simplified. This relationship
will simplify analyses when many different combinations of alleles are tested. By obtaining the
homozygous condition from gametes directly a given breeding program aiming at homozy-
gozity can be shortened by 5 to 6 plant generations. It can be visualized that there are decided
advantages for the breeder and geneticist in applying the monoploid method to resolve
possible recombination products by sampling and selecting on chromosome doubled gametes
302 C. J. JENSEN
(8) monoploid cells as protoplast provide a unique material for gene transfer;
host-pathogen reactions; and cytoplasmic and! or chromosomal incompatibility.
Some of these advantages are of direct practical value (CHASE, 1969, 1974;
MELCHERS, 1972; KASHA and REINBERGS, 1975). Figure 1 depicts this by compar-
ing conventional breeding methods with the monoploid method, though some-
what simplified (see also WALSH, 1974; Chap. 11.3).
F or breeding purposes one of the main advantages of using monoploids is that
completely homozygous lines are produced directly from gametes of F 1 hybrids
or from later (advanced) selections. This allows for a direct fixation of quantitative
characters (as used in studies on yield in high lysine mutants and on pleiotropic
effects of mildew-resistant mutants and also on short straw mutants at Risf6).
The utilization of monoploidy in genetics and breeding requires that a ran-
dom sample of gametes is obtained and that the gametes are converted into
sporophytes at relatively high frequencies.
In terms of practical plant breeding the most important aspect of using mono-
ploids and doubled monoploids in barley is a saving of time in several plant
generations and the ease of recognizing the desired product among lines for
selection. These points have been described and discussed in more detail by PARK
et al. (1974) and KASHA and REINBERGS (1975). REINBERGS et al. (1975) have
provided evidence from a comparative study on field-grown barley lines that
about 20 lines are sufficient to characterize a given cross. The material consisted
of 52 doubled monoploid lines from two crosses which were compared with
pedigree lines and lines obtained at random (single seed descent). The comparisory
which was made on two locations for two years clearly showed that the argu-
ments brought forward for the monoploid method in maize (see CHASE, 1%9,
1974) can also be brought forward for a self-pollinating species like barley. These
workers found a decided advantage of the monoploid method in saving time for
the breeder and providing a reliable material on which to base selections.
Other areas in which monoploids could be advantageously used are: seed of
doubled monoploids would make a good material to start mutation studies in
barley. Monoploid cell and tissue cultures, especially the protoplasts, will provide
the geneticist, biochemist and cell biologist with a material that might prove a
powerful tool in plant modification and somatic hybridization (see Chap. IV.).
4. Production of Monoploids in Barley
For the artificial induction of monoploids in barley there are now two routes
which can be illustrated as follows:
(1) Based on potential male gametes (microspores, see also Fig. 10), and (2) on the
female gametes (megaspores). Potentially the first route, via anther or microspore
culture, has the advantage because there are far more potential monoploids per
spike in the form of male gametophytes than there are female gametophytes. Both
methods are based, however, on embryogenesis and the deVelopment of plants
from monoploid embryos followed by chromosome doubling to obtain homozy-
gous diploids.
Monoploid Production l:>y Chromosome Elimination 303
- - - -.... x H.bulbosum
c:f
GENESIS
I[
n o CJ
ANTH Ell I POLLEN
CULTUltf
n
EMBRYO
/
CULTURE
1
CALLUS
Fig. 2. Diagram relating two main
routes of monoploid (n) and n/2n
doubled monoploid production in
i~~~
barley. One route is sketched via
anther or pollen culture and the MONOPLOID ( n)
PLANTS
other via use of the female gamete,
which after fertilization by another
Hordeum species and zygote for- .
mation, develops into a monoploid
embryo by somatic chromosome
1 CHAO_OSOitf
DOU.U ••
HOMOZYGOUS
elimination of H. bulbosum chro- DIPLOIDS (2 n)
mosomes
5. Anther and Microspore Culture

Anther culture has been described in barley by CLAPHAM (1971, 1973), GRESS-
HOFF and Doy (1972a), BOUHARMONT (1974), ZENKTELER and MISIURA (1974),
MALEPSZY and GRUNEWALDT (1974), GRUNEWALDT and MAU!PSZY (1975),
PEARSON and NILAN (1975), DALE (1975, personal communication). It is evident
from these reports that anther culture in barley has, at present, considerable
limitations. There are still major obstacles to be solved before the anther or
micros pore cultures of barley can be as effectively handled for haploid production
as those of the Solanaceae (see references in NITSCH, 1974 b-, 1975; SUNDERLAND,
1974; BAJAJ, 1976). These limitations derive from three main areas: the physiolog-
ical condition, the growing of donor plants, and plant induction via callus. Non~
synchronous development of micros pores at the culture stage in Gramineae (WEN-
ZEL et al., 1975), and differential behavior of genotypes (GRESSHOFF and Doy,
1972 b) lead to low frequency and variability in embryogenesis and organogenesis
from callus and the formation of chlorophyll-deficient and karyotypically abnor-
mal plants. If these limitations can be overcome this method would be potentially
more attractive than the interspecific hybridization method.
304 C. J. JENSEN
6. Interspecific Hybridization-The Bulbosum Method
The alternative method of obtaining monoploids in barley is based on interspe-

cific hybridization. The first report on utilizing hybridization and somatic chro-
mosome elimination (see KASHA, 1974a) came from crosses between tetraploid
H. vulgare (2n=4x=28) and tetraploid H. bulbosum (2n=4x=28). Soon after
their first report on haploid barley induction (KAO and KASHA, 1969), KASHA and
KAO (1970) demonstrated that the method of crossing diploid barley with diploid
H. bulbosum could be used to produce high frequencies of barley monoploids.
Although other reports had already pointed out the cytological consequences of
this cross combination (SYMKO, 1969; LANGE, 1971), it was the application of
embryo culture following growth of donor plants in controlled artificial environ-
ments, that made these relatively high frequencies of monoploid production possi-
ble. Following this report other workers became interested in the production of
monoploids(JENsEN, 1973, 1974a; FEDAK, 1973; ISLAM and SPARROW, 1974; FINCH,
1974). This method is currently used by one private plant breeding firm in Canada
and by more than a dozen major research institutions throughout the world. The
technique is recommended as a tool in breeding and genetics and allows for:
(1) Induction and production of large numbers of normal monoploids,
(2) monoploids and doubled monoploids occur randomly from all possible geno-
types (3) genetically unaltered and stable doubled monoploids (homozygotes)
are made in the shortest possible time.
7. Principles of the Bulbosum Method
In essence the Bulbosum technique is based on making an interspecific cross with

H. vulgare as the female and H. bulbosum as the male. The method utilizes the
female gametes of barley and is schematically representd in Figure 3.
Fertilization of H. vulgare by H. bulbosum pollen proceeds readily. Zygote
induction is high and the chromosomes of H. bulbosum are rapidly eliminated
from the cells of the developing embryo. The endosperm develops for two to five
days and then aborts. In the developing monoploid embryo cells the division and
increment is slower than in diploid cells (JENSEN, unpublished). This compara-
tively slow growth of the monoploid condition, together with the disintegration of
the endosperm, leads to the formation of small embryos which have to be dis-
sected out of the fruits and,.provided with nutrients in vitro in order to complete
their development. Following in vitro embryo culture the developing plantlets are
raised under normal greenhouse conditions and chromosome doubling is induced
on established plants.
The method has the advantage that very high frequencies of female gametes
are induced to form embryos from which all H. bulbosum chromosomes are
usually lost. Hybrids, although easily recognized, are normally rare. The mecha-
nism by which chromosome elimination proceeds has not been established. How-
ever, it is genetically controlled by chromosomes two and three of H. vulgare
(KASHA, 1974a; Ho and KASHA, 1975).
SCHEME OF MONOPLOID BARLEY PRODUCTION

