Leaflets 12: 577-85. 2008.
and Plant Regeneration of Vigna mungo (L.) Hepper via Half
*Harisaranraj, R., S. Saravana Babu and Suresh, K.
of Plant Biology and Plant Biotechnology
College, Erode. (T.N.) INDIA
11 August 2008
The present study optimized the regeneration
protocol by using half seed explant in Vigna mungo (L) Hepper organogenesis. Half seed explants were inoculated
onto B5 medium supplemented with kinetin (4.7 M
to 23.5 M), 6- benzyladenine
(4.4 M to 22.2 M),
naphthaleneacetic acid (5.4 M
to 27.0 M), indolebutyric
acid (4.9 M to 24.5 M)
and 2,4- dichlorophenoxyacetic acid (4.5 M to 22.5 M).
Callus initiation was observed in all media evaluated and the highest cell
proliferation was obtained from explants cultivated in the presence of 13.3 M BAP and 13.5 M 2,4-D. Shoot induction
was obtained from callus induced on 13.3 M BAP
and 13.5 � 2,4-D at 6 weeks after
transferring the callus to a B5 medium supplemented with 13.3 M BAP. Roots
were induced from shoots on B5 media with indolebutyric
acid (IBA, 14.7 M)
and then regenerated plants were hardened and acclimated in greenhouse
Words: Vigna mungo,
callus induction, half seed, rooting, shoot regeneration.
Vigna mungo (L) Hepper (Black
gram) is considered to have been domesticated in India from its wild
ancestral form V.mungo var.silvestris
(Lukoki, Marechal & Otoul, 1980). Center of genetic diversity is found in
India (Zeven and de Wet. 1982). Natural
distribution of V.mungo var.silvestris
ranges from India to Myanmar (Tateishi. 1996). Vigna mungo (L) Hepper (Black Gram or Urd Bean)
is one of the most widely used pulse crop in India. It is a highly prized
pulse, very rich in phosphoric acid. It is cultivated as fallow crop
after rice cultivation in India. It is grown in various agro-ecological
conditions and cropping systems with diverse agricultural practices (Sanjeev Gupta et al). The improvement and
optimization of Vigna mungo
characteristics, such as increasing resistance to pests, lowering
allergenic protein levels in seeds, drought and salt tolerance is therefore
The crop improvement was done by breeding methods
in early days. However, breeding is difficult due to the fact that Vigna mungo is
self-pollinating crop and the genetic variation among the Black gram
varieties is narrow. The regeneration system used to generate genetically
modified plants was somatic embryogenesis from immature seeds (Christou et al., 1989) or organogenesis from cotyledonary nodes (Finer and McMullen, 1991; Shan et
al., 2005). In Vigna mungo, genetic
modification is also based on organogenesis from the cotyledonary
nodes, a high efficient regeneration system was obtained by sub culturing
nodes on Thidiazuron (TDZ) supplemented medium (Tzitzikas�������� et
al., 2004). Medium components with quantity of plant growth regulator
influence the regeneration of plants (Shan et al., 2005). Callus
regeneration is advantageous over direct regeneration for transformation
since effective selection of transgenic cells can be achieved. However, the
efforts made to regenerate plants from callus have yielded poor results since
plants could not be regenerated from any type of Vigna
mungo callus (Hu and Wang, 1999). In attaining this goal, we owe much
to transformation techniques for producing new breeding materials that would
not be available in the germplasm among
cross-compatible species. A number of successful regeneration protocols have
been developed, especially with a view to facilitate genetic transformation.
The earlier reports
focused on the cotyledonary node since the
morphogenetic potentiality is confined to that region (Hu
and Wang, 1999). Eventhough plants have been
successfully transformed by using cotyledonary
nodal explants, the major problem of chimerism
still persists (Meurer et al., 1998). This
is primarily due to pre-existing meristematic shoot buds, which continue to
grow effectively on a medium containing a selectable agent like antibiotic or
herbicide. Christou et al. (1988) could
effectively select transgenic calli after
bombarding protoplasts but they failed to produce transgenic plants. The callus
induction and plant egeneration of Indian soybean (Glycine max (L.) Merr.
cv. CO3) via half seed explant
culturewas carried out by (B. D. Ranjithakumari et al).
In this present investigation was to standardize the optimum protocol for
callus and shoot regeneration from half seed explant
of the Vigna mungo.
