Factors Affecting Germination and Conversion...

8/11/2019 Factors Affecting Germination and Conversion...

1/5

g

J.

Plant hysiol.

59.1317

1321

2002)

@

Urban Fischererlag

http://www.urbanfischer.de/journals/jpp

K

JOURNATF

T

PTANT HYSIOLOGY

Factorsffecting erminationndconversionrequencyf somatic

embryos

f

Tea

Camellia

inensis

1.)

O.

Kuntzel

Introduction

The ever increasing

emand or a

popular

non-alcoholic

beverage .e. tea

lCamellia

rnensis

L.)

O.

Kuntzel

of the

familyTheaceaeequires rop mprovementhroughbiotech-

nologicalmeans,

especially ecause mprovement

hrough

conventional ethodss l imited

y several

roblemsBarua

1989) However,

he

prerequisite

or

such a beverage s

an

efficient nd eproducible

egeneration

rotocol.

Somatic

mbryogenesiss considered

o be the mosteffi-

cient regeneration

ystem

or

tea,

yet

it

comes with serious

problems

ncluding

bnormal evelopment,

oor

or abnormal

germination

nd

low

frequency f normal

conversionnt o

Tapan

umar ondal*,mita hattacharya,

nilSood, aramvir

ingh huja

lnstitute

f Himalayanioresourceechnology,

alampur-176061,.P, ndia

Received

uly 1, 001

Accepted

June 9,

00 2

Summary

The

causeof

poor

and abnormal

ermination

f tea somaticembryoswas nvestigated

ith respect

to

(a)

the different actors hat affect eserve

mobilisation iz.

chilling, esiccation

r GA3and

(b)

those

hataffect he maturation

rocess

nd

reserve

ccumulation iz.

ABA.

Teasomatic mbryos ere

sensit iveo desiccation

nd heirnormal

evelopmentr

germination

couldnot

be evoked y external gents ike

hilling nd

GA3Supplementationith

external ources

of nutrient

recursors

nd readily vailableorms

of carbohydratesike

sucrose

r

maltose

ogether

with rans-cinnamic

cid

mproved

he

germination

f hesomatic mbryos

ignificantly.

Key words:

Camellia inensis-

germination

somatic mbryos

tea

Abbreviations : BA

=

abscisic cid.

GA3

=

gibberellic

cid

-

MS1

=

basalMS medium

upple-

mented

ith

GAr. t-CA

=

trans innamic cid

.

E-mail onesponding

uthor: [email protected]

IHBT ubl ikat ion

umber: 102

plantlets Vieitez

1995).While ABA is important

or

th e

accumulation

f storage eserves

nd acquisit ionf desicca-

tion olerance uring mbryomaturation,

ormal

ermination

requiresmobilisation

f the reserves

hat

were

accumulated

during hematurationrocessMarion-Poll997)

In

thisstudy,we

aim o

investigate

hether he

(i)

causeof

poor

or abnormal

ermination

s

due o

inadequate

eserve

mobilisation

r

(ii)

due to the lack of reserve

ccumulation

and desiccation

ensitivity uring he embr yo maturation

phase

Different actors

affecting

a)

reserve mobilisation

such as chilling, esiccation

r GA3and

(b)

the maturation

process

and reserve ccumulation

uch as ABA have, here-

fore,

been nvestigated. e hope

o resolve hese

problems

by s upplementing obilised eserves

nto he

germination

medium or

a high frequency f normal

onversion f tea

somatic mbryos.

0176-1611021159/12-13171500/0


2/5

1318

TaPan

umar

Mondal

t

al

Material

nd

Methods

supplemented

ith

433pmol/L

GA3

MS1)All

experiments

ere

e-

peated hrice

and

cultures

ere

maintained

I25'C

f

2 C

under

ool

f luorescent

ight

52pmolm-'s

)

with

a

photoperiod

fabout

2h '

A| |observa t ionsWere taKenat regu la r in te rva |so f15daysaf te r the

initial

eriod

of

treatment

nd

he

maximum

umber

f

embryos

hat

underwent ormalmaturatronnd germinationere ecorded ftera

total

period

f

90

days

While

he

rue

orpedo

haped

green

embryos

wereconsideredasnormal ,matureembryos,germina t ionWaSchar-

acterised

y

the

emergence

f

a

radicle

wrth

simultaneous

reening

and

elongation

f

hypocotyl

oupled

with

he

opening

of

the

coty-

ledonarY

eaves.

