CHAPTER - IIIshodhganga.inflibnet.ac.in/bitstream/10603/5355/14/14_chapter 3.pdfscale propagation 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” 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



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



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



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



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.



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).



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



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



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



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.



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.



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.



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



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.



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



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



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



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.



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



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).



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.



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



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



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



3.4. RESULTS


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.



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.



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



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).



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



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).


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).



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.



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)



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.


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



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



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



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.



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.



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.



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



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.


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



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.

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