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Cotton Leaf Curl Virus Epidemic in Pakistan: Virus Characterization, Diagnosis

and Development of Virus Resistant Cotton through Genetic Engineering

Article · January 1997

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 Cotton Leaf Curl Virus Epidemic in Pakistan: Virus
Characterization, Diagnosis and Development of Virus
Resistant Cotton through Genetic Engineering
Y. Zafar1, A. Bashir1, S. Mansoor1, M. Saeed1, S. Asad1, N.A. Saeed1, R. Briddon2,
P.G. Markham2, C.M. Fauquet3 and K.A. Malik1
1
National Institute for Biotechnology and Genetic Engineering (NIBGE), Pakistan
2
Department of Virus Research, John Innes Center, UK
3
The Scripps Research Institute, California, USA
(Presented by Yusuf Zafar)

The cotton leaf curl virus (CLCuV), a whitefly-transmitted symptoms produced on the cotton plant are leaf curling, thick-
geminivirus (Mansoor et al, 1993) has caused heavy losses to ening of veins, enations and stunted plant growth. Cotton leaf
the cotton crop and still remains the most important constraint curl disease was recorded as early as 1967 from Pakistan
for the development of the cotton sector in the country. The (Hussain et al, 1991). Since 1991/92, cotton leaf curl disease
34

that was only curiosity previously is now a major threat to the Biological Properties
sustainability of this crop. The epidemic of CLCuV in Pakistan
Insect Transmission Studies
is one of the best examples of the dramatic shift in importance
of an unimportant endemic disease in the past. It is estimated Our research has shown that CLCuV can be transmitted from
that the disease has resulted in a loss of 4.98 million bales of cotton to cotton and tobacco by whiteflies. Factors affecting
cotton with an estimated value of US$7.4 billion. insect transmission efficiency have been studied. The efficiency
of virus transmission was greatly affected by temperature. At
Recent advances in molecular biology and genetic engineering higher temperatures (40-45Cº) the virus efficiency was increased
have opened new avenues in understanding and controlling the ten fold and CLCuV could be transmitted by a single whitefly.
disease epidemics. Genetic engineering of crop species such as Aphids and jassids were also tried but were unable to transmit
cotton allows introduction of a specific character such as dis- the virus, thus whitefly is the only insect vector of CLCuV.
ease resistance to be incorporated in existing varieties without
compromising other agronomic characters. This technology is Experimental Host Range
superior to conventional plant breeding as breeding for disease Traditionally plant viruses are experimentally transmitted to
resistance using resistant germplasm may result in some unde- some indicator host plants for the observation of symptoms pro-
sirable characters contributed by resistant germplasm. duced. Tobacco plants which were infected by whitefly trans-
The National Institute for Biotechnology and Genetic Engi- mission were used in graft transmission. Infected tobacco leaves
neering (NIBGE) is a federal research institute with a mandate were grafted onto Nicotiana benthamiana, N. tabacum var.
to apply modern and innovative techniques in agriculture, health, Samsun, datura and tomato var. moneymaker. Grafting from
environment and energy. Realizing the potential of molecular cotton to cotton was done to maintain virus infected plants. For
biology and genetic engineering in solving the CLCuV prob- sap inoculation a young infected leaf was ground in 0.06 M
lem in the country, NIBGE initiated the research program with phosphate buffer pH 6.5, centrifuged in an Eppendrof tube for
the following objectives. 3 minutes and supernatant was collected. Sap mixed with
carborundum was rubbed on young leaves of tobacco, N.
• Biological and molecular characterization of CLCuV benthamiana and tomato plants. CLCuV was successfully trans-
which includes virus purification, cloning and sequencing mitted by grafting from tobacco to N. benthamiana, tomato
of the genomic components and generation of infectious and datura and all these plants showed identical symptoms.
clones. Mechanical inoculation did not show symptoms on indicator
• Development of PCR/DNA probe-based diagnostic test plants suggesting that CLCuV could not be transmitted by sap.
for the detection of virus in insects and plants and use of
Preparative Scale Purification of Geminivirus
this diagnostic test for the identification of alternate hosts
Particles from Infected Plants
of CLCuV.
