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Int. J. Med. Arom. Plants, ISSN 2249 – 4340RESEARCH ARTICLE

Vol. 1, No. 3, pp. 306-312, December 2011

*Corresponding author: (E-mail) [email protected] http://www.openaccessscience.com©2011 Open Access Science Research Publisher [email protected]

Development of Protocol for in vitro Propagation and Conservation of

Vitex negundo L.

M.M. RAHMAN*, S.K. BHADRA

Department of Botany, University of Chittagong, Chittagong, Bangladesh

Article History: Received 22nd October 2011, Revised 27th November 2011, Accepted 28th November 2011.

Abstract: Shoot apex, leaf and nodal segments of field grown plants of Vitex negundo L were aseptically cultured on MSand B5 medium fortified with different concentrations of cytokinin (BAP and Kn) and auxin (NAA and IAA) at differentcombinations. Nodal segments underwent direct organogenesis producing MSBs very efficiently. Maximum number ofMSBs (3.6±0.51) per explant was induced on MS medium supplemented with 2.0 mg/l BAP + 0.5 mg/l NAA. Shootapex though produced MSBs, the magnitude was meagre and the leaf segments failed to produce MSBs. Of the two basalmedia, MS was found superior to B5 for promoting response of the explants. The highest elongation of MSBs(2.17±0.17cm) took place in MS medium containing 2.0 mg/l BAP + 1.0 mg/l IAA. Elongated multiple shoot buds wererooted on rooting media. The maximum number of roots (35.60±1.63) per shoot was observed in half strength MS me-dium supplemented with 1.0 mg/l NAA. In vitro developed complete seedlings were acclimatized in outer environmentwith 82% survivability. Experiment on in vitro culture aided ex situ conservation was based on induced growth retardantand for this purpose abscisic acid (ABA) at different concentrations and low nutrient conditions were used in MS me-dium to keep the growth of in vitro seedlings stunted. It was observed that the growth of seedlings was minimum(0.71±0.10cm) in half strength MS medium containing 0.4mg/l ABA which is about three times less than that of fullstrength MS medium and the seedlings can be stored more than eight months without any subculture.

Keywords: Conservation; In vitro; Propagation; Protocol; Vitex negundo.

Introduction

Vitex negundo L. (Bengali name: Nishinda)is a widely used medicinal plant of the familyVerbenaceae. Leaves of this plant are antipara-sitic and used as alterative, vermifuge and ano-dyne, effective against inflammatory swellingsof joints in rheumatic attack, relieve catarrh andheadache. Juice of fresh leaves removes foetiddischarges and worms from ulcers. Flowers areastringent and cooling. Fruits are nervine stimu-lant, emmenagogue and vermifuge. Root is ton-ic, febrifuge, expectorant and diuretic. It regu-lates hormones and increases breast-milk pro-duction (Ghani 2003).

The availability of this plant species is re-ducing day by day due to indiscriminate collec-tions by the local herbalists and habitat destruc-tion. Still now there is no step for commercialcultivation of this medicinal plant species. Un-der such a situation we need to develop technol-ogy for mass scale propagation and conservationof this important medicinal plant species forsupporting the demand of the related industries.

In vitro culture based micropropagation tech-nique has been successfully used for rapid andmass propagation of many medicinal plants (Pu-rohit and Dave 1996; Sudha and Seeni 1996;Hassan and Roy 2005; Sultana and Handique2005; Biswas et al. 2009; Baksha et al. 2007;Roy 2008; Bhadra et al. 2009). In vitro regene-ration of V. negundo has also been reported bysome authors (Sahoo and Chand 1998; Hiregou-dar et al. 2006; Jawahar et al. 2008) In vitro cul-ture technique has also been used in ex situ con-servation of many plant species (Kartha et al.1988; Bajaj 1988; Kondo et el. 1989). It is nowestablished that in vitro culture in conjunctionwith cryopreservation or the use of growth re-tardants can be adopted for conservation ofplant species, which are at threatened or rarestate. The present study was therefore underta-ken with the objectives to develop a more effi-cient and reliable protocol for rapid in vitro mul-tiplication and ex situ conservation of this im-portant medicinal plant species.