H. VULGARE (VV) H. BULBOSUM (BB)
(2n - 2x -1') 12n - zit - 1')
GAMETES
~IIOS' (V-B)
I-----~ X 4-----f
~~ (B"VI
1
B
V-B CHROMOSOME
ZYGOTE ELIMINATION
V
EMBRYO
CULTURE V
MONOPLOID
BARLEY
PLANT
2n. x ·1
VV
DOUBLED
MONOPLOID
2n - 2x · 14 GAMETE SELECTION
FERTILE
HOMOZYGOTE
Fig. 3. Scheme of monoploid and doubled monoploid barley production via the interspecific
hybridization method followed by somatic chromosome elimination of one of the parents
chromosome sets, here H. bulbosum
8. The Technique of the Bulbosum Method
8.1 Growing Barley for Crossing
The H.·vulgare to be used as female parents should be reared under the most
favorable conditions for the respective genotypes. It is also important to maintain
a continuity of flowering plants over a period. Figure 4a lists the main working
steps in producing monoploids and doubled monoploids.
At Ris~ the seeds are sown in pots filled with a peat soil (commercial "K-soil")
to which fertilizers have been added. Flats containing 24 pots are placed on glass-
wool mats on low glasshouse benches. When the seedlings are established (about
two weeks after sowing) water is supplied twice daily to the flats by an automatic
drip and additional fertilizer is provided every two weeks. An 18 h-a-day light
source consists ofHQIL-Osram-400 W lamps. The light intensity at plant pot level
306 C. J . JENSEN
STEPS IN PRODUCING MONOPLOID BARLEY

& COMPLETELY HOMOZYGOUS BARLEY
EMASCULATE
POLLINATE
12
DOUBLE
CHROMOSOMES
Fig. 4a. Steps in producing mono-

ploids and doubled monoploids in
barley. The various steps are listed
POT £ GROW
in sequence of procedure (G. A.
hormone treatment). Approximate
MULTIPLY
figures in man-days per 1000 mono-
HOMOZYGOTES ploids are given for the various
steps indicating various grades of
ease of execution
is 46 X 10 3 lux and at the level of the spikes about 84 x 10 3 lux, with three lamps
per m 2 of growing area. STOSKOPF et al. (1970) give a description of growth-room
facilities which permit control of environmental conditions throughout the year
and provide relatively large growing areas. The day (18 h) temperature is about
+ 18° Cand the night (6 h) temperature + 13° C. (Growing conditions at Ris~ are
shown in Figure 6a, b.)
There is evidence that spring type barley adapted to one growing area (e.g. six-
rowed Canadian cultivars) behave differently and need quite different tempera-
ture conditions than for example Northern European two-rowed cultivars. Ex-
amples of environmental influences on the ge\1otype are given by GUSTAFSSON
and DORMLING (1972), and DoRMLING et al. (1975).
8.2 Hordeum bulbosum L.
Diploid H. bulbosum is a wild, perennial, cross-fertilizing species found in some

hilly parts of Morocco, Tunisia, Cyrenaica, Italy, and Spain (LEIN, 1948; KATZNEL-
SON and ZOHARY, 1967) and vernalization is needed to induce flowering (KOLLER
and HIGH KIN, 1960). It is self-sterile (LEIN, 1948 ; LUNDQVIST, 1962), but it is easy
to propagate plants vegetatively from bulbils produced at the bases of stems. At
OLLEN PRODUCTIOM
HORDEUM BULBOSUM (2n t 2x =14
11---------
AQUISITION Of MATERIAL CHECK
I~--------~------~·
I COUNT
CHROMOSOMES
L
VERNALIZATION
• wk_, + SOC, .h II ht
GROWING FOR POLLEN PRODUCTION

+ U"C, ,. h light, + 12"C, • h d.,k
--iI
... .
MULTIPLY
CHECK
_ II
_ iiI.I
Fig. 4b. Scheme of handling H. bulbosum
material
Ris~ we find it practical to plant H. bulbosum material out in the field in autumn.
After a good year's growth in the field bulbs are taken in, potted and placed in a
cool room. This material then gives good flowering shoots when moved to the
crossing area. Figure 4 b presents the scheme of handling H. bulbosum material. As
expected with a wild, allogamous species there is considerable variation within
families or ecotypes.
8.3 Emasculation of Barley
F or ease of handling, the potted plants are removed to a crossing area where the
florets can be emasculated in one of the conventional ways (POPE, 1944;
HAMILTON, 1953; WELLS and CAFFEY, 1956).
1. Sideway (slit) removal of the anther with forceps, the forceps pierce the
palea and lemma lengthways forming a slit through which the three anthers are
easily extracted without damaging the stigma (Fig. 5 b).
2. Cutting across the top third of the flower above the anthers ("egg-topping")
with a pair of scissors, care being taken not to damage the stigma. If this is done in
the afternoon the next morning the anthers will have begun to push through the
cut opening and can easily be removed.
308 C.J.JENSEN
Micropylor end
Megagametophyte
(e)
Fig.5a--e. Stages of Hordeum floral structures for monoploid production: (a) Three anthers
and ovary with stigma of a barley flower almost at the selfpollinating stage. (b) Hand emascu-
lation of barley spike (inset : emasculated floret). (c) ,Highly schematised ovule of barley. (After
JENSEN, 1973). (d) Flowering spike of H. bulbosum shedding pollen
The following procedures increase the level of seed set:

a) Emasculation is best done on fully mature flowers preferably about one day
before natural pollen release. By leaving emasculation to a late stage increases the
risk of accidental selfpollination but selfed fruits can easily be distinguished from
those of H. bulbosum-crossed fruits at harvest, as the latter are devoid of a proper

endosperm (Fig. 61).
b) On two-rowed cultivars four to six of the basal florets are left to self-
pollinate, all other florets are emasculated except poorly developed ones at the tip
of the spike (which is removed) in all about 25 florets per spike. Six-rowed culti-
vars are treated intact because removal of the outer florets damages the normal
functioning of the spike.
c) After emasculation the spike is enclosed in a parchment bag which is in turn
covered by a cellophane bag. Closing the base of the bag with a clip ensures the
maintenance of a. high level of humidity which is of particular importance for
good fruit set (Fig. 6c).
8.4 Pollen Production and Collection in H. bulbosum
Unfortunately at present there is no method to store H. bulbosum pollen satisfac-

