Chemical Studies on the Development of Plant Embryos Citation Siu, Ralph Gun-Hoy (1943) Chemical Studies on the Development of Plant Embryos. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/y6p0-4b68. https://resolver.caltech.edu/CaltechTHESIS:03142025-221133774 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Haagen-Smit, Arie Jan Thesis Committee: Unknown, Unknown Defense Date: 1 January 1943 Record Number: CaltechTHESIS:03142025-221133774 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:03142025-221133774 DOI: 10.7907/y6p0-4b68 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17066 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 19 Mar 2025 16:43 Last Modified: 19 Mar 2025 16:48 Thesis Files PDF - Final Version See Usage Policy. 41MB Repository Staff Only: item control page

CHE:.HCAL STUDIES ON THE DEVELOPMENT OF PLANT ELIBRYOS Thesis by Ralph Gun-hoy Siu In Pa rti a l Fulfilmen t of the Require □ ent s for the De gree of Doctor of Ph1losophy Ca lifornia Instit u te of Techn ol ogy Pasadena, Ca lifornia 1943 TABLE OF C01'1 TE!\!TS PREF ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pe. . g e 1 I NTRODUCTIO~ ................................... pag e _2 EXPERI;;:El'! TAL SECT I OH ........................... p age 6 Cha,pter 1. Techn i qu e for culture of Datura emb r yos in vitro .......... page 6 C - - - - - Chap ter 2 . Effec t of coc '.Jnut mil ~ on grov1t h of Da tur a embr yos in vitro ....... cage - Cl-:apter 3. Vari ab ilit y in grovrt h of embryos . p2.ge - -- 11 20 Chap ter 4 , Enviro nmen t a l f a ct ors inf l uencing growth of Datura embryos ......... page 32 Chap ter 5 . Effect of che mic a l subst an ces on gr owth of Datura embryo s ...... p age 45 Chap ter 6. E}n1:r yo fe,c t or in na tural e xtre.cts . page 47 Chap ter 7. Chemica l studies on emb ryo fact or in c oconut milk .................. page 51 DI SC USS I OtJ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p e.ge 84 5 UMlVIJ~Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . page 94 REFERENCES ..................................... page 97 1. PREFACE Although considera ble a dvances have been made in pla nt embryo geny fro m t h e morp holo gic a l st a ndp oint, the knowledge of the phy siol ogi c a l a nd t he chemic a l f a ctors involved i n the development of t h e e~b r yo is me ager. Th e present wor k is a study of t h e conditions a nd the chemic a l sub st a nces, whic h are necess ary for the development of plant embryos. During the c ourse of t h is inv e sti ga ti on, t he au t h or ha s been g iven much help an d enc our agement. He ha s received va luRble a dvice a nd many p ers ona l f a vors fro m Professor A.J. Haa gen-Smit. Many of t he ideas in t h is wor k have orig- ina ted fro m Pr ofessor J. van Overbeek, who also has given muc h of h is ti me and energy to t he p roject. Helpful sug- gestions hav e b een te ndered by Professor F. Went, Profess or J. Banner, a nd Dr. Harriet Bonner. Messers. S. Gottlieb, ~ .D. Bonner Jr., P.R. Sa unders, Dr. J. Kirchner and Dr. H.D. ~ ic h e ne r hav e been ve ry ge ner ous with t h eir help durin g va rious phases of t his project. ~rs. Gertr ud e .iilson as- sisted in the culturing of the t hous an ds of embryos, which was necess ary to ar r ive a t t h e conclusi on s dr awn in this thesis. To all of t h e a.bove p ers ans t h e a uth or vi shes to express hi s sincere gratitude and a;ipreciati on. The a uthor also wi s hes t o e xpress his indebtedness to the Americ a n Cyani mid Co. and t o t he Americ a n Philos ophic a l Societ y for gr a nts received durin g the c ourse of this investi ga tion. 2. INTRODUCTION Investi r;e.tions on t h e culture of p l an t ei7bryos in vi...:. · tro have been stimulated by the study of the nutrition of plant emb r yo s, the question of lethal fe.ctors E,nd incompatibility in ~lant breeding, the germination and the development of otherwise abortive seeds, and artificial parthenogenesis. Of t h e many investi ga tors who studied t~e nutrition of plant embryos over a decade ago ( 2, 13, 14, 16, 21 , 22, 25 , relatively fe w