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In vitro plant regeneration in brinjal from cultured seedling explants

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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 fortified 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 fortified 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 fortified 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 1/2 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 efficiency 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, flowered and set fruits.
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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 fortied 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 fortied 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 fortied 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 efciency 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 inuenced 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 efcient 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 fortied
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 signicance 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 signicant 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 fortied
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 fortied 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 fortied 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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63
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 efciency 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 sufcient 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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Received: October, 2009; Revised: January, 2011;
Accepted : Febraury, 2011
... (Franklin et al. 2004, Xing et al. 2010, Shivraj et al. 2011, Mir et al. 2011, Bhat et al. 2013. Kaur et al. 2011 reported direct plant regeneration from cultured seedling explants in brinjal. Hypocotyl, cotyledon, leaf and root are most widely used as an explant (Franklin et al. 2004, Xing et al. 2010, Shivraj et al. 2011, Mir et al. 2011, Kaur et al. 2011. ...
... Kaur et al. 2011 reported direct plant regeneration from cultured seedling explants in brinjal. Hypocotyl, cotyledon, leaf and root are most widely used as an explant (Franklin et al. 2004, Xing et al. 2010, Shivraj et al. 2011, Mir et al. 2011, Kaur et al. 2011. ...
... While using mercuric chloride, there was decrease in seed germination (44-50%). Kaur et al., 2011 observed that seed germination with commercial bleach was quite good as compared to HgCl 2. The results of in vitro plant regeneration using cotyledon, hypocotyl and root explants of Manjari Gota at different hormone concentrations and combinations are given in Table 2. Explants cultured on MS + 2 mg/ l zeatin + 0.1 mg/l IBA show quick callus initiation in cotyledons (8.03 days) followed by hypocotyl (9 days) and root explants (10.83 days). 100 % callus induction was observed on cotyledon and hypocotyl explants on all medium supplemented with zeatin, BAP and IAA. ...
Article
Full-text available
The present investigation was undertaken for developing a high frequency regeneration system, as an essential pre-requisite for the genetic transformation in brinjal. Regeneration of multiple shoots via callus induction and organogenesis was achieved using cotyledons, hypocotyls and roots as explants at various concentrations and combinations of growth regulators. Hundred percent callus induction was observed in cotyledon and hypocotyl explants when cultured on medium supplemented with BAP or Zeatin. Earliest callus induction was observed in cotyledon explants (8.03 days). The MSB5 media supplemented with 2 mg/l zeatin + 0.1 mg/l IBA found to be the best medium with regards to number of days requires for callus induction and shoot induction, number of shoot buds formed per explant and regeneration frequency. Among explants used cotyledons found to be give best response to shoot induction and number of shoot bud formation (87.50 % and 24) and followed by hypocotyls (83.33 % and 22) and root explant (69.17% and 12). In vitro rooting (53.33%) was obtained within 13 days on MSB5 media supplemented with1mg/l IBA. The in vitro rooted plantlets were successfully established in polycabonated polyhouse with 92% survival rate.
... However, they reported that higher BAP level had negative effect on organogenesis leading to shoots vitrification. Kaur et al. (2011) had also observed that increase in BAP concentration above 2.5mg/L resulted in decreased regeneration capacity and number of buds on all the explants. Pawar et al. (2012) and Sagare and Manty (2012) also observed the shoot regeneration mediated through callus on medium containing BAP@ 1.0 -2.0 mg/L with highest shoot regeneration at 2.0 mg/L BAP. ...
... These findings are in line with (Sagare and Manty, 2012;Bhat et al., 2013) who also observed the increase in shoot regeneration with increase in kinetin level using cotyledonary leaves as explant. On the contrary to this Kaur et al. (2011) had reported that addition of kinetin decreased the regeneration capability and number of buds on all the explant. ...
... The lower concentrations caused callusing (Fig. 2C). Similar results have also been reported by Kanna and Mayabalan (4), and Kaur et al. (5). ...
