Green page: Research Article

IPC code; Int. cJ.7 - AOlH 4/00, AOlH 5/04, AOlH 5/06, A61K 35/78

Keywords: Adenine snlphate, Bacopa monnieri, Callusinduction, Micropropagation, Mnltiple

shoots, Benzyl Amino Purine (BAP), Indole-3-Acetic Acid (lAA), Naphthalene Acetic Acid

(NAA),Kinetin, Nodal explants.

..In vitro multiple shoot and root induction of Brahmi [Bacopa monnieri

(Linn.) Penn.] was achieved from nodal explants. Shoot segments obtained from field grown

plants were surface sterilized and cnltured on Murashige and Skoog's (MS) medium without any

hormones to get regular supply of node explants. In order to find out the suitable conditions for

propagation from nodal explants, MSmedium with different combinations of auxins and cytokinins

were tested. All the treatments were found effective in inducing shoots except those with auxin!

cytokinin alone. The percent response of 92-1 00 was observed in 90% of treatments. The effect

of special cytokinin like adenine sulphate on multiple shoot formation from the nodal explantsof B. monnieri was tested on MSmedium fortified with different hormonal concentrations of

auxin and cytokinin. High percentage of multiple shoot formation (96.3) and maximum numberof shoots (16.33) was observed in the combinational treatmentoflAA (0.2 mg/l) and BAP(1.5

mg/l) . Addition of adenine sulphate at 60mg/l along with lAA(0.2mg 11)and BAP(1. 5mg/l) was

found most effectivein inducing highest number of shoots (18). Allthe other tested combinations

have little effect on increasing the number of shoots. In vitro propagated plants were successfully

transplanted to plastic pots containing sterile soil with 85% survival rate and later establishedunder ex vitro condition.

Bacopa monnieri (Linn.)Penn. (Hindi-Brahm i) is a creeping,

succulent herb belonging to the familyScrophulariaceae. It is one of the few

Ayurvedicherbs with high economic and

scientific values with potential forresearch in tissue culture and

phytochemistry. In India whole plant or

aerial part o~the plant is used to support

healthy blood sugar levels, to prevent the

effect of ageing, to develop intelligence,

to promote mental health and to increase

the body resistance against diseases

(Kapoor, 1990). The principal active

factors that have been reported from this

plant are two steroidal saponins, bacoside

Aand B (Chatterji et ai, 1963). Bacoside

Aand B are optical isomers and bacoside

B may be an artefact, formed during theisolation of bacoside A (Rastogi &

M Ramesh*, R M Saravanakumar and S Karutha PandianDepartment of Biotechnology, Alagappa University,

Karaikudi 630 003, Tamil Nadu, India

*Correspondent author, E-mail: ramesh_biotek@yahoo.co.in; Phone: (04565) 225215

Received 15 March; Accepted 12 September 2005

Mehrotra, 1990). Other bacosides have

been identified as dammarane type

saponins, known as bacosaponins (Garaiet aI, 1996). Other constituents include

alkaloids (herpestine and brahmine),

flavonoids, glycosides, betulic acid and

phytosterols (Jain & Kulshreshtha, 1993).

In addition, Bacopa extract has anxiolytic,

cognition enhancing (Bhattacharya &

Ghosal, 1998), relaxing (Dar & Channa,1997), antioxidant (Mukherjee & Dey,

1996), anticancer (Elangovan et aI,

1995) and immuno-modulatory activities(Dahanukar & Thatte, 1997).