Cotton is a woody plant and upon homogenization of tissues
• Development of virus-resistant cotton through genetic produces a lot of phenolic compounds which interfere with vi-
engineering. rus purification. Conditions were optimized for the purifica-
• Molecular diversity and distribution of virus in cotton tion of intact geminivirus particles from infected cotton plants.
growing areas of Pakistan. One to three months old healthy and infected cotton plants were
The Cotton Group of NIBGE extensively studied leaf curl dis- used in this study. The geminivirus particles isolation was per-
ease and made vital contributions to solve this problem. formed according to Czosnek et al. (1988) with some modifi-
cations. The samples were treated with 2% aqueous uranyl ac-
Biological and Molecular Properties etate (negative staining) and observed on JEOL transmission
electron microscope at a power of 40 Kv. Intact geminivirus
A basic understanding of the causative agent is essential for
particles were observed in one of the fractions. The optimiza-
devising control strategies. Some of the biological properties
tion of isolation procedures of CLCuV from the cotton plant
such as insect transmission, transmission of virus to indicator
itself is the first report of its kind in the world. The purification
host plants, detection of natural alternate hosts, virus particle
of intact virus particles has allowed development of antisera
morphology and detection of virus coat protein by Western and
which can be used for the detection of virus in infected plants
ELISA was carried out. The molecular characterization of vi-
by ELISA.
rus is essential for genetically engineered resistance as well as
development of molecular diagnostic methods. The work on Molecular Properties of CLCuV
molecular characterization of CLCuV includes cloning of full
length genomic components, determination of complete nucle- DNA Isolation, PCR Amplification and Cloning of
otide sequence, generation of infectious clones and evaluation PCR Amplified DNA
of molecular diversity of CLCuV. Total DNA was isolated from tobacco leaves by the Kirby
 35

method. For the isolation of total DNA from cotton a method Table 1. The Genome Organization of CLCuV Pak-1. EMBL
modified from Doyle (J.K.Brown, personnel communication) Accession No. X98995 (Mansoor et al, 1996)
was used. Universal primers for dicot-infecting geminiviruses ORF Reading frame Start Stop No. of a.a
were used in PCR for the amplification of CLCuV DNA. Afri- AV1 +1 124 432 103
can Cassava Mosaic Virus (ACMV) and Indian Cassava Mo-
AV2 +2 284 1051 256
saic Virus (ICMV) were used as a positive control where as
AC1 -3 2594 1506 363
DNA isolated from healthy N. benthamiana was used as a nega-
tive control. The PCR product of the expected size was ob- AC2 -1 1606 1155 150
tained both from infected cotton and tobacco plants. The am- AC3 -3 1461 1060 135
plification of viral DNA was confirmed by using ACMV DNA AC4 -1 2437 2137 100
A as a probe. PCR amplified DNA was digested with SaII and AC5 -1 592 70 174
EcoRI and a fragment of about 1.2kb was cloned in SaII-EcoRI
site in Bluescript vector. For the amplification of DNA B de-
generate primers reported by (Rojas et al, 1993) were used in Sequence of several clones identified two geminivirus species.
PCR. ACMV which is known to be amplified by these primers The comparison of these viruses is given in Table 2. The two
was used as a positive control. However, no amplification was viruses are named CLCuV PK-1 and CLCuV-PK2.
obtained with primers for DNA B.
Cloning of Components DNA-1 and DNA-2 Table 2. Putative Open Reading Frames (ORFs) of
Cotton Leaf Curl Virus (CLCuV) species
Double stranded replicative form of CLCuV was purified from
ORFs Start Stop No. of Amino Protein Similarity
infected cotton plants. Clones of DNA 1 of CLCuV were se-
Acids Mr (kD) Index (%)
lected by southern hybridization. Full length clones of genome
A 1 2750 71
A were obtained either by cloning at unique restriction site or
by combining two cloned fragments in such a way that desired AV2 139 (124) 495 (435) 118 (103) 13.7 (12.1) 90
open reading frames (ORF) remained intact. Sequencing of AV1 299 (284) 1069 (1054) 256 (256) 29.7 (29.7) 74
several clones was carried out and two variable clones of DNA AC1 2600 (2594) 1518 (1503) 360 (363) 40.3 (40.8) 87
1 were identified named as CLCuV Pak-1 and CLCuV Pak-2. AC2 1615 (1606) 1211 (1154) 134 (150) 15.3 (17.4) 66
Analysis of clones identified another clone which did not hy-
AC3 1470 (1461) 1066 (1058) 134 (134) 15.7 (15.6) 66
bridize to DNA-1clone but hybridized to viral DNA. The se-
AC4 2445 (2680) 2144 (2135) 100 (181) 11.1 (20.6) 72
quence analysis of this clone did not show homology to know
geminiviruses. The clone did not hybridize to intergenic probe. AC5 814 (592) 290 (68) 174 (174) 19.9 (19.3) 68
This clone has been named as CLCuV DNA-2. PCR primer Values outside parenthesis represent CLCuV-Pk2/Fsd/1.