torily. Thus, an ample supply of freshly collected pollen has to be available
throughout the crossing period. To achieve a continuous supply of pollen, vernal-
ized plants of H. bulbosum are moved at regular intervals from the cool room to
the glasshouse or growthroom.
Normally one H. bulbosum plant is needed to pollinate two to five barley
plants. However, this depends on the productivity of the H. bulbosum material, on
its mode of flowering and on the timing of the flowering peaks. The best pollen-
producing plants are obtained from large, well formed bulbs. At Ris~ plants
producing these bulbs are obtained from field-grown H. bulbosum material.
Pollen must be collected from freshly dehiscing anthers (Fig. 5d). This is best
done about 1 h after the lights have been on in the morning. A sheet of aluminum
foil is placed underneath the dehiscing spike which is loosely tapped. The pollen
caught on the foil is transferred to a clean petri dish protected from light. Pollen
should be used immediately after collection.
Figure 6 depicts some of the stages of monoploid production from growing
the plants for the crosses to fruit production.
8.5 Pollination
Pollen is applied carefully and amply to the barley stigmas by: (a) dipping a
sharpened toothpick into the pollen and applying it to the stigma, (b) dipping a
fine camel hair brush into the pollen and lightly flicking it into the stigma region,
(c) blowing the pollen on to the stigma with an aspirator.
The optimum stage of pollination depends on the time at which the flower is
emasculated, temperature and genotype. Under Ris~ conditions and with North-
ern European barley cultivars, late emasculation can be followed by pollination
the next day. But normally, the spikes are pollinated two days after emasculation.
Pollen tube growth of H. bulbosum appears to proceed as normally as that of
H. vulgare pollen on H. vulgare stigmas.
.,' 1'_".
... .'~l~~f,
.. .~. i...
(a)
Fig.6a-f. Stages of monoploid production: (a) Light regime and sources (HQIL - Osram -
400 W) and humidity for the growth of Hordeum. (b) Section of growth room with barley at
flowering. (c) Enclosed spike of emasculated and pollinated in glassine and parchment isola-
tion bag. (d) Spraying spikes with hormones. (e) Arrangement of cut shoots of barley in water
culture (see text), left: pollinated spikes of barley, right : H. bulbosum and barley shoots mixed
for pollination. (I) Right : Barley spike, with 15 florets with fruits from pollinations with
H. bulbosum, 6 florets (bottom of spike) showing selfpollinated fruits (more plump due to
presence of endosperm). (Spike 14 days after pollination.) Left: Petri dish with harvested fruits
ready for sterilization
8.6 Hormone Treatment of Pollinated Spikes
Hormone treatment of pollinated spikes from one or two days after crossing does
improve fruit set (LARTER and CHAUBEY, 1965; KASHA, 1974a; JENSEN, 1974 a).
The application should be repeated once or twice and the isolation bags left on
the spikes to prevent the florets from drying out. Hormones may be applied to the
florets b)' brush, syringe or by a fine spray as depicted in Figure 6d.
It has also been suggested that the hormone can be sprayed onto the foliage of
the plant rather than the single spike, and a single application of 75 mgfl of
gibberellic acid applied two days after pollination was found to be effective (SUB-
RAHMANYAM and KASHA, 1973). But applying the hormone repeatedly after polli-
nation is probably more effective. Kinetin (ISLAM and SPARROW, 1974) may have a
similar effect. JENSEN (unpublished) experimented with a number of hormones in
mixtures (25 mg/l G A3, 10 mgfl 6-benzylamino purine = BAP; 10 mgfl indole-3-
flcetic acid=IAA, 15mgfl naphthalene acetic acid = NAA) and found no notice-
able difference in fruit set; but embryo formation was advanced in comparison
with that of GA3 alone.
Morphactin, 2,3,5 iodobenzoic acid-TIBA (50 mgfl) in combination with cyto-
kinin 6(y,y-dimethyl-allylamino)-purine= 2iP or kinetin at 10-20 mg/l and GA3
(25 mgjl) and NAA (25 mgfl) was beneficial to rapid fruit development but with a
large amount of liquid endosperm. This increased the chances of damage and
embryo infection. The embryos, though, were comparatively large and developed
well'in culture. There was also an indication that spraying the hormone mixture
more than once enhanced the development of the fruits. More work on these lines
is needed before definite conclusions can be drawn.
8.7 The Cut-Shoot or Detached Tiller Method
At the time of or just before emasculation, shoots are detached at the base and
placed in a water culture solution (modified Hoagland solution, see Appendix 2).
It is advisable to burn about 3 cm of the cut end of the shoot base. This allows
shoots from field-grown barley to be handled under environmentally controlled
conditions and also provides for controlled nutrient feeding to spikes (JENSEN,
1973). One container can be used for about 60-70 shoots held in place by a
perforated top of plastic or styrofoam (Fig. 6e). Air from the bottom of the contai-
ner stirs and aerates the solution which is changed weekly. A plastic bag held by a
support enclosing all spikes of a container will retain a high humidity level.
Pollination is done ,by hand or by placing shoots of H. bulbosum about to shed
their pollen beside and above the emasculated spikes of barley. The air stirring the
water culture solution is enough to vibrate the spikes of H. bulbosum to ensure
pollen release. Hormone treatment may be applied via the culture soluti"on by
adding 20 mg/l of gibbereliic acid (GA 3 ) on the second day after pollination.
Table 1 shows that cut shoot and nutrient-cultured spikes produce as many
embryos and monoploids as undetached shoots (see Sect. 11.2.6).
312 C.J.JENSEN
8.8 Harvest and Handling of Fruits
The optimum stage of harvesting the fruits for embryo culture varies with con-
ditions and genotype. Embryos left too long in the fruits may show brown spots or
become infected. Fruits which are dried up are difficult to dissect. There is a
considerable variation in optimum harvest time due to: (a) Differences in
nutrition ofthe embryos and fruits on the spike, (b) Fruit position on the spike in
relation to distance from the artificial light source, (c) Genotypic differences in
fruit and embryo development.
In practice the optimum stage for dissection is reached at 13-15 days after
pollination when grown at + 23° C (conditions for Guelph) and about 18-21 days
when grown at + 18° C (conditions for Risj1l).
For harvesting, the florets are pealed off the spike and care is taken to avoid
damage to the fruit coat (Fig. 6f). Palea and lemma are removed and the intact fruit
placed in a petri dish. The fruits are sterilized for five min. in a 5% calcium hypo-
chlorite solutions plus a drop of Tween-20 or -80. Sterilization is followed by
five consecutive washings with sterile water.
If not used immediately, the fruits may be stored at + 5° C in a sterile dish on
filter paper moistened with culture medium (see Sect. 9). When stored thus for
one week no reduction in viable embryos could be found.
9. Embryo Culture
9.1 Embryo Dissection
Instruments for dissection can readily be made following the descriptions given
by CUTrER (1967), and ROMBERGER (1966). Dissection of embryos from sterilized
fruits is done under 10 x magnification in a stream of sterile air on a laminar flow
bench. The fruits are placed in a sterile cover of a petri dish and slit open at the
side with a dissecting needle to expose the liquid contents of the fruits. The
embryo, often freely suspended in this liquid, is lifted out by a second needle and
placed.on the culture medium. Care should be taken not to damage the embryo
and to place it with the ventral surface on the medium (NORSTOG, 1965). Callus
growth can often ensue if the embryo is orientated upside down on the medium.
9.2 Media
Stocl(solutions should be prepared as 10 x or 100 x strength of the final concen-

tration and stored cold (5° C). A stock solution can be checked by testing it
against a standard cultivar (e.g. performance of 3-week-old Bomi embryos).
Three main media i.e. Bs (GAMBORG et al., 1968), Bll (NORSTOG, 1973), and R-
M-IS (ISLAM et al., 1974) have been used as agar solidified media. At Risj1l we have
had more success with a floating culture system using a liquid medium
(Appendix 1).
This system has one disadvantage in requiring the transfer of the small devel-
oping plantlets to an agar solidified medium for good root and shoot growth. It
should eventually be possible to eliminate reculturing.
Liquid Media. Following the formula in Appendix 1, C-17 and C-2r, the stock
solutions are mixed, water added, pH adjusted and sterile filtered via a membrane
filter unit as shown in Figure 7b (e.g. Millipore filter-type: GS 0.22 IJ., filter holder
type: XX4304700). The sterile medium can be stored in the cold. For the floating
embryo technique (JENSEN, in preparation) about 8 ml of medium are put in 5 em
diameter sterile plastic dishes, a sterile millipore filter is floated on top of the
medium. The excised embryos are placed on these filters and the dishes are sealed
with strips of parafilm (See Fig. 7 d).
Solid (Agar) Media. Appendix 1 gives the formula for the medium Bs (see also
GAMBORG et aI., 1968; KAO and KASHA, 1969). After mixing stock solutions and
agar the medium is boiled to dissolve and the pH adjusted to 5.5. Glass vials are
filled to one third of their capacity with· medium, loosely capped and autoclaved
in bulk. The autoclaved, sterilized medium can also be dispensed via a sterile
funnel into sterile glass or plastic tubes or dishes.
9.3 Variation in Number of Embryos per Spike and Embryo Size
Often, in a production of monoploids, some spikes of a plant do not develop

many fruits following these crosses with H. bulbosum. In most cases the first three
to four formed shoots have the best fruit-setting ability. Fruits not properly
formed rarely have a culturable embryo. Occasionally even well-formed fruits
have no embryo. However, up till now we have no explanation for these short-
comings.
The embryos from the H. vulgare x H. bulbosum crosses grow and develop
differently than those from normal diploid barley (Fig. 7a). They are monoploids
and have a slower mitotic cycle (analogous to differences in mitotic cycle as
described by BENNETT et al., 1973); their nutrition during in situ growth is differ-
ent since reduced endosperm formation disrupts normal nutrition.
9.4 Culture Conditions
A culture cabinet with low light intensity (500-1000 lux) and temperature control
from ISO C-25° C within a differential of ± 1°C is adequate for embryo culture. A
useful and relatively inexpensive growth cabinet has been described by DAVIES
(1973). The first phase of culturing for one or two weeks is done in the dark at
18° C. Light encourages precocious germination. When properly differentiated
(Fig. 7 e, 2) the embryos can be moved into a 12 h light regime. At this stage a
temperature of 20° C is used at Ris!ll and 22'J C at Guelph. When differentiated
(Fig. 7 e, 3) the embryos are moved from liquid to solid medium where they de-
velop into plants with good roots within one'or two weeks.
Once plants are established (Fig. 71), the vials are transferred to growth room
conditions with higher (about 40 x 10 3 to 50 x 10 3 lux) light intensities.
LEmbryos
.-:-------+:---;'1 *1
'L" ~~~"~~3~~:~~: lid (sterile)