The increase in the number of

people with poor body health has made

the alternative medical practitioners andpharmaceutical companies to overexploit

many plants. Unscrupulous and

unscientific management practices and

ever increasing demand for

phytochemicals have threatened the

existence of most of the medicinal plantsincluding B. monnieri. In vitromicropropagation techniques paved the

wayfor multiple shoot and root induction

in large number of medicinal plants in ashort span of time to meet their demand

in drug industries. Brahmi has been

previouslypropagated using tissue culturetechniques. Thakur et al C1976a)

Abstract

Introduction

m Natural Product Radiance

=============G=--re=--e=--ft==l@!!le:ResearchArticle

Fig. 1: In vitro micropropagation from nodal explants of Bacopa monnieri (A) Shoot tipculture of B. monnieri; (B) Nodal explant developing callus like growth with large number ofshoot buds; (C) Explant showing large number of shoot buds with developed shoots;(D) Micropropagated shoots of 9cm on MS medium with 0.2 mg/l NAA and 1.5mg/l BAP;(E) Effect ofNAA (O.lmgll) and BAP (left panel) and IAA (0. Img/l) and BAP (I.Omg/l) on callusformation; (F) Micropropagated shoots (16.33 per explant) onMS medium with 0.2mg/l IAA and

1.5mg/l BAP; (G) Root initiation from micropropagated plant on MS medium with 0.2mg/lIAA,1.5mg/1 BAP and 60mg/l adenine sulphate after 28 days .in culture; (H) Plantlets

micropropagated in the presence (left) and absence (right) of adenine sulphate.

observed the role of benzyl amino

purine on shoot bud regeneration

from single epidermal cells of

Brahmi. The same group later

reported the development of

abnormal plantlets with alternate

phyllotaxy, instead of opposite

arrangement (Thakur et al, 1976b).

Totipotent ability of distal ends of

B. monnieri internode segments in

regenerating shoots has been

reported on medium without any

hormones (Thakur & Ganapathy,

1978). Morphogenic potential of

node, inter node and stem explants

have been reported successfully(Tiwari et aI, 1998; Shrivastava &

Rajani, 1999; Allet aI, 1999; Tiwari

et aI, 2001). In these reports the

shoots established were again

transferred to full or half strengthmedium with auxin alone for

rooting. Recently, Mohapatra and

Rath (2005) achieved

micropropagation from leaf and

nodal segments and analyzedbiochemical parameters to establish

the sustainabilityofplants. Regardingits genetic improvement, only one

group reported successful protocol

for genetic transformation of this

species using Agrobacteriummediated transformation (Nisha etaI, 2003). The present study

established reliable and reproducible

protocol for rapid one step multiple

shoot and root regeneration from

nodal explants of the herb usingvarious concentrations and

combinations of auxins and

cytokinins.

Vol 5(1) January-February 2006 m

Green @!Be: Research Article

Effect of NAAlIAA and BAP

Results and Discussion

The nodal explantswere cultured

on MS media supplemented with variousconcentrations of NAA(0.1-0.5mgll) or

1AA(0.1-0.5 mg/l) along with the

combination of BAP (1.0-3.0 mgll). The

effectiveness of nodal segments on

in vitro micropropagation was analyzed

after 30 days of culture initiation. Multipleshoot induction (more than two) was

observed on the 12th day of incubation inall concentrations of hormones tested.

Among the different auxin-cytokinin

In medicinal plants like

B. monnieri, reliable and reproducible

propagation from different explants is a

prerequisite for genetic manipulations liketransformation.

Statistical analysis

All the experiments were

repeated thrice (each with 9 explants) and

the response of explants on multiple shoot

induction was recorded after 28 days of

culture initiation. The data pertaining to

mean percentage of culture showing

response, number of shoots per explants

and shoot length were scored and

presented in mean ± standard error.

to Sigma polypropylene boxes (with two

holes at the bottom) containing

autoclaved garden soil. The plants were

covered with polythene bags with fine

holes and kept in a culture room at

26±2°C and 60-65 % relative humidity

for 8-12 days. After 12 days, the polythene

covers were removed and transplanted to

the field for further growth.