designed on the basis of this clone suggest that this genomic Values inside parenthesis represent CLCuV-Pk1/Fsd/3.
V in an ORF represents virion sense strand and C denotes the complementary
component is associated with both whitefly-transmitted strand.
geminiviruses associated with the disease.
Biological Variability in Symptom Expression
Complete Nucleotide Sequence of CLCuV Pak-1
Biological and molecular variability of CLCuV was also stud-
CLCuV Pak-1 was completely sequenced by dideoxy chain ter-
ied. The important variation observed in the field is the upward
mination method using radioactive 35S or 33P dATP. The se-
or downward curling of the leaves. It is not known whether the
quence data were assembled and open reading frames were iden-
curling is determined by different viruses or is a plant response
tified. The two variable clones have genome organization typi-
to virus infection. To study this phenomenon, cotton plants
cal of old world geminiviruses. There were two open reading
showing upward or downward curling were passaged by graft-
frames AV1 and AV2 in the virus sense where as five open read-
ing to healthy cotton plants and the symptoms on the plants
ing frames in complementary sense namely AC1, AC2, AC3,
were recorded. It was found that plants showing downward
AC4 and AC5 were identified. The two strains (Pak-1 and Pak-
curling during passage produced plants with upward curling.
2) differed in their genome size and predicted amino acid se-
Similarly, one of the plants where graft was showing upward
quence of complementary sense genes. However, the most strik-
curling produced downward curling symptoms. The results show
ing differences were in the intergenic or common region. The
that symptom phenotype was not maintained and that curling is
sequence data has been submitted to EMBL data bank and this
either a plant response or may be due to the dominance of one
is the first sequence of CLCuV in databank.
strain.
Total No. of nucleotides = 2749
Length of common region = 278
36

Molecular Diversity: Evidence of Presence of of virus in the plant. Recently, we have developed a multiplex
Two New Geminivirus Species in CLCuV Pak-1 PCR for the detection of two geminiviruses species associated
with cotton leaf curl disease in Pakistan. A simplified method
For the evaluation of molecular diversity several clones were
is used for the isolation of template suitable for PCR. A rapid
obtained either by ccc DNA cloning or PCR. As discussed ear-
profile used PCR primers designed to specifically detect these
lier, the common region is the most diverse region among
two virus species. The two species could be found indepen-
CLCuV isolates. A common region of six clones was completely
dently or co-infecting the same plants. This is a unique example
sequenced. Four of the isolates have a common region sequence
where two geminivirus species could cause the same disease in
of PK-1 strain while 2 of the clones had a sequence of Pak-2.
a geographical area.
The data suggested that considerable variability exists in
CLCuV. Based on the sequence data, Pak-1 and Pak-2 specific Screening by Southern Hybridization
primers were designed and the desired product has been con-
CLCuV samples were collected from different cotton growing
firmed by cloning and sequencing. These primers are being used
districts of the Punjab and full length cloned viral DNA was
for the assessment of distribution of two strains of CLCuV.
used as probe in southern hybridization. The cloned DNA probe
Detection and Differentiation of Geminiviruses hybridized with the samples and thus can be used for screening
purposes.