Thet rea tmentstha tWereusedfor increas ing theconvers ion f re .

quency f

somatic

mbryos

re

as

ollows

Treatments

or

reserve

obilisation

Desiccation

the

control

Chil l ing

Globular

omatic

mbryos

noculated

n

EIM

n sealed

Petri

dishes

were

hi l led

t

4 C

under

ark

ondit ions

or

periods f

15'

30'

45'

60

or 90 days.The embryos hatweremaintainedn EIMat 25'C lo r

similar

eriods,

rior

o

their

ransfer

o

MSO

and

MS1

media

served

as

he

control

GAr

Normal

germination

s

generally

ght

about

by

GA3

hrough

he

action

of

cr-amylase

n

the

mobi

on

of

starch

stored

during

he

embryo

maturation

hase Therefore,

lobular omatic

mbryos

were

treated

or

different

eriods f 3,

4 and

5

weeks

with

different

oncen-

t ra t ionso f f i l te rs te r i l isedGA3(O29pmoUl '144pmol /L '289pmol /L '

14

44Pmol/L)

n

EIM

Treatment

or

reserve

ccumulation

uring

normal

embryo

maturation

Process

ABA

The

globular

omatic

mbryos

were

reated

or

3'

4 or

5 weeks

with

diffeLnt

concentrations

f

lter

terilised

BA

1

89

pmol/L'

46

pmol/

L,

47.29pmollL,236.47VmollL)

n EIM norder o testwhether BA n-

fluenced

he

normal

mbryo

development'

cquisition

f

desiccation

tolerance

nd

subsequent

ermination

Supplementation

f

mobilised

eserves

o

germination

medium

Sucrose

S u c r o s e a l 3 % , 6 % 0 1 9 % W a S a d d e d a s S u p p | e m e n t t o t h e E | M i n

der

o

understand

ts

nfluence

n

normal

mbryo

evelopment'

atu-

ration

nd

germination

Maltose

nd

rans-cinnamic

cid

Effect

f

ilter

terilised

altose

t

3,

4

or 5

%

either

lone

or

n combi-

nationwithtrans-crnnamrcacid(918pmol/L'1836pmol/L'36'72pmo

L)

or

3,

4 or

5

weeks

was

ested

n

he

normal

evelopment

nd

ger-

mination

f he

somatic

mbryos

Acclimatisation

nd

eld

ransfer

Germinated

mblings,

hen

2-3cm

high'

were

ransferred

o

Hikko-

trays

containing

mixture

f soil:

sand:

arm

yard

manure

n the

ratio

of

9:

1

1

(pH

5

6),

accl imat ised

n

hardening

hambers

or

8

months

and inallywere ransferredo a greenhouse

n

poly

sleeves

Statistical

nalYses

Data

were

ransformed

sing

square

oot

ransformation

or all

experi-

ments

and

then

analyseo

sing

Analysis

f

Variance

ANOVA)

Dun-

can's

Multiple

Range

Test

was

used

n all

experiments

o test

he

dif-

ferences

mongst

he

means

t

P


3/5

1320

Tapan

Kumar

Mondal

t al.

of ABA.

Despite

he

fact

that

ABA

plays

an

important

ole

n

accumulation

f

storage

eserves

nd acquisition

f

desicca_

tion

olerance

uring

embryo

development

nd

maturation,

ABA

arrested

normal

development

f

the

embryos

as

has

been

observed

n

hybrid

arch,

Lelu

and

Label

1994),

hite

spruce

(Artree

et

al. 1991)

and

mango

(pliego_Alfaro

t al .

1996).

Effect

of

supplementation

of

mobilised

reserves

o

the

germination

media

Sucrose

The

concentration

f

sucrose

n

the

culture

medium

and

the

duration

f the

culture

reatment

ffected

he

germination

f

the

embryos.

