in vitro mass propagation of andrographis lineata

J. S W A M Y B O T - C L . 25 : 1 - 10 ( 2 0 0 8 ) PRINTED IN INDIA. ALL RIGHTS RESERVED

IN VITRO MASS PROPAGATION OF ANDROGRAPHIS LINEATA NEES. THROUGH NODAL EXPLANT - AN IMPORTANT ETHNOMEDICINAL PLANT. A LAKSHMIPRABHA, B.R CHANDRASEKAR, V. NANDAGOPALAN AND N. JAYABALAN. Department of Plant Science, Bharathidasan University, Tiruchirappalli - 620 024. •Department of Botany, National College, Tiruchirappalli. 'Corresponding author - [email protected]

ABSTRACT In this present investigation, in vitro mass propagation was successfully achieved using developed through nodal segments of Andrographis lineate. It is widely used for the treatment of liver ailments. The explants were tested with different medium, amino acids, carbohydrates in various concentration and combination with plant growth regulators. The highest percentage (94.2%) of proliferation of multiple shoots were obtained by placing nodal segments on MS salts with B vitamins (mMS) supplemented with 1.5 mg/L 2ip in combination with 0.6 mg/L Kin and Glutamine (20 mg /L) which produced 7.8 shoots per explant. The elongated shoots were excised and transferred onto mMS medium supplemented with various concentrations (0.5 to 3.0 mg/L) of IAA, IBA and NAA. Among the three auxins 2.0 mg/L IB A supplemented medium produced highest percentage (95.6%) of root length. The well rooted plantlets were transferred to pot containing garden soil and farm yard manure (2:1) and placed in environmental plant growth chamber for proper acclimatization and subsequently transfer to the green house condition. The in vitro propagated plants were established successfully in vivo and the survival rate was 78%. 5

Key

wards:

cytokinin,

Mass propagation,

nodal segments,

Andrographis

lineate,

mMS

medium,

Amino

acids,

auxins.

INTRODUCTION Medicinal plants, since time immemorial have been used in virtually all civilization as a source of medicine (UNESCO, 1996). In the world medicinal market, India plays a vital role after China in the herbal medicine preparation. Andrographis lineate is a medicinally important erect herb widely found in South India, belonging to the family Acanthaceae. Most of the species of Andrographis were widely used in Indian systems of medicine as a stomachic, tonic, antipyretic, alterative, febrifuge and c h o l a g o g u e ; for liver disorders, general debility and colic pains (Bentley and Trimen 1983; Gupta el al, 1990; Aminuddin et ah, 1997). Andrographolide, neoandrographoloide and Kalmeghin are some of the active principles present in the plant (Handa et al., 1986). Now-a-days, for the preparation of required drugs, several medicinal plants are cultivated in the agriculture lands for large scale production as effectively achieved in Catharanthus rosesus,

Ocimum sanctum etc., (Chu and Kurtz, 1990). The cultivation of some endangered medicinal plant species in agriculture land is found to be difficult due to several problems, like loss of seed viability, unfavourable situation for seed germination and damages caused by insect pests. The plant breeding techniques are widely used for the improvement of economically important crop plants, e s p e c i a l l y the plants with low adaptation to different environment conditions. The development of medicinal plants with required character also faces several practical problems. Hence, the scientists are searching for alternative tool or method for improvements in medicinal plant cultivation. Recent advances in medicinal plant cultivation proved that large scale multiplication of economically important plant through in vitro regeneration (Hussey, 1993; Varshney et al., 2000) is feasible. Several economically important medicinal plants are cultivated all over the world for their high c o m m e r c i a l i m p o r t a n c e based on their herbal

LAKSHMIPRABHA, ET AL,

medicine production but reports on large-scale multiplication of medicinal herbs are scarce (Shah et al., 1988). Based on the literatures available, we find that Andrographis lineate as one such potential plant needs more exposure to society.

multiple shoot proliferation, i.e., Alanine, Glutamine, Serine and Proline (10, 20, 30, 40 and 50 mg/L) (Himedia Laboratories Ltd. India). The experiments were carried out u s i n g a m i n o acids with best concentration of BAP, 2ip and Kin for obtaining more number of multiple shoots from nodal explants.

