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S L J Tea Sci 5 9 (2) , 62-64 ,1990 .

Printed

n Sri L anka

P R O G R E S S T O W A R D S T H E C O M M E R C I A L P R O P A G A T I O N

O F

T E A B Y T I S S U E C U L T U R E T E C H N I Q U E S

T. M.

Sa ra thchandra ,

P. D.

Upa l i and P. V . A ru lp ragasam

T e a

Res earch Inst i tute

o f S r i L anka ,

Talawakele ,

S r i

Lanka )

A tissue c ulture method is described for m icropropagation of tea using single node explants of

field grown plants of clone TRI 2025. Pre-treatment with PVP (polyvinyl pyrolidone) reduced

browning of explants and prevention of contamination was successful with 95% ethanol and

mercuric chloride. Nodal explants showed best establishment and growth response o n

modified MS medium containing BAP 2.5 mg

I

1

and IBA 0.1 mg

I '

1

.

Shoots and nodes

excised from 8-week-old established cultures showed about 2-3 fold multiplication every 6

weeks on modified MS medium containing BAP 2.5 mg

I

1

and IBA 0.01 mg

I

1

.

From

5 0

established cultures 36153 shoots were obtained after 12 months. Further attempts are being

made to improve the multiplication ability of the shoots of various clones and to induce root

formation.

INTRODU TION

C o n s i d e r a b l e p r o g r e s s

h a s

b e e n a c h i e v e d

in t h e

r e c e n t p a s t

in t h e

micropropagat ion

o f

m any herbace ous p lan ts (Longer , 1981 ) and f ru it t rees . How ever ,

s i m i l a r a d v a n c e s h a v e

n o t

b e e n a c h i e v e d

in t h e

p r o p a g a t i o n

o f

w o o d y p l a n t s

o f

e c o n o mi c v a l u e . W o o d y p l a n t s w e r e a s s u me d

to b e

intractable

in

cu l ture and research

wo rkers therefore pa id on ly scant a t ten t ion to them (Abbot , 197 7) .

A l t h o u g h t h e r e h a v e b e e n

a

few repor ts on the regenerat ion

o f C a m e l lia s i n e n s i s

f r o m c o t y l e d o n , e m b r y o , s t e m a n d l e a f c a l l u s , v e r y l i t t l e w o r k h a s b e e n d o n eo n

m i c r o p r o p a g a t i o n

o f t e a

u s i n g n o d a l e x p l a n t s a n d s h o o t t ip s .

T h e

p o s s i b i l it y

o f

regenerat ion o fshoots f rom nodal ex p lants o ft e a h a s b e e n r e p o rt e d b yP h u k a na n d

M i t r a ( 1 9 8 4 ) .

In v i t r o

proli feration

o f

shoots

o f

t e a h a s b e e n r e p o rt e d

b y

A r u l p ra g a s a m

and Lat i f f (1986) .

T h e a i m o ft h is s t u d y w a s tod e v e l o p a s u c c e s s f u l me t h o d fo re s t a b li s h me n to f

nodal exp lants o f

C a m e l lia s i n e n s is

in

v i t r o

wi thout b rowning a nd co ntaminat ion an d for

the m ass propagat ion o f shoots th rough ax i ll a ry branc h ing .

M A T E R I A L S A N D M E T H O D S

Plant mater ia l (c lone TRI 2025) was co l lected d i rect ly f rom the f i e ld . S ing le node

exp lants were prepared for cu l tu re b y removing the leaves wi th the pet io les . The nodal

exp lants

2 - 3

c m

in

s i z e , w e r e d i p p e d

in 1 . 5 %

PVP (polyvynyl pyrol idone)

f o r 3 0

m i n .

