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Indian J. Hort. 68(1), March 2011: 61-65
In vitro plant regeneration in brinjal from cultured seedling explants
Mohinder Kaur*, Ajmer S. Dhatt*, Jagdeep S. Sandhu** and Satbir S. Gosal**
Department of Vegetable Crops, Punjab Agricultural University, Ludhiana 141 004
ABSTRACT
In vitro plant regeneration of brinjal genotype BL-3 was tried using hypocotyl, cotyledon and leaf explants
from in vitro raised seedlings on Murashige and Skoog medium fortied with 6-benzylamino purine (BAP) and
kinetin (kin) combination (2.0-3.0 mgl-1 BAP with or without 1.0 mgl-1 kin). The cotyledon explant gave cent percent
regeneration on MS medium fortied with 2.0 mgl-1 BAP, 2.5 mgl-1 BAP, or 2.5 mgl-1 BAP + 1.0 mgl-1 kin, while the
highest numbers of buds on 2.5 mgl-1 BAP (24.90), followed by 2.0 mgl-1 BAP (17.90). Leaf explant also induced cent
percent regeneration on MS medium fortied with 2.0 mgl-1 BAP and maximum number of buds (9.53) regenerated
with 2.5 mgl-1 BAP. Hypocotyl had the maximum regeneration (66.53%) and maximum buds (3.96) on MS with
2.5 mgl-1 BAP. Maximum bud elongation (58.73%) was obtained on ½ MS medium supplemented with 0.3 mgl-1BAP
+ double agar. MS basal medium induced maximum rooting of 61.11% plantlets. The hardening with 0.2% bavistin
solution enhanced the survival efciency of plantlets to 81.81%. The plantlets were established in the polythene
bags and then transferred to earthen pots in the glasshouse, where they grew, owered and set fruits.
Key words: Egg plant, in vitro, regeneration, hypocotyl, cotyledon.
INTRODUCTION
Brinjal (Solanum melongena L., 2n = 2x = 24)
also known as egg plant, aubergine or Guinea
squash, is a widely adaptive and highly productive
vegetable of tropical and subtropical regions. For
the improvement against biotic and abiotic stresses
as well as quality improvement through genetic
transformation, standardization of plant regeneration
protocol is the prerequisite. The direct organogenesis
is the formation of plantlets directly from explants on
the culture media. The various factors like explant and
growth regulators inuenced the in vitro regeneration
through organogenesis in eggplant (Magioli and
Mansur, 8). The concentration and combination of
exogenous auxin and cytokinin in the process of
bud differentiation as well as the tissue system had
variable response on plant regeneration in brinjal
(Prakash et al., 11). Generally, high cytokinin to auxin
ratio leads to shoot formation and intermediate callus
production (Sarker et al., 13). Variable response
of genotypes, explants and media for regeneration
have also been substantiated by Sharma and Rajam
(15), Jahan and Syed; Magioli et al. (7), Picoli (10),
Dobariya and Kachhadiya (5), and Sarker et al. (13).
As successful application of in vitro techniques for
crop improvement rests upon reproducible plant
regeneration protocol, the present investigation deals
with an efcient method of direct plant regeneration
from cultured seedling explants in brinjal.
MATERIALS AND METHODS
The investigation for plant regeneration in brinjal
was carried out during 2005-2008 in Tissue Culture
laboratories of School of Agricultural Biotechnology,
PAU, Ludhiana. Seeds of BL-3 were rst washed
with TeepolTM (Labolene). Then bold seeds were
disinfected with 50 and 75% commercial bleach
i.e., ‘Ala Bleach®’ (sodium hypochlorite 4%, sodium
hydroxide and amine oxide 1%) for 20 and 25 min.
Disinfected seeds were then cultured on half-strength
MS (Murashige and Skooge, 9) solid medium for
germination and incubated at 25 ± 2°C in dark for 20
days. The seed germination (%) was calculated from
the number of seeds germinated over total number
of seeds cultured in vitro. Cotyledon, hypocotyl and
leaf explants were excised aseptically from 15 to
20-day-old seedlings, cultured on MS medium fortied
with different concentrations of BAP and kinetin
(2.0-3.0 mgl-1 BAP with or without 1.0 mgl-1 kin) and
regenerated at 25 ± 2°C for 16/8 h light and dark
cycles. Plant regeneration (%) was calculated from the
number of explants regenerated after 20 days over the
total number of explants cultured for regeneration. The
number of buds per explant was calculated from the
average number of buds from 10 regenerating explants.