undertook the culturing of i mma ture embryos. Th e earliest of these was HG,nn i z. ( 23 ) in 1904. He excised immature eobr yo s, which we re only 1.2 mm . long , from t :ie ovules of ~ an~ and Cochle ar ia and grew the m to ma tur- it y in a mixture of g lucose, a s;2ragine, leucine , g l ycoco ll, t yrosine, mine r s l s a lt s , a nd peptone digest . Ae a result of i mprov ementsin cult ur a l techniqueE, small er embryos were grown in Vitro by l a ter workers ( 26 , 29, 42 ). This culturing of imm ~ture embryos in vitro became a very useful technique for the propagation of otherwise aborWhen Li num austriacum was crossed with L. ti ve embryos . perenne, for example, viable seeds were not obtained . How- ever, Laibach ( 26) excised the embryos, which were about a fortni gh t old and 1.5 mm. long, and )la ced t~em on a cotton wadding watered b y a 10 to 15 p er cent sucrose solu ti on in a test tube . After a wee k t hey were tr a nsferred to a ~oist blottin g pe.pe r. Ger mina tion occurred in ,.'. f ev.: d"1~'S an d the embr;:;os subse qu e ntl y grffw into n <)r:TID,1 ma ture p lants, wh ict were inter-f er tile a nd c apab le of being p ollinated by either 3 parent. Tho us a nds of imu~ t ure hybr id embryos of cherry, apricot , peach , ano_ ot her :-1'.:n... tic ul t u r :d y l on ts r:_;:1.ve b een cu ltured. sirnil e,rly 41, 42 ). ( 26 , As wa s t te ca s e wit h t h e e ~rlier investi ga tors Tu ~rny (39) f ound t hnt t t. e II sterili ty 11 of s o;n e varie ties Jf sweet cherry (Pr~ avi um ~J v;c E due to nutritional and cultur a l conditions. He a lso suggested (40 ) that the ab ortion of the embryos migh t hav e been correlated with the ear liness with which s or:1e va rieties ripened treir fruit. 11 Abund ,::rnce 11 cherry, 1,·: hi ch ri pene d in early August, produced nesrly 100 per cent viable seeds. 11 BL-,_ck Ta rtar 11 che1" ry, which ripened in early Jul y , g.s.ve on ly 70 to 85 pe1. . cent viable seed c0 , ivh ile t he 11 Earl y Purple Gui gn e 11 cherry, wr:, ich ri;ened in e arly J une , produced only abortive seeds. Although Tukey was not ab le to grow cherry embryos v.-~icr, vrnre iri the e~~::'."'1:,- st a ges of development, he was a1le to cult~re those which were in the period of rapid growth prior to their collapse i n·the seed. From t he standp oint of the p l an t bre eder, then, t h e realization that the e arly-ripeni ng va rieties J roduced abo rtive seeds might have argued against the use of ~n e Brly-ri p ening variety as a fe ma le parent in & cross. But through the cul- ture of excised embryos i n vi~ lt \vas p ossible to obtain offspring from crosses involving two parents with e arlyripening char ac teristics. T~e newly-bred variety, if satis- f a ct ory , could be perpetuated by the usua l ve get a tive methods of propagation. All of tr,e imma ture embryos i:-1hich were c ult ured in vitro by the a.b ove mentione d workers were rel pti vely l ar ge. 4. Th e f a ilure of t h ese workers t o r:::row s mall embryos in vi.tro made p re v a le n t t he ide a of a li mit in t h e size of e xcis ed embryos which could be cul t ur_ed in vitr,2. Dietrich ( 20) , for examp le, a sserted t h8.t e mb r yo s less thEJ.n one t h ird of their full size we1°e inc D.pable of development outside of the seed. LaRue , who v_;a s ab le t0 cuJ_ t 1Jre imrT:a ture enbryos of Chrys an t:-, e_~~~ Leuc a nthemum and Bryo,e__hyll:::!::~ cren a tum, wh ich 1ive r e only 0 . 5 m ,,1 . i n len f tl1, sug 6 e stec. ( 29) t he. t h~ve re a c hed a new lower t h res ho l d wh ic ~ 11 •••• it i t will b e does diff ic ult t o 1) 8.S S • 11 La.R ue I s s 'u-,te ::i e:-,t we :,t unc},.[11 ler, ,~:eo. ;.rn t il v ;:·;-! Ov e r b eek., Con\lJ ,1 c'. nd BL1.