... Thereby, root induction was observed on hormone-free MS medium (Fig. 2E). It was also demonstrated by many researchers (Kaur et al.,5). Rooting was also induced on quarter-strength hormone-free MS medium (Sammaiah et al.,12). ...
Article
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Direct plant regeneration of three eggplant genotypes, viz., BL-5, BR-14 and BSR-23 was studied with hypocotyl, cotyledon and leaf explants on Murashige and Skoog (MS) medium fortified with different concentrations BAP and Kin. The interaction of genotype, medium and explant gave 80.36% regeneration from cotyledons of BL-5 on MS medium fortified with 3.0 mgl-1 BAP and 75.19% for cotyledons along with 3.0 mgl-1 BAP and 1.0 mgl-1 Kin. However, the same genotype induced 21.76 buds per cotyledon on MS medium supplemented with 2.5 mgl-1 BAP + 1.0 mgl-1 Kin. In general, maximum shoot elongation (%) was recorded in BSR-23 (58.53%) followed by BL-5 (56.16%), whereas, maximum rooting (%) was induced in BL-5 (57.50%), followed by BSR-23 (57.14%). The rooted plantlets were hardened on wet filter paper with 0.2% carbendazim solution for 20 days, transferred to polythene bags in greenhouse and thereafter, at 4-5 leaf stage to the earthen pots for growth and flowering.
... Although the first part of the process (induction of microspore embryogenesis) is highly efficient, little progress has been made in the second part (organogenesis from calli). In literature, different authors working with different eggplant explants proposed different protocols to regenerate plantlets (Franklin et al. 2004;Gisbert et al. 2006;Borgato et al. 2007;Xing et al. 2010;Kaur et al. 2011). Only two studies addressed the particular case of regeneration from microspore-derived callus (Miyoshi 1996;Corral-Martínez and Seguí-Simarro 2012), and their results were not conclusive. ...
... All of them included MS medium (pH 5.8), 0.8 % plant-agar and different growth regulators, as follows. M11 included no regulators, M12 was supplemented with 0.3 g/l BA according to Kaur et al. (2011), M13 was supplemented with 1.5 mg/l GA 3 (Shivaraj and Rao 2011) and M14 was supplemented with 1.5 mg/l GA 3 and 8 mg/l AgNO 3 (Xing et al. 2010). A minimum of 124 shoots were cultured in each of the four media. ...
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Doubled haploid (DH) technology allows for the production of pure lines, useful for plant breeding, through a one-generation procedure that reduces considerably the time and resources needed to produce them. Despite the advantages of microspore culture to obtain DHs, this technique is still insufficiently developed in eggplant, where DHs are produced from microspore-derived calli through organogenesis. At present, very little is known on the best in vitro conditions to promote this process. This is why in this work we addressed the optimization of the process of regeneration of eggplant DH plants from microspore-derived calli. We evaluated the effect of different media compositions in the induction of organogenesis, in the promotion of shoot growth and elongation, and in root growth. According to our results, we propose the repeated subculture of the calli in MS medium with 0.2 mg/l IAA and 4 mg/l zeatin to produce shoots, and then the repeated subculture of the excised shoots in basal MS medium to promote their conversion into entire plantlets. This procedure yielded 7.6 plants per 100 cultured calli, which represents a ~4× increase with respect to previous reports. We also evaluated by flow cytometry and SSR molecular markers the effect of these in vitro culture conditions in the rate of DH plant production, finding that ~70 % of the regenerated plants were true DHs. These results substantially improve the efficiencies of DH recovery published to date in eggplant, and may be useful to those working in the field of eggplant doubled haploidy and breeding.
... MS media with 3 mgl -1 2,4-D and 1 mgl -1 BAP and MS basal with 0.5 mgl -1 IAA and 2 mgl -1 BAP resulted in 55% callus induction using cotyledonary leaf explants ( Table 2). The profuse callus proliferation was observed from cotyledon explants in MS medium containing 2 mgl -1 BAP or 2.5 mgl -1 BAP (Mohinder Kaur et al., 2011) and in MS with 3 mgl -1 2, 4-D and 0.05 mgl -1 BAP (Alim et al., 2014). ...
