Kurze Mitteilungen· Short Communications

Departm ent of Botan y, University of Delhi, Delh i 110007, India

Chilling Induces Development of Embryoids into Plantlets inEschscholzia

A. K . K AVATHEKAR, P. s. G ANAPATHYa nd B. M . ]OHRI

With 1 figure

Received July 26, 1976 . Accepted September 15, 1976

Summary

Somewhat comparable to the dormant embryo in seed, the in vitro-produced embryoid sof Eschscholzia californica could develop into plantlers when given a chilling treat ment.

Key w ords: chilling, embryoids, in vitro culture, germination, dormancy , Eschscholzia .

Introduction

In Daucus carota and Ranunculus sceleratus in vi tro-fo rmed embryoids de velop

into normal plantlets capable of flo wering (see H ALPERIN, 1970 ; VASIL and VASIL,

1972). The em bryoids of Eschscholzia calijornica, ho we ver , fail to develop into

plantlets in the same medium in which th ey differentiate, or a fte r subculture on freshmedium of the same constitution. Even omission of casein hydrolysat e from the me­

d ium ; supplementing wi th growth regul ators ; decotylization of embryoids; or even

culture of different stages of embryo ids failed to produce normal pl ant lets(KAVATHEKAR, 1974). In th is communic at ion a re described ex periments deal ing withplantlet fo rmation from embryoids subjected to chilling treatments.

Materials and Methods

Embryoids differentiating from the proliferated placental tissue (see KAVATHEKAR andGANAPATHY, 1973) were used. Individual embryoids (Fig. 1 A) were subcultured on thebasal medium (NB) used by N ITSCH and N ITSCH (1969), and NB + casein hydrolysate (CH;500 ppm). NB + gibberellic acid (GA3 ; 10-6 M), and NB + 6-benzylaminopur ine (BAP ;10-6 M) were used in experiment s dealing with continuous chilling.

The embryoids on NB and N B + CH were subjected to two types of chilling as describedbelow. One set from each medium was maintained althou gh under culture room conditions(22 ± 3 °C; 10 hr photop eriod- 300-S00 lux at bench level) as control :

Z. Pflanzenphysiol. Bd . 81. S. 358-363. 1977.

Development of embryoids 359

a) Alternate Chilling: The cultures were kept under culture room conditions and thenin dark for 14 hr at 6 DC.

b) Continuous Chilling: The cultures were exposed to 2 treatments:

1. Set 1: The cultures were kept continuously at 6 DC in dark for 4 wk, and then transferredto culture room conditions. The experiment was terminated at the end of 8 wk.

2. Set 2: The cultures were kept continuously at 6 DC in dark for 8 wk. They were thentransferred to culture room conditions. The experiment was terminated at the end of16 wk.For each set the controls were maintained in the culture room for 8 and 16 wk.

Results and Discussion

a) Alternate Chilling: The response of controls was similar to what has beendescribed earlier (KAVATHEKAR and GANAPATHY, 1973): formation of callus at radi­cular pole, secondary embryoid differentiation therefrom, and cotyledonary moundformation (Fig. 1 B). There was no plantlet formation.

Unlike in controls, where plantlets were not formed, the cold-treated culturesshowed the following responses, after 8 wk:

The radicle did not develop the primary root, but adventitious roots emergedfrom the callus at the radicular pole. The elongation of hypocotyl was poor. Thecotyledons became chlorophyllous and elongated normally. There was inhibition ofmound formation from the cotyledons. Foliage production at plumular pole didoccur. There was no secondary embryoid differentiation. Nevertheless, when chillingwas discontinued, and cultures maintained under culture room conditions, there wasdifferentiation of secondary embryoids.

Thus, alternate chilling of mature embryoids could overcome partially their in­ability to form plantlets. Presumably, alternate chilling is inadequate to obtain nor­mal development and differentiation of embryoids into plantlets.

b) Continuous Chilling: The results of continuous chilling are given in Table 1.Set 1 - At the end of 4 wk of continuous chilling, the development of callus

at the radicular pole, in all the media tested, was rather poor. About 16 per centof the cultures showed primary root formation (in all the treatments). The elongationof the hypocotyl approached that of the in vivo seedlings (on NB + BAP and NB +GA3) , whereas in embryo ids raised on NB and NB + CH, the elongation of hypo­cotyl was insignificant. The embryo ids in nearly 50 per cent cultures, in all themedia, developed leafy cotyledons by the end of 4 wk of chilling; less than 10 percent formed leaves. The differentiation of secondary embryo ids was appreciably lowor absent.