MATERIALS AND METHOD Rooting and Hardening

Plant Material and explant preparation

Well elongated single shoots were excised and transferred to various concentrations of auxins containing medium for root induction. The medium containing various concentrations of IAA, IBA and NAA (0.5 to 3.0 mg/L) were used to optimize the concentration of auxins for root induction. The well rooted plantlets were transferred to the pot c o n t a i n i n g garden soil and farm yard m a n u r e mixture in 2:1 ratio. Then the pots were kept in the environmental plant growth chamber in the relative humidity and temperature for proper acclimatization of the in vitro raised plants. Then the plants were transferred to the green house and later to the field condition and the survival rate was calculated.

Whole plants of Andrographis lineate was collected from the forest area of kolli hills,Tamil nadu and maintained in the green house condition for explant sourcing. The nodal explants were used as an explant for further mass propagation studies. The nodal explants were cut into 0.5 to 1.0 cm sizes. Then the e x p l a n t s were washed thoroughly in the tap water 3 to 5 times, followed by soaking in 2% soap solution (Teepol) for 2 to 6 minutes and washed in running tap water for 10 to 30 minutes followed by washing in distilled water for 3 to 5 times. Then the nodal explant was treated with 7 0 % ethanol for 30 s e c o n d s and followed by 0 . 1 % (W/V) Mercuric chloride (HgCL,) for 3 to 10 m i n u t e s . After the d e c a n t i n g the mercuric chloride solution, the explants were rinsed 3 to 5 times with sterile distilled water. The surface sterilized explants were placed in MS medium with B vitamins (mMS) medium containing various concentration and combination of plant growth regulators (BAP, 2ip and Kin) for standardizing the mass p r o p a g a t i o n protocol for Andrographis lineate.

Statistical analysis All experiments consisted of 20 explants and the experiments were repeated in triplicate. The cultures were observed periodically and percentage of r e s p o n s e was r e c o r d e d . A c o m p l e t e by r a n d o m i z e d block design w a s used in all experiments and analysis of the variance and mean separation were carried out using Duncan's Multiple Range Test ( D M R T ) and the significance was determined at 5% level (Gomez and Gomez, 1976).

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Cytokinins, Medium and Carbohydrate The MS medium was supplemented with various cytokinins like BAP, 2ip (0.5 to 1.75 mg/L) and Kin (0.2 to 1.0 mg/L) alone and in combination for multiple shoot induction and proliferation. After standardizing the hormone concentration the explant was tested in various mediums like M S , Nitsch, mMS and Streward. Then different types and different c o n c e n t r a t i o n of c a r b o h y d r a t e s w e r e tested in the form of sucrose, maltose, fructose and glucose (10, 20, 30, 40 and 50 mg/L).

Shoot multiplication from nodal explants from green house grown plants which were often prone to contamination, responded more favourably with the formation of high frequency than other explants. Results of this study show that nodel explants p o s s e s s a higher s h o o t o r g a n o g e n i c potential of the green house grown plants (Sudha et al, 2005).

Amino acids

Influence of culture Media

RESULTS AND DISCUSSION

The following amino acids were used to

and carbohydrates

The explants were testdd with four different medium like M S , m M S , Strewart and

study the influence of various amino acids for

2

IN VITRO MASS PROPAGATION OF ANDROGRAPHIS LINEATA

Nitsch. Among the four mediums the mMS medium showed highest percentage of response ( 9 2 % ) , followed by MS m e d i u m , Nitsch medium and Strewart medium where in they have showed percentage r e s p o n s e of 7 8 , 60 and 4 2 % respectively (Fig.- 1). Other media tested showed very low frequency and reduced percentage of multiple shoot induction and still lower number of nodes per shoot with significantly high frequency of abnormalities. To support our results, most of the medicinal plants had superior regeneration percentage in MS + B vitamin treated culture (Juliani et al, 1999; Bais et al, 2000; Kelley et al, 2002, Nuri, 2004).

mg/L) showed highest mean number of shoots (6.6) per e x p l a n t , followed by B A P ( 1 . 0 m g / L ) in combination with Kin (0.6 mg/L) which showed 83.8 percentage response with 4.6 shoots per explant (Table - 1). At the end of shoot multiplication stage, there was a significant influence of the hormone type used and the c o n c e n t r a t i o n w h i c h play important role on number of multiple shoots and shoot elongation. Between the different cytokinins treatments used in this study, the combination of both cytokinins has proved to be superior to the use of each cytokinin alone in other species too. Likewise high frequency of multiple shoot induction has been observed in Syzygium alternifolium by using BAP, Kin and 2 i p (Khan et al., 1999). Similarly the combinations of two cytokinins in Vicia faba and Phaseolns vulgaris were effective in inducing direct multiple shoot proliferation from shoot tip and nodal explants (Mohamed et al., 1992; Khalafalla and Hottori, 1999).

