CHAPTER 4

DISCUSSION

I n t h e p a s t two decades c o n s i d e r a b l e p r o g r e s s h a s been

made i n improvement o f o r n a m e n t a l s , p a r t i c u l a r 1 y i n

d i s t i n c t i v e p l a n t s l i k e o r c h i d s i n which b a s i c r e t h o d o l o g j e s

have a l r e a d y been e s t a b l i s h e d . However, new phase of

s t r a t e g y i s s t i l l t o be e s t a b l i s h e d t o p r o p a g a t e

r e c a l c i t r a n t s p e c i e s i n O r c h i d a c e a e i n v i t r o c o n d i t i o n .

Kri k o r i an (1982 1 and Ammi r a t o (1983 1 have emphasi zed

t h a t t h e problem of i n d u c i n g morphogenesis i s fundamenta l

d e s p i t e t h e manipu la t ion by growth r e g u l a t o r s o r a d d i t i v e s .

I t i s sometimes d i f f i c u l t t o s e e any response o r even

s t i m u l a t i o n of c e l l d i v i s i o n .

I n t h e p r e s e n t s t u d y d i f f e r e n t morphogenetic r e s p o n s e s

a r e d i s c u s s e d under t h e f o l l o w i n g heads jn of t h e

p r e s e n t work and a v a j l a b l e l i t e r a t u r e . Wherever p o s s i b l e

r e l e v a n t examples have been c i t e d .

4.1 FRUIT SETTING

F r u i t s e t t i n g i s n o t s e e n i n a l l t h e o r c h i d s main ly

because of t h e absence o f p o l l i n a t i n g a g e n t s . This h a s been

done e f f e c t i v e l y by a r t i f i c i a l p o l l i n a t i o n i n P e r e s t e r i a

e l a t a and Pha ius l u r i d u s . P e r s i s t a n t f a i l u r e i n s e t t i n g t h e - f r u i t i n P e r e s t e r i a e l a g a i n d i c a t e d t h a t no i n s e c t c o u l d

90

b r i n g about p o l l i n a t i o n . P e r e s t e r i a e l a t a i s a member of the

S t a n h o p i a e , a c t u a l l y a d a p t e d t o p o l l i n a t i o n by male

e u g l o s s i n e which a r e endemic t o South America (Robert

D r e s s l e r 1981). T h e i r absence i n t h i s a r e a of c u l t i v a t i o n

and a l s o t o a t t r a c t any o t h e r i n s e c t s of s i m i l a r type

i n d i c a t j n g t h e i r s p e c i f i c i t y f o r e u g l o s i n e bees t o b r ing

about p o l l i n a t i o n h a s r e s u l t e d i n no seed s e t . I n Phaius

l u r i d u s and Coelogvne nervosa though seed s e t t i n g occurs

n a t u r a l l y i n t h e i r own h a b i t a t , hand p o l l i n a t i o n j s

i n e v i t a b l e t o s e t more f r u i t s . Matur i ty of t h e f r u i t t a k e s

60-70 d a y s I n P e r e s t e r i a e l a t a and u p t o 90 d a y s f o r Phaius

l u r i d u s . The n a t u r e f r u i t s a r e h a r v e s t e d normal ly a f t e r t h e

appearance of r i d g e s on t h e f r u i t s i n d i c a t i n g t h e i r m a t u r i t y .

4 . 2 ORCHID SEEDS

Orchid s e e d s a r e minute , d u s t !i ke and v a r y i n t h e i r

s i z e and s h a p e . There a r e f i v e b a s i c forms of s e e d ( C l i f f o r d

and Smith 1 9 6 9 ) .

I n t h e p r e s e n t l y i n v e s t i g a t e d s p e c i e s , c o l o u r and

n a t u r e of t h e s e e d s e x h i b i t d i v e r s i t y . The c o l o u r of ?he

s e e d s i n P e r e s t e r i a e l a t a and Pha ius l u r i d u s i s c reamish

i n d i c a t i n g t h e f r m a t u r i t y . The g r e e n i s h n a t u r e o f t h e seeds

was found t o be prominent i n mature f r u i t s o f Coelogyne

nervosa .

All t h e s e e d s l a c k endosperm resulting i n unique

u n d i f f e r e n t i a t e d embryos e n c l o s e d w i t h i n t r a n s p a r e n t seed

91

c o a t . Embryonal c e l l s p o s s e s s prominent d e n s e l y g r a n u l a t e d

c y t o p l asm.

4 .3 SEED CULTURE

S ince long t i m e i t i s a f a m i l i a r f a c t t h a t a l l o r c h i d s

whether t e r r e s t r i a1 o r e p i p h y t i c , a u t o t r o p i c o r h e t e r o t r o p i c

and tempera te o r t r o p i c a l a r e a s s o c i a t e d w i t h mycor rh iza l

fungus which i s i n d i s p e n s a b l e f o r s u r v i v a l of t h e i r embryo

and g e r m i n a t i o n . The a s s o c i a t i o n i s main1 y re1 a t e d t o t h e

s u p p l y of s imple s u g a r s t o d e v e l o p i n g embryo ( A r d i t t i 1967

a , b , Har r i son and A r d i t t i 1 9 7 8 ) . E a r l i e r i t was d e m o n s t r a t e d

t h a t o r c h i d seeds c a n be g e r m i n a t e d under i n vi t r o c o n d i t i o n

w i t h o u t t h e a s s o c i a t i o n o f mycor rh iza l fungus (Knudson 1922 ,

C a r l s o n 1935, Ardi t t i and B i l l s 1965, Chennaveerai ah and

P a t i l 1975, Devi e t a l . 1990 and Pathak g c. 1 9 9 2 ) .

E a r l i e r , i t was b e l i e v e d t h a t o r c h i d s e e d s were s t e r i l e

and i n c a p a b l e of g e r m i n a t i o n b u t l a t e r i t was conf i rmed by

v a r i o u s workers t h a t t h e y c a n g e r m i n a t e ( C o n s t a n t i n 1 9 1 3 ) .

There a r e d i f f e r e n t v i e w s r e g a r d i n g t h e g e r m i n a t i o n of

e p i p h y t i c and t e r r e s t r i a l o r c h i d s . According t o S t o u t m i r e

(1963 , 1 9 6 4 a ) , t h e r e i s no problem i n g e r m i n a t i n g e p i p h y t i c

o r c h i d s . However, Wareup ( 1 9 7 1 ) and McIntyre e t a l . ( 1 9 7 4 )

have p o i n t e d o u t t h a t d i f f i c u l t y was encounte red j n t h e

g e r m i n a t i o n of t e r r e s t r i a l s p e c i e s , t h e r e a s o n b e i n g

a t t r i b u t e d t o t h e f a c t t h a t t h e y a r e endemic and g r e a t e r

dependence of s o i l growing o r c h i d s on mycor rh iza l f u n g u s f o r

g e r m i n a t i o n which r e n d e r t h e g e r m i n a t i o n of t e r r e s t r l a1

o r c h i d s i n v i t r o very d i f f i c u l t ( S t o u t a m i r e 1 9 7 4 ) . F u r t h e r ,

d u r a t i o n of dormancy p e r i o d i s l o n g e r t h a n i n t h e e p i p h y t i c

s p e c i e s .

The advantages of g r e e n pod c u l t u r e i n o r c h i d seed

g e r m i n a t i o n was emphasised by Sagawa ( 1 9 6 7 ) . I n t h e p r e s e n t

s t u d y t h e s u c c e s s of g r e e n pod c u l t u r e was r e s t r i c t e d t o

e p i p h y t i c s p e c i e s l i k e Coelogyne nervosa and P h o l i d o t a

i m b r i c a t a . Mathews and Rao ( 1 9 8 0 ) showed i n Vanda h y b r i d ,

o n l y 30% of seeds f r o r r i p e pods g e r m i n a t e d , whereas i t was

80-100% from green pods. V i j and Pa thak ( 1 9 8 8 ) r e p o r t e d

s f m f l a r t y p e of response ~ n Cymbidium macrorhizon ~ n which

s e e d s from mature pods f a ] l e d t o g e r m j n a t e i n a1 1 t h e media

t e s t e d . However, i r - a t u r e s e e d s germina ted on basal medium

w i t h o u t growth r e g u l a t o r s .

Sagawa (19631 i s of t h e o p i n i o n t h a t i n c r e a s e d

p e r c e n t a g e of g e r r i n a t i o n i s due t o t h e f a c t t h a t d u r i n g

s u r f a c e s t e r i l i z a t i o n b e f o r e i n o c u l a t j o n i t i s t h e

s u r f a c e of t h e pod t h a t i s a f f e c t e d by s t e r i l a n t and n o t t h e

i n n e r s e e d s . However, i t s h o u l d be a s c e r t a i n e d whether t h e

s e e d s of t h e o l d e r pods accumula ted any of t h e c h e m i c a l s

t h a t would i n t e r f e r e wi t h t h e p r o c e s s of immediate

g e r m i n a t i o n a s i s wel l known i n most of t h e ang iosperms .

A s t h e immature and mature s e e d s can h a r d l y be

d i s t i n g u i s h e d morphologi c a l . l y , some of t h e i r hi s t o c h e m i c a l

and biochemi ca! f e a t u r e s a r e needed f o r s e l e c t i n g

pod c o n t a i n i n g t h e r i g h t t y p e of s e e d s . ,A s t u d y of t h e

d e g r e e of m a t u r i t y of o r c h i d s e e d s w i t h i n a pod r e v e a l s

changes i n t h e i r enzyme complements (Raghavan 1 9 6 7 ) . Withner

( 1 9 5 5 ) i n h i s s t u d i e s on o v u l e c u l t u r e of V a n i l l a s p e c i e s

s u g g e s t e d t h a t v a n i l l i n e l i k e s u b s t a n c e s which a c c u n u l a t e

d u r i n g m a t u r a t i o n of s e e d s may i n h i b i t t n e g e r m i n a t i o n of

normal ly mature pods . Burgeff ( 1 9 5 4 ) a l s o c o n s i d e r e d t h e

p r o d u c t i o n of i n h i b i t o r s d u r i n g r r a t u r a t l o n of s e e d s of

European o r c h i d s and Paphiopedi lum. He s u g g e s t e d t h a t

i n h i b i t o r s c o u l d be removed by p l a c i n g t n e s e e d s i n 1 j q u l d

~ e d i u r n f o r few d a y s . I n many s p e c i e s , s e e a s from mature pods

have been found t o e x h i b i t poor g e r m i n a t i o n a l s o due t o

dormancy of s e e d s .

4.3.1 S t a n d a r d i s a t i o n of Media

Knudson ( 1 9 2 1 ) f o r m u l a t e d f i r s t a new s i m p l e n u t r i e n t

rredium f o r g e r m i n a t i o n of o r c h i d s e e d s wi t n o u t any s y m b i o t i c

a s s o c i a t i on w i t h mycorrhi z a l f u n g u s . A f t e r t h i s s e v e r a l

~ e d i a have been c r i t i c a l l y e v a l u a t e d . To d a t e , a t l e a s t 25

media s u i t a b l e f o r t h e g e r m i n a t i o n of o r c h i d s e e d s and

growth of t h e s e e d l i n g s have been proposed and u t i l i z e d .

Four t y p e s o f media (MS, N H B M , VW and K N C ) a r e employed

f o r i n v i t r o s e e d g e r m i n a t i o n i n t h e p r e s e n t i n v e s t i g a t i o n .

I n c o r p o r a t i on of m i c r o n u t r i e n t s i n t h e c u l t u r e media i s

recommended even i f i t i - a s not b e n e f i c i a1 but were n o t

harmful (Ard i t t i 19671. The b e s t s t a n d a r d i s e d media \;ere i n

t u r n u t i l i s e d f o r t h e s e e d l i n g s t u d i e s . The number of l e a f ,

r o o t and h e i g h t produced b-ere t a b u l a t e d . Some of t h e media

c o n t a i n o n l y t h e major n u t r i e n t s w h i l e o t h e r s c o n t a i n n i n o r

e l e m e n t s a l s o .

The s e e d s of P h a i u s l a r i d u s g e r m i n a t e d on NHBH, !!S and

Knudson's C media. The g e r - i n a t i o n was s low on Vacin L Went

medium. However, NHBM was found t o be s u p e r i o r . Pha ius

l u r i d u s i s a t e r r e s t r i a l s o e s i e s and g e n e r a l l y requires h i g n

s a l t c o n c e n t r a t i o n f o r ger - : n a t i o n . N H B M c o n t a i n s high s a l t

s t r e n g t h when compared t o K:;C o r VW ( s e e T a b l e 2 ) . Tne q u i c k

enhanced germi n a t i on may 3e a1 so due t o t h e p resence of

v i ab1 e o r p a r t i a l l y d i f f e r o n t i a t e d embryos a s made out by

Burgeff ( 1 9 3 6 ) and Harl ey 1951 ) . R h i z o i d s a r e normall-y 2roduced a t t h e p o s t e r i o r end of

t h e p r o t o c o r m s , a s n o t i c e d i n A e r ~ d e s c r i spm, Denorobiurn

~ a c r o s t a c h y u m (Ekanthappa 1961 ) . However, i n Cvmbjdiu- t h e y

a r e produced on t h e e n t i r e s u r f a c e of t h e protocorm. S ~ m i l a r

f e a t u r e were a l s o r e c o r d e d i n V a n i l l a (Knudson 19501 . Many

workers have shown a d i r e c t c o r r e l a t i o n between r h i z o i d s

f o r m a t i o n and c h l o r o p h y l l development i n t h e p ro tocorms .

There a r e s p e c i e s which a c q u i r e c h l o r o p h y l l i n t h e i r

g e r m i n a t i n g embryos e a r l y and b e a r l e s s e r number of r o o t

h a i r s . I n c o n t r a s t t o t h o s e i n which c h l o r o p h y l l development

i s l e s s , more number of r h i z o i d s a r e a e v e l o p e d on t h e

p r o t o c o r - s . I n t h e p r e s e n t s t u d y t h e protocorms of Pha ius

l u r i d u s posses more number of r h i z o l d s a s we!! a s

c h l o r o p h y l l p igments . The development of rh i z o i d s i n Phai us

l u r i d u s - a i n t a i n e d r o o t c h a r a c t e r of protocorm. I n n a t u r e

r h i z o i d s a s s j s t t h e e n t r y of mycorrhiza! f u n g u s Bernard

( 1 9 0 2 ) e s t a b l i s h e d t h e f a c t t h a t f u n g a l hyphae can p e n e t r a t e

t h r o u g h :he r h i z o i d s and l a t e r i t was a1 s o conf i rmed by

Harvai s and Hadley (19671 .

