g

J. Plant Physiol. 159.1317

-1321
(2002)
@ Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp
K
JOURNAT OF
T
PTANT PHYSIOLOGY
Factors affecting germination and conversion frequency of somatic
embryos of Tea
lCamellia
sinensis
(1.)
O. Kuntzel
Introducti on
The ever i ncreasi ng demand for a popul ar non-al cohol i c
beverage i.e. tea
lCamellia
srnensis (L.) O. Kuntzel of the
family Theaceae requires crop improvement through biotech-
nol ogi cal means, especi al l y because i mprovement through
conventi onal methods i s l i mi ted by several probl ems (Barua
1989) However, the prerequisite for such a beverage is an
effi ci ent and reproduci bl e regenerati on protocol .
Somatic embryogenesis is considered to be the most effi-
cient regeneration system for tea, yet it comes with serious
probl ems i ncl udi ng abnormal devel opment, poor or abnormal
germi nati on and l ow frequency of normal conversi on i nto
Tapan Kumar Mondal*, Amita Bhattacharya, Anil Sood, Paramvir Singh Ahuja
l nsti tute of Hi mal ayan Bi oresource Technol ogy, Pal ampur-176061, H. P, Indi a
Recei ved Jul y 31, 2001
.Accepted
June 19, 2002
Summary
The cause of poor and abnormal germination of tea somatic embryos was investigated with respect
to (a) the different factors that affect reserve mobilisation viz. chilling, desiccation or GA3 and (b)
those that affect the maturation process and reserve accumulation viz. ABA.
Tea somati c embryos were sensi ti ve to desi ccati on and thei r normal devel opment or germi nati on
coul d not be evoked by external agents l i ke chi l l i ng and GA3 Suppl ementati on wi th external sources
of nutrient precursors and readily available forms of carbohydrates like sucrose or maltose together
wi th trans-ci nnami c aci d i mproved the germi nati on of the somati c embryos si gni fi cantl y.
Key words: Camellia sinensis- germination
-
somatic embryos
-
tea
Abbrevi ati ons: ABA =
absci si c aci d.
-
GA3
= gi bberel l i c aci d
-
MS1 =
basal MS medi um suppl e-
mented wi th GAr.
-
t-CA
=
trans ci nnami c aci d
.
E-mail conesponding author: dihbt@csir.res in
I HBT Publ i kat i on Number: 2102
pl antl ets (Vi ei tez 1995). Whi l e ABA i s i mportant for the
accumul ati on of storage reserves and acqui si ti on of desi cca-
ti on tol erance duri ng embryo maturati on, normal germi nati on
requires mobilisation of the reserves that were accumulated
duri ng the maturati on process (Mari on-Pol l 1997)
In this study, we aim to investigate whether the (i) cause of
poor or abnormal germi nati on i s due to i nadequate reserve
mobi l i sati on or (i i ) due to the l ack of reserve accumul ati on
and desi ccati on sensi ti vi ty duri ng the embryo maturati on
phase Different factors affecting (a) reserve mobilisation
such as chi l l i ng, desi ccati on or GA3 and (b) the maturati on
process and reserve accumulation such as ABA have, there-
fore, been investigated. We hope to resolve these problems
by suppl ementi ng mobi l i sed reserves i nto the germi nati on
medi um for a hi gh frequency of normal conversi on of tea
somatic embryos.
0176-1611021159/12-1317 $ 15 00/0
1318 TaPan Kumar Mondal et al
Materi al and Methods
suppl ement ed
wi t h 433pmol / L GA3 (MS1) Al l experi ment s
were re-
peated thrice and cultures were maintained aI25' C f 2"C under cool
f l uorescent l i ght (52pmol m-' s
t )
wi t h a phot operi od of about 12h'
A| | obser vat i onsWer et aKenat r egul ar i nt er va| sof 15daysaf t er t he
initial period of treatment and the maximum number of embryos that
underwent normal maturatron and germination were recorded after a
total period of 90 days While the true torpedo shaped
green embryos
wer econsi der edasnor mal , mat ur eembr yos, ger mi nat i onWaSchar -
acterised by the emergence of a radicle wrth simultaneous
greening
and elongation of hypocotyl coupled with the opening of the coty-
ledonarY leaves.
