3. Organogenesis - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/4792/9/09_chapter 3.pdf ·...
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3.1 INTRODUCTION
Plant cell cultures are initiated through the formation of a mass of
undifferentiated cells called “callus.” Plant regeneration through callus cultures
(indirect organogenesis) is an effective strategy for successful exploitation of
in vitro techniques for somaclonal variation induction, genetic transformation
and protoplast culture (Sarasan et al., 1994; Lusia and Rojas, 1996; Ahroni et al.,
1997). Recent reports proved that the organogenesis protocols provided useful
systems for the study of regulating mechanisms of plant growth and
development (Castillo and Jordan, 1997). There are several plant species
adopted for the successful plant regeneration through indirect shoot
organogenesis and some of the plant species are recalcitrant to in vitro indirect
organogenesis. Castor is one among the plant species showing extremely low
percentage of callus induction and successive plant regeneration. There are
notable problems remained to be overcome for the callus induction and
subsequent plant regeneration in castor. The extrinsic and intrinsic factors like
type of explants, age of explants, type of media and exogenous and
endogenous plant growth regulators, the amount of carbon sources and
additives present in the media are the main factors to be analyzed.
Despite research efforts over the last three decades, whole plants still
could not be regenerated with reproducible frequencies from friable callus
cultures of castor. The sporadic appearance of shoots from callus cultures of
castor implies that the calli contain at least a few morphogenic cells
interspersed in several non-morphogenic tissues. Failure to isolate a competent
cell line might result in its suppression by the overgrowth of non-competent
cells. Alternatively, the occasional appearance of shoots could be owing to the
activation of recalcitrant calli to undergo caulogenesis caused by a rare
inductive stimulus resulting from the interaction between exogenous and
endogenous conditions. In castor, callus initiation and plantlet regeneration
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31
from vegetative explants are reported by (Athma and Reddy 1983; Sarvesh
et al., 1992). However, regeneration of plants from callus cultures has been
problematic. There are only few reports on plantlet regeneration in castor and
in most of the cases regenerated plantlets were obtained from apical meristems
and shoot tip callus (Reddy et al., 1986; Reddy et al., 1987a, Sangduen et al.,
1987, Genyu et al., 1988; Sujatha and Sailaja, 2005; Malathi et al., 2006). As
genetic transformation involves several manipulations for gene introduction
followed by selection for 2 – 3 subculture cycles, the efficiency of these
regeneration systems for genetic transformation of castor need to be
established.
In this present investigation, several challenging difficulties were
highlighted during the regeneration of castor from callus cultures. The
excretion of secondary metabolites from the explants into the medium,
browning of callus after a short period of culture, a low frequency of green
compact callus formation, formation of loosely arranged organogenic callus,
yellowing of organogenic callus within a short period of culture and very slow
response for shoot proliferation from the selected organogenic callus cultures
were recorded as main regeneration difficulties to be overcome. Hence, this
experiment was undertaken for the production of a good regenerative and
reproducible protocol for castor organogenesis through callus induction by
using a different type of explants.
3.2 MATERIALS AND METHODS
3.2.1 Explant Obtention
Castor in vivo seed germination was achieved by ten days. After seedling
growth hypocotyl (HL) and cotyledonary leaf (CL) explants were taken from
the 10 days old seedling for the organogenic callus studies.
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3.2.2 Initiation, proliferation and selection of organogenic callus
Cotyledonary leaf and Hypocotyl were used as explants and placed
horizontally in callus initiation medium which contained mMS medium salts
and varying concentrations of BA (0.5 – 4.0 mg/l), KN (1.0 to 3.0 mg/l) for
organogenic callus induction. After the identification of suitable concentration
of cytokinin for callus induction, combinations of different concentrations of
NAA (0.2 – 2.0 mg/l) were tested for enhanced callus production. After 6
weeks of culture, callus formation was observed from the cut end of the
explants. From the obtained mass, the organogenic nature of the callus was
identified by the presence of green colour with compact nodular texture
(GCN). The organogenic portions were isolated and subcultured in the same
medium. Greenish friable (GF), brown compact (BC), brown friable (BF) and
yellowish green friable (YGF) colored non-organogenic callus were also
observed and they were not selected and discarded for further studies due to
nil response. The selected organogenic callus was weekly subcultured for
another two weeks for the induction of well developed green compact
organogenic callus. For callus induction, maximum of 50 explants were tested
and these experiments were repeated for three times with five replicates.
3.2.3 Adventitious shoot proliferation
8-week-old organogenic callus (250 mg) was transferred to 200 cm3
narrow bottles containing 50 ml of shoot initiation medium. Then the cultures
were subcultured for 2 months with weekly subculture for the initiation of
shoots. During each subculture removal of dead, dark brown cells was done.
