BAM . I

E ffec t of c e r ta in p la n t growth re g u la to rs

oo th e growth and metabolism of i s o la te d

co ty ledons.

CHAPTER 1 I

IITKQDUCTIGU

C otyledons, which p r io r to seed germ ination

o ften weigh a hundred tim es as much as t h e r e s t of the

seed, a c t p rim arily as s to rag e organs* The p r in c ip a l

func tion o f cotyledons i s to supply the embryonic ax is

w ith re s p ira to ry su b s tra te s by a u to ly s is during: th e ea rly

s tag es of seed g e m in a tio n . A f te r the n u tr ie n t re se rv es

have been exhausted th e cotyledons g en e ra lly s h r iv e l and

drop o f f . In p la n t spec ies w ith ep igea l seed lin g s the

coty ledons o ften develop in to l e a f l i k e organs with

considerab le p ho to syn the tic a c t iv i ty , to sumaort th e i n i t i a l

growth o f th e seed lin g , bonn ier (1982) has c h a ra c te r ise d

the n u tr i t io n a l re la tio n sh ip s between ootyledons and the

embryonic ax is in bean ( Phaaeolus v u lg a r is ) seeolinrro .

Ecotyledonized embryos cu ltu red on supplemented a e d ia s<p?ew

le s s ra p id ly com^&rsd to embryos w ith in ta o t cotyledons

2

cu ltu red on an agaxvwater medium only* The eootyledonized

eabryos showed more growth when detached cotyledons were

placed nea r them o r when e x tra c ts o f detached cotyledons

were added to th e medium* fhe growth of the embryo during

germ ination depends upon re se rv es s to red in th e cotyledons*

The rese rv es a re broken down by hydro lysing enzymes and

tra n s lo c a te d to th e embryo ax is (Opik, 1965; Walton and

S oofi, 1969).

Depending upon the n a tu re o f food re se rv es n resen t

in the co ty ledons, seeds f a l l m ainly in to two groups* Those

where the s to rag e m a te r ia l i s carbohydrate and those where

the p r in c ip a l s to rag e m a te ria l i s l ip id * Seeds r ic h in

p ro te in s may belong to e i th e r group* Some seeds such as

soyabean may co n ta in p ro td in as th e predominant s to rag e

m aterial* In ad d itio n to carbohydrates, p ro te in s and l ip id s ;

cotyledons may co n ta in re se rve m inera ls and phosphorus

compounds such as phy tin and sometimes tannins*

S tarch i s a common s to rag e form o f carbohydrate

present in cotyledons o f many seeds such as those o f legumes*

But pentosans and hexosans a re s to red in cotyledons of

Isnlsaa# and Me&ksm*

Proteins co n stitute the main form of nitrogenous

reserves of the seeds. H yd ro lytic breakdown of stored

3

proteins into peptides and amino-acids makes a va ila b le

substrates f o r re s p ira to ry a c t iv i t y and furnishes b u ild in g

blocks fo r the synthesis of new enzyme p ro te ins.

In many nuts ( Jiyqans. Prunus e t c . ) , acorns

( kuerions) and other seeds, o i ls are stored in the

cotyledons but the la t t e r remain underground during

ge m in a tio n . In seeds of sunflower* curcu rb its and

c ru c ife rs the o i l r ic h cotyledons, during the course of

germination, become seedling leaves and assume photosynthetic

fu n ctio n . G lycerides o f fa t ty acids ar© g e n e ra lly the

reserve form o f l ip id s present in most o i l r ic h seeds. Upon

germination, break-down of reserve tr ig ly c e rid e s y ie ld s

products which support the growth o f the seedling.

T rig ly c e rid e s are f i r s t hydrolysed to give f a t ty acids and

g ly c e ro l. F a tty acids are oxidized to actyl-COA and the

la t t e r may be metabolized through the TCA cycle to y ie ld

energy. A d is t in c t iv e feature of f a t ty seedlings, however,

is the conversion o f l ip id reserves to carbohydrates through

glu co/eo genesi s *

In ce rta in types of epigeal seedlings such as those

of c ru c ife rs and legumes, marked expansion of the cotyledons

occurs before the f i r s t leaves appear. These cotyledonary

leaves may support the i n i t i a l growth of the seedlings by

supplying photosynthates. Factors such as l ig h t , cations and

hormones ex e rc ise a s tim u la tin g in flu en ce on th e expansion

of cotyledons (iCursanov £$, aJL., 1969). The a b i l i t y to

s tim u la te growth o f cotyledonary leav es i s a ty p ic a l

m an ife s ta tio n o f th e p h y sio lo g ica l a c t iv i ty o f cy to k ln in s

(M ille r , 1956).

Cotyledons which a re a u to tro p h ic , a re capable o f

ro o tin g . The l i f e span o f C urcu rb ita cotyledons g e ts

considerab ly extended on ro o tin g (Mohamad 1975)*

The pro longation o f l i f e so an o f roo ted cotyledons may be

due to th e supply by the ro o ts o f e s s e n t ia l f a c to r s such

as cy tok ln in s and /o r o th e r hormones ( W ollgiehn, 1967), o r

by the in c re a se in the m ineral uptake e ff ic ie n c y r e s u l t in g

in improvement o f p ho to syn the tic performance ( L o v e ll .e t a l .

1973).

Excised cotyledon i s a c losed system in which no

tra n sp o r t o f o rgan ic m a te ria l occurs and lo s s o f dry

weight by r e s p ir a t io n i s n e g lig ib le (Longo <gt a l . . 1979).