The in i t i a l sur face s ter i l i za t ion was done b yi mme r s i n g t h e m in 9 5 % e t h a n o l f o r 4 5

seconds, fo l lowed

b y a

second ster i l izat ion

in

0 .1

%

mercur ic chlor ide solut ion

f o r 1 5

min and then r insed 3t im e s inster i le d isti lled w ater . Establ ishm ent o fexp lants in

v i t r o

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was done on modi f ied

M S

(Mu r a s h ig e a n d Sk o o g , 1 9 6 2 ) m e d iu m (T ab l e

1 )

containing

V

2

strength M Ssa l ts ,0 . 4 m g I

1

T h i a m i n e H C I , 2 . 0 m g I

1

ascorbic a c i d , 1 0 m g I

1

m y o

inositol,3 0 g I

1

sucrose ,0 . 1 m g I

1

IB Aan d 2 . 5 m g I '

1

B A P .T h e p H w a sadjustedt o

5.3 before autoclaving.

After 8 w e e k s inculture 5 0 establ ished noda l explants were se lec ted fo r further

multiplication

( F i g . 1 ) .

Sh o o ts p r o d u c e d

b y

these explants we re asept ica lly rem ove d,

l e a v e s t r i m m e d

a n d

e a c h n o d e s e p a r a t e d

a n d

p l a c e d

o n M S 2

m e d i u m ( T a b l e

2 )

consisting o f V

2

strength M Ssal ts ,0 . 4 m g I '

1

T h i a m i n e H C I ,2 . 0 m g I '

1

ascorbic

acid ,

1 0 m g\~* myo- lnos i to l ,3 0 g I

1

sucrose , 0 .01 mgI

1

I BA a n d2 . 5m gI '

1

B A P a n d t h ep H

w a s a d ju s ted t o 5 . 3 before autoclaving. A ll cul tures were g iven 2000 L U Xlight intensity

a n d te m p e r a tu r e w a s m a in ta in e da t 2 4 2 Cinagrowth room wi th 1 6 hphotoper iod.

Subsequent ly ,

fo r

further shoot p rol iferat ion, s ingle n odes with axi llary b uds without

leaves w ere separa ted f rom solita ry shoots an d t ransfer red

a t 6

we ek in te rva ls .

T A B L E

1 - Th e compos i t ion o f med ium used fo r es tab l i shmen t o f noda l exp lan ts

o f t e a

Componen t

Concen t r a t ion m g l~)

Major e lements

Minor e lements

myo-lnosi tol

T h i a m i n e

- H C I

Ascorbic acid

Sucrose

IBA

B A P

Ag a r

half strength

1 0 0

0 .4

2 .0

3 0

g

0.1

2 .5

5 g

p H 5 . 3

* Murashige and Skoog (1962) minera l sa l ts

T A B L E 2 - Th e com pos i t ion o f m ed ium used fo r p ro l if e ra t ion

Componen t

Concen t r a t ion m g t

1

Major e lements

Minor e lements

myo-lnosi tol

T h i a m i n e

- H C I

Ascorbic acid

Sucrose

IBA

B A P

Ag a r

M S *

M S *

1 0 0

0 .4

2 . 0

3 0g

0 .01

2 .5

hal f strength

p H

5 . 3

* M urashige and Skoog (19 62) minera l sa lts

63

5 g

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RESULTS

A N D

DISCUSSION

P r e - t r e a t m e n t w i t h P V P b e f o r e s u r f a c e s t e r i l i z a t i o n r e d u c e d t h e b r o w n i n g

of

explants during the preparation for cul ture. However, once establ ished browning did not

occur when leaves and basa l ends were t r immed or nodes were separa ted for t ransfer .

O n t h e o t h e r h a n d , ith a s b e e n o b s e r v e d th a t th e p r e s e n c e ofascorbic ac id in the

m e d i u m g r e a t l y d e c r e a s e d o x i d a t i o n

of

p l a n t m a te r ia l

in

cul ture . S ince h igher sa l t

concentrat ion a lso caused browning and sometimes even k i l led plant t issues in previous

exp erim ents , reducing the sa l t concentrat ion to hal f strength could a lso b e a co ntr ibutory

factor in reducing the browning of t issues. Simi lar effect of low sal t concentrat ion in

A z a l e a s

w as a lso repor ted by And erson (1975 ) .