The regenerated buds were then elongated on half-
strength MS with different BAP concentrations. The
shoot elongation (%) was calculated. The elongated
plantlets were excised aseptically and transferred to
different MS basal medium for root induction. The
rooting (%) was calculated after 15-20 days from
number of plantlets rooted over the total number
*Corresponding author’s E-mail: mkaur97@rediffmail.com
**School of Agricultural Biotechnology, Punjab Agricultural University,
Ludhiana 141 004
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Indian Journal of Horticulture, March 2011
of plantlets cultured. The rooted plants were then
hardened for different days on moistened cotton and
lter paper with sterilized water as well as with 0.2%
bavistin solution. Hardened plants were transplanted
to polythene bags lled with mixture of sand, soil and
FYM in 1:2:1 ratio and kept in greenhouse for further
growth at 25°C. The, plants with 4-5 expanded leaves
were grown to owering and fruiting. At least three
repeats were maintained for each treatment. Statistical
analysis was done in CRD factorial design using
CPCS-1 software package developed by Cheema
and Singh (4). Least square differences at 5 percent
level of signicance were calculated and interpreted
accordingly.
RESULTS AND DISCUSSION
The seeds disinfected with commercial bleach in the
present investigation showed quite good germination.
The interaction with time duration of treatment
(Table 1) imply that 50% commercial bleach
encouraged seed germination (85.80%), while increase
in concentration limited it to 51.91%. Commercial
bleach disinfection for 20 min. had signicant effect
on seed germination (74.58%) that declined to 63.13%
on 25 min. treatment. The interaction of concentration
and treatment duration indicate that the highest seed
germination (%) was obtained from 20 min. disinfection
with 50% commercial bleach. As per Sarker et al.
(13), seed treated with 0.1% (w/v) mercuric chloride
for 5-6 min. showed germination in brinjal. Seed
germination with commercial bleach was quite good
as compared to HgCl2 and exhibited the normal
growth and development of the seedlings (Fig. 1a).
Commercial bleach contains 4% sodium hypochlorite
(NaOCl), which acts as sterilizing agent.
The interaction of explant and medium composition
for plant regeneration and number of buds explant-1
(Table 2) indicates that there was cent percent
regeneration of cotyledon on MS medium fortied
with 2.0 mgl-1 BAP, 2.5 mgl-1 BAP, 2.5 mgl-1 BAP +
1.0 mgl-1 kin, while the highest number of buds were
developed by cotyledon explant on 2.5 mgl-1 BAP
(24.90). MS medium fortied with 2.0 mgl-1 BAP also
induced 100 percent regeneration in leaf explant,
whereas hypocotyl had highest regeneration (66.53%)
potential with 2.5 mgl-1 BAP. MS medium fortied with
2.5 mgl-1 BAP induced maximum number of buds in
leaf (9.53) and hypocotyl (3.96) explant also. Increase
in BAP concentration above 2.5 mgl-1 BAP as well as
addition of kin decreased the regeneration capability
and number of buds on all the explants and lead
to the browning of explants that could not elongate
their buds into shoots. Leaf did not regenerate with
3.0 mgl-1 BAP. Also, higher concentrations of BAP
could not initiate regeneration on hypocotyl explant.
In general, MS medium supplemented with 2.0 mgl-1
BAP (87.07%) was the best combination for direct
regeneration and 2.5 mgl-1 BAP for the highest number
of buds (12.80). Different concentrations of BAP and
kin had differential response for adventitious shoot
formation. The direct regeneration potential depends
upon the proportion of auxin and cytokinin. Also, the
requirement for exogenous auxins and cytokinins
in the process of bud differentiation varies with the
tissue system and apparently depends on endogenous
level of two hormones in the tissue (Sasan et al., 14).
In present study, callus induction was observed at
lower concentrations of cytokinins and regeneration
response increased with augmentation to an optimum
level in a particular genotype. It may be due to higher
concentration of auxins in the explant itself and was
balanced to a desired level by addition of cytokinins
in the culture medium for better plant regeneration.