This system has been widely used to study growth and

m etabolic changes induced by c e r ta in growth re g u la to rs

( Sankhla 1970 ; Kulaeva £ t a l . . 1972 j Gordon and L et ham,

1975 I S e rv e ttaz jgt a l . ,1976 ; Longo g t a l . . 197 8 ).F acto rs

which a re known to in flu en ce th e expansion growth of

excised coty ledons independently as w ell as by in te r a c t in g

m utually a re l i g h t , ca tio n s and growth re g u la to rs .

5

Detached cotyledons expand more ra o id ly in l ig h t

than in the dark ( B anerji and L aloraya, 1956 ; Letham

1971 ; Blfven , 1971)* The cy to k in in induced expansion

growth i s a lso h ig h er in the presence o f l ig h t than in i t s

absence, B anerji and Laloraya (1965) observed a sy n e rg is tic

e f fe c t o f k in e tin and l ig h t on the expansion o f pumpkin

cotyledons* Letham (1971) found th a t growth o f detached

rad ish ootyledon was considerab ly more i n l ig h t than in

darkness. Huff and Boss (1975) rep o rted th a t red l ig h t

stim ulated the enlargement o f ra d ish cotyledons and

increased th e con ten t o f reducln sugars compared to dark

co n tro ls .

C erta in ca tio n s have been found to s tim u la te th e

expansion growth o f detached cotyledons o f some p lan t

sp ec ies . Of tbe v a rio u s ca tio n s which have been stud ied

potassium ( £+)appe r s to p lay an im portant ro le in the

expansion growth o f detached cotyledons* Thus, Knypl and

Rannerfc (1970) showed th a t expansion of is o la te d cucumber

cotyledons in 0,1 13 KUl was n e a rly th re e fo ld ov er w ater

con tro l. Moreover, KCl a t 0,01 II s tim u la ted ch lo rophy ll

accum ulation. Both expansion and ch lo rophy ll development in

iso la te d cotyledons were in h ib ite d by Calcium (Ca++) and

the in h ib ito ry e f f e c t of Ca'H’ was com pletely reversed by F+,

lo change in ch lo rophy ll accum ulation was brought about by

la * , a lth o u tjh i t a c ce le ra te d the growth. Growth as ^ e l l as

6

ch lo rophy ll accum ulation were in h ib ite d by Li* and "Iff* .

Marked in c re a se in the tu rnover o f p ro te in s and M a as

a lso increased D'lA syn thesis was observed in th e t>renence

o f potassium , suggesting th a t a c c e le ra te d sev e ra l

m etabolic p ro cesses .

Various p la n t growth re g u la to rs , n a tu ra l as w ell

as sy n th e tic , have been found to a f f e c t the expansion

growth of excised cotyledons i a a number of p lan t sp ec ie s .

She e f fe c t of cy tok in ins appears to have been more

ex tensive ly in v e s tig a te d . C ytokinins have been found to be

more e f fe c t iv e in promoting th e expansion growth o f

detached cotyledons compared to g ib b e re ll io ac id and auxins

(B anerji and L alo raya,l967 ; Udayakuaar $£ a l . . 1 973 ;

Letham 1971 { Kursanov a l . . 19 69).

Sankhla (19 69, 1970) rep o rted th a t m orohactin,

which resem bles o th e r growth re ta rd a n ts in i t s o v e ra ll

growth re ta rd in g p ro p erty , somewhat enhanced th e exnaafelon

of coty ledons in Ioomoea pentaoh.vlla. Both GA and

m orphactin s tim u la ted cotyledon expansion when given alone ;

but in combination they acted a n ta g o n is t ic a l ly and s tro n g ly

in h ib ite d expansion. Moreover, cy to k in in s (k in e t ln and BAP )

and 2 ,4-D (an auxin) acted s y n e rg is t ic a l ly w ith m orohactin

in in flu en c in g expansion o f detached cotyledons of Ittomoeag,fia,tegkylj.a»

7

..9y * p * & a * a g

The a b i l i t y o f cytoledons to en large in response

to k in e tin trea tm en t was f i r s t dem onstrated by Ikuaa and

Thi mann (1963) in ger?ainating le t tu c e seeos and l a t e r

eonfirmed by Scheibe and Lang (1965)• In v e s ti a tio n s on

oytokinin Induced expansion of i s o la te d cotyledons

reoeived im petus from the work o f B aaerji and L aloraya

(1965) who discovered th a t k in e tin induced expansion of

is o la te d pumokin cotyledons was not on ly in flu en ced by

l ig h t but was a lso determ ined by th e s tag e o f germ ination

at which cotyledons were excised from the seedling* Thus,

k in e tin induced expansion was more marked in th e presence

of l ig h t , p a r t ic u la r ly when th e cotyledons had been excised

at the s tag e in young seed lings where hypocotyl had not

yet fu l ly d if fe re n tia te d * K in e tin induced expansion in

l ig h t was about tw ice th a t observed in dark , in d ic a tin g

sy n e rg is tic e f f e c ts o f l ig h t and k inetin*

The is o la te d cotyledon has s in ce become a popular

te s t system fo r studying hormone Induced growth and

accompanying m etabolic changes. A good d ea l of in fo rm ation

has been gathered employin' excised ootyledone o f various

p lan t spec ies such as Xaathlum ( iisashi and Leopold, 11 S'?);

f la x (Sveshnikova and Ehoklova, 19 69) f mustard ( L ovell and

3

Moore, 1973) * Sunflower ( tiilad g t a l . . l9 7 0 ) bean (u e p s ta in

and I la n , 1970) ; fenugreek ( R ijven and Prakash, 1970) *

rad ish (Letham, 1971) ; watermelon ( Singh ^ a l . . 1973 i

Longo e$. a ^ . , 1 T78) and cucumber ( l a r a in and L aloraya,

1974 | Tsui s i §1** 1930).