The seeds of P e r e s t e r i a e l a t a g e r r n ~ n a t e d w e l l on a l l

t h e f o u r - e d i a . However, q u i c k germina t ion and b e t t e r growth

was observed on MS medium. N H B M a l s o e q u a l l y f a v o u r s

g e r m i n a t i o n . The p e r c e n t a g e of germina t ion i s near1 y t o t a l ,

which r a y be due t o t h e i n c o r p o r a t i o n of p e p t o n e , c a s e i n

hydro1 y sa r e ( C H ) and coconut milk I n a d d i t i o n t o o t h e r

f a c t o r s ! ike c o n c e n t r a t i o n of s a l t and v i a b l e embryo.

The a d d i t i o n o f pep tone t o t h e mediur, s t i m u l a t e s seed

g e r m i n a t i o n . C a s e i n h y d r o l y s a t e has been s p a r i n g l y used i n

o r c h i d cu l - tu re . I t s add j t i o n t o n u t r i e n t medium f a v o u r s

germj n a t i o n of s e e d s of S p a t h o g l o t t i s pl i c a t a

(Chennaveera iah and P a t i l 1 9 7 5 ) , Cymbidium m a s t e r s i ( P r a s a d

and Mi t ra 19751, A e r i d e s m u l t i f l o r u m , R h y n c h o s t y l i s r e t u s a

and Vanda p a r v i f l o r a (Vi j e t a l . 1 9 8 1 ) , Goodyera b i f l o r a

( P a t h a k e t a l . 1 9 9 2 ) .

Peptone i s no t e f f e c t i v e d u r i n g t h e e a r l y s t a g e s of

seed g e r m i n a t i o n but promotes protocorm growth and

p r o l i f e r a t i o n a s i n L'anda (!lathews and Rao 1 9 8 0 ) , Dendrobiu-

and Vanda (Morel 1 4 7 4 ) . In t h e p r e s e n t work p e p t o n e a t

v a r i o u s c o n c e n t r a t i o n h a s s t i m u l a t i n g e f f e c t on seed

g e r m i n a t i o n i n P e r e s t e r i a e l a t a .

The m i c r o n u t r i e n t i n t h e medium promotes r a r e of

g e r m i n a t i o n and g rov th of t h e s e e d l i n g s . These r e s u l t s agree

w i t h t h e r e p o r t s of A r d i t t i ( 1 9 6 7 a , b ) .

Coelopyne i s a r e p l p h y t l c o r c h l d which responds well to

Knudson ' s C medlur f o r germination. I t was found t o b ~

s u p e r i o r t o r e s t of t h e medla. B e t t e r g e r m i n a t ~ o n i n ~t ma>

be due t o t h e 1 ow s a l t concentration of medium and v a r i o u s

o t h e r a d j u v e n t s ( C a s e i n h v d r o l y s a t e , Peptone and Coconut

r l i l k ) . NHBM was found t o be poor ~ n a c c e l e r a t i n g t h e r a t e of

g e r m j n a t i o n . The e f f e c t i v e r o l e of Knudson's C medjum In

g e r m i n a t i o n i s r e p o r t e d i n C o e l o ~ v n e p u n c t a t a .

Coconut mi lk a t v a r i e d p e r c e n t a g e enhanced p e r c e n t a g e

of g e r m i n a t i o n and c a l l u s i n g of s e e d s . C a l l u s f o r m a t i on i s

a l s o n o t i c e d on b a s a l redium supplemented w i t h Casein

h y d r o l y s a t e and pep tone . The c a l l u s i s w h i t i s h t o c reamish

and f r i a b l e i n n a t u r e . During s e e d germina t i on o r g a n i s a t i o n

of c a l l u s has been recorded i n Vanda t r i c o l o r ( C u r t i s and

Nichol 1948) and Vanda h y b r i d (Goh 1 9 7 0 ) . I n t h e s e s p e c i e s ,

c a l l u s f o r m a t i o n was i n v a r i a b l y produced i n p resence of

a u x i n and coconut milk.

I n d u c t i o n of c a l l u s i n p r e s e n c e of coconut milk i s

main ly due t o i t s compos i t ion . N a t u r a l growth r e g u l a t o r s

l i k e I A A and Z e a t i n a r e commonly found i n t e n d e r coconut

mi lk . I n a d d i t i o n o t h e r f a c t o r s l i k e amino a c i d s o u r c e s

s u p p o r t c a l l u s f o r m a t i o n . Rao ( 1 9 6 3 ) observed c a l l u s

i n i t i a t i o n even I n t h e absence o f supplements . According t o

Ekanthappa ( 1 9 8 1 ) c a l l u s i n i t i a t i o n i s g r e a t e r i n c a s e s when

immature s e e d s a r e used f o r f l a s k i n g . I n mature seeds c a l l u s

was formed more f r e q u e n t l y on medium s u p p l e r e n t e d w i t h

coconut mi lk o r 2,L-D. C u r t i s and Nicho? ( 1 9 & 8 ) conc luded

t h a t immature and u n d i f f e r e n t i a t e d o r c h i d seeds probably

caused t h e c a l l u s i n g of s e e d s . Narayanaswamy and Norstog

( 1 9 6 4 ) a r e of t h e o p i n i o n t h a t c a l l u s f o r m a t i o n i n c u l t u r e

of immature s e e d s i s p robably due t o t h e u n s t a b l e p o l a r i t y

w i t h i n t h e embryo and t h e p o l a r i t y w i l l be r e g a l n e d i n t h e

c a l l u s , s imultaneous!y w i t h t h e d i f f e r e n t i a t i o n of r o o t and

s h o o t p o l e s . However, i n t h e p r e s e n t work c a l l u s was

produced o n l y from mature s e e d s i n Coelogyne n e r v o s a . T h i s

i s c o n t r a r y t o t h e o b s e r v a t i o n s of o t h e r works.

P h o l i d o t a i m b r i c a t a an e p i p h y t i c o r c h i d i s wel l s u i t e d

f o r g e r m i n a t i o n i n Knudson's C medium which c o n t a i n s major

and few minor e l e m e n t s of low s a l t s t r e n g t h . The p e r c e n t a g e

98

of g e r m i n a t i o n i s near t o t a l due t o low s a l t s t r e n g t h I n t h e

media i n a d d i t i o n t o t h e p r e s e n c e of v i a b l e embryo and

v a r i o u s o t h e r components of t h e media. The g e r r i n a t i o n r a t e

was found t o be low i n high s a l t s t r e n g t h media l i k e MS and

NHBM. The p r e f e r e n c e i s !ow s a l t s t r e n g t h d u r i n g

g e r m i n a t i o n .

C a l l u s f o r m a t i o n f r o r t h e s e e d s of P h o l i d o t a i - b r i c a t a

i n t h e p r e s e n t work was s i g h t e d i n p r e s e n c e of h igh

c o n c e n t r a t i o n of coconut ri!k ( 2 0 % ) . I t i s f r i a b l e and non-

p h o t o s y n t h e t i c . I t s i n d u c t i o n may be due t o a v a i l a b i l i t y of

endogenous a u x i n s i n t h e coconut m i l k .

Dur ing s t a n d a r d i s a t i o n of t h e media i n t h e p r e s e n t

s t u d y , i t was evidenced t h a t s e e d s of t e r r e s t r i a l s p e c i e s

p r e f e r r e d t o grow i n high s a l t s t r e n g t h composi t i o n , whereas

s e e d s o f e p i p h y t i c s p e c i e s seem t o be c o m f o r t a b l e i n

p r e s e n c e o f low s a l t s t r e n g t h . However, t h e r e a r e ' r e p o r t s of

g e r m i n a t i o n of e p i p h y t i c s p e c i e s on hi gh s a l t s t r e n g t h

~ e d i u m s u c h a s Nitch a s i n Dendrobium moschatum and 2.

f imbr ia tum (Devi e t a l . 1 9 9 0 ) .

The h i g h s a l t s t r e n g t h i n t h e media i s mainly due t o

t h e p r e s e n c e of micro-elements and s t e a d y i n c r e a s e of

n i t r a t e c o n c e n t r a t i o n . Both MS and NHBM a r e h i g h s a l t

s t r e n g t h media p r e f e r r e d by two t e r r e s t r i a l s p e c i e s such a s

P h a i u s l u r i d u s and P e r e s t e r i a e l a t e .

Low s a l t s t r e n g t h i s due t o t h e absence of minor

e l e m e n t s and r e d u c t i o n i n n i t r a t e a s wel l a s phosphorous

c o n c e n t r a t i o n . Reduct ion i n p h o s p h a t e l e v e l can i n c r e a s e

g e r m i n a t i o n , t h e reason b e i n g t h a t o r c h i d seeds a r e

s e n s i t i v e t o phosphorous. S i m i l a r l y t h e g e r n i n a t i o n of

V a n i l l a p l a n i f o l i a seed was g r e a t l y improved f o l l o w i n g a

t e n f o l d r e d u c t i o n i n n i t r o g e n c o n t e n t of Rnudson's B medium

b u t was t o t a l l y i n h i b i t e d i f c o n c e n t r a t i o n s were doubled

(Lugo-Lugo 1955 a , b ) . I n g e n e r a l i t appears t h a t low

c o n c e n t r a t i o n s of n i t r o g e n s t i m u l a t e d germina t ion of o r c h i d

s e e d s whereas h i g h c o n c e n t r a t i o n s a r e i n h i b i t o r y (Ardi t t i

1 9 6 7 ) .

4.4 SEEDLING STUDIES

The pro tocorms o b t a i n e d d u r i n g t h e germina t ion of s e e d s

were s u b j e c t e d t o s e e d l i n g s t u d i e s . Both auxins and

c y t o k i n i n s e i t h e r a l o n e o r i n combina t ion showed many

c o n t r a d i c t o r y r e s u l t s . The l i t e r a t u r e p e r t a i n i n g t o s e e d l i n g

s t u d i e s r e v e a l s t h a t a u x i n s o r c y t o k i n i n s a l o n e can enhance

t h e g r o w t h of t h e s e e d l i n g s .

4.4.1 Effect of Auxins

The most commonly employed a u x i n s f o r t h e p r e s e n t

s t u d i e s a r e I A A and NAA. 2,4-D h a s been r e p o r t e d t o i n h i b i t

seed g e r m i n a t i o n and i s even d e t r i m e n t a l t o s e e d l i n g growth.

The u s e of 2,4-D i n o r c h i d c u l t u r e l e a d s t o unnecessary

c a l l u s i n g i n s e e d s and may l e a d t o g e n e t i c i n s t a b i l i t y .

I n t h e p r e s e n t work, P e r e s t e r i a e l a t a d ~ d not

p r o l i f e r a t e f u r t h e r a f t e r s u b j e c t i n g t o d i f f e r e n t a u x i n s .

However, I A A h a s been found t o be a f a v o u r a b l e aux in f o r

p ro tocorm pro1 i f e r a t i o n . E l o n g a t i o n of protocorm was

o b s e r v e d a t v a r i o u s c o n c e n t r a t i o n s . I n a d d i t i o n shoots

emerged from nany protocorms bu t f a i l e d t o grow f u r t h e r .

Hadley and Harva is ( 1 9 6 8 ) have r e p o r t e d t h e i n e f f i c i e n t

a c t i o n o f I A A on seed!ing growth . However, p r o l i f e r a t i o n i s

r e s t r i c t e d t o e l o n g a t i o n of p ro tocorms .

C o n t r a d i c t o r y t o i t s n e g a t i v e r o l e t h e r e a r e r e p o r t s on

t h e p o s i t i v e a c t i o n of I A A on s e e d l i n g growth (Boesmann

1 9 6 2 ) . IAA a t 1 mg/! o r 2 mg/l i s e f f e c t i v e on s e e d l i n g

growth of C a t t l e y a ( H i r s h 1 9 5 9 ) and of o t h e r o r c h i d

s e e d l i n g s (Hayes 1 9 6 9 ) .

The r e p o r t on t h e p r o l i f e r a t i o n of p ro tocorms under t h e

i n f l u e n c e of 2,4-D i s s c a n t y . Chennaveera iah and Pa t i !

( 1 9 7 5 ) r e p o r t e d t h a t t h e g e r m i n a t i o n r a t e was maximum i n

p r e s e n c e o f 2,4-D. I n t h e p r e s e n t s t u d y 2,4-D enhances

f u r t h e r p r o l i f e r a t i o n , b u t s e e d l i n g growth was tota?!y

a r r e s t e d . A t h i g h e r c o n c e n t r a t i o n ( 5 m g / l ) browning of t h e

p ro tocorms was observed . I n d u c t i o n of s h o o t growth a t lower

l e v e l s of 2 , 4 - D h a s been r e p o r t e d by Veda and T o r i k a t a

( 1 9 6 9 ) . S i m i l a r l y 0 . 1 ppm promoted s h o o t growth i n C a t t l e y a

( I c h i h a s h i and Kako 1 9 7 3 ) . From t h e p r e s e n t s t u d y i t was

evident that 2,4-D can be used for per-ination but for

proliferation of seedlings growth 2,4-D czn be replaced by

any other suitable growth regulator.

During studies in Phalus luridus it Kas evident that

auxin alone can sufficiently support the protocorm

proliferation and further growth. Among various auxins

tested NAA stood first. At all the concentration i t was

found to be excellent for proliferation and growth of the

seedlings. This synthetic auxin exhibited diverse response

among different species of orchid.

The growth promoting effect of NAA has been reported in

many of the orchid species such as Cysripedium (Boesman

1962) and Vanda (Mathews and Rao 1 9 8 0 ) . However,

concentration above 1 ppm seems to stimulate production of

less nunber of seedlings and more amount of callus tissue.

Such tissue does not differentiate into pl-antlets at any

stage of growth. Bose and Mukherjee ( 1 9 i 6 ) reported that

lower concentration of NAA (1 ppm) induces callus and higher

concentration results in roots.

Fonnesbach (1972) emphasised the role of NAA and its

vigorous proliferation of protocorm. Higher concentration

resulted in the inhibition of chlorophyll synthesis.