Otherwise, the whole callus tissues become necrotic and dead. The plant
growth regulators like, BA, KN and TDZ (0.5 – 2.5 mg/l) with different
concentrations and combination of IBA and IAA (0.05 – 0.8 mg/l) were tested
for proliferation of shoots. During multiple shoot proliferation, maximum of
30 callus cultures were tested and these experiments were repeated for three
times with five replicates.
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33
3.2.4 Effect of carbon source, additives and amino acids on multiple shoot
proliferation
The effect of sucrose, glucose, fructose and maltose (10 – 50 g/l) were
tested for organogenic callus induction and multiple shoot proliferation. In that
same way, the influence of different amino acids like alanine, glutamine,
proline and serine (5 – 25 mg/l) were also tested. During whole plant
regeneration of castor-bean, browning of explants as well as the medium was
noticed. To control phenolic exudation process, different concentrations of
additives like, activated charcoal (50 – 250 mg/l), citric acid, ascorbic acid and
PVP (5 – 25 mg/l) were tested.
3.2.5 Elongation, Root induction and hardening
The multiple shoots were transferred to shoot elongation medium
containing different concentration of GA3 (0.1 – 0.5 mg/l) with PF - 68
(1.0 mg/l) and root induction from the elongated shoots were obtained from
the mMS medium fortified with IBA (0.1 – 0.5 mg/l) and AgNO3 (0.2 –
1.0 mg/l), sucrose 30 g/l and agar (0.8%). After complete regeneration of
shoots with tertiary roots (35 - 40 days after root induction) the regenerated
plants were transferred to plastic pots containing sand, soil and vermiculate in
1:1:1 ratio for hardening The hardened plants were maintained in
environmental plant growth chamber (SANYO, JAPAN) for proper
acclimatization and then the in vitro regenerated plants were transferred to
green house condition for 15 days and successfully transferred to the field.
The survival percentage of all the hardened plants was recorded regularly. For
root induction studies, 30 elongated shoots were tested for each concentration.
The experiments were repeated for three times with five replicates.
3.2.6 Statistical analysis
Means and standard errors were used throughout the study and the
values were assessed using a parametric Moods median test (Snedecor and
Cochran, 1989). The data were analysed for variance by Duncan’s multiple
Organogenesis
34
range test (DMRT) using the SAS programme (SAS Institute, Cary, N.C.). For
organogenesis from the cotyledonary leaf and hypocotyl explants, 50
explants were tested with 5 replicates and each experiment was repeated
three times. During multiple shoot induction 30 callus were tested for each
treatment and the each experiment was repeated 3 times with 5 replications.
3.3 RESULTS AND DISCUSSION
3.3.1 Organogenic callus induction
The HL and CL explants were taken from the 10 days old well
established seedlings. After one week of inoculation, callusing was observed
from both the explants. Individual treatment of KN and BA showed low
response for callus induction. Among the two cytokinins tested media
comprising BA (2.0 mg/l) showed superior response (28.7%) with CL and
25.3% with HL explants. The callus from both the explants was green colour
with compact nature. However, nodular nature is absent in both the explants.
Hence, along with BA (2.0 mg/l) different concentration of NAA (0.2 –
2.0 mg/l) was tested for enhanced callus production. So, to enhance the nature
of the callus combinations of BA with NAA was tested. High proficiency callus
induction of CL explants was noticed on the mMS medium comprising BA
(2.0 mg/l) and NAA (0.8 mg/l) with the maximum of 69.5 % response whereas,
HL explants showed 54.2% of response in the same concentration (Table 3.1). In
this concentration, maximum amount of organogenic callus was observed
when compared with other concentrations in the mMS medium fortified with
30 g/l sucrose and 8 g/l agar. The induced calli from the explants were green,
compact and nodular in nature (Plate 3, 4).
Similar to our results, Sarvesh et al. (1992) reported that when epicotyls
and cotyledonary explants supplemented with BA (2.5 mg/l) + NAA
(0.1 mg/l) produces 96.5% of callus formation. Genyu (1988) by his experiment
proved that BA (1.0 mg/l) + NAA or IAA (1.0 mg/l) developed callus from
young stem explants of castor. Athma and Reddy (1983) speculated that
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35
cotyledonary leaf explants of castor treated with BA (0.5 – 2.0 mg/l) with NAA
(0.5 mg/l) produced 90 – 98% of callusing. Suresh and Rao (1994) achieved
callus from the MS medium supplemented with BAP (4.0 mg/l) + NAA
(4.0 mg/l) from axillary bud and terminal buds of castor.