Materials and methods
Matured Vigna mungo seeds were utilized in the efficient production
of in vitro Vigna mungo
plant production experiments. Vigna mungo seeds were washed under continuous flashing of
running tap water for 30 min and then treated with a solution of the Tween 20 (5% v/v) for 10 min and finally surface
sterilized with HgCl2 (0.1% w/v) for 10 min. Lastly, the seeds were washed
three times with autoclaved distilled water to remove any trace of
Half seed explants preparation
Disinfected seeds were soaked in sterile distilled
water for about 4 h and a longitudinal cut along the hilum
was made to separate the cotyledons, and the seed coat was removed. The
embryonic axis found at the junctions of the hypocotyl
and cotyledon was excised to obtain the half- seed explants.
Initial explants consisted of half seed (ie, the part of the explant
from where the embryonic axis was removed) was cultured in flate side up in the medium with the base of the explant was embedded condition. The callus initiation
medium contained B5 salts and vitamins (Gamborg et
al., 1968), 30 g l-1 sucrose. The pH was adjusted to 5.8 and solidified
with 0.7% agar before autoclaving at 121�C for 20 min. The effect of growth
regulators was tested using KIN (kinetin),
BAP (6-benzyladenine), NAA (naphthaleneacetic acid), IBA
(indolebutyric acid), 2,4�D (2,4�dichlorophenoxyacetic acid) as follows: KIN
(4.7 M to 23.5 M), BAP (4.4
M to 22.2 M) and NAA (5.4 M to 27.0 M) or IBA
(4.9 M to 24.5 M) or 2,4�D (4.5 M to 22.5) in
combination with BAP (4.4 M to 22.2
M). Medium without plant growth regulators was used as a control.
Cultures were maintained under light 16h or dark 8 h conditions at 26�2�C. 50
half seed explants were inoculated per treatment and repeated 3 times and the
frequency of callus formation was determined 4 weeks after culture initiation.
Shoot induction and multiplication
To induce and proliferate shoots, the calli were transferred to shoot induction medium
containing B5 salts, B5 vitamins, 30 g l-1 sucrose, pH 5.8, 0.7% agar and
fortified with BAP (4.4 M to 22.2
M). Calli were kept under the same light/
darkness conditions from the callus initiation for 4 weeks. Depending on the
treatment, the number of shoots per treatment was recorded at 6 and 10 weeks
after transferring the calli to shoot induction
and Hardening of plantlets
For root induction, 14.7 M IBA
was tested. Elongated shoots (1.5 to 2.5 cm long) were isolated and
transferred to B5 medium, supplemented with B5 vitamins, 30 g l-1 sucrose, pH
5.8, 0.7% agar and 14.7 M IBA.
After 3 weeks, rooted plantlets were rinsed with water to wash off the agar
medium and then, transplanted to soil containing cups for hardening. The
hardened plants were maintained at 24�2�C with 18 h photoperiod (140 moles s-1) for 2 weeks then, transferred to the
The various concentrations of plant growth
regulators (KIN, BAP,
BAP+ NAA, BAP+
IBA and BAP+
2, 4 �D) were tested in callus induction and plant regenerations. Frequency
of callus and shoot initiation, number of shoots were analyzed by SPSS
software, in which statistical significance was determined at the 0.05
Half seed explant is an
efficient source for the Vigna mungo callus initiation and shoot regeneration. The
half seed derived cotyledonary nodal callus could
serve as an ideal starting material for developing an efficient Vigna mungo transformation
system. (Sairam et al., 2003).
The regeneration of callus induction and plant
regeneration were influenced by different kinds of plant growth regulator (KIN,
BAP, NAA, IBA
and 2,4�D). Callus was induced in all media tested
and significant differences were observed in the induction frequency between
different plant growth regulators (Fig. 1 to 5). Among the various plant
growth regulators, the highest frequency of callus induction was observed to
be when the half seed explants were cultured in the presence of BAP
(13.3 M) + 2,4�D (13.5 M) (Fig. 5). These observations indicate
that the morphogenetic potential is confined to only the cotyledonary
nodal cells as reported by Hu and Wang (1999).
Fig. 1. The effect of different
concentrations of KIN on callus
Fig. 2. The effect of different
concentrations of BAP on callus
The low frequency of callus induction was observed
in KIN (14.1 M) exposed explants
cultured on the B5 medium. The explants grown in B5 medium supplemented with BAP
(4.4 M to 22.2 M) and different auxin
combination such as NAA (5.4 M to 27.0 M),
IBA (4.9 M to 24.5 M) and 2,4�D (4.5 M to 22.5) were given the better yield
than that of KIN (4.7 M
to 23.5 M). Barwale et al. (1986a)
reported that the organogenic callus cultures were
obtained from immature soybean embryos grown on the medium with a high
6-benzylaminopurine (BAP) concentration
(13.3 M) and 0.2 M NAA.
