Vol. 7(2), pp.

8-14, April, 2015

DOI: 10.5897/IJGMB2015.0108
Article Number: B8D441352705
ISSN 2006-9863
International Journal of Genetics and Molecular
Copyright © 2015 Biology
Author(s) retain the copyright of this article
http://www.academicjournals.org/IJGMB

Full Length Research Paper

Effects of cytokinin types and their concentration on in

vitro shoot induction and multiplication of korarima
Rahiel Hagos1 and Hailay Gebremdhin2*
1
Department of Dry Land Crop and Horticultural Sciences, Mekelle University, P. O. Box 231, Mekelle, Ethiopia.
2
Department of Plant Science, Debre Berhan University, P. O. Box 445, Debre Berhan, Ethiopia.
Received 18 January, 2014; Accepted 10 April, 2015

Korarima (Aframomum corrorima (Braun) P.C.M. Jansen) is a herbaceous perennial plant that belongs
to the family Zingiberaceae in which lack of a steady supply of quality planting material is one of the
bottlenecks for the exploitation of the export potential. Thus, the use of micropropagation is suggested
to alleviate these problems. In micropropagation, in vitro seed germination and multiplication of
hypocotyls/shoot tips to regenerate shoots is preferred because it minimizes sterilization cost. So far,
there was no available efficient protocol optimization on in vitro propagation of korarima in Ethiopia.
Therefore, an experiment was conducted to determine the optimum concentration of Kinetin (KIN) and
N-6-benzyladenine (BA) on in vitro regenerating of korarima explants from hypocotyls/shoot tips.
Multiplied shoots were subcultured on hormone free medium for four weeks to avoid carryover effect.
The different concentrations of KIN (0, 0.5, 1.0, 1.5 and 2.0 mg/l) and/or BA (0, 1.5, 3.0, 4.5 and 6.0 mg/l)
were arranged in randomized complete design (CRD) with four replications. Results indicate that
medium containing 6.0 mg/l of BA alone was found to be optimum for shoot induction after six weeks.
High level of BA alone was found the best for excess shoot proliferation, but high level of KIN mostly
enhances longer shoots and regenerates minor roots. Generally, it can be concluded that Korarima can
successfully induce shoot from hypocotyls that originated from seeds without any contamination on the
initial inoculation of the explants and higher concentration of BA (6.0 mg/l) could be used to obtain
desirable shoot regeneration of korarima.

Key words: Cytokinins, N-6-benzyladenine (BA), hypocotyls, kinetin (KIN), shoot induction, shoot Multiplication,
in vitro.

INTRODUCTION

Korarima (Aframomum corrorima (Braun) P.C.M. Jansen), under the monocotyledonous crops. Morphologically, the
which is native to Ethiopia, is one of the renowned spices plant resembles Indian cardamom (Elettaria cardamomum)
and medicinal plants of the family Zingiberaceae. It is a and consists of an underground rhizome, a pseudostem
herbaceous, perennial and aromatic species classified and several broad leaves (Eyob, 2009). Mature korarima

*Corresponding author. E-mail: hailushgm@yahoo.com

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0
International License
 Hagos and Gebremdhin 9