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307Int. J. Med. Arom. Plants In vitro Propagation and Conservation of Vitex negundo

Rahman and Bhadra http://[email protected]

Materials and Methods

Shoot apex, leaf and nodal segments fromjuvenile twigs of field grown plants were col-lected and thoroughly washed under running tapwater. The materials were then surface sterilizedwith 1% savlon and liquid soap for 5 - 10 mi-nutes with constant shaking. The materials werethen washed 3 - 4 times with distilled water forcomplete removal of detergent and taken underrunning laminar airflow cabinet and transferredto 500 ml sterilized conical flask. After rinsingwith 70% ethanol for less than 60 seconds, theywere immersed in 0.1% HgCl2 for 5-7 minutes.To remove every trace of the sterilant, the mate-rials were then washed 4 -5 times with steriledistilled water. Then the explants were cut intopieces (0.5 - 1.0 cm) and grown on MS and B5

media supplemented with different concentra-tions and combinations plant growth regulators(PGRs). For elongation, the induced shoot budswere individually grown in aseptic condition ona wide range of PGR supplemented MS mediaand the vessels were maintained in the cultureroom in the same light and temperature condi-tions. In order to promote root formation themultiple shoot buds at a height of 2 - 3 cm wereseparated and individually cultured on rootingmedia. At first half and one fourth strength MSmedium without any PGR were used and thenhalf strength MS medium supplemented withdifferent concentrations and combinations ofauxins (IBA, IAA and NAA) were used for pro-fuse rooting.

In all cases the media were solidified with0.8% (w/v) agar (Sigma) and pH was adjustedto 5.8 before autoclaving for 30 minutes at121°C under a pressure of 1.1 kg/cm2. All cul-ture vessels with inoculated explants were incu-bated in a culture room at 25 ± 20C under a reg-ular cycle of 14 hr light and 10 hr dark

After sufficient growth the well rooted plan-tlets were transferred to outside environmentand planted in pots containing a mixture of soiland compost (1:1) through successive phases ofacclimatization. For ex situ conservation the invitro developed seedlings were cultured on full,half and one fourth strength MS medium sup-plemented with different concentrations of ab-scisic acid (ABA).

Results and Discussion

Shoot apex, leaf and nodal segments of fieldgrown plants were aseptically cultured on MSmedium supplemented with different concentra-tions of cytokinins (BAP and Kn) and auxins(NAA and IAA) in different combinations.Nodal segments underwent direct organogenesisproducing multiple shoot buds (MSBs) in dif-ferent PGR containing media (Table 1).

Table 1: Effect of different concentrations andcombination of PGRs in MS medium on induc-tion of MSBs in nodal explants of V. negundo.

PGR supplementsin the media (mg/l)

Daysto

response

% of explantsgiving

response

No. of shootbuds* /explant

( x ±SE)BAP 1.0 10-12 82 1.8±0.20

2.0 10-12 85 2.0±0.323.0 9-12 85 2.2±0.20

Kn 1.0 10-14 78 1.67±0.212.0 10-14 80 1.80±0.203.0 10-13 80 2.20±0.20

BAP+NAA 2.0+0.2 8-10 86 2.2±0.202.0+0.5 8-10 93 3.6±0.512.0+1.0 8-11 90 3.4±0.513.0+0.2 8-12 88 2.4±0.243.0+0.5 8-12 90 2.8±0.373.0+1.0 8-10 92 3.0±0.32

BAP+IAA 2.0+0.2 7-10 88 1.8±0.202.0+0.5 8-10 90 2.2±0.372.0+1.0 8-12 92 2.2±0.203.0+0.2 8-10 86 2.2±0.203.0+0.5 8-12 91 2.6±0.243.0+1.0 8-10 91 2.6±0.24

Kn+NAA 2.0+0.2 10-14 82 2.33±0.212.0+0.5 9-14 85 2.60±0.242.0+1.0 8-14 87 2.50±0.223.0+0.2 10-13 82 2.40±0.243.0+0.5 8-14 88 2.66±0.213.0+1.0 8-14 87 2.60±0.24

Kn+IAA 2.0+0.2 10-14 81 2.00±0.262.0+0.5 8-13 87 2.20±0.202.0+1.0 8-12 86 2.17±0.173.0+0.2 10-12 80 2.17±0.303.0+0.5 8-12 85 2.40±0.243.0+1.0 8-12 86 2.20±0.20

* Values are the mean of three replicates with 15explants.