2h& expansion induced by cy tok in ins i n excised

cotyledons of a number o f p lan t sp ec ie s , i a so ch r a c t e r t s t i c

and c o n s is te n t th a t b ioaesays based on th is response have

been developed by some workers fo r te s t in g cy to k in in

a c tiv ity ( Esaehi and Leopold, 1969 ; R ijven e t a l . . 1970 ;

Lethaaa, 1971 ; Udayakumar £ t al. . 1973 f fa ra in e t al. , 1974).

In th i s system a m easurable response to oy tok in lns i s

observed w ith in a sh o rt period which a ffo rd s an advaata^e

over the conventional time consuming, t i s s u e c u l tu ra l

b ioassays.

Sxclsed Xanthlum cotyledons ex h ib ited a la rg e

and rao id expansion in response to cy tok in in s even when

immersed in sm all volumes o f th e t e s t so lu tio n . The s,stem

was found to be s e n s i t iv e to BAP concen tra tion as low as

10 (jisash i and Leopold,l9 69).

Lethaa (1971) developed a bioaesay fo r cy to k in in s

based on th e i r a b i l i t y to nromote markedly th e expansion of

excised rad ish coty ledons and s tud ied the e f fe c t o f a number

9

o f n u tr ie n ts on cy tok in in induced expansion o f th e se

ootyledons. While KHgPO d id not promote expansion growth

of coty ledons in the absence o f cy to k in in , i t markedly

enhanced the cy to k in in induced response* However*additiono

of owsrose, myoinositol, in d o l-3 -y l-a c e t ic a d d (6 and 60 p i ) ,

GA5 (10 uM), 0 0 3 (2ma), MgS04 o r Ca(S03) 2 to th e medium

did not ap p rec iab ly enhance th e cy tok in in evoked response.

Excised cotyledons a lso expand in response to GA

only in l ig h t (Letham,117l { Esashl and Leopold, 1969 ;

Udayakumar § t a l .« 1973). A ddition o f m annitol (0 .25 0 to

the medium was found to e lim in a te GA induced expansion in

Xanthium coty ledons. Cytokinin induced expansion, though

considerably reduced in magnitude was not e lim inated by th e

add ition o f m annitol (S sash i and Leopold, 1969). Letham (1971)

found th a t in Iso la te d ra d ish cotyledons,enlargement was

induced not on ly by cy tok in ins but a lso by G-A . The growth

responses evoked by cy tok in ins and GA were found to be

ad d itiv e . The response to GA was, however, minimi zed In

the presence of m ann ito l, w ithout a f fe c tin g ap p rec iab ly the

response to cy to k in in s .

H arain and L aloraya (1974) found th a t the a b i l i t y

o f is o la te d cucumber cotyledons to expand q u a n ti ta t iv e ly in

response to in c re a s in g concen tra tion of cy tok in in s in the

dark was an e f fe c t s p e c if ic to cy to k in in s . O ther growth

10

re g u la to rs te s te d by th ese workers f a i le d to Induce

expansion in th e Iso la te d cucvunber cotyledons In th e dark .

S im ila rly the expansion o f fenugreek cotyledons In response

to k in e tin occurred only in the dark ( B ijven and P rakash ,1970).

In most experim ents c a rr ie d ou t to study th e

e f fe c t o f growth re g u la to rs on i s o la te d co ty ledons, th e

cotyledons have been m aintained In continuous co n tac t w ith

the hormone so lu tio n . Lon/*> a l . .H 9 7 8 ) . however,showed

th a t continuous presence o f BAP was not e s s e n t ia l f o r f u l l

e ffe o t on the growth o f watermelon co ty ledons. They showed

th a t a s in g le trea tm en t fo r one hour equals the e f fe o t o f

a continuous c o n ta c t.

The q u a n ti ta t iv e response to cytoklnins,varies With

th e p la n t sp ec ies and th e type o f cy tok ln in tested.U c yakumar

and K lrshn& sastry (1973) compared cy tok ln in induced response

In is o la te d cotyledons o f ra d ish , ho rse gram and cucumber.

She expansion In case o f cucumber cotyledons in response to

cy tok ln ins a t a given co n cen tra tio n was found to be much

g re a te r than ra d ish and horse gram. Moreover th e e f f e c t of

BAP was more marked than k in e t in .

Esashl ami Leopold (1969) found th a t th e expansion

growth o f excised Xanthium cotyledons Increased w ith

11

in c re a s in g cy tok in in co n cen tra tio n . These workers

compared the s tim u la tio n o f cotyledon enlargement fey

seven d i f f e r e n t cy to k in in s v iz . benzyladenine, z e a tln ,

SD 8339, S S - d im e th y la lly l adenine, k in e tin , d ipheny lurea

and adenine. BAP was found to be most a c tiv e o f th e

cy tok in ins te s te d , producing more than fo u r fo ld In c rease

over co n tro l a t 1<T*m, follow** by SD 8339, z e a tin ,

d im e th y la lly l adenine, k in e tin and d iphenylurea . Adenine

was, however, in e f f e c t iv e .

C ytokinin evoked responses have been found to

in c re a se fu r th e r w ith potassium trea tm en t. C ytokinins have

also been found to s tim u la te the up take of monovalent ions

in detached co ty ledons.

