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Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 56
CHAPTER - III
DIRECT ORGANOGENESIS
3.1. INTRODUCTION
The ability of individual cells to develop into whole plants, known as totipotency
has formed the basis for regeneration and propagation of selected genotypes through tissue
culture. The totipotency theory was first proposed and clearly formulated by the German
botanist Haberlandt in 1902. Unfortunately his attempt to regenerate plants from palisade
mesophyll cells of Daucus carota in a simple hormone-free medium was not successful. In
the 30 years following Haberlandt’s work very little progress was made in this regard. The
major breakthrough in tissue culture came after the discovery of the cytokinin and kinetin
in 1956 (Miller et al., 1956). It was demonstrated that they stimulated cell division.
Addition of cytokinins and auxins to the culture media rendered plants to form callus
tissue, roots, and/or shoots de nono.
In vitro culture studies include aspects such as direct organogenesis and
organogenesis through callus pathway. These studies have significant advantages over
traditional clonal propagation techniques. They also exploit the potential of combining
rapid large-scale propagation of new genotypes, the use of small amounts or original
germplasm and the generation of pathogen-free propagules (Altman, 1999). In recent years
there has been an increased interest in in vitro culture techniques which offer a viable tool
for mass multiplication and germplasm conservation of rare, endangered and threatened
medicinal plants (Ajithkumar and Seeni, 1998 and Tiwari et al., 2000).
In vitro morphogenesis and plant regeneration was carried out by Cheepala et al.
(2004) using various seedling explants such as cotyledonary node, axillary branch node,
hypocotyl, epicotyl cotyledon and stem segments from green house grown plants. Tissue
culture enables mass propagation of uniform plants and overcomes the problems of
propagation in the hot summer in Israel when stock plants die from heat stress or were
weakened and were thus more vulnerable to endemic contaminations. A method for large-
scale propagation of Achillea filipendulina cv. ‘Parker’ through clean meristem culture is
described by Evenor and Reuveni (2004). Although there are a number of reports of
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 57
in vitro propagation of Rubus species (Harper, 1978; Anderson, 1980; Welander, 1982;
Finne, 1986; Reed, 1990; Bobrowski et al., 1996 and Debnath, 2003), none is available for
R. pubescens being propagated by tissue culture. In vitro culture is an efficient method for
ex situ conservation of plant diversity, because with this technology many endangered
species can be quickly preserved from a minimum of plant populations (Cuenca et al.,
1999).
There are reports of adventitious shoot regeneration through direct organogenesis
using various explants of medicinally important species like leaf explants of Paulownia
portunei (Kumar et al., 1998a), Pyrus communis (Caboni et al., 1999), Pothomorphe
umbellata (Pereira et al., 2000), Tagetes erecta (Vanegas et al., 2002), Echinacea
purpurea (Koroch et al., 2002), Achras supota (Purohit et al., 2004), Charybdis numidica
(Kongbangkerd et al., 2005) and Hagenia abyssinica (Feyissa et al., 2005), Internodal
explant of Euphorbia tirucalli (Uchida et al., 2004), Centella asiatica (George et al.,
2005), Feronia limonia (Hiregoudar et al., 2005) and Mentha piperita (Shasany et al.,
2006), internode and root explants of Bacopa monnieri (George et al., 2006), root explants
of Echinops spp. (Dalia et al., 2006) and Solanum melongena (Sarker et al., 2006), node
explants of Enicostemma littorale (Shanthi and Xavier, 2003), Ficus benghalensis
(Rahman et al., 2004a), Zehneria scabra (Anand and Jeyachandran, 2004), Caesalpinia
bonduc (Kannan et al., 2006) and Eclipta alba (Tejavathi et al., 2006), axillary explants of
Excoecaria agallocha (Rao et al., 1998) and Ceropegia candelabrum (Beena et al., 2003),
cotyledon explants of Brassica juncea (Gua et al., 2005) and Cucumis sativus (Mohiuddin
et al., 2005), hypocotyle explants of Vigna subterranea (Lacroix et al., 2003), Brassica
oleracea (Sasaki, 2002) and Bixa orellana (Neto et al., 2003), petiole explants of Piper
colubrinum (Kelkar and Krishnamurthy, 1998) Thapsia garganica (Makunga et al., 2005)
and Solanum violaceum (Raghu et al., 2006b). Cotyledonary node explants of Gossypium
hirsutun (Rauf et al., 2005) and Cicer arietinum (Sarker et al., 2005) and inflorescence
explants of Zea mays (Leon et al., 2002) and Clivia spp. (Ran and Simpson, 2005).
Shasany et al. (2006) carried out a high effieiency regeneration system in Mentha
piperita by multiplying screened somaclones with desired morpho-chemotypic traits in the
mint development program. Clonal propagation of Curculigo orchioides, for commercial
purposes requires a simple, economical, reproducible and rapid multiplication protocol
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 58
which was standardized through in vitro technique using shoot tip and rhizome disc
explants (Nagesh, 2008). Direct plant regeneration from leaf explant of Enicostemma
axillare was obtained using different concentrations of BAP with stable concentration of
KN (Jeyachandran et al., 2005).
Tissue culture techniques are being used globally for the ex situ conservation of the
plants; reports of in vitro plant regeneration from tissue of medicinal plants are Ocimum
gratissimum (Gopi et al., 2006), Teucrium stocksianum (Bouhouche and Ksiksi, 2007),
Exacum sp., (Unda et al., 2007), Dendrobium candidum (Zhao et al., 2007), Artemisia
vulgaris (Sujatha and Ranjithakumari, 2007), Bambusa glaucescens (Shirin and Rana,
2007), Salvia africana-lutea (Makunga and Staden, 2008), Ophiorrhiza prostrata (Martin
et al., 2008), Swertia chirata (Chaudhuri et al., 2008), Paulownia tomentosa (Corredoira
et al., 2008), Prunus serotina (Liu and Pijut, 2008), Daphne sp., (Noshad et al., 2009),
Huernia hystrix (Amoo et al., 2009), Cornus canadensis (Feng et al., 2009), Drymaria
cordata (Ghimire et al., 2010) and Phaseolus vulgaris (Kwapata et al., 2010).
The present study accomplishes the development of adventitious shoots using
different explants of P. barbatus for the reliable production and rapid large-scale
propagation of these species in order to conserve populations of P. barbatus.
3.2. REVIEW OF LITERATURE
Kumar et al. (1998b) evolved a reliable protocol for multiple shoot induction and
plantlet regeneration. Apical buds of Ficus carica were multiplied on MS medium
supplemented with 2.0mg/l BAP and 0.2 mg/l NAA and an average multiplication rate of
four per subculture was established with 90% success and excised shoots were rooted in
liquid half strength MS medium supplemented with 2.0mg/l IBA and 0.2% activated
charcoal.
Large number of shoots were propagated from single embryonic axis of sainfoin
on MS medium supplemented with 2.0mg/l BAP either 0.05 or 0.1 mg/l IBA and on the
media containing 2.0mg/l BAP and 0.05mg/l NAA. The highest shoot length was observed
on a medium containing 2.0mg/l BAP only. Shoots were rooted at a frequency of 60%
within 4 weeks in half strength MS medium containing 1.0mg/l IBA and grew into normal
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 59
fertile plants (Sancak, 1999). Tissue culture technique have been established as a useful
approach for ex situ conservation of rare, endemic or threatened plant species (Cuenca
et al., 1999). Inflorescence nodal segments of Centaurea paui showed optimum shoot
proliferation on MS medium supplemented with 0.5mg/l BA or with 2.0mg/l KN. The
combination of 2.0mg/l IAA + 2.0mg/l IBA on MS medium yielded the optimum root
induction.
Shirin et al. (2000) described a rapid and large-scale in vitro clonal propagation of
Kaempferia galanga by enhanced rhizomes as explants. In vitro plantlet production has
been achieved on 0.75 x MS medium supplemented with 12.0µM BA, 3.0µM NAA and
3% sucrose. Hardened plantlets produced normal storage roots as the plants. Tiwari et al.
(2000) described a rapid and large-scale in vitro clonal propagation of Centella asiatica
by enhanced axillary bud proliferation in nodal segments. The synergistic combination of
22.2µM BA and 2.68µM NAA induced the maximum frequency of 9.1% shoot formation;
subculturing of nodal segments harvested healthy shoots with similar frequency. MS
medium supplemented with 6.7µM BA and 2.88µM IAA was found most suitable for
shoot elongation. In vitro shoots were rooted when cultured on MS medium containing
2.46µM IBA.
Higher proliferation of shoots and multiplication coefficient was obtained from
inflorescence nodal segments of Centaurea spachii on MS medium supplemented with
1.0mg/l BA. The optimal shooting result (60%) was obtained on MS medium with a
combination of 2.0mg/l IAA + 2.0mg/l IBA. High survival rate of over 80% was obtained
when the plantlets were transferred to green house conditions (Cuenca and Marco, 2000).
Arous et al. (2001) investigated the optimum shoot bud induction on MS medium fortified
with BAP (0.5mg/l) and NAA (1.0mg/l) through zygotic embryo hypocotyls of Tunisian
chili. Shoot bud development was enhanced by the addition of GA3 (0.5mg/l) to the
medium. Plants were rooted in MS medium at half strength and transferred into pots.
Nodal explants of Anisochilus carnosus produced best multiple shoot induction
and shoot elongation, when cultured on MS medium fortified with BAP (0.5mg/l). The
highest percentage of rooting was observed with IBA (1.0mg/l). Regenerated plantlets
were transferred to the field with 61% survival rate (Jeyachandran et al., 2004). Arulmozhi
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 60
et al. (2001) established adventitious shoot buds from nodal explants of Paulownia
fortunei, in which MS medium supplemented with BAP (4.0mg/l) + AdS (20mg/l).
Microshoots with 2 - 3 leaves developed roots on MS medium containing IAA or IBA or
NAA, though NAA at 2.0mg/l was best.
Rapid tissue culture systems were developed for Piper longum through shoot tip
multiplication and direct regeneration on MS medium supplemented with 8.9µM BA and
4.64µM KN. Adventitious shoot regeneration from leaf segments was achieved on MS
medium containing 17.76µM BA and 8.28µM picloram. Elongated shoots were separated
and rooted in MS medium supplemented with 2.46µM IBA (Soniya and Das, 2002a).
In vitro direct shoot regeneration from leaf and nodal explants of Enicostemma
hyssopifolium were reported by Seetharam et al. (2002). Multiple shoots were elicited
from leaf explants on MS medium supplemented with BAP (1.5mg/l) and IAA (0.5mg/l),
while from nodal explants on BAP (1.0mg/l) and IAA (0.5mg/l), shoot elongation was
developed from leaf derived shoots on MS media with KN (1.0mg/l) and BAP (1.0mg/l),
while node derived shoots on media with KN (1.0mg/l) and BAP (0.5mg/l). In vitro shoots
were rooted on half strength MS medium supplemented with IAA 1.0mg/l. Dickinson
(1978) effected bud formation on the leaves (i.e., de novo morphogenesis on the leaves) of
angiosperms, which occurs spontaneously under natural conditions in some
monocotyledonous and many dicotyledonous species. Ephiphyllous bud formation on
intact leaves under in vitro conditions has been described by Paterson (1984). The
stimulatory effect of KN + BAP on growth of stem enhances the rate of multiplication and
elongation of shoots 4 - 5 folds (Saxena, 1990 and Bejoy and Hariharan, 1993).
Organogenesis was induced directly without any intervening callus phase on MS
medium supplemented with BA (4.0mg/l) and indole propionic acid (IPA 1.0mg/l) from
the cotyledonary leaf discs of Cucumis sativus cv. and elongated shoots were rooted in
basal medium with 1.0mg/l IBA (Soniya and Das, 2002b). Cotyledonary explants cultured
on basal medium in the absence of any exogenous growth regulators never differentiated
to produce shoot buds. According to Wehner and Locy (1981) the Cucumber lines and
varieties differ from one another in their plant regeneration using hypocotyls and
cotyledons.