plants can reach a height of 1-2 m and set seeds after 3- korarima growers using the conventional vegetative pro-
5 years of planting, depending on the planting materials pagation (Tefera and Wannakrairoj, 2004; Eyob, 2009).
used and it continues to bear seeds for several years. Therefore, enhancement of korarima seed germination
Different plant parts of korarima (seeds, pods, leaves and and multiplication is essential in propagation and breeding
flowers) are locally used in traditional medicine to treat program, as well as for testing and using available germ-
humans and cattle as well. Since its rhizomes and leaves plasm. Particularly, proper tissue culture procedures, which
spread well on the ground, korarima plants could also assure successful and efficient propagation from seedling,
serve a lot for soil and water conservation, especially by need to be developed. To date, only two tissue culture
covering and protecting the soil from erosion and drying studies have been reported by using of either lateral buds
all year round in the mountainous areas where the crop is from rhizomes of the Jimma local cultivar (Tefera and
commonly cultivated (Eyob et al., 2008). Wannakrairoj, 2004) or by using pretreated seeds of
The production and productivity of korarima is conti- cultivar Mume collected from the southern part of the
nuously decreasing mainly due to absence of expansion country (Eyob, 2009).
of their cultivated areas and destruction of their forest Even though korarima has much advantage, lack of a
natural habitat, which had been and still is the major steady supply of quality planting material is one of the
source of korarima production. In line with this, results bottlenecks for the exploitation of the export potential of
from a recently undertaken formal survey carried out in korarima in Ethiopia. However, only limited efforts have
parts of Southern Ethiopia, attempts of the Ethiopian been made so far to improve the crop using traditional
government to motivate farmers to expand their korarima and modern biotechnological approaches. Thus use of
plantations were not successful due to the associated micropropagation is suggested to alleviate the problem of
varied production constraints thereof. This mainly includes shortage of planting materials. In vitro seed germination
lack of improved korarima varieties with high yielding and multiplication of hypocotyls/shoot tips to regenerate
potential and product quality, together with suitable shoots is preferred because it minimizes sterilization cost.
agronomic practices, like best techniques of propagation So far there was no available efficient protocol optimization
(Jansen, 2002; Eyob, 2009). on in vitro propagation of korarima. Hence, this study was
Korarima can be propagated either by seed or by initiated to come up with a suitable in vitro propagation
cutting of its clumps, though the latter is by far the most protocol for the local Jimma cultivar in the absence of
common method, as it yields earlier and ensures a true- coconut water (CW) and Imazalil (IMA), which gave the
to-type propagation than the former. During in vivo best result when used as media components by Tefera
propagation of korarima, the suitable propagation technique and Wannakrairoj (2006), but that are not readily available
is by using seed (Eyob, 2009). However, vegetative at the local market. In addition, inclusion of CW and TDZ
propagation through cuttings results in the destruction of to the culture medium resulted in reduction of both
the productive garden, on top of the commonly associated number of roots, root length and shoot length. Therefore,
shortage of planting materials to cover wider areas of the present study was envisaged to focus on optimizing
land. Consequently, seed propagation of korarima is under- and/or developing an efficient protocol for the in vitro
taken to cover large areas of land retaining the mother regeneration of korarima from pretreated seeds/hypocotyls
productive stand intact, however, it is essential to give the of Korarima; Jimma local cultivar.
utmost care while preparing the seeds (Endashaw, 2007).
Nevertheless, the slow seed germination and growth of
the subsequent seedlings are concerns of korarima MATERIALS AND METHODS
growers. The germination of korarima seeds faces certain
Treatments and experimental design
problems due to the presence of dormancy, possibly
associated with its hard seed coat like were reported on
The establishment of contamination free seeds (capsules) of Jimma
seeds of Elettaria species (Sulikeri and Kololgi, 1977) local cultivar, cultivated around the Jimma located areas was selected
and presence of low food reserve in the seed endosperm. from the Jimma Agricultural Research Center (JARC), which was
More importantly, as korarima is mainly propagated through apparent disease-free planting material and fits agronomically. Well
the vegetative means using one year old rhizomes, the ripened fresh capsules (red capsules) were selected and then used
need for bulk of rhizomes as planting materials and slow as a starting material for the subsequent experiments. Extracted
seeds were treated with 50% H 2SO4 for 60 min and soaked in 250
multiplication rate of the rhizomes is a serious bottleneck mg/l of gibberillic acid (GA3) for 24 and 48 hrs before sterilization
observed. Also the destructive harvesting of the rhizomes and then sterilized by 3.0g/l Kocid for 30 min, 70% ethyl alcohol for
associated with vegetative propagation is a serious threat 3 min and 25% NaOCl + 2-drop of Tween-80 for 15 min during
as there is always the possibility of losing the mother sterilization.
plant during this process. The problem becomes much Every sterilization steps were washed by distilled water. Plant
more aggravated when one has to transport the rhizome growth regulators (PGR) used for the experiment were N-6-
benzyladenine (BA) and Kinetin (KIN). To identify the best
clumps to distant areas due to their perishable nature and combinations of these hormones for shoot induction and multiplication,
relatively costly transportation. Susceptibility to unknown various concentrations of KIN (0.0, 0.5, 1.0, 1.5, and 2.0 mg/l)
diseases is also among the major problems faced by and/or BA (0.0, 1.5, 3.0, 4.5 and 6.0 mg/l) were treated with four
10 Int. J. Genet. Mol. Biol.