In the present study on Phaius luridus any notable

change like callus formatton and inhibition of chlorophyll

s y n t h e s i s was not observed i r r e s p e c t i x ~ e of t h e

c o n c e n t r a t i o n s used. I n t e r e s t i n g l y shoot growth was

s u p p o r t e d even a t h lgher c o n c e n t r a t i o n . The induced r o o t s

were s t o u t and h e a l t h y . I n a d d i t i o n , p r o f u s e r o o t f o r n a t i o n

was a l s o n o t i c e d a t rhe b a s a l r e g i o n of t h e s h o o t s . Probably

i t i s d u e t o t h e a c c u m ~ l a t i o n of endogenous auxin i n

a d d i t i o n t o t h e exogenous s u p p l y . The d e l e t e r i o u s e f f e c t of

2,4-D a t h i g h e r c o n c e n t r a t i o n h a s been observed i n many

s p e c i e s o f o r c h i d s . I n P h a i u s l u r i d u s p ro tocorns were

n e c r o s e d and d ied i i i t h i n few weeks of c u l t u r e a t h i g h e r

c o n c e n t r a t i o n s . IA;. i s no t e f f e c t i v e i n growth of t h e

s e e d l i n g though i t enhanced s h o o t i n i t i a t i o n from t h e

p r o t o c o r m s . The p r o t o c o r - s o f Coelogyne nervosa i n t h e

p r e s e n t work responaed w e l l t o I A A . The s e e d l i n g s e x n i b i t e d

h e a l t h y growth and b e t t e r h e i g h t i n p resence of IAA a t t h e

c o n c e n t r a t i o n 1 and 2 mg/l . A t t h e c o n c e n t r a t i o n of 3 mg/l

i t r e s u l t e d i n c a l l u s i n g of p ro tocorms . A t t h e same

c o n c e n t r a t i o n ( 5 g / l ) of I A A i t enhances protocorms

p r o l i f e r a t i o n and h e a l t h y growth of t h e s e e d l i n g s i n Pha ius

w a l l i c h i ( N a i r 1 9 8 2 1 . I n C a t t l e y a IAA h a s a l s o been found t o

be e f f e c t i v e f o r growth and p r o l i f e r a t i o n of protocorms

(Boesman 1 9 6 2 ) . Hadley and H a r v a i s ( 1 9 6 8 ) r e p o r t e d t h a t IAA

i s n o t s a t i s f a c t o r y f o r growth of o r c h i d s e , e d l i n g s , whereas

IBA o c c u p i e d a t o p p o s i t i o n i n i n d u c i n g p r o l i f e r a t i o n .

C a l l u s formation from t h e s e e d l i n g s i s n o t a r a r e

phenomenon. The s t i m u l a t i o n o f c a l l u s fo rmat ion under t h e

103

i n f l u e n c e of auxin h a s been n o t i c e d i n s e e d l i n g s o f

Dendrobium t r a n s p a r e n c e and subsequent ly ? : a n t l e t f o r m a t i o n

from c a l l u s t i s s u e u n d e r t h e i n f l u e n c e of IAA and N A A .

However, m a j o r i t y of t h e e x p e r i m e n t s wi tn IAA, KAA and IBA

showed a c c e l e r a t i o n of s e e d germi n a t i on and s e e d l i n g growth

( W i t h n e r 1 9 7 4 ) .

Among v a r i o u s a u x i n s t e s t e d f o r Phol ido ta i n b r i c a t a i n

t h e p r e s e n t s t u d y IAA ranked f i r s t i n p r o l i f e r a t i o n and

growth of t h e s e e d l i n g s . E l o n g a t i o n of protocorms o c c u r e d

u n d e r t h e i n f l u e n c e of I A A b u t c o n t i n u e d t o s u p p o r t i t s

g rowth . NAA a l s o e q u a l l y s u p p o r t s h e a l t h y growth of t h e

s e e d l i ng. The t r e a t m e n t s w i t h 2 , 4 - D a t v a r i o u s c o n c e n t r a t i o n

r e s u l t e d i n n e c r o s i n g o f p r o t o c o r m s .

4.4.2 E f f e c t of C y t o k i n i n s

The most commonly used c y t o k i n i n s a r e Kn, BAP and 2 - i p .

The l i t e r a t u r e p e r t a i n i n g t o s e e d l i n g s t u d i e s r e v e a l s t h a t

c y t o k i n i n a l o n e can enhance t h e growth of t h e s e e d l i n g s .

I n t h e p r e s e n t s t u d y , t h e i n d i v i d u a l a c t i o n o f

d i f f e r e n t c y t o k i n i n s on P e r e s t e r i a e l a t a r e v e a l e d t h e

s i g n i f i c a n t r o l e of 2 - i p f o r protocorm p r o l i f e r a t i o n and

h e a l t h y growth of t h e s e e d l i n g s . The s u p e r i o r i t y of 2 - i p

o v e r t h e o t h e r c y t o k i n i n s i n d i c a t e d t h e s p e c i f i c r e q u i r e m e n t

of p ro tocorms f o r p r o l i f e r a t i o n . A t h igher c o n c e n t r a t i o n

c a l l u s i n g of p ro tocorms a l s o i n d i c a t e d t h a t c r i t i c a l

c o n c e n t r a t i o n i s r e q u i r e d f o r p r o l i f e r a t i o n i n t h i s s p e c i e s .

I n t h e p r e s e n t s t u d y on Pha ius l u r i d u s , k i n e t i n a l o n e

i n d u c e d a s t r i k i n g response and BAP ( 1 mg/l r e s t r i c t e d t h e

development of shoot . A t h igher c o n c e n t r a t i o n ( 5 mp/! ) i t

l e a d s t o s w e l l i n g of protocorms and even f u s i n g of a d j a c e n t

p ro tocorms r e s u l t i n g i n t u b e r l i k e s t r u c t u r e . The !eaves

were e n l a r g e d and looked abnormal. Abnormal s t r u c t u r e s have

been r e p o r t e d d u r i n g t h e germi n a t i on of c e r t a i n h y b r i d

embryos (Rao and Avadhani , 196h, Goh 1970 a , b ) . Though e x a c t

r e a s o n f o r t h i s abnormal s t r u c t u r e I s unknown, a p o s s i b i l i t y

i s due t o accumulat ion of r e s e r v e food mater i a!.

The protocorms of Coeloayne nervosa i n t h e p resen t work

responded we! 1 t o ki n e t i n and 2 - i p . However, k i n e t ~ n

enhanced seedl-i ng growth. I n a d d i t i o n , con cent rat^ on of

k i n e t i n was a1 s o found t o be c r i t i c a l . D e l e t e r i o u s e f f e c t a t

h i g h e r c o n c e n t r a t i o n i n d i c a t e d t h e d i f f e r e n t i a1 requ i rement

of g rowth r e g u l a t o r s a t p a r t j c u l a r c o n c e n t r a t i o n .

Among v a r i o u s c y t o k i n i n s t e s t e d on P h o l i d o t a i m b r i c a t a

i n t h e p r e s e n t work k i n e t i n ranked f i r s t i n p r o l i f e r a t i o n

and growth of t h e s e e d l i n g s . K i n e t i n k e e p s p r o l i f e r a t i n g

pro tocorms i n t o h e a l t h y s h o o t s . BAP d i d no t show any

p o s i t i v e e f f e c t on s e e d l i n g growth. Many protocorms t u r n

brownish and even l e a d t o c a l l u s i n g .

4.4.3 Effect of Combination of Auxins and Cytokinins

The combinat ion o f aux ins and c y t o k i n i n s d i d not show

f a v o u r a b l e e f f e c t of s e e d l i n g growth i n t h e p r e s e n t s t u d y .

However, p r o l i f e r a t i o n was c o n t i n u e d i n -;ny c o ~ b i n a t i o n s .

NAA i n combina t ion wi th k i n e t i n e l o r p a t e s protocorms

p a r t i c u l a r l y when NAA c o n c e n t r a t i o n i s r e l a t i v e l y h i g h i n

P h a i u s l u r i d u s . A t t h e same t i m e r e l a t i v e l y h i g h - - c o n c e n t r a t i o n s of k i n e t i n p roduced s t o u t protocorm b e a r i n g

ep idermal h a i r s . IAA i n combina t ion w i t h k i n e t i n o r BAP d i d

no t show any marked e f f e c t on p r o t o c o r r growth, The b e s t

growth r e s p o n s e was o b t a i n e d i n O r c h i s p u r s u r e l l a when b a s a l

medium i s supplemented w i t h 0 . 5 IAA and 5 m g / l k i n e t i n

(Hadley and Harva is 1 9 6 8 ) .

I n t h e p r e s e n t s t u d y on P h o l i d o i a i m b r i c a t a t h e

i n i t i a t i o n of r o o t s i n t h e s e e d l i n g s i s due t o t h e

i n t e r a c t i n g e f f e c t of I A A and k i n e t i n . The i n t e r a c t i n g

e f f e c t o f I A A and k i n e t i n on s h o o t / r o o t ba lance i s an

e s t a b l i s h e d f a c t i n p l a n t t i s s u e c u l t u r e ( K e f f o r d and

Goldecre 1 9 6 1 ) .

4 . 4 . 4 E f f e c t o f Complex O r g a n i c Supplements

I n c o r p o r a t i o n of complex a d d i t i v e l i k e coconut milk and

banana j u i c e i s s t i l l p o p u l a r i n o r c h i d c u l t u r e . Most of t h e

commercial l a b o r a t o r i e s i n t h e t r o p i c a l c o u n t r i e s u s e

coconut m i l k f o r o r c h i d s e e d c u l t u r e t o s t i n u l a t e s e e d

g e r m i n a t i o n and a l s o h e a l t h y growth of t h e s e e d l i n g s

(Wi thner 1 9 5 3 , A r d i t t i 1968 , E r n s t e t a l . 1 9 7 0 ) . Coconut

milk c o n t a i n s s e v e r a l f a c t o r s ( s e e T a b l e 1 9 ) .

I n t h e p r e s e n ? i n v e s r i g a t i o n d i f f e r e n t concent r a t i o n s

( 5 , 10 , 15 and 2G of co-p lex a d d i t i v e s were e - p l o y e d .

Coconut mi lk a t 59 i s b e n e f i c i a l f o r s e e d l i n g growth i n

P h a i u s l u r i d u s , whereas ~ n P e r e s t e r i a e l a t a both 5 and 10

p e r c e n t s a r e s t i m u l a t o r y . The s e e d s o f P h o l i d o t a i - b r i c a t a

responded w e l l t o a i l t h e c o n c e n t r a t i o n s . B e n e f i c i a l e f f e c t

of c o c o n u t milk f o r o r c h i d seed g e r m i n a t i o n a s we!! a s t h e

g rowth o f t h e s e e d l i n g was r e p o r t e d i n V a n i l l a and

Cypripedium ( H e g a r t y 1 9 5 5 ) . Improvement of s e e d l i n g

deve lopment of c e r t a i n t e r r e s t r i a l and ephyphyt ic o r c h i d s

w i t h t h e a d d i t i o n of coconut milk t o t h e b a s a l medium was

a l s o r e p o r t e d by v a r i o u s workers (Lawrence and A r d i t r i 1 9 6 4 ,

McIn tyrc e t a l . 1 9 7 4 , .

I n t h e p r e s e n t s t u d y 15 and 20% c o n c e n t r a t i o n p l a y e d

d e l e t e r i o u s i n Coeloeyne nervosa . I n i t i a l l y shoot i n i t i a t i o n

was pronounced f r o n t h e p r o t o c o r p s b u t f u r t h e r growth of t h e

s e e d l i n g s was a r r e s t e d and n e c r o s e d . The d e l e t e r i o u s e i f e c t

of c o c o n u t mi lk a t n i g h e r c o n c e n t r a t i o n may be due t o t h e

u p s e t of o s m o r e g u l a t i o n . The coconut m i l k normal ly c o n t a i n s

s imple s u g a r s ( 2 0 % ) l i k e g l u c o s e and f r u c t o s e . When t h i s i s

added t o b a s a l medium which a l r e a d y c o n t a i n s 3% sugar t h e

t o t a l s u g a r c o n t e n t becomes h i g h , a d v e r s e l y a f f e c t i n g t h e

Osmoregula t ion r e s u l t i n g i n n e c r o s i s . S i m i l a r t y p e of

r e s p o n s e was n o t i c e d i n Vanda h y b r i d when 40% coconut mi lk

and 2% s u c r o s e was used i n t h e medium (Mathews and Rao

1985) .

107

Coconut r i l k a t v a r i o u s concentration i n t h e p r e s e n t

work i n h i b i t e d germina t ion of s e e d s of Coelogvne n e r v o s a .

The i n h i b i t o r y e f f e c t was c o n t i n u e d even vnen t h e medium was

supplemented wi th c a s e i n h y d r o l y s a t e and peptone. The

i n h i b i t o r y e f f e c t of t h i s u n d e f i n e d compound has a l s o been

r e p o r t e d i n Dendrobium h y b r i d (Kotomori and Murashige 1 9 6 5 ) .

The growth e f f e c t by t h e coconut m i l k , vnen inc luded i n t h e

g e r m i n a t i o n medium may r e s u l t i n o rgan d i f f e r e n t i a t i o n by

g i b b e r l i n t h a t i t c o n t a i n s . However, i n major i ty of t h e

c a s e s i t enhanced t h e r a t e o f s e e d germina t ion .

C o n t r a d i c t o r y t o t h e above r e s p o n s e , t h e i n f l u e n c e o f

c o c o n u t milk and seed g e r m i n a t i o n o f Phol ido ta i m b r i c a t a was

pronounced i n almost a l l t h e c o n c e n t r a t i o n s used . Though t h e

e x a c t r e a s o n f o r t h e c o n t r a d i c t o r y resu!:s i s unknown, j t

may be a t t r i b u t e d t o t h e c o m p o s i t i o n of t h e milk which

i n c l u d e s d i f f e r e n t amino a c i d s , v i t a m i n s and c y t o k i n i n s j n

p a r t i c u l a r and t h e c o n c e n t r a t i o n of G i b b e r l i c a c i d p r e s e n t

i n i t . The h i g h e r c o n c e n t r a t i o n o f coconut milk 115 and 2 0 % )

r e s u l t s i n f r i a b l e , c reamish n o n p h o t o s y n t h e t i c c a l l u s .

The i n c i d e n c e of c a l l i I n seed c u l t u r e has been

o b s e r v e d by rany workers ( B u r g e f f 1936, C u r t i s 1947a ,

Wi thner 1 9 5 9 , Rao 1963, M j t r a g. 1 9 7 6 ) . According

t o t h e s e i n v e s t i g a t i o n s t h e immature c o n d i t i o n of seeds and

u n d i f f e r e n t i a t e d n a t u r e of t h e embryo a r e t h e p r o b a b l e

f a c t o r s f o r t h e c a l l u s i n g of t h e s e e d s . I n t h e p resen t s t u d y

t h e c a l l u s i n g of seeds i n p r e s e n c e o f 20% coconut r i ? k may

be due t o t h e p robable a v a i l a b i l i t y o f h i g h e r auxin t o t h e

mature s e e d s .