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 61
Govindaraju et al. (2003) standardized a protocol for Withania somnifera. Direct
differentiation of multiple shoots from leaf, nodal segments and shoot tips occurred within
two weeks on MS medium containing BAP (0.5 - 3.0mg/l) in combination with IAA
(0.5mg/l). Shoots were elongated on MS medium fortified with GA3 (0.5mg/l) when the
explants were rooted successfully in half strength MS media with IBA (0.5-10mg/l) alone
or along with IAA (0.5mg/l).
Ramula et al. (2003) developed a protocol for plant regeneration from seedling
excised root segments of Hemidesmus indicus. Formation of shoots (5.02 ± 1.01) was
from the proximal end of root segments, when placed on MS medium supplemented with
2% sucrose, BAP 3.0mg/l and NAA 0.5mg/l. Rapid elongation of shoot buds were
observed upon transfer of the responding root segment to half strength MS medium.
Formation of roots was observed from the basal region of the regenerated shoots. An
effective in vitro regeneration method was carried out by Banu and Handique (2003)
through nodal explants of Phyllanthus fraternus. Maximum multiplication
(98shoots/explant) was achieved on MS medium supplemented with 1.0mg/l BAP and
0.2 - 0.3mg/l IAA. Rooting was achieved with 87% of the microshoots on MS medium
containing 1.0mg/l IAA and 0.5mg/l IBA.
An efficient protocol has been developed for direct shoot organogenesis from
embryo axes explants of two different landraces of Bambara groundnut. Shoot
regeneration frequently was 100% and from five to eight shoots per explant were obtained,
when the embryo axes were placed on media containing BAP (1.0mg/l) and NAA
(1.0mg/l) and then cut transversely and transferred onto a medium containing 1.5mg/l
BAP. The regenerated shoots were rooted on a medium containing 1.0mg/l NAA and then
transferred to the greenhouse (Lacroix et al., 2003).
Anand and Jeyachandran (2004) developed a protocol for in vitro propagation of
Zehneria scabra. Nodal explants placed on MS medium fortified with 5.0mg/l BAP and
0.5mg/l IAA was the optimum for multiple shoots followed by elongated shoots which
were subcultured for rooting on MS medium supplemented with 2.0mg/l NAA. Direct
shoot bud differentiation was achieved in leaf segments Achras sapota by culturing on
Schenk and Hildebrandt (1972) medium supplemented with 5.0μM thidiazuron and
8.88μM BAP (Purohit et al., 2004).
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 62
An in vitro protocol was developed for rapid multiplication of plantlets via., direct
organogenesis from leaf segments of Embelia ribes (Shankarmurthy et al., 2004). The
frequency of shoot bud production was the highest at the concentration of 3.0mg/l FAP
(6-fufurylaminopurine) and 0.4mg/l NAA. Rooting of microshoots was on the same
medium in a single phase culture. The rooted plantlets were well accomplished with a
survival frequency of 96%. Uchida et al. (2004) reported that the successful plant
regeneration was obtained from internode explats of Euphorbia tirucalli on LS medium
supplemented with 0.02mg/l thidiazuron. They were rooted on LS medium containing
0.02mg/l NAA.
In vitro organogenesis of Citrullus lanatus, by the induction of adventitious buds
in cotyledonary segments has been reported (Krug et al., 2005). In vitro organogenesis of
watermelon occurred with higher efficiency, when cotyledon segments from the proximal
region collected from three days old seedling which were cultured in MS medium
supplemented with BAP (1.0mg/l) and coconut water (10%). The histological study
showed that the organogenesis occurred directly, without callus formation on epidermal
and subepidermal layer of the explants. Dong and Jia (1991) reported an improvement in
shoot bud development in watermelon cotyledons, when combining cytokinin and auxin
in the media, but Srivastava et al. (1989) detected an inhibition of shoot organogenesis,
when NAA or IAA was added to the induction medium.
In vitro studies carried out by Jeyachandran et al. (2005) using leaf explants of
Enicostemma axillare on MS medium supplemented with various plant growth regulators
at different concentrations. BAP 3.0mg/l and KN 2.0mg/l produced microshoots from the
leaf surface through direct organogenesis. Twenty microshoots were obtained after 45
days. The elongated shootlets were rooted MS medium fortified with IBA (2.5mg/l) and
BAP (0.5mg/l). The rooted plantlets were transferred to the field condition, after
hardening.
An in vitro protocol was developed for nodule formation from leaf sections of
Charybdis numidica on MS liquid medium supplemented with 20μM BA under dark
conditions. Nodules were cultured on semisolid MS medium with factorial combination of
BA (0 - 40μM) and NAA (0 - 10μM) under continuous light. The highest number of
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 63
shoots were formed on medium containing 2.5μM NAA and 20μM BA. Regenerated
shoots were successfully rooted on semisolid MS medium supplemented with 10μM IAA.
The plantlets were transferred to the green house after hardening (Kongbangkerd et al.,
2005). In vitro clonal propagation protocol (Baskaran and Jayabalan, 2005) was achieved
from aromatically important Indian cereal crop of Sorghum bicolor (L.) Moench. Plantlet
production system has been induced on MS medium with synergetic combination of BA
(22.2μM), KN (4.6μM), AdS (2.8μM), 5% coconut water (CW) and 3% sucrose, which
promoted the maximum number of shoots as well as beneficial shoot length, when the
healthy shoots clumps were cultured on MS medium fortified with BA (22.2μM), KN
(4.6μM), AdS (2.8μM), NAA (2.7μM), ascorbic acid (30.0μM) and 5% CW, a rapid
production of axillary and adventitious buds. Rooting at highest (100%) were obtained on
MS medium containing 22.8 μM IAA.
Anupama et al. (2005) evolved protocol for the in vitro propagation of Cayratia
pedata. Shoot tip explants were cultured on MS medium supplemented with various
concentrations of NAA (0.5mg/l) and BAP (1.0 - 4.0mg/l). Maximum shoot regeneration
was obtained in 3.0mg/l BAP + 0.5mg/l NAA on medium. Maximum shoot proliferation
was achieved at 2.0mg/l of IBA on medium. The rooted plantlets were hardened
subsequently. Yasmeen and Rao (2005) developed a method for regeneration of multiple
shoots from cotyledons of Vigna radiata (L.) Wilezek. The direct shoot formation was
recorded on MS medium fortified with BAP at 2.25mg/l and 5.0mg/l in combination with
IAA and KN (0.5mg/l each). The maximum of 9.1 shoot buds were induced from
cotyledons, when cultured on MS medium with BAP (5.0mg/l) and KN (0.5mg/l).
Ran and Simpson (2005) have developed a protocol for in vitro propagation of the
genus Clivia. The optimal media for peduncle-pedicel (PP) junction were MS basal
medium containing 10μM BA and 10μM of 2,4-D or MS supplemented with 5.0μM BA,
10μM NAA, 250mg/l glutamine and 500mg/l casein hytrolysate and their usage depended
on the breeding lines. Bhatia and Ashwath (2005) studied a method for effect of medium
pH on shoot regeneration from the cotyledonary explants of Tomato. The explants were
inoculated onto MS medium which was maintained wide range of pH (4.5 - 7.5).
Percentage of shoot regeneration and number of shoots produced/explant were not
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 64
significantly affected by the medium pH. Shoot height was significantly affected and
further shoots were only produced at a pH range of 5.5 - 6.0 lower (4.5 - 5.0) and higher
(6.5 - 7.0) medium pH significantly reduced shoot height. The best regeneration and
growth occurred only in the pH range of 5.5 - 6.0.
Cotyledonary nodes of Gossypium hirsutum produced maximum number of
3.43 shoots/explant, when cultured on MS supplemented with 0.25mg/l KN. Ninety three
percentage of rooted plantlets with 5.85 cm shoot length was obtained when shoots were
cultured on MS supplemented with 0.5mg/l NAA and 0.1mg/l KN (Rauf et al., 2005). Gua
et al. (2005) reported that the highest frequency of shoot formation was 61.3 - 67.9% in
cotyledon and 40.7 - 52.4% in leaf segments of mustard when 2.27 or 4.54µM TDZ was
combined with 5.37µM NAA. Next to TDZ, CPPU was also very suitable for induce shoot
formation frequency which was 45.0% in cotyledon and 36.4% in leaf segment, when
cultured 1.61µM CPPU was combined with 2.69µM NAA, respectively.
Laskar et al. (2005) evolved a method for adventitious shoot bud regeneration from
leaves of Potentilla falgens. Explant browning, a major hurdle in establishment of culture,
was overcome by treating leaves with a combination of antioxidant such as 100mg/l
ascorbic acid, 100mg/l citric acid and 20mg/l L-cysteine HCl. Adventitious bud
differentiation and shoot regeneration (80%) was observed on modified MS medium
supplemented with 0.1mg/l BAP and 0.1mg/l NAA. Rooting was induced on MS basal
medium. The regenerated shoots had 70% survival rate.
Vidya et al. (2005) standardized a micropropagation protocol for an endangered
species of Entada pursaetha using cotyledonary node explants. The synergistic effect of
BAP (5.0mg/l) with NAA (0.5mg/l) induced (9.8 shoots/explant) adventitious shoots from
the cotyledonary node. The microshoots rooted well on MS medium supplemented with
2.0mg/l IBA.
Santos et al. (2006) showed shoot multiplication on half salt MS liquid medium
supplemented with 2.5mg/l KN, 1.5mg/l BA from inflorescence apices of yellow king.
The highest shoot multiplication rate (9.0 shoots/explant) was obtained on a liquid MS
medium at full strength supplemented only with BA at 1.0mg/l. Shoot multiplication was
achieved by releasing apical dominance of the single elongated shoot on WPM
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 65
supplemented with 0.7mg/l BAP and 0.05mg/l NAA. The highest rooting percentage was
recorded on half strength WPM containing 1.0mg/l IBA (Durkovic, 2003). Wann and
Gates (1993) and Orlikowska and Gabryszewska (1995) worked with different genotype
of mature red maples and common feature was the use of MS medium and low
concentration of TDZ during axillary bud multiplication. In both reports best quality and
highest number of shoots produced after addition of 0.01mg/l TDZ. Combined TDZ +
BAP treatment produced callus formation only and replacing TDZ by BAP in combination
with NAA decreased and retarded shoot proliferation and led to necrosis.
Cultured leaf explants of Sugarcane exhibited swelling followed by direct shoot
regeneration which occurred on MS medium supplemented with NAA (5.0mg/l) and KN
(0.5mg/l). Therefore increased concentration of NAA reduced the percentage of plant
regeneration (Gill et al., 2006). Adventitious shoot buds from leaves of Santalum album
were reported by Mujib (2005). Bud formation occurred on WP solid basal medium
containing 2.22µM BAP. Regenerated shoots were rooted on WPM containing 5.71µM
IAA. Adventitious shoots from leaves were obtained in BAP supplemented media
(Welander, 1988).
Gavhane and Mukundan (2006) standardized in vitro propagation protocol for
Acorus calamus L. using rhizome explants. Maximum shoot multiplication was obtained
on MS medium supplemented with BAP (11.09μM) with NAA (5.37μM). The highest
percentage of rooting was achieved on MS medium containing 4.9μM IBA. Plantlets with
well developed roots were successfully transferred to the soil with 90 - 95% survival rate.
In vitro rhizomes were produced. Analysis revealed that the content of asarone in mother
plant and in vitro field grown plants was similar.