*lMillipore,MF, ~47mm,O.45f.1
on 8ml liquid medium
(dl
Fig.7a-f. Stages in monoploid production: (a) 4 embryos of barley 14days old from the
same spike. 2n=selfpollinated, diploid. embryo; n=monoploid embryos. 2n embryo.mea-
sures 2.7 mm in length. (b) Arrangement for sterilizing liquid culture medium using a modi-
fied pressure cooker vessel, a glass beaker, a filter holder and Erlenmeyer flask for the
collection of the medium which is forced through a sterile 0.22 Il filter using compressed air
(see RIDDLE, 1973). (c) Ten 147day-old embryos from the same spike following crosses with
H. bulbosum on barley. All except X are monoploid. X is a hybrid and measures 1.6 mm in
length. (d) Floating embryo culture: sketch of method used at Risl/l. (e) Three dishes of
floating embryo culture with lids removed: 1 at start of culture, 218 day of culture, 3 25 days
old culture. (0 Monoploid sporophyte in culture tube
Fig. 8a-d. Schematic view for chromosome doubling: (a) Growing monoploids into sturdy
plants. (b) Treating shoot-plants wjth colchicine solution plus a carrier (DMSO). (c) Treat-
ment of a monoploid plant with colchicine via the capping method (inverted vial). (d) Differ-
ent categories of spikes on a colchicine treated monoploid; 1 almost completely fertile spike
(bearing chromosome doubled embryos) ; 2 a chimeric spike, one half with normal seeds
(doubled), the other half with no seed set (monoploid, non-doubled); 3 and 4 typical spikes
with monoploid character, sterile florets
316 C.J.JENSEN
10. Plant Culture and Multiplication

10.1 Transfer to Soil Culture
Plantlets cultured in vitro require hardening and good root development to cope
with transplat:ltation to the soil. About a week before transplanting the culture
vials are placed in a greenhouse and receive about 15 x 10 3 lux of light at 20° C
18 h light, 12° C 6 h dark. .
They are lifted carefully with some agar on the roots to small clay pots filled
with a light soil mixture (sand: peat: soil in 1: 1 : 1 by volume). The potted plants
are put in a tray with peat and covered by a plastic tent to prevent desiccation.
Growth commences from a few days to a week later and the piants can be
adjusted to ordinary greenhouse conditions. Care must be taken not to oyerwater
the freshly potted plants and to protect them from desiccation. The balance
between dry and moist conditions results in a good survival of plants (Fig.8a).
10.2 Screening Plants for Hybrids and Off-Types

When the plants have-grown to the two to three tiller stage the following grouping
can be made on morphological characters:
Barley Monoploids. Upright shoots, narrow leaves, no visible hair, slender but
vigorous in tillering.
Hybrids. Type 1: Prostrate growth habit, leaves slightly curled and dark green,
visible hair on leaves, excessive tillering. Type 2: Upright growth, foliage slightly
broader than monoploids, hair distinctly visible on leaves and stems.
Off-Types. (Includes aneuploids, triploids, chimeras etc.). Plants very irregular,
a range of intermediate types from between previous two groups.
Before chromosome doubling the material is screened for monoploids. This
morphological grouping has been found to be reasonably accurate. Possible hy-
brid plants need not be discarded as the H. bulbosum spike characters seem to
dominate over the H. vulgare characters.
103 Chromosome Doubling

Vig<;;>rous plants at the three to four tiller stage are treated as follows: Soil is
washed ofT the roots, which are cut back to about 3 cm below the crown. The
plants are placed in a glass vial (Fig. 8b). The colchicine solution (2.5 g colchicine
dissolved in 20 ml dimethyl sulfoxide (DMSO) is made up to 1 litre with water.
A few drops of Tween-20 or -80 are added. Enough of this solution is placed in
the vials to cover the plant crowns and part <1fthe leaves. (See Sect. 11.2.7 for plant
stages and colchicine dosage).
The plants under treatment (Fig. 8b) are placed in a growth chamber (+20° C
in the light) for 5 h, and potted directly after treatment into a light soil.
At Guelph plants are treated whilst still in the culture vial. The method is
essentially the same except for a washing and temperature treatment at + 32° C
for three hafter colchicine application.
Plants not responding to this treatment may be cut back and treated by the
inverted vial method (see JENSEN, 1974 b and Fig. 8 c). At Adelaide this tiller-
capping method (on plants with 2-3 tillers) has proved highly successful for
chromosome doubling (ISLAM and SPARROW, 1974).
10.4 Rogueing and Seed Harvesting of Doubled Monoploids
Given optimum conditions for flowering the colchicine treated plants flower from
one to two months later. The first formed shoots are usually monoploid (sterile
and two third the size of normal diploids). Later formed shoots bear from a few
seeds per spike to almost full seed set (Fig.8d). Cross pollination is negligible
and thus isolation (by bagging) of the spikes is not necessary.
During flowering and seed harvest the material should be rogued for abnor-
mal spike and other plant characters.
After harvesting, the seed is sown for multiplication and again a further
screening for off-types is practised. Tetraploids and doubled monoploids are
distinguished either by their broader leaf shape, darker color, growth habit or
irregular flowering. When sown in the field as lines the doubled monoploids are
found very uniform in plant character within the respective lines demonstrating
their homozygosity.
11. Discussion and Remarks
11.1 General
The problems of producing monoploids and doubled monoploids in barley are

mainly technical. The advantage of anther and microspore culture techniques are
obvious, but extensive research is needed before these methods can be as efficient
as the Bulbosum method. Recent research in micros pore methods (NITSCH,
1974a,b, 1975; REINERT et al., 1975; Bajaj et al., 1975) points to advances with
simple, technical adjustments of procedures (e.g. preconditioning anthers in situ,
centrifugation and cold shock treatment, washing of microspores and addition of
amino acids promoting embryogenesis). There is also the possibility of separation
and selection of viable microspores as for rye (WENZEL et al., 1975) and the
induction in situ of a type of micros pore prone to form embryos (SUNDERLAND,
1974). It has been observed that in vitro culture of barley cells and tissues is often
carried out on media with too high a concentration of solutes. Decreasing the
levels of solutes by one half of the standard media and adjusting the osmolarity
with mannitol gives better organogenesis (JENSEN, unpublished).
Experience has shown that the Bulbosum method is highly reproducible on
almost any barley material even though conditions for growing and treatment are
not identical.
The method of hybridization followed by chromosome elimination proVes to
be of general interest for haploid induction in other species of Hordeum (RAJ-
HATHY and SYMKO, 1974) and also of cultivated hexaploid wheat, Triticum aestivum
318 C.J.JENSEN
(BARCLAY, 1975). These recent reports no doubt will contribute to a broader

interest in this method. At the same time work on the chromosome elimination
mechanisms (DAVIES, 1974; KASHA, 1974a, SAGAR and KITCHIN, 1975) will be-
come of high priority with a view to utilize haploid induction in general.
11.2 The Bulbosum Technique

11.2.1 Growing Conditions
The following discussion concerns the problems of the Bulbosum technique and
suggestions for its improvement. The importance of the growing conditions for
barley have been stressed (JENSEN, 1974a; KASHA, 1974a). Barley is photoperiod-
sensitive and under suboptimal conditions short days can lead to sterility (BATCH
and MORGAN, 1974).
Work on vegetative and reproductive growth phases under controlled envi-
ronmental conditions (phytotron studies by DORMLING et al., 1975) show that
even in related genotypes of barley the plants express themselves differently under
similar conditions. These studies point to the need for more detailed studies on
optimum growing conditions of the genotypes for monoploid induction. Also
the nutrition of the plant should not be neglected (see HEWITI, 1966; MACLEOD
and CARSON, 1969).
11.2.2 Hordeum bulbosum and Pollen

Handling H. bulbosum plants for pollen production usually causes no problems
when well developed bulbs are available. Studies on the reproductive development
of tetraploid H. bulbosum, indicate that the photoperiod as well as vernalization
at low temperatures determine phase changes (KOLLER and HIGHKIN, 1960).
Storage of H. bulbosum pollen is difficult, so that a continuous supply of pollen-
producing plants must be available during crossing. As indicated before, H. bulbo-
sum as on outcrossing species is variable in terms of visible morphological char-
acters. Even though we have made our observations on comparatively limited
material (less than 100 genotypes) it seems types adapted to our growing condi-
tions and pollen-producing requirements can be selected.
11.2.3 Hybridization
In crossing work, fruit induction and embryo development on the spike the
following comments can be made: emasculation, although costly, cannot be neg-
lected and methods to increase the efficiency of emasculation should not damage
the stigma. Scissors emasculation is more expedient than sideways emasculation
but tends to give a lower fruit set. (For a discussion of emasculation techniques
see: POPE, 1944; HAMILTON, 1953; WELLS and CAFFEY, 1956). Some cultivars of
barley are sensitive to mechanical damage to the flower. which might result in
complete female sterility (QUALSET and SCHALLER, 1968). .
Low humidity perhaps more than anything else, in the period between emas-
culation to the embryo development is the main cause of reduced fruit set. Even a
relative humidity of 80% in the crossing area may be insufficient to prevent the
desiccation of emasculated flowers. The type of isolation bag may also be critiCal
in obtaining good fruit set.
11.2.4 Fruit Induction and Ovular Growth