replications of each treatment. Wannakrairoj (2004). Then after culturing on hormone free media
for one month, new regenerated shoots were cultured on fresh
medium supplemented with different hormonal combinations of KIN
Preparation of growth culture conditions and BA in jams jars containing 40 ml of MS media with different
concentration of each hormone. In each experimental unit, three
Culture media were prepared by taking the recommended amounts shoots (1.5-2.0 cm) were cultured per jar/replication with a total
of MS (Murashige and Skoog, 1962) stock solutions supplemented observation unit of nine explants per treatment. The experiment
with 3% (w/v) sucrose as a carbon source, and 0.7% (w/v) agar (for was repeated two times, though only data from the last one was
shoot multiplication) and 0.8% (w/v) agar (for root induction) as used for statistical analysis. Cultures were incubated in the culture
solidifying agents. In each experiment, the desired concentrations room at a constant temperature of 25 ± 2C and relative humidity of
and types of PGR were added before autoclaving and 40 ml of the 50-60%, under cool white fluorescent light of 28 μmol m-2 s-1 (1500-
respective medium was poured into 350 ml jams jar and covered 2000 lux) photosynthetic photon flux density with 16 hper day
with a cap for shoot multiplication. After mixing up all media photoperiod for six weeks. After six weeks, data were recorded from
components together with the combined PGR and adjusting the individual emerged new shoots obtained from each cultured shoots
volume, the pH of the culture medium was adjusted to 5.70 with and subsequently cultured on free hormone media.
either 1% N HCl or 1% N NaOH. Later, the respective solidifying
agent (agar) was added into the medium. Medium was autoclaved
at 15 pounds per square inch (psi) that is 121°C for 20 min. Data analysis
Autoclaved media was allowed to cool in sterile environment after
which it was ready for use. Finally, all the autoclaved culture media Mean values of the parameters were subjected to analysis of variance
were retained in the media room for a maximum of three days prior (one-way ANOVA and two-way ANOVA) using the SAS software
to use. packages (SAS, 2008, version 9.2). The significant differences
among treatments were compared using Least Significance
Difference (LSD) for the one-way ANOVA and Ryan-Einot-Gabriel-
Plant material, surface disinfection and inoculation of explants Welch range test (REGWQ) for the treatment combinations of the
two-way ANOVA at a 5% of probability level.
Fresh capsules (seeds) (2–5 mm in diameter) of Korarima were
obtained at the peak harvestable stage of capsules and were
collected from apparent disease free growing parents. The capsules RESULTS AND DISCUSSION
were collected thoroughly from the agronomically desirable parents
and harvested fresh capsules seeds were extracted and then
sterilized before culturing on the prepared germinated media. Then Selection of shoot tips for the subsequent experiments
seeds were washed five times immediately with tap water at least from in vitro germinated seeds
for 15 min and then they were kept (soaked) under 3.0 g/l Kocid
[Cu(OH)2] for 30 min, and rinsed five times with sterilized distilled In order to break seed dormancy, seeds were soaked in
water. The rinsed seeds were also soaked in 50% of H2SO4 for 60 50% of H2SO4 for 60 min and in 250 mg/l of GA3 for one