High frequency rooting of microshoots was obtained from apical and axillary buds
of suckers of banana (Musa sapientum L.). It was achieved by using liquid medium
(Akbar and Roy, 2006). When the explants were cultured on MS medium fortified with
BA, KN and NAA (0.5mg/l each), a large number of shoots developed. Addition of 10%
coconut milk to the medium increased the numbers of shoots per culture. Shoots rooted
well within two weeks. Then shoots were separated and subcultured on half strength MS
liquid and agar gelled medium fortified with IBA (1.0mg/l) for rooting. The rooted
plantlets were transferred to small polythene bags containing sterile sand, soil and humus
(1:2:1) and maintained with a high humidity for acclimatization.
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 66
Sarker et al. (2006) have reported the in vitro regeneration of egg plant (Solanum
melongena L.). Cotyledonary leaf was found to be the best for multiple shoot regeneration
on MS medium supplemented with BAP (1.0mg/l) and KN (1.0mg/l). Proliferation and
elongation of such shoots were obtained in hormone free MS medium. Moreover, the
regenerated shoots produced healthy roots when they were cultured on MS medium
without hormone supplements. The in vitro raised plantlets were successfully established
in soil.
Kannan et al. (2006) established an in vitro plant regeneration through direct
organogenesis from mature stem explants of Caesalpinia bonduc. High frequency
adventitious multiple shoot regeneration response (89%) and maximum number of shoots
of stem segment (9.0 shoots) were recorded on MS medium supplemented with optimal
concentration of BAP (2.0mg/l) and IAA (1.0mg/l). The excised shoots were transferred to
MS medium supplemented with IBA (2.0mg/l) for rooting. About 90% of fully
regenerated plantlets were gradually hardened and transferred to field with 85% survival
rate in natural conditions.
An efficient clonal propagation was developed for large-scale production of Costus
speciosus from rhizome in a medium containing adenine sulphate (Raghu et al., 2006a)
alone and in combination with BA, KN and NAA, on multiple shoot and root induction.
Improvements were made by adding adenine sulphate in quality and an increasing quantity
of cultures were reported. In vitro plant regeneration was obtained from leaf, petiole,
internode and somatic embryo explants of Solanum violaceum (Raghu et al., 2006b). The
direct regeneration of shoot buds was achieved using certain plant media with various
concentrations of BA (2.22 – 13.31μM). In vitro shoots were rooted using half MS
medium containing IAA (2.85μM).
Tejavathi et al. (2006) obtained multiple shoots from nodal segments of Eclipta
alba and shoot apices of Evolvulus alsinoides cultured on MS medium augmented with
auxins and cytokinins. Initiation of terminal flower buds were observed on branches of
multiple shoots on MS medium supplemented with IBA (13.8μM) + BAP (0.44μM) in
Eclipta alba and IBA (4.92μM) in Evolvulus alsinoides.
Chapter – IIІ Direct Organogenesis
“In Vitro Propagation of Plectranthus barbatus Andrews – A Valuable Medicinal Plant in the Palni Hills of the Western Ghats, South India” 67
Nithiya and Arockiasamy (2006) evolved a protocol for in vitro mass
multiplication of Datura metel L. through root cultures. Maximum shoots were formed on
MS medium supplemented with 4.0mg/l BAP, after 15 days. Shoot elongation was noticed
on MS medium containing BAP 2.0mg/l and GA3 1.0mg/l. The well rooted plantlets were
hardened then successfully transferred to field after two weeks, with an impressive
survival rate of 60%. George et al. (2006) established an in vitro propagation of Bacopa
monnieri. Direct multiple shoot regeneration coupled with rooting was obtained in MS
growth regulator free medium from leaf, internode and root explants. Leaf explants
showed superior response while root was the least responsive. The plantlets showed 100%
survival without only hardening step. The micropropagated plants resembled mother
plants in morphological characters.
An efficient protocol has been achieved for in vitro regeneration and
transformation of Echinops cv. through axillary bud and root segment cultures. Best
conditions for propagation were those using MS medium supplemented with 3% sucrose
and IAA (1.0mg/l) + BA (0.5mg/l) under 16 hrs of cool fluorescent light. Root cuttings
produced shoots all along the root but each growth regulator combination affected where
roots would sprout shoots. Rooted plantlets were successfully acclimatized after
propagation. Agrobacterium mediated transformation of Echinops was successfully
achieved and expression of GUS reporter gene was tested under the control of two
constitutive promoters (Dalia et al., 2006).
Shasany et al. (2006) have reported an efficient in vitro multiple shoot regeneration
from internode explants of Mentha piperita. Internodal explants were cultured on MS
medium supplemented with NAA (0.53μM) and BAP (4.43μM) and the best response in
terms of shoot regeneration and biomass increase was observed. An average of 74.4 shoots
per explant was scored on medium after a period of 12 weeks. In vitro multiplication of
Amomum microstephanum Baker (Thoyajaksha and Ray, 2006) was effected by culturing
small and active rhizome buds on MS medium supplemented with 2.0mg/l BA and 3%
sucrose. Highest multiplication of well developed plantlets (14 shoots/bud) was obtained
on this medium containing 1.0mg/l IAA and 3% sucrose per liter. In vitro derived plants
performed well under poly house conditions and were morphologically identical to the
mother plant.
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Sharma et al. (2006) developed a rapid clonal propagation system for medicinally
potent tree species Vitex negundo L. through in vitro culture of mature nodal explants.
Maximum number of (8.5) shoots were developed on MS medium fortified with 6.0mg/l
BAP. Shoots elongated after subculturing shoots on MS medium augmented with GA3
(2.0mg/l). In vitro raised elongated shoots were excised and transferred on half strength
MS medium fortified with 0.5mg/l IBA. Rooted shoots were successfully acclimatized in
pots containing vermicompost and sterilized soil (1:3) and after 15 days, these plantlets
were finally transferred to environment and had 75% survival rate.
A reproducible and feasible protocol was achieved for Arachnis labrosa using
aerial roots from in vitro source. Better morphogenetic response was recorded from
explants cultured in slanted condition (45º) on MS medium containing sucrose (3%w/v)
and IAA (4.0μM) + KN (6.0μM) in combination where about 80% of the explants
responded positively. The protocorm like bodies and shoot buds differentiated into rooted
plantlets on regeneration medium containing NAA (10μM) and BA (8.0μM) in
combination where 10 - 12 shoots/bud developed in each passage of four weeks interval.
Well rooted hardened plantlets could be obtained for transferring to the potting mix after
25 weeks of culture initiation (Ranjandeb and Temjensangba, 2006).
Gopi et al. (2006) developed a rapid system for regeneration of the important
medicinal plant of Ocimum gratissimum from nodal explants. Single node explants were
inoculated on MS basal medium containing 3% (w/v) sucrose, supplemented with
different concentrations and combinations of BAP, KN, IAA or IBA for direct plant
regeneration. Maximum number of shoots (14.3 ± 1.5) were observed on the medium
containing 0.5mg/l BAP and 0.25mg/l IAA after four weeks of culture. Regenerated
shoots were separated and rooted on half strength MS medium supplemented with 0.5mg/l
of IAA alone for three weeks. Normally, other species like O. basilicum showed positive
response towards plant regeneration in MS medium in the presence of BAP combined with
auxins as reported by various authors (Dode et al., 2003; Begun et al., 2002 and Phippen
and Simon, 2000).
An efficient protocol for rapid in vitro propagation through multiple shoot
formation from cotyledonary node explants were evolved for Capsicum annuum. Multiple
shoots were obtained, when frequently subcultured on MS medium augmented with
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1.5μM TDZ and 0.5μM IAA. The elongated shoots were rooted best on MS medium
containing 1.0μM NAA. Ex vitro rootings were achieved when basal cut ends of the
in vitro regenerated shoots dipped with 200μM IBA for half an hour. Of all the
micropropagated plants, about 95% survived after hardening following transfer to soil
(Siddique and Anis, 2006).
Premkumar et al. (2007) have reported an efficient direct regeneration from
immature embryo axis explants of Pigeonpea (Cajanus cajan). Immature embryo axis
explants displayed 70% frequency of shoot bud induction and 16.4 shoots, when cultured
in MS medium supplemented with 1.5mg/l BAP. Addition of 50.0mg/l L-cysteine along
with 1.5mg/l BAP on the MS medium enhanced the induction of shoot bud and
development of multiple shoots. Shoot elongation was achieved, when microshoots were
transferred on MS medium supplemented with 0.3mg/l GA3. Elongated shoots rooted,
when cultured in half MS medium fortified with 1.0mg/l IBA. Well developed plantlets
were acclimatized carefully and transferred to the field with 75 - 80% survival rate.
Clonal propagation was carried out by Luna et al. (2007) using juvenile shoot
cuttings of Melia composita. Effect of IBA (3000ppm) gave the maximum sprouting,
rooting, number of leaves, roots, shoot length and root length from the 9.0 - 12 mm
diameter juvenile shoot cuttings. Varadarajan et al. (2007) designed a biopreserver method
for in vitro culture of Solanum trilobatum L. using nodal explants. High frequency of
multiple shoots were noticed in cultures incubated in biopreserver when cultures incubated
normal incubation room conditions. Maximum number of shoots were formed in MS
medium with 2.0 – 3.0 mg/l of BAP.
A clonal propagation system has been standardized by Debnath et al. (2007) for
Chlorophytum borvillianum Sant. Maximum callusing (100%) was obtained from young
shoot buds and short segments of inflorescence axis bearing flower buds grown on MS
medium supplemented with a combination of 2.0mg/l 2,4-D and 0.2mg/l BA. The calli,
when subcultured on MS medium supplemented with 4.0mg/l BA showed multiple shoot
proliferation. Maximum shoot multiplication was observed after 60 days of the second
subculture on MS medium containing 5.0mg/l BA. Shoots were rooted on MS medium
containing 2.0mg/l IBA. The plantlets were transferred to the field after acclimation and
showed 60% survival.
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Datta et al. (2007) achieved an in vitro clonal propagation of Jatropha curcas
through nodal explants. Shoot bud formation was optimum on MS medium supplemented
with 22.2µM BA + 55.6µM AdS, in which culture produced 6.2 shoots/node. The rate of
multiplication (30.8 shoots/node) was significantly enhanced on MS medium
supplemented with 2.3µM KN, 0.5µM IBA and 27.8µM AdS. About 52% of root
induction occurred in MS basal medium supplemented with 1.0µM IBA. The plantlets
were successfully acclimatized in soil with 87% survival frequency.
An efficient protocol was developed for mass propagation of Curcuma longa L.
(Sudhersan, 2007). Multiple shoots were induced in MS medium with 1.0 - 5.0mg/l BA.
Microrhizomes were induced in MS medium with 3% sucrose under light or total dark
culture conditions. Root initiation and plantlet growth of microshoots and microrhizomes
were readily achieved on growth hormone free MS media with 3% sucrose.
Micropropagated plantlets and microrhizomes were acclimatized and successfully grown
in the green house. An efficient and rapid plant regeneration system via., direct
organogenesis was established by Bouhouche and Ksiksi (2007) for Teucrium
stocksianum. Hypocotyl explants excised from seedlings germinated in vitro were cultured
on MS medium supplemented with different concentrations of KN and IAA to induce
shoot formation. Optimal regeneration was achieved on medium containing 3.0mg/l KN
and 0.5mg/l IAA. Root induction was achieved on half strength MS medium containing
IBA. Rooted plantlets were successfully acclimatized, with a survival rate of 75 – 80%.
An in vitro propagation system for Artemisia vulgaris which is a traditional
medicinal plant has been developed by Sujatha and Ranjithakumari (2007). The best
organogenic response, including adventitious shoot number and elongation was obtained
when hypocotyl segments were cultured onto MS medium supplemented with 4.54µM
TDZ. Regenerated shoots formed roots when subcultured onto a medium containing
8.56µM IAA. Healthy plantlets were transferred to garden soil, farmyard soil and sand in
the ratio of 2:1:1 to mixture for acclimatization, which was successful and subsequent
maturity was achieved under greenhouse conditions over a six-month period.