... Another important factor to consider is the morphogenetic pathway induced for the production of the clones, as it is known that in indirect organogenesis or embryogenesis, callus development reduces the genetic stability of the clones, due to higher mutation rate as a consequence of a differentiation phase followed by uncontrolled cell divisions (Krishna al., 2016). Therefore, is a need to assess the genetic stability of these plant materials, regardless of the purpose of the work (Kaur et al., 2011a;Kaur et al., 2011b). ...
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Many factors may influence the genetic stability of plant in vitro clones, among which the genotype and the regenerative process induced by plant growth regulators. The resulting somaclonal variations may be useful for breeding projects, but may be detrimental to germplasm conservation. The objective of this work was to evaluate the genetic stability of Solanum melongena cv. ‘Florida Market’ clones, obtained in response to different plant growth regulators. For the production of clones, leaf explants were used from commercial seed germinated plants. The explants were inoculated in Murashige and Skoog medium supplemented with different plant growth regulators at pre defined concentrations. The DNA was extracted by the CTAB method from leaves of complete plants obtained by somatic embryogenesis induced by naphthaleneacetic acid (NAA) or indirect organogenesis induced by benzylaminopurine (BAP) or thidiazuron (TDZ). For the RAPD, 117 DNA samples were amplified by ten decamer primers and 49 specific bands were selected among the products for the comparative study. A total of 5733 fragments were obtained, with a rate of 5.37% polymorphism. NAA did not generate polymorphism and the BAP was responsible for the highest rate obtained (14.28%). Two RAPD primers were identified as markers for monitoring the genetic stability of eggplant. The polymorphic pattern was observed only in clones originating from indirect organogenesis. These results indicate the usefulness of a monitoring protocol for studies using in vitro cloned eggplant.
... The study by Sharma and Srivastava (2014) observed best shoot regeneration at 1.0 mg L -1 kin + 1.0 mg L -1 IAA. Therefore, optimum ratio of cytokinin to auxins is required for bud formation and shoot regeneration (Kaur et al. 2011). However, the reports from Mohamed et al. (2010) showed BAP alone can induce shoot organogenesis without auxin. ...
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Tomato (Solanum esculentum L.) is an important vegetable crop cultivated throughout India. The crop is extensively damaged by alepidopteran insect, Helicoverpa armigera Hubner (fruit borer). To mitigate this problem, an attempt was made to generate transgenic tomato plants resistant to fruit borer. The regeneration protocol being pre-requisite for any transformation studies, 1.0 mg L⁻¹ BAP (Benzyl amino purine) in combination with 0.2 mg L⁻¹ IAA (Indole-3-acetic acid) was standardized as shoot regeneration media for tomato cv. Pusa Ruby using leaf explant. The highest callus initiation (100%) and an average number of shoots (3.9) were obtained in the above media. Rooting was observed at 2.0 mg L⁻¹ of IBA(Indole-3-butyric acid) with a highest average number of roots (1.06). The transformation system has been established by Biolistic Gun method using plant binary vector pCAMBIA2300 mobilized into the Agrobacterium strain LBA4404 carrying cry2AX1 under the control of CaMV 35S promoter. The callus of tomato was bombarded twice from three different distances between tissue and macro carrier assembly. The highest transformation efficiency (75%) was obtained at distance 9 cm and transformants were screened using 50 mg L⁻¹ of kanamycin. Out of 72 explants bombarded, only six explants developed completely but only two putative transformed plants were successfully hardened in the greenhouse. The integration and expression of the cry2AX1 gene in transformed plants were confirmed by PCR and Quantitative ELISA. The cry2AX1 protein accumulated in leaf quantified by ELISA has recorded 38.9 and 79.9 ng ml⁻¹ respectively compared to 129 ng ml⁻¹ of protein sample in positive control.