Similar to our results, in Melia azedarach, organogenic callus cultures
were initiated by using a combination of BA (4.4 µM) and NAA (0.46 µM) and
successful regeneration of plantlets was obtained by using the callus cultures
(Vila et al., 2003). Like our result, in all dicot plants, combination of the high
amount auxin (2, 4 - D or NAA) with a low amount cytokinin (BA or Kin) was
widely used for the initiation of organogenic callus (Caboni et al., 2000; Rugini
and Muganu, 1998; Rani et al., 2006; Haliloglu, 2006) and sometimes cytokinins
alone (BA or KN) was also used for the induction of organogenic callus (Yam
et al., 1990). Our experiments proved that combined effect of BA and NAA
showed best response for organogenic callus induction.
During organogenic callus formation, calli with variation in texture were
noticed and only the hard and compact green calli responded well for the
induction of shoots. Hence, shoot induction and multiplication was achieved
by using the above said calli. Individual effect of different concentrations of
BA, KN and combined effect of KN + NAA were also tested for the induction
of organogenic callus, unfortunately, in these concentrations, abnormalities
like, induction of roots, bulging of explants and necrosis of tissues were clearly
observed. It is also observed that BA or KN when applied alone induced only
waste brownish friable callus from cotyledon explants (Sudha et al., 1998; Tang
and Guo, 2001; Martin, 2002). In our studies also combination of cytokinin with
auxins showed best response because, the nature of the callus varied according
to the exogenous application of the cytokinin and also for the auxin: cytokinin
ratio (Marks and Simpson, 1994; Soniya and Sujitha, 2006). In Tylophora indica,
organogenic callus induction was achieved by the supplementation of
cytokinin alone (Manjula et al., 2000; Handro and Floh, 2001). In these cases,
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36
endogenous hormone level (auxin level) plays a vital role in callus induction
(Fracaro and Echeverrigary, 2001).
In Iris ensata, I. setosa and I. sanguinea supplementation of BA with NAA
showed best response for callus induction and they also proved that
supplementation of auxin and cytokinin alone showed poor response for callus
induction (Boltenkov and Zarembo, 2003). Callus induction from the different
explants of apple (Malus domestica) for organogenesis was also obtained by the
combined treatment of BA and NAA (Caboni et al., 2000; Martin, 2002). In
Dianthus caryophyllus also organogenic callus induction was successfully
induced by the supplementation of auxin and cytokinin synergistically (Kallak
et al. 1997). Previous experiments in our laboratory proved that phenolic
excretion and oxidation is a severe problem during callus induction and callus-
mediated regeneration and this problem was recovered by the addition of
additives along with plant growth regulators (Ganesan and Jayabalan, 2005).
Unexpectedly, in this present investigation, excretion of phenolic compounds
from explants to the medium was strictly avoided by regular sub-culturing of
callus and without addition of additives.
3.3.2 Shoot proliferation from the callus
During shoot proliferation, all the treatments of BA + NAA (Graph 1a),
TDZ + NAA (Graph 1b) and KN + NAA (Graph 1c) showed shoots
proliferation from the obtained cotyledonary leaf and hypocotyl derived callus
cultures. NAA (0.05 – 0.8) was tested in combination with the cytokinins After
one month, cotyledon callus and hypocotyl callus were successfully
regenerated and produced maximum of 17.8 shoots (CL) and 15.4 (HL) shoots
from the callus in the mMS medium fortified with TDZ (1.0 mg/l) and NAA
(0.4 mg/l) (Graph 1b, e; Plate 3). In the case of BA and NAA combination, BA
(2.0 mg/l) and NAA (0.4 mg/l) produced 16 shoots (CL) and 14.1 (HL) shoots
from the callus cultures (Graph 1a, d). At the same time, KIN - NAA
combination showed poor response for multiple shoot induction. Only 7.0 (CL)
Organogenesis
37
and 6.4 (HL) shoots were induced from the callus culture (Graph 1c, f) after 45
days of culture (Plate 3, 4).
From the callus supplemented with IAA, 12.5 and 11.6 shoots were
successfully regenerated from CL and HL callus in the mMS media fortified
with TDZ (1.0 mg/l) and IAA (0.2 mg/l) (Graph 1b, e). While from BA and
IAA combination, BA (1.5 mg/l) and IAA (0.3 mg/l) produced 12.1 and 10.3
shoots from the CL and HL callus cultures (Graph 1a, d). At the same time, KN
and IAA combination showed poor response for multiple shoot induction.