B ijven §& a l . . (Vi72) s tu d ied th e e f fe c t of a

number of a lk a l i and ammonium s a l t s on k in e tin induced

expansion of excised fenugreek co ty ledons. K in e tin induced

expansion was s tim u la ted by and io n s , bu t KC1 alone

was in e f f e c t iv e i n promoting expansion.

Green £ t a l . »(1978) repo rted th a t expansion

response o f cucumber cotyledons to cy tok in in s was g re a tly

enhanced in the presence o f potassium* A d im in ish ing response

to k in e tin with in c re a s in g a^e o f th e cotyledons was

a t t r ib u te d by th ese workers to low er le v e ls o f potassium

12

a tta in in g in th e oytoledon. They argued th a t th e presenoe

of a h igher le v e l o f KC1 in the in cu b a tio n medium o f f s e ts

the lo v e r potassium conten t o f th e t i s s u e and enables a

much la rg e r response to cy to k in in s . At 40 mM KOI, response

to k in e tln was n ea rly 4 tim es more than in the absence o f

KOI. Ca f u r th e r Increased th e e f fe c t o f potassium on the

response to k in e t ln .

Green and H ulr 0 3 7 9 ) found th a t a hi^h e te rn a l

concen tra tion o f potassium a lso promotes th e lig h t- in d u c e d

growth o f cucumber co ty ledons. The response to w hite l ig h t

was over th re e tim es g re a te r when 40 mM KC1 was p re se n t in

the medium. Calcium d id not enhance th e l ig h t Induced growth

of cotyledons, a b a c is ic a d d s tro n g ly in h ib ite d th e responses

to l ig h t and cy to k in in . The in h ib i t io n was g re a te r in the

presence o f KC1. K in e tic s tu d ie s showed th a t response to

l ig h t promoted by potassium was in h ib ite d com p etitiv e ly by

ab sc is ic acid and response to cy to k in in was in h ib ite d non

com petitively . These re su lts suggest th a t c y to k in in s ,lig h t

and ab so is io a d d have prim ary p ro p e r tie s a f f e c t in g aanj^aae

perm eab ility . The in te ra c t io n of l i g h t and cy to k in in s w ith

potassium exp la in s many s im i la r i t i e s between the e f fe c ts

o f l ig h t and cy to k in in s as a lso t h e i r antagonism w ith

ab se is io ac id .

I la n fit njL.,(1171) stud ied th e e f fe c t of k in e t ln

on the uptake o f c e r ta in monovalent io n s in detached sunflow er

13

cotyledons and found an in creased uptake o f X+ and Rb+ in

the presenoe of k in e t in . Uptake o f Li* was only s l ig h t ly

promoted w hile th a t of Ja+ was not a ffe c te d by k in e t in . The

oapaoity of coty ledons to absorb potassium was found to be

depressed by l i g h t .

In cucumber co ty ledons, S astry e t &1.# (1973)

observed an enhanced uptake o f potassium in the presence

of BAP accompanied by a marked in c re a se in the fre?h w el^ it

of coty ledons. The increased uptake of potassium in the

presence of BAP was fu r th e r enhanced by l i g h t .

The coty ledons o f sev e ra l p la n t spec ies undergo

d ra s tic changes in fun c tio n and in c e l l s t ru c tu re during

germ ination a f t e r being exposed to l i g h t . Reserve m a te r ia ls

are g ra d u a lly dep le ted and th e sto rag e fu n c tio n i s superseded

by th e p h o to sy n th e tic func tion (Kagawa §£ a l . . 1175).

P la s t id s and micro bodies ( glyoxysomes and peroxysomes)

are deeply involved in th ese tran sfo rm atio n s , A good d ea l

of evidence shows th a t ad m in is tra tio n o f cy to k ln in s to dark -

grown cotyledons Induces the appearance o f some fe a tu re s

th a t a re norm ally a sso c ia te d w ith il lu m in a tio n ( S e rv e ttaa

e t a l . ,1 9 7 6 ) . Longo £ t a l . , (1979) s tud ied th e e f f e c t o f

BAP on th e development o f p la s t id s and m icrobodies in excised

watermelon co ty ledons. Study of u l t r a s t r u c tu r e in c e l l s of

p a lisad e oarenohyma showed th a t a t the beginning o f th e

treatm ent th e se o e l ls were packed with re se rv e m ate ria ls*

m ostly f a t g lo b u les and p ro te in bodies* A fte r fo u r days,

p ro te in bodies showed sign of p a r t i a l d ig e s tio n and some

atreohes o f membranes were observed In p la s t id s th a t were

f i l l e d w ith s ta rc h grains* In t r e a te d cotyledons#however,

both degradation o f re se rv e m a te r ia ls and development o f

o rg an e lle s were aocelerated* A fte r fo u r days o f trea tm en t

most of th e s to rag e m a te ria l was broken down and la rg e

Vacuoles rep laced p ro te in bodies* L ipid g lobu les were

replaced by cytonlasm and o rg a n e lle s . Development of

p la s t id s was promoted by BAP*

A la rg e in c re a se in fre sh weight i s known to

accompany expansion of excised coty ledons fo llow ing

treatm ent w ith cy to k in in s , but no ap p rec iab le in c re a se in

dry weight occurs ( Letham, 197* ; O epstain and I la n , 1970 ;

Udayakumar and K rlshn asastry ,l9 7 3 )* Excised d is c s from

leaves o f young ra d ish p la n ts e x h ib it a s im ila r response*

Promotion o f growth by cy tok in ins in such t is s u e s appears

to be through s tim u la tio n of c e l l expansion ra t: e r than

by promoting c e l l d iv is ion* C ytokinins promote th e su rface

growth and the fre sh weight o f l e a f t is s u e s but have l i t t l e

e f fe c t on dry weight ( K u rash i,l9 5 9 )*

In cucumber co ty ledons, trea tm en t w ith BAP had

no ap p rec iab le e f fe c t on th e dry vvei^ht e i th e r l a l ig h t

o r l a th e dark ( Udayakumar and S as try , 1175) • The cy tok in in

induced In c rea se in f re sh weight i s be lieved to be due

p rim arily to an in c reased uptake o f w ater ( L etham ,l97 l) .

