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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

    62

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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 .

    64

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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


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