It was also observed that higher concentration of
hormones caused browning of explants and hampered
the regeneration and growth of buds. Therefore,
optimum ratio of cytokinin to auxin is required for shoot
regeneration. Hypocotyl, cotyledon and leaf explants
also demonstrated differential response for direct plant
regeneration on different media concentrations of the
cytokinins (BAP and kin). It can be due to inherent
differences in the level of expression in the explants
on a particular medium. As a whole, cotyledon was the
best explant with 69.16% regeneration and 11.48 buds,
followed by leaf (45.65%, 4.47) and hypocotyl (24.18%,
1.29) explants. The differences for regeneration
hypocotyl, cotyledon and leaf explants on can be seen
visually in Fig. 1b, c, d respectively. It was observed
Table 1. Effect of commercial bleach on seed germination.
Bleach conc.
(%)
Time duration (min.) Mean germination
25 20
75 44.63 (41.90)* 59.19 (50.27) 51.91 (46.08)
50 81.64 (64.60) 89.97 (71.52) 85.80 (71.42)
Mean germination (%) 63.13 (53.25) 74.58 (60.90)
LSD (P = 0.05) Conc.= 0.92; Time duration = 0.92; Conc. × time duration = NS
*Figures in parenthesis indicate arc sine transformation of values.
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In Vitro Plant Regeneration in Brinjal
that cotyledon expanded to almost double size in a
week and then developed small buds, which elongated
further into the shoots (Fig. 1e). The differences
among explants for direct regeneration have also been
reported ( Bora et al., 2; Prakash et al., 11; Sharma and
Rajam 15; Sarker et al., 13; Magioli et al., 7; Taha and
Tizan, 16). Even the difference within different portions
of hypocotyl for morphogenetic potential has also been
detected (Sharma and Rajam, 15). The formation of
shoot buds was characterized by the appearance
of shoot apex with the developing leaf primordial
(Sarker et al., 13).
Elongation of buds into plantlets was experimented
with four medium compositions trong Maximum bud
elongation (58.73%) resulted from half-strength ½ MS
medium supplemented with 0.3 mgl-1 BAP and double
agar. The least effect was seen when with 0.5 mgl-1
BAP and the addition of double agar increased the
elongation (26.31%) of buds. However, it was 28.88%
on hormoreifree MS (Fig. 2). There was excessive
callus proliferation with 0.5 mgl-1 BAP, which converted
most of buds into callus and did not let them elongate
into plantlets. The addition of double agar reduced
this proliferation. Decrease in BAP concentration
also lowered the callus induction and increased
the bud elongation as visible in Fig. 1e. Most of the
scientists like Sarkar et al. (13) and Borgato et al. (3)
reported that the shoot buds upon subculture to MS
basal medium elongated into healthy shoots after
organogenesis. Small shoots were elongated on MS
medium containing zeatin and augmentin by Billings
et al. (1).
Among different media compositions (Fig. 3), the
maximum rooting of plantlets (61.11%) was observed
on MS basal medium followed by MS liquid (54.83%).
The addition of 0.5 mgl-1IBA reduced the rooting to
4% only. The lowered concentration of IBA (0.1 mgl-1)
increased it to some extent, whereas only 14.70%
rooting was observed on half-strength MS medium.
IBA (weak auxin) is generally used for root induction
in most of the plants, induced callusing at cut ends of
brinjal plantlets that inhibited the differentiation of roots.
This might be due to the reason that it has high auxin
level in the plant tissue itself, which is increased further.
Thus, MS basal medium best was root induction
(Fig. 1f). Here, in brinjal, the inherent level of auxins
seems to be high and its application for rooting
revert the tissue towards callus. The root formation
in brinjal was reported in half-stength MS medium
(Taha and Tizan; 16 Sarker et al., 13), ½ MS medium
Fig. 1. In vitro regeneration in brinjal: (a) germinated
seedlings, (b) regenerated hypocotyl, (c) regenerated
cotyledon and , (d) regenerated leaf, (e) elongated
plantlets from cotyledonary buds, (f) rooted
plantlets, (g) hardened plantlets, (h) establishment
in polybags, and (i) well established plants with fruits
in greenhouse.
Fig. 2. Shoot elongation on different MS media compositions.
M1–1/2 MS, M2 – 1/2MS + 0.5 mgl-1 BAP, M3 – 1/2MS
+ 0.5 mgl-1 BAP + double agar, M4 – ½ MS + 0.3
mgl-1 BAP + double agar.