Bewli and tfitham (1976) stud ied th e k in e tln

Induced uptake o f w ater in detached cotyledons o f e ig h t

v a r ie t ie s of ra d ish ( Ranhanus s a tiv u s L .) • Fresh weight

Of cotyledons in c reased in a l l th e c u l t iv a r s by k in e tln

trea tm en t. Dry weight and c e l l number was, however, not

a ffec ted by k in e t ln trea tm en t. In h ib it io n by m ann ito l,

s im ila r r a te s o f H ) e f f lu x from k in e t ln tre a te d and

con tro l cotyledons and th e k in e tln stim ulated in c re a se in

reducing sugars in d ic a te d th a t in Iso la te d co ty ledons,

weight gain due to w ater uptake was dependent upon an

In c rease in th e con cen tra tio n o f o sm otloa lly a c tiv e c e l lu la r

components. The cotyledon enlargement and reducing sugar

build up was In h ib ite d by chloram phenicol, streptom ycin ,

oycloheximide and 6-m ethylpurine. This was sug^e^tive of a

requirem ent fo r RTA and n ro te in sy n th es is in cotyledon

expansion.

G epstain and H an (1970) showed th a t k in e t ln

promotes th e a c t iv i ty o f s ta rc h hydro lysing enzymes In bean

cotyledons, as a r e s u l t of which th e le v e ls of o sm o tica lly

M tiv e m etab o lites a re Increased lead in g to more w ater

uptake* The a c t iv i ty o f i s o c i t r a t e ly a se was found to

increase by BAP treatm ent in squash cotyledons ( Peaner

and Ashton, 1167). Thus, cy tok ln ins in flu en ce th e conversion

Of sto rage components to o sm o tlca lly a c tiv e compounds,

possib ly v ia t h e i r e f f e c ts on th e sy n th es is o f h y d ro ly tic

tasyms. The In c rea se in co n cen tra tio n o f sugars le a d s to

water uptake and cotyledon enlargem ent.

In excised watermelon cotyledons BAP has been

shown to promote the conversion of re se rv e l ip id s to so lub le

sugars. The osm otic p o te n t ia l of BAP t r e a te d cotyledons i s

always low er than th a t o f u n tre a te d ones sugg estin g th a t

BAP in c re a se s th e cap ac ity o f cotyledons fo r w ater uptake

not only through a bu ild up of so lu te s but a lso by d i r e c t ly

In fluencing c e l l w all e x te n s ib i l i ty ( Loacre e t a l . . 1978).

This view i s su b s ta n tia te d by the f a c t th a t w hile BAP

trea ted coty ledons bend w ithout breaking, w ater co n tro ls

break e a s ily when ben t.

In cucumber cotyledons BA? has been shown to

Increase th e r a te o f d isappearance o f l i p id re se rv es

concomitant w ith an in c re a se in th e concen tra tion of so lub le

sugars ( i'su i jgt a i . ,1 9 3 0 ) . Chloramphenicol and actinoaycin-D

in h ib ite d th e Ba? induced expansion but had 11 . t i e e f fe c t

17

on the expansion o f u n trea te d c o n tro ls . This s u g e s t s

th a t d i f f e r e n t biochem ical p rocesses a re involved in th e

expansion o f u n tre a te d cotyledons and those tre a te d w ith

BA?* The BAP Induced expansion was re la te d to in creased

re sp ira tio n and in c reased sy n th esis o f p ro te in s and n u c le ic

adds* A h ig h er conten t o f 3HA and MA p e r cotyledon

was found in BAP tre a te d cotyledons than u n d a te d ones*

H uff and Hose (1975) s tud ied th e promotion o f

enlargement by a e a tin and by red l ig h t in excised ra d ish

cotyledons* Z eatin caused g re a te r accum ulation o f reducing

sugars than in l i g h t c o n tro ls , continuous f lu o re sc e n t

l ig h t trea tm ent o r a b r ie f red l ig h t trea tm en t Increased

both growth r a te and the conten t of reducing sugar compared

to dark contro ls* Reducing sugar le v e ls were , however* not

affec ted by z e a tln in darkness* Amylase a c t iv i ty vas h ig h er

in continuous w hite l ig h t o r a b r ie f red l ig h t trea tm en t,

au f^esting th a t reducing sugar b u ild up was due to Increased

amylase a c tiv ity * Zeatin t re a te d cotyledons* however,showed

lover amylase a c t iv i ty compared to l ig h t c o n tro ls suggesting

an accumulation through a d i f f e r e n t ro u te presumably v ia

conversion o f f a t s to sugars in lig h t*

Longo £ t &L» 0 9 7 9 ) reported th a t in watermelon

cotyledons* trea tm en t w ith BAP in th e dark ac ce le ra te d

breakdown o f re se rv es and stim u la ted development o f

organelles* The a c t iv i ty o f HUBP carboxylase and IADPB-

gljroxylate reductase,tw o marker enzymes fo r p la s t id s ,

was increased th re e fo ld by BAP. BAP increased th e peak

lev e l of i s o c i t r a t e ly a se and ac ce le ra te d i t s decay phase.