\:es lee (4: 6 , 47) , v:>) v1ere i n t er ested i n a r ti- on t h e c ul t ure sf s ~a ll D~ t ura emtrJo s. cJ t :ie inciuc e;';ei: t :Jf Thes e wo r k ers ~ivl- V--e e ,~:~: cell t o 6.i v i de an d b) the de- velopm ent of t h e r esu lti ng pro e cbryo into a viabl e p l a nt . I n t h ei r c:.t te :,1pts t 1J grow sm D. 11 L,L12.t u re Da t ur.1:: embry os Rnd pr o e mbryo s t h ey d iscov ere d t h e folloKi ni f a cts : a) Excised ~a tur e e7b ryos c o~ld be t rown i n d i ~ li ~h t on~ medi um of O. S t o 1 ,::; e r ce :i t e.1: : J_r, 1 iJer ce n t C: . ext r ose , er a l s ol u ti 8n . D. t t he Tu~ey ::1 i n- Roo t, hyy ocot y l, end cot y l ed ons ~eveloped , wh ich resulted in normal seedling s. bryos D.t,d II tor~edo 11 b) La r ge r i mma t u re em- stEu: : e fo r med ro •J ts 2.nd hyp ocotyl bu.t no cotyledons in t h e above med i um . But they co ul d be grown into normal J l an ts with the addition of g lycine, t ~ i am i ne, 5. ascorbic a cid, nicotinic a cid, vitami n B5, adenine, s u ccinic acid, end p antot h e n ic a cid to the Etbove medium. c) Embryos s ma ller tha n 0.5 nm . in len g th were no t c apable of gr owth even in t h e mo difi e d medi um . Wit h t h e a dditi on of non - autoclaved coconut milk, h owever, embryos a s small a s 0.15 mm. in len g th could be grown to maturit y . It was a t this p oint th a t the p resent investiga tion begc. n. 6. EXPERI ~,LENTAL SECTION Chapter 1 --- ,!~chnique for cul tu.re of Da t ura e~brvos in vitro --·~ The meth od devised by van Overbeek et al (47) was used i n t h is investi 6 c-.tion. Capsules v:ere picked 12 to 15 d.s,ys after p C> llin Dti on fr om. Dn. t _~ and in the field. p l a nts ~.,:r own in f I'eenhour:- es They wer e brough t t o t he dissectin g r oom where the embryos were is ol a t ed und grown 1-.l nder aseptic condi tl ons. The c aps1..1.les 1;_;ere first so a ked in 70 p er cent ethyl alcohol f or ab ou t one 8 inu te. T~ey we re t ~en held wit h ster- ile forceps a nd ab out 200 cc. of sterile water p oured over e a ch capsu le, i n order t o wash of f t he a lcoho l. After the wa s hin ;:; process, t h e capsv les wer e ;) l a ced e 2. ch in a sterile Petri dish a~d t he c 2r pel s cu t wi t h t h e a id of a s terile sc a l p el a nd a sterile p a ir of f or ceps . then disc a rded. Th e c a r pe ls we re Th e ovules were re~oved fr om t he p lace nt a l tis su es a nd □ ixed in a sterile Petri dish. This made po ssible a r a ndom s::twpli n~: of the ov ul es, which wo. s necess a ry f or the qua n tit a tive assay of t h e e~bryo f a ct or pres e nt i n different medi u . As a rule, a sin g le c apsule, wh ic h c ont a ined abou t 300 ovules , wa s used for e 2ch test.· Abou t 5 t ei 10 ov u l e s were 9 L.--,,c ed ctn d held between 2 mic 2:> oscor) e slide s , v1·hich ha d been c-J.i _,_) ped previously in 70 per cent e thyl a lc oho l and fl a med . In order to avo id t h e use of ho t slides, 2 pairs ~e re nee ded. Wh ile 1 pair wa s being ernp loyed to l-1o ld the ovules, t he other 02,ir, which had 7. been dipped previously in a lcoho l and flamed, was be ing cooled. Th e ovules be twe e n t he glass slide s were cut longi- tudinall y by drawing a sterile t hin razor blade t hrough them. It W&S found t hat althou gh the us e of ho t razor blades had to be avoided, gre[1_te r c u t tin €_: fc:1.cili ty was obtE:_ined wi th a HD.rm b l 3.6.e t han a cold one . ·:n10n sma ll embryos had to be isol a ted, the ovules were cut into c1.ppro xi :rna tel? equal parts. Howe ver, when embr yos ove r 0 . 5 mm . in len g t h had to b e isolated, the ovu les were c u t into 2 unequ a l parts in order to av oid injury to the embryo . The halves were p l&ced on t he slides with t h e cut surf3_ce facing upwards. These were then examined under a pre - para tio n microsc ope . The back of t he microscop e wa s fitt~d with a celluloid shield t o protect the expo sed embryos from c on t am in ~tion by the o~er a t or 1 s b re a t h . If the cu t had been mc1.de '.J r ol_,er l y an er;-1br~ro con~.d be seer: in one c orner of t he erTib r ro E~_c of each 0f the c11t ()ViJ.les . The embr yo WF,S re - m0ved wit h t ~e aid 0 f dissecti ng ne e dles, w~ ich ha d be en 2 pa ir s 0f nee dl es were used ~lt ern~t e l y , a s i n t he case of the microsco pe slides . The enbryos wer e tr Rn sferred direct- l y to st erile shell vi~ls (retempe red, short -st yle type , ·3.6 cm. l ong x l cm. diamete r), c ontaining the cultur e med i um . T~e cult ur e cedium (he re afte r referrRd t o a s 1 pe r cent dextrose medium), which was used a t t he be g innin g of t h is inv es ti ga t ion , wa s made up a ccordin g t o t able 1. As a result of new findings during t h e c ourse of t h is work, thj_s culture medi um we.s subjected to n odif ic o. ti ons , which 8. will be described in t h e app r op ri a te subse ouen t parts of t h is t he sis . Tab le 1 . Co~p osition cf 1 u ~r cent dextr ose culture me di um. We i gh t in ~;l ., 8~l!1S C::-ie mic a l O. G80 pot a s s i um c h lori de 0 . 