Very little work has been done .on fruit induction following difficult crosses or
abnormal fertilization in barley. The classical work of VAN OVERBEEK et al. (1941)
on chemical stimulation of ovule development has had no success with Gramineae.
In situ experiments by JUNGFER (1952) in barley have been negative. In vitro
experiments in culturing ovaries or ovular structures of barley have been unsuc-
cessful (WALKER and DIETRICH, 1963; KLASSEN and LARTER, 1967). A study of the
ovular sap under embryo development led SMITH (1973) to compose an analytical
culture medium-i.e. one that was of similar composition as the analysis of the
sap had shown. His interesting approach will be applied to our barley material.
Normal embryo development is tightly linked to development of the flower
(LA CROIX et al., 1962). Treating fertilized flowers with different hormones (Gib-
berellic acid, indole-3-acetic acid, naphthalene acetic acid), alone or in combina-
tion with cytokinins, does not seem to be effective. One exception, however, was
found at Risf/l when spraying with a mixture of GA 3 , cytokinin, and morphactin.
Here, larger embryos resulted and harvesting of fruits for embryo dissection could
be delayed by almost one week without a decline in plant production.
11.2.5 Endosperm Development and Nutritional Aspects

An interesting case in endosperm/embryo relationship is found in barley. Here
mutants are known that either develop only embryos (without any endosperm) or
endosperm without any embryo development (HARLAN and POPE, 1925). At Risf/l
we have recently found a mutant with no endosperm formation but with embryo
development. This type has been termed "watery kernel" (HARLAN and POPE,
1925), and for perpetuation it has to be excised from the fruit about two to three
weeks after pollination or else it dies. Its embryos grow readily on any of the
media listed in Appendix 1. Besides being of use in studies indicated by RICE and
CARLSON (1975) the endosperm-less diploid mutant will be used in a comparative
study on embryo development in barley in conjunction with the monoploid em-
bryo from interspecific crosses. A better understanding of nutritional require-
ments for the embryo might be obtained by studying this mutant.
RUTISHAUSER (1969) has provided a detailed analysis of genome balances in
different cross combinations and the different endosperm-genomic make-ups. On
similar grounds a tabulation is given for the different crosses of cytotypes of
H. vulgare and H. bulbosum (see Fig. 9). The fate of the endosperm development in
these interspecific hybrids has not been fully examined although there are indica-
tions (SUBRAHMANYAM and KASHA, 1973) that it may be similar to barley x rye
crosses (ODENBACH, 1965) and wheat x rye crosses (Moss, 1972; ,BRINK and
COOPER, 1947).
Observations at Ris¢ indicate. however, that the decline in endosperm devel-
opment is abrupt and varies in time due to genotype combination rather than to
320 C.J.JENSEN
GAMETE EXPECTED GENOMIC CONSTITUTION
COMBINATION
cJ
EMBRYO
ENDOSPERM
9 SPOROPHYTE
Fig. 9. Possible cross combina-
tions of various cytotypes of
V B V (VV B)- H.vulgare and H. bulbosum.
The combinations are listed as
B V V iBBVi® genomes; Vone H. vulgare ge-
nome; Bone H.bulbosum ge-
nome. Endosperm formation:
V BB V BB (VVBB)-
+ normal; - endosperm
~B V BB V BBBBV+ disintegrates; x endosperm
formation not always stable.
·VV BB VV (VVVVBB)-
(After data of SUBRAHMANYAM
BB VV VV (BBBBVVi· and KASHA, 1973)
environmental conditions. This leads to the question of how to influence chromo-

some elimination in the embryos without disrupting "normal" development of the
endosperm.
11.2.6 Zygote Formation and Embryo Development in sit.u

After pollination with H. bulbosum, fertilization in barley seems to proceed in a
way similar to that described for selfed barley (POPE, 1943; LUXOVA, 1967; CASS
and JENSEN, 1970; and W.A.JENSEN, 1973). However, after zygote formation and
central cell fertilization a different set of conditions operate (SUBRAHMANY AM and
KASHA, 1973). At times more than one monoploid embryo develops in these fruits.
Several times we have extracted three embryos from, a single ovule, giving viable
monoploid plants. The origin of these extra embryos is not known. As Figure 5 c
shows, there are several possibilities for extra embryo sac-derived embryos from
synergids, central cell or antipodals. Under conditions at Ris9l, Bomi (a cultivar of
H. vulgare) x H. bulbosum embryos have 10, 19, and 242 cells after two, three or six
days of pollination, respectively. This corresponds roughly to the figures given by
SUBRAHMANY AM and KASHA (1973). It is not known whether the differences in envi-
ronmental conditions, the genotype of barley or of H. bulbosum, or all factors
together determining the rate of chromosome elimination. Certainly, temperature
is a decisive factor in rate of embryo growth.
Observations show that the rate and mode of development for the monoploid
embryo and selfed diploid barley embryo differ, i.e. monoploid emQryos have cells
smaller than those of diploids, their mitotic cycle and rate of growth is also
comparatively slower. Morphologically the m6noploids differ from normal di-
ploids in shape, especially the scutellum which often has an irregular heart-shaped
form in monoploids (Fig. 7 a). Very often the suspensor is not as prominent in
monoploid embryos as in normal diploids. Selfed diploids of the endosperm-
lacking mutant (see Sect. 7.3.5), on the other hand have a shape very similar to
monoploids yet are somewhat larger. NORSTOG (1972a) has described the early
development of selfed diploid barley embryos;. and has subsequently shown
Table 1. Comparison between greenhouse and field-grown barley when treated as detached
and normal shoots in monoploid production
Location Spike Florets Fruits Embryos Monoploids Monoploids as %

treated pollinated harvested cultured obtained of embryos
Field Detached 456 388 171 59 34.5

Field On plant 392 121 42 10 23.7
Glasshouse Detached 422 359 182 98 53.8
Glasshouse On plant 430 371 169 85 50.3
(NORSTOG, 1972b, 1974a) that the presence of a suspensor, which firmly attaches
the developing 'embryo to the ovular tissues, restricts early embryo dissection.
Monoploid embryos, often even at very early stages of development, appear to
float in the sap of the fruits on dissection. As mentioned in various reviews
(NORSTOG, 1972b; KAPIL, 1974; DURE, 1975) little is known about the mechanism
of nutrition of the embryo and ovular tissues. In barley KIRBY and RYMER (1974)
have described the vascular system supplying the single florets. Much more work
is needed to understand and find ways of influencing the nutrition of embryos in
situ. A comparison has been made (Table 1) between cut-shoot and plant devel-
oped embryos, both from field material and under glasshouse conditions.
The results of detached and non-detached shoot treatment are not consistent
for the different cultivars. It can be concluded, however, that in general practice
there is little difference between the two methods. The advantage of the detached
method is that field-grown material can be brought indoors, and the simultaneous
treatment of large numbers of spikes with pollen and hormones is possible.
The best time of harvesting the fruit seems to vary with type of cultivar and
growing conditions. (At Guelph the optimum time is about two weeks after
pollination, whilst at Ris0 it is some five to seven days later.)
11.2.7 Embryo Culture, Genotypic Effects, and Chromosome Doubling

Diploid Hordeum vulgare Embryos. The main emphasis in application of embryo
culture in genetics and plant breeding has always been in helping to raise plants
from embryos developed under incomplete endosperm development (MAHEsH-
WAR I and RANGASWAMY, 1965). Work by NORSTOG (1965, 1970, 1972b, 1973)
concentrates mainly on developmental aspects of the barley embryo per se. How-
ever, diploid embryos of barley have different culture requirements for optimum
growth than monoploid embryos.
Monoploid H. vulgare Embryos. The media listed in Appendix 1 give good
results with embryo cultures and especially medium C-17 supports substantial
growth of both monoploid and diploid embryos.
Table 2 shows the results of embryo growth and plant production on agar-
solidified liquid medium C-17. There are clear advantages of using the liquid form
as both genotypes have performed better on the liquid culture media than on the
solid. In our ex perience irregularly developed small embryos have a better chance.
of growth on liquid than on agar media.
322 C.J.JENSEN
Table 2. Growth of monoploid embryos on liquid and agar-solidified C-17 medium.