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The carbon sources also played a very important role in the tissue culture studies. Several tissue culture reports refer to the influence of carbon sources on the in vitro morphogenesis of different plant species. Among the many available carbon s o u r c e s , s u c r o s e has been extensively reported (Peterson and Smith, 1999). The carbon sources were supplied in the form of carbohydrates like sucrose, m a l t o s e , fructose and glucose in different ranges (10, 20, 30, 40 and 50 mg/L) to the mMS medium. Among the carbohydrates with various c o n c e n t r a t i o n t e s t e d , the highest percentage of response was found in sucrose 30 mg/L (88%) followed by maltose, glucose and fructose (58, 46 and 29%) respectively (Fig.-3). Influence of carbohydrates, in this study agrees with similar findings in other plants (Nhut, 1998, Khalafalla and Hatlori, 1999; Benson, 2000 and Mialoundama et al, 2002).

Influence of Amino acids Usually, in tissue culture experiments, the amino acids are used as nitrogen source. These nitrogen s u p p l e m e n t s are involved in the production of metabolites which are used in plant growth development (Bais et al., 2000). After the identification of suitable cytokinin combination for multiple shoot induction, we planned to improve the multiple shoot induction percentage by means of addition of amino acids along with multiple shoot induction media. Different concentration of alanine, glutamine, serine and proline were tested. Among the different concentrations tested, the improved multiple shoot induction was recorded in glutamine (20 mg/L) treated cultures. In this concentration, maximum of 7.8 shoots were initiated with 9 2 % r e s p o n s e from the n o d a l e x p l a n t s . In other concentrations too increased multiple shoots were r e c o r d e d and a m o n g t h e m glutamine showed superior response. Next to glutamine treatment, proline showed best response and produced 5.8 shoots per explant. Apart from the multiple shoot induction, the percentage of basal callus formation was low and completely absent in glutamine treated explants. At the same time, it was more in other amino acid treated cultures (Fig. - 2).

Influence of cytokinins The explants of Andrographis lineate nodal explant were tested with various concentration and combination of cytokinins like BAP, 2ip and Kin. Among the different concentration cytokinins alone (2ip 1.25 mg/L) showed highest percentage of response ( 8 0 . 2 % ) with 2.9 shoots per explant followed by BAP 1.0 mg/L and Kin 0.6 mg /L which showed 79.8 and 60.4 percentage with 2.4 and 1.6 shoots per explant respectively. To improve the multiple shoot induction percentage, the effect of combination a m o n g the cytokinin was t e s t e d . Among the cytokinin 2ip (1.5 mg/L) with Kin (0.6

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LAKSHMIPRABHA, ET AL., Similar to our o b s e r v a t i o n , glutamine mediated enhanced multiple shoot formation was noticed in several d i c o t p l a n t s . In Daphen cneorum, the n u m b e r of multiple shoots were increased up to 12 by g l u t a m i n e (20 mg/L) supplementation along with multiple shoot induction medium (Mala and Bylinsky, 2 0 0 4 ) similarly, glutamine (25-250 mg/L) mediated high frequency of multiple shoot induction was also achieved in Morus alba and M. clustralis (Anis et al., 2003), Prosopis (Green et al., 1990) and in Santalum album (Rao and Bapat, 1993). In most of the plant tissue culture studies, the amino acids were used primarily for callus induction and somatic embryogenesis studies (Xie and Hong, 2001). Our results proved that, addition of amino acids also accelerates the plant recovery percentage through direct organogenesis (Thorpe, 1993). Rooting and hardening The e l o n g a t e d shoots w e r e separated individually and transferred to rooting medium. The medium containing various concentrations of IAA, IB A and NAA (0.5 to 3.0 mg/L) was used for

optimizing the concentration of auxins for root induction. Of the three auxins, the effective root induction were achieved after two weeks by using 2.0 mg/L IBA treatment in the medium fortified with MS salts, B vitamins, sucrose 30 g/L and agar 7 mg/L at pH 5.8. Root induction from the elongated shoots was also obtained by using NAA, IBA and IAA. In IBA (2.0 mg/L) maximum percentage of r e s p o n s e ( 9 5 . 6 % ) with r o o t s per shoot were successfully initiated from the elongated shoots having an average of 3.4 cm in length (Table - 2). 5