Thet r eat ment st hat Wer eusedf or i ncr easi ngt heconver si onf r e.
quency of somatic embryos are as follows
Treatments
for reserve mobilisation
Desiccation
the control
Chi l l i ng
Globular somatic embryos inoculated on EIM in sealed Petri dishes
were chi l l ed at 4"C under dark condi t i ons f or peri ods of 15' 30' 45' 60
or 90 days. The embryos that were maintained on EIM at 25' C lor
similar periods, prior to their transfer to MSO and MS1 media served
as the control
GAr
Normal
germination is generally
ght about by GA3 through the
action of cr-amylase on the mobi on of starch stored during the
embryo maturation
phase Therefore,
globular somatic embryos were
treated for different
periods of 3, 4 and 5 weeks with different concen-
t r at i onsof f i l t er st er i l i sedGA3( O29pmoUl ' 144pmol / L' 289pmol / L'
14 44Pmol / L) i n EI M
Treatment for reserve accumulation
during normal
embryo maturation Process
ABA
The globular somatic embryos were treated for 3' 4 or 5 weeks with
diffeLnt concentrations
of f ilter sterilised ABA ( 1 89 pmol/L' 9 46 pmol/
L, 47.29pmollL,236.47VmollL)
in EIM in order to test whether ABA in-
fluenced the normal embryo development' acquisition of desiccation
tolerance and subsequent
germination
Supplementation
of mobilised reserves to germination
medium
Sucrose
Suc r oseal 3%, 6%019%WaSaddedasSupp| ement t ot heE| Mi nor -
der to understand its influence on normal embryo development'
matu-
ration and germination
Mal tose and trans-ci nnami c
aci d
Effect of filter sterilised maltose at 3, 4 or 5 % either alone or in combi-
nat i onwi t ht rans-crnnamrcaci d(918pmol / L' 1836pmol / L' 36' 72pmol l
L) for 3, 4 or 5 weeks was tested on the normal development and
ger-
mination of the somatic embryos
Acclimatisation
and f ield transfer
Germi nat ed embl i ngs, when 2-3cm hi gh' were t ransf erred t o Hi kko-
trays containing a mixture of soil: sand: farm yard manure in the ratio
of 9: 1 : 1 (pH 5 6), accl i mat i sed i n hardeni ng chambers f or 8 mont hs
and finally were transferred to a green house in poly sleeves
Statistical analYses
Data were transformed using square root transformation
for all experi-
ments and then analyseo using Analysis of Variance
(ANOVA) Dun-
can' s Multiple Range Test was used in all experiments
to test the dif-
ferences amongst the means at P<0 05
Resul ts and Di scussi on
Treatments
for reserve mobilisation
for normal and
higher
germination
Desiccation
Dryi ng
(up to 65 %) and death
(51-72"/") coupl ed wi th
poor
gei mrnati on (1.2 to 1 6ok) of the embryos were observed
wi thi n a week when desi ccated for 3 weeks under hi gh rel a-
ti ve humi di ty created by ei ther of the chemi cal s
Al though
desi ccati on
re-di rects the bi ochemi cal
and
physi ol ogi cal
pathways towards
germi nati on (Kermode et al 1986) and i n-
creases the sensi ti vi ty of the devel opi ng
embryos to GA3
(Le-
Page-Degivry
et al. 1990) the adverse effect of desiccation
1320 Tapan Kumar Mondal et al.
of ABA. Despite the fact that ABA plays an important role in
accumulation of storage reserves and acquisition of desicca_
ti on tol erance duri ng embryo devel opment and maturati on,
ABA arrested normal development of the embryos as has
been observed i n hybri d l arch, (Lel u and Label 1994), whi te
spruce (Artree et al. 1991) and mango (pliego_Alfaro
et al.
1996).
Effect of supplementation of mobilised reserves to the
germi nati on
medi a
Sucrose
The concentration of sucrose in the culture medium and the
duration of the culture treatment affected the germination
of
the embryos. Maximum germination (29 and 35
o/o)
was ob_
served after 4 and 5 weeks of treatment on 3 % sucrose ano
this decreased with any increase or decrease in sucrose con_
centration or the period
of treatment. Starch reserves in the
embryos are important for successful germination (Bewley
and Bl ack 1985). In thi s case, sucrose probabl y
substi tuted
for the lack of starch reserves Osmotic shock due to higher
concentrations of sucrose is probably
the reason that em_
bryos did not germinate
at these concentrations.
Mal tose and trans-ci nnami c aci d
Whi l e none of the untreated embryos germi nated,
a si gni fi _
cantl y (P<0.05) l ow percentage
of germi nati on (3_6%) was
observed when any concentration of either maltose or lrans_
ci nnami c aci d al one were used. However, normal and si gni fi _
cantl y hi gh germi nati on percentages
of about 70.6 %
(P <0.05) were observed when the embryos were cul tured
on a medi um suppl emented wi th trans-ci nnami c aci d
(18 36 pmol /L) and mal tose (4%) for 4 weeks fol l owed by
transfer to MS1 medi um (Fi g 1 B, C). Thi s i s at par wi th ger_
mination results after treatments for 3 and 5 weeks (Fig. 2).
The success achieved with maltose and its superiority over
sucrose probably is due to the difference in their breakdown
products.
Maltose, as compared to sucrose, is not onlv bro_
A
0 uM t-CA
*
o
o
.E
tr
o
(,
@
Itr3o/"
1.4v"
l sso/o
9.18 uM t-CA
I
c
e
6
c
E
o
ct
18.36 ull tA
s
E
.9
6
q
E
o
(,
UM t.CA
*
g
o
6
s
F
o
gt
Figure2' Effect of different concentrations of maltose in combination with trans-cinnamic acid on the germination
of somatic embryos (Data were
transformed by square root transformation prior to graphic representation. Different letters in superscript above the bars indicate statistically sig-
nificant differences at P<0 05)
on tea somati c embryos i s probabl y due to thei r sensi ti vi ty to
desi ccati on.