Only 7.6 and 6.4 shoots were induced from CL and HL callus (Graph 1c, f)
culture after 45 days of culture. In this present investigation, we confirmed that
along with any type of cytokinin, addition of NAA favors shoot organogenesis
from the callus cultures of castor-bean.
Callus mediated regeneration is reported from hypocotyl sections
(Reddy et al., 1987a), young stem segments (Genyu, 1988), young leaves
(Reddy and Bahadur, 1989a) and epicotyl / cotyledons (Sarvesh et al., 1992).
However, differentiation of callus into shoots and shoot buds was reported to
be either occasional of low. Similar to our results Reddy and Bahadur (1989b)
found shoot regeneration from leaf callus but produced only 3 – 4 shoots in the
medium fortified with KN 2.0 mg/l + IAA 1.0 mg/l, which is contrast to our
result where 17.8 shoots were produced from cotyledon leaf derived callus
treated with TDZ (1.0 mg/l) with NAA (0.4 mg/l). Suresh and Rao (1994)
achieved shoots from the axillary bud callus when MS medium supplemented
with BAP (0.5 mg/l) with NAA (0.5 mg/l) whereas terminal bud callus was
also obtained when the MS medium supplemented with KIN (0.5 mg/l) with
NAA (0.5 mg/l) in Castor TMV5. Studies by Sarvesh et al. (1992) proved that in
castor 20% of the cultures with shoot buds induced on B5 medium (Gamborg,
1968) supplemented with 2.5 mg/l BA with 0.1 mg/l NAA on transfer to the
same medium produces 6 – 8 shoots per callus which is also very low
comparatively to our result.
Organogenesis
38
Similar to our result, TDZ mediated-shoot proliferation was obtained in
several plants (Fiola et al., 1990; Malik and Saxena, 1992). Usually TDZ used for
somatic embryogenesis and it was reported in many plant species, either alone
or in combination with other growth regulators (Murthy et al., 1998; Faisal and
Anis, 2006). But in our experiments, TDZ along with NAA were proved as best
cytokinin for organogenesis from cotyledon callus cultures. In some cases,
shoot multiplication and regeneration was efficiently achieved by 2iP (0.17 μM)
from the callus derived rhizomes of Cymbidium ensifolium (Chang and Chang,
2000). In Fraxinus angustifolia and Carica papaya also 2iP showed a vital role in
the shoot organogenesis from different explants derived callus (Tonon et al.,
2001; Khatoon and Sultana, 1994). Usually, BA or KN was widely used for
multiple shoot initiation from the callus cultures (Martin, 2002). In our study,
combination of TDZ with NAA yielded notable results in shoots proliferation
form the obtained callus mass.
3.3.3 Effect of carbon sources, additives and amino acids on shoot
proliferation studies from callus cultures
During multiple shoot proliferation, effects of different concentrations of
carbon sources were tested. Among them, sucrose 30 g/l showed best response
for both the explants (Graph 2a, b). In the other concentrations and forms of
carbon sources tested reduced percentage of multiple shoot induction
frequency was observed. At the same time, severe browning of callus was
noticed in other concentration of glucose, fructose and maltose tested. Similar
to our report, in Coyylus avellana sucrose-mediated shoot multiplication was
effectively achieved and maximum of 3 – 4 shoots were regenerated (Yu et al.,
1993). As per previous tissue culture reports, the most commonly used
carbohydrate for plant tissue culture is sucrose. In nature, carbohydrate is
transported within the plant as sucrose and the tissue may have the inherent
capacity for uptake, transport and utilization of sucrose (Sul and Korban, 2004).
Organogenesis
39
During multiple shoot proliferation from callus cultures of CL and HL
explants, severe browning of callus was noticed. The addition of additives
controlled the browning of callus and tissues and simultaneously increased the
percentage of multiple shoot induction (Amin and Jaiswal, 1988). Hence, in this
present investigation, we have evaluated different type of additives to control
the phenolic oxidation. All the four additives tested showed best response for
the suppression of browning of callus tissues. Among the four different
additives tested, supplementation of PVP (15 mg/l) showed superior activity to
control the browning process. At the same time, addition of PVP along with
multiple shoot induction medium enhanced the multiple shoot induction
percentage (72.2%) with CL and 57.8% with HL callus. Maximum of 20 shoots
/callus were initiated by the addition of PVP and in the case of controls only
17.8 shoots were obtained from cotyledonary leaf explants whereas, in
hypocotyl explants 12.2 shoots were obtained which is also higher than the
shoots obtained from control (10.4 shoots/explant) (Table 3.2). Like our results,
control of phenolic compounds by the addition of additives showed best
response in several crops (Amin and Jaiswal, 1988; Quraishi and Mishra, 1998).