At the end of 8 wk (4 wk after transfer to culture room condition) the cultures re­vealed the following significant responses: On NB and NB + CH secondary em­bryoid differentiation occurred. Adventitious roots differentiated from the callus atthe radicular pole and increased greening of the cotyledons was noticed. In additionin some of the cultures callusing was noticed from leafy cotyledons. On NB + GA3

and NB + BAP callusing from the cotyledons was absent. Differentiation of foliage

Z. Pflanzenphysiol. Bd. 81. S. 358-363. 1977.

360 A. K. KAVATHEK AR, P. S. G AN APATHY and B. M. ]OHRI

Fig. 1

Z. Pflanzenphysiol. Ed. 81. S. 358-363.1977.

Development of embryoids 361

occurred on all the med ia tested . In addition, on NB + BAP there was pronouncedbud formation in the axils of cot yledons as well as leaves.

The embryoids which developed into plantlets resembling the in vivo seedlingswas onl y about 15 per cent (Fig. 1 C- F).

Set 2 - At the end of 8 wk of continuous chillin g, the cultures showed the fol­lowing responses :

Inhibition of root initiation, and slow grow th of the callus at th e radicular pole.The growth of hypocotyl and cot yledons was comparable to that observed at theend of 4 wk of chilling in Set 1. The differentiation of foliage, how ever, was poorand differentiation of secondary embryoids was completely inh ibited.

Four wk after transfer to cultu re room conditions from the continuous 8 wkchilling treatment, the cultures showed the following changes: Rhizogenesis occurredfrom the call us at radicular pol e on NB and N B + CH. The elon gation of hypo­cetyl was comparable to in v ivo seedlings on CH, GA g and BAP -supplementedmedia. The cotyledons tu rn ed deep-green foll ow ed by formation of numerous leaves,on N B + CH, NB + GA 3, and N B + BAP. On NB + BAP ax illa ry bud formationwas quite extens ive. Secondary embryoid differentiat ion (from the callus at ra dicularpole) was much more on NB than on NB + CH. Differentiation of secondaryembryoids did not occur on media supplement ed with BAP and GA 3 • At the end of16 wk, about 30 per cent no rmal plantlet s were formed, but the primary root wasabsent or poorly developed.

Although chilling inhibited different iation of secondar y ernbr yoids, the initiationof primary root could not be promoted. This cou ld be du e either to a more st ringentrequirement, or the inadequacy of the pr esent treatments. Th e absence of histologicalstructures no rmally associated wi th roo t-apical-me ristem in the emb ryoids (the typeof which was used as explants in th e pre sent study) could also be the reason forth e inability of embryoids to develop normal primary roots.

Irrespective of the treatment s, th e near-normal development of cot yledons wasalways accompanied by increased foliage production. This is also the situ at ion innature (with E. cali/arnica) where th e differentiation of leaf sta rts onl y after thenormal cot yledons ha ve developed . That chill ing results in normalization of

Fig. 1: Plantler formation from dor mant embryo and embryoids.

A. Th e embryoid, the type of which was used as exp lant. X 3. B. Control culture on NB(at the end of 8 wk). Note callus formation and differentiation of secondary embryoids. X 3.C. A plantlet dev eloped from excised 25-day-old seed-embryo cultured on NB (after 8 wkof culture). The seed had been pre tr eat ed with 25 ppm of GAg for 12 hr. X 1.8. D. Plantleton NB at the end of 4 wk of continu ous chilling. N ote primary root, elongated hypocoty l,green and bifid coty ledon, and emerg ing leaves. X 4. E. Simila r plantlet as (D) on N B,on transfer to culture room conditi ons 4 wk later. Note ext ensive fol iage and rooting.X 1.8. F. Same as (E), but on N B + GA3• Note the long slender pr imar y root, elongatedhypocotyl, lon g and bifid cot yledon , and elongated leaves. X 1.2.(c, cot yled on ; h, hypocotyl, I, leaves; p, pr imary root ; se, secondary embr yoids.)

Z. P'[lanzenphy siol. Bd . 81. S. 358-363. 1977.

~ 't:l

-... '";:: N '";:: ":

> "" ""<.., ~ ~ ?--

00 ..... Y'> w '" :!: 0\~ ..... \0 "~

Ta

ble

1;E

ffec

to

fco

ntin

uo

usch

illi

ngo

np

lan

tlet

form

atio

nfr

om

emb

ryo

id")

.