Chi l l i ng
Chi l l i ng general l y i ncreases the l evel s of GA3 and GA l i ke
substances and bri ngs about a-amyl ase medi ated mobi l i sa-
ti on of starch reserves for embryo germi nati on (Wood 1982).
Germi nati on (4.8 and 10 I %) was recorded i n tea embryos
that were treated at 4'C for 30 or 45 days as compared to
very l ow (2%) or no germi nati on i n the untreated somati c em-
bryos or in the ones that were treated at 4
"C
for 15 or 60
days. However, i ncrease i n the ti me of chi l l i ng treatment
beyond 15 days resulted in increase in vitrification and a si-
mul taneous decrease i n the normal devel opment of torpedo
shaped embryos
Germi nati on of somati c embrvos from Tea 1319
GAr
As compared to the GA3 untreated embryos (control ), whi ch
fai l ed to germi nate, but remai ned fresh, the treated ones un-
derwent bl eachi ng and necrosi s at al l concentrati ons of GA3
used. GA3 brings about de novo synthesis of oc-amylase and
hydrolases for the mobilisation of starch reserves and normal
embryo germi nati on (Bewl ey and Bl ack 1985). Yet, GA3 fai l ed
to mobi l i se reserves duri ng germi nati on because the requi si te
reserves probabl y had not accumul ated duri ng the embryo
maturation phase.
Treatment for reserve accumulation
ABA
No embryo germination was observed in any of the untreated
embryos or in those subjected to the different concentrations
D c
Figurel. A Globular secondary somatic embryos derived form tea cotyledons B Normal germination of tea somatic embryos treated with mal-
tose and trans-cinnamic as compared to untreated control C Emblings derived from germinated somatic embryos of tea. D Hardened healthy
plants with normal leaves and vigorously growing root system derived from somatic embryo germination
ken down more sl owl y, bui al so can provi de a steady suppl y
of more readi l y metabol i sabl e carbon source (gl ucose) to the
embryos. Thi s provi des the requi si te metabol i tes requi red for
germi nati on i n vi ew of the fact that the embryos
probabl y
l acked the stored carbohydrate reserves due to thei r i neffi -
ci ency i n accumul ati ng these reserves duri ng thei r maturati on
Dnase.
Ci nnami c aci d i s an i mportant compound i nvol ved i n a
wi de vari ety of metabol i c
pathways l i ke the fl avonoi d bi osyn-
thesi s
pathway, the phenol i cs synthesi s
pathway and, most
i mportantl y, i n the synthesi s of Mal onyl -CoA, the key pre-cur-
sor of fatty acid synthesis
pathway An absolute requirement
for a speci fi c combi nati on of mal tose and t-CA i n the case of
tea i n thi s study further i ndi cates the cri ti cal requi rement of
sugars and fafty aci ds duri ng maturati on and subsequent
germi nati on.
Accl i mati sati on and fi el d transfer
Pl antl ets wi th young l eaves and stout roots (Fi g 2D) grew up
to a hei ght of about 4-5 cm wi thi n 6-8 weeks of transfer to
Hi kkotrays, whi ch, upon transfer to pol y-sl eeves after B
months. devel oped i nto heal thy and vi gorousl y
growi ng
ol ants wi thi n a
Year
Concl usi on
The somati c embryos of tea, Ii ke thei r zygoti c counterparts'
appear to exhi bi t desi ccati on sensi ti vi ty and fai l to accumu-
l ate storage reseryes Fai l ure of chi l l i ng and GAs to cause a
hi gher percentage of normal l y devel oped embryos and thei r
germi nati on i s i nteresti ng as thi s i s i ndi cati ve of the faci that'
si nce reserves were not stored by the embryos duri ng the
maturati on
phase, thei r mobi l i sati on cannot be brought about
by external agents
l mprovement of the conversi on frequency by suppl ements
from external sources l i ke the readi l y avai l abl e forms of car-
bohydrates such as sucrose and maltose further prove that
tea somati c embryos fai l to accumul ate carbohydrates i n the
form of storage reserves Al though ABA hel ps i n the accumu-
l ati on of reserves and i n the acqui si ti on of desi ccati on tol er-
ance by the embryos duri ng the maturati on
phase, fai l ure of
Germination of somatic embryos from Tea 1321
ei ther to take
pl ace i n thi s study i s probabl y because of appl i -
cati on of ABA duri ng phases other than embryo maturati on'
Acknowledgements.
The authors acknowledge the kind help of Dr
Subedar Pandey for the statlstical analyses and Tapan lvlondal ac-
knowledges the financial assistance of the University Grants Commis-
si on duri ng t he course of t hi s st udy
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