Maximum ermination

29

and

35

o/o)

was

ob_

served

after4

and

5 weeks

of

treatment

n

3 %

sucrose

ano

this

decreased

ith

any ncrease

r decrease

n

sucrose

on_

centration r the periodof treatment. tarch eservesn the

embryos

are

important

or

successfulgermination

Bewley

and

Black

1985).

n

this

case,

sucrose

robably

ubstituted

for

the

lack

of

starch

eserves

Osmotic

hock

due

to higher

concentrations

f

sucrose

s

probably

he

reason

hat

em_

bryos

did not

germinate

t hese

concentrations.

Maltoseand trans-cinnamic

cid

While

none

of

the

untreated

mbryos

erminated,

signif i_

cantly

P


4/5

on easomatic mbryoss

probably

ue o their

ensit ivityo

desiccation.

Chi l l ing

Chilling

enerally

ncreases

he levels

of GA3and

GA

like

substances

nd brings bout

a-amylase ediated

mobil isa-

t ionof starch eservesor

embryo

ermination

Wood

1982).

Germination

4.8

and 10

%) was recorded

n tea

embryos

that were

reatedat 4'C for

30 or 45 days

as compared

o

very

ow

2%)

or

no

germination

n

heuntreated

omatic m-

bryos or in

the ones hat were

treatedat 4

C

for

15 or 60

days. However,ncrease

n the

time of

chilling reatment

beyond15 days resulted

n increase n

vitrification

nd a si-

multaneous

ecreasen

the normal evelopment

f

torpedo

shaped mbryos

Germinationf somatic

mbrvosrom

Tea 1319

GAr

As

comparedo

the GA3untreated

mbryos

control),

hich

failed

o

germinate,

ut

remained

resh,

he reated

nesun -

derwent

leaching nd necrosis

t

all concentrations

f GA3

used.GA3bringsabout de novosynthesis f oc-amylasend

hydrolases

or

he

mobilisation

f starch eserves

nd normal

embryo

ermination

Bewley

nd Black

1985). et,

GA3 ailed

to mobiliseeserves

uring

ermination

ecausehe equisite

reserves

robably

ad

not accumulated

uring

he

embryo

maturation

hase.

Treatment

or reserve

ccumulation

ABA

No

embryo

germination

as

observed n

any of

the untreated

embryos

r in thosesubjected

o the

different oncentrations

D

Figurel. A

Globular econdary

omatic mbryos

erived orm

ea cotyledonsB

Normal

ermination

f tea somatic

mbryos reated

withmal-

tose

and rans-cinnamics

comparedo untreated

ontrol

C Emblings erived

rom

germinated

omatic mbryos

f tea. D Hard ened

ealthy

plants

withnormal

eaves

nd

vigorously

rowing

ootsystem

erived rom

somatic mbryo

ermination


5/5

ken

down

more

slowly,

ui

also

can

provide steady

upply

of

more

eadily

metabolisable

arbon

ource

glucose)o

th e

embryos.

his

provideshe

requisite

etabolites

equired

or

germination

n view

of the

fact

that

the

embryos

robably

lacked he stored

arbohydrate

eserves

ue

to their

neffi-

ciency

n accumulating

hese

eserves

uring

heir

maturation

Dnase.

Cinnamic

cid

is

an

important

ompound

nvolved

n

a

wide

variety

f

metabolic

athwaysike

he

lavonoid

iosyn-

thesis

pathway,

he

phenolics ynthesis

athway nd,

most

importantly,

n

he

synthesis

f

Malonyl-CoA,

he

key

pre-cur-

sor

of

fatty

acid

synthesis

athwayAn absolute

equirement

for

a specific

ombination

f

maltose

nd

-CA

n he

case

of

tea

in this

study

urther

ndicates

he

crit ical

equirement

f

sugars

and

fafty acids

during

maturation

nd

subsequent

germination.

Acclimatisation

nd

ield

transfer

Plantlets

ith

young eaves

nd

stout

oots

Fig

2D)

grew

up

to

a

height

f about

4-5 cm

within

-8

weeks

of

transfer

o

Hikkotrays,

hich,

upon

transfer

o

poly-sleeves

fter

B

months.