Hardening

Micropropagated plantlets

(7 -9cm in height with 8-10 leaves and8-12 roots) with and without adenine

sulphate treatment were gently washed

under running tap water and transferred

Multiple shoot induction and rooting

About 1.5-2cm long nodal

explants were aseptically excised from

4 week-old axenic seedlings and used as

explants for shoot induction on MS

medium supplemented with various

concentrations and combinations of

hormones. Results were recorded at a

regular interval and final data were scoredafter 4 weeks of culture to study the effect

of auxin and cytokinin and in

combination with adenine sulphate to

induce multiple shoots and buds at

26 ± 2°C with a 16hr photoperiod under

cool white fluorescent light. For each

treatment, a total of nine explants were

used in three different experiments. To

study the role of auxins and cytokinins

alone, same number of explants weretransferred to MS basal medium andtreated as control.

were used for multiple shoot induction.

Along with the above mentioned media,

adenine sulphate (20-100 mgll) was used

for enhancing multiple shoot induction.

The pH of all media was adjusted to 5.7­

5.75 before adding 0.8% agar gel (Sigma,

USA). Moltened media of about 30m! was

dispensed into polypropylene magenta

vessels (Sigma, USA)and was autoclavedat 15lb and 121°C for 18 minutes.

Autoc1avedmedia were kept at 26± 2°Cbefore use.

Culture medium and conditions for

propagation

Plant materials and maintenance

Materials and Methods

Actively growing and healthyshoot material of B. monnieri were

collected from the field grown plants in

the morning. After removing the leaves,

the shoots were cut into pieces (2-3cm),

each containing a single nodal region.

Explants were thoroughly washed under

running tap water for 20 minutes and with

3% (v/v) laboline for 3min followed by

brief washing with sterile distilled water.

Shoot segments were surface-sterilized,

in 70 % (v/v) ethyl alcohol for 30sec

followedby 0.1% mercuric chloride (wlv)for 3min and then rinsed four times withsterile distilled water. After surface

sterilization explants were finely trimmed

again and placed on to 30ml of basalmedium consisted of MS salts and

vitamins (Murashige & Skoog,1962)

supplemented with 3% (w/v) sucrose and

0.8 % (w/v) agar gel (Duchefa, The

Netherlands) in magenta boxes (Sigma,

USA).All cultures were kept at 26±2°C

temperature and at a relative humidity of

70% under 16hr photoperiod. In vitroestablished shoots served as mother stock.

Nodal segments were separated after

28-30 days of culture initiation for

multiple shoot induction and rooting withdifferent concentrations of auxins and

cytokinins (Fig.1A).

Murashige and Skoog's mediumfortified with different combinations of

auxin NAA (0.1-0.5mgll) and IAA(0.1­

0.5 mg1) and q10kininBAP(1.0-3.0J11g1)

m~ --'Natural Product Radiance

============G=,=e=en=palle: Resea,thA,';tle

combinations tried, IAA and BAP

formulations exhibited a better response

than NAAand BAP.In general, number of

shoots multiplied per explant and its

length was slightly more on media with1M and BAP than with NAAand BAP'We

found an increase in the percent response

of explants (96.3-100) showing multiple

shoot regeneration with an increase in the

concentration ofBAP from 1-2.0mgll and

NAAfrom 0.1-0.3mgll. However, a furtherincrease in the concentrations of BAP has

no effect on shoot induction. Between the

two auxins (1M and NAA)tested, 1M (0.1,

0.2, 0.3mgll) with BAP (1.0, 1.5 and 2.0

mg/I) was found to be superior in

increasing the number of shoots (14-16

shoots/explant) than NAA (0.1, 0.2,

0.3mgll) and BAP (1.0, 2.0, 3.0 mgll)

(11-12 shoots/explant). It was observed

that there was no significant variation in

shoot length between the media fortified

with 1M and NAA.Highest average lengthof shoot was 9.43cm on the medium

containing 0.2mgll 1M and 1.5mgll BAP

(Table 1). The percentage of explants

exhibiting multiple shoot formation was

found to be 96-100 in all the combinations

tested. Similar kind of observations were

reported from the leaf, inter node and node

explants of the species (Tiwari et at,

1998). Callus-like growth containing largenumbers of adventitious shoot buds with

some developed shoots were observed at

the cut ends of nodal explants (Fig.1B).