Well rooted plantlets were transferred to the pot containing garden soil and farm yard manure in the ratio of 2:1 for h a r d e n i n g p r o c e s s . T h e hardened pots were kept in the environmental plant growth chamber for proper acclimatization. After three weeks the pots were transferred into the green house condition for further growth and later were established in the field and the survival percentage was 7 8 % . Our results reveal that in vitro mass propagation of Andrographis lineate through nodal culture is feasible for the mass p r o d u c t i o n of plantlets for further field cultivation.


Table - 1:

Effect of different concentrations of cytokinins and combination on shoot induction from nodal explants of Andrographis lineate.

Plant growth regulators mg / L BAP

2ip

% of response

Mean No. of shoot / explants

52.4°

1.5*

Kin

0.50 0.75

-

72.6'

1.8"

1.00

79.8*

2.4

125

62.4'

2.0

1.50

50.4P

1.4"

58.6""

2.6

0.50 0.75

K

64.8

1.00

72.8

125

80.2 *

1.50

69.8'

HL

F

1.75

1

M

K

2.8"

3.2

h

2.9' M

2.0

LM

1.8"

B

0.8'

PTL

IJC

60.6

020

44.4

0.40

49.8

0.60

60.4

LM

i.6°

0.80

50.8*

1.4"

R

1.00

46.6

OiO

0.6

79.2*

0.75

0.6

8I.0

1.00

0.6

1.0"

2.4

1

F

3.2

H

83.8

E

4.6

F

H

3.2

H

125

0.6

79.0

1.50

0.6

72.8*"

2.8*

0.75

0.6

82.5*'

52

1.00

0.6

89.6*

5.6

125

0.6

90.4

C

5.4*

A

6.6

A

AB

4.8

E

1.50

0.6

94.2

1.75

0.6

92.6

2.00

0.6

88.6

D

D

B

4.0s

Values are mean 20 explants per treatment and repeated three times. Mean values within a column followed by different letters are significantly different from each other at 5 % level comparison by DMRT.

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LAKSHMIPRABHA, ET AL.,

Table - 2:

Effect of different concentrations of auxins on root induction of elongated shoots obtained from nodal explants of Andrographis lineate.

Plant growth regulators mg/L BAP

2ip

% of response

Mean No. of shoot / shoot

Root length (cm)

Basal Callus

Kin

00 0.5 J

1.0

32.0

1.5

30.5* h

2.0

36.8 '

2.5 3.0 0.5

h

1.2

1.2

e

1.4*

0.8«

k

28.4

0.6

h

0.4

1.9*. 1.5

f

0.8*

e

1.4*

1.0

62.48"

i.2

1.8

C

1.8

+ ++

f

18.6'

dc

h

++

e

1.0

76.6

1.5

90.8

b

2.0

b

3.0

b

2.0

95.6

a

2.4

a

3.4

a

2.5

89.8

bc

1.6

d

2.8

C

3.0

78.2

d

1.2

e

2.0

d

1.0

59.4

h

1.5

70.8

2.0

64.28

+

0.5

2.5

0.88

f

1.2

67.8*

o.6

u

e

i.2«

0.9J

+

f

k

+

1.0

0.7

3.0

Values are mean 20 explants per treatment and repeated three times. Mean values within a column followed by different letters are significantly different from each other at 5 % level comparison by DMRT

6


Figure - 1.

Effect of different media on multiple shoot on nodal explants of Andrographis

lineate.

100 90 80 70

8 co

60

|

50-

o

5

4030 20 10 0 MS

mMS

Strewart

7

Nitsch

LAKSHMIPRABHA, ET AL.,

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