The influence of various amino acids like alanine, glutamine, proline
and serine were also evaluated for the multiple shoot initiation from the
obtained callus. All the four amino acids showed enhanced activity on multiple
shoot induction. From the four, 15 mg/l glutamine showed best response for
multiple shoot proliferation and maximum of 22.1 shoots and 17.9 shoots /
callus clump was regenerated from cotyledonary leaf and hypocotyl explants
(Table 3.3; Plate 3,4). Similar effect of glutamine-mediated plant regeneration
was obtained in barley by using microspore explants (Ritala et al., 2001).
Generally, glutamine has been used for the induction of embryogenic callus
and direct and indirect induction of somatic embryos (Kim et al., 1997; Ipekci
and Gozukirmizi, 2002). But in our work, multiple shoot initiation was
effectively supported by the addition of glutamine (15 mg /l) as one of the
media component.
Organogenesis
40
3.3.4 Effect of Pluronic F68 on shoot multiplication and elongation
When the calli were transferred to the regeneration medium containing
TDZ (0.3 mg/l), NAA (0.4 mg/l), PVP (15 mg/l), Glutamine (15 mg/l) and
Sucrose (30 g/l), they showed signs of shoot regeneration by producing tiny
green meristems on the surface of the calli within 6 weeks of culture which
later formed shoot buds. To increase the frequency of shoot regeneration from
the callus PF - 68 (0.5 – 2.0 mg/l) was supplemented along with the above
mentioned PGRs. The percentage of shoot regeneration increased to 94.5% and
90.4% and produced 25.8 shoots and 19.4 shoots /callus on 1.0 mg/l of PF - 68
with CL and HL derived callus (Table 3.4; Plate 3, 4). This suggested that PF -
68 had the potential to promote organogenesis of castor.
There are several standardized protocols are existing for callus-mediated
somatic organogenesis and embryogenesis of economically valuable crops
(Wilkins et al., 2000, 2004). Organogenic callus induction and plant
regeneration from the callus cultures of castor-bean was reported by Ganesh
kumari et al. (2008) from CL explants. The influence of PF – 68 on organogenic
was high compared with the other PGRs. Our results proved that organogenic
callus induction and direct shoot regeneration was possible in castor-bean.
Usually, addition of GA3 with shoot proliferation medium was used for the
elongation of shoots (Caboni et al., 2000). In our studies also shoot elongation
was achieved by combined treatments of PF - 68 (1.0 mg/l) with GA3 (0.3 mg/l)
(Table 3.5). Regular weekly sub-culturing of induced shoots was done on the
same medium for complete elongation and maturation and complete
maturation of shoots required total of 2 – 4 weeks (Plate 3, 4). Wilting of leaves
was totally avoided by this weekly interval subculture (Pretto and Santarem
1997; Reddy et al., 2002).
3.3.5 Root induction and hardening
Induction of rooting is an essential step in plant propagation in vitro. The
classical root induction method uses a shock of high auxin concentration.
Organogenesis
41
However, the roots are stunted and malformed (Moncousin, 1991; Rao and
Purohit, 2006). In the present investigation, root induction was achieved after
7 days of culture. During root induction we have tested different
concentrations of auxins and AgNO3. Our results proved that root induction in
castor-bean was difficult compared to other crop plants. Hence, along with
different auxins we have tested AgNO3 for root induction. In the present study,
compared with individual treatment of auxins and AgNO3, combination of IBA
(1.5 mg/l) with AgNO3 (0.6 mg/l) showed best response (72.5%) for root
induction from elongated shoots and in this concentration, maximum of
5.9 roots with 5.6 cm in root length was induced (Table 3.6; Plate 3, 4). These
results demonstrated that AgNO3 can influence root emergence and growth
and can improve rooting efficiency (Bais et al., 2000). Similar to our results, it
has been accepted that interaction of thiol compounds stimulate rooting in vitro
(Biddington, 1992). He demonstrated that the use of ethylene inhibitors such as
AgNO3 might promote root formation in shoot cultures of apple.
The use of AgNO3, a potent ethylene action inhibitor, for promoting
in vitro rooting (Bais, 2000). In the case of IBA and IAA also root induction was
noticed but in all the concentrations of IBA tested showed low response for
root induction (data not shown) when compared with combined treatment of
IBA and AgNO3. Usually, IBA mediated root induction has been reported for
several plants including Hemidesmus indicus (Sreekumar et al., 2000) and Cunila
galiodes (Fracaro and Echeverrigary, 2001). Our results proved that combination
of IBA with AgNO3 was needed for the high percentage of root induction.