Previously we have used the polymerase chain reaction (PCR)
for the identification of alternate hosts of cotton leaf curl virus. Development of Polyclonal Antisera
Surveys were conducted for the collection of additional plant
Purification of intact geminate particles paved the way for the
species which were not reported previously to be infected with
production of polyclonal antisera against CLCuV. For this pur-
geminivirus. Samples from infected cotton plants were also
pose rabbits were immunized with viral particles mixed with
collected from different cotton growing areas of Pakistan. DNA
Freund’s complete adjuvant (first dose). Subsequent subcuta-
A of ACMV or CLCuV was used as a probe for the detection of
neous booster doses were given with Freund’s incomplete ad-
whitefly transmitted geminiviruses. A number of well charac-
juvant at an interval of 15-20 days. Polyclonal antisera has been
terized geminiviruses both from the old and new world were
distributed to Ayub Agriculture Research Institute — AARI and
used as positive control while DNA extracted from healthy
National Agriculture Research Center — NARC for evaluation
plants of various species was used as negative control. It was
by ELISA. We received feed back from both research institutes
found that use of ACMV or CLCuV as probe was able to detect
and they recommended that this material needs further purifi-
geminiviruses by dot blot hybridization both from the old and
cation for reliable testing for the amplification such that the
new world viruses. Similarly the use of ACMV or CLCuV as
process is completed in two hours. This method is being used
probe detected geminivirus in 30 plants species out of 40 plant
to assess the distribution of two geminivirus species in cotton
species suspected of whitefly-transmitted geminiviruses.
growing areas of the Punjab, Pakistan.
For the differentiation of an alternate rate of CLCuV from other
geminiviruses, a probe of CLCuV common region was prepared. Development of Monoclonal Antibodies and
The common region of whitefly-transmitted geminiviruses is ELISA Test
the most diverse region and may serve as a virus specific probe. As a first step for the development of rapid immunological test
It was found that under high stringency level the use of a com- for screening, sets of primers based on the sequence to genome
mon region as probe specifically detected CLCuV both in cot- A were synthesized for PCR amplification and cloning in ex-
ton and alternate hosts and further confirms that members of pression vectors. These primers amplified 1.1kb fragment from
the Malvaceae family are the alternate hosts of CLCuV. cloned as well as from total DNA isolated from infected cotton
leaves. This PCR product has been cloned in bacterial expres-
Development of a Diagnostic Test sion vector for large scale protein production and purification.
Development of PCR Based Diagnostic Test The expressed protein has been used for the generation of mono-
clonal antibodies. Two clones of hybridoma cell lines produc-
PCR/DNA probe-based test has been developed for CLCuV
ing antibodies against CLCuV have been identified and are being
by designing two sets of primers. One set of primers is capable
tested for the specificity of monoclonal antibody by ELISA.
of amplifying a portion of viral genome from common region
These studies are being done in collaboration with Department
to N-terminal sequence of coat protein. The other set amplifies
of Pharmacology, John Hopkins University, Baltimore, USA.
part of viral genome from replication associated protein (AC1)
to common region. Newly developed sets of primers were used Development of Virus-resistant Cotton
to detect presence of CLCuV in cotton plant leaves collected
from different cotton growing areas. The amplification of viral
Through Genetic Engineering
genome was confirmed by cloning and sequencing of ampli- Several approaches have been reported for the development of
fied product. This is a highly sensitive assay for the detection transgenic resistance against whitefly transmitted geminiviruses.
 37

The approaches that are being used for CLCuV at NIBGE are rus. One of them is the production of antisense RNA against
the following replication associated protein or over-expression of ACI gene
in a transgenic plant driven by CaMV S35 promoter.
• Expression of antisense RNA against complete or frag-
ments of AC1 gene Open reading frame coding for ACI was identified on clone
PS1. Primers (V2417, V2418) were designed such that an NCoI
• Over-expression of AC1 in transgenic plants site was incorporated at 5' end of both primers. A product of
• Expression of a virus-induced cytotoxin gene in expected size (1.2 k) was obtained. The PCR product was di-
transgenic plants gested with NCoI enzyme and was cloned in PJIT 166 (a plant
expression vector) both in the sense and antisense orientation.
Tissue Culture of Cotton The orientation of clones was confirmed by restriction analy-
The genetic engineering of a plant is heavily dependent on trans- sis. The clone was also confirmed by sequence analysis. GUS
formation technology whereby a functional foreign gene could gene from the vector was removed by digestion with SmaI and
be inserted into the genome of the cotton plant. Currently, the relegated to give pSJITAC1. The construct was lifted from
two most widely used methods for plant transformation involve pSITAC1 by digestion with Sph1 and Sst I and ligated in pBin
agrobacterium-mediated transformation and bombardment of plus vector digested with the same enzyme and vector pSBinJIT
cells with DNA coated particles. These two methods appear to AC1 was obtained. Electro-competent cells of agrobacterium
be most important for genetic engineering of cotton both in strain C58 (disarmed strain) were used for electro transforma-
terms of success and current efforts. Unfortunately, transfor- tion and transformed cells were selected by kanamycin resis-
mation with Agrobacterium requires that the cotton genotype, tance.