Treatment w ith BAP in creased the t o t a l mass o f th e

■iioc-frondrta, The le v e ls o f m itochondria l enzymes in creased

to the same ex ten t as th e t o t a l m itochondrial p ro te in so

th a t p ro te in r e la te d s p e c if ic a c t i v i t i e s o f m itochondria l

enzymes in t r e ted and co n tro l cotyledons were th e same.

Only the sp e c if ic a c t iv i ty o f cytochrome oxidase was h ig h er

in m itochondria from BA? tre a te d cotyledons*

In watermelon cotyledons,LOrigo (1980) observed

th ree types o f in te r a c t io n between l i g h t and BAP ( i ) the

increase In fre sh weight and decay o f g ly o x y la te cycle

enzymes was s tim u la ted by BAP, w hile l i ^ h t was w ithout any

e ffe c t, illu m in a tio n did no t modify the responses to BAP

( i i ) both l i g h t and BAP in c reased th e weak a c t iv i ty of

ie o o i t r a te ly a se but th e e f fe c t o f th e two was not m utually

add itive ( i l l ) l ig h t and BikP were both e f fe c t iv e in

ln o re a r in / the enzymes o f p la s t id s and peroxisomes* The

e ffeo t o f l i g h t and BAP to g e th e r being more than add itive*

The sy n e rg is tic in te r a c t io n between l ig h t and BAP suggests

th a t the two c o n tro l th e development o f p la s t id s and

19

miorobodies through two independent a c tio n mechanisms.

1 lim ited p o rtio n o f th e two mechanisms may, however* be

m utually common.

There i e evidence to show th a t l ig h t causes an

increase in endogenous cy tok in ins in sev era l p la n t m a te r ia ls

Ineluding watermelon co ty ledons, work o f Longo s i a l« .( l3 8 0 )

■•mbe to exolude th e hypothesis th a t th e l ig h t Induced

Stim ulation of p la s t id and microbody development i s mediated

by a r i s e in endogenous cytokinins* L igh t has a promotive

action even when th e maximal response to BAP has been

attained*

S e rv e tta z £& gl**(l976) showed th a t in i s o la te d

sunflower cotyledons BA? s tim u la te s th e development o f

several m itochondria l and microbody enzymes, and th a t i t s

e ffe c t on peroxyeomal and glyoxysomal enzymes i s very

s im ila r to th a t of w hite l i ^ i t .

Karavaiko and ftish ra (1976) s tu d ied th e e f f e c t of

cytokinin , GA and XM on the a c t i v i t y o f a d i and a lk a lin e

Inorganic pyrophosphatases and m alic enzyme in is o la te d

gourd cotyledons* Each hormone increased , enzyme a c t iv i ty

to various degrees but had no e f fe c t on th e i n i t i a t i o n of

•nzyme a c tiv ity * Maximum a c t iv i ty o f a lk a lin e phosphatase

was found in cy tok in in tre a te d cotyledons* The e f fe c t on

a lk a lin e phosphatase and m alic enzyme was found to be

correlated w ith i t s e f fe c t on th e con ten t o f ch lo rophy ll

in co ty ledons.

Dendsay and Sachar (1973) repo rted th a t i n mung

bean coty ledons trea tm en t w ith I aa brought about a 2 to

4 -fo ld in c re a se in th e a c t iv i ty o f peroxidase and changed

the e le c tro p h o re tic p a t te rn o f iso p ero x id ase s .

B harti e t a l (1979) s tud ied changes in th e a c t iv i ty

of IM oxidase and peroxidase % ascorb ic acid u t i l i z a t i o n

and the le v e ls o f param agnetic manganese during k in e tin

induced growth o f the is o la te d cucumber cotyledons in l ig h t

and in the dark . K in e tin s tim u la ted th e a c t iv i ty of

peroxidaae both in l i # t and In th e d&ric. K in e tin tre a te d

cotyledons exposed to l i g h t had a h ig h e r a c t iv i ty o f Iaa

ox ida te but a low er le v e l o f param agnetic manganese (Mn++) .

The le v e l of oaramagn t i c manganese in cotyledons m aintained

in th e dark was not a f fe c te d by k in e tin trea tm en t. I t was

suggested by th ese workers 1hcd. phenolic co fac to rs requ ired

fo r o x id a tio n o f manganese and IAA were l im it in g in the

k in e tin t r e a te d cotyledons in darkness.

S tim u la tion o f expansion by k in e tin in excised

fenugreek co ty ledons accompanies an in c re a se in MA con ten t

( iiijv e n g t a t . ,1 9 7 1 )• Treatment w ith analogues o f n u c le ic

ac id bases showed a requirem ent fo r mHlA sy n th es is In k in e tin

induced expansion. Thus 8-azaguanine in h ib ite d bo th

expansion and n e t MA sy n th e s is , whereas 5 -f lu o ro u ra c il

in h ib ite d only th e n e t MA sy n th e s is . A pyrim idine analogue,

th io u ra o il was found to in h ib i t both expansion and n e t MA

in c re a se . Key and In g le (1968) repo rted th a t f lu o ro u ra c il

In h ib i ts n e t MIA sy n th e s is , but n o t th e expansion in

fenugreek co ty ledons, sugg estin g th a t the req u ired sy n th es is

i s th a t o f ttMAf sinoe f lu o ro u ra c il i n h ib i t s th e sy n th es is

of s-EIA and t-M A . Thus, in fenugreek cotyledons k in e t in

appearsiio e x e rc ise i t s c o n tro l a t th e le v e l of t r a n s c r ip t io n .