Banana j u i c e a t v a r i o u s l e v e l s o f c o n c e n r r a t i o n was

found t o be u s e f u l i n h e a l t h y growth o f t h e s e e d l i n g s . The

h i g h e r c o n c e n t r a t i o n ( 2 0 : ) was c o n s i d e r e d as e f f e c t i v e

c o n c e n t r a t i o n f o r h e a l t h y growth of t h e s e e d l i n g s i n P h a i u s

l u r i d u s and a l s o i n P e r e s t e r i a e l a t a , The l e a v e s becore d a r k

g r e e n and p o s s e s s b e t t e r p l a n t h e i g h t when c o r ~ a r e d t o o t h e r

t r e a t m e n t s .

Wi t h n e r (1953 , r e p o r t e d i t s s t i m u l a t o r y e f f e c t on

Paphiopedi lum s e e d s . Anderson ( 1 9 6 7 ) i s of t h e op in ion t h a t

a d d i t i o n o f 2-6 n! of g r inded banana p u l p i n Knudson's C

medium s t 1 mulated t h e groi i th of C a t t l e y a seedl i nps.

The b e n e f i c i a l e f f e c t of banana j u i c e i s probab1.y due

t o t h e i n t e r a c t i o n of v a r i o u s f a c t o r s l i k e b u f f e r i n g e f f e c t

of banana which he!ped t o m a i n t a i n an opr inur pH. I n

a d d i t i o n , t h e c h e l a t i n g of i r o n t h u s making i t a v a i l a b l e t o

t h e g rowing s e e d l i n g s and p r e s e n c e o f v a r i o u s m i n e r a l s

p a r t i c u l a r l y potassium and magnesium f a v o u r h e a l t h y growth

of t h e s e e d l i n g s .

4 .5 ENCAPSULATION OF PROMCORMS

The encapsulation of somatic embryos, orchid protocorms

has been attempted recently in several plant species to

produce synthetic seeds (Kitto and Janick 1985, Fujija

al. 1987, Bapat and Rao 1988, Datta and Potrykus 1989). - Production of artificial seeds can be considered as a

substitute for the real seeds, particularly it is of immense

value in those species which exhibit seed sterility. It

also helps in storage and efficient delivery system. The

encapsulation technique shows pronise in rapid

micropropagation of valuable and commercial crops like

orchids. Singh (1990) has encapsulated and regenerated

plantlets from the orchid protocorms. Though the methodology

of encapsulation is easily accessable the difficult task is

found to be encountered during conversion frequency or

reverse frequency.

In the present study encapsulation of protocorms and

their reverse frequency have been atterpted in Phaius

luridus, Peresteria elata and Coelogyne nervosa. The most

commonly used encapsulating agents are alginate and agar.

Among these, alignate is found to be suitable for orchids.

There are various concentrations of 1 to 57* sodium alginate

used for encapsulation. It is found that 22 is more suitable

for embedding of protocorms. A t the concentration of 1%

pro tocorms t e n d t o b reak t h e b e a d s , whereas i t was t o o hard

when t h e c o n c e n t r a t i o n used was a t 5%.

Proper h a r d n e s s of che s e e d c o a t p l a y s an i - p o r t a n t

r o l e d u r i n g r e v e r s e f r e q u e n c y . I n t h e p r e s e n t

s t u d y , f o r p r o p e r h a r d n e s s of t h e s e e d c o a t , calcium

c h l o r i d e was used a t c o n c e n t r a t i o n of 50 mM. The beads were

formed w i t h i n 1-2 minutes . The t i m e t a k e n f o r t h e fo rmat ion

of beads d i f f e r s f o r d i f f e r e n t s p e c i e s . I n b a r l e y , i t was

formed a f t e r 15-12 minutes a t 2 5 ' ~ on r o t a r y s h a k e r a t 60

rpm ( D a t t a and Pot rykus 1 9 8 9 ) . T h i s d i f f e r e n t i a ? t i - i n g i s

p r o b a b l y due t o t h e c o r r e l a t i o n between t h e c o n c e n t r a t i o n s

of sodium a l g l n a t e and ca lc ium c h l o r i d e .

4.5.1 Convers ion Frequency

Conversion of e n c a p s u l a t e d embryos o r p r o t o c o r m s i n t o

s e e d l i n g s i s an e s s e n t i a ? need f o r commerci a? e x p l o i t a t i o n

of t h e t e c h n i q u e .

Conversion o r r e v e r s e f r e q u e n c y v a r i e s c o n s i d e r a b l y i n

d i f f e r e n t p l a n t sys tems . The f r e q u e n c y of embryo t o p l a n t

c o n v e r s i o n was v e r y low f o r m u s t a r d , c a r r o t , c o t t o n and corn

b e c a u s e of t h e poor q u a l i t y of embryos. I n v i t r o c o n v e r s i o n

a s h i g h a s 86% was r e p o r t e d f o r a l f a - a l f a a r t i f i c i a l s e e d s

( M i z r a h i 1988). A r t i f i c i a l s e e d s were a l s o a b l e t o germina te

when t h e y were p l a n t e d d i r e c t l y i n g r e e n house . T h e i r

c o n v e r s i o n f requency was found t o be 20% (Redenbaugh c. 1986 1 .

111

Singh (1990) r e p o r t e d t h a t h i g h e s t f r e q u e n c y was

o b t a i n e d from e n c a p s u l a t e d o r c h i d p r o t o c o r m s , s t o r e d

e v e n a f t e r 180 d a y s a t 4 ' ~ . However, i n t h e p r e s e n t s t u d i e s ,

t h e convers ion f r e q u e n c y was found t o be low i n P e r e s t e r l a

e l a t a Pha ius l u r i d u s and Coelogvne n e r v o s a . The r, - e n c a p s u l a t e d pro tocorms were s t o r e d u p t o 1 month and

s u b j e c t e d f o r c o n v e r s i o n f requency . The p e r c e n t a g e of

c o n v e r s i o n f r e q u e n c y was found t o be more t h a n 50 p e r c e n t

a f t e r a week, whereas i t was reduced t o l e s s t h a n 10% a f t e r

5 weeks. The p r o b a b l e r e a s o n f o r t h e l o w e s t c o n v e r s i o n

f requency i s due t o t h e e n c a p s u l a t i o n of n o n - p r e t r e a t e d

pro tocorms , The p r e t r e a t i n g of somat ic embryos o r p ro tocorms

i s normally done by mixing them i n t h e n i x t u r e of sodium

a l g i n a t e + MS b a s a l medium.

4.6 TETRAZOLIUM CHLORIDE TEST

The t e t r a z o l i u m t e s t was deve loped by Lakon i n 1949 a s

an a l t e r n a t i v e t o p o i s o n o u s Selenium s a l t . The d i s c o v e r y of

d i f f e r e n t i a ! s t a i n i n g method t o t e s t t h e v i a b i l i t y of o r c h i d

s e e d , was c o n s i d e r e d a b i g boon t o o r c h i d i n d u s t r y , because

seed c u l t u r e of o r c h i d i t s e l f i s a b i g i n d u s t r y . The

forehand knowledge a b o u t t h e v i a b i l i t y of t h e s e e d sown w i l l

go a long way i n h e l p i n g t h e commercial g rowers and

r e s e a r c h e r s t o c u t down t h e c o s t of p r o d u c t i o n by

e l i m i n a t i n g t h e sowing o f non-v iab le s e e d s .

11 2

I n t h e p r e s e n t s t u d y , v i a b i l i t y o f t h e s e e d s xas

d e t e r m i n e d by u s l n g t o p o g r a p h i c a l t e t r a z o l i u r t e s t i n a ' ?

t h e s p e c i e s . The s e e d s t u r n r e d d i s h a f t e r 20 h r s In

P e r e s t e r i a e l a t a and P h a i u s l u r i d u s , whereas i n C o e l o ~ \ , n e

n e r v o s a , P h o l i d o t a i m b r l c a t a and Acampe praemotsa c o l o ~ r

change was n o t i c e d a f t e r 30 h r s . The changes I n c o l o u r t o

r e d d i s h i n d i c a t e d t h e a c t ~ v i t y of dehydrogenase enzyme. Tne

t e t r a z o l i u m s a l t i s a c o l o u r l e s s chemical when imbibed by

t h e s e e d a s c o ? o u r l e s s s o l u t i o n i s reduced by t n e

dehydrogenase enzyne t o red c o l o u r . I n t h e absence o f any

a c t i v e enzyme, dead t i s s u e remains u n s t a i n e d .

The t i n e t a k e n f o r v i a b l e embryo t o t u r n r e d wi th

t e t r a z o l i u m s a l t v a r i e s w i t h t h e s p e c i e s . Singh ( 1 9 8 i )

r e p o r t e d t h a t Dendrobi um dens i f l o r u m , B l e t i 1 l a hyaci n t h i ~

and A e r i d e s odoratum s t a i n b e a u t i f u l l y w i t h i n 16 h r s a f t e r

a p p l i c a t i o n . While s p e c i e s l i k e Epidendrum r a d i c a n s and

S p a t h o ~ l o t t i s p l i c a t a r e q u i r e 2 2 - 2 4 h r s t o t a k e t h e s t a i n .

Near1 y 80-907e of t h e s e e d s were v i a b l e i n a l l t h e

p r e s e n t l y i n v e s t i g a t e d s p e c i e s , i n d i c a t i n g t h e a c t i v j t y of

t h e enzyme dehydrogenase and i n t u r n a v o i d i n g t h e r i s k of

sowing non-vi a b l e s e e d s .

4.7 CLONAL PROPAGATION

Now-a-days e f f o r t s a r e b e i n g made t o i n c r e a s e t h e

number of s p e c i e s which can be p r o p a g a t e d from s h o o t t i p

(Vajrabhaya and Vajraghaya 1 9 7 0 , Sagawa anc Shoji 1967 , Goh

1 9 7 3 ) .

Efforts have also been made to use otner explants such

a s leaf tip in Cattleya and Epidendrum (Ardittj et al. 1971 ,

Ball c. 197:) and flower stalk in Pha!aenopsjs (Intuwong

et al. 1 9 7 2 , Rotomori and Murashige 1 9 6 5 ) . - -

The main drawback with shoot tip culture is the

sacrificing an entire new growth tip or whole plant. In

order to overcome this problem one of tne most suitable

explant for clonal propagation in orchids is that of nodal

segment (Scull? 1 9 6 6 ) . The orchjds which nave been clonally

propagated in vitro is given in Table 20.

4.7.1 Nodal Culture

The nodal. explant from in vitro grown seedlings of

Peresteria elata and Phaius luridus compr!sed of 1 to 2

axillary buds, The length of the explant Kas short and stout

due to the arrest of shoot growth in presence of cytokinin.

One more additional in vitro step of shoot elongation was

employed in order to increase the length of the explant.

Most suitable growth regulator was Gkg for internodal

elongation at lower concentration. Initially the

concentration of cytokinin in the medium was reduced. Shoot

elongation was noticed after 2 weeks in culture, After

obtaining sufficient length the nodal explants were

? r e p a r e d and i n o c u l a t e d on d i f f e r e n t media. Lakshmi i i t a et - a ? . ( 1 9 7 9 ) have employed GA3 t o i n c r e a s e t h e l e n g t h of 7

vi t r o d e v e l o p e d crowned shoots i n Santalum *. - The noda l e x p l a n t s of Pha ius l u r i d u s and P e r e s t e r i a

e l a t a were p l a c e d on d i f f e r e n t media l i k e MS, Vacin ;nd Went

and Knudson's C. ?IS nedium was found t o i n i t i a t e

p r o l i f e r a t i o n a t t h e e a r l i e s t i n b o t h t h e s p e c i e s .

Mosich g s. (1974) have used s e v e r a l media Lor t h e

c u l t u r e of nodal e x p l a n t but growth o c c u r r e d i n t h e n i g h e s t

p e r c e n t a g e on s o l j d Knob's medium which was supp'emented

wi th BAP and t rans -c innamic a c i d ( t C A ) .

The a x i l l a r y buds , p r e s e n t e a r l i e r on t h e noda' e x p l a n t

s p r o u t e d t o form l e a f y shoots under t h e i n f l u e n c e of h i g h

c o n c e n t r a t i o n of c y t o k i n i n . A s a concept h i g h

c o n c e n t r a t i o n o f c y t o k i n i n n o r m a l l y s u p p r e s s e s a p i c a l

dominance and f a v o u r s t h e s p r o u t i n g of ax j l l a r y bud, which

o t h e r w i s e remains dormant . In t h e p r e s e n t s t u d y , of v a r j o u s

c y t o k i n i n t e s t e d , BAP a t h i g h e r c o n c e n t r a t i o n was noted t o

be e f f e c t i v e i n s p r o u t i n g t h e dormant a x i l l a r y buds. The

e f f e c t i v e r o l e of BAP i s p robably d u e t o i t s powerfu l

a c t i o n . BAP i s a powerful s y n t h e t i c c y t o k i n i n and i s s e v e r a l

t imes s t r o n g e r t h a n k i n e t i n . However, i n Phaius l u r i d u s 2 - j p

was a l s o found t o be e f f e c t i v e i n s p r o u t i n g of t h e buds and

f u r t h e r deve lopment . A c l u s t e r of s h o o t s was formed due t o

t h e t r a n s f o r m a t i o n of sprou ted buds i n t o l e a f y s h o o t s .

The r e s u l t s of p r e s e n t s t u d y conf i rmed w i t h o t h e r

r e p o r t s on Dendrobium (Mosich g &. 1974) and

Epidendruc ( S t e w a r t and But ton 1 9 7 6 ) . In Dendrobium

p l a n t l e t s were developed from buds on nodal s e c t i o n s w i t h i n

45 d a y s . I n t e r e s t i n g l y bud development i n t h i s s p e c i e s

o c c u r e d o n l y on media c o n t a i n i n g t - c i n n a n i c a c i d s . During

t h e c u l t u r e of nodal segment w i t h dormant buds ,

i n c o r p o r a t i o n of t -c innamic a c i d i n t h e medja i s of common

p r a c t i c e a s i t e x h i b i t s a n t j - a u x i n a c t i v i t y . The t -c innamic

a c i d r e v e r s e s i n h i b i t i o n caused by a u x i n t r a n s p o r t e d down

from s h o o t t i p and i n d i r e c t l y f a c i l i t a t e s c y t o k i n i n t o

s p r o u t t h e dormant buds. However, t h e p r e s e n t r e s u l t s r e v e a l

t h a t t h e a x i l l a r y buds can be t r i g g e r e d t o s p r o u t even i n

t h e a b s e n c e of t -c innamic a c i d i n t h e medium. The p r o b a b l e

r e a s o n nay be due t o e i t h e r low amount o r absence o f

endogenous a u x i n s i n t h e e x p l a n t and p r e s e n c e of h i g h

c o n c e n t r a t i o n of c y t o k i n i n i n t h e medium. The f a c t o r s can

s u f f i c i e n t l y overcome t h e a p i c a l dominance e x h i b i t e d by

The r e p o r t of a d v e n t i t i o u s s h o o t f o r m a t i o n on t h e nodal

e x p l a n t w i t h o u t the u s e of t -c innami c a c i d was n o t i c e d i n

Dendrobium ( A r d i t t i e t a l . 1 9 7 3 ) .