After 28 days of culture in the same

medium, most of the shoots attained a

length of 6-9cm. Shoot buds were foundin all tested concentrations and buds were

visually seen in all three replicates. These

results agree with the earlier study on

Bacopa using BAP/KN (Tiwari et at,1998). In this experiment we observed

induction of large number of shoot buds

after 18-22 days of inoculation. Among

the numerous shoot buds induced around

12-14 shoot buds regenerated into shoots

and attained the height of 6-9cm (Fig. 1C).

However, in the presence of 2.5 and 3.0

mgll BAPalong with 0.4 and 0.5mgllNAA/

IAA, only 5-6 shoot buds regenerated

into shoots with a height of 6-7cm

(Fig. ID). The nodal explants cultured on

medium supplemented with 0.2mgll 1Mand 1. 5mg/l BAP facilitated the

development of maximum number of

shoots (16.33) followed by 0.3mgll 1M

and 2.0 mg/l BAP (15.66). Similarly

medium supplemented with 0.2mgll NAA

and 1.5 mgll BAPregenerated a maximum

of 12.33 shoots per nodal explants with

shoot length of 9.43 (Table 1). There wasenormous number of shoot buds that did

not attain the measurable height, as they

spread throughout the surface of the

explants very closely. In general, the nodal

explants cultured on medium with various

concentrations of auxin and cytokinin in

combination have developed healthy dark

green shoots with medium sized leaves

than medium with auxin/cytokinin alone.

Explants cultured on media fortified withIAA/NAA/BAP alone induced shoots at

lesser number (2-3) at alesserfrequency

(72-92) compared to the media

supplemented with combination

treatments of cytokinin and auxin

(Table 1).

We observed greenish callus with

different concentrations of 1M (0.1-0.5

mgll) andBAP (1.0-3.0mgll).Thequantum

of callus-like growth was not much in the

presence of NAA(0.1-0.5 mgll) and BAP

(1.0-3.0 mgll) than that of 1M (0.1-0.5

mgll) and BAP(1.0-3.0 mgll) (Fig. IE) .

Shrivastava and Rajani (1999) studied

callus response from stem and leaf

explants on MS medium supplemented

with NAA,IAA and 2, 4-D. They observed

all the hormones studied, exhibited a

tendency to regenerate in to shoots or

roots rather than callus. Among the levels

of NAA (0.1-0.5 mgll) and IAA (0.1-0.5

mg/I) along with BAP (1.0-3.0 mg/l)

tested, the shoot-bud induction response

was higher in the media containing 1M

(0.1-0.5mgll) and BAP (1.0-3.0mgll). The

percent response of nodal explants

developing the number of shoot buds was

high with increasing the concentration of

BAP up to 1.5mg/l. Among the media

tested, MS medium supplemented with

0.2mg/l IAA and 1.5mg/l BAP gave

maximum number of shoots (16.33 per

explant) and the shoots reached 6.03cm

within 30 days of culture (Fig. IF) . In

B. monnieri the stimulatory effect of

BAP on high frequency shoot bud

regeneration was earlier observed from

leaf, internode and axillary buds of node

explants (Tiwari et ai, 1998), internodes

(Thakur & Ganapathy, 1978), leaf

explants (Shrivastava & Rajani, 1999).

Tiwari et al (1998) reported the influence

of BAP on generating large number of

shoot buds from leaf explants followed

by node and internode. The same group

(2001) reported the role of BAP,

thidiazuron, kinetin and 2-isopentyl

adenine on shoot bud induction node,

inter node and leaf explants. Mohapatra

and Rath (2005) used BAP alone for

multiple shoot proliferation with 95%

frequency with 9.4 mean numbers of

shoots per node culture. In the present

investigation, it was observed that 1M incombination with BAP was more suitable

than NAAin combination with BAP.Similar

effect ofBAP in e~hancing multiple shoots

Vol 5(1) January-February 2006 m

Green ~e: 'eseGrth Art;tle

from the different explants were reported

in many medicinal and other plant species

like Withania somnifera Dunal (Sen

&Sharma,1991), Ocimum sp. (Pattnaik

&Chand, 1996), Vitex negundo Linn.(Chandramu et aI, 2003), Psidiumguajava Linn. (Loh & Rao, 1989), and

Citrus sp. (Maggon & Singh, 1995).