Multiple shoot buds were initiated within 8– 12 days of culture. The response of shoot apexwas very poor. In a few combinations of mediaone shoot apex produced only 1 - 2 MSBs. Sothe results of induced MSBs from shoot apicesare not presented here. Leaf segments failed togive response in these media combinations. This





finding indicates the specificity of explant andPGR interaction. Maximum number of nodalexplants (93%) produced MSBs on 2.0 mg/lBAP + 0.5mg/l NAA. The average number ofMSBs (3.6±0.51) per explant was also highestin this media combination (Figure 1A). Thelowest number of MSBs (1.67±0.21) per explantwas recorded in 1.0 mg/l Kn and in this mediacomposition the minimum number of explant(78%) produced MSBs. Such positive response

of BAP in combination with NAA on inductionof MSBs has been noted in other medicinalplant species such as Gloriosa superba (Hassanand Roy 2005), Rauvolfia serpentina (Baksha etal. 2007) and Boerhaavia diffusa (Roy 2008;Biswas et al. 2009). On the other hand, Sahooand Chand (1998) and Islam et al. (2009) re-ported that MS medium supplemented with BAindividually enhanced the induction of MSBs inV. negundo.

Figure 1: Different steps of in vitro propagation and conservation of V. negundo.(A) Initiation and proliferation of MSBs on MS medium containing 2.0 mg/l BAP + 0.5 mg/l NAA.(B) Elongation of MSBs on MS medium containing 2.0 mg/l BAP + 1.0 mg/l IAA.(C) Induction and proliferation of roots in ½ MS medium containing 1.0 mg/l NAA(D) In vitro developed complete seedling after transplantation in small plastic pot.(E) Growth of shoot buds in 0.4 mg/l ABA supplemented half strength MS medium.(F) Growth of shoot buds in MS medium (as control).





In order to examine the efficiency of thePGR combinations in the background of differ-ent nutrient compositions, both MS and B5 me-dia were used separately as background of somePGR combinations and the results are depictedin Figure 2. Comparative study revealed that inmaximum cases MS was better than B5 in termsof in vitro culture in V. negundo. Baskaran andJayabalan (2005) in their experiment on Ecliptaalba recorded the higher efficiency of MS me-dium compared to that of B5 for in vitro culture.

70

75

80

85

90

95

BN-1 BN-2 BN-3 BN-4 BN-5 BN-6 BI-1 BI-2 BI-3 BI-4 BI-5 BI-6

Media composition

% o

f exp

lant

s gi

ving

res

pons

e MS medium B5 medium

00.5

11.5

22.5

33.5

44.5

BN-1 BN-2 BN-3 BN-4 BN-5 BN-6 BI-1 BI-2 BI-3 BI-4 BI-5 BI-6

Media composition

No. o

f MSB

s/ex

plan

t

MS medium B5 medium

Figure 2: Comparison between MS and B5 me-dium in terms of induction of MSBs in nodalexplants of V. negundo.BI-1=2.0 mg/l BAP+0.2 mg/l IAA, BI-2=2.0mg/l BAP+0.5 mg/l IAA,BI-3=2.0 mg/l BAP+1.0 mg/l IAA, BI-4=3.0mg/l BAP+0.2 mg/l IAA,BI-5=3.0 mg/l BAP+0.5 mg/l IAA, BI-6=3.0mg/l BAP+1.0 mg/l IAA,BN-1=2.0 mg/l BAP+0.2 mg/l NAA, BN-2=2.0mg/l BAP+0.5 mg/l NAA,BN-3=2.0 mg/l BAP+1.0 mg/l NAA, BN-4=3.0mg/l BAP+0.2 mg/l NAA,BN-5=3.0 mg/l BAP+0.5 mg/l NAA, BN-6=3.0mg/l BAP+1.0 mg/l NAA.

Although nodal segments gave rise toMSBs, they did not grow rapidly. In order toinduce rapid elongation, these MSBs were iso-lated and individually grown on different spec-trum of same PGR combinations. The resultsobtained from this experiment are summarizedin Table 2. Here also the role of PGR combina-tion in growth and development of plant organwas proved. The highest elongation(2.17±0.17cm) of MSBs was developed on 2.0mg/l BAP + 1.0 mg/l IAA (Figure 1B) and thelowest elongation (1.13±0.08cm) was on 1.5mg/l Kn + 0.5 mg/l NAA.