min then seeds were crushed by hands using nylon cloth and
immediately soaked in 250 mg/l of GA3 for 24 and 48 h. Finally,
to two days. Seeds were not germinated in all media
they were washed with sterilized distilled water for 20 min and more strengths except one-fourth MS strength liquid and solid
scales were then removed, followed by washing with detergents. media. Germination was started after 24 days and the
Inside the laminar air-flow cabinet, seeds were rinsed with 70% germination percentage was 66.7% (data not shown).
ethanol for 3 min; followed by one-step surface sterilization using Therefore, one-fourth MS medium strength was more
25% sodium hypochlorite so called commercial bleach (which preferable for seed germination of korarima and the use
contains 5.1% active chlorine) mixed with two drop (2.0 ml/l) of
Tween-80 (wetting agent) by vigorous shaking for 15 min. Then, the of a single capsule for a given basal MS medium strength
seeds were washed five times using sterilized distilled water and was also important to obtain uniformly emerged hypocotyls
were further trimmed to remove dead seed coat and sulfuric acid than mixed seeds. Seeds soaked on GA3 for 1 to 2 days
affected scales. Before culturing, the explants (sterilized seeds) were best for breaking of the dormancy and encouraged
were soaked in sterile distilled water for 20 min to remove traces of seeds to start their germination within 24 days, but they
chlorine and other remnants in the seed.
had erratic seed germination. Hypocotyls emerged from
The surface sterilized seeds were inoculated into different liquid
MS basal media strengths in an aseptic condition that contained full the one-fourth solid MS media and treated in 250 mg/l
MS (FMS), half MS, one-fourth MS, one-eighth MS and free water GA3 for 48 h were highly elongated (thinner) and abnormal
as a control. 20 ml of each liquid media was dispensed into 100 ml in their performance than one-fourth liquid MS medium
baby food jar embedded with cotton pad and were adjusted to pH of treated for 24 h (Figure 1).
5.70 prior to autoclaving. On top of this, solid one-fourth MS Seeds placed in sterile cotton pad soaked with distilled
medium was also prepared for germination and it followed the
above procedures except with the addition of 7.0 g/l agar. Each
water have the ability to germinate better than FMS, half
germination media strength was inoculated by seeds that emerged MS and one-eighth MS but it took longest mean days (for
from a single capsule. more than five months). On top of this, emerged hypocotyls
from free distilled water were highly sensitive to bacterial
contamination as compared to hypocotyls emerged from
Effect of cytokinins on shoot induction and multiplication one-fourth MS medium. Liquid media including sterile
Induced shoot tips obtained from hypocotyle explants were cultured cotton pad was better than solid media for facilitating the
on MS medium fortified with 1.0 and 3.0 mg/l of KIN and BA in imbibitions of water to induce enzymatic activities of the
combination for further shoot induction and multiplication, according seed and minimize the contamination during inoculation
to Balachandran et al. (1990), Sanghamitra (2000) and Tefera and of the hypocotyls in test tubes for further shoot induction
 Hagos and Gebremdhin 11