Effect of adenine sulphate

To study the effect of adenine

sulphate in the presence of auxin and

cytokinin on the induction of multiple

shoots, the nodal explants were cultured

on medium supplemented with adenine

sulphate (20-100mg/l) along withdifferent hormonal concentrations of NAA

(0.1-0.5mg/l), IAA (0.1-0.5 mg/l) with

BAP(1.0-3.0mgll). Additionof 20-40mgll

of adenine sulphate to media with

0.1-0.5mg/1 NAA/IAAalong with 1.0-3.0

mg/l BAP had no significant effect on

percent response of shoot induction and

number of shoots produced per explants.

Only the presence of adenine sulphate at

60 mg/l along with all the concentrations

of NAA/IAAalong with BAP (1.0-3.0 g/l)

enhanced the average number of shoots

per explants (Tables 2 & 3) . Similar effect

of adenine sulphate in enhancing shooting

response to a limited extent was reported

in aromatic endangered medicinal plantHemidesmus indicus R. Br. Addition

of 15mg/1 adenine sulphate to mediurr,

with 1.0 mg/l BAP and 0.5mg/1 NAA

enhanced the average number of shoots

from 4.82 to 5.13 (Misra et aI, 2003).

Among the different concentrations of

adenine sulphate tested, addition of 60

mg/l adenine sulphate with 0.2 mg/l IAA

and 1.5 mg/l BAP induced the highest

number (mean of 18) shoots. On medium

with 0.2mg/l IAAand 1.5mg/l BAP only

16.33 shoots were seen after 30 days of

culture initiation. Similarly medium with

0.2mgll NAAand 1.5mg/l generated meanof 12.33 shoots in contrast to 14 on

medium with 0.2 mg/l IAA,1.5 mg/l BAP

and 60mg/l adenine sulphate. Addition of

80 and 100QIg/1adenine sulphate had no

beneficial effect.The increase in multiple

shoot induction in the present study may

be attributed to the synergistic effect of

adenine sulphate with the combinationsof IAAand BAP.

Rooting

The in vitro grown shoots in the

concentrations tested developed bunch of

short, white root system in the same

medium after 18-22 days. Rootinduction

Table 1: The effect of auxins (IAA & NAA) along with cytokinin (BAP) on multiple shoot induction from

the nodal explants of Bacopa monnieri on MS medium

Auxin! Concentration% of explant responseNo of shoots/Shoot length

Cytokinin

(mgll)for multiple shootexplant(em)

induction (Mean±SE)

(Mean:tSE)(Mean:tSE)

1!*

I 72.6 ± 0.41I2.33 ± 0.41 6.33 ± 0.41I

NAA0.1- 82.6 ± 0.41I2.33 ± 0.41 I5.66 ± 0.41

I

iBAP

1.096.3 ± 0.414.33 ± 0.416.03 ± 0.50!NAA+BAP0.1+1.096.3 ± 0.41!

11.00 ± 0.35 8.16 ± 0.08!