In order to promote root formation the mul-tiple shoot buds at a height of 2 - 3 cm were se-parated and individually cultured on rootingmedia. Response of rooting was very much de-pendent on type and concentration of auxinssupplements in the media. Among the threetypes of auxins, NAA was superior to that ofIBA and IAA in terms of induction of numberof root per shoot (Table 3). The maximum num-ber of roots (35.60±1.63) per shoot was formedin half strength MS medium supplemented with1.0 mg/l NAA (Figure 1C) and the minimumnumber (2.60±0.40) was in half strength MS +2.0 mg/l IAA. The highest and lowest elonga-tion of root (2.62±0.11 and 1.33±0.12cm) wasrecorded in half strength MS medium containing1.0 mg/l IBA and 0.5 mg/l IBA respectively.The efficiency of NAA in in vitro rooting hasbeen reported in many other medicinal plantspecies including Lavandula vera (Andrade etal. 1999), Curculigo orchioides (Wala and Jasrai2003), Vitex trifolia (Hiregoudar et al. 2006)and Rauvolfia serpentina (Baksha et al. 2007).On the other hand, Jawahar et al. (2008) andIslam et al. (2009) reported that IBA (0.5 mg/l)supplemented half strength MS medium wasbest for induction of root system in V. negundo.After rooting the complete plantlets were trans-ferred to outside environment and planted inpots (Figure 1D) containing garden soil andcompost (1:1) through successive phases of ac-climatization. Survival percentage of the regene-rated plantlets under natural conditions wasabout 82% and the plantlets were morphologi-cally uniform having normal growth.





Table 2: Data on elongation of MSBs of V. negundo when cultured on 0.8% (w/v) agar solidifiedMS medium supplemented with different PGR combinations.

PGR supplements in the medium(mg/l)

Initial length* (cm) ofindividual shoot buds

( x ±SE)

Length* (cm) of individualshoot buds after 30d of cul-

ture ( x ±SE)

Increase in length* (cm) ofshoot buds after 30d of culture

( x ±SE)

BAP+NAA

1.5+0.5 1.33±0.11 2.58±0.08 1.25±0.111.5+1.0 1.42±0.15 2.84±0.11 1.42±0.152.0+0.5 1.33±0.11 2.75±0.17 1.42±0.152.0+1.0 1.25±0.11 2.83±0.17 1.58±0.152.5+0.5 1.42±0.15 2.84±0.21 1.42±0.152.5+1.0 1.42±0.15 2.75±0.17 1.33±0.11

BAP+IAA

1.5+0.5 0.92±0.05 2.17±0.15 1.25±0.111.5+1.0 1.33±0.11 3.16±0.21 1.83±0.212.0+0.5 1.33±0.11 2.58±0.08 1.25±0.112.0+1.0 1.50±0.11 3.67±0.20 2.17±0.172.5+0.5 1.42±0.08 2.83±0.21 1.41±0.152.5+1.0 1.42±0.15 3.25±0.34 1.83±0.21

Kn+NAA

1.5+0.5 1.25±0.11 2.38±0.08 1.13±0.081.5+1.0 1.30±0.12 2.55±0.17 1.25±0.112.0+0.5 1.40±0.12 2.60±0,19 1.20±0.122.0+1.0 1.30±0.10 3.10±0.19 1.80±0.122.5+0.5 1.30±0.12 2.65±0.10 1.35±0.102.5+1.0 1.20±0.12 2.60±0.18 1.40±0.19

Kn+IAA

1.5+0.5 1.40±0.10 2.60±0.10 1.20±0.121.5+1.0 1.30±0.12 2.75±0.16 1.45±0.122.0+0.5 1.20±0.12 2.80±0.20 1.60±0.192.0+1.0 1.30±0.12 3.00±0.16 1.70±0.122.5+0.5 1.10±0.10 2.35±0.19 1.25±0.112.5+1.0 1.60±0.10 3.05±0.17 1.45±0.12

* Values are the mean of three replicates with 15 explants.

Table 3: Effect of auxin, low nutrient conditions and their combinations on induction and growth ofroots in in vitro developed shoot buds of V. negundo.PGR supplements in the media (mg/l) Days to root induc-

tion% of rooting No. of root* /shoot bud

( x ±SE)

Length* of individualroot (cm)

( x ±SE)

¼ th MS without PGRs 8-10 70 2.80±0.22 1.55±0.12½ MS without PGRs 6-7 72 3.60±0.40 1.54±0.16

