S ince ovules are no t a lways arranged in the same p lane in the ovary , some ser ial sec t ions show o n l y one ovule in the y o ung ovary even though the l a t ter ac t ua l l y conta ins two ovu l es . However , in l a t e r harve s t s , s ta r t ing f r om f o ur days .:1 f te r po l l ination, mo s t ovaries con s i s t o f
only one ovule w i t h the deve lop ing emb r y o . S imilar ob servat ions have b een repo r t ed by Chen and Gib son ( 1 9 7 4 ) that unf e r t i l i zed ovules in TY'ifo l iwn nigrescens b e g i n to d e g e n e ra t e three days a f t e r pollina t ion . Ovar y d i s s e c t ions also p r ove t hat a l l o r t. l on n f an ovule o ccurs w it h in
1 0 days a f t e r p o l l ina t i on as s ta t ed b y Pandey ( 1 9 5 5 ) , Pov i l ai t is and Boyes ( 1 9 6 0 ) and Ev�ms ( 1 9 6 1 ) . Therefore , the r e s u l t s in the presen t s t udy agree w i th the f in d i ngs o f thesu workers a n d Ai tken ( 1 9 3 9 ) who
repor t ed tha t only one seed is formed in each ovary . However , as in
d ip l o i d red c lover ( Pandey 1 9 5 5 ; Evans 1 96 1 ) it is only rarely in
the t e t raploid variety ' Pawe ra ' red c l over t ha t two d eveloped seeds are
found in one ovar y . There f o r e , t h e f orma t ion of two seeds in an ovary may no t be a u n ique f e a t u r e o f t e t ra p lo L dy .
The ovule i s camp y l o t ropous <:� s found in o t he r 'l'rifo l-iwn s p e c i e s (Hindmarsh 1 9 64 ; Chcn and G i b s on 1 9 7 1 ; Hh i t e a n d W i l l i ams 1 9 7 6 ; W i l li ams and White 1 9 7 6 ; Remb e r t 1 9 7 7 ) . The numb e r o f c e l l layers
in both t he outer and inner integumen t s are in accordance with the general p a t t e rn d e s cribed by Corner ( 1 9 5 1 ) f o r leguminous seeds . W i th respe c t t o developmen t , f unc t i on and s t a i n a f f ini ty , endo the l i um appears t o b ehave s imilarly t o t ha t of d i p l o i d red c love r 'I'!.•'ifo l iwn pratense ( H indma r s h 1 9 6 4 ) , 'l'rifo l 'iu.m n-t-gr•e;_;c .c:!lc (Chen and G ibson 1 9 7 4 ) and .1'. arnbiguwn (Wi l l iams and Wh i t e 1 9 7 6 ) Jnd 'I'. semipi loswrt (Wh i t e and W i l l iams 1 9 7 6 ) .
Degenerat ion o f the endo t h e l i u m l > L'g i ns a t the hear t - s haped s tage o f emb ryo developme n t as found b y ChL'n a nd G l bson ( 1 9 7 1 ) , Bha t nagar and Johri ( 1 9 7 2 ) , and Whi t e and H i ll i mns ( 1 9 7 6 ) . Chen and G i b s on ( 1 9 7 1 ,
1 9 7 4 ) f ound t h e endothe l i um t o b e almo s t comp l e te l y diges ted i n the ir mos t a dvanced samples c o l le c t ed 1 0 d .. y s a f t e r po l l inat ion . However ,
in the present s tudy t r aces o f t h e �ndo t he l i um are f o und t o p e r s i s t i n t o the la t e r s t ages o f d c u 2 l opm� n t .
The p a t tern o f d i ge s t i o n of t hL: i nner integumen t i s very s i mj l a r
I t i s somewhat l e s s s imi lar t o that described by Chen and Gibson ( 1 9 7 4 ) and ID1i t e and W i l l iams ( 1 9 7 6 ) . Small d i f ferences in t iming may b e
due t o spec ies d i f ferences o r t o d i f ferent env i ronmental cond i tions dur ing seed developmen t .
The vascular sys tem o f the ovule shows a s imilar p a t tern t o that of d i p l o i d red c l over (Hindmar sh 1 9 6 4 ) . However , in t e t rap l o i d ' Pawera ' red c l over , the micropy l e is a curved canal which does no t have the zig- zag c on f igura t ion a s described by Hindmarsh . In the la t e r s tages o f seed
devel o pmen t , as t he mi cropy lar cana l .i s sur rounded by t he outer in teg ume n tary t is sue , it might be describt-;J by t he t e rm " exos tome " as
d e f ined by Remb e r t ( 1 9 6 7 ) . This con t ras t s w i th t he observat ion o f
three s e c t ions o f micropylar canal s u rrounded by inner and outer integumentary t i s s ue in Wis teria sinensis .