Shirin and Rana (2007) carried out nodal segments from field grown culms as
explants to develop a method of in vitro plantlet regeneration in Bambusa glaucescens
through axillary bud proliferation. A synergistic effect of the two cytokinins was observed
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and the best interaction giving the highest rate of shoot multiplication was obtained for a
combination of 5.0µM BA and 15µM KN. The MS medium supplemented with 25µM
IBA was the most suitable for rooting of shoots. Saxena (1990) and Das and Pal (2005)
recommended the use of KN along with BA in the medium in order to get an enhanced
rate of shoot multiplication through forced axillary branching in Bambusa tulda and
B. balcooa, respectively. A synergistic effect of 15µM BA and 15µM KN combination
was also reported by Shirin et al. (2003), which resulted in a high rate of shoot
multiplication in Bambusa vulgaris from axillary buds of mature culms.
An efficient protocol is described for rapid in vitro multiplication of the vulnerable
medicinal herb Drosera indica by enhanced axillary bud proliferation from shoot tips as
explants. Multiple shoot production was independent of different strengths of MS, various
percentages of sucrose and also when pH was altered. Although the maximum number of
multiple shoots developed on MS medium supplemented with Zn (0.5mg/l) and KN
(0.5mg/l), respectively, which clearly depicts that there is not much difference
comparatively with a variation in hormone concentration in case of Zn. High cytokinin
concentrations resulted in retardation of shoot growth. Rooting was best achieved on MS
basal medium which has been reported by Jayaram and Prasad (2007). Nalini and Murali
(2002) in D. indica and Kawiak et al. (2003); Kim and Jang (2004) in D. rotundifolia,
D. ramentacea and D. peltata stated that higher concentrations of BA or NAA are not
desirable and generally induce a red pigmentation and necrosis.
Unda et al. (2007) regenerated plantlets through direct organogenesis from leaf
explants of Exacum sp. Four genotypes were evaluated on MS media supplemented with
combinations of BA and NAA for direct shoot organogenesis without an intervening
callus phase. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per
explant across media (10.2 and 6.6, respectively) while the 4.44µM BA + 0.54µM NAA
treatment induced the greatest number of shoots among the genotypes evaluated. The
advantage of using adenine based cytokinins were reported on other herbaceous plants,
including Dianthus caryophyllus (Messeguer et al., 1993), Gerbera jamesonii
(Nongmanee and Kanchanapoom, 1995), Gloxinia perennis (Wutisit and Kanchanapoom,
1995) and Saintpaulia ionantha (Sunpui and Kanchanapoom, 2002). Reports of auxin and
cytokinin combinations supporting organogenic differentiation have been well
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documented for several species (Lisowska and Wysokinska, 2000). These were produced
in higher concentrations of BA combined with lower concentrations of NAA and resulted
in greater numbers and higher quality shoots.
An efficient in vitro propagation protocol for Dendrobium candidum using
transverse thin cell layer (tTCL) culture system was established by Zhao et al. (2007). The
frequency of shoot regeneration and the number of adventitious buds produced from the
regenerated shoots on MS half strength macronutrients and 2% sucrose, supplemented
with 1.2mg/l NAA and 1.2mg/l BA. Upon this medium, the youngest explant inoculated in
the upright orientation exhibited a high frequency of shoot regeneration (92%) and the
highest number of adventitious buds (24.5) per explant. Rooting of shoots and adventitious
buds were achieved on MS medium with half strength macronutrients and 2% sucrose
with 1.0mg/l NAA and 1.0mg/l IAA. The BA has been reported to be effective in
regeneration of a number of orchid species, such as Dendrobium nobile and Cymbidium
aloifolium (Nayak et al., 2002) and a combination of BA with an auxin such as NAA was
more effective in regeneration of Dendrobium firmbriatum via., PLBs (Roy and Banerjee,
2003).
Tejavathi and Sharadamma (2007) analyzed morphogenetic potential of shoot tip
and nodal explants of Antirrhinum majus cvs. by culturing on MS medium supplemented
with PGR. MS + IBA (9.8μM) + BAP (8.87μM) for shoot tip cultures in cv. Rocket
golden and MS + BAP (8.87μM) or 1/2 MS + IAA (5.71μM) in cv. Rocket redtone and
MS + IAA (28.54μM) or KN (4.65μM) in cv. Rocket golden for nodal cultures were found
to be best suited for multiple shoot induction. Tewary et al. (2008) obtained an in vitro
plant regeneration from old seedlings cotyledon and hypocotyl segments of mulberry. MS
medium + BAP (2.0mg/l) with NAA (0.1mg/l) gave rise to 30 - 40 shoots per explant and
subsequent subculturing of regenerating shoots in liquid MS medium + BAP (2.0mg/l) +
NAA (0.5mg/l) induced 40 - 70 shoot buds per explant. Shoots were excised and cultured
on MS + NAA (1.0mg/l) for root induction.
Formation of multiple shoots occurred when leaf segments were cultured on MS
medium fortified with BAP 2.0mg/l and NAA at 3.0mg/l and with increase in the level of
BAP 2.0 – 3.0mg/l to enhance multiple shoots. Among the concentration of coconut water
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15% (v/v) along with (0.5mg/l) BAP proved to be ideal for multiple shoot induction. MS
medium fortified with 1.0mg/l BAP or 2.0mg/l L-glutamic acid also induced shoot buds
on leaf segments of Coccinia indica (Venkateshwarlu, 2008).
Ghnaya et al. (2008) described a procedure that allows for the easy and rapid
induction of caulogenesis in four cultivars of Brassica napus L. from transverse Thin Cell
Layers (tTCLs). The tTCL explants were excised from hypocotyl and petiole of two weeks
old seedlings and cultured on a solid basal MS medium supplemented with NAA
(0.1 – 0.4mg/l), BAP (1.0 – 4.0mg/l) and sucrose (20 – 40g/l). The comparison between
the regeneration ability of different explants showed that the hypocotyls exhibited a high
rate of shoot organogenesis when they were cultured on MS medium supplemented with
3.0mg/l BAP, 0.3mg/l NAA and 30g/l sucrose. Tang et al. (2003) showed that the PGR
content and the sucrose concentration affected significantly the regeneration process from
traditional explants. Silva (2003) provided evidence of the capacity to efficiently produce
non chimeric transgenic plants using similar methods.
A high frequency in vitro shoot bud differentiation and multiple shoot production
protocol from hypocotyl of cotton has been reported by Divya et al. (2008). Murashige
and Skoog basal medium with Nitsch and Nitsch vitamins was found to be optimal in
shoot regeneration. A combination of 2.0mg/l thidiazuron and 0.05mg/l NAA was the
most effective for shoot regeneration (76%) and an average of 10.6 shoots per explant.
Optimal rooting was obtained on half strength MS medium supplemented with 1.0mg/l
IBA and activated charcoal. The induction of roots on shoots obtained from TDZ
containing media was delayed and they did not elongate further as reported by Ouma et al.
(2004). Lower concentrations of TDZ yielded more shoot primordia compared to higher
concentrations, and resulted in hyperhydricity of shoots associated with water soaked
callus. Ouma et al. (2004) reported that TDZ concentration higher than 0.5mg/l resulted in
callusing in contrast Divya et al. (2008), which noticed TDZ concentrations up to 2.0mg/l
appeared to be optimal and did not induce significant amounts of callus. To counteract
this, silver nitrate, an anti-ethylene compound, was used to enhance regeneration in many
recalcitrant species including cotton. Silver nitrate incorporation was beneficial in
increasing regeneration response by lowering hyperhydricity, as reported in sunflower
(Mayor et al., 2003) and potato (Turhan, 2004) and resulted in better quality shoots.
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A regeneration system was developed by Liu and Pijut (2008) for Prunus serotina
from a juvenile (F) and two mature genotypes (3 and 4). Adventitious shoots regenerated
from leaves of in vitro cultures on woody plant medium with thidiazuron, NAA. The best
regeneration for genotype F (91.4%) was observed on medium with 9.08µM TDZ and
1.07µM NAA. The highest mean number of shoots (8.2) was obtained on medium
containing 9.08µM TDZ and 0.54µM NAA. Hammatt and Grant (1998) and Espinosa
et al. (2006) reported that Prunus serotina can be regenerated from juvenile source leaf
explants by using TDZ and NAA with a regeneration efficiency of 62% and 38.3%
depending on the study.
Adventitious shoot induction and plant development in Paulownia tomentosa
explants were derived from mature trees. Optimal shoot regeneration was obtained in leaf
explants when cultured on induction medium containing TDZ (22.7 or 27.3µM) in
combination with 2.9µM IAA for 2 weeks and subsequent culture in TDZ-free shoot
development medium including 0.44µM BA for a further 4-week period. The addition of
IAA to the TDZ induction medium enhanced the shoot forming capacity of explants. The
highest regeneration potential (85 – 87%) and shoot numbers (upto 17.6 shoots/explant)
were obtained in leaf explants harvested from the most apical node exhibiting unfolded
leaves. An analogous trend was also observed in intact petiole explants, although shoot
regeneration ability was considerably lower, with values ranging from 15% for petioles
isolated from node. Rooting frequency was significantly increased up to 90% by a 7 days
treatment with 0.5µM IBA, regardless of the previous culture period in shoot development
medium (Corredoira et al., 2008).
A successful protocol for multiple shoot induction of Curculigo orchioides was
evolved using shoot tips and rhizome discs. Proximal rhizome discs were optimal for high
frequency shoot bud formation than shoot tips and distal rhizome discs. Synergistic effect
of BAP (1.0mg/l) and KN (1.0mg/l) was evident on the regeneration of shoot buds from
proximal rhizome disc than shoot tip explant. Optimum root induction was achieved on
half MS liquid medium supplemented with 1.0mg/l of IBA. In vitro raised plantlets were
acclimatized in green house and transferred to natural condition with 90% survival
(Nagesh, 2008). Chaudhuri et al. (2008) formulated a reproducible protocol for the rapid
propagation and conservation of Swertia chirata using leaves taken from in vitro shoot
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cultures. Direct induction of more than seven shoot buds per explant was achieved for the
first time when the explants were placed on MS medium supplemented with 2.22µM BA,
11.6µM KN and 0.5µM NAA. Direct organogenesis was noted exclusively from the
adaxial surface of the basal segments of leaves. Plants raised through direct organogenesis
were evaluated for their clonal fidelity by chromosomal analysis and DNA fingerprinting.
An efficient and rapid method for in vitro clonal propagation of Huernia hystrix
was developed by Amoo et al. (2009), resulting in shoot regeneration within 3 weeks of
culture. A 100% shoot response with a multiplication rate of four shoots per explant was
obtained on MS medium containing 5.37µM NAA and 22.19µM BA. Callus produced at
the base of the explant on the same medium showed root organogenic potential. The
in vitro regenerated shoots produced roots when transferred to half strength MS medium
with or without auxin.
Feng et al. (2009) reported an efficient regeneration system for a dwarf dogwood
species Cornus canadensis through organogenesis from rejuvenated leaves, and
characterize the development of the plantlets. Micropropogated shoots were quickly
induced from axillary buds of node on an induction medium consisting of basal MS
medium supplemented with 4.44µM BAP and 0.54µM NAA. The new leaves of
adventitious shoots were used as explants to induce calli on the same induction medium.
Nearly 65% of leaf explants produced calli, 80% of which formed adventitious buds.
Gibberellic acid (1.45µM) added to the same induction medium efficiently promoted
quick elongation of most adventitious buds and 0.49µM IBA added to the basal MS
medium promoted root formation from nearly 50% of the elongated shoots. GA3 is widely
used to promote elongation of adventitious shoots in plant regeneration, e.g., for in vitro
shoot proliferation of Cassava (Bhagwat et al., 1996) and regeneration of Acacia mangium
via,. organogenesis (Xie and Hong, 2001).