Embryos from HP40 and F I (HP4O x 1508) after crossing with H. bulbosum
Medium Cultivar Number Plants from % of cultures with:

of embryos
embryos Roots only Shoots only Callus only
cultured Number %
Liquid HP40 242 108 44.6 9.1 5.3 8.4

Solid HP40 245 67 27.3 7.5 8.7 15.6
Liquid FI 231 129 55.8 7.3 7.5 10.9
Solid FI 236 72 30.5 6.2 11.2 18.3
Table 3. Embryo culture and plant production in an F I (HP4O x 1508) compared to 1508
and HP40 as the parents
Combination Number of Monoploid Total plants

or cultivar
Spikes Flow- Fruits Em- Embryos Plants Plants No. Em-
ers bryos per fruit % of bryos
% embryos %
HP40 24 415 372 224 60.2 89 39.7 106 47.3

1508 25 412 325 170 52.3 53 31.2 69 40.6
F I (HP4O x 1508) 23 420 403 312 77.4 196 62.8 218 69.9
Washing the freshly dissected embryos in the medium before transfer to the
culture dish has resulted in increased development (JENSEN, in preparation),
however it is not clear whether washing removes some inhibitor from the embryo.
In some instances dormancy can set in, even in not fully developed embryos (see
also RYCZKOWSKI, 1971).
Despite the many steps in technique of monoploid production (Fig.4a) it has
been possible at Guelph, RiSjll, Aberystwyth, Cambridge and Adelaide to produce
monoploids by this method at ever increasing frequencies. By our continued
experience with this technique we have increased our production from 5 or 6%
monoploids per embryos cultured in 1971/1972 to about 62% of success in 1973/
1974, which is very encouraging.
Genotypic Effects on Monoploid Production. The differential behavior of geno-

types as exemplified in Table 3 affects the success of embryo culture. Here, the F 1
between two cultivars (HP40 x 1508) gives consistently higher yields of mono-
ploids than the parent varieties. There is also a great variation within cultivars.
Whether these differences are determined genetically or are purely an expression
of the physiological status of the donor plant at the time of treatment or of .the
embryo per se is not known.
We also have evidence that the pollen parent, H. bulbosum, influences the
development of embryos and determines in part whether chromosomes are elimi-
nated completely or whether hybrids result. This interaction of H. vulgare and
Table 4. Response of different barley genotypes in crosses with a clone of H. bulbosum

presented as percentages of monoploids and hybrids
Genotype Embryos Plants

cultured
H. vulgare H. bulbosum Total % of Monoploids Hybrids
'i? 6 embryos
% of %of
embryos embryos
HP40 102 288 159 55.2 130 45.1 29 10.1

Bomi 102 257 145 56.4 138 53.7 7 2.7
Sultan 102 270 159 58.9 118 43.7 41 5.2
Mutant 1508 102 172 83 48.3 75 43.6 8 4.7
H. bulbosum in determining frequency of monoploids and of hybrids, needs further

experimentation.
Table 4 shows the influence of the female genotype (here H. vulgare) and
H. bulbosum on hybrid frequencies (i.e. incomplete chromosome elimination). The
cultivar Sultan shows a much higher frequency of hybrids than the other cultivars
tested. There is some evidence that embryos can be graded at dissection into
hybrid and monoploid embryos. Although high frequencies of hybrids in a pro-
duction series mean reduction in number of monoploids, the screening of hybrids is
a comparatively easy task as the morphological differences of hybrids and mono-
ploids are very distinct.
Precocious Development in vitro. Immature embryos when cultured in vitro do
not normally go through all stages of embryogenesis to become dormant, but
germinate precociously (NORSTOG and KLEIN, 1972; NORSTOG, 1972b). This
means that the less an embryo is developed at the time of excision the more likely
it is that a weak, underdeveloped plantlet is obtained. To get viable plantlets from
immature embryos means culturing them into mature embryos and it seems that
our liquid medium C-17 is favorable for prolonged embryo growth.
From in vitro Culture to Soil Culture and Chromosome Douhling. Transfer of
monoploid plants from in vitro culture systems to soil and glasshouse growing
conditions requires sturdy plants, a well aerated soil and the right amount of
moisture.
Emphasis should be placed on treating the material uniformly on a rational
scale. The sooner the plants are established and ready for colchicine treatment the
quicker the homozygous seed will be set. Doubling treatments commence when
three to five shoots have been produced on the vigorously growing monoploids.
Depending on the size, age and state of the monoploids various concentrations of
colchicine dissolved in 2% dimethyl sulfoxide can be administrated for a period
of up to five hours in the culture tube, or in petri dishes 0.01-0.05% for plants
with 2-3 shoots, 0.05-0.1% for plants with 3-5 shoots, 0.2-0.5% for 4-12-week
old plants. Following this treatment the plants must receive good growing condi-
tions to stimulate vigorous growth. On average about one half of the treated
shoot initials will double and consequently set seed. An encouraging observation
324 C.J.JENSEN
is that the chromosome doubling can be made efficient by relatively small changes
in treatment. As WALSH et al. (1973) have pointed out, a possible seed source effect
is circumvented by increasing seed from a doubled monoploid in a further genera-
tion and using this in replicated field trials.
A proposal has been made (JENSEN, 1974 b) to term the various generations
after monoploid induction. At the same 'time it was pointed out that different
ploidy levels can arise directly after colchicine application and for this reason any
plants other than diploid can easily be screened off.
11.3 The Process of Monoploid Induction, Chromosome Elimination

and Somatic Reduction
11.3.1 The Bulbosum Process of Chromosome Elimination

The process by which monoploids via fhe Bulbosum method in barley are ob-
tained is one of somatic chromosome elimination of the H. bulbosum parent,
however the mechanism of preferential chromosome elimination is not fully un-
derstood. KASHA (1974a) has reviewed this subject, and Ho and KASHA (1975)
have reported on the genetic control of the mechanism. Using a test system of a
trisomic series in H. vulgare, crossing with tetraploid H. bulbosum and finally
testing with telotrisomics of chromosome 2 and 3 revealed that the genes for
elimination are most likely situated in 3 L, and also that some genetic factors are
found in both arms of chromosome 2. Cytologically, the process of elimination is
a gradual one. Elimination is most prominent in actively dividing tissue such as
embryos, young endosperm or meristems. Three possible hypotheses for the eli-
mination mechanism have been proposed (KASHA, 1974a). (1) Asynchrony of
mitotic cell cycle times due to differences between the potential species.
(2) Spindle or centriole abnormalities. (3) A system similar to "modification-
restriction" in bacteria and bacteriophage.
The first s~tem would work in much the same way as in mammalian cell
hybrids grown in culture (KAO and PUCK, 1975). Here the preferential elimination
works by cell cycle times, the type with the slower cell cycle time tending to be
eliminated (SUBRAHMANYAM and KASHA, 1973). This system has also been pro-
posed fm: chromosome elimination in certain Nicotiana hybrids (GUPTA and
GUPTA,1973).
The second system involves spindle or centromere attachment problems as
described for example in certain insects (CAMENZIND, 1974). In this connection it
is worth pointing out that barley sperm cells reach the 2C condition (D'AMATO et
al., 1965) before pollen dehiscence and the sperm cells are probably at the G2
stage when fertilization takes place. If this is the same for H. bulbosum the argu-
ment is against this system as only stages after the S-phase would affect elimina-
tion of zygotic chromosomes.
The third possibility has been proposed by DAVIES (1974). In this system
foreign DNA is recognized by a nuclease compound and can then be inactivated.
SAGAR and KITCHIN (1975) in a recent account have treated this subject in detail
bringing forth their results of selective destruction of DNA in Chlamydomonas
and explained chromosome elimination in other plant and animal systems.
11.3.2 Chromosome Elimination and the Influence of Parent Genotypes,

Species and Other Genera
Whatever the systems involved, the application of it points to a stable mechanis~.
Recently it has been observed that monoploid and especially hybrid frequencies
in H. vulgare x H. bulbosum crosses depend on the genotype of both parents
(Table 4). Work is in progress to try and isolate H. bulbosum genotypes which
induce hign frequencies ofmonoploids and also those which give high frequencies
of hybrids which in turn can be used in gene transfer programs. Likewise, recent
experiments at Cambridge (FINCH, personal communication) and at Risl1l indicate
that the genotype of H. vulgare in combination with H. bulbosum genotypes deter-
mines the rate and efficiency of chromosome elimination.
The elimination of chromosomes is not restricted to H. vulgare x H. bulbo-
sum crosses but seems to be widespread in Hordeum species (ISLAM and SPAR-
ROW, 1974; KAsHA, 1974a; RAJHATHY and SYMKO, 1974). In this way haploids
have been produced in intcr~pecific and intergeneric crosses between Hordeum
species (PRICE, 1968; AHoKAs,1970; KAsHA, 1974a).
Recently, wheat haploids have been produced in large frequencies by crossing
wheat with pollen of H. bulbosum (BARCLAY, 1975). The system requires embryo
culture and works essentially on experimental procedures similar to the produc-
tion of barley monoploids by the Bulbosum method.
11.3.3 Consequence of the Bulbosum Method of Somatic Chromosome