As harves t ing was done on a l ternat e days , s ta r t ing f rom the date o f p o l l i na t ion , the ear ly s t ag e s o f f e r t i l iza t ion a s descr ibed by o ther worke r s ( Pandey 1 9 5 5 ; Fos t er and G i f f ord 1 9 5 9 ; Chen and Gib s on 1 9 7 1 ,
1 9 7 4 ; Fahn 1 9 7 4 ; W i l l iams and Whi t e 1 9 7 6 ) were not detec ted . Fur ther more , s ince the numb er of ovar i e s used for s e c t ioning was no t large ,
the chances o f f inding s u f f i c ient corre c t ly or iented sec t ions f o r analysis
of the contents of the embryo sac j us t before and after fer t i l i za t ion were very s l im . Remnan t s o f nucellu s 1vere de tec ted a s reported by Whi te and Wi l l i ams ( 1 9 7 6 ) . Howeve r , i n the present s t udy t hese nuce llar r emnan t s were apparen t l y mo re pers i s tan t t han those descr ibed by Whi t e
and W i l l iams .
Endosp erm d ivi s ions are in i t i a t e d e a r l i e r than those o f the zygo te a s repor ted b y Dnyansagar ( 1 9 5 4 , 1 9 5 6 ) , Pandey ( 1 9 5 5 ) , Schopmeyer ( 1 9 7 4 )
and Whi te and W i l l iams ( 1 976) . The genera] pa t t ern of endosperm
d evelopmen t agrees w i t h t , � f ind ings o f prev i ou s wo rke r s in s ome l egume spec i es including Tr>ifoli�un p1•ateu. -� and 1'. repens ( Pandey 1 9 5 5 ;
Raven and Cur t i s 1 9 7 0 ; Chen and G ibson 1 9 7 1 , 1 9 74 ; Cu thber t son 1 9 74 ; Wh i te and W i ll iams 1 9 7 6 ; \Vi l l iams <mu Hhi t e 1 9 7 6 ) .
The f o rmat ion o f t h e endo s p erm haus t o r ium and t he ini t iation o f
c e l lu lar i t y is s l ower in ' Pawera ' r e d clover than i n '!.'rifo liwn r.2perzs
(Chen and Gib son 1 9 7 1 ) or T. arnb,iguwn (Wi ll iams and Whit e 1 9 7 6 ) .
Howeve r , c e llu lar endosperm deve loprn2n t occ urs ea r l i er in ' Pawera '
red c lover than i n 1'. oetrd;;-iz.oswrt ( Wh i t e and W i l l Lams 1 9 7 6 ) 1-1he r e wa l l f ormat ion i s de layed up t o 14 days a f t e r pol lina tion.
99
S ince who l e seeds were s e c ti oned only up to 1 0 days af ter pol linat ion
owing t o d i f f icul t ie s in sec tioning o lder seeds , no ob s e rv a tions wer e mad e o f cL i l u lar endosperm deve lopme n t b eyond t h i s t ime . However , i t i s p o s s i b l e t h a t thi s p r o c e s s f o l lows t he pa t t ern o bserved i n Trifolium �.-vnb-iguwn ( W i l l iams and Wh i t e 1 9 7 6 ) .
The zygo t e appa r e n t ly b � g ins to d i v ide b e tween two and f our days a f t er p o l l ina tion , s inc e mo s t four day o l d ovules contain early g lob ular emb ryo s . Compar ed w i th othe c Tr>ifo liwn spec i e s s tudied b y Chen and
Gibson ( 1 9 7 1 , 1 9 74 ) and l.H l l l ams and I·Jh i te ( 1 9 7 6 ) , th is s low dev e lopme n t of t h e emb ryo may b e d u e t o a s low ra t e o f p o l len tube growth ( Evans
1 9 6 1 ) o r to a longer zygo t � res t i ng p � t iod (Maheshwar i 1 9 5 0 ;
Fahn 1 9 7 4 ; S p rone 1 9 7 4 ) . T h e s e two factors may b · · inf l uenced to some
e xten t b y e nv ironme n tal c o nd i t ions (Hi.l h e shwar i 1 9 5 0 ) .
As repor ted b y Haheshwa r i ( 1 9 5 0 ) , B l1a tnagar and Johri ( 1 9 7 2 ) , Chen and G ib s on ( 1 9 7 4 ) , Fahn ( 1 9 7 4 ) and Wh i te and W i l liams ( 1 9 7 6 ) ,
the f i r s t division o f the zygo t e o c c ur s a f ter that o f the prima ry endosperm
nucleus . The e a r ly s tages o f emb r y o development up to the globular-shaped
s tage wer e no t o b s e rved i n this s t udy b u t are a s s umed to f o l low t he
general p a t tern d es c r ibed f o r o t h e r angiosperms in general texts , such
as tho s e b y Maheshwar i ( 1 9 5 0 ) , Ward law ( 1 9 5 5 ) , Fos t e r and G i f f ord ( 1 9 5 9 ) , Fahn ( 1 9 7 4 ) and S prone ( 1 9 7 4 ) .