The s i g n i f i c a n t r o l e of BAP i n s p r o u t i n g t h e dormant

buds and i t s f u r t h e r growth i n t o p l a n t l e t s was r e p o r t e d by

Kononowicz and J a n i c k ( 1 9 8 4 ) i n V a n i l l a p l a n i f o l k . I n t h i s

s p e c i e s BAP a t 1 mg/l c o n s i d e r a b l y e f f e c t e d shoot

p r o l i f e r a t i o n by cnb,ancing a x i l l a r y b r a n c h i n g on noda! stem

e x p l a n t .

The c a l l u s was i n v a r i a b l y formed on t h e noda? e x p l a n t

of P h a i u s l u r i d u s under che i n f l u e n c e of 2 ,4-D. The c a l l u s

i n i t i a t i o n was c r i t i c a l a t c o n c e n t r a t i o n 2 rng/?. I n

a d d i t i o n , MS s a l t s t r e n g t h s u p p o r t e d t h e growth of t h e

c a l l u s .

There a r e s e v e r a l r e p o r t s on s u i t a b l e media f o r c a l l u s

f o r m a t i o n and proliferation of nodal e x p l a n t . Severa l

s p e c i e s o f Dendrobiums t h r i v e d w e l l on Knudson's medium

(Va!mayer 1 9 7 4 ) . 1:acin and Went medium was s u i r a b l e f o r

Dcndrobium nobi l e [Sagawa and Valmayer 1 9 6 6 ) . C a l l u s

p r o l i f e r a t i o n i n t h e above s a i d s p e c i e s was c o n t r i v e d only

when s u b c u l t u r e d on f r e s h media s u p p o r t e d w i t h coconut mi lk .

The _il v i t r o developed s h o o t s , when subcul t u r e d on

r o o t i n g medi urn, p a r t i cul a ~ l y of P e r e s t e r i a e l a t a , pseudobul b

f o r m a t i o n was observed a t t h e b a s a l r e g i o n . The pseudobulb

f o r m a t i o n was r e p o r t e d by Ekanthappa (1981). However, ~n

m a j o r i t y o f t h e s p e c i e s i t was d e v e l o p e d d u r i n g auxln

g e r m i n a t i o n of s e e d s r a t h e r t h a n d u r i n g c l o n a l p r o p a g a t i o n .

The pseudobulb f o r r a t i o n ~ n a few c u l t u r e s may be a t t r i b u t e d

t o t h e p r e s e n c e of a u x i n s and low s a l t s t r e n g t h of t h e

media.

117 4.7.2 Shoot Apex Culture

The most popula r and commonly used e x p l a n t f o r c l o n a l

p r o p a g a t i o n i n o r c h i d i n d u s t r y i s shoot apex. Shoot t i p

e x p l a n t remained t h e most commonly used e x p l a n t f o r

monopodials (Goh 1970, 1973 , Vajrabhaya and Vajrabhaya 1970,

Kunisak i and Kin 1972, Intuwong and Sagawa 1 9 7 4 ) .

High p r i o r i t y f o r s h o o t t i p e x p l a n t by commercial

g rowers i s t o produce g e n e t i c a l l y uniform c l o n e s . The

g e n e t i c s t a b i l i t y of t h e m e r i s t e m a t i c l i n e i s wel l known and

i s an es tabLished f a c t t h a t t h e m e r i s t e m a t i c c e l l s e x h i b i t

two n o t a b l e p r o p e r t i e s . There i s a s t r i c t c o n t r o l over t h e

sequence of DNA s y n t h e s i s and m i t o s i s which does not a l l o w

e x t r a d u p l i c a t i o n of DNA t h a t a r e r e s p o n s i b l e f o r somaric

p o l y p l o i d y and secondly due t o t h e con t inuous d j vi s i ons

which e l i m i n a t e a t l e a s t p a r t of t h e spontaneous ly o c c u r r i n g

chromosome s t r u c t u r a l changes and o t h e r g e n e t i c d e f e c t s .

T h i s t e c h n i q u e of s h o o t apex c u l t u r e was f i r s t

t r a n s g e n e r a t e d by Morel i n 1960.

The e x p l a n t used i n t h e p r e s e n t s t u d i e s comprised bo th

s h o o t apex and a x i l l a r y bud r e g i o n . N o r r a l l y t h e s e buds

d e v e l o p i n t o i n f l o r e s c e n c e . However, i n most o r c h i d s t h e

buds remain dormant o r p o o r l y developed (Morel 1 9 7 4 ) .

C u l t u r e s d e r i v e d from s h o o t a p i c e s were found t o be most

l i k e l y t o form somat ic p ro tocorms o r protocorm l i k e b o d i e s ,

w i t h a h i g h p o t e n t i a l f o r p r o l i f e r a t i o n . So-et imes a p i c a l

s h o o t t j p s o f t e n grev and r o o t e d t o produce s i n g l e p l a n t l e t s

d i r e c t l y . Even protocorm l i k e bodj e s f o r m a t ] on c o u l d be

s t i m u l a t e d by s u b c u l t u r i n g s h o o t s formed i n c u l t u r e a f t e r

c u t t i n g them 1 ongi t u d i n a l l y o r t r a n s v e r s e 1 y .

I n t h e p r e s e n t s t u d y t h e s h o o t apex e x p l a n t of

P e r e s t e r i a e l a t a , Phaius l u r i d u s and Coelogyne nervosa when

cu! t u r e d on b1S medi urn supplemented w j t h grok-th regu l a t o r s

d i f f e r e n t morphogenetic r e s p o n s e s were observed .

I n P h a i u s l u r i d u s t h e e x p l a n t s s t a r t e d p r o l i f e r a t i o n

a f t e r a week i n t h e c u l t u r e . Many p r o t o c o r - l i k e b o d i e s

( P L B ' s ) were formed a t t h e a p i c a l r e g i o n . The ?rotocorm l i k e

b o d i e s a r e covered w i t h r h i z o i d s and l a t e r t h e s e a r e

t r a n s f o r m e d i n t o l e a f y s h o o t s .

The e x p l a n t s of P e r e s t e r i a e l a t a exhi p it d i f f e r e n t

morphogene t ic e f f e c t a s i t p roduced m u l t j p l e snoots d i r e c t l y

on t h e e x p l a n t . P ro tocore l i k e b o d i e s were not s i g h t e d i n

t h i s s p e c i e s i r r e s p e c t i v e o f v a r i o u s growth r e g u l a t o r s b e j n g

used i n t h e medlurn.

F o r m a t i o n of protocorm l i k e b o d i e s on shoot apex was

e a r l i e r s i g h t e d by v a r i o u s workers i n C a t t l e y a (Morel 1964)

Cymbidium (Wirnber 1963, Fonnesbech 1 9 7 2 ) .

Growth r e g u l a t o r s p l a y a d e c i s i v e r o l e i n i n i t i a t i o n of

somat ic p ro tocorms . In t h e p r e s e n t s t u d y t h e r e j s c o n f i n e d

119

e f f e c t of I A A but k i n e t i n e n h a n c e s prorocorm l i k e b o d i e s

i n Pha ius l u r i d u s . Whereas, i n P e r e s t e r i a e l a t a BAP a l o n e

i n d u c e s d i r e c t shoot deve lopment . I n h i b i t i o n of p ro tocorm

l i k e b o d i e s by NAA i s enhanced by BAP a s r e p o r t e d by Kim and

Kako ( 1 9 8 4 ) d u r i n g s h o o t apex c u l t u r e i n Cymbidium. The

e f f e c t of p l a n t growth r e g u l a t o r s on organ f o r m a t i o n i n

Cymbidum shoot apex c u l t u r e was r e p o r t e d by K i m and Kako

( 1 9 8 2 ) , i n which BAP encouraged t h e f o r - a t i o n of p ro tocorm

l i k e b o d i e s and s h o o t s .

The r e p o r t on i n i t i a t i o n of m u l t i p l e shoots d i r e c t l y on

s h o o t t i p e x p l a n t i s s c a n t y . However, r h i s r e s p o n s e was

n o t i c e d i n Habenaria d i s c o l o u r ( T e o 1 9 7 8 ) .

The s p r o u t i n g of b o t h a p i c a l and a x i l l a r y buds on t h e

same e x p l a n t under t h e i n f l u e n c e of growth r e g u l a t o r s ,

p a r t i c u l a r ! y low a u x i n t o h i g h c y t o k i n i n . The c y t o k i n i n

a l o n e can b r i n g about m u l t i p l e branched p l a n t l - e t s . The

b r a n c h e s can be e x c i s e d and c u l t u r e d s e p a r a t e l y and f u r t h e r

p r o l i f e r a t i o n can be induced i n c u l t u r e s . T h e o r e t i c a l l y t h e

p r o c e s s of i n v i t r o s h o o t p r o l i f e r a t i o n can be c o n t i n u e d

i n d e f i n i t e l y .

M u l t i p l e shoot f o r m a t i o n by s t i m u l a t i n g a x i l l a r y a s

w e l l a s a p i c a l buds i n s h o o t apex c u l t u r e has been r e p o r t e d

i n Dendrobium (Sagawa and S h o j i 1 9 6 7 ) , P h a l a e n o p s i s

( I n t a w o n g and Sagawa 1 9 7 4 ) , Rhynchos ty l i s g i g a n t e a

1 2 0

(Vaj rabhaya and Vajrabhaya 1 9 7 0 ) , Vanda Cv i n s i e n i s X y. t a s s e l l a t a hybr id (Teo e t a l . 1 9 7 3 ) .

I n t h e p r e s e n t i n v e s t i g a t i o n , m u l t i p l e shoot f o r m a t i o n

was observed i n Pha ius l u r i d u s and P e r e s t e r i a e l a t a due t o

s p r o u t i n g of bo th a p i c a l and a x i l l a r y b u d s . P r i m a r i l y a p i c a l

bud p r o l i f e r a t e d i n t o s i n g l e s h o o t . A t t h e s a - e t i m e

a x i l l a r y buds a1 s o p r o l i f e r a t e and t r a n s f o r m e d i n t o 1. e a f y

s h o o t s . Many new buds were i n i t i a t e d a t p r o l i f e r a t i o n s i t e .

I n t h i s way shoot m u l t i p l i c a t i o n by enhanced a x i l l a r y

b r a n c h i n g was c o n t r i v e d i n p r e s e n c e of c y t o k i n i n a t h i g h e r

c o n c e n t r a t i o n s u i t a b l e f o r s h o o t m u l t i p l i c a t i o n . SAP a t

v a r i e d c o n c e n t r a t i o n ( 2 and 5 mg/ l ) was found s u i t a b l e f o r

s h o o t m u l - t f p l i c a t i o n . The combined e f f e c t of BAP ( 2 mg/!)

and 4-phenyl u r e a ( 1 0 mg/! 1 enhanced s h o o t m u l t i p l i c a t i o n i n

P e r e s t e r i a e l a t a . The c y t o k i n i n when combined with ?ow a u x i n

had a s i g n i f i c a n t r o l e i n e x p l a n t p r o l i f e r a t i o n .

The e a r l i e r r e p o r t on shoot m u l t i p l i c a t i o n by enhanced

a x i l l a r y branching was observed i n Dendrobium autenuatum by

Kukulzanka and Wajsciechowska ( 1 9 8 3 ) . I n t h i s s p e c i e s

h i g h e s t d e g r e e of s h o o t m u l t i p l i c a t i o n was n o r i c e d i n

p r e s e n c e of BAP a t v a r i e d c o n c e n t r a t i o n . Kononowicz and

J a n i c k (1984) ach ieved shoot p r o l i f e r a t i o n by a x i l l a r y

b r a n c h i n g on BAP. I n Vanda h y b r i d , Mathews and Rao ( 1 9 8 5 )

found n e a r l y 50% of t h e e x p l a n t e x h i b i t i n g p r o l i f e r a t i o n

a i d e d by t h e p r e s e n c e of BAP a t 1 n g / l . The p e r c e n t a g e o f

p r o l i f e r a t i o n was same when BAP combined with I A A .

Shoot m u l t i p l i c a t i o n by a x i l l a r y branching can a l s o be

a c h i e v e d on t h e media c o n t a i n i n g lo^ s a l t s t r e n g t h .

S u c c e s s f u l p r o p a g a t i o n of Cymbidiur,, C a t t l e v a and Dendrobium

was r e p o r t e d by Sagawa and Kunisak i (19681 on Vacin and

Went medium, which c o n t a i n s h a l f t h e s a l t s t r e n g t h of MS

medium, which was a l s o employed f o r t h e p r e s e n t s t u d y . When

e x p l a n t s w i t h b o t h a p i c a l and a x i l l a r y bud were c u l t u r e d on

Vacin and Went w i t h NAA and BAP, a x i l l a r y b r a n c h i n g was

enhanced c o n s i d e r a b l y i n A r a n t h e r a James 1: Widi a s t o r e t y 5

a l . 1 9 8 6 ) . -

M u l t i p l e s h o o t f o r m a t i o n from e n l a r g e d a x i l l a r y buds i s

n o t a r a r e phenomenon. Kunisak i and K i m ( 1 9 7 2 ) r e p o r t e d

m u l t i p l e shoot f o r m a t i o n from e n l a r g e d axi l l a r y buds o f

Vanda CV Jaaquim. I n t h e p r e s e n t i n v e s t i g a t i o n , s i m i l a r t y p e

of response was n o t j c e d i n P e r e s t e r i a e l a t a i n 2 - i p .

From t h e l i t e r a t u r e and p r e s e n t s t u d y i t i s e v i d e n t

t h a t h i g h e s t d e g r e e o f s h o o t m u l t i p l i c a t i o n can be a c h i e v e d

w i t h e x p l a n t c o n t a i n i n g b o t h a p i c a l and a x i l l a r y meris tems.

C y t o k i n i n e i t h e r a l o n e o r i n combinat ion w i t h low a u x i n

c o n c e n t r a t i o n p l a y s a d e c e s s i v e r o l e i n t r i g g e r i n g d i f f e r e n t

morphogenet ic phenomena.