Bhuvaneswari et al. (2009) determined a simple and efficient protocol for Ocimum
sanctum through in vitro techniques. The treatment of BAP (2.5mg/l) showed the best
response and produced an average of 12 shoots per shoot tip explant with 84% of
response. Nodal explant responded at the concentration of 2,4-D + BAP (1.0 + 1.0mg/l)
produced maximum result of 74% responses. IAA alone was effective for induction of
roots (12 mm).
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An efficient in vitro plant regeneration system has been developed for rapid
propagation of shoot buds from leaf explants of Stevia rebaudiana. Best response in terms
of multiple shoot bud formation was obtained on MS medium with 0.5mg/l BAP +
5.0mg/l KN + 5.0mg/l IAA. The maximum number of roots were induced in half strength
medium containing 0.5mg/l IBA. The in vitro plantlets were successfully acclimatized and
transferred to the field (Saravanakumar et al., 2009).
An efficient regeneration protocol were developed from the leaves of an 11-year-
old Phtinia x fraseri ‘‘Red Robin’’ tree. A high frequency of adventitious buds and the
highest maximum mean number of adventitious buds per explant were obtained in light
conditions on MS medium containing 2.0mg/l BAP and 0.2mg/l NAA. After preculturing
for 6 days, over 95% of the shoots successfully rooted on ½ MS medium supplemented
with 0.3mg/l IBA within 3 weeks (Zhu and Wei, 2010).
A protocol for plantlet regeneration through shoot formation were developed for
the neotropical shrub Brunfelsia calycina (Liberman et al., 2010). Explants from young
and mature leaves were incubated on MS medium with various combinations of IAA and
BA. Shoot emergence was best at 4.44µM BA and 2.85µM IAA for young leaf explants,
and at 8.88µM BA, 2.85µM IAA for mature leaf explants. When shoots were transferred
to MS medium supplemented with 1.23 - 2.46µM IBA, they developed roots.
An efficient protocol for high frequency in vitro regeneration of multiple shoots
and somatic embryos from the embryonic axis of common bean (Phaseolus vulgaris) were
developed by. Kwapata et al. (2010). Olathe pinto bean performed the best producing over
20 multiple shoots per explant while cv. Condor black bean was the poorest with nine
multiple shoots per explant. The optimum media for regeneration of multiple shoots was
4.4mg/l MS containing 2.5mg/l BA and 0.1mg/l IAA supplemented with 30mg/l silver
nitrate. Adventitious shoots and somatic embryos were regenerated on MS medium
containing 1.0mg/l TDZ and 0.05mg/l NAA supplemented with 30mg/l silver nitrate or
activated charcoal. Efficient and effective rooting of plantlets was achieved by dipping the
cut end base of in vitro regenerated shoots in 1.0mg/l IBA solution and culturing on media
containing 4.4mg/l MS supplemented by 0.1mg/l IAA, NAA or IBA.
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An efficient and reproducible procedure were described by Ghimire et al. (2010)
for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on
MS medium supplemented with NAA and BAP. The highest mean number of shoots per
explant (10.65 ± 1.03) were recorded on MS medium containing 3% sucrose and 0.8%
agar supplemented with 0.1mg/l NAA and 1.0mg/l BAP. The plantlets that regenerated
from the basal parts the leaf explants were rooted successively on MS medium alone or in
combination with IBA. The highest mean number of root organogenesis, with 25.67 ± 3.68
roots per leaf segment, were obtained in the presence of 1.0mg/l IBA.
3.3. MATERIALS AND METHODS
3.3.1. Source of Explants
Both in vitro derived and in vivo field grown explants were used as the source of
explants. Non meristematic explants like the first fully expanded and second leaf over
1.0 cm long from apical buds and petiole, internode, root and inflorescence rachis explants
were made into 0.5 - 0.8 cm long segments, the stem segments were transversely sliced
into pieces of about 0.5 mm in thickness and the slices from stem were used as transverse
thin cell layer (tTCLs) explants for regeneration in the present investigation.
3.3.2. Sterilization of Explants
All the explants were excised with sterile blade and collected in a beaker. The
excised explants were thoroughly washed with running tap water for 10 - 15 minutes.
Thereafter, the explants were washed with detergent (Teepol 5% v/v) solution for
3 minutes, fungicide (Bavistine 2% w/v) treatment for 2 minutes then soaked in 70% (v/v)
ethanol for 30 seconds and finally disinfected with 0.1% (w/v) HgCl2 for 2 minutes and
rinsed with sterile distilled water five times.
3.3.3. Inoculation of Explants
The laminar air flow chamber was sterilized with 70% ethanol and by
UV-irradiation for 15 minutes. The leaf explants were inoculated in such a way that either
the adaxial or abaxial surface was touching the agar stands of culture tubes and petiole,
internode, root and inflorescence stem segments were placed both in vertical or horizontal
position, while transverse thin cell layers (tTCLs) of stem segments were cultured on the
medium containing different concentrations with combination of growth regulators. By
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means of a long stainless steel forceps, one explant per tube was placed. It was routine
process to flame the mouth of the test tube after uncapping and before recapping the tubes
to reduce contamination. To facilitate planting, two forceps were used alternatively to
allow adequate time to cool, furthermore, to prevent burning the fingers and explants.
Each treatment consisted of 7 explants and the experiments were repeated five times.
3.3.4. Culture Condition
All the cultures were maintained at 25 ± 2˚C under 16/8 hrs light/dark condition of
80µEms-2s-1 irradiance provided by fluorescent lamps (TL 40W/54 cool-day light).
In vitro response of inoculated explants was assessed every week in culture by counting
the proliferated shoots which attained 2.0 cm in length and above. The subsequent
subculture was made only on the medium containing maximum shoot proliferation rate.
3.3.5. Shoot Proliferation and Multiplication
Based on the preliminary experiments with both BAP, KN and TDZ only the latter
was selected for shoot bud regeneration. Different explants were cultured on MS basal
medium containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations BAP,
KN, and TDZ (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/l) alone or in combination with auxins
(0.1 - 2.5 mg/l) were used for shoot proliferation. After two weeks the clumps of shoots
were subcultured on MS medium containing 3% (w/v) sucrose and 0.8% agar (w/v) with
suitable growth regulators for multiplication and maturation of the shoots. Activated
charcoal (0.5 - 3.0%) and coconut water (10 - 15%) were added to promote the production
maximum shoots and to prevent microbial contamination.
3.3.6. Shoot Elongation
Proliferated multiple shoots were divided into small clusters of 2 - 3 shoots. They
were subcultured on shoot elongation medium containing GA3/KN (0.2 - 1.2mg/l) alone or
in combination with cytokinins like BAP/KN (0.1 - 2.5mg/l) or NAA (0.1 - 2.5mg/l). The
cultures were incubated at 25 ± 2˚C under 16/8 hrs light/dark photoperiod. After two
weeks, shoots longer then 3.0 cm were counted and transferred to rooting medium.
3.3.7. Root Induction and Transplantation
The longer shoots (3cm length) were excised and transferred to MS basal medium
containing 3% (w/v) sucrose, 0.8% (w/v) agar and different concentrations of IBA and
IAA (0.1 - 2.5mg/l) alone or in combination of cytokinins such as IBA + KN, IBA + BAP
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and IAA + KN (1.5 + 0.1 - 2.5mg/l) for root induction. Rooting was observed from 15 to
20 days. Plantlets with well developed roots were removed from the culture tubes and after
washing their roots in running tap water, they were grown in the mixture of river sand
garden soil and saw dust in the ratio of 1:1:1 in paper cups for a month and subsequently
transferred to pots. Potted plants were covered with transparent polythene membrane to
high humidity and watered every three days with half strength MS salts solution free of
sucrose for two weeks.
3.3.8. Acclimatization and Hardening of Regenerants
As described in chapter -2
3.3.9. Statistical Analysis
3.3.9.1. Observation of Culture and Presentation of Results
The cultures were examined periodically and the morphological changes were
recorded on the basis of visual observation. Whenever possible the effects of different
treatments were quantified on the basis of percentage of cultures showing the response per
culture. The experimental design was Completely Randomized Design (CRD) and
factorial with auxin and cytokinin as independent variables. Each treatment consisted of at
least 7 explants and all the experiments were repeated five times. The data pertaining to
frequency of shoot proliferation and multiplication, shoot elongation and root induction
cultures were subjected to standard deviation. Mean separation was conducted by using
Duncan’s new Multiple Range Test (DMRT) and means were compared with P < 0.05 at
level of significance.
3.3.9.2. Calculation
The experimental results were calculated as follows:
No. of shoots proliferated
Frequency of shoot proliferation : ----------------------------------------- x100
No. of explants cultured
No. of shoots rooted
Frequency of root induction : ---------------------------------------- x100
No. of shoots cultured
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3.4. RESULTS
3.4.1. Shoot Proliferation and Multiplication
Among the different explants like non-meristem containing leaf, internode,
inflorescence rachis, petiole, root and transversely sliced thin cell layers (tTCLs) of stem
explants were tested. Even different concentrations and combinations of growth regulators
were used to define an efficient regeneration medium. Direct bud formation was observed
in both non meristem containing explants, cultured on MS medium supplemented with
appropriate plant growth regulators. The earliest visible signs of growth were seen after 2
weeks of inoculation in the form of swelling at one or both the cut ends of the explants on
MS fortified medium, as this study that would easily and readily promote direct
organogenesis in Plectranthus barbatus. The treatment if failed to induce regeneration
within 30 days, they were unsuitable and their regeneration potential was not pursued
further.
The combinations that were used in the subsequent cultures were those that elicited
adventitious shoots 20 days from culture initiation and are presented in appropriate Tables.
The highest percentage of culture response and optimal number of shoots from different
explants of direct organogenesis is given in Fig. – 3.1. All the explants with best response
in terms of shoot proliferation and biomass increase was observed in the MS medium
containing KN (1.5mg/l) followed by combination of NAA (1.0mg/l). BAP (2.0mg/l) and
KN (1.5mg/l) resulted in optimum level of responding culture and shoot numbers per
explant. The concentration of BAP and TDZ alone performed the poorest in both
regeneration percentage and number of shoots per explant when compared to KN alone or
in BAP + NAA and KN + NAA combinations. In all the six explants (non meristem
containing tissue) that were tested, leaf showed highest number of (19.7) shoot at 1.5mg/l
KN followed by internode (13.5), inflorescence rachis (11.5) and petiole (11.2) showed
optimum number of shoots in same media compositions. Yet comparatively low number
of (10.32 and 9.32) shoots were observed in both transverse TCL section of stem and root,
respectively and were observed in KN (1.5mg/l) fortified medium. Following main steps
in regeneration of plants through direct organogenesis is given in Fig. – 3.2.
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3.4.1.1. Leaf Explant
Leaf explants grown on media supplemented with different concentrations of KN,
BAB and TDZ alone or in combination with auxin (NAA) initially responded with the
enlargement and swelling of the leaf tissue. They started differentiating multiple shoot
buds within 2 weeks of inoculation, especially at the petiolar ends, where bud initials
began to appear, produced through the leaf tissue. Following transfer of original explants
to shoot development as same medium allowed further development and multiplication.
Among the various concentrations of cytokinins like KN, BAP and TDZ (0.5 – 3.0mg/l)
were tested for the leaf segments, the highest adventitious shoot regeneration percentage
(80%) was observed on MS medium at 1.5mg/l KN and average maximum number of 19.7
shoots per explant, followed by BAP 2.0mg/l and this was 60 % response with 9.11 shoots
per explant, and TDZ (1.5mg/l) showed less frequency (57.1%) and minimum number of
7.89 shoots per explant compared to other cytokinins (Table - 3.1). KN was the most
important factor for adventitious shoot regeneration, as no adventitious shoot developed
on explants exposed to media without KN. Higher concentrations (above 3.0mg/l) of both
BAP and KN alone produced green compact and brown friable callus, respectively. Lower
concentrations of cytokinins had no adventitious shoot formation, however only the
optimum concentration showed adventitious shoots from wounding points of leaf explants,
after 2 - 3 weeks. The media containing other growth regulators such as IAA, NAA and
2,4-D did not induce shoot, otherwise they produced either callogenesis or rhizogenesis.