... In recent years, a mass propagation and reliable regeneration procedure for medicinal plants have been established, because it would be a primary step to facilitate gene introduction and transferring new traits (Siahsar et al. 2011). In the development of any regeneration protocol, growth hormone combination and choice of explant play a major role (Kaur et al. 2011). In the present study, combination of BAP 2.0 mg L -1 and NAA 0.5 mg L -1 proved to be the best. ...
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An efficient selection and plant regeneration protocol for biolistic gun transformation using leaf derived callus of Coleus forskohlii has been developed. Highest regeneration frequency 90% with 50 shoots per callus clump was obtained on Murashige and Skoog (MS) media supplemented with benzylaminopurine (BAP) 2.0 mg L-1 + naphthalene acetic acid (NAA) 0.5 mg L-1 The rate of shoot multiplication was increased with each subculture. Rhizogenesis was obtained on the same media composition. The in vitro raised plants were established successfully in sand and cocopeat (1:1). Callus of C. forskohlii was bombarded using biolistic gun with pABC plasmid DNA which contains β-glucuronidase (GUS) reporter gene and Arabidopsis thaliana white brown complex homologs (AtWBC19) as selectable marker gene. Kanamycin in the shoot induction medium was compared qualitatively and quantitatively for its efficiency as a selection agent for the selection and regeneration of transgenic plants after biolistic gun transformation. Kanamycin levels at or above 50mg L-1 completely inhibited growth of untransformed shoots. The integration of selectable marker gene GUS and AtWBC 19 into the genome of transgenic plants was confirmed using histoenzymatic GUS assay and polymerase chain reaction (PCR) respectively. These results pave the way for the transformation of Coleus forskohlii with desirable genes. Highlights • Regeneration and Biolistic gun transformation protocol was standardized for Coleus forskohlii.
... However, Kinetin gives very poor response for shoot induction. These results were in accordance with Mohinder et al., (2011) andSharmin et al. (2008). MS medium supplemented with 0.01 mg/l NAA produced 95% of rooting (Table 2) (Plate 2). ...
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An efficient and reproducible in vitro regeneration protocol was developed with high frequency regeneration of shoot buds from shoot-tip in MS medium + BAP (2.0 mg/l) in Brinjal cv. Utkal Anushree. Rooting was obtained on MS medium+ NAA (0.01 mg/l). The Agrobacterium tumefaciens strain GV3107 containing a binary vector pCAMBIA2300 carrying rd29A::DREB1A gene has been used for transformation. The shoot-tip from in vitro grown seedling was pre-cultured for 72 hrs and co-cultivated for 24 hrs. Shoot buds were produced on the regeneration medium containing kanamycin (100 mg/l) and cefotaxime (250 mg/l). A transformation frequency of 6.40% was achieved after PCR analysis with gene specific primer. The gene expression for moisture stress tolerance was assessed through morpho-physiological and biochemical analyses.
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Eggplant is an agronomically important non-tuberous solanaceous crop grown primarily for its large oval fruit. In popular medicine, eggplant is indicated for the treatment of several diseases, including diabetes, arthritis, asthma and bronchitis. Eggplant is susceptible to a number of diseases and pests capable of causing serious crop losses. This problem has been addressed by hybridizing eggplant with wild resistant Solanum species, which present a wide genetic diversity and are source of useful agronomic traits. The application of in vitro methodologies to eggplant has resulted in considerable success. Eggplant tissues present a high morphogenetic potential that is useful for developmental studies as well as for establishing biotechnological approaches to produce improved varieties, such as embryo rescue, in vitro selection, somatic hybridization and genetic transformation. Taken together, these characteristics also make eggplant a complete model for studies on different areas of plant science, including control of gene expression and assessment of genetic stability of somaclones derived from different morphogenetic processes. In the present study, important factors that affect the efficiency of in vitro regeneration through organogenesis and embryogenesis as well as genetic transformation are analyzed. The potential of this species as a model plant for studying various aspects of plant genetics and physiology is also discussed.