Concentrations of BAP+
3. The effect of different concentrations of BAP
+ NAA on callus regenerations.
Half seed explant derived
calli were cultured on the medium fortified with
various concentrations of BAP supplemented
B5 medium (SI medium). In Vigna mungo, BAP was
found to be the most efficient in shoot formation when excised parts of
mature and immature cotyledones were used (Sairam et al., 2003). 13.3 M BAP
+ 13.5 M 2, 4 �D treated callus produced the maximum number of shoots
than other concentrations of BAP alone
(Table 1). The marked difference observed between the average number of
shoots obtained from callus induced from different concentrations and
combinations of BAP with 2, 4 - D could be
related to the media used for callus induction and multiplication. Callus
induced on 13.3 M BAP + 13.5
M 2, 4 �D formed the highest number of shoots after 6 weeks in the presence
of BAP treated SI medium (Table 1). The
former calluses were completely covered with shoot buds were easily
disintegrated during manipulation. However, the shoots could be excised from
their surface. The calli could be kept for six
months of subcultures producing an average of 6 shoots per callus in every
month. The procedures that have been established for shoot induction and
multiplication of Glycine species
have reported an average from 2 to 5.5 shoots, depending on the explant used (Mederos et al.,
1997; Moura, 1998). These calli
could be maintained in a long term process of shoot induction. The maximum
number of shoots was regenerated to 13.3 M BAP
supplemented with SI medium. It is in contrast with BAP,
alone or in combination with NAA, has previously been reported as being
efficient in promoting shoot differentiation in several species (Blakesley and Constantine, 1992).
B5 medium+(BAP and 2,4 - D)
No. of shoots
BAP+2,4 � D
4.4 M + 4.5 M
���������� 8.8 M +9.0 M
M + 13.5 M
M +18.0 M
������� 22.2 M +22.5 M
22.8 � 2.2
32.4 � 2.9
28.8 � 6.2
30.8 � 1.8
Use of 5.0 M BAP
significantly increased the regeneration frequency of shoots from the Vigna mungo cotyledonary node. Wright et al. (1986) and Hinchee et al. (1988) also obtained shoot
regeneration on cotyledonary explants at 5.0
M BAP. Use of 7.5 M BAP
[as routinely used in cotyledonary node protocol; Olholf et al., (2003); Paz et al. (2004)]
gave the lowest regeneration rate when applied to the half- seed system.
However, for H. canariense and H. foliosum, the best results for shoot induction were
obtained directly from apical or axillary buds
cultured in media supplemented with BAP
and NAA (Mederos, 1991; Moura,
Concentrations of BAP+IBA
4. The effect of different concentrations of BAP
+ IBA on callus regenerations.
Barwale et al. (1986b)
reported that, the best multiple shoot induction was observed in B5 medium
supplemented with 1.0 or 5.0 M BAP
in several Vigna mungo
genotypes. Paz et al. (2006) suggested that culture condition
optimized in the cotyledonary node system were not immediately
applicable to the half-seed protocol and by improving an optimum hormonal
environment to the explant in vitro, the
ability of transformed cells to regenerate into plants may be improved. In Vigna mungo,
elongated shoots obtained from callus were rooted in B5 medium with 14.7
M IBA. The IBA
was an efficient auxin to produce the shoots (Paz et
Concentrations of BAP+2,
5. The effect of different concentrations of BAP
+ 2,4 �D on callus regenerations.
Similar results have been described for some
species of Hypericum (Mederos, 1991; Moura, 1998).
The presence of IBA on the rooting media
seemed to have an effect on the number of roots induced per shoot. Three
weeks were enough to develop strong and healthy plantlets with good root
systems, ready to be transplanted and acclimated. The plantlets developed in
vitro were transferred to sterile soil and maintained in controlled
conditions in a growth chamber for one week. In this period of time, the pots
were watered regularly. After 2 week, the plants were taken to the
greenhouse. In conclusion, using plant growth regulators, the efficient
callus mediated regeneration from half seed explant
of Vigna mungo
has been standardized. The half seed callus could serve as an ideal starting
material for developing an efficient Vigna
mungo transformation system.
gratefully thankful to Prof. K. Sekar, Lecturer
(SG), A.A Government
Namakkal, Tamilnadu, India
for constant encouragement and timely help during the present work. We are
also greatful to the Directors, BIIOGENEIC �
Institute of Biotechnology, Namakkal, Tamilnadu, India, for providing laboratory facilities.
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