The combinations of BAP (2.0mg/l) or KN (1.5mg/l) with NAA (1.0mg/l) improved the
shoot proliferation rate significantly (15.05 and 11.63 shoots, respectively) compared to
BAP and TDZ alone, but shoot height and internodal length was reduced especially at high
NAA concentration (Table - 3.1) (Plate - 3.1).
3.4.1.2. Internode Explant
Surface disinfected internode explants were cut into small pieces and cultured on
MS medium. Internode explants failed to respond morphogenetically in hormone free MS
medium. The addition of cytokinins (KN, BAP and TDZ) alone or in combination with
auxin (NAA) to the medium was essential to induce adventitious multiple shoots from the
internodal explants. Two fold increase in its size and swelling of explants from the cut
ends were observed within 15 days.
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Multiple shoots developed directly from the cut ends of internode explants.
Multiple shoots were obtained without intervening callus phase, when the internode
segments were cultured on MS medium fortified with KN, BAP and TDZ alone or in
combination with NAA in different concentrations. Maximum number of 13.58 shoots
emerged out from single explant within 15 days of inoculation on MS medium
supplemented with KN (1.5mg/l) and 77.1% of shooting response was seen, this was
followed by 10.3 and 9.1 shoots per explant on MS medium with NAA (1.0mg/l) in
combination with BAP (2.0mg/l) and KN (1.5mg/l) with 71.4% and 62.9% of shooting
regeneration response, respectively. Both of BAP (2.0mg/l) and TDZ (1.5mg/l) alone
produced minimum of 7 shoots (each) per explant comparatively higher in KN (1.5mg/l)
concentrations. A higher and low hormone concentration of both BAP and TDZ (0.5 and
3.0mg/l) hormones produced few shoots, as well as this concentration also resulted in
callus formation. The inhibition of organogenic induction caused by higher concentration
of NAA (2.5mg/l) with cytokinins was observed on MS fortified medium. The shoots were
multiplied repeatedly subculturing the original internodal explants on shoot multiplication
medium (KN 1.5mg/l) after each harvesting of newly formed shoots. Activated Charcoal
(AC) enhanced the recovery of shoot primordia into multiplication and minimum or
without contaminations (Table - 3.2 and Plate - 3.2).
3.4.1.3. Inflorescence rachis Explant
Inflorescence rachis explants were cultured on PGR free MS medium and media
containing various concentrations of KN, BAP and TDZ individually for shoot
regeneration. The PGR free media did not respond well for shoot induction. Among the
different concentrations tested, KN (1.5mg/l) produced maximum number of 11.53 shoots
per explant with maximum frequency (74.3%) followed by BAP + NAA (2.0 + 1.0mg/l)
and KN + NAA (1.5 + 1.0mg/l) showed optimum of 8.8 and 8.7 shoots per explant,
respectively. On the other hand, in BAP and TDZ alone supplemented medium at 2.0mg/l
and 1.5mg/l induced 6.7 and 6.4 shoots per explant, respectively. However, in the study
they were able to induce more shoots in KN containing medium. KN is among the most
active of the cytokinin like substances and it induced greater in vitro shoot proliferation
than other cytokinins. A significant decrease in shoot number and shoot length was
observed upon increasing concentrations of both of KN, BAP and TDZ from the optimum
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level. A significant effect of BAP + NAA and KN + NAA interaction was also observed
on shoot multiplication (Table - 3.3). All the interactions between BAP + NAA and KN +
NAA significantly increased shoot proliferation than BAP (2.0mg/l) and TDZ (1.5mg/l)
alone. Increasing concentration of NAA (above optimal level) with KN (1.5mg/l) or BAP
(2.0mg/l) significantly reduced number of shoots and proliferation of the callus from
wounding part of the explant (Plate - 3.3).
3.4.1.4. Petiole Explant
The morphogenic responses of petiole explants to various cytokinins (KN, BAP
and TDZ) are summarized in Table - 3.4. Placing explants in a medium without growth
regulators failed to produce shoot multiplication. However, the multiplication rate and
shoot numbers were higher in cultures supplemented with plant growth regulators. The
percentage of response varied with the type of growth regulators used and its
concentrations or in combination. Various concentrations of KN, BAP and TDZ (0.5, 1.0,
1.5, 2.0, 2.5 and 3.0mg/l) alone facilitated shoot bud differentiation. Among the various
cytokinins tested, KN 1.5mg/l was found to be more efficient than others with respect to
initiation and subsequent proliferation of shoots. Maximum 11.2 shoots per explant were
observed on MS medium supplemented with KN (1.5mg/l) followed by TDZ (1.5mg/l)
7.0 shoots and BAP (2.0mg/l) 6.7 shoots per explant were observed. Upon lowering
concentration of each cytokinin, a reduction in number of shoots per culture was recorded.
Similarly, at a higher concentration (3.0mg/l) the number as well as the percentage of
response was drastically reduced. A callus occasionally formed at the base of the explant
at the higher concentration and subsequently retarding shoot bud formation. BAP with
NAA and KN with NAA were found to be most suitable combinations for shoot bud
regeneration and multiplication. Upon increasing the concentration of NAA (above
optimal level), a gradual decrease in regeneration frequency and number of shoots per
explant was recorded. The BAP (2.0mg/l) with NAA (1.0mg/l) and KN (1.5mg/l) with
NAA (1.0mg/l) combination was the optimal of 9.1 shoots per explant in both
combinations. The elevated concentration of NAA at 2.0, 2.5mg/l resulted in little
callusing at the cut ends, thus reducing the percent shoots regeneration and the number of
shoots per explant (Plate - 3.4).
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3.4.1.5. Root Explant
The regeneration of adventitious shoots of P. barbatus from root explants were
dependent on cytokinins alone or in combination with auxin being present in the medium.
Root explants inoculated into hormone free MS medium and MS media supplemented
with different concentrations of either cytokinins or in combination with auxin. Hormone
free MS medium did not show any organogenesis, callogenesis or rhizogenesis. The slow
response of root explants were due to the reorganized development of lateral meristem in
the root into shoot meristem. The root explant grown on MS medium supplemented with
different concentrations of KN, BAP and TDZ alone, initially responded with the
enlargement and swelling of root tissue. They started differentiating multiple shoot buds
within 20 days of inoculation, especially at the cut ends of roots, where bud initials began
to appear, protruding through the root explant. Following transfer of original explants to
same medium allowed further development into multiplication. Adventitious buds were
produced in all the concentrations of KN, BAP and TDZ and maximum number of
9.3 shoots per explant were produced at 1.5mg/l KN followed by TDZ (1.5mg/l) and BAP
(2.0mg/l) individually produced 7.0 and 6.2 shoots per explant, respectively. The
regeneration efficiency increased on increasing concentration at appropriate level and then
showed a decrease at increasing higher concentration. NAA individually had no
significant effect on shoot proliferation. An addition of NAA (1.0mg/l) with BAP
(2.0mg/l) or KN (1.5mg/l) enhanced higher shoot number of 8.8 and 7.4 shoots per explant
with higher frequency response of 68.6% and 57.1%, respectively (Table - 3.5). The
percentage of bud forming explants represents the response capability of the tissue to the
medium and the number of shoots per explant represents the capacity of the explants to
produce shoots (Plate - 3.5).
3.4.1.6. Transverse Thin Cell Layers (tTCLs) of stem Explant
The stem segments were transversely sliced into pieces of about 0.5 mm thickness
and the slices were used as tTCLs explants for plant regeneration. Transverse thin cell
layer segments were cultured on MS medium without any plant growth regulators,
remained green for 2 weeks and gradually turned brown and dried up later without shoot
formation. However, shoots could be successfully induced from tTCLs segments
inoculated on MS medium with different concentrations of either cytokinins or in
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combination with auxin (NAA). Among the different concentrations of cytokinins
(KN, BAP and TDZ - 0.5 to 3.0mg/l) were tested. A small green protuberance gradually
emerged on the brim of the explants within two weeks of culture containing MS medium
fortified with KN (1.5mg/l). An average of maximum 10.3 shoots per explant was
observed with maximum frequency (71.4%) response, followed by stable concentration of
KN (1.5mg/l) with different concentrations of NAA (0.1 - 2.5mg/l), KN (1.5mg/l) + NAA
(1.0mg/l) produced a maximum 8.7 shoots per explant with 68.6 percentage of response.
BAP (0.5 – 3.0mg/l) alone or in combination with NAA (0.1 – 2.5mg/l) was chosen for
promoting the shoot induction. As inoculation on MS medium supplemented with BAP
(2.0mg/l) alone and BAP (2.0mg/l) in combination with NAA (1.0mg/l), the explants
exhibited a higher frequency (68.6% and 62.9%) of shoot regeneration with 6.9 and 6.7
shoots per explant, respectively (Table - 3.6). Thidiazuron (TDZ) at 1.5mg/l produced 6.1
shoots with maximum of 54.3% response, comparatively less than other cytokinins.
Sometimes a small amount of light green callus proliferation was observed when in the
above increasing concentration at appropriate level of NAA (1.0mg/l). Non regenerating
explants could show either callus or browning and necrosis after 15 days of culture
(Plate - 3.6).
3.4.2. Shoot Elongation
For shoot elongation, 30 days old multiple shoot mass were transferred to half
strength MS medium containing of GA3/KN (0.2 - 1.2mg/l) alone or various combination
with different concentrations of KN, BAP and NAA (0.2 - 1.2mg/l). Among the various
combinations used, GA3 0.6mg/l + KN 0.5mg/l combination supported maximum shoot
length (8.1 cm length/shoot) and mean number of average node 7.84 with 15.2
leaves/explant within 20 days of culture (Plate- 3.1.f, 3.2.g, 3.3.e, 3.4.g and 3.5.e). When
increasing the concentration of GA3 the shoot length also increased up to optimum level
(0.6mg/l), afterwards it was decreased with further increase. The optimum concentration
of GA3 (0.6mg/l) + BAP (1.0mg/l) or NAA 0.5mg/l also proved best shoot elongation and
increasing BAP (1.0mg/l) and NAA (0.5mg/l) enhanced the number of multiple shoots or
basal callusing. Optimum concentration of KN at 0.6mg/l alone also evaluated shoot
elongation, further increased hormone concentration to regenerate multiple shoots
(Table - 3.7).
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3.4.3. Rooting of in vitro shoots
Well developed shoots (above 3 cm) were excised and grown in half MS medium
supplemented with either IBA or IAA (0.1 - 2.5mg/l) in combination with KN/BAP
(0.1 -2.5mg/l). The 15 days of culture in rooting medium resulted in maximum percentage
of rooting and 4 to 6 days of dark treatment stimulated maximum rooting in IBA alone or
IBA + KN supplemented medium. The percentage of rooting increased with the increasing
concentrations of IBA/IAA up to 1.5mg/l and it decreased with further increase of the
above. The hormone concentration with respect to percentage of rooting per shoot and
nature of roots were controlled by auxins and their concentrations.
a. Effect of IBA
Shoots were subcultured in half strength MS medium with different concentrations
of IBA (0.1 - 2.5mg/l) individually for rooting. Among the various concentration used,
IBA at 1.5mg/l was found to yield higher percentage of rooting in in vitro derived shoots.