Reduction
As illustrated in Figure 9, as a general rule, the chromosome elimination process
depends partly on a genome balance. Mainly for this reason endosperm forma-
tion can be ruled out in systems where a diploid barley is used as the female
parent and a diploid H. bulbosum as the male parent.
Another irregularity is the malformation of the antipodal cells which often
become highly polyploid before disintegrating. What is important to stress, how-
ever, is that since embryo induction is obtained in high .frequencies, a random
sample of gametes can be expected from this system of monoploid induction.
Another question concerning the formation of monoploids by the Bulbosum
method is that there might be a chance during fertilization and early zygotic
stages for H. bulbosum material to influence the development of the H. vulgare
monoploid. The evidence to-day is that although this chance does exist, it nev-
ertheless does not manifest itself in the material from the monoploid productions.
On the contrary, most attempts to transfer genetic material from H. bulbosum to
H. vulgare have so far been unsuccessful.
CASS and KARAS (1975) have shown on an ultrastructural level that barley
sperms (gametes) are totally naked and possess no mature plastids. Cytoplasmic
transfer from H. bulbosum, supposing it has a similarly formed sperm cell .and
fertilization proceeds normally, must therefore be unlikely.
Another interesting aspect of the preferential chromosome elimination pro-
cess is that it is possible to produce monoploids of barley in a cytoplasm of
326 C.J.JENSEN
H. bulbosum by using H. vulgare as the male and H. bulbosum as the female. In this
instance valuable material for cytoplasmic studies can be produced, again using
embryo culture as a vehicle to obtain high frequencies of "foreign cytoplasm"
monoploids.
11.3.4 Somatic Chromosome Elimination in Other Genera and by Other Pro-

cesses
With regard to the process of somatic chromosome elimination information has
been given by workers hybridizing somatic animal cells (EPHRUSSI, 1972). The
ability to culture protoplasts of H. vulgare and of H. bulbosum followed by so-
matic hybridization would make an ideal system to study chromosome elimina-
tion. These studies could be supplemented by techniques to differentiate the
chromosomes of the two species by banding patterns and the ability to regenerate
from the fused somatic cells (see Chap. IV.1.2 of this Volume).
As already mentioned in Section 11.3.1. Hordeum species may not be the only one
where chromosome elimination is found in higher plants, KASHA (1974a) has
listed a number of other cases, JACKSON and JORDAN (1975) describe a possible
case for Haplopappus gracilis where a monoploid has been examined cytologically
with only two chromosomes.
Whether or not chemical agents can be used as efficiently to affect somatic
chromosome reduction remains to be seen. KASHA (1974a) and ZENK (1974) give
examples where chemicals such as: colchicine (in Sorghum); 3-fluorophenylal-
anine (in Lolium x Festuca hybrids) or parafluorophenylalanine (in Nicotiana
cultures); chloramphenicol (in Hordeum root tips) have resulted in reduced chro-
mosome numbers in the plant cells or tissues. However, both these authors con-
clude that somatic chromosome reduction via chemicals is not properly under-
stood and should therefore not be viewed too optimistically as a practical method
of inducing haploidy.
12. Anther and Pollen Culture
The status on anther culture can be summed up as follows: CLAPHAM (1973) (see
also Chap. 11.3) obtained relatively high callus formation with cultivars Sabarbis
and Akka or with various hybrids of barley cultivars. The anthers were cultured
on medium B (essentially LINSMAIER and SKOOG, 1965) with high sucrose (12%)
levels and various levels of" antiauxins (0.02 mgjl tri-iodobenzoic acid, TIBA).
Under favorable conditions 29.6% of anthers showed callusing. However, the
number of plants produced were few and showed various ploidy; out of 25 plants
three were predominantly haploid; one partly haploid, 17 diploid and four pre~
dominantly tetraploid.
These results were reproduced by MALEPSZY and GRUNEWALDT (1974), who
found in addition a genotype effect in that only one cultivar of the various
genotype tested, "Amsel", yielded plants of various ploidy levels. PEARSON and
NILAN (1975) also report a genotypic response, as the spring cultivars Akka,
Zephyr, Unitan, Traill, and Trebi formed callus and even some plantlets. The
winter cultivar Hudson, on the other hand, did not produce callus or plantlets.
One major handicap was that the plantlets produced were chlorophyll-deficient
and could not be kept alive for long. The recent result of GRUNEWALDT and
MALEPSZY (1975) point out the real difficulties of anther culture in barley, i.e.
relatively high callus formation with differences between cultivars. In cultivar
Vogelsanger Gold 32% of the anthers, yielded calli, however, even though about
1000 plantlets could be formed from three-week-old callus, almost all except four
were chlorophyll-deficient, aneuploids and/ or polyploids. ZENKTELER and MISI-
URA (1974) have also reported the formation of pollen embryos in barley.
Chlorophyll deficiency and different levels of ploidy make the plantlets from
anther culture of little use in the production of monoploids in plant breeding and
genetics.
Hopefully, the inspiring work on the pollen or microspore culture in Solana-
ceae species (NITSCH, 1974a) can one day be successfully extended to barley.
WENZEL et al. (1975) have described an interesting technique of separating viable
rye micros pores from non-viable ones. They argue that the asynchronous devel-
opment of microspores might playa role in diminishing the chances of micros-
pores dividing.
13. Monoploids via Protoplasts
Work on the culture of protoplasts isolated from tetrads is still at the very
beginning (BAJAJ, 1974). The potentials of using this scheme are obvious as mono-
ploids obtained directly from the process of meiosis would have many advantages
over those from later stages, e.g. early m~rospores (potential male gametes) (see
Method Hybr,ds
Potential f;yJ - 8 - - -....~ Hybnds (2 n )
gametes /
X H9u:bo.um
! i
F.. ~',.o~on
Bulbosum . ~ _ _---o",o;.
UII do'o'opmo"'
cross n • r ., .... c
Ch,,~,~••"moM',," ~ ~
- - - - -_ _ _ _ _
Anther
pollen
• EmbryogenesIs _
'"t "'V
Embryo MonoPIO'dI.......
~."', '"!-' f
~ ~
I
o
n --
~o -
-co-
ndl-
II.-
" -------- r
culture
~
nther/m lcrospore
---_.......--
culture Hetero zygotes (2 n )
Chlorophyll defects
OrganogenesIs
....._, (n.2n)
Callus
Fig. 10. Schematic representation showing possible routes of monoploid and doubled mono-
ploid production
328 C.J.JENSEN
Fig. 10). KAO et al. (1974) have established a method for handling diploid barley
protoplasts from leaves.
The system described here-the Bulbosum method, which is based on a ga-
metic fusion followed by somatic chromosome elimination in the early embryonic
cells should make an interesting subject for somatic cell fusion. For example
protoplasts of monoploids of barley and H. bulbosum can be fused and by a
chromosome banding procedure recently developed for barley (unpublished) the
fate of the single chromosomes can be studied in these fusion products.
14. Retrospect and Prospect
For anther and pollen culture the main obstacle in barley seems to be the inability
to obtain normal green plants with high frequency from induced callus. This lack
of normal chlorophyll production seems to be common to many Graminae in
vitro cultures (GAMBORG et al., 1970; CLAPHAM, 1973; Chap. 11.3). CLAMPHAM'S
observations and the report by TULECKE (1967) indicate that alterations in the
culture medium might bring about different frequencies of chlorophyll-deficient
plants. It is noteworthy that plants regenerated from callus obtained from the
scutell um of monoploid H. vulgare embryos were mostly with normal chlorophyll
development (JENSEN, unpublished). There was no change in frequencies of nor-
mal green monoploid plants from this callus when cultured on other media than
those listed in Appendix 1 (i.e. media of LAMPORT, 1963; SCHENK and HILDE-
BRANDT, 1972; KRUSE, 1974). This leads one to speculate whether the origin of the
callus or the mode of its initiation may also playa role in chlorophyll-deficiency
as found in barley.
The Bulbosum method depends in the first place on ability to hybridize and to
induce high frequencies of embryos. H. bulbosum need not be the ideal partner for
H. vulgare to induce monoploids of barley via somatic chromosome elimination.
There can well be a range of Hordeum (KASHA, 1974a; PRICE, 1968) that might be
tried as a more efficient partner than H. bulbosum. On the other hand, there seems
to be much variation in different genotypes of H. bulbosum regarding ease of
chromosome elimination. It is our aim to try and select for this variation in
H. bulbosum collections.
Soma.tic chromosome reduction with the aim of inducing haploidy might also
be achieved by applying, for example, halogenated amino acids. As yet no conclu-
sion can be reached whether this methods is of practical value to induce high
haploid frequencies.
The main advantage of the monoploid method is seen as a speeding-up of
material for testing recombinants. Whether the monoploids for this purpose are
derived from potential male or femal gametes is of no importance in barley. What
is important is that normal monoploids can be produced in high frequencies.
Acknowledgments. I am grateful to the editors for their outstanding patience and guid-
ance, and to my colleagues for help at various stages of this work. I am most grateful to Dr.
K.J. Kasha and Dr. K. Norstog for their cooperation and help with many a problem. The
additional comments on the manuscript by Dr. D. Clapham and Dr. M. P. Coutts are greatly
appreciated. Personal communications with Dr. P.J.Dale, Dr. R.A.Finch, Dr. O.L.Gamborg,
and Dr. D.B.H.Sparrow have been very helpful and thanks are expressed for the interest
shown.
Appendix 1. Composition of media used in embryo culture of monoploid barley
Chemical B5 BII R-M-IS C-17 C-21