multiple shoots were obtained for the subsequent

experiments.

Effects of different concentrations of KIN, BA and their

combination on shoot induction and multiplication of
A B C stage of korarima

Number of days to shoot induction

As shown in Table 1, KIN alone results in a high significant

difference on number of days to shoot induction (P =
D E F 0.005), but both the interaction effects of BA and KIN nor
KIN alone significantly affected the number of days to
Figure 1. Seed germination of korarima in MS liquid and solid
media. A) Hypocotyls emerged from the culturing of korarima seeds shoot induction. Hence, 1.0 mg/l of KIN was found to
in one-fourth MS liquid media. B) Hypocotyls emerged on solid one- have the shortest number of days to shoot induction but it
fourth MS media; C and F) Inoculation of hypocotyls on induction had no significant difference with 0.5 and 1.5 mg/l
media; D and E) Hypocotyls generated from free distilled water. treatment levels of KIN. The highest number of days to
shoot induction was observed in 2.0 mg/l of KIN but there
was no significant difference with the PGR free medium.
Table 1. Mean and probabilities of the independent effect of KIN
and BA on korarima shoot growth after six weeks of culture. Number of shoots
Mean (main effect) Number of shoots was not statistically different among
Treatment No. of days to the treatment combinations of KIN and BA, except with
No. of leaves
shoot Ind. the treatment combination of PGR-free medium, 0.5, 1.0,
0 18.07
a
12.6
a 1.5 and 2.0 mg/l of KIN alone, 1.5 mg/l of BA alone, 0.5 +
0.5 16.21
ab
13.6
a 1.5, 1.0 + 1.5 and 2.0 + 3.0 mg/l of KIN and BA in
1.0 15.3
b
12.2
a combination, respectively and was significantly different
KIN (mg/l) ab a from the other treatment combinations (Table 2). But, the
1.5 17.27 12.47
a a best medium for shoot multiplication was obtained on 6.0
2.0 18.2 11.47
mg/l of BA alone (which is similar with findings of
a b Kochuthressia et al. (2010)) and the combination of 1.5
0 17.80 9.8 mg/l of KIN with 3.0 mg/l of BA, had a good response to
a a
1.5 16.40 15.2 regenerate the largest number of shoots with an average
a a
3.0 16.87 13.8 number of 10.33 and 9.67 shoots per plantlet,
a ab
BA (mg/l) 4.5 17.67 11.67 respectively. But the combination of 1.5 mg/l of KIN with
a ab
6.0 16.33 11.87 3.0 mg/l of BA was not significantly different from the
NS
KIN 0.005*** 0.650 combination of 0.5 mg/l of KIN and 3.0 mg/l of BA as well
NS
BA 0.268 0.003*** as 3.0 mg/l of BA alone in number of shoots.
KIN*BA 0.073
NS
0.193
NS The lowest number of shoot multiplication (3.33 in
CV(%) 13.57 30.38 average) was observed at higher concentration of KIN
(2.0 mg/l), which is not similar with the findings of
*** = highly significant difference at 1% probability level and *
significance at 5% probability level (REGWQ). Means within a
Kochuthressia et al. (2010) in which 6.4 shoots/explant
column followed by the same letter are not significantly different at regenerated on red ginger (Alpinia purpurata) and the
P < 0.05 level of significance (REGWQ). No, number; Prob., PGR free MS medium was used as a control also
probability; Ind., induction; NS, non significance. induced shoots at a rate of 4.33 shoots per explants. The
result from the present study on the shoot multiplication
of korarima (Jimma local cultivar) agrees with those of
Sanghamitra (2000) and Naz et al. (2009) on shoot
and multiplication. After eight weeks, hypocotyls (cotyle- multiplication of turmeric with high concentration of BA
donary nodal segments) that emerged from the seed (5.0 mg/l) and Kavyashree (2009) on ginger var. Varada
culture was taken and cultured in test tube containing MS with high concentration of BAP (17.76 μM). But it had no
medium fortified with 1.0 mg/l of KIN and 3.0 mg/l BA significance different with other treatment combinations,
according to their germination date for further shoot and hence as the concentration of KIN increases to 2.0
proliferation. Hypocotyls were also subcultured every month mg/l and BA at zero, the average numbers of shoots per
on the same medium of KIN and BA until the desired plantlet become lowest. In general, the sole use of BA in
12 Int. J. Genet. Mol. Biol.

Table 2. Effects of different concentration of KIN and BA in shoot induction and multiplication after six weeks of culture.