170 c a J.c i um su lf a te 0 . 170 ' '11 1-1ke? ' s S8 l t magn esi um s ulfa te :·~ i ~,~t u.r e _ ,._. ) ( Ll.0 0 . 17 (J c a lci um phosph~te Bake r ' s c. P . Bake r ' s C. P . c. P . :a ,?Jrn r I s c. p . Bc=1-ker I s Baker ' s C. P . .J . l? O Difeo b r. ct o- c'. extrof',e Dife o o. oo ~ Merck lJ . S . P. 0 . 00 1 0 .0002 n i cotinic r_cj_d l,; er ck U. S . F . h::,rclr o c::l or j_ oe 0 . 025 su c cini c c.ci d 0 . 0005 c :o. lci -u.m d-p_j1D to the n.s. te l<erc k 1 liter 3/4 cc . of t h is ~ed i u7 , t ~e pH of w~ ic h ~as 5 . 5, was p l a ce d in e ~c h c ul t ure vi& l, tthich w~s fitted with a c otton p l ug an d ~u t ocl a ved a t 15 ~ ounds ) re s sure f or 20 ~in~t es . ·,vhi le t ;~e ;:;,g1:1.r W[;S s ti ll liquid, P;_e t es t s olu ti. on , wh ich 9. had b e en sterilized previ ous l y by me ans of a Seit z bacteriolo gic a l filt er, wa s introduced into t h e vi a ls . Pa ste ur pi- pettes, delivering 27 drop s p er cc., were u s ed . Th e aga r □ edium wa s allowed to solidify on a slantin g r a c k , after which one enbr yo we s intr oduced jnt o e3 oh vial. Unless ot her- wise st a ted , 10 embr yo s were used for e 2ch t es t solution . For greater un if or mity ~f gr owt h , t he embr yo s were a ll i nse rted abou t 1 nm . be low t he surf &ce of the ag ar. The emtryos we r e cultured in the da r k a t c 0nst a nt te ~pera t ur es . During t h e e Rrly part ~f this i nvesti ea ti on , th_e e mb r yo s were ~rown a +u t o 32±0.5° C. ., ,.,. +~~ ,) C • ~~ Me a s ure ments of ·the le ng t h of t~e embr yo s were made a fter different lengths of time by me an s of an ocul ar ~icro meter. Bu t si n ce the measure~e nt s were Da~e t hr ough the curved walls of t h e vi a ls, t he fi gur es give n i n -- t ~is t heei e ar e 1 ar g er t ha n t he a ct ua l dimensi ons of t he embryo s. - The ~ve r age relative gr ow t h in the l eng t h of t ~e embry os (fi nal/initi a l ~en g t h ) an d the pe rcent ag e of t he embry os which fin a lly de vel cped (here a ft er re f erred t o as 11 )e r ce nt viabili t y 11 ) we re t D.:rn~ .s. s indices of the amount of embr yo factor i n t he medium . Tt e above is J ~.s: ti on D.. nd c ul t Tcln~: of t he e:~1br::,·os were c2rried ou t in a room w~ic~ was kep t cl0sed 2nd free of wind curre n t s . It ~as fo und t ha t if t je descr i bed manipilla ti on s were p erf or me d wi t ~ a re a s onable speed c on t a mi na ti on w&s ne ~:li g ible. s eptic spra ys and f ac e masks, we re necesE Rry . Illumi na ti on 10 . wa s fur n i shed ty ~n 85- wa tt fluo re scent l a @; , hung a b ou t 2 f ee t abov e t h e d is se cti on t ab le. I ncand esce n t lim~s were n o t used b ecause the ~e a t pr oduc ed by t hen dr ied out t h e e xposed e mb r y0 s r a t her r a) id.ly . 11. Effect of c oconu t ,"1 :i. l k on gro1., ;r t h of Datur & e ~bryosJ~ vitro-a) Growth of e;:itrvos i n l ,2er cen t d.ex tl"'OSe ;:·rnd i ura . ----Embryo s ve.ryin 6 fro m 0. 1 t o 1.7 mm . in len g tt wer e iso- lL.ted fr'o r:1 Datur ro ca..'._) S'J.les of d. :;_fferent :::_g es , :. nd cultured 1 le :1:;:_: t L of t h e e,;fb r~· cs were ta i.e:1 a fter d i fferen t number of Ab ou t 1 0 per ce n t of t ~,e embryo s betueen 0 . 5 2,ncl 1.0 mm. in le ng t h and r~rely embryos less t han 0 . 5 mm. in len g th were capab le of t evel o9 ment in t h is ~ed ium. It is also evi den t t ~·w t grow t ~ c ou ld be e.etected a fter onlr 2. days of culture . E~bryos which f a iled to s h ow any signs of gr owt h within 2 day s did not dev elop a.ny further. I n f a ct , a sli gh t shrink- age of t h e e mb r y o was observed i n most of the f a ilures . b) Growth :)f .