mgjl mgjI mgjI mgjl mgjI
Macro- KN0 3 2500 150 300 300

nutrients CaCI 2 ·2H 2O 150 740 250
MgS0 4 ·7H 2O 250 750 325 300
(NH 4hS04 134
NaH 2P04 · H 2O 150 100
KCI 750 150 300
KH 2P04 910 150 500
Ca(N0 3 h 350 500
NH 4N0 3 200
Micro- KI 0.75 0.10

nutrients H 3 B0 3 3.0 0.5 0.5 5.0 15.0
MnS04 ·4H 2O 10.0 3.0 3.0 0.5
ZnS04 ·7H 2O 2.0 0.5 0.5 0.25
Na2Mo04·2H20 0.25 0.025 0.025 0.012
CuS04· 5H20 0.025 0.025 0.025 0.012
CoC1 2 ·6H 2 O 0.025 0.025 0.025 0.012
Na2EDTA 37.3
FeS04· 7H 2 0 27.8
Ferric citrate 10.0 10.0 3.0 20.0
Fe EDTA 17.5 10.0
Vitamins Nicotinic acid 1.0
Thiamine HCI 10.0 0.25 0.25 0.25 10.0
Pyridoxine H CI 1.0 0.25 0.25 0.25
Inositol 100.0 50.0 50.0 50.0 150
Ca-pantothenate 0.25 0.25 0.25
Glycine 0.75
L-ascorbic acid 0.50
Amino L-glutamine 400
acids L-glutamic acid 200 150 300
L-alanine 50 30
L-cysteine 20
L-arginine 10 20 50
L-Ieucine 10 10
L-phenylalanine 10 20
L-tyrosine 10
330 C.J.JENSEN
Appendix I.(continued)
Chemical B5 8 11 R-M-IS C-17 C-21

mgl mgl mg'l mg!1 mg!1
Amino L-tryptophan 10
acids L-aspartic acid 30 100
L-proline 50 50
L-valine 10
L-Iysine 10
L-serine 25 25
L-threonine 10
Sucrose 20000 34200 20000 60000 45000
Agar (DIFCO) 7500 6000' 7000
pH 5.5 5.0 5.0 5.5 5.5
a Purified.
In addition: Media BII, C-17 and C-21 have the following additives peril medium:
BII: Malic acid, 1 g dissolved in 50 ml H 2 0, pH 5.0.
C-17: Citric acid, 500 mg in 50 ml H 2 0, pH 5.3. tri-potassium citrate, 300 mg, added
to final medium, pH of medium adjusted with K 0 H. Filter sterilize.
C-21: Citric acid, 50 mg in 50 ml H 2 0, and tri-potassium citrate, 250 mg, pH 5.0.
Added to medium and final pH brought 5.5 with KO H. Filter sterilize.
Abbreviations used for Media:

Bs: Medium described by GAMBORG et al. (1968).
BII : Medium described by NORSTOG (1973).
R-M-IS: Medium used by ISLAM and SPARROW (1974) based on Randolph's
medium described in MORRISON et al. (1959).
C-17: Medium used by Jensen based partially on BII and other media used as
liquid culture on small and non-uniform monoploid embryos.
C-21: Medium used by JENSEN (1974b) on uniform, well developed embryos.
Appendix 2. Hoagland solution, modified for barley shoot culture
Chemical Stock solution Final solution use

gmll ml/I
KH 2 P04 38.0
MgS04 ·7H 2 O
KN0 3
Ca(N0 3 lz4H 2 O
52.0
66.0
94.0
} 1 mljl
H 3 B0 3 2.86
ZnS04 ·7H 2 O
CuS04· 5H 2 0
Na2Mo04 ·2H 2 O
0.22
0.10
0.05
} 1 mill
FeEDTA 12.0 1 ml/l
ButTer: 2-morpholino ethanesulfonic acid MES 19.5g+2.0g NaOH, in one 1 H 20.

Adjust to pH 6.5 with NaOH. Use 5 mill nutrient solution. For original Hoagland solution
see HEWITT, 1966.
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Chapter n Haploids

Haploid plants have the gametophytic number of chromosomes, that is a single

(A) (8) (C)

Fig.2A-1. In vitro induction of androgenesis from microspores of Nicotiana tabacum. (A)

3. Culture Media and Nutritional Requirements

WHITE MURASHIGE NITSCH and NITSCH

Fig. 3. Effect of various concentrations of charcoal

medium. Although charcoal has highly increased the efficiency of adrogenesis in

In culture, microspores undergo various modes of androgenesis (Fig. 4) which

Control Charcoal medium

Fig. 4A-E. Diagrammatic illustration of microsporogenesis, and various modes of develop-

6. Stability of Haploid Cell Cultures

Table 4. Effect of various concentrations of PFP on the growth (percentage of control) of

Weeks PFP (mgjl)

Hap!. Dip!. Hap!. Dip!. Hap!. Dip!.

1 140 130 150 110 160 130

7. Significance and Uses of Haploids

References see page 331.

2. Induction towards aNon-Sexual Pathway

By modifying their normal development, we hope to trigger isolated micros-

100 0 Colchicine 10- 3 M. Colchicine

3. Technique for the Isolation of Microspores

The unopened buds are sterilized by a 2 min immersion in a freshly prepared

4. Requirements for the Growth of the Embryo

4.1 Nutrient Requirement

production of haploid plants from anther culture was a two-step procedure,

4.1.1 Effect of Sugar

4.1.2 Effect of Amino Acids

for the production of haploid plants by in vitro culture of isolated microspores.

4.2 Effect of the Environment

3 days 10days 21 days

10 days 21 days 35days

Fig. 8. Effect of various types of

5. Advantages of Isolated Microspore Culture

5.1 The Influence of the Anther Tissue Can Be Detrimental

1. Although anther culture of about two dozen species as different as N icotiana

5.2 A Homogeneous Population Will Allow Better Experimentation

1. Unlimited numbers of cells which develop freely into embryos in a liquid

References see page 331.

2. Methods of Haploid Induction in Cereals

hybnds Ploidy x-4x, ~

Table 3. Influence of genotype on the growth response of wheat anthers cultured at

Cultivar Number of % of anthers

1. Maris Ensign 180

2.1.2 Pathways to Multicellular Pollen Grains

2.1.3 Pollen Embryos, Pollen Calli, and Somatic Calli

2.1.4 Testing for Homozygosity of Diploid Plantlets from Anther Culture

2.1.5 The Albinos

therefore reasonable to exclude "mutation" in the ordinary sense of the word as

2.1.6 Approaches to Cereal Anther Culture

2.2 Other Means of Haploid Induction in Cereals

2.2.1 Use of the Cross H. vulgare x H. bulbosum

2.2.3 Androgenesis and the "Indeterminate Gametophyte" Mutation in Maize

2.2.4 Effect of Aegilops Cytoplasm on Cultivar Salmon of Triticum

2.2.6 Colchicine-Induced Somatic Reduction

2.2.7 Fluorophenylalanine-Induced Reduction

3. Uses of Haploids in Cereal Breeding

3.1 Production of Homozygous Varieties in Self-Pollinated Cereals

The obvious application of haploid techniques to cereal breeding is as a quick

The newly-developed doubled haploid stocks would initially be highly uniform, a

'L" ~"3:: lid (sterile)