The p r o l i f e r a t i o n of a x i l l a r y bud i n t o r r u l t i p l e shoot

e x h i b i t e d i n p r e s e n t i n v e s t i g a t i o n may be a t t r i b u t e d t o t h e

phenomenon of a p i c a l dominance. A p p l i c a t i o n of h i g h e r

c o n c e n t r a t i o n of c y t o k i n i n i n t h e medium removes a p i ca!

dominance. Apical dominance i s due t o t h e s y n t h e s i s of

endogenous auxin i n t h e shoot apex and t r a n s p o r t e d downwards

r e s u l t i n g i n s u p p r e s s i o n of s p r o u t i n g of a x i l l a r y bud.

Exogenous supply of c y t o k i n i n r e s u l t s i n s p r o u t i n g and

p r o l i f e r a t i o n of axi l l a r y buds i n P e r e s t e r i a e l a t a and

P h a i u s l u r i d u s . -- 4 . 7 . 2 . 1 Sub c u l t u r e

There i s a l i r r ~ t t o which s h o o t m u l t i p l i c a t i o n c a n be

a c h i e v e d i n a s i n g l e p a s s a g e , a f t e r which f u r t h e r a x i l l a r y

branching s t o p s . A t t h i s s t a g e t h e m i n i a t u r e s h o o t s a r e

e x c i s e d and p l a n t e d on a f r e s h medfum of t h e same

compos i t ion . The shoot mu1 t i p l i c a t i on c y c l e can be r e p e a t e d

i n t h i s manner and multiplication c y c l e c a n go on

i n d e f i n i t e l y . Boxus ( 1 9 7 4 ) e s t i m a t e d t h a t w i t h i n a y e a r

s e v e r a l m i l l i o n p l a n t s c o u l d be r a i s e d s t a r r i n g from a

s i n g l e mother p l a n t of s t r a w b e r r y .

I n t h i s s t u d y , a i.n v i t r o r a i s e d m i n i a t u r e s h o o t s of

P e r e s t e r i a e l a t a and P h a i u s l u r i d u s were s e p a r a t e d and

p l a c e d on c y t o k i n i n r i c h medium. Shoot m u l t i p l i c a t i o n c y c l e

was found t o b e enhanced due t o t h e i n i t i a t i o n of new

s h o o t s . Hundreds of s h o o t s were ob ta ined i n t h e above two

s p e c i e s due t o s u b - c u l t u r e on f r e s h mediur.

4.7.2.2 C a l l u s induction

The r e p o r t on c a l l u s fo rmat ion from shoot apex e x p l a n t

i s s c a n t y . I n v i t r o c a l l u s may be ob ta ined from almost any

t y p e of p l a n t . I n Cymbidium c a l l u s was i n i t i a t e d from t h e

shoot apex. The c a l l u s may i n t u r n u t i l i s e f o r c e l l

suspens ion c u l t u r e (S teward and Mapes 1 9 7 1 ) . Mathews and Rao

( 1 9 8 5 ) r e p o r t e d c a l l u s growth from shoot t i p e x p l a n t of

Vanda hybr id i n NAA and BAP a t equal c o n c e n t r a t i o n . In t h e

p r e s e n t s t u d y , c a l l u s fo rmat ion was observed from shoot apex

under t h e i n f l u e n c e of 2,6-D. S i m i l a r l y i n Vanda h y b r i d ,

2,6-D i n f l u e n c e s c a l l u s i n i t i a t i o n from shoot t i p (Mathews

and Rao 1 9 8 3 ) . The f r i a b l e nonphotosynthe t ic c a l l u s d i d not

p r o l i f e r a t e f u r t h e r and d r i e d a f t e r a few weeks i n c u l t u r e .

However, i n Paphiopedjlum i n s i g n e out of 1 2 6 e x p l a n t s on ly

5% produced c a l l u s and p l a n t l e t s i n presence of 2,4-D. Such

c o n t r a d i c t o r y r e s u l t s perhaps a r e determined by v a r y i n g

An a t tempt t o r e - d i f f e r e n t i a t e f u r t h e r from t h e c a l l u s

i r r e s p e c t i v e of manipu la t ion w i t h v a r i o u s growth r e g u l a t o r s

d i d no t y i e l d any p o s i t i v e r e s u l t . A t t h i s t i m e , t h e problem

was n o t pursued any f u r t h e r a s g e n e t i c v a r i a b i l i t y i s

commonly pronounced i n t h e p l a n t s which a r e r e g e n e r a t e d from

t h e c a l l u s .

4.7.3 Leaf C u l t u r e

S i n c e t h e beginning o f c l o n a l p r o p a g a t i o n v a r i o u s

o r g a n s h a v e been and a r e b e i n g used f o r t h e micro-

p r o p a g a t i o n . Leaves f o r example have been used f o r a long

t ime ( C u t l e r 1 9 6 2 , I sbe l ! 1 9 3 1 , S t o u d t 1 9 3 6 ) .

The e a r l i e r works Kere mainly c o n t a i n e d on Beponi a

l e a f . L a t e r on i t was n o r i c e d t h a t i n o r c h i d s t o o , young

l e a v e s c o u l d be used t o p r o p a g a t e d e s i r a b l e c l o n e s . The l e a f

t i s s u e c u l t u r e i s f e a s i ~ l e f o r p r o p a g a t i o n i n c e r t a ~ n

o r c h i d s l i k e Aranda (Loh e m . 1 9 7 5 ) . C a t t l e v a (Cnarpagna t

e t a l . 1 9 7 0 ) , Epidendrua end & e l i o c a t t l e y a Churchi l l e t - - - a l . 1 9 7 1 ) . Number of p r e l i - i n a r y e x p e r i m e n t s j n d i c a t e d t h a t - c u l t u r e o f l e a f t i p was a l s o a p romis ing p o s s i b i l i t y .

S e e d l i n g s l e a f t i p s proved e a s y t o c u l t u r e ( C n u r c h i l ! e t a l .

1 9 7 0 ) .

The d e - d i f f e r e n t i a t i o n and r e - d i f f e r e n t i a t i on -ode of

morphogenesis i s i n t e r e s t i n g and i s most p o p u l a r method of

m i c r o p r o p a g a t i on. This mode of morphogenesi s i s i c p o r t a n t

f o r c o m m e r c i a l l y i m p o r t a n t s p e c i e s l i k e o r c h i d s . I n most

p l a n t s d e - d i f f e r e n t i a t e d t i s s u e l i k e c a l l u s can be sub-

c u l t u r e d and s e r i a l l y p r o p a g a t e d on medium c o n t a i n i n g t h e

hormone l e v e l s t h a t were used t o induce i t . The c a r e f u l

b a l a n c e o f a u x i n and c y t o k i n i n r a t i o c o u l d induce d i f f e r e n t

o r g a n o g e n e t i c p a t t e r n i n t h e c a l l u s . Skoog and M i l l e r (1957)

have comrented on the hormonal contro! of organ formation.

High cytokinin to low auxin induced shoot bud, whereas low

cytokinin to high auxin induced roots or enbryoids and equal

concentration lead to di sorganised growth. Mass production

of callus followed by shoot regeneration would seem to be an

ideal method for large scale propagation. However, the most

serious drawback such as somaclona! variation and loss of

morphogenetic capacity limits its use to few species on1 y

(Hussey 1983).

The de-differenti ation and re-differenti ation mode of

development was reported in several orchid genera like

Cattleya and Laeliocattleya (Churchill et a l . 1971) and

Aranda (Loh &. 1975). Callus has been found to be

initiated from leaf bases of Aranda and Laeliocattleya.

Later they re-differentiate into shoots. These workers also

reported i ni ti ation of protocorm-like bodies on the callus

and their proliferation into shoots. Similar mode of

response was observed in the@ present study on Coel ogyne

nervosa. Callus was initiated from the leaf base and also on

all the surface of leaf explant in a few cultures. The

presence of auxin (2,4-D and IAA) was found to aid callus

formati on. Sub-culturing of f ri able callus on fresh medium

on the same composition results in more proliferatjon. The

proliferated callus when subjected to second sub-culture on

cytokinin (Kinetin 1 mg/l) containing medium, many localised

r e g i o n s on t h e c a l l u s were o r g a n i s e d i n t o protocorm l i k e

b o d i e s . L a t e r t h e s e d i f f e r e n t i a t e d i n t o s h o o t s .

I n t h e p r e s e n t s tudy i t i s e v i d e n t t h a t r e g e n e r a t i o n

was more f r e q u e n t a t basal r e g i o n t h a n a t any o t h e r p a r t of

t h e l e a f . The l e a f base i n nonocot g e n e r a l l y i s m e r i s t e m a t i c

and on i s o l a t i o n and c u l t u r e i t d i f f e r e n t i a t e s i n t o

pl a n t l e t s ( Z i mmer and PI e p e r 1 9 7 5 ) . I n C a t t l e v a (Champagnat

e t a l . 1 9 7 0 ) , Aranda (Loh e t a l . 1 9 7 5 ) , I A A , k i n e t i n and CM -- induced ca l l -us f r o ~ t h e l e a f b a s e and l e a f t f p . However,

i n t h i s s t u d y 2,b-D a lone c o u l d b r i n g about c a l l u s .

P l a n t r e g e n e r a t i o n from 5 v i t r o c u l t u r e s o c c u r s

f r e q u e n t l y t h r o u g h advent i t i o u s s h o o t f o r m a t i o n , b u t r a r e ! y

th rough embryo f o r n a t i o n !Murashige 1 9 7 4 ) . R e g e n e r a t i o n of

s h o o t s accompl i shed vI a protocorm l j ks b o d i e s was qu j t e

f r e q u e n t i n t h e p r e s e n t s t u d y on Acampe praemorsa . T h i s mode

of development cor responds t o o n e of t h e embryonal s t a g e i n

o r c h i d s e e d g e r m i n a t i o n .

The l e a f e x p l a n t of Acampe pracmorsa i s h i g h l y

p o t e n t i a l f o r r e g e n e r a t i o n , However , the p l a n t from mature

l e a v e s d i d n o t respond i n any c u l t u r e medium, t h e reason

be ing a t t r i b u t e d t o t h e e x u d a t i o n of p h e n o l i c s which I n h i b i t

growth of any t i s s u e .

P h y s i o l o g i c a l age of t h e p l a n t i s a c r i t i c a l f a c t o r f o r

r e g e n e r a t i o n (Vajrabhaya and ~ a j r a b h a ~ a 1 9 7 0 ) . It was a l s o

127

o b s e r v e d i n t h e p r e s e n t s t u d y t h a t t h e exp!ant t a k e n from

v i t r o grown s e e d l i n g s p r o l i f e r a t e d , whereas i t was no t s o i n

mature p l a n t s . S i m i l a r r e p o r t s were recorded i n Vanda h y b r i d

(Elathews and Rao 19851, r e r u s a (Vi j &. 1 9 8 4 ) . Apar t from Orchidaceae n o t a b l e i n s t a n c e was ev idenced

i n P u n i c a c e a e . J a i d k a and Mehra ( 1 9 8 6 ) have made s i m i l a r

o b s e r v a t i o n i n e x p l a n t s of Punica grana tum, t h e younger

s e e d l i n g s showing g r e a t e r and e a r l i e r p r o l i f e r a t i o n a s

compared t o t h e o l d e r o n e s . The above r e p o r t s a r e confirmed

i n t h e p r e s e n t i n v e s t i g a t i o n on Acampe praemorsa i n which

pro tocorm l i k e b o d i e s a r e d e r i v e d d i r e c t l y f r o r young fi vi t r o grown s e e d l i n g s .

Many c u l t u r e s exhi b i t i n l t i a t i on of protocorm l i k e

b o d i e s on l e a f t i p , l e a f base and e n t i r e r e g i o n of t h e l e a f .

The number of protocorm l i k e b o d i e s produced a t t h e basa l

r e g i o n was more t h a n from t h e t i p . The c e n t r a l p a r t of t h e

l e a f a l s o had t h e p o t e n t i a l t o d e v e l o p i n t o protocorm l i k e

b o d i e s , p a r t i c u l a r l y on t h e d o r s a l s i d e of t h e l e a f .

The o b s e r v a t i o n i n t h e p r e s e n t i n v e s t i g a t i o n r e v e a l s

t h a t f o r m a t i o n of protocorm l i k e b o d i e s i s s p r e a d o v e r t h e

e n t i r e s u r f a c e of t h e e x p l a n t . T h i s o b s e r v a t i o n s u g g e s t s

t h a t a l m o s t t h e e n t i r e s u r f a c e of t h e j u v e n i l e l e a f i s

p o t e n t i a l a s m e r i s t e m a t i c i n Acampe praemorsa. These r e s u l t s

a r e c o n f i r m e d e a r l i e r on R h y n c h o s t y l i s r e t u s a ( V i j &.

198C) where in t h e e n t i r e l e a f i s p o t e n t i a l l y m e r i s t e m a t i c

128

and pro tocorm l i k e bodies were produced on t h e e n t i r e

s u r f a c e w i t h t h e i n t e r v e n i n g c a l l u s . Other r e p o r t s of

p ro tocorm l i k e b o d i e s from t h e l e a f e x p l a n t a r e i n

P h a l a e n o p s i s a m a b i l i s hybr id (Tanaka and Sakanish i 1 9 7 7 ) and

P h a l a e n o p s i s (Tanaka e t a l . 1 9 7 6 ) .

I n many c u l t u r e s of Acampe p r a e m o r s a i n t h e p r e s e n t

s t u d y r e g e n e r a t i o n of protocorm 1 i k e bod1 e s was r e s t r i c t e d

t o e i t h e r ex t reme t i p o r l e a f base and sometimes a t bo th t h e

r e g i o n s when e n t i r e l e a f was c u l t u r e d . C h u r c h i l l a?. ( 1 9 7 3 ) o b s e r v e d protocorm l i k e b o d i e s on t h e t i p p o r t i o n and

c o n s i d e r e d t h e s e a s merisematic l o c i . R e s t r i c t i o n of t h e

r n e r i s t e m a t i c p o t e n t i a l i n t h e ep idermal c e l l s of t h e b a s a l

r e g i o n of t h e !eaf lamina was d e m o n s t r a t e d i n C a t t l e v a

(Champagnat &. 1970) and i n Aranda (Loh pt 2. 1 9 7 5 ) .

The r e s t r i c t i o n of r e r i s t e - a t i c a c t i v j t y a t t h e l e a f t i p by

C h u r c h i l l &. ' 1970 , 1971 and 1 9 7 3 ) o r l e a f b a s e s by

Champagnat ( 1 9 7 1 ) Fay be a t t r i b u t e d t o g e n e t i c make up and

phys j o l o g i c a l age of t h e e x p l a n t . I n a d d i t i o n , n u t t i t i ona l

f a c t o r s a l s o d e t e r m i n e t h e i r p o t e n t i a l i t y .