£!Eb ryos in 1 pe r ce n t dextrose ,-: . edi ui71 .£.QI2t a inin ~: coconut :71 11: . Heart - shap ed e mbry o s smaller than 1 r,1n . in len g t r. were excised o.nC cultured in t Le 1 per cent a.extrose mediu1;1 with and with ou t the a ddition of coconut ~il k ( liquid in t h e nut of Cocos nucifera ). Measurements of the length of the em- 12 . bryos were t ake n a fter 0 , 2, e.n d 6 days o f growt h a t 25 ± 1° C. Th e growth do. t 8. f or the indi vidue. l e mbryos are shown in t ab le 3 and figure 2 . Tab le 2 . Growth of Da t ura embryos in 1 per cent dextrose medium . Le ng· t h of embr yo in 2 days l7l m. "Embr yo no . 0 days 1 1. 2 1. 2 1.1 2 0.4 0 . 35 0 . 35 3 0 . 25 0.2 0.15 4 0 . 75 0.6 0. 6 5 0.1 0 . 15 0.15 6 0.9 o. ;3 0 .8 7 1.7 2.9 5. 0 8 1.4 1.5 5. 0 ,~ ;j 1. 6 1. 9 5.0 10 1.1 1. 3 4.0 11 1. 0 1.2 5 5.0 12 1.7 2.8 7.0 1 0~· 1.0 1.0 1.0 14 .1. 2 l.l 1.2 15 0 . 55 0.5 0.5 16 0.6 0.55 0 .55 17 0 .7 0.7 0.6 18 0.9 0. 9 0.3 19 0 .35 0 .3 0. 3 20 0.5 0 . ( 3 d.ay s ) 1.8 ( 7 days ) Q t:; ..,._, I 6 day s I 13. t• tt ·t·t-,-, +-t- .~. ' + -1->4..). 1-l-- j-: j- + -'+- -11-,--~ + + -1-::j:f r-,., -.l q _, -!1- -+-'-~ J.. t:! rl ·• t-+ ; - +- :t ,+ -H- :+ .j +,- -H trt- -~ t~ - ·+ + ~ ·'- r + + -++ 'i + !:' ++ ...+ +- ::t.=14 - +:_ + -:::::: t - +- ·!+ i ·+ ' ~:t ·::t - . - -+· ~ !-1- -lI r+-· ,- l:!:M ,--1- · - tta -, =ht ...: .,.. ·'-· ~ + ~Lt -, + >----- .;.., C +~ •i-i .... -+-- ::;. ::.t .. -..... -++ - .... 1-i+ ..; j-~ i--.:: f,-, -Fl c...W I.+ H-< t-+ f+-+1 ·r:- - ~+ ·t-+ .l ·+ + 1-<+ -~=f ~ ±·; 1 ,- 11 .! ..). 4 1: .L ':;..J. .. :tt -:+ '1 .'j e+. ...). :f ! '-B t?I+ ~ . ,; +,. L,~ + +q: + +++ 1..1 ' ··- ~-I , t ll : i-, LL :m h--- .. 14 . Tabl e 3 . Effect of c oc onut milk on growt :1 of Da t ura embr yos a t 25 ° C. ,...., Medi um -· No c oconut mil k e,dded 5 drop s coconut milk p er v i e.1 Embryo No . Le n;-i;th of e mbr yo i n mm . 0 day s 2 days 6 days 1 0 . 95 1. 0 1. 0 2 0 . 25 0. 2 0.2 3 0.4 0 . 35 0 . 35 4 0 . 25 0. 2 0 .15 5 0.4 0 . 35 0 .3 6 0. 5 0. 6 1. 5 7 0.1 0 . 15 0 . 15 8 0 .15 0. 2 0.2 9 0 . 45 0 . 45 0 . 35 10 0. 9 0.8 0.8 Av . 0 . 44 0 . 43 0 . 50 1 0 . 25 1. 0 5. 0 2 0.6 1. 4 3. 3 3 0. 6 1. 9 8.0 4 0 . 35 1. 55 6. 5 5 0 . 25 0 . 25 0 . 25 6 0 . 35 0 . 55 2. 5 7 0. 6 1.15 3.0 8 0 .7 5 1. 8 6. 5 9 0.6 1. 7 4.0 10 0.6 2. 0 5. 0 Av . 0 . 50 1. 33 4. 4 J :+± ~!t:nt ±t;m g \Oc ..... ni1:a: t-1 -,. f-+-h .c .,+ - 1:t+t I• H H rt I -+- + uu 1::: I+ "T ,- t-- W- +H· ..... r -~ } t&¥ :j I+ W- +-+- +-+ -·· :tj:+,..t . +-tt tt =1: ts. I:!:: r:: J _l_ ~ .u. j I-, -:1,.... 'f il± ➔ l ., f :1t!r1 t ± H- ... I '.rtt r + +~ I J ·t-+ T t ~-i- :t t-1· + -~ ~ - -r ·tt I+ ' ; t! ~ H-· H- . + ! :;+ ::.. -W- -++ 4 ~ ·t-- ·• t-- '·• :t .:. 16. The necessity of c oconut milk for the growt h cf s ma ll imma ture embryos is obvi ous fr om figure 2. Withou t t te ad- diti on of coconu t ;T tilk only 1 out of 1 0 embryos gre w. How- ever, t he gr owt h of t 11is one embryo was abnormal a nd did no t give rise t o a normal diff eren ti ated seedlin g ~ When c oconut milk was added t o the dext ros e medi um , 9 ou t of t he 1 0 embryos develop ed in to no r mal viable seedlin€s · c) Effects of different conce ntr a tio ns of c ocon ut milk on 1.t.§. ·growth of embryos . He art-shaped embr yo s, 0 .3 t o 0 . 6 mm. i n len gth, wer e is ol a ted a nd grown i n t h e 1 p e r cent dext r os e me di um co ntaining different conce ntr a ti ons of c oco nu t 7il k . Coconut milk was c on centra ted in va cuo at 50 to 55° C. to one fifth of its ori gina l volume. St ock s oluti on s were pr epare d by dilutin g this co ncentr a te. One t d 5 drops 0f t he re spective stock s ol utions were added t o e a ch of the cult ure vials t o give t he desired concentrations . The embryos were cultured a t 25±1° C. a nd measurements were ~ade af t er 0, 2 , 4, an d 7 days , respectively; The growt h da t a of t hese embryo s ar e pr e sen ted in table 4 and fi gur e 3. There were no si gnific a nt differences in t h e o erc ent a ge s of vi abi lit y in t h e embryos grown in medi a containing the eo uiv a le nt of 3 t o 1 5 dro os of c oc onu t ~ilk per vial . Opti mum gro wth of t he embr yo s, however, t ook place in midia c ontaining 3 t o 7 drops of c ocon ut E' ilk p er vi a l. Wit h in- creasi ng co ncentra tions t h e growth of t h e embryos decre as ed rap idly, so t ha t a t a c oncentr a ti on equivalent to ·20 drops /✓/He of ori ginal coc onut mil k p er vlal ~ growth wa s obtained. 