KIN BA NS /explants SL/explants SFW SDW

(mg/l) (mg/l) (cm) (mg) (mg)
hgf a ba ba
0 0 4.33 3.11 390 30
c-h abc ba ba
0 1.5 6.00 2.65 413 33
abc abc ba ba
0 3 9.33 2.62 333 27
a-d bc ba ba
0 4.5 8.33 1.9 290 21
a abc a ba
0 6 10.33 2.086 567 42
hg a ba ba
0.5 0 4.0 3.10 267 23
d-h abc ba ba
0.5 1.5 5.33 2.52 310 23
abc bac ba ba
0.5 3 9.00 2.35 390 29
a-d abc ba ba
0.5 4.5 8.33 1.82 370 28
a-d c ba ba
0.5 6 8.33 1.82 290 22
c-h ab ba ba
1 0 5.75 2.89 500 39.6
b-h abc a a
1 1.5 6.33 2.257 550 50
a-d abc b b
1 3 8.33 2.253 177 16.7
a-g abc ba ba
1 4.5 7.33 2.367 357 34.7
a-d bc ba ba
1 6 8.67 2.053 353 34.7
d-h abc ba ba
1.5 0 5.33 2.10 487 30
a-e bac ba ba
1.5 1.5 8.00 2.21 413 24
ab bc ba ba
1.5 3 9.67 1.88 430 26.7
a-e c ba ba
1.5 4.5 8.00 1.756 377 32.3
a-d abc ba ba
1.5 6 9.00 2.077 373 33.3
h abc ba ba
2 0 3.33 2.383 477 45
a-d bc ab ba
2 1.5 8.33 1.843 510 31
e-h c ba ba
2 3 4.67 1.633 483 43
a-g bc ba ba
2 4.5 7.33 1.836 320 30
a-f c ba b
2 6 7.67 1.820 233 16.7
Means 7.24 2.21 0.386 0.0307
CV (%) 13.15 13.13 30.06 31.24
SE (±) 0.549 0.168 0.067 0.0055
SE mean (±) 0.21919
NS NS
KIN 0.0001*** 0.0001*** 0.243 0.062
NS NS
Prob. BA <.0001*** 0.0001*** 0.108 0.537
*** ***
KIN*BA <.0001*** 0.0124* 0.009 0.003
*** = highly significant difference at 1% probability level and * significance at 5% probability level (REGWQ). Treatment mean values with the
same letter in a column are not significantly different at P < 0.05 level of significance (REGWQ). NS, non significance; SE, standard error, CV
(%), coefficient of variance; Prob., probability; NS/explants, mean number of shoots per explants; SL/explants, mean shoot length per
explants; FW, mean shoot fresh weight, DW- mean shoot dry weight.

shoot induction and multiplication medium is applicable The highest number of leaves was found on 1.5 mg/l of
for better multiplication of shoots than it is combined with BA and it was not significantly different with the three
KIN and it is similar with the results of Nayak (2000), 5 concentrations of BA (3.0, 4.5 and 6 mg/l), but the lowest
mg/l BA in C. aromatica Salisb and Sharma and Singh number of leaves was observed on PGR-free medium.
(1995), 8 mg/l BA in ginger enhanced microrhizome
production.
Shoot length
Number of leaves
Both KIN and BA alone highly significantly affected the
The number of leaves was not affected by the concen- shoot length with P≤.0001, but their interaction (KIN*BA)
tration of KIN alone (P = 0.65) and their interactions was affected significantly (P = 0.01). Hence the PGR-free
(KIN*BA) (P = 0.19), but BA alone highly significantly medium had the longest mean shoot length (3.11 cm)
affected the number of leaves at P = 0.0033 (Table 2). followed by 0.5 mg/l of KIN alone (3.10 cm) than other
 Hagos and Gebremdhin 13