The l e a f e x p l a n t of P e r e s t e r i a e l a t a and Phaius l u r i d u s

d i d n o t show any morphogenetic r e s p o n s e excep t s w e l l i n g of

t h e e x p l a n t . S e v e r a l miss ing f a c t o r s l i k e composi t ion of t h e

medium, s u i t a b l e growth r e g u l a t o r s o p e r a t e i n t r i g g e r i n g

morphogene t ic p o t e n t i a l . The n e g a t i v e r e s p o n s e i n t h i s s t u d y

may be due t o miss ing f a c t o r s i.n t h e medium.

Physiological age and nature of the explant play vital

role in bringing morphogenesis. The succulent leaves show

greater growth potential than non-succulent ones. This has

been confirmed in the epiphytic orchid Aranda (Loh &.

1975). In Laeliocattlcya (Churchill g &. 1971)

physiological age of the explant determines morphogenesis.

This was found to be important since only tissue obtained

before the development of notch responds, whereas tissue

removed after notch formation did not. respond.

In the presently investigated species, few are

terrestrial and their leaves are non-succulent in nature.

This may be one of the reasons for negative response in

culture.

4.7.4 Root Culture

In recent years root tips have been used as a material

for propagation of orchids vitro (Champagnat 1971,

Churchill et al. 1972, Tanaka et al. 1976). Development of

buds or shoots from root in orchids is well known and has

been reported earlier in many genera like Habenaria repens

(Holm 1925), Listera cordata and Phalaenopsjs staurtiana

(Scully 1971).

Some of the orchid roots are potential to form callus

and further proliferation into plantlets. However, formation

of protocorm-like bodies on the root tip and their further

d i f f e r e n t j a t i o n i n t o p l a n t l e t s i s a l s o e s t a b l i shed (Tanaka

e t a l . 1 9 7 6 , Kraus and Monteiro 1989). - -

Most monocoty?edonous r o o t s which have been c u l t u r e d

a r e grown on r e l a t i v e l y d i l u t e medium of White ( B u t c h e r and

S t r e e t 1 9 6 4 ) . I n t h e p r e s e n t s t u d y on r o o t cu!rure of P h a i u s

l u r i d u s and P e r e s t e r i a e l a t a , c a l l u s forr,a:ion and i t s

f u r t h e r p r o l i f e r a r i o n was o b t a i n e d on MS s a l t s t r e n g t h wi th

2,4-D. From t h e above f a c t i t was e v i d e n t t h a t o r c h i d r o o t

can grow and p r o l i f e r a t e even on h i g h s a l t s t r e n g t h nedium.

A comparab le response was n o t e d i n t h e c u l t u r e of e x c i s e d

r o o t of P h a l a e n o p s i s (Tanaka e t a l . 1 9 7 6 ) gro;,:ing well on

h a l f s t r e n g t h MS medium. However, c o n t r a d i c t o r y r e s u l t s were

o b s e r v e d i n Epi dendrum 0 ' b r i enanum i n whi c h t h e e x c i sed

r o o t s were found t o d i e when c u l t u r e d on s o l i d o r l i q u i d

forms o f Murashi ge and Skoog medium (Churchi! ! e t a l . 1 9 7 2 ) .

They s u g g e s t e d s p e c i f i c r e q u i r e m e n t o f each t i s s u e i n p l a n t

sys tem.

Among v a r i o u s growth r e g u l a t o r s t h a t were t e s t e d f o r

r o o t c u l t u r e , o n l y 2,4-D was found t o be f e a s i b l e f o r

p r o l i f e r a t i o n i n Pha ius l u r i d u s . The h i g h e s t d e g r e e of

p r o l i f e r a t i o n was found a t t h e c o n c e n t r a t i o n of 2 mg/ l ,

i n d i c a t i n g t h e s p e c i f i c r e q u i r e m e n t of a t i s s u e . In t h e

p r e s e n t s t u d i e s 2,4-D induced c a l l u s from r o o t e x p l a n t of

P h a i u s l u r i d = . Even a few p r o t o c o r m - l i k e bodies were a l s o

initiated on the callus. The presence of 2,4-D is an

essential factor for the initiation and proliferation of

protocorm-like bodies.

In monocotyledons 2,4-D is an essential constituent in

the culture medium for induction of callus from excised

roots (Carter et al. 1967, Klein and Edsall 1968, Krikorian

and Katz 1968). The concentration of 2,A-D seems to be

critical for differentiation in several species of orchids.

Monocotyledons generally require high concentration of 2,4-

D, for instance 6 mg/l was required for tne culture of root

of Rye grass (Mullin 1970) and 2-6 mg/l for Allium seedling

root (Klein and Edsall 1968, Krikorian and Katz 1968).

The present study reveals that 2,4-D at 2 mg/l is

beneficial whereas it is detrimental to root explant at 5

mg/l. In Epidendrum 0.5 mg/l is optimal for the

proliferation of callus, whereas at 1 and 2 mg/l it shows

toxic effect on root explant. Further, higher concentration

( 5 mg/l) leads to lethal effect. The varying degree of

requirement of 2,4-D concentration in root culture may be

attributed to the endogenous accumulation of auxin and also

physiological nature of the explant. Many cultures did not

proliferate except growth being extended and branched under

the influence of NAA (0.5 mg/l) and BAP (2 mg/l). There are

a few reports on the formation of protocorm-like bodies from

r o o t e x p l a n t a s i n P h a l a e n o p s i s (Tanaka e t a!. 1976, Kraus

and Monte i ro 1 9 8 9 1 and Catasetum (Kerbauy 1 9 8 h ~ .

An a t t e m p t t o induce c a l l u s o r p r o t o c o r r - 1 i k e b o d i e s

from r o o t t i p e x c i s e d from m a t u r e p l a n t s d i d n o t y i e l d

p o s i t i v e r e s u l t s . Though t h e e x a c t r e a s o n s f o r n e g a t l v e

r e s p o n s e of mature e x p l a n t s i s unknown, i t may be r e l a t e d

t o t h e a g i n g of t h e t i s s u e and a l s o accumula t ion of p h e n o l i c

compounds i n t h e t i s s u e .

4.7.5 Pseudobulb C u l t u r e

The b e n e f i t s of meristem c u l t u r e u t i l i s i ne b u l b s , s h o o t

t i p e t c . a t t r a c t e d t h e a t t e n t i o n o f many r e s e a r c h e r s . The

a p p l j c a t i on of m e r i s t e r c u l t u r e i s most1.g e i t h e t t o

e l i m i n a t e v i r u s i n f e c t i o n o r l a r g e s c a l e p r o d u c t i o n of

asexua l s e e d l i n g s . The t e c h n i q u e s were e x p l a j n e d by I s 1 ey

( 1 9 6 5 ) and Russon ( 19653 f o r c u l t u r i n g of meris tem

c o n t a i n i n g pseudobulb. Pseudobulb c u l t u r e was made i n

C a t t l z ( S c u l l y 1 9 6 7 ) and Cymbidium (Morel 1 9 6 0 ) . I n t h e

p r e s e n t i n v e s t i g a t i o n m u l t i p l e s h o o t s were induced from

t h e pseudobulb of P e r e s t e r j a e l a t a w i t h o u t i n t e r v e n i n g of

p r o t o c o r m - l i k e b o d i e s . The r e s p o n s e was f a v o u r a b l e when 5

v i t r o grown pseudobulbs were s l i c e d i n t o t h i n s e c t i o n s and - c u l t u r e d on t h e medium s u p p o r t e d wi th BAP a t v a r i e d

c o n c e n t r a t i o n . Exposing of meris tem t i s s u e a f t e r s l i c i n g t h e

pseudobulb , may b e , i s one of t h e r e a s o n s f o r p r o l i f e r a t i o n

and f u r t h e r d i f f e r e n t i a t i o n i n t o p l a n t l e t s . The d i r e c t shoot

formation without the intervening of protocorm-like bodies

has been reported in Cattleya (Scully 1967). However,

regeneration vi a protocorm-like bodies was reported in

Cymbidium (Morel 1960).

An attempt to culture pseudobulbs of other species in

the present study was found to be futile due to high rate of

contamination and exudation of phenolics.

4 . 8 ROOTING

Adventitious and axillary shoots developed in culture

in the presence of a cytokinin generally lacking roots. To

obtain complete plants the shoots must be transferred to a

rooting medium which is different from the shoot

multiplication medium.

A very dilute medium is found to be satisfactory for

rooting in a 1 arge number of plant species. Sometimes salt

concentration is reduced to half (Garland and Stoltz 19811,

or a quarter (Skirvin and Chu 1979) for rooting. Bulblets

and shoots of Narcissus rooted only when the culture medium

contained in MS salt at half strength (Seabrook G. 1976).

In the present study, in vitro crowded shoots were

separated and placed on rooting medium. The rooting was

established after reducing MS salt strength to half. Profuse

r o o t s were i n i t i a t e d f r o r t h e b a s a l p o r t i o n of t h e s h o o t s ,

when h a l f s t r e n g t h r,edium was supplemented w i t h NAA.

B a s a l media supplemented w i t h low c o n c e n t r a t i o n of

a u x i n i s b e n e f i c i a 1 f o r r o o t i n g o c c a s i o n a l l y a s i n Narc i ssus

( S e a b r o o k g &. 1976) and S t r a w b e r r y (Boxus 1 9 7 4 ) . Many

c u l t u r e s i n t h e p r e s e n t s t u d y r o o t e d w e l l a f t e r s h i f t i n g

u n r o o t e d s h o o t s t o hormone f r e e media. However, from t h e

a v a i l a b l e l i t e r a t u r e i t i s e v i d e n t t h a t f o r most of t h e

s p e c i e s a u x i n a t low c o n c e n t r a t i o n i s e s s e n t i a l f o r t h e

i n d u c t i o n o f r o o t s . NAA and I B A ( 0 . 1 t o 0 .5 mg/! ) a r e a l s o

commonly employed f o r t h e i n i t j a t i o n o f r o o t s . In t h e

p r e s e n t s t u d y a l s o NAA was found t o be s u i t a b l e f o r r o o t

i ni t i a t i o n .

4.9 FACTORS AFFECTING GROWTH AND MORPHOGENESIS

The hormonal concept on o r g a n f o r m a t i o n was proposed by

Skoog and M i l l e r ( 1 9 5 7 ) and t h e y s t a t e d t h a t h i g h c y t o k i n j n

and low a u x i n induced shoot f o r m a t i o n , whereas low c y t o k i n j n

and h i g h a u x i n promoted r o o t i n g . However, v a r i o u s r e p o r t s on

hormonal c o n c e p t on organ f o r m a t i o n r e v e a l s t h a t t h i s

p a t t e r n o f r e s p o n s e i s n o t u n i v e r s a l .

The a b i . l i t y of c e l l s i n c u l t u r e t o d i f f e r e n t i a t e i n t o

s h o o t s , r o o t s o r embryos i s t r i g g e r e d by v a r i o u s f a c t o r s .

F a c t o r s a f f e c t i n g r e g e n e t a t i o n have been i n v e s t i g a t e d

i n many s p e c i e s . F a c t o r s a f f e c t i n g morphogenesi s i n t h e

p r e s e n t scudy a r e d i s c u s s e d below.

4.9 .1 Basa l Media

Knudson's C (19461 , Vacin and Went (19491 , Murashige

and Skoog (19621 , N i t s h ' s ( 1 9 6 9 ) , Linsmaier and Skoog ( 1 9 6 5 )

Gamborg e t a l . ( 1 9 6 8 ) media have been s u c c e s s f u l l y employed

t o i n d u c e growth and morphogenesis , p a r t i c u l a r l y f o r c l o n a l

p r o p a g a t i o n of o r n a m e n t a l s . E s s e n t i a l i t i e s of media f o r each

s p e c j e s a r e dependent on s p e c i f i c requ i rement of n u t r i e n t s .

I n t h e p r e s e n t s t u d i e s , e p i p h y t i c o r c h i d s p r e f e r r e d media

c o m p r i s i n g low s a l t s t r e n g t h , whereas t e r r e s t r i a l s p e c i e s

t h r i v e wel l a t h i g h s a l t s t r e n g t h media l i k e MS and N H B M .

However, !!S a l o n e was i n e f f i c i e n t i n i n d u c i n g morphogenesi s .

R o o t i n g of u n r o o t e d s h o o t s was a f f e c t e d by h a l f

s t r e n g t h nedi-ur i n t h e p r e s e n t s t u d y . Half s t r e n g t h MS was

a l s o e f f e c t i v e i n i n d u c i n g r o o t i n g i n N a r c i s s u s (Seabrook g

al.. 1 9 7 6 ) . The f a v o u r a b l e e f f e c t of reduced macroelement - c o n c e n t r a t i o n on r o o t i n g i s p robably due t o t o t a l i o n i c

c o n c e n t r a t i o n t h a n t o t h e need f o r o n l y a smal l amount of

t o t a l n i t r o g e n .

4.9 .2 Carbon Source

The most commonly employed carbon source i n p l a n t

t i s s u e c u l t u r e i s s u c r o s e . Addi t ion of g l u c o s e and f r u c t o s e

can r e p l a c e sucrose i n some c a s e s . Germinat ion and s e e d l i n g

growth a r e s i g n i f i c a n t ! y a f f e c t e d by t h e qua! i t y and

q u a n t i t y o f ca rbohydra te s o u r c e l i k e s u c r o s e , D-glucose and

D - f r u c t o s e i n t h e medium (Sharma and Tandon 1 9 9 0 ) . I n

L i l i a c e a e h i g h l e v e l of s u c r o s e i n h i b i t e d organogenes i s

(Takayama and M i sawa 1 9 7 9 ) . A t 30 g / l , 10070 s u r v i v i n g s h o o t

t i p e x p l a n t s produced b u l b l e t s , b u t a t 90 g/! only 3 of 28

e x p l a n t s produced b u l b l e t s . The remain ing expl .ants p roduced

c a l l u s .

The sporophyt j c d i f f e r e n t i a t i o n depending on

c o n c e n t r a t i o n of s u c r o s e was d e m o n s t r a t e d by Sul ik ! yan and

Yehra ( 1 9 7 7 ) i n Nephro lep is .