To our knowledge, this is the first report for indirect organogenesis of
castor-bean through cotyledonary leaf and hypocotyl explants. Root induction
was also highly increased when the medium was supplemented with IBA and
AgNO3. The increased root length leads to increase in the survival percentage
of hardened and field grown plants. For hardening process sand, soil and
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vermiculated soil were used in 1:1:1 ratio (Plate 3, 4). After proper
acclimatization the hardened plants were transferred to field.
3.4 CONCLUSION
In conclusion, an efficient and simple protocol for in vitro adventitious
shoot multiplication from callus cultures, and whole plant regeneration has
been described. The protocol was optimized by manipulations of different
PGRs, amino acids, carbohydrates and additives for enhanced multiplication.
In conclusion, protocol explained in this research paper provides a rapid plant
regeneration system from callus for castor-bean which could be used for the
somaclonal variation induction, and producing transgenic plants in castor-bean
through Agrobacterium and biolistic methods.
Table 3.1
Effect of cytokinins and auxins on organogenic callus induction from Cotyledonary leaf and Hypocotyl explants on mMS medium supplemented with B5 vitamins.
Cotyledonary leaf explant Hypocotyl explant Concentrations of growth regulators
(mg/l)
Percentage of organogenic
callus formation
Type and nature of callus
Percentage of organogenic
callus formation
Type and nature of callus
KN 1.0 1.5 2.0 2.5 3.0
12.3 ± 0.4p 14.1 ± 1.2op 17.6 ± 1.5mn 15.2 ± 0.5o 10.9 ± 1.1po
GF GF
GLC BF BC
11.5 ± 0.7o 12.5 ± 0.6mn 15.5 ± 1.2l 13.5 ± 1.6m 11.4 ± 1.9op
YGF YGF YWF
BF BC
BA 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
8.5 ± 0.5q 24.7 ± 0.5l 25.3 ± 0.3j 28.7 ± 1.3jk 20.5 ± 0.6lm 18.1 ± 0.4m 13.2 ± 1.4p 7.6 ± 0.5qr
GF GC GC GC GC GC
GLC BF
16.7 ± 0.6k 18.5 ± 0.5j 20.5 ± 0.6i 25.3 ± 1.6h 17.3 ± 1.5jk 14.7 ± 1.7lm 12.4 ± 1.5mn 11.7 ± 1.1no
GF GC GC GC GF GF
GLC BF
BA + NAA 2.0 + 0.2 2.0 + 0.4 2.0 + 0.6 2.0 + 0.8 2.0 + 1.0 2.0 + 1.2 2.0 + 1.4 2.0 + 1.6 2.0 + 1.8 2.0 + 2.0
46.5 ± 1.0f 52.6 ± 1.5e 55.5 ± 0.5d 69.5 ± 1.5a 61.5 ± 1.7b 59.3 ± 1.4bc 53.5 ± 0.7de 44.8 ± 0.5fg 41.5 ± 0.7h 38.5 ± 0.4i
GC GC
GCN GCN GCN GC GC GC GC GC
40.4 ± 1.6g 51.2 ± 1.5c 52.3 ± 1.1b 54.2 ± 1.7a 53.1 ± 0.7ab 52.3 ± 0.2b 51.7 ± 1.4bc 50.4 ± 1.4cd 48.9 ± 1.6e 44.5 ± 1.5f
GC GC
GCN GCN GCN GCN GCN GCN GC GC
GF – Green Friable; GC – Green Compact; GCN – Green Compact Nodular; YWF – Yellowish White Friable; YGF – Yellowish Green friable;
GLC – Greenish Less Compact; BC – Brown Compact; BF – Brown Friable
Number of explants tested = 50. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Table 3.2
Effect of different concentrations of additives on multiple shoot proliferation from callus cultures of castor (Ricinus communis L.) on the mMS medium fortified with