0 . 97 1. 08 0 . 52. 0 . 58 0 , 80 0 . 48 100 37 . 5 50 ,-, r"' 7.5 0 . 44 0 . 50 o. 3;::, 0 . 48 0 . 35 0 . 43 1 00 100 40 20 75 1 00 1 25 15 20 25 ~ 0 ,/, . ;) ~ 0 . 56 1. 66 2 . 10 2 . 10 o. ·94 1. 2 1 1. 7 2 0 . 69 ..L i:::, . ::) r 0 . 44 100 C' r i::; 0 . 93 0 . 42 100 ,:::, ::) 5 10 0 . 38 90 15 1. 43 3 0 . 64 0 . 47 70 v 0 . 33 t::; 0 . 34 4 days 1 0 . 37 0 2 day s 0 0 de.y s Pe r cen t v i ab il i ty • - 0. 53 0 . 64 1.51 1 . 63 2 . 42 3 . 24 3 . 59 2 .58 2 . 15 0 . 33 7 day s 1. 2 '7 1 • <.) 1.7 1. 8 2. 0 2.8 2.2 1.8 1. 4 0. 9 2 days 1. 3 1. 5 2.3 2. 9 3. 5 4. 8 5.0 4. 5 • 7 J.. 1. 2 1. 8 '7V 4. 9 5.0 7.4 8.5 6.8 4.6 0.9 0.9 3. 0 7 days . 4 days Gr owt h me asure me n ts Av. l eng t h i n mm . Av . r e l a t ive gro·wt h Gr ow t h of D&t ur a e mbryo s i n 1 pef ce n t dextros e med i um c on t a i nir.g differen t c oncen t ra t ion s of coconut mil k . 0 Cocon u t ui l k pe r vi al no . of mg . drops dry wt. Tabl e 4 . -(I 1---' . t n: •t~ • T t.: ± + :.• ·t++ .; ·; +t ., td 3 , l::J: -H .i t-'~u~~1-1~~ ::r ]: -- +- r ..::. :t; ~ /8 . 't h- (._ i ~ 1-,'-+ !i= i- .,.-+· JI _c._ ff :t'" t+ ·!- . . t >-+ rt. ...... ti -+ i t+t t+ H ., ·1 .:.J.. ... + -- '(JIJ I ~~ ·H- ;... -~ tIm •-+ -+•+ -+~ - < 1 + :f:I= t rr 1 -, ., I~ + -'- -t -+ t ... • H H- I- :Ji ..! + r 18 . d) Eff ect of cocon ut Dilk on the gr owth of Datura embryos £.EOVided wi th .2.£ ;!!thou t sup:o lement_?-Fl _ll1:!trients . Embryos between 0 . 2 an d 0 . 6 me . in len gt h we re grown in the a ) 1 per c en t dextrose mediura , D.S 0o'l ven i n t ab le 1, b ) l per cent dextrose medium, a s g iven in t ab le 1, p l us 5 drops of cocon ut ~i l k ) er vial, and c) 0 . 8 per cen t a ga.r , 9lus l l) er cent c1 ex t rose , p lus 5 d.ro) S of coconut. mil k per vial. lvl easureiJ.ents of tte lengt::: of t he embryos we re t ake n afte r O, 3 , and 7 days of culture a t 25±1° C. and ar e give n in t h e t ab le below. Table 5 . - · \1edi um Growth of Dat ur a emb r yos in coconut milk with an d withou t su~plement a ry nu trie n t s . Gr·owth me asur e ments of embr yo s Per ce n t re l a t i vegr·owth viability ~ e n.,,;th i n mm . Odavs--· 3 da rs 7 da.ys 3 days ·------~ 7 davs ·-- 1 ~ .- : J t - - ~ - i - - - - - - - ~ - · --- 0. 9 --- 1.74 3 . :)7 ~-? 8.2 1 . 24 l . 8~1 4. 3 6. 5 '--'• 0 0 . 38 0 b 100 0 . 41 C 100 0 . 28 ,.::, 0 r;a_;,r. LL ,_ - i.:edium II c 11 , t.Jn ich 2.::,cJrn cJ t t e i nor ~:~,.n lc s ,, lts o.nd the vi g orous a bl'01vtt of tl:e e nbryos a s <.'l. i d meo. i w-:-i 11 b 11 , uh ich c on t o.ine d all of tr~e L1gredi e n t~, ,~-f t te 1 p er ce nt dextr os e was ~a t observed durin g t he first 3 d8.ys of gr owth tu t b ecame pro minen t on l y a fter abou t e wee k . viabilit y 1:1as t he s a,n e i n bo t h c r,.ses . T~e pe rcent a~e 20 . Chap ter 3 Before a ny qua ntitative estimation of the embryo factor -I~ in a test solution c ould be obt a i ned fr om t he assay method described in t he previ ous pages t he de gree of variability in t he growth of t he embryos had to be known. Personal vari a tions among different workers were tested in the followin g wa y. Ovules from a single cap- sule were mixed to gethe r in a sterile Petri dish. Ten ovules wer e t aken randoml y from the dish an d tr en sferred to v i a ls c on t a i ning 1 per cent de xtrose medi um by each of t hree pe rs on s. The enbr yos were eub se o_uently cult ured side by si de a t 25±1 ° C. However, all measurements of t he lengths of t h e embr :ro s we re ma.de by on e c:nd t te s ~1.,T1 e p erson . The res ult s ar e recorded in t at. le 6. Bec au se of t h e uns uit ab il ity of t h e 1 p er cent de xtr ose medi u2 