In vitro shoots produced, the higher frequency of rooting (85.7%) response with 20.1
roots/shoot and an average of maximum root length (8.95 cm) were observed within
15 - 20 days of culture (Plate- 3.2.h, 3.4.h and 3.6.g) (Table - 3.8).
b. Effect of IAA
In vitro raised shoots were transferred to half strength MS medium fortified with
various concentrations of IAA (0.1 - 2.5mg/l) for rooting. Among the various
concentrations of IAA used, 1.5mg/l of IAA was found to be the most suited for maximum
rooting response. The maximum (68.6%) frequency of rooting was noticed, when the
shoots were cultured in half MS medium fortified with optimum level of IAA (1.5mg/l)
and produced 13.32 roots with 5.95 cm root length/shoot.
c. Effect of Auxins with Cytokinins
The optimum concentration of IBA/IAA 1.5mg/l for rooting selected in the
previous experiment was tested in combination with various concentrations of KN/BAP
(0.1 - 2.5mg/l). The combination of IBA 1.5mg/l + KN 0.5mg/l showed maximum
percentage (80%) response with 17 roots and average of 8.58 cm root length, followed by
IBA (1.5mg/l) + BAP (0.5mg/l) showed 71.4% responsive culture, 13.9 roots with 7.11
cm root length. Less number of 7.84 roots with 4.89 cm root length was obtained, when
culturing on half MS medium containing with IAA (1.5mg/l) + KN (0.5mg/l) as shown in
the Table - 3.8.
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3.4.4. Acclimatization and Hardening
Rooted plantlets of 2 weeks old cultures were transferred to soil under shade for
in vitro hardening. The plantlets were taken out from the flasks, washed with sterile water
removed from agar/medium and transferred to paper cups containing river sand, garden
soil and saw dust in the ratio of 1:1:1. These plantlets were irrigated with half strength MS
medium twice for 20 days in controlled conditions and covered with polythene bags
(Plate - 3.1 to 3.6).
The plants were maintained under controlled temperature (25 ± 2˚C) for a week,
subsequently they were transferred in the nursery bags and successfully established to the
field. The regenerated plants did not show any detectable variation in morphology or
growth characteristics with the respective donor plants. The survival percentage was
90 - 95%.
3.5. DISCUSSION
In vitro techniques can facilitate molecular genetic manipulations. However, the
successful application of in vitro method is greatly dependent on a reliable regeneration
system (Koroch et al., 2002). The objective of the study reported here was to establish an
in vitro propagation method for Plectranthus barbatus for conservation purposes as wild
natural populations are becoming sparse. Among the six (leaf, internode, inflorescence
rachis, petiole, root and tTCLs of stem) explants used, all the explants were capable of
directly regenerating large number of plantlets in standard (Full strength) MS medium
containing cytokinins (KN, BAP and TDZ) and an auxin (NAA) with KN at 1.5mg/l being
maximum frequency of response and number of shoots in all the explants. There was good
shoot bud induction and proliferation response only in the presence of cytokinins and no
response in the basal medium. Similar results are well documented in several medicinal
plants in Catalpa ovata (Lisowska and Wysokinska, 2000), Ficus benghalensis (Rahman
et al., 2004a), Cicer arietinum (Sarker et al., 2005), Gossypium hirsutum (Rauf et al.,
2005), Costus speciosus (Raghu et al., 2006a), Artemisia vulgaris (Sujatha and
Ranjithakumari, 2007), Swertia chirata (Chaudhuri et al., 2008), Basilicum polystachyon
(Amutha et al., 2008), Paulownia tomentosa (Corredoira et al., 2008), Cornus canadensis
(Feng et al., 2009), Huernia hystrix (Amoo et al., 2009) and Phaseolus vulgaris (Kwapata
et al., 2010)
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In order to increase regeneration efficiency from leaf, internode, inflorescence
rachis, petiole, root and tTCLs of stem explants derived from in vitro or in vivo raised
plantlets were used in the present study. The use of in vitro raised plants were previously
reported in Piper colubrinum (Kelkar and Krishnamurthy, 1998), Tagetes erecta (Vanegas
et al., 2002) and Ophiorrhiza prostrata (Martin et al., 2008), for in vivo in Charybdis
numidica (Kongbangkerd et al., 2005), Enicostemma axillare (Jeyachandran et al., 2005),
Bacopa monnieri (George et al., 2006) and Paulownia tomentosa (Corredoira et al., 2008).
The present study was undertaken as an efficient method for developing
adventitious shoot bud regeneration from both of in vitro and in vivo raised explants of
Plectranthus barbatus. The observations showed the explants response with respect to
hormone and its combinations for shoot buds proliferation, multiplication, shoot
elongation, rooting and hardening process.
3.5.1. Shoot Proliferation and Multiplication
The present study demonstrated the suitable method for in vitro proliferation of
multiple shoots and complete plantlet, development for shoot bud proliferation and
multiplication, the hormone concentrations used individually or in combinations. By using
this method, multiple shoots have been induced directly from different explants like leaf,
internode, inflorescence rachis, petiole, root and tTCLs of stem. Among the cultures, leaf
explant produced maximum number of shoots followed by internode and inflorescence
rachis on MS medium containing KN, BAP and TDZ alone or in combination with NAA.
Other explants like petiole, root and tTCLs of stem proved moderate shoot bud
proliferation.
Two major properties of cytokinins useful in culture are stimulate of cell division
and release of lateral bud dormancy. The most commonly used cytokinins are BAP, KN
and TDZ. Cell division is regulated by the joint action of auxins and cytokinins, each of
which influence different phase of cell cycle. The variations in the regeneration potential
among explants are attributable to the differences in their physiological and genetic
makeup of cells. Auxins affect DNA replication, whereas cytokinins seems to exert some
control over the event of mitosis and cytokinesis. Thus auxin and cytokinin level in
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cultures need to be carefully balanced and controlled. The use of plant growth regulators
with high cytokinin activity, such as thidiazuron would be an alternative for the induction
of adventitious buds from mature material (Corredoira et al., 2008).
3.5.1.1. Leaf Explant
Among the cytokinins, KN is more effective for plant regeneration from leaf of
Thapsia garganica, when compared to BAP during the micropropagation of Fennel
(Makunga et al., 2005). Leaves showed direct multiple shoot formation first from the cut
end of the petiole within two weeks of inoculation as was reported by George et al. (2006)
and Kelkar and Krishnamurthy (1998). Morphogenetic potential of various explants of
family Lamiaceae was evaluated by Sudhakaran and Sivasankari (2003), Shasany et al.
(2006) and Makunga and Staden (2008). Plant regeneration from leaf explant was reported
in Pyrus communis (Caboni et al., 1999), Achras sapota (Purohit et al., 2004), Hagenia
abyssinica (Feyissa et al., 2005), Charybdis numidica (Kongbangkerd et al., 2005),
Enicostemma axillare (Jeyachandran et al., 2005), Solanum violaceum (Raghu et al.,
2006b), Exacum sp. (Unda et al., 2007), Saussurea involucrata (Guo et al., 2007), Swertia
chirata (Chaudhuri et al., 2008) and Prunus serotina (Liu and Pijut, 2008).
Among the cytokinins, TDZ proved to have the highest shoot regeneration
frequency and comparatively KN indicated poor response of shoot formation in cotyledon
as reported by Gua et al. (2005), Corredoira et al. (2008) and Liu and Pijut (2008). Zeatin
was the most effective cytokinin for shoot proliferation whereas the higher number of new
microshoots was obtained with 4.7mg/l zeatin (Grigoriadou et al., 2002). This is in
contrast, to the present study where maximum number of shoot bud proliferation and
multiplication was observed in MS medium supplemented with KN 1.5mg/l. Similar
results were obtained in Enicostemma hyssopifolium (Seetharam et al., 2002). NAA had
no significant effect on mean number of shoots as reported by Liu et al. (2008). Similar
results were also observed in present study.
A combination of NAA (1.0mg/l) with BAP (2.0mg/l) induced shoot proliferation.
Similar results were observed in leaf explant of Echinacea purpurea (Koroch et al., 2002)
and Charybdis numidica (Kongbengkerd et al., 2005). With increasing or decreasing
concentration of NAA from the level of 1.0mg/l, shoot induction was also dramatically
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decreased to 20%. So, at the range of NAA 1.0mg/l combined with BAP 2.0mg/l or KN
1.5mg/l was the best combination for shoot regeneration from the leaf explant. Similar
results were also reported in Brassica juncea (Gua et al., 2005), who reported that the
shoot regeneration frequency from leaf segments was also obviously enhanced in the
presence of NAA in all the cytokinins used, when 0.5mg/l NAA was added in the medium
combined with other cytokinin 1.0mg/l BAP and 0.5mg/l KN gave the highest shoot
regeneration potential. Thus, these are in accordance with the present investigation.
3.5.1.2. Internode Explant
Plant regeneration from internode explants were reported in Euphorbia tirucalli
(Uchida et al., 2004), Feronia limonia (Hiregoudar et al., 2005), Ophiorrhiza prostrata
(Martin et al., 2008) and Mentha piperita (Shasany et al., 2006). Stem explants failed to
respond morphogenetically in hormone free MS medium. Addition of a cytokinin alone or
in combination with auxin to the medium was essential to induce adventitious multiple
shoots from the stem explants as has been reported by Kannan et al. (2006). This study is
in accordance with the present reports, whereas multiple shoot developed directly from the
cutting point of the internode explants. Multiple shoots were obtained without intervening
callus phase on MS medium containing KN, BAP and TDZ (0.5 – 3.0mg/l) alone or in
combination with NAA. The regeneration frequency for internode explant gradually
increased with an increase in concentration, reaching its maximum at 1.5mg/l KN, 2.0mg/l
BAP and 1.5mg/l TDZ individually, then decreased with any further increasing
concentration.
The present study corroborates with the previous findings Amoo et al. (2009) and
Jayaram and Prasad (2007) as well as shoot multiplication rate generally increased with
increased BA at optimum level. With further increasing of concentration of BAP, shoot
number also decreased. In contrast, Noshad et al. (2009) have shown that in higher
concentration of BA, KN and TDZ alone showed higher number of shoots at lower
concentration there was minimum number of shoots. Its mode of action may be attributed
to its ability to induce cytokinin accumulation was reported by Sujatha and
Ranjithakumari (2007). Kumar et al. (1998a) reported that maximum shoot regeneration of
60% was obtained on MS medium supplemented with 2.25mg/l BAP and 0.2mg/l NAA,
which was significantly higher than other combination and further increase in PGP
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concentration resulted in heavy callus formation on the explant. In the present study also
BAP (2.0mg/l) combination with NAA (1.0mg/l) was most effective in inducing
adventitious shoot regeneration from internode explants.
3.5.1.3. Inflorescence rachis Explant
Inflorescence rachis segments have been proved successful as starting material for
direct adventitious shoot formation of field grown plants of P. barbatus. Inflorescence
rachis segments were used to get higher rate of shoot multiplication of several plants
(Cuenca et al., 1999; Cuenca and Marco, 2000; Leon et al., 2002 and Ran and Simpson,
2005). The cytokinins are an efficient growth regulators for shoot multiplication as has
been reported by Cuenca et al. (1999) and Cuenca and Marco (2000). Ran and Simpson
(2005) cultured explants on MS medium + 10mg/l BAP which produced maximum shoots.
Divya et al. (2008) showed that TDZ induced high frequency production of shoot
primordia from hypocotyl sections. The 2.0mg/l KN and NAA 0.5mg/l + BAP 1.5mg/l
treatments, were the only combinations which were able to induce direct regeneration
from the explants, unaccompanied by callus production as was reported in Thapsia
garganica (Makunga et al., 2005).
In the present study it was revealed that the concentration at 1.5mg/l KN alone was
effective for shoot bud proliferation and multiplication, BAP (2.0mg/l) and TDZ (1.5mg/l)
was found to have less shoot regeneration than KN. Increased cytokinin concentration
(above the optimum level) had a negative effect of shoot regeneration. These results were
in agreement with the previous reports on Hibiscus cannabinus by Herath et al. (2004),
Brassica juncea by Gua et al. (2005) and Mentha piperita by Shasany et al. (2006). Ouma
et al. (2004) reported that TDZ concentration was higher than 0.5mg/l which resulted in
callusing. In contrast to our observations TDZ and BAP concentration up to 2.0mg/l
appeared to have optimum regeneration and did not induce significant amount of callus.