be regenerable from callus tissues, a feature which so far ap- Development of Recombinant DNA Construct
pears to be limited to some Coker lines and an Australian culti-
for Resistance Against CLCuV Based on Virus-
var Siokra 1-3 among the commercial cultivars. Because of
this limitation, researchers have strong interest in alternative induced Expression of a Cytotoxic Gene
transformation processes such as direct transformation of mer- During recent years, several techniques have been reported for
istems with Agrobacterium. the development of genetically engineered resistance against
geminiviruses. Pioneering work on the use of ribosome-inacti-
Tissue Culture of Local/Exotic Varieties of Cotton vating proteins (RIPs) has been initiated at John Innes Centre.
Nineteen local/exotic cultivars of cotton were evaluated for in- RIPs are naturally occurring plant toxins that are presumed to
vitro callus induction and plant regeneration. Varieties S-12, provide a defense mechanism against pathogens or predator by
NIAB-78, AEM-1-85, FH-682 and BH-36 produced signifi- disrupting protein synthesis in damaged eukaryotic cells. One
cantly better calli than other varieties. However, induction was of these RIP, dianthin has been exploited in these studies. The
observed to be highly variable not only among different geno- expression of dianthin is driven by viral coat protein promoter
types but also among various explants of the same genotype. and is activated in-trans by one of the viral gene product (AC2).
Successful attempts were made to control contamination, auto- The activation of transgene expression during virus infection
inhibitory response and decay of calli. Embryoids were observed avoids the constitutive expression of the transgene.
in some varieties but regeneration was obtained only in Coker- A set of primer was designed to amplify the coat protein pro-
312 and Sikora 1-3 varieties. Further work is in progress. moter of CLCuV. Necessary restriction sites were introduced
Meristem Tip Culture of Local Varieties of Cotton to ensure that the promoter is in frame with the dianthin gene.
The primers were successfully used and the desired product
Meristem shoot tips of ten cultivars of cotton Gossypium hirstum
was cloned in pGEMT vector and checked by restriction analy-
were cultured on several media formulated for shoot and root
sis. The cloned fragment was further checked by sequence analy-
development. The best shoot development was observed on
sis. The clone was sequenced by use of a T7 sequencing kit
media containing 0.1 mg/liter Kinetin, while rooting was ob-
using 33P labeled nucleotide. The sequence analysis confirmed
served on media containing 0.5 mg/l NAA and 0.1 mg/l Kine-
that the desired product has been cloned. A plant expression
tin. No inter-varietal variability was observed. A complete pro-
vector pJIT163 was used for the expression of transgenic
tocol was developed from meristem tip culture to field transfer
dianthin. Double 35S promoter in the vector was replaced by
for biolistic gun transformation of cotton.
coat protein promoter and dianthin was cloned in the correct
Development of a Recombinant DNA Construct reading frame. The construct was lifted by digestion with Sst I
Based on Sense or Antisense Expression of and ECoRV and cloned in plant transformation vector pBin
AC1 Gene Plus. The clone was transferred to agrobacterium strain C58 by
electroporation.
Several technologies have been reported for the development
of genetically engineered resistance against cotton leaf curl vi-
38

Development of Constructs Based on represented by only a few loci because there are not enough
Antisense RNA Expression of Parts of number of characters available. Moreover, they can also be af-
Complementary Sense Genes fected by environmental factors and growth practices. To have
an accurate and reliable estimate of genetic relationships and
To express the viral antisense RNA in transgenic plants, an ex-
genetic diversity, a large number of polymorphic markers are
pression cassette was constructed. A ~1.5 kb fragment contain-
essentially required.
ing the double CaMV 35S promotor and poly A terminal se-
quence was isolated from the plasmid pJit 60 (gift from Dr. P. Random amplified polymorphic DNA (RAPD) technique of
Mullineaux, J11, Norwich, UK) with Xho1 and Sst1 and Williams et al (1990) provides an unlimited number of markers
subcloned into plasmid pBluescript 11 KS to get pSQMW1. which can be used for various purposes. In addition to techni-
The fragments of ~ 460 bp (Start of AC1-end of AC4 gene), cal simplicity and speed of RAPD methodology, its level of
~520 bp (end of AC4- start of AC2 gene) and ~ 540 bp (Start of genetic resolution is equivalent to restriction fragment length
AC2-end of AC3 gene) were isolated from the pYASF clone polymorphism (RFLP) for determining genetic relationships.