f or t h e growth of small embry os , those whi ch s howed a n i niti a l devel opmen t did not c on tinue their grow th after re a ching a size of ab out 1.5 to 2 ti mes t he ir ori gina l le ng t h . However, 80 per ce nt of t he e rab r yo s isolated by person A s howed t he i niti a l growt h , a s c ompared to 10 to 20 pe r ce nt i n t he case of ~ ersons B. and C. Exam ina tion of t he cu lture vials af t er the t e sts sh owed t ha t co ns iderable quan tities of t he e mbr yo sac c on tent s we re transferre o. t e r m II e;nb ryo f a ctor II , as used i n t h is t hes is, includes a ll ·the unknown che mica l substances, -il(·.ich are ne cessary for the growt h cf s mall Da t ura embryos i n vitro. ,ff, mh L.e 21. along with the embryos by person A but not in the case of persons Band C. The presence of these substances in the 1 per· cent dextrose medium might have stimulated the development of the embryos, which ordinarily would not have gr>own in the 1 per cent dextrose medium. Table 6. Person A B Embryo no. C Embryo length in mm. 0 days 2 days 1 2 3 4 5 6 7 8 9 10 0.3 0.6 0.35 0.4 0.6 0.5 0.65 0.25 0.25 0.35 0.8 1.05 dead 0.65 0.7 0.9 1.0 dead dead 0.5 Av. 8 9 10 0.42 0.35 0.3 0.25 0.40 0.50 0.4 0.3 0.25 0.5 0.3 0.80 dead dead dead dead 0.65 dead 0.55 dead dead dead Av. 1 2 3 4 5 6 7 8 9 10 0.36 0.4 0.25 0.7 0.5 0.4 0.45 0.25 0.3 0.5 0.45 0.60 0.8 dead dead dead 0.6 dead dead dead dead 0.6 Av. 0.42 0.67 1 2 3 4 5 6 7 - Growth of embryos isolated by different persons. %via- Av. rel. growth bility (failures not counted) 80 1.7 20 1.5 30 1.6 22. However, pr eli rri inary experiments by van Overbee k (unpublish ed da.ta) di d no t sh ow any incre 2cse in the growth of embryos, when the endosperm W[~S tr a nsferred t ogether with the embryos fro m the ovules to the culture vials. Nevertheless, in order to avoid per son al variations, a ll subsequent tests wer e performed each by one and t h e same person. The following test was carried out to determine the effect of emb ryo size as a cause of variability. Ten em- bryos, which were about 0.5 and 1 mm . in length , respectivel y , were cultured in t he 1 per ce nt dextr os e medium cont a inin g different amoun t s of embryo factor. Measurements of the growth of the e mb ryos were t aken a fter 2 days of culture a t 25tl° C. The results a re sh own in t abl e 7 and p lotted in fi gure 4. Tab le 7. Gr o1,,;t h of embryos of differ e nt sizes. Per cent Incre a se Av.relative Rel a tive Dry wt. of I nitial embryo e mbryo viability c one . of in g rowt h (fa il length e mbr yo le ng th factor ures not ffi O' • in mm. in mm. c ounted) f a ctor _ in _ ___!2 0 .56 0 0 1 0 .59 2 1.18 6 v • 8 ...i.. 12 7.08 ~~ 0 1.lS r-: LI. 0 .50 0 .50 100 0.26 0.68 1. 5:2 1. 62 0 . 43 1. 92 l.90 70 1.02 100 1.12 0 . 51 0.20 0 . 46 1.4 100 40 1.10 0 .47 0 30 1.18 --- -0. ·J l -0. 06 100 0 . 49 --1.4 l.96 -From the da t a 9 resen te d it can be seen t ha t although .. ...., i + ~m:m++l C: II ~ . :::i 1 T lli. 24. the percen t age vi a bility a nd t he tb solute incre as e in embryo len gth were differen t fo r embryos of different sizes , t he relative .growths of the viab l e embr y os were the s ame in both c ases . From this point of v i ew , then, t he re l at ive in- crease (f inal / i niti a l length) in the len ~th of the viable e mb r yos wou!d s erve as a better index of t he amoun t of emb ryo f a ct or t han the other 2 criter i ~ . This index will be used in t he sub sequent p arts of this thesis. Two f a ctors , vlz ., pH of t he me di a and t he t emp erature i n wh ich the e~bryos wer e c~ltur ed , were foun d to be of gre a t importance in influenci ~~ the growth of the embryos. · These ~111 b e tre a t ed in det a il in Chap ter 4 . \li t h t he c ontr o l o f t µ e se VG.ri ou s factors a satisfact or y bio- a ss CLy f or t he e:".'lbr?b f c~ctor' 11,0.. s ob t a i ned . T:"le VG.ri- ation in t he tests w~s rel ~tively sma ll un d the results 1) . . T. -:e s e .) t.i:eeCi sc t s nents of t ~e growt h of t he e ~bryos were sad e ~ft er ab ou t 2 02- irc d s e ts ,:-;_ nd t ~eir differences a re listed in t able 8 . 