Krawiarz a l . . (1977) s tu d ied th e in te ra c t io n

between abaci s i c a d d and BAP in th e expansion and greening

o f squash co ty ledons. ABA in h ib ite d th e expansion o f

co ty ledons, accum ulation o f oh lo rophy ll and th e a c t iv i ty o f

ac id and a lk a lin e pyrophosphatases and o f m alic enzyme,

a f fe c t o f ABA was re la te d to i t s co n c en tra tio n . Synthesis

o f ch lo ro p h y ll and th e a c t iv i ty o f th e enzymes were in h ib ite d

b efo re in h ib i t io n o f growth was m an ife s t, suggesting th a t

th e a c tio n of aba on metabolism vas independent o f i t s e f fe o t

on growth. In h ib i t io n o f growth by ABA was more pronounced

In co ty ledons tre a te d w ith BA? than th o se re c e iv in g no BAP.

F arineau a l . .(1 9 7 8 ) s tu d ied th e In te ra c t io n

between chloram phenicol and BAP in cucumber co ty ledons. BAP

was found to s tim u la te bo th ch lo rophy ll sy n th esis and p i as t id

9u

d if f e r e n t ia tio a and in creased the a rea of appressed

thylakoids. Chloramphenicol decreased the rate o f ch lo ro p h yll

synthesis o nly when i t was given continuously du ring the

incubation u erio d .

Effect of frib b e re llin s

G ibbere llic acid (GA^), a t r e la t iv e ly higher

concentrations, has been shown to cause expansion o f iso la ted

cotyledons in puapkin ( ^aner j i and la loraya, 1 9 67). ’Canthium

( ^saehi and Leopold, 1969), radish (Lethara, 1971) , watermelon

(bia,s»h cjt a l . . 1973)» oucuaber ( Udayakuraar £t a l. .1973)

and Itaoaoea ( Gankhla, 19 69) • The promotive e f fe c t o f GA

on expansion o f excised cotyledons is , ho waver, mo»aifest

only in the presence o f l ig h t .

A comparison of th e e f fe c ts o f indole— a c e t i c ac id ,

k in e tin and GA- on the growth of pumpkin ootyledon3 was

made by B anerji and L aloraya (1967)* Auxin was found

in e f f e c t iv e in enhancing the growth o f Is o la te d co ty ledons.

K ine tin and OA, both caused enlargement of cotyledons but jthe n a tu re of growth and the u lt im a te fo m they produced were

d i f f e r e n t .

Expansion growth o f lanthium cotyledons was shown

to be stim u la ted by in th e ra^gc. o f 10"*® to 1 0“ ®fl,

i>ut the responses were low er than those induoed by BAP

( -icashi and Leopold, 19 69). M annitol a t 2 . 5 x 1 1 com pletely

ro

23

inh ib ited the response to GA .

Singh ^1 . (1913) s tu d ied th e e f fe c t o f IAA,

and k in e t in on th e expansion and ch lo ro p h y ll syn thesis

of iso la te d iratexmelon ( C itru l lu s v u lg a ris ) ootyledons

in l ig h t . G ib b e re llic aold was found most e f fe c t iv e in

increasing th e s iz e o f ooty ledons, whereas IAA was

in e ffe c tiv e . Expansion growth due to GA and k in e t in was

associated w ith in c reased ch lo rophy ll foxm atlon. GA caused

aore ch lo rophy ll accum ulation than k in e t in . Chloramphenicol

aaft oyclohexlmide were t e s ted to d i f f e r e n t i a t e between th e

effeo t o f GA and k in e t in on p ro te in sy n th es is In oytoplasm lo

and c h lo ro p la s t f ra c tio n s in th e expanded co ty ledons.

Chloramphenicol s tro n g ly in h ib ite d expansion, ch lo ro p h y ll

content and n u c le ic a d d sy n th es is whereas oyclohexlmide

was r e la t iv e ly le s s e f fe c t iv e . GA was more e f fe c t iv e than

k in e tin in revers ing th e in h ib ito ry e f f e c ts o f chloram phenicol.

The a b i l i t y o f GA and k in e t in to re v e rsa the in h ib ito ry

e ffec t o f chloram phenicol was in te rp re te d as to suggest

that GAj and k in e t in in c re a se the amount o f oh lo rophy ll in

Iso la ted ootyledons by c o n tro l l in g th e sy n th es is of n u c le ic

aoids and p ro te in s i n th e c h lo ro p la s t.

Sankhla (1969) s tu d ied th e e f fe o t o f GA a t 1 and

10 ppm on the expansion o f Ioomea ootyledons GAj a t 10 pp®

was more e f fe c tiv e than GA a t 1 ppm in causing expansion.

SStel..

Auxins have g en e ra lly been found to be in e f f e c t iv e

l a promoting expansion growth o f i s o la te d coty ledons

(B anerji and Lai oraya* 1967 I Esashi and Leopold, 1969}

Bingh §& a i,.l973 ; Udayakuraar §£ g ^ ., 1973).

S&akhla (1970) studied Hie e f f e c t o f IAA, IAA and

2,4«*D on th e expansion growth o f detached Iuomea o en tao h v lla

cotyledons in l i g h t . Both 31AA and IAA had no e f fe o t on

ootyledon expansion. However, 2,4-D a t 10 ppm enhanced

expansion growth o f detached co ty ledons. In com bination

with m orphactin, 2,4-D acted s y n e rg is t ic a l ly in prom oting

the expansion growth o f co ty ledons.