½ MS+IBA0.5 15-20 75 3.60±0.40 1.33±0.121.0 4-5 82 4.20±0.37 2.62±0.112.0 4-5 88 11.20±0.73 2.60±0.14

½ MS+IAA0.5 12-15 72 3.60±0.40 1.62±0.191.0 15-20 78 3.80±0.37 1.72±0.132.0 20-25 77 2.60±0.40 1.69±0.10

½ MS+NAA0.5 8-12 85 33.00±2.24 1.48±0.131.0 8-12 85 35.60±1.63 1.96±0.172.0 7-12 90 32.80±2.44 2.29±0.22

½ MS+IBA+IAA

2.0+0.5 -- -- -- --2.0+1.0 -- -- -- --2.0+2.0 12-15 75 8.60±1.08 2.44±0.15

½ MS+IBA+NAA

2.0+0.5 8-13 92 6.00±0.32 2.26±0.112.0+1.0 7-12 95 13.20±0.58 2.53±0.142.0+2.0 8-12 95 6.80±0.37 2.37±0.19

* Values are the mean of three replicates with 15 explants; --= No response

In vitro regeneration of plants ultimately re-sults in development of complete plantlets andbecause of their rapid growth it becomes verydifficult to keep the culture for long period forconservation. There are several methods of exsitu conservation such as cryopreservation, algi-

nate encapsulation, use of growth retardants andlow temperature storage for in vitro conserva-tion of genetic resources. But in the presentstudy only ex situ conservation based on in-duced growth retardant was followed. And forthis purpose abscisic acid (ABA) at different





concentrations and low nutrient conditions wereused in MS medium to keep the growth of invitro seedlings stunted so that they can be keptfor longer period. Full strength MS medium wasused here as control and the other media compo-sitions were used to examine the poor growth ofseedlings. The data on elongation of seedlingswere recorded after 30 days of culture and theresults are summarized in Table 4. It was foundthat the growth of seedlings was lowest(0.71±0.10cm) in half strength MS medium con-taining 0.4mg/l ABA (Figure 1E) followed bythat in one fourth MS + 0.4 mg/l ABA and onefourth MS + 0.2 mg/l ABA. In control medium

the growth was 1.92±0.15cm (Figure 1F). Incomparison, growth in half strength MS +0.4mg/l ABA was about three times less thanthat of full strength MS medium. It was foundthat the seedlings could survive normally underthis stunted growth condition for more than 8months without nay subculture. Agarwal et al.(1992) succeeded in slow growth storage of invitro developed shoots of Vanilla walkeriae anendangered orchid using low nutrient media.Charoensub and Phansiri (2004) found thatmannitol added to the media could reduce thegrowth of in vitro derived plantlets of Plumbagoindica.

Table 4: Data on elongation of in vitro developed shoots of V. negundo grown on different low nu-trients and ABA containing media.Media with or without ABA

(mg/l)Initial length*

(cm) of shoot buds( x ±SE)

Length* (cm) of shootafter 30d of culture

( x ±SE)

Increase in length* (cm) of shoot after30d of culture

( x ±SE)MS (Control) 1.25±0.11 3.27±0.17 1.92±0.15½ MS 1.58±0.15 3.33±0.17 1.75±0.21

¼ MS 1.66±0.11 3.08±0.08 1.42±0.15MS+0.1mg/l ABA 1.75±0.11 3.33±0.11 1.58±0.15MS+0.2mg/l ABA 1.42±0.15 2.71±0.10 1.29±0.19MS+0.4mg/l ABA 1.33±0.17 2.41±0.14 1.08±0.14½ MS+0.1mg/l ABA 1.58±0.15 2.75±0.16 1.17±0.12½ MS+0.2mg/l ABA 1.58±0.08 2.41±0.08 0.83±0.11½ MS+0.4mg/l ABA 1.67±0.17 2.38±0.13 0.71±0.10¼ MS+0.1mg/l ABA 1.17±0.11 2.21±0.14 1.04±0.10¼ MS+0.2mg/l ABA 1.42±0.08 2.21±0.14 0.79±0.10¼ MS+0.4mg/l ABA 1.50±0.18 2.25±0.09 0.75±0.09* Values are the mean of three replicates with 15 explants.

Conclusion

From the overall results of this researchwork it can be concluded that the protocol forproduction of complete seedlings under in vitrocondition could be developed in V. negundo andthis protocol could be used for mass propagationby our herbal industries. It was proved that in-duction of MSBs through direct organogenesiswere dependent on PGR supplements in the me-dium. The results of in vitro aided ex situ con-servation indicated that it is possible to go forthis kind of conservation strategy on short termbasis until cryopreservation facilities can be de-veloped. Here the selection of growth retardant,its concentrations as well as low nutrient condi-tion of the medium need to be identified proper-ly.

Acknowledgement

The first author gratefully acknowledges thefinancial support of the University GrantsCommission of Bangladesh in the form of Ph.D.scholarship.

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