TDZ (0.3 mg/l) and NAA (0.4 mg/l)
Cotyledonary leaf explant Hypocotyl explant Additives (mg/l ) Percentage of
response Mean number
of shoots / callus Percentage of
response Mean number
of shoots / callus
Charcoal 50
100 150 200 250
58.4 0.2mn 62.1 0.3k 65.6 0.5hi 63.2 0.2j 60.4 1.0l
16.8 0.4j 17.2 0.8i 17.6 0.5h 17.0 0.2ij 16.5 0.6k
34.0 ± 0.6op 44.5 ± 0.4k 50.4 ± 1.2ef 55.1 ± 0.6c 50.3 ± 1.3fg
7.6 ± 1.2j 8.0 ± 0.5i 8.7 ± 0.2f 8.5 ± 0.5fg 7.4 ± 1.3jk
PVP 5
10 15 20 25
69.3 0.6cd 71.8 0.5ab 72.2 0.2a 70.2 0.1b 67.3 0.3de
18.4 0.2ef 19.2 1.0c 20.0 0.3a 19.0 0.1cd 18.6 0.9e
52.3 ± 1.6d 55.3 ± 1.4b 57.8 ± 1.5a 55.0 ± 0.5cd 50.1 ± 0.4g
9.3 ± 1.6de 10.6 ± 1.4b 12.2 ± 1.8a 9.7 ± 0.1d 8.3 ± 0.4gh
Ascorbic acid 5
10 15 20 25
67.5 1.0f 68.7 0.6e 69.1 0.3d 66.2 0.7g 65.0 0.1i
17.1 0.5ij 17.9 0.1g 19.5 0.2b 18.9 0.1d 17.6 0.2h
51.2 ± 0.3e 52.3 ± 0.2e 54.3 ± 1.4d 50.0 ± 1.6h 48.1 ± 0.7i
8.4 ± 0.6g 9.5 ± 1.5de 10.5 ± 1.7bc 9.7 ± 0.3d 8.4 ± 0.5g
Citric acid 5
10 15 20 25
56.5 1.0p 59.6 0.5m 60.1 0.2lm 58.2 0.1n 57.4 0.3op
15.4 0.5m 16.0 0.2l 16.5 0.1k 15.0 0.4n 14.4 0.3o
41.6 ± 0.4n 43.8 ± 0.5l 45.8 ± 0.6j 42.7 ± 1.5m 39.7 ± 0.6o
6.2 ± 0.3m 7.0 ± 0.5l 7.6 ± 1.2j 7.0 ± 1.7l 6.1 ± 1.5mn
Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Table 3.3
Effect of different concentrations of amino acids on the multiple shoot proliferation from the callus on the medium supplemented with on the mMS medium fortified
with TDZ (0.3 mg/l), NAA (0.4 mg/l) and PVP (15 mg/l)
Cotyledonary leaf explant Hypocotyl explant Amino acids
(mg/l) Percentage
of callus formation
Mean number of shoots / callus
Percentage of callus
formation
Mean number of shoots / callus
Alanine 5
10 15 20 25
56.3 0.2m 57.6 0.1k 57.2 0.5l 52.2 0.1n 49.3 0.6o
18.7 0.3o 19.3 0.1kl 19.0 0.2n 18.3 0.5p 17.5 0.1q
52.7 ± 0.5lm 56.8 ± 0.2j 54.7 ± 0.6kl 55.4 ± 0.1k 52.4 ± 0.3m
8.4 ± 0.3o 10.8 ± 0.6hi 9.4 ± 1.3m 8.0 ± 0.5p 7.4 ± 0.1q
Proline 5
10 15 20 25
64.6 0.7i 65.7 0.4gh 66.5 0.2g 65.4 0.1h 63.2 0.2j
19.2 0.1l 19.9 0.4i 20.5 0.2fg 19.6 0.3jk 19.0 0.7n
58.4 ± 1.4hi 70.3 ± 0.4b 62.7 ± 0.6ef 56.4 ± 0.6jk 53.8 ± 0.5l
8.9 ± 0.7n 11.0 ± 0.6h 12.5 ± 0.2f 9.7 ± 0.5l 8.1 ± 0.5op
Serine 5
10 15 20 25
73.5 0.1e 74.0 0.5de 73.5 0.3e 74.3 0.6d 72.7 0.8f
20.3 0.4h 21.5 0.3c 20.6 0.1fg 21.7 0.2b 19.7 0.1j
59.4 ± 0.3h 61.2 ± 0.1fg 62.1 ± 0.3f 63.7 ± 0.6e 50.3 ± 0.7n
9.0 ± 0.1mn 10.3 ± 0.6j 11.5 ± 0.1g 13.0 ± 0.3de 9.5 ± 0.5lm
Glutamine 5
10 15 20 25
84.3 0.2b 84.9 0.5ab 85.2 0.1a 83.6 0.3c 83.1 0.4cd
21.1 0.3e 21.4 0.1cd 22.1 0.2a 21.0 0.4de 20.7 0.2f
65.4 ± 0.5d 67.6 ± 0.3bc 72.3 ± 0.2a 68.4 ± 0.6b 63.2 ± 0.1ef
10.2 ± 0.1jk 13.4 ± 0.6d 17.8 ± 0.1a 15.4 ± 0.4c 16.5 ± 0.5b
Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Table 3.4
Effect of different concentrations of pluronic F68 on regeneration of shoot buds from organogenic callus cultured on mMS medium fortified with TDZ (0.3 mg/l),