NAA+BAP

f0.2+1.5 96.3 ± 0.4112.33 ± 0.419.43 ± 0.04

NAA+BAP

0.3+2.0100.0±0.076.33 ± 0.417.03 ± 0.04

NAA+ BAP

0.4+2.596.3 ± 0.414.33 ± 0.418.00 ± 0.07

NAA+BAP

0.5+3.096.3 ± 0.412.33 ± 0.416.90 ± 0.07I

I 92.6 ± 0.41 3.43 ± 0.407.56 ± 0.06IAA 0.1 -IlAA + BAP

0.1+1.0I96.3 ± 0.41 14.66 ± 0.419.40 ± 0.07

lAA + BAP

0.2+ 1.596.3 ± 0.4116.33±0.418.03 ± 0.05

IAA+ BAP

0.3+2.092.6 ± 0.4115.66 ± 0.419.56 ± 0.04

IAA+ BAP

0.4+2.596.3 ± 0.418.33 ± 0.417.56 ± 0.06

lAA + BAP

0.5+3.092.6 ± 0.415.66 ± 0.41I7.06 ± .04

Each value represents the mean ± SE of 3 replicates with 25-35 nodal explants.

*MS basal medium devoid of any hormonesm____ Natural Product Radiance

Green !!lle: Researlh Arl;lle

Table 2: The effect of adenine sulphate along with different combinations

of NAAand BAPon multiple shoot formation from nodal explants ofBacopa monnieri

Auxin! Concentration Cone.of adenine % of explant responseCytokinin (mgll) ; sulphate for shoot induction

(mg/l) (Mean:l:SE)

1No.of shoots!jexplant(Mean±SE)

was found to be 100% in all

concentrations tested with an average of

14-21 roots per explants with 16-18 shoots

(Fig. 1G). Average length of root system

was 2cm. Rooting did not occur onmedium without IAAlNAA.

Vol 5(1) January-February 2006

Each value represents the mean ± SE of 3 replicates with 30-35 nodal explants.Values given in bold are control for each treatment

NAA+BAP I 0.1+1.0

NAA+BAP I 0.2+1.5

NAA+BAP, I 0.3+2.0

NAA+BAP 0.4+2.5

NAA+BAP 0.5+3.0

o

20

40

60

80

100

o

20

40

60

80

100

o

20

40

60

80

100

o

20

40

60

80

100

o

20

40

60

80

100

96.3:1:0.41

96.3±0.41

96.3±0.41

100.0±0.35

96.3±0.41

92.6±0.41

96.3:1:0.41

96.3±0.41

96.3±0.41

92.6±0.41

100.0±0.35

92.6±0.41

92.6:1:0.41

92.6±0.41

96.3±0.41

96.3±0.41

100.0±0.35

92.6±0.41

96.3:1:0.41

96.3±0.41

96.3±0.41

92.6±0.41

92.6±0.41

92.6±0.41

96.3:1:0.41

92.6±0.41

96.3±0.41

92.6±0.41

96.6±0.41

92.6±0.41

11.00:1:0.35

12.33±0.41

12.66±0.41

13.00±0.35

12.33±0.41

11.00±0.35

12.33:1:0.41

13.00±0.35

13.66±0.41

14.00±0.35

13.66±0.41

12.33±0.41

6.33:1:0.41

6.66±0.41

7.00±0.35

7.33±0.41

6.66±0.41

6.33±0.41

4.33:1:0.41

4.66±0.41

5.00±0.35

5.33±0.41

5.00±0.35

4.33±0.41

2.33:1:0.41

2.33±0.412.66±0.41

3.00±0.35 .

2.33±0.41

2.33±0.41

Transplanting

In vitro propagated plantlets

transplanted into the polypropylene boxes

with autoclaved garden soil recovered

well after 8-12 days and exhibited 80%

survival. Alltransplanted plants showed a·

similar growth habit to field grown plants

(Fig.IH). Plantlets micropropagated andhardened in the presence and absence of

adenine sulphate did not exhibit any

phenotypic difference.Acclimatizedplants

established in the field condition grew

normally and did not show any variationson growth characteristic features when

compared to the control plants.