T h e e x p l a n ts e s ta b l i s h e d w e l l in m o d i f i e d MS m e d i u m . A f te r 8 weeks , the f i rs t

t r a n s fe r w a s d o n e to th e s a m e m e d i u m . Sh o o ts w i th 24 leaves were produced f rom

axi l lary buds of nodal explants after 6 weeks in cul ture. Wel l establ ished explants in the

i n i t i a l m e d i u m s h o w e d r a p i d p r o l i f e r a t i o n a f t e r t h e 2 n d a n d 3 r d t r a n s f e r o n t h e

p r o l i f e r a t i o n m e d i u m . Ho w e v e r , ifth e e x p l a n t b e a r i n g th e o r i g in a l b a s a l n o d e w a s

transfer red to f resh m edium , it a lso deve loped few addi tional shoots . The combinat ion of

a u x i n s a n d c y to k i n i n s in th e e s ta b l i s h m e n t m e d i u m h a savi ta l effect on th e ini tia l

es tabl ishment of the explants . The best combinat ion was 0 .1 mg

I

1

IBA and 2 .5 mg I

1

B A P Cal l i formed at the basal end of some cul tures were cut off before transference to

t h e p r o l i f e r a t i o n m e d i u m ( M S

2)

s i n c e t h e c a l l i r e t a r d e d a x i ll a r y b u d f o r m a t i o n

(Sara thchandra , Upa l i and Wi jewardena , 1988) . The tendency for ca l l i format ion a t the

basa l end was reduced apprec iably by decreas ing the aux in concentra t ion in MS 2 .

It

s e e m s th a t th e p r e s e n c e ofBAP in th e m e d i u m a n d t r im m i n gofthe leaf during the

transfer , induce d axi llary bu d formation an d elong ation; the axi llary shoot, reach ed

a

m a x i m u m l e n g th of 3 cm a f te r 6we eks . From 50 es tabl ished cul tures 361 53 shoots

were obta ined a f te r 12 months (F ig . 2 ) . In conc lus ion,

in v i t r o

propagation

of C a m e l l i a

s i n e n s i s

is feasible through axi llary bud p rol iferation. Further attempts are being m ad e to

improv e th e mult ipl icat ion abi li ty of the shoots of var ious c lones a nd to induce their root

formation. This work can be regarded as the f i rst successful organized study towards

the commercia l micropropagation of tea using nodal explants.

REFERENCES

ABBOT,

A. J .

1977) .

Propagating temperate

woodyspecies

in

tissue

culture.

Sc ient ia

Hor t . ,

28,

155 .

ANDERSON, W. C. 1 9 7 5 ) .Propagation of Rhododendrams bytissueculture Part1.Development

of a culture method for multiplication of shoots.

Proc. Int. PI. Prop.

2 5 , 1 2 9 - 1 3 5 .

ARULPRAGASAM, P. V. and

LATIFF,

R.

1986) .

Studies on the tissue culture of tea

Came l l i a

s inens is L.)

O. Kuntze). 1.

Development

of a culture method for the multiplication of shoots.

S.LJ. Tea Sc i.

55,44-47.

LONGER, B. V 1981) .

Cloning

of Agricultural Plants via nvi tro techniques, p. 273 Boca Raton,

Florida,

CRC Press.

SARATHCHANDRA, T. M., UPALI, P. D. and WIJEWARDENA, R. G. A.

1 9 8 8 ) . Studies

on the

tissue

culture of tea

Cam el l ia s inens is ,

L.) O. Kuntze) 4. Somatic embryogenesis in stem

and leaf callus cultures. S.

L. J. Tea Sci.

5 7 , 5 0 - 5 4 .

MURASHIGE,

T. and SKOOG, F.

1962) .

A revised medium for rapid growth and bioassays

with

tobaccotissueculture.Physiol. Plant . 15,473-497.

PHUKAN M. K. and MITRA, G. C

1984) .

Regeneration of tea shoots from nodal explants intissue

culture,

Cur ren t Sc i ence ,

53

16),

874 -876 .

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S.

L

J

Tea Sci 9

2), 62-6 4.

Printedin Sri

Lanka

Fig. 1

-

We l l es tab l ished noda l exp lan ts se lected

fo r

further m ultiplication

Fig.2 - Thousands o fshoots obta ined af ter 12 mon ths

T. M. Sarathchandra , P. D. Upali and P. V. Arulpragasam