NAA (0.4 mg/l), PVP (15 mg/l ) and glutamine (15 mg/l)
Cotyledonary leaf derived callus cultures
Hypocotyl derived callus cultures Concentrations
of PF – 68 (mg/l) Percentage of
response Mean no. of
Shoots / callus Percentage of
Response Mean no. of
Shoots / callus
Pluronic F - 68 0.5 0.6 0.7 0.8 0.9 1.0 1.2 1.4 1.6 1.8 2.0
90.5 ± 0.4f 91.5 ± 0.2d 92.4 ± 0.1c 93.6 ± 0.2b 94.0 ± 0.5ab 94.5 ± 0.6a 93.2 ± 0.8b 91.4 ± 0.2de 89.5 ± 0.4g 89.0 ± 0.6gh 87.5 ± 0.8h
19.6 ± 0.4g 21.5 ± 0.2f 22.5 ± 0.5e 23.6 ± 0.2c 25.0 ± 1.0ab 25.8 ± 0.6a 24.8 ± 0.3b 23.5 ± 0.5cd 23.3 ± 0.6d
22.5 ± 0.2e
21.5 ± 1.7f
77.4 ± 0.8fg 80.3 ± 0.4f 82.5 ± 0.5de 83.5 ± 0.3d 89.5 ± 0.6ab 90.4 ± 0.1a 89.4 ± 0.5b 85.6 ± 0.1c 82.1 ± 0.6e 78.5 ± 0.5f 77.4 ± 0.7fg
12.4 ± 0.5i 13.6 ± 0.4h 15.7 ± 0.1fg 17.4 ± 1.0de 18.4 ± 0.6bc 19.4 ± 0.3a 18.5 ± 0.6b 18.0 ± 0.3c 17.4 ± 0.5de 16.2 ± 0.5f 15.4 ± 0.5g
Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Table 3.5
Effect of GA3 in combination with PF - 68 on shoot elongation of castor (Ricinus communis L. cv TMV 5)
Growth regulators (mg /l)
Percentage of response
Shoot length (cm)
PF - 68 + GA3 1.0 + 0.1 1.0 + 0.2 1.0 + 0.3 1.0 + 0.4 1.0 + 0.5
51.8 0.5e 56.7 0.6ab 62.3 0.5a 57.4 0.8b 50.5 0.4ef
4.5 0.5cd 5.1 0.8c 5.6 0.6a 5.4 0.7e 4.9 0.3ef
Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Table 3.6
Effect of IBA and AgNO3 on root induction from elongated shoots of Castor (Ricinus communis L. cv TMV 5)
Growth regulators (mg/l )
Percentage of Response
Mean No. of roots / explant
Average root length (cm)
IBA + AgNO3 1.5 + 0.2 1.5 + 0.4 1.5 + 0.6 1.5 + 0.8 1.5 + 1.0
65.0 1.2c 69.5 1.9b 72.5 2.2a 70.5 3.2b 68.5 2.5b
5.2 0.25c 5.4 0.32bc 5.9 0.51a 5.5 0.16b 5.1 0.25d
5.2 0.5bc 5.5 0.9ab 5.6 0.8a 5.3 0.4b 5.0 0.5c
Number of explants tested = 30. Values are means SE of 5 replication of 3 repeated experiments. Means within a column followed by the same letters are not significantly
different according to Duncan's Multiple Range Test (DMRT) at 5 % level.
Adventitious shoot proliferation from cotyledonary leaf and hypocotyl explants of Castor (Ricinus communis)
Effect of carbohydrates on multiple shoot proliferation from cotyledonary leaf and hypocotyl explants of Castor (Ricinus communis)
Plate 3 Organogenesis from cotyledonary leaf explants of Ricinus communis L.
a. Callus initiation (1.5 x) b. Callus proliferation (1.5 x) c & d. Shoot bud initiation (1.5 x & 1.0 x) e. Multiple shoot initiation (0.5 x) f & g. Shoot elongation (2.0 x & 1.0 x) h. Root initiation (0.5 x) i & j. Hardening of in vitro derived plants (0.1 x & 0.2 x)
Plate 4 Organogenesis from hypocotyl explant of Ricinus communis L.
a. Callus initiaion (1.5 x) b. Callus proliferation (1.0 x) c, d & e. Shoot bud initiation and proliferation (2.0 x, 1.5 x & 1.5 x) f, g & h. Shoot elongation (0.5 x, 0.5 x & 0.3 x) i. Root initiation (0.5 x) j. Hardened plants (0.2 x) k. Well grown plants (0.1 x)