From s ix days af ter p o l l i n a t ion the embryo begins to p roduce co ty ledonary ou tgrowths f o l l owing t h e norma l p a t tern of d icotyledonary
deve lopmen t as d e s c ri bed by a num b e r o f worker s ( Raven and Cur tis 1 9 7 0 ; Fahn 1 9 7 4 and Neushul 1 9 7 4 ) . The emb r y o p r o g r e s s e s through the hea r t shaped t o the torp edo-shaped s tages 11s o u t l i ned by Raven and C ur t i s ( 1 9 7 0} Fahn ( 1 9 7 4 ) a n d W i l l iams a nd White ( 1 9 7 6 ) . A t t he early c o ty ledonary s tage , the emb ryo also b e g i ns to Lend to adapt to t he shape of the
emb r yo sac as in o ther sp e c i e s ( Ha h e shwa r i 1 9 5 0 ; Fos t e r and G i f ford 1 9 5 9 ) . A t t h i s s tage , the a p i ca l nK� r b tem ( p lumul e ) i s conspicuousl y d i f ferent f rom t h a t o f the monoc o t y l edonous emb ryo (Raven and Curt i s
1 9 7 0 ; Fahn 1 9 7 4 ; Neushul 1 9 7 4 ) .
A l t hough i t was not p o s s i b le t o d e t e rmine the s tage a t \vh ich the
emb r y o reaches i ts f i nal c e l l numbe r- , i t a ttained maximum s i .:: e 1 4
days a f ter p o l l i na ti on . A t t h i s s toge , the co tyleJons haJ r e a c hed the
cha 1azal end o f the emh r y o ;; a c !J u t t h L d ry \vt:: i g h t o f tile seeJ �vas only
/
MASSEY UNIVEitStT1
abo u t 40% o f i t s maximum dry wei gh t . ( Page 40 ) . These f indings appear to b e in accordance wi th the resul t s of Loewenberg ( 1 9 5 5 ) and McKee e t a l . ( 1 9 5 5 ) and also show that t h e maximum s i ze o f .the emb ryo i s reached much ear lier than the maximum wei gh t of the s eed .
100
The p a t tern o f embryo development in t e t rap loid ' Pawera ' r ed c lover i s s imilar to tha t s een in d i p loid red c lover by Evans ( 1 9 6 1 ) , and a l though developm�n t is s l ightly s l ower in ' Pawera ' , the p a t tern i s also s imi lar to tha t r ep o r t ed for certain d i p loid Tr•ifo liwn species
( Chen and G ib son 1 9 7 1 , 1 9 7 4 ) and for t e t ra p l o i d Tr>ifo liwn amb-iguwn
(Will i ams and Wh i te 1 9 7 6 ) .
The early s tages o f suspensor f o rma tion were no t c learly observed in this s t udy , b u t is a s s umed tha t deve lopmen t f o l lows the general p a t tern seen i n o ther l egume s ( Ward l aw 1 9 5 5 ; Fos ter and G i f ford 1 9 5 9 ) . The s i ze , shape and numb er o f c e l l s i n the s u s p ensor can vary be tween s pe c i e s ( Bha tnagar and J ohri 1 9 7 2 ) even wi thin the Legumino sae
(Maheshwar i 1 9 5 0 ) . However , in the l a ter s tages of emb ryo deve lopmen t , the s us p ensor o f ' Pawe ra ' appears a s a globu lar shaped s truc ture whi ch resembles the Trij'o liwn minus s u s p enso r d e s c ri bed by J ohansen ( 1 9 5 0 ) .
B . CELL STRUCTURE CHANGES IN THE SEED COAT DURING SEED DEVELOPMENT LITERATURE REVIEW
The important role o f the impermeab le seed coa t in sma l l- seeded legumes in ma intaining seed v iab i l i ty and caus ing delay in germina t ion has been wel l recogn i zed . It has been s ugges ted by var ious workers that the fai lure of germina t ion of viable legume seeds was mainly due to the imp ermeab l e layer i n the seed coa t . Al though p revious workers have inve s t igated th e c' Ll s truc t ure in the seed coa t of d i f f eren t plan t s pec ies , including sma l l-seeded l egumes , no detai led accoun ts o n the seed coa t s truc ture of red c lover d u r i n g d i f f e rent seed deve lopmental s t age s have appeared i n the l i tera ture . Theref ore , inves t i ga ti on o f changes in cell s t ruc t ure i n tl1e seed coat during s e e d developmen t i n
1 Pawera 1 red clover wa s \va r ran ted .
As the ovule ma tures into a seed , the integuments undergo consp icuous change s . In le gume seeds , the inner o f the two integuments disappears during the on togeny of the seed , whe reas the o u ter integumen t d i f fer entua t e s into a varie ty o f dis t inc t layers ( Esau 1 9 6 0 ) . Mos tly these·