The c o n c e n t r a t i o n of s u c r o s e r e q u i r e d f o r t h e a x e n i c

g e r m i n a t i o n of seeds v a r i e d c o n s i d e r a b l y acong d i f f e r e n t

t a x a j n O r c h i d s , Lee e t a l . ( 1 9 8 3 ) observed t h a t g e r m i n a t i o n

of Laeliobrlegesi seeds was low when 2-4 p e r c e n t s u c r o s e was

i n c o r p o r a t e d i n t o t h e medium. However, s e e d l i n g growth was

optimum a t 3-4 p e r c e n t .

I n t h e p r e s e n t i n v e s t i g a t i o n 30 g / l of sucrose proved

t o be more e f f e c t i v e t h a n 20 g / l . However, h i g h e r

c o n c e n t r a t i o n ( 5 0 g / l ) r e s u l t e d i n d e l e t e r i o u s e f f e c t on

s e e d l i n g growth probably due t o t h e osmot ic i n b a l a n c e .

4 . 9 . 3 pH and Agar C o n c e n t r a t i o n

Morphogenesis i s pH dependent . pH i n f l u e n c e s t y p e s of

c a l l u s f o r m a t i o n . Maintenance of pH i s v i t a l f o r even

s u r v i v a l of t h e t i s s u e . pH i s normally a d j u s t e d between 5 . 6

t o 6 b e f o r e a u t o c l a v i ng . Genera l ly o r c h i d s e e d s p r e f e r a c i d i c medium. There a r e

r e p o r t s t h a t c e r t a i n s p e c i e s r e q u i r e h i g h range of pH.

Holtum (1964) r e p o r t e d t h a t a pH of 6 . 0 was i d e a l f o r

S p a t h o g l o t t i s and 5.2 was found t o be wel l s u i t e d f o r Vanda

and Dendrobi um . I n t h e p r e s e n t s t u d y t h e pH of t h e media was a d j u s t e d

between 5 . 2 t o 5 . 8 f o r s e e d germina t ion and 5 .8 f o r c l o n a l

m u l t i p l i c a t i o n . E p i p h y t i c s p e c i e s showed r e s p o n s e a t t h e pH

range of 5 . 5 t o 5 .6 .

The c o n c e n t r a t i o n of agar p l a y s an i n d i s p e n s a b l e r o l e

i n growth and morphogenesis a s i t d i r e c t l y i n f l u e n c e s t h e

h a r d n e s s of t h e media. I n t i s s u e c u l t u r e t h e c o n c e n t r a t i o n

o f t h e agar used i n between 0.6 t o 1%. I n t h e p r e s e n t work

0.62 was b e t t e r t h a n 0.8% f o r seed c u l t u r e . The h e a l t h y

growth of t h e p ro tocorms was o b t a i n e d when t h e medium was

supplemented w i t h 0 .6% a g a r . The p r o l i f e r a t i o n of t h e

protocorms was found t o be r e t a r d e d a t t h e c o n c e n t r a t i o n

0.8%. The n e g a t i v e i n f l u e n c e of agar c o n c e n t r a t i o n

p a r t i c u l a r l y d u r i n g s e e d g e r m i n a t i o n i s mainly due t o t h e

h a r d n e s s of t h e medium which r e s u l t s i n b l o c k i n g of

m o b i l i z a t i o n of n u t r i e n t s and growth r e g u l a t o r s i n t h e

medium.

4 . 9 . 4 Genotype

The work w i t h t o b a c c o c e l l c u l t u r e l e d t o t h e concept

t h a t a l l t h e c a l l u s I n p l a n t system i s t o t i p o t e n t , b u t i n

p r a c t i c e t h i s i s dependent on v a r i o u s f a c t o r s . The a b i l i t y

t o p roduce c a l l u s , embryo and a d v e n t i t i o u s buds v a r i e s

between one s p e c i e s t o a n o t h e r but a l s o from var ie t -y t o

v a r i e t y and even t i s s u e t o t i s s u e .

The l e a f e x p l a n t s of e p i p h y t i c s p e c i e s i n t h e p r e s e n t

s r u d y a r e more p o t e n t t h a n t e r r e s t r i a l s p e c i e s . T h i c k ,

s u c c u l e n t n a t u r e of t h e l e a f i n p a r t i c u l a r i s a ? s o more

p o t e n t . The r e g e n e r a t i o n p o t e n t i a? i s r e s t r i c t e d t o both

b a s a l and t i p r e g i o n , when e n t i r e l e a v e s were used a s i n

Acampe praemorsa . Re-di f f e r e n t i a t i o n p o t e n t i a1 was more

pronounced i n t h e l e a f e x p l a n t s of Coelogyne n e r v o s a . The

e x p l a n t c o n t a i n i n g b o t h a p i c a l and a x i l l a r y buds i s more

l i a b l e t o produce m u l t i p l e s h o o t s i n P e r e s t e r i a e l a t a and

P h a i u s l u r i d u s . However, t h e l e a f e x p l a n t s of t h e s e s p e c i e s -- a r e n o t p o t e n t enough t o r e g e n e r a t e i r r e s p e c t i v e of t h e

m a n i p u l a t i o n .

4.9.5 E x p l a n t O r i e n t a t i o n

The placement o f t h e e x p l a n t on t h e medium g r e a t l y

i n f l u e n c e s morphogenesis . Leaf e x p l a n t s of ~ a s t e r t i a and

Hovarthia produced more somatic embryos when grown on agar

and placed upright (Bey1 et al. 1983). The explant of Sapium

produced more number of shoots when placed in horizontal

position than in vertical position (Mridula et al. 1983). In

Morus upright position of leaf explant proved benefjcial for - maximum induction of shoots (Mhatre 1988).

In the present work leaf proliferation was prominent

when placed in horizontal position. The explant with

axillary buds was found to be proliferating well when placed

in upright position.

4.9.6 Pretreatment of Explants

Pretreatment of the mother explant intensifies Its

potenti a1 to regenerate. The soaking of explants obtained

from physiologically mature parts of the tree in water or

preculturing them in unsupplemented media for several days

cannot only be effective in preventing phenolics but also in

di luting toxic substances.

Bonga (1977) obtained adventi ti ous shoots from dormant

buds of Abies balsamea after pretreating in sterile water or

dilute solution of caffeic acid for 10 min to 24 hrs before

culturing. Further, he noticed improvement of response In

buds after treating with 0.1% malonic acid for 15 min (Bonga

1981 1 .

The buds of Eucalyptus when soaked in water for 3-4 hrs

removes inhibitory phenolic substances (Crosswal l and Nj tsch

1975). Growth and morphogenesis in tissue culture can be

influenced by pretreating explants with growth regulators

before they are cultured (George and Sherington 1986).

Multiple shoot formation was observed fron excised leaf

explants of Annona squamosa cultured on MS medium without

pretreatment. In Catheranthes (George and Rao 1982) it was

reported that pretreatment of seeds either by soaking or

germination on cytokinin containing media was a pre-

requi site for high frequency i nducri on of multiple buds fror

the explants.

Oka and Ohyama (1981) emphasised the beneficial effect

of pretreatment. Pretreatment of rrulberry leaf explant ~n

BAP resulted in adventitious shoot buds. Subsequent results

found that mulberry seedlings germ] nated on medium

containing high levels of cytokinln (4-pu), shoot formation

being strongly stimulated. Similar type of results was

substantiated in mulberry by ?!hatre ( 1 9 8 8 ) when leaf

explants were pretreated initially in liquid media w ~ t h BAP

for 24-48 hrs, later moved to so11d medium of the same

composition, resulted in multiple shoot induction. The non-

soaked leaves did not yield any response.

The leaf explants of Acampe praemorsa in the present

study when soaked in liquid MS medium with BAP for 48 hrs,

yielded better results. Similarly nodal explant of Phaius

luridus proliferated we!! when pretreated with high

cytokinin in the medium. The favourable response may be

attributed to the diluting of toxic leaches and also to

better uptake of the components of the media.

4.10 HISTOLOGY

Histological studies during seed germination in the

presently investigated two taxa Phaius luridus and

Peresteria elata have revealed that there is a distinct

apical and basal region in the embryo. The basal region is

mainly of radicular axis consisting of larger vacuolated

cells. The distinctive structures like cotyledon and

epicotyledons are present while typical radicle is wanting

in the two taxa studied. There is no controversy regarding

the absence of radicle and procambium in mature enbryo

(Swamy 1949, Mitra 1971). However, exceptionally Rao ( 1 9 6 7 )

reported the presence of procambial cells below the shoot

apex in the embryo of Arundina gramifolia.

The embryo of the presently investigated taxa and those

of Aerides crispum, Polystachya flavescence (Ekanthappa

19811, Coelogyne braviscapa, Dendrobium ovatum (Gurudeva

1989), do not possess an exomorphically evident cotyledon.

The existence of polarity as evidenced by

promeristematic activity at terminal end provide an added

142

e v i d e n c e t o t h e c o n c e p t t h a t o r c h i d embryo i s a l s o

d i f f e r e n t i a t e d .

A r d i t t i (1967 a , b ) compared o r c h i d s e e d g e r m i n a t i o n t o

d e v e l o p i n g dormant buds , bu t t h e r e i s ample e v i d e n c e t o

p rove t h a t t h e p h y s i o l o g y of seed g e r m i n a t i o n i s d i f f e r e n t

from t h e meris tem c u l t u r e i n o r c h i d s (Wimber 1 9 6 5 ) .

The o r g a n i s a t i o n of c o t y l e d o n a r y s h e a t h fo l lowed by

f i r s t l e a f p r i m o r d i a ev idenced i n t h e p r e s e n t 2 t a x a

conf i rms e a r l i e r r e p o r t s ( C a r l s o n 1943 , M i t r a 1 9 7 1 ) .

Rao ( 1 9 7 7 ) r e p o r t e d t h a t shoot apex i s e j t h e r t e r m i n a l

o r 1 a t e r a l i n p o s i t i o n . However, i n t h e p r e s e n t s t u d i e s

s h o o t apex i s t e r m i n a l i n p o s i t i o n . The o c c u r r e n c e of

l a t e r a l meris tem may p r o b a b l y due t o o b s e r v a t j o n made on non

median s e c t i o n . S i m i l a r l y Batygina and Vasi l y e v a ( 1 9 8 3 ) a r e

of t h e o p i n i o n t h a t t h e s h o o t apex i s endogenous i n o r i g i n .

T h i s r e p o r t i s based on t h e j r o b s e r v a t i o n on

p a r a c o t y l e d o n a r y non-median s e c t i o n . However, i n t h e p r e s e n t

s t u d y s h o o t apex i s exogenous i n o r i g i n . T h i s i s i n

c o n f i r m a t i o n w i t h t h e r e p o r t s on A e r i d u s c r i spum,

P o l y s t a c h y a f l a v e s c e n c e (Ekanthappa 1 9 8 1 ) and Dendrobium

ovatum (Gurudeva 1 9 8 9 ) .

4.11 ELECTROPHORESIS

The l i t e r a t u r e concerned w i t h t h e p h y s i o l o g y of t h e

o r c h i d s e e d h a s been comprehensively reviewed by A r d i t t i

( 1 9 6 7 a ) . I n most o f t h e c a s e s such s t u d i e s have been d e a l t

with exogenous factors which affect seed germination, growth

and development of the enbryo. Endogenous bioche-ica!

changes occurring during development of the orchid esbryo

and seedling have been studied by histochemical methods. It

is now well established that differentiation of cellular and

tissue system when progressed to bring about morphological

development, there is continuous synthesis or degradation of

specific enzymes and structural protein.

The present electrophoretic study shows that the number

of isozyme bands was found to be increased during

developmental stage. Furthermore, isozyme pattern is

different for each stage. In the present study on Pholidota

imbricata and Phaius lurjdus reveals that the number of

isozyme bands was increased in seedling stage. These

observations are in agreement with the view that protein

synthesis including synthesis of various enzymes is an

important process of germination (Mayer and Paljakoff

1963).

The seeds of Pholidota imbricata in the present study

are devoid of esterases while they appeared in the seedling

stage. This indicated their role of metabolic activity in

the progressive or developmental stage. Keshwani g . (1969) are of the opinion that the number and activity of

esterase isozyme increases progressively with the

germination period probably due to the specific i soenzymes,

either activated or synthesised de novo at the right stage

by t h e s e e d . I n t e r e s t i n g l y s e e d s of Pha ius l u r i d u s i n t h e

p r e s e n t s t u d y showed d e c r e a s e i n t h e number of e s t e r a s e

bands i n s e e d l i n g s t a g e . Near ly 2 bands d i s a p p e a r e d i n t h e

s e e d l i n g s t a g e though t h e e x a c t r e a s o n i s unknown, b u t i t

nay be c o r r e l a t e d t o s p e c i f i c metabol ic r o l e of c e r t a i n

e s t e r a s e s d u r i n g deve lopmenta l s t a g e s . S i n c e i t i s a

t e r r e s t r i a l s p e c i e s , t h e r e i s a p o s s i b i l i t y t h a t i t may be

r e l a t e d t o t h e n a t u r e of h a b i t a t .

I n b o t h p r e s e n t l y s t u d i e d t a x a t h e number of p e r o x i d a s e

bands i n c r e a s e d c o n s i d e r a b l y i n s e e d l i n g s t a g e i n d i c a t i n g

t h e i r h i g h e s t metabol ic a c t i v i t y . During t h e developmental

c o u r s e p e r o x i d a s e a c t i v i t y i n c r e a s e s c o n s i d e r a b l y due t o

t h e i r p h y s i o l o g i c a l r o l e i n l i g n i n s y n t h e s i s , s u b e r i s a t i o n

of c e l l s e t c . a s r e p o r t e d by E s p e l i c , Kola t tukundy

( 1 9 8 5 ) .

O x i d a t i o n of I A A by p e r o x i d a s e h a s been of p a r t i c u l a r

i n t e r e s t . Marvin &. ( 1 9 6 9 ) r e p o r t e d t h a t exogenous

supply o f I A A i n c r e a s e d p e r o x i d a s e a c t i v i t y i n t h e s e e d l i n g

of Vanda o r c h i d . I n t h e p r e s e n t s t u d y bo th t h e t a x a conf i rm

i n c r e a s e i n p e r o x i d a s e a c t i v i t y i n t h e s e e d l i n g s t a g e i n

p resence o f I A A i n t h e medium. I t s u p p o r t s t h e o p i n i o n t h a t

auxin i s c a p a b l e of enhanc ing t h e a c t i v i t y o f an enzyme

though i t can be i n v o l v e d i n i t s d e s t r u c t i o n . However,

f u r t h e r s t u d i e s may r e v e a l t h e e x a c t r o l e of t h i s a u x i n on

deve lopmenta l s t a g e s and i n t u r n t o manipu la te growth

r e g u l a t o r s d u r i n g s e e d g e r m i n a t i o n .