Similar results were also reported by Divya et al. (2008) in cotton. BAP with NAA was
found to be the most effective combination for shoot regeneration and multiplication as
was reported by Faisal et al. (2007), Koroch et al. (2002) and Kongbangkerd et al. (2005).
Analogous with these reports, the present study also exemplifies the positive modification
of shoot induction efficacy that was obtained by employing BAP at 2.0mg/l in
combination with NAA at 1.0mg/l.
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3.5.1.4. Petiole Explant
Successful in vitro regeneration of plants from petiole explant cultures were
reported in Piper colubrinum (Kelkar and Krishnamurthy, 1998), Thapsia garganica
(Makunga et al., 2005), Solanum violaceum (Raghu et al., 2006b), Paulownia tomentosa
(Corredoira et al., 2008) and Brassica napus (Ghnaya et al., 2008). The incubation of
hypocotyl explants on MS medium supplemented with BAP (1.0mg/l) or TDZ (1.0mg/l)
resulted in organogenic frequency of 98.6 and 99.7% (Sujatha and Ranjithakumari, 2007).
Corredoira et al. (2008) showed that TDZ played an essential role in inducing adventitious
shoot regeneration in petiole explant derived from Paulownia tomentosa. A comparison of
cytokinin activity showed that both zeatin and TDZ resulted in higher frequency of
organogenesis than BAP as has been reported by Neto et al. (2003). The higher number of
shoots per explant occurred at 2.5mg/l BAP from petiote explant of Solanum violaceum as
was reported (Raghu et al., 2006b) followed by BAP 2.0mg/l which induced higher
number of shoots in Cunila galioides (Fracaro and Echeverrigaray, 2001). This is contrary
to the present research, since more shoots are reported in KN (1.5mg/l) medium. KN is
among the most active of the cytokinin-like substances and induced greater in vitro shoot
proliferation than other cytokine. This corroborates with the previous finding of Martin
et al. (2008), Jayaram and Prasad (2007) and Makunga et al. (2005). Regeneration
frequency for leaf explants gradually increased with an increasing BAP concentration,
reaching its maximum at 1.0mg/l, then decreased with any further increase in BAP
concentration (5.0mg/l) as has been reported in Piper colubrinum (Kelkar and
Krishnamurthy, 1998). Similarly BAP up to 2.25mg/l showed an increase in shoot
production and further increase to the level of BAP 2.25mg/l resulted in a decrease in the
extent of shoot regeneration (Raghu et al., 2006b).
Thus, these results are analogous to the present observations where the higher
number of shoots occurred at KN 1.5mg/l, BAP 2.0mg/l and TDZ 1.5mg/l individually
and further increase at the concentration of appropriate level resulted in decrease in the
shoot regeneration. The best results for bud induction were obtained in MS medium
supplemented with cytokinin with auxin (NAA) as has been reported (Arous et al., 2001;
Faisal et al., 2006 and Liu and Pijut, 2008). This is in accordance with the present
investigation, where BAP 2.0mg/l + NAA 1.0mg/l followed by KN 1.5mg/l + NAA
1.0mg/l was the best formulation for shoot proliferation and multiplication.
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3.5.1.5. Root Explant
After two weeks of culture, the roots became intense green and swollen in all the
concentrations tried. Similar results were also reported by Kelkar and Krishnamurthy
(1998) using root explant of Piper colubrinum. These results showed the incubation period
after 4 weeks small nodule like structures appeared predominantly on the proximal end
and rarely on the distal end. Similar results were observed in root on MS medium with KN
(1.5mg/l) supplementation. Such a polarization of morphogenetic response in root explants
is common in other plants (Neto et al., 2003 and Dalia et al., 2006). Root showed direct
multiple shoot formation first from the cut end of the petiole within two weeks of
inoculation without PGR which was reported in Bacopa monnieri (George et al., 2006).
This is unlike the results of the present study, which shows that phytohormone are
essential for shoot organogenesis. Similar results were also reported in root cultures of
Solanum melongena (Sarker et al., 2006).
Dalia et al. (2006) showed optimum adventitious shoot regeneration from root
organogenesis on MS medium supplemented with 0.5mg/l NAA and 1.0mg/l BAP. Sarker
et al. (2006) reported that the higher percentage of regeneration response in Solanum
melongena was obtained on MS supplemented with 1.0mg/l BAP and 1.0mg/l KN. In the
present investigation high percentage of regeneration response was observed in root
segment cultured on MS medium fortified with KN 1.5mg/l followed by BAP (2.0mg/l)
TDZ (1.5mg/l) with moderate shoot regeneration frequency. Same kinds of results also
were obtained in Centaurea paui (Cuenca et al., 1999); Feronia limonia (Hiregoudar
et al., 2005) and Gossypium hirsutum (Rauf et al., 2005). The synergistic effect of NAA
with BAP enhanced the induction of shoot bud have been reported (Kumar et al., 1998a;
Arous et al., 2001; Koroch et al., 2002; Sarker et al., 2005; Unda et al., 2007 and Martin
et al., 2008).
However, these results are analogous to the present observation where BAP
2.0mg/l along with NAA 1.0mg/l induce higher rate of shoot multiplication. A
combination of a higher amount of BAP (2.0mg/l) and small amount of NAA (1.0mg/l)
induced shoot proliferation. Higher concentration of BAP and KN showed growth
retardation (Nalini and Murali, 2002 and Jayaram and Prasad, 2007). Similar results were
observed in present study, where increasing concentration of above 2.0mg/l BAP, 1.5mg/l
KN, TDZ and1.0mg/l NAA gradually decrease in regeneration frequency and number of
shoots per explant were recorded.
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3.5.1.6. Transverse Thin Cell Layers (tTCLs) of stem Explant
The success of rapid and direct shoot regeneration without an intermediate callus
phase from transverse thin cell layers (tTCLs) of stem explant opens another efficient way
to mass propagation of P. barbatus. In the tissue culture of the younger stem segments of
Dendrobium candidum, one or two buds were induced from stem explants with nodes after
30 - 50 days and four to six buds were produced from each regenerated bud after they
were subcultured for 30 days (Chen and Cun, 2002). Similar result was observed in
present study within 20 days of inoculation. High frequency of shoot regeneration
occurred from cultured tTCLs of stem segements on MS medium supplemented with
1.0mg/l NAA and 1.5mg/l KN. The efficiency of NAA and BAP on shoot and adventitious
bud induction from the explant was no better than that of KN. According to Zhao et al.
(2007) among the PGR, BAP had the higher induction efficiency while that of KN had the
lowest with NAA.
The previous results shown that the frequency of responsive tTCLs segments
increased as the concentration of NAA ranged from 0.0 to 1.0mg/l and decreased from 1.0
to 3.0mg/l. This is in conformity with the present investigation. Auxins and cytokinins
were required for shoot induction and development, the BAP (Nayak et al., 2002), BAP +
NAA (Zhao et al., 2007 and Ghnaya et al., 2008), BAP and KN alone or in combination
(Nagesh, 2008) have been reported for adventitious shoot formulation through the tTCLs
explant, the maximum of shoot induction probably due to the synergistic activity of BAP
and KN as observed in Pisonia alba (Jagadishchandra et al., 1999). In present study the
KN, BAP and TDZ alone was efficient in shoot bud induction, which was highest at
1.5mg/l, 2.0mg/l and 1.5mg/l, respectively while further increases in the appropriate level
of cytokinins progressively decreased shoot bud numbers. This result is in agreement with
reports on Brassica sp. (Tang et al., 2003; Kennedy et al., 2005 and Ghnaya et al., 2008).
Silva (2003) provided evidence of the capacity to produce non-chimeric transgenic plants
using tTCLs segments.
The tTCLs of one week old seedlings produced higher number of somatic embryos
and regenerated somatic embryos developed into plantlets in Geranium hybrid and direct
bud primordia without intermediate callus phase on the surface of tTCLs explants of
Gladiolus spp. The advantage of the tTCLs system is to produce a high frequency of shoot
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regeneration and reduce the time interval required, with potentially more than 80,000
plantlets produced from a single tTCLs explant of orchid and the primary somatic embryos
that differentiated on sunflower tTCLs gave rise to secondary embryos, which developed
into normal fertile plants (Silva, 2003). So tTCLs can be proposed as suitable for high
frequency of shoot regeneration in tissue culture system which probably is attributed to
efficient utilization of nutrients and plant growth regulators.
3.5.2. Shoot Elongation
Mangal et al. (2003) have reported that MS medium supplemented with BAP
(1.0mg/l) and GA3 (0.5mg/l) resulted in desirable shoot elongation in Chrysanthemum
plants. MS medium fortified with 0.5mg/l of BAP and 1.0mg/l of GA3 promoted good
shoot elongation on Pothomorphe umbellata (Pereira et al., 2000). Length of the shoots
was more in KN supplemented media whereas BAP supplemented media showed reduced
shoot length as were reported in Ocimum basilicum (Sudhakaran and Sivasankari, 2003).
The great shoot elongation was obtained on MS medium without plant growth regulator
(Vanagas et al., 2002 and Cuenca et al., 1999).
The present observation has shown that directly regenerated microshoots were
subcultured on half strength MS medium supplemented with GA3 (0.6mg/l) alone or in
combination with KN (0.5mg/l) for maximum shoot elongation, similarly regenerated
shoots were further elongated on half strength MS medium without PGR as has been
reported by Kelkar and Krishnamurthy (1998). This is in accordance with reports of
Govindaraju et al. (2003), Arous et al. (2001) and Sharma et al. (2007) who reported that
shoot bud development was enhanced by the addition of GA3 to the medium.
3.5.3. Root induction, Hardening and Acclimatization
Half strength MS medium with plant growth regular induced more roots compared
to full strength MS and of the three auxins, IBA was best suited for inducing roots and this
is followed by IAA and the NAA was poor and it was characterized by callus formation.
Similar results were also reported by Beena et al. (2003). This successfull application of
IBA root inducing experiments has also been documented in various in vitro protocols
viz., Psoralea corylifolia (Faisal and Anis, 2006), Curculigo orchioides (Nagesh, 2008),
Cotton (Divya et al., 2008) and Ficus benghalensis (Rahman et al., 2004a). In contrast, to
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the previous reports of Pereira et al. (2000), Shasany et al. (2006), Sarker et al. (2006) and
Ghnaya et al. (2008) the higher frequency of rooting with the highest root length occurred
without PGR supplementation medium.
The present study has shown that the half strength MS medium with IBA at
1.5mg/l individually produced a greater number of healthy roots than IAA (1.5mg/l). The
combinations of IBA at 1.5mg/l with KN or BAP (0.5mg/l) produced moderate root
frequency. A crucial aspect of in vitro propagation is to acquire regenerated plants that are
capable of surviving outside the sterile and protected in vitro environment. 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 condition found ex vitro
(Nagesh, 2008).
In present study, the successfully rooted plantlets were transferred to paper cups
containing river sand, garden soil and saw dust for hardening. Plantlets were maintained in
the culture room (25 + 2˚C) conditions initially for 3 - 4 weeks and later transferred to
normal environment conditions and maintained for about 4 weeks. Similar pattern of
hardening was observed by Jeyachandran et al. (2005) and Nagesh (2008). This study
shows that shoot buds can be successfully regenerated on P. barbatus (non-meristemoid
tissue) through direct organogenesis. By using this protocol, a number of plants of
P. barbatus can be effectively multiplied. This protocol enabled conservation of natural
populations of this valuable aromatic and medicinal herb.