developed

nto

healthy

and

vigorously

rowing

olants

ithin

Year

Conclusion

The

somatic

mbryos

f

tea,

ike

heir

zygotic

ounterparts'

appear

o

exhibit

esiccation

ensit ivitynd ail o accumu-

late

storage

eseryes

ailure

f

chil l ing

nd

GAs

o

cause

a

higher

ercentage f

normally

eveloped

mbryos

nd

heir

germination

s

interesting

s

his

s ndicative

f

the

aci

hat'

since

eserves

ere

not

stored

by

the

embryos

uring

he

maturation

hase,

heir

mobilisation

annot

e brought

bout

by

external

gents

lmprovement

f

the

conversion

requency

y supplements

from

external

ources

ike

he

readily

vailable

orms

of

car-

bohydrates

uch

as

sucrose

and

maltose

urther

prove

hat

tea

somatic

mbryos

ail

o

accumulate

arbohydrates

n the

form

of storage

eserves

lthough

BA

helps

n

he

accumu-

lation f reservesnd n the acquisition

f

desiccation

oler-

ance

by

the

embryos

uring

he

maturation

hase, ailure

f

Germination

f somatic

mbryos

rom

Tea

1321

either

o

ake

place

n

his

study

s

probably

ecause

f

appli-

cation

f

ABA

during

hases ther

han

embryo

maturation'

Acknowledgements.

he

authors

cknowledge

he

kind

help

of Dr

Subedar

Pandey

or the

statlstical

nalyses

nd

Tapan

vlondal

c-

knowledgeshe inancial ssistancef the University rantsCommis-

sion

uring

hecourse

f

his

tudy

References

Artree

SM,

Moore

D,

Sawhney

K,

Fowke

C

(1991)

nhanced

matu-

ration

and

desiccation

olerance

f

white

spruce

Picea

glauca

(Moench)

oss

Somatic

mbryogenesis

n cotyledon

ulture

f ea

(Thea

sinensisL.).

Hort

Sci

66:

465-470

Barua

DN

(1989)

Science

nd

Practice

n tea

culture

Tea

Research

association

alcutta'

P

161-222

Bewley

D,

Black

M

(1985)

Seeds:

Physiology

f Development

nd

Germination

lenum

ress,

ew

York

Deng

DM, Cornu

D (1992)Maturation nd germinationf walnut o-

matic

embryos

Plant

Cell

TissOrgan

Cult

28:

195-202

Kermode

A, Bewley

D,

Dasgupta

, Misra

S

(1986)The ransition

from

seed

development

o

germination:

key

role

or

desiccation?

Hor t

c i

21 :

113-11

8

LePage-Degivry

T,

Barthe

P,Garello

G

(1990)

nvolvement

f

endog-

enous

abscisic

cid

n onset

and

release

f

Helianthus

nnus

em-

bryo

ormancy

lant

hysio l

2:

1164-1168

Lelu

MA,

Label

P

(1994)

hanges

n he

evels

f abscisic

cid

and

ts

glucose ster

conjugate

uring

maturation

f

hybrid

arch

Larix

leptoeuropaea)

omatic

embryos'

n

relatron

o

germination

nd

plantlet

ecovery.

hysiol

lant

2:

1164-1168

Marion-Poll

(1997)

BA

and

seed

development

rends

n Plant

Sci-

ences

2:

447-448

Mondal

TK,

Bhattacharya

, AhujaPS 2001) nduction f synchron-

ous

secondary

omatic

mbryogenesis

n Camellia

rnensis

L

)

O

Kuntze

Plant

hYsio l

58: 45-951

Murashige

,Skoog

F

(1962) revised

medium

or rapid

growth

and

bioassays

ith obacco

issue

ultures

Physiol

lanta

5:

473-497

Pliego-Alfaro

Monsalud

MJR,

Litz

RE, Gray

DJ,

Moon

PA

(1996)

Effect

of abscisic

acid

osmolarity

nd

partial

desiccation

n the

development

f

recalcitrant

ango

somatic

embryos

Plant

Cell

Tiss

Org

Cult

44:63-70

Vieitez

AM

(1995)

Somatic

mbryogenesis

n Camellia

pp

In: Jain

SM,

Gupta

PK,

Newton

R

(eds)

Somatic

mbryogenesis

n

woody

plants,

Vol

2.

Angiosperm

Kluwer

Academic

Publishers'

ether-

lands

pp

235-276

Wood

BW

(1982)

Gibberellrn

ike

substances

n developing

ruits

t

pecan

Hortsci

17:70-71

Factors Affecting Germination and Conversion...

Documents