In all, the result shows that, with an increased in

concentration of KIN alone in the medium, the number of
shoots induced decreased significantly. In this study,
higher concentration of KIN alone regenerates roots and
longer shoots (Figure 2). On the contrary, BA alone was
A C very responsive to the shoot induction and multiplication
but not true for shoot elongation on korarima. The
application of 6.0 mg/l of BA alone resulted in significant
increased number of shoots, shoot length, shoot fresh
weight and dry weight per explant.
B D This result agrees with works of several authors
(Sanghamitra, 2000; Sivakumar and Krishnamurthy,
Figure 2. Explants cultured on different cytokinin combinations 2000; Tefera and Wannakrairoj, 2004, 2006). In addition,
of KIN and BA. A) Explants cultured on KIN (2.0 mg/l) + BA 3.0 mg/l of BA alone was found to be not significantly
(3.0 mg/l). B) Explants cultured on MS + 2.0 mg/l of KIN and different from 6.0 mg/l on number of shoots, shoot length,
regenerates roots. C) Explants cultured on KIN (1.0 mg/l) + BA shoot fresh and dry weight. Lastly, from the present
(6.0 mg/l). D) Explants cultured on MS + 6.0 mg/l of BA. study, the combination of BA and KIN developed fewer
multiple shoots, which was similar with the results of
Purohit and Dave (1996) and Sivakumar and Krishnamurthy
(2000) and emerged few roots.
treatment combinations, and it had significant difference From this study, it can be concluded that, 0.5 mg/l of
with 0.5 + 6.0, 1.5 + 3.0, 1.5 + 4.5, 2.0 + 1.5, 2.0 + 3.0, KIN including MS were more responsive for regeneration
and 2.0 + 6.0 mg/l of KIN and BA treatment combinations. of roots and elongation of shoots and also shoots were
When 2.0 mg/l of KIN combined with the four levels of more vigorous than other treatment combinations in
BA, the shoot length became decreased and had no korarima. On the other hand, shoots cultured on 6 mg/l
significant difference among them. But as the concen- BA including MS, responded more for induction of new
tration of KIN increases from 0.5 to 2.0 mg/l and combined microshoots. As the concentration of BA increases
with 6.0 mg/l of BA, there was no significant difference on throughout the hormonal combination of the medium, the
shoot length (Table 2). Whereas the shortest shoots was number of shoot induced was increased. All the shootlets
obtained on the treatment combination of 2.0 + 3.0 mg/l regenerated survived (100%) without contamination.
of KIN and BA (which were reported by Kochuthressia et However, some physiological senescence like drying on
al. (2010) on red ginger exhibited shoot regeneration rate the bottom of the shootlets and tip burn in the shoots
up to 6.4±0.32 shoots/explants), but it had no significant cultured with more concentration of KIN were observed.
difference with all treatment combination, except with
PGR free medium, 0.5 and 1.0 mg/l of KIN alone, which
had a long shoots. Conclusion

Generally, different concentration of cytokinins type and

Shoot fresh and dry weight concentration influenced the in vitro propagation of korarima
in shoot induction and multiplication. The use of KIN resulted
The interaction of the two factors (KIN*BA) was highly in significantly lower shoot regeneration as compared to
significant (P = 0.009) on shoot fresh and dry weight with BA. Higher concentrations of BA increased the number of
P = 0.003, but not their alones (Table 2). In the case of regenerated shoots but decreased shoot length. The
shoot fresh weight, all treatment combinations were not application of BA in the medium as compared to KIN
significantly different among themselves. But there was stimulates the rate of shoot regeneration and the greatest
significant difference between 6.0 mg/l of BA alone or 1.0 shoot regeneration was found on media with 6.0 mg/l of
+ 1.5 mg/l or other treatment combinations of KIN and BA BA alone. But at high concentration, it has impacts on
in combination with 1.0 + 3.0 mg/l of KIN and BA (which destroying plantlets via vitrification andshoot tip necrosis.
had a lowest shoot fresh weight). On the other side, Therefore, higher concentration of BA (6.0 mg/l) could be
highest shoot dry weight was obtained on 1.0 + 1.5 mg/l used to obtain desirable shoot regeneration of korarima.
(50 mg per shootlet) of KIN and BA and had significant
difference with 1.0 + 3.0 mg/l and 2.0 + 6.0 mg/l (16.7 mg Conflict of interest
per shootlet) of KIN and BA in combination that is the
lowest shoot dry weight was found on them. Generally, The authors did not declare any conflict of interest.
there was no significant difference in almost all the
treatment combinations, but there was significance
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