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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