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Among the in vitro derived different explants such as cotyledonary leaf, hypocotyl, shoot tip and root of two local varieties, namely Singhnath and Kazla (BARI Begun-4) of eggplant (Solanum melongena L.) cotyledonary leaf was found to be the best for multiple shoot regeneration. High frequency direct organogenesis of shoots was achieved from cotyledonary leaf in MS supplemented with 1.0 mg/l BAP and 1.0 mg/l Kn. Anatomical studies using freezing microtome supported the formation of shoots through organogenesis. Proliferation and elongation of such shoots were obtained in hormone free MS. Moreover, the regenerated shoots produced healthy roots when they were cultured on MS without hormonal supplements. Following the formation of roots the in vitro raised plantlets were successfully established in soil. Viable seeds were obtained from the in vitro raised mature plants.
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Micropropagation of carnation cv. Lipstick was tried using shoot-tip of greenhouse raised plants and nodal segment of in vitro raised shoots by culturing them on MS medium supplemented with various growth regulators. High rate of multiplication of shoot tip was recorded in the medium containing basal MS salts supplemented with 0.5 mg l(-1) NAA, 1.5 mg l(-1) kinetin and 0.5 mg l(-1) GA(3) while that of nodal segment on MS medium supplemented with 0.5 mg l(-1) NAA, 0.5 mg l(-1) kinetin and 0.5 mg l(-1) GA(3). The best rooting of the microshoots was obtained on MS medium with 0.5 mg l(-1) IBA. The rooted plantlets were transferred to the ambient condition using different substrates. Vermiculite recorded a high survival percentage of 90% while the lowest was recorded in the substrate soil. The hardened plantlets were finally planted in beds inside greenhouse.
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Micropropagation techniques play an important role to conserve elite or rare plants threatened with extinction. A study was carried out to find a regeneration protocol for an elite seedless clone of pummelo (Citrus grandis [L.] Osbeck.), BLRV, using mature nodal explants. 6-benzyl adenine (BA) with and without α-naphthalene acetic acid (NAA) was used to induce multiple shoots. 0.75 mg I-1 was found as the best concentration of BA in induction of multiple shoots whereas use of 0.1 mg l-1 NAA in BA-containing media made no significant changes in the measured characteristics. Among half-and full-strength Murashige and Skoog (MS) media, Murashige and Tucker (MT) medium, and Woody Plant Medium (WPM); full-strength MS medium yielded the best results in shoot induction from in vitro nodal explants inducing about 94% of explants to regenerate averagely 3.5 shoots and 3 usable shoots with mean length of 1.8 cm. Cent per cent rooting in micro-shoots was achieved by individual application of indole-3-butyric acid (IBA) at 4 mg I -1 in half-strength MS medium within two weeks after culture initiation. This treatment resulted in the highest root length (average 7.25 cm), too. NAA did not yield satisfactory results since it caused formation of short and deformed roots, which was probably the main cause for stunted shoot growth. In this study, a two-stage regeneration protocol was optimized for in vitro production of pummelo plantlets.
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A report about stable transformation office (Hiri et al.. IMaiit ,1. J: 271) by rnean> of Agrobacterium allows us to suppose that combining of an optimal regeneration system with a suitable transformation technique can lead to successful transformation of other cereals, for instance barley. The ex périment- were carried out with 6 Estonian and Finnish cultivais of barlev (Kymppi, Anni. Toele. Mette, Klo and Ape). To assay the optimal régénéra tion sysîeni, explants from different tissues (anthers, microspores. snitelhirn. immature embryos with and without scutellum) and different developmental stages were cultivated on different callus induction media and the obtained calli were transferred to shoot regeneration medium. The highest embryogenie callusogenesis rate characterized the immature embryos with scutellum. The best embryogénie callus inducing and regeneration media were selected. The three morphologically normal plants regenerated on MS medium containing maltose and different amino acids. A supervirulent Agrobacterium strain LBA4404 (pTOK233), successfully exploited in rice transformation, was used in transformation experiments. The binary vector pTOK233 contains GL'S intron construct and consequently GUS expression occurs only after DNA in tegration to barley genome. The calli were cocultivated with. Agrobacterium 2-3 days, washed with Claforan and transferred to callus-induction medium containing ihe antibiotics to select transformed tissues. The calll were tested hist ocho mil ally (t ho number nf blue CJUS expressing foci were counted ).