Fig. 3. Root induction on different MS media compositions.
M
1 - MS, M2 - MS + 0.5 mgl-1 IBA, M3 - MS + 0.1 mgl-1
IBA, M4 - ½ MS, M5 - MS liquid.
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Indian Journal of Horticulture, March 2011
Table 2. Effect of medium composition and explants on direct plant regeneration and number of buds per
explant in brinjal.
Treatment Plant regeneration No. of buds per explant
Hypocotyl Cotyledon Leaf Mean Hypocotyl Cotyledon Leaf Mean
2.0 mgl-1
BAP
61.23
(51.47)
100.00
(89.96)
100.00
(89.96)
87.07
(77.13)
3.03 17.90 8.06 9.66
2.0 mgl-1
BAP + 1.0
mgl-1 kin
0.00
(0.00)
35.56
(36.59)
9.66
(18.07)
15.07
(18.22)
0.00 7.03 2.93 3.32
2.5 mgl-1
BAP
66.53
(54.63)
100.00
(89.96)
73.13
(58.75)
79.88
(67.78)
3.96 24.90 9.53 12.80
2.5 mgl-1
BAP + 1.0
mgl-1 kin
41.50
(40.08)
100.00
(89.96)
58.33
(49.77)
66.61
(59.94)
2.06 10.16 4.83 5.68
3.0 mgl-1
BAP
0.00
(0.00)
75.80
(60.50)
53.73
(47.12)
43.17
(35.87)
0.00 11.93 3.93 5.28
3.0 mgl-1
BAP + 1.0
mgl-1 kin
0.00
(0.00)
52.63
(46.49)
24.70
(29.78)
25.77
(25.42)
0.00 4.23 2.03 2.08
3.5 mgl-1
BAP
0.00
(0.00)
20.13
(26.64)
0.00
(0.00)
6.71
(8.88)
0.00 4.20 0.00 1.40
Explant mean 24.18
(20.88)
69.16
(62.87)
45.65
(41.92)
1.29 11.48 4.47
LSD
(P = 0.05)
Medium = 0.59; Explant = 0.38;
Medium × explant = 1.02
Medium = 0.23; Explant = 0.15;
Medium × explant = 0.39
*Figures in parenthesis indicate arc sine transformation of values.
supplemented with 0.6 mm IAA (Magioli et al., 7), ¼
MS medium (Dobariya and Kachhadiya, 5) and MS
medium containing 1.0 mgl-1 3-indole butyric acid
(Borgato et al., 3).
Hardening of rooted plantlets in wet cotton resulted
in softening and killing of plants. That may be due to
the excess of water supplied by wet cotton during
hardening. Hardening of rooted plantlets on wet lter
paper increased the survival (Fig. 1g). The plantlets
were transferred to polythene bags (Fig. 1h) after 7, 10,
15 and 20 days of hardening and kept in greenhouse
at 25 ± 1°C. No plant survived after 7 and 10 days of
hardening. However, survival increased to 40% (4 out
of 10) and 65% (13 out of 20) when plantlets hardened
for 15 and 20 days, respectively. The addition of
0.2% bavistin to the tap water further enhanced the
survival efciency of plantlets to 81.81% (8 out of 11).
The plants with 4-5 healthy leaves were transferred
to the earthen pots in the glasshouse, where they
grown up, owered and set fruits (Fig. 1i). The in vitro
regenerated plantlets are very sensitive and prone
to the attack of microorganisms, when subjected to
external environment. Treatment of bavistin checked
the fungal infection and longer duration of hardening
made plantlets acclimatized to the external conditions.
Most species grown in vitro require acclimatization
process in order to ensure that sufcient number
of plants survive and grow on transferring to the soil
(Hazarika 6). Taha and Tizan (16) achieved 80%
acclimatization in eld-transferred plantlets. Successful
acclimatization and transfer of plants to the soil were
stated by different workers (Salih and Al-Mallah, 12;
Sarker et al., 13; . Dobariya and Kachhadiya (5) also could
established rooted shoots in polythene bags lled with a
potting mixture of sand, soil and FYM in 1:2:1 ratio.
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In Vitro Plant Regeneration in Brinjal
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Received: October, 2009; Revised: January, 2011;
Accepted : Febraury, 2011