%cu

ltur

esw

hich

sho

wed

Cal

lus

atth

eP

rim

ary

Cal

lus

Elo

ng

ated

Cal

lus

Gre

enL

eaf

Me

diu

mC

ateg

ory

radi

cul

arro

ot

fro

mh

yp

oco

ryl

fro

mle

afy

form

atio

npo

leh

ypoc

otyl

coty

ledo

nsco

tyle

do

nsin

exp

lan

t

Co

ntr

ol

993

)0

918

990

0N

BSe

t1

8316

8316

8316

16Se

t2

990

8316

8316

0

Co

ntro

l9

10

910

910

0N

B+

CH

2)(5

00p

pm

)Se

t1

8316

3366

8016

66S

et2

990

3366

3366

66

Co

ntr

ol

91

056

41

8316

16N

B+

GA

3(l

0-O

M)

Set

183

1649

4933

6666

Set

28

316

168

316

8333

Co

ntr

ol

91

08

918

91

49N

B+

BA

P(I

O-OM

)Se

t1

8316

1683

3366

83Se

t2

910

1683

4949

83

!)12

cult

ure

spe

rtr

eatm

ent;

one

emb

ryoi

da

cult

ure

;d

ata

giv

enob

tain

edaf

ter

8w

kin

con

tro

lan

dS

et1,

and

afte

r16

wk

inS

et2

.2)

Gly

cine

was

del

eted

fro

mN

B.

3)

Fra

ctio

nfi

gu

res

om

itte

dfo

rsa

keof

con

veni

enc

e.

<;> C1'

N ~ r ::>::

> <: > ...;

:I: '":>< > 1" :-0 Y"

Cl

~ > "" > ...;

:I: .-.: .,., ::l 0- ?' ~ '--

--< o :I: :::

D evelopment of embryoids 363

cotyledons to a greater degree is substant iated by anatomical prepar ations (un­published observations).

Thus, the chilling tr eatment leads to partial development of the embryoids intoplantlets. Th e requirement of chilling is also true of seeds of E. californica unde rin vivo conditions ( COOK, 1962 ; K AVATHEKAR, 1974), and germination of seedswhi ch are shed in Marchi Ap ril (under Delhi conditions) occurs after 4-5 months(i.e. they remain dormant for th is period). Thi s dormancy can be overcome by eitherin vivo or in vitro exposure to cold trea tments. Th e cold-requiremen t can be replacedby GA 3 appl ication aft er the seeds are shed. Interstingly, this is also true ofembr yoids wh ich do not «germinate» unless given a period of cold tre atment, th eeffect of GA 3 and BAP being synergistic. We conclude that the embryo ids formedfrom the placental callu s of E. calif ornica (under th e cond itions described ) exhib ita phenomenon very similar to dorm ancy encountered in seeds.

References

COOK, S. A.: Genetic systems, var ianon and ad aptation in Eschschalzia califarnica. Evolu­t ion, Lan caster, Pa. 16, 278-299 (1962).

H ALPERIN, W.: Embryos from somatic plant cells: 169-1 91. In : P ADYKULA, H ELEN A. (ed.),Co ntrol mechanisms in the Expression of Ce llula r Phenotyp es. Symp. Int. Soc. Cell Bio!.,Aca demic P ress, New Yo rk and London , 1970.

KAVATHEKAR, A. K.: Studies on Origin, Development and Dormancy of Embryoid s ofEschschalzia califarnica. Ph . D. Th esis, Un iv. D elhi, 1974.

KAVATHEKAR, A. K., and P. S. GANAPATHY: Emb ryoid diff erentiation in Eschschalzia cali­farnica. CurroSci. 42, 671- 673 (1973).

N ITSCH, ]. P ., an d C. NI TSCH: H aploid pl ant s fro m poll en grains. Science, Wash. 163,85-87 (1969).

VASIL, 1. K. , and VIMLA, VASIL: To ti potenc y and embr yogenesis in plant cell and ti ssuecult ures. In vitro 8, 117-1 27 (1972).

Dr. A. K. KAVATHEKAR, D epartment of Bot any , Sri Venkateswara College, Unive rsity ofD elhi, New Delhi 110021, India.

Z. Pflanzenphysial. Ed. 81. S. 358-363.1977.