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A protocol with a high rate of transformation and regeneration of 'Hibush' eggplant (Solanum melongena L.) has been developed. This protocol used leaves of in vitro-grown seedlings as a source of explants. The shoot regeneration culture medium contained 0.1 μM thidiazuron (TDZ) combined with 10 to 20 μM N6-[isopentyll adenine (2iP). Adding TDZ significantly improved regeneration efficiency and produced a mean of 15 buds and 3 to 4 shoots per explant. When explants were cocultivated with Agrobacterium tumefaciens strains Q10, Q20, Q30, Q40, Q201, Q202, Q203, or Q204 containing the native cryIIIB Bacillus thuringiensis (Bt), neomycin phosphotransferase (NPTII), and β-glucuronidase (uidA) genes, a callus/bud regeneration frequency of 38.8% was observed on the selection medium. Kanamycin at 50 μg·mL-1 was most effective in selecting for transgenic buds and shoots. Augmentin at 300 μg·mL-1 was used to eliminate A. tumefaciens. Augmentin also enhanced shoot proliferation. A transformation/regeneration efficiency of 20.8% was observed for shoot production. More than 400 putative transgenic plants have been produced with this method. From 50 putative transgenic plants, gene integration has been confirmed with Southern blot analysis and progeny tests.
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The relative importance of genotype and explant, and their interactions for in vitro plant regeneration via both organogenesis and somatic embryogenesis in Solanum melongena (eggplant) has been studied. Hypocotyl, cotyledon and leaf explants of four commercially grown Indian cultivars, Pusa Purple Long, Long White Cluster, Pusa Kranti, and Pusa Purple Cluster were used in the study. A combination of benzyladen-ine (11.1 μM) and indoleacetic acid (2.9 μM) was found to be optimum for shoot regeneration. Naphthalene acetic acid induced embryogenesis in all the three explants; 32.2μM was optimum for hypocotyl explants while 10.7μM yielded maximum number of somatic embryos from cotyledon and leaf explants. Genotype, explant and genotype-explant interaction had highly significant effects on both organogenesis and somatic embryogenesis with genotype exerting maximum effect on both these processes. Pusa Purple Long was found to be the most responsive genotype for regeneration of both adventitious shoots and somatic embryos among the cultivars. Among the explants, hypocotyls yielded the maximum number of adventitious shoots followed by cotyledons and leaves. The embryogenic response of leaves and cotyledons was, however, significantly higher than that of hypocotyl explants. Significant differences for morphogenetic potential were also observed within a single explant (hypocotyl). There was a basipetal gradient for organogenesis (i.e. decrease in number of shoots from base to apex) while the terminal hypocotyl segments showed better embryogenic potential than the median segments.
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A substantial number of micropropagated plants do not survive transfer from in vitro conditions to greenhouse or field environment. The greenhouse and field have substantially lower relative humidity, higher light level and septic environment that are stressful to micropropagated plants compared to in vitro conditions. The benefit of any micropropagation system can, however, only be fully realized by the successful transfer of plantlets from tissue-culture vessels to the ambient conditions found ex vitro. Most species grown in vitro require an acclimatization process in order to ensure that sufficient number of plants survive and grow vigorously when transferred to soil. This article reviews current and developing methods for the acclimatization of micropropagated plantlets.