Conclusion

The nodal explants asepficallyobtained from in vitro multiplied shoot

segments were used for multiple shootinduction using MS basal media

supplemented withvarious concentrations

and combinations of auxins like IAA,NAAand cytokinins like BAP and Adenine

sulphate. Micropropagation was achieved

with the percent response of 92-100 in

most treatments. Ofthe two auxins tested,we found IAAto be more suitable than

NAAfor highest percentage of multipleshoot induction with maximum numberof shoots. Maximum number of shoots

wereobsetvedonMSmediumsupplemented

with O.2mgllIAAand 1.5mgllBAP (16.33

per node). Thepresence of60mg/1adenine

m

Green l!!!Ie: Researth Art;,'e

Table 3: The effect of adenine sulphate along with different combinations

of IAAand BAPon multiple shoot formation from nodal explants ofBacopa monnieri

Each value represents the mean ±SE of 3 replicates with 30-35 nodal explants

Values given in bold are control for each treatment

m

IAA+BAP 0.1+1.0

Acknowledgements

sulphate along with 0.2 mgll 1M and 1.5

mg /1 BAP enhanced the number of

multiple shoots from 16.33 to 18 per

explant. Micropropagated plantlets

transferred to plastic pots grown to

maturity under ex vitro condition.

Although there are several reports on

multiple shoot regeneration from node

explants of B. monnieri, there is no

report on in vitro multiplied shoots with

not more than 18 shoots per explants and

shoot length up to 9cm after 30 days of

culture period. Another significant aspect

of this work is that all the multiplied

shoots developed roots in the same

medium itself and therefore, no need to

transfer them to separate root regeneration

medium. Since this plant appears to

have high potential in drug industry to

prepare high quality drugs on large

scale, supply of pharmaceutically

important raw :naterial can be ensuredthrough this Kind of rapid one step

micropropagation technique. Further

work is in progress on standardizing the

parameters for Agrobacte riurn mediatedtransformation using node explants as

starting material.

1. Ali G, Srivastava PS and Iqbal M,

Proline accumulation, protein pattern

and photosynthesis in Bacopa monnieri

regenrants grown under Nacl stress,

Bioi Plant, 1999, 42, 89-95.

The authors are thankful to Tamil

Nadu State Council for Science and

Technology(TNSCS&T),Chennai, India for

financial support.

Natural Product Radiance

References

No. of

explant(Mean:l:SE)

5.66:1:0.41

5.66±0.416.o0±0.41

6.33±0.35

5.66±O.41

5.33±0.41

8.33±0.41

8.66±o.41

9.33±O.41

9.66±0.41

9.60±0.40

8.66±0.41

15.66:1:0.41

16.33±O.41

16.33±0.41

17.33±0.41

16.66±0.41

15.66±0.41

16.33:1:0.41

16.66±0.41

17.33±0.41

18.00±0.35

17.66±0.41

16.66±0.41

14.66:1:0.41

14.66±o.41

15.33±O.41

15.66±0.41

14.66±0.41

14.33±0.41

96.3:1:0.41

96.3±0.41

96.3±0.41

92.6±0.41

92.6±0.41

92.6±0.41

92.6:1:0.41

92.6±0.41

96.3±0.41

92.6±0.41

96.6±0.41

92.6±0.41

92.6:1:0.41

92.6±0.41

96.3±0.41

96.3±0.41

100.0±0.35

92.6±0.41

96.3:1:0.41

96.3±0.41

96.3±0.41

92.6±0.41

100.0±0.35

92.6±0.41

96.3:1:0.41

96.3±0.41

96.3±0.41

100.0±0.35

96.3±0.41

92.6±0.41

% of explant responsefor shoot induction

(Mean:l:$E)

o

20

40

60

80

100

o

20

40

60

80

100

o20

40

60

80

100

o

20

40

60

80

100

o

20

40

60

80

100

Cone. of adenine

sulphate(mgll)

0.3+2.0

i0.2+ 1.5,

1

IAA+BAP 0.5+3.0

IAA+BAP 0.4+2.5

IAA+BAP

IAA+BAP

Auxin! Cone.

Cytokinin (mg/l)

Green l!!!!le: Researth Artit'e

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