Bendsay £ t a l . .(1 1 7 8 ) rep o rted th a t supraoptim al

concen tra tions o f IAA (1 (T^;i and 1CT^i) b ro u ^ it about 2 to

A-fold in c re a se in peroxidase a c t iv i ty over the w ater

con tro l in excised iaung bean co ty ledons. S y n th e tic auxin,

2,4-D proved su p e rio r to IAA in enhancing peroxidase

a c t iv i ty . CrAj and k in e tin had no e f f e c t e i th e r on perox idase

a c t iv i ty o r on th e p a t te rn o f iso p e ro x id ase s . ABA a t 1 00

fig/ml s tro n g ly suppressed perox idase a c t iv i ty , and th i s

e f fe c t was s u b s ta n t ia l ly reversed by auxin . Actinoraydn-D

a t 50 jir/rnl and cyclohexinide a t 5 p g /o l reduced peroxidase

a c t iv i ty both in c o n tro ls and in auxin tre a te d co ty ledons.

These r e s u l t s sag eated th a t In aung bean coty ledons auxins exercised th e i r e f f e c t on perox idase a t th e le v e l o f

t r a n s c r ip t io n and t r a n s la t io n .

E ffect of Fueloocoin

Fusicoocin (FC)a fungal to x in produced by th e

fungus Fusicoocum amvodall. has a lso been shown to be

e f fe c t iv e in causing th e expansion growth of detached

cotyledons ( Marre e t a l . . 1974). Like aux ins, FC s tim u la te s

th e e longation growth o f excised stem segments ( B a llio

e t a l . . 1971 f Lado jgt a l . . 1973). Besides growth, a

number o f im portan t p h y sio lo g ica l 0roc esses a re in fluenced

by iC. Most o f th e se e f fe c ts such as c e l l enlargem ents H+

ex tru s io n , potassium uptake and stom atal opening have been

observed in t i s s u e s o f a number o f h ig h er p la n t sp ec ie s .

Ihe cap ac ity to respond to FC seesas to be p re sen t g en e ra lly

in a l l green s la n ts from th e Charophyceae to h ig h e r p la n ts .

Fungi, b a c te r ia and a n ia a ls a re , however, not known to be

responsive to FC.

R ijven (1976) s tud ied th e e f fe c ts o f FC and k in e tin

in is o la te d fenugreek ootyledons. Both FC and k in e t in

s tim u la ted the expansion grovrth of co ty ledons. C erta in

im portan t d iffe re n c e s were, however, observed between the

e f fe c ts o f k in e t in and FC. FC and k in e tin added to g e th e r

a t optim al co n cen tra tio n s promoted growth s y n e rg is t ic a l ly .

K.C1 doubled the fre sh weight in c re a se induced by 1C and

Induced a s ig n if ic a n t drop in pH of th e medium by 2 u n i ts .

On the o th e r hand KOI stim ulated the fre sh weight response

o f k in e tin by only 20% and had no s ig n if ic a n t e f fe c t on th e

pH of th e medium. O ther monovalent ca tio n s v i z . , ^a+, L i+

and could not rep lace I* in enhancing th e PC Induced

growth o f detached cotyledons* £C mimics the e f f e c t o f BA?

on cotyledon expansion, but f a i l s to d u p lic a te i t s a c tio n

on microbody enzymes. Suggesting th a t the e f fe c t on

microbody enzymes i s not c lo se ly lin k ed w ith th e mechanism

of growth promotion.

.la r re (1980) sug-rested th a t PC d i r e c t ly a c tiv a te s

the ATP driven e lec tro g en ic pro ton pump a t the plasmalesnma.

This would lead to the in c re a se in the H+ co n cen tra tio n

g rad ien t ac ro ss th e plasmalemma and to the h y p e rp o la riza tio n

of p o te n t ia l d if fe re n c e . Consequently, a l l o f th e tra n sp o r t

p rocesses deoending on th e proton g ra d ie n t and /o r p o te n t ia l

d if fe re n c e a re s tim u la ted . Among th ese are K* uptake,

symport o f an ions, jp^sjpfcovyCaieds. sugars and aminoacids

xdLth n ro to n s, and H+/^ a + count e r tra n sp o r t .

Aims and o b je c ts o f th e study

The review o f l i t e r a t u r e presen ted in the ^receding

ptiges revealed th j.t although consider;.b le in fo rm ation e x is ts

on the e f fe c ts o f cy tok in ins on expansion growth and

ar> so e la te d m etabolic changes th a t occur in is o la te d

cotylecions, in fo rm ation about the e f f e c ts produced by

g ib b e re ll in s rend fu s ico c c in i s meagre, in s p i te of t h e i r

reported a b i l i t y to induce expansion l a is o la te d co ty ledons.

The p resen t study was th e re fo re aimed a t studying th e

e ffe c ts of C rlbberellins ( Ga^ and ) and of fusi occin

in ad d itio n to th o se o f cy tok in in s (k in e t in and benzyl-

aminopurlne) on expansion growth and on c e r ta in a sp ec ts

of metabolism in cucumber oytoledons. The e f fe c ts have

been stud i d both w ithout and ?ith 5 mM potassium (su p p lied

as KOI) to fin d out how f a r prepence o f potassium which

also induces expansion, can modify the e f fe c t of the re

growth re g u la to rs . The desifja of exoeriraents has been kept

uniform to perm it comparisons. B esides, an experiment has

been e a rr ie a out to compare th e e f fe c t of a g lb b e re ll in ,

a cytokinin and fu s ic o c c ia .