using specific primers (SH1 and SH2, and SH3, SH4, SH5 and RAPD analysis was used to evaluate the genetic diversity of
SH6 respectively) by PCR. The amplified DNA fragments were elite commercial cotton varieties in addition to the intravarietal
end filled with T4 DNA polymerase. The individual fragments studies. Twenty individual plants of cotton variety S-12 were
were then cloned in PSQW1. More than 60 recombinants were analyzed with 10 primers for any polymorphisms. No poly-
screened for sense and antisense insertions of the above men- morphisms were observed with any of the ten primers indicat-
tioned gene sequences. Confirmation was obtained by cutting ing that the technique can be used for the analysis of purity of
the plasmid insert with restriction enzymes. PstI (AC1-AC4 seeds in cotton.
and AC1/AC2/AC3 gene) and Sa11 (AC1 middle region)
Twenty-two varieties belonging to Gossypium hirsutum L. and
The fragments of 2.0 kb harboring the above mentioned genes one to G. arboreum L. were analyzed with 50 random decamer
in both sense and antisense orientation were isolated by cutting primers using polymerase chain reaction (PCR). Forty-nine
the pSQW1 with Sst1 and EcoRV. Finally these fragments were primers detected polymorphism in all 23 cotton varieties, while
cloned into the plant transformation vector PGA 482 at Sst1/ one produced monomorphic amplification profiles. A total of
Hpal site to yield PGS clones. Later these plasmid carrying the 349 bands were amplified and 89.1% of which were polymor-
respective genes were transformed into agrobacterium tumefa- phic. Cluster analysis by unweighted pair group method of arith-
cient strain LBA4404 by electroporation. metic means (UPGMA) showed that 17 varieties can be placed
in two groups with a similarity ranging from 81.51% to 93.41%.
Transformation of Constructs in an Elite
G. hirsutum L. varieties S-12, V3 and MNH-93 showed a simi-
Pakistani Cotton Variety S-12 larity of 78.12, 74.46 and 69.56% respectively with rest of the
A protocol was developed for Agrobacterium mediated trans- varieties. One variety CIM-1100 showed 57.02% similarity and
formation of an elite cotton variety S-12. The method that uses was quite distinct. The diploid cotton G. arboreum L. var. Ravi
3-day-old mature embryos for transformation and selection was was also very distinct from rest of its tetraploid counterparts
made on kanamycin. Analysis of the first batch of plants sug- and showed only 55.7% similarity. The analysis revealed that
gests that the gene has been integrated in these putative the intervarietal genetic relationships of several varieties is re-
transgenic plants. The plants are being tested for the presence lated to their center of origin. The results also showed the ge-
of genes and the ability of plants to resist geminiviruses. netic relationship of elite commercial cotton varieties with some
standard “Coker” and diploid G. arboreum L. var. Ravi (old
Cotton Genome Project world cotton). As expected, most of the varieties have a narrow
A majority of the present day commercial cotton varieties grown genetic base. The genetic similarities obtained can be used for
in Pakistan belong to Gossypium hirsutum L. (upland cotton) a the selection of possible parents to generate a mapping popula-
very few to diploid species G. arboreum L. Breeders have tion. The polymorphic profiles can be used for the identifica-
evolved these varieties through selection based on morphologi- tion of different varieties and the protection of breeders propri-
cal and physiological features (yield, fiber quality, resistance ety rights.
against certain pests and diseases, etc.). Acknowledgements
Most of the varieties grown in Pakistan originated from intraspe- Financial assistance for the present tripartite project (Pakistan, UK
cific crosses of G. hirsutum L. at various research centers around and USA) was provided by the Common Fund for Commodities (the
the country. These hybridization practices resulted in narrow Netherlands) through the International Cotton Advisory Committee
genetic base of the new varieties. Any crop with a narrow ge- (ICAC), Washington, DC, USA.
netic base is more prone to natural disasters such as outbreak References
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 39

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