25 . Va :c i 3.b ili t y in t he ;::.: rowt:-1 of To.ble 8 . embryos i n 2 p er ce n t s u crose me~i um . fiS"air 3/o vi a bility no • .,____,,,o_e_,_ t--,-A -se:E--.l:l-r-,;,::D-,i~f-:,:f-.--t Av. relative growtn" .o e t A be t 1:3 Di f •..,,f-.- - - t 1 50 50 0 2 .9 2 60 70 10 2.0 0 .2 3 80 60 20 2.3 o.o 4 90 80 10 5 80 70 10 6 70 80 10 7 80 70 10 8 90 80 10 9 50 70 10 60 11 3. 0 0.1 2 .4 0.3 2.6 2.8 0 .2 2.8 2 .7 0.1 3.4 0.3 2. 6 2.4 0 .2 20 2.0 2.3 0.3 60 0 3.1 2. 6 0 .5 70 70 0 2.3 2 .4 0 .1 12 60 40 20 2. 9 0 .4 13 80 100 so 2.8 2 .4 0.4 14 60 50 10 0 /..J • /__, 0 2 .4 0.2 15 50 50 0 2.2 2 .4 0. 2 16 40 40 0 2 .7 2.8 0 .1 17 80 40 40 S .4 2.6 0 .2 18 30 70 40 2 .0 2.2 0 .2 19 60 90 30 2.• 5 2. 6 0 ,1 20 70 80 10 0 2. 3 o.o t:..., . '7 u • • UereafteY' t ''~"' ·· V81"'• ~J'8 l ''C ~1 c, t , ,,re r,·r o', t 1 ()1 on.1 c; t'r .e vi able e~brvo s will be o~e seri t e( in tte t &b lee . Dead em-I~ J..L ~ - ' J• ..!.,;.:-, C. •. ~- ' · • - . • ,,_, '- • ·- . ~ ' 7 •• . "". . bryos wi l l \ 0t b a inclu~ed i~ t~e C£ lcu l a ti ons . n •, 26. Th e av erage difference b etwee n t h e rel a tive gr ow ths of t h e paired s e ts wa s 0 . 20 , which was on l y 7 . 7 per ce nt of t h e a v c r ege re l ~tiv e growt h 0f t he 4 0 s e ts . Howe v er , t h e rel a tiv e growt h v a lues am on g t ~e ~~d! v idua l sets differed hav e bee n du e in par t t o t b e fact t ha t t h e p H and t h e time af me c s ure men t were no t ide n tic a l i n a ll c ases . F~rt he r- i,1 ore , t h e ~es) ~1nse of enbr yos fr oli1 01ffere:1 t capsu le s , which had. be en: '. ·,i c ked fr om )J. c1.:1 t s ~:roi.,m un c e:t' un c on trolle d coi1- diti an s in gre e nh ous es, mi sh t h ove v aried wit ~ t h e cap s u le s use c"i . T:.--i i s v ,._rlabi lity cci.:, t e seen from tect le 9 , wh ic 1,. p:ce- sents da t a t a ken av er a period : f 2 ~ontis . Tc.\b le ·J . r i c\f.,, i J_i t :? .j_r. t~-~e :·. rc 'T,._: t ~.1 o f \T ~•1.. 1 e:·t1br':,,.os fi,,orn f i fferen t CR9sules . i\:v . l'")e l c.~. t i ,_r c 1.r1c1·G ~1. f_- c ·i r1 1 eri :· t t . t _-;~ t e i.., 2 CL\.~_y·s 1 1 - - - - - - · - - - ~ - - . : . . : ;..,;;,.;:.._;:..;:_;_.._:.__.;__ _ ,_;__;,_,_--.-_.:..;.,::___;___ _ __;,,:_.;,._--1 ?);, s u.c:r·os e z;·v S'.J C:e:) SG :·:'! e 0. i u.n + .:) c.'!r op s cocon u t T ll t 1.:,e r v i c1. l l -1 . 5 3 5.5 Dl f :r ere Y-'. c e 0,..._ , • Iry S .7 B l. S 8 2. 0 ;j 1.5 10 2.5 4.4 ________ _ _____....,____________ _ ,._ Aver .::i.z_:e Z7. Th e fi gures in t h e l ae t c ol~mn of t a ble 9 give a me a s ure of t h e am0un t of e mbryo f a ct or a ctiv ity in cocon ut milk wh e n tested on a 2 µ er c en t su c rose medium . Th e v a l ue s f or t h e same s c ~ple of co c onu t ~i l k , as t e sted on diff ereD t day s , r anged from 1 . 4 t o 2 . 7 . Bec ause of t h is v~i_ riab il i ty in the re spon se of e:r:1bryos f r om different ca9 s u les, a st andard act i ve soluti on , usua l l y 3 dr op s of c oc onut Qilk )e r vi a l of culture medi um , wa s in c luded in e a c h te s t . Also included in ea ch test were dup lic a te c on tr ol s of 10 e mb r yo s e a ch . I n t h is way , a s a tisf a ctory quan tit a - t i ve v a l ue f or t he am oun ts of emb ryo f a ct or i n a given test s ol uti on co uld b e ob t a ined . An i de a as to t he qwrn ti t a tive v a ri a t i on s wl.2. ich mi gh t be exp ec t ed fr o,n -=~ t y,) i c a l e::1b r yc f s. ct or b i o- assay c ou:!..d be obt a ined from t h e fi g ur es i n t abl e 10 . I n t h is t es t a 2 p er cent s ucr ose ille di wa , wh ic h was buff e red a t p H 6 . 7 wit h 0 . 01 rao l o,r ,n rmo s od 5 '.J.m- di µ ot a s s i um 0:: oso ha te , vms us ed an d t he e mb r yo s we re c ul t ured for 3 days a t 3 2t 0 . 5° C . Teble 10 . S et Re l ':' t iv e c n:ic . cf Gr owt t ~f e ~br yos suop li ed ~ it h dif ~e r en t c 0nce ntr a t ions of e~bryo f a ct or . 1 i! 0 ■ of .. cTrieff ic i ...:Av . re l ei. ti ve e n t of 1h 1 ri e..b il~rov, t h ±st d . error ity e::1-~}::c :\-o f c ctor) e :-;;l;r~:l o s A 0 36 55 0 . 45 4 . 0±0 . 2.4 6 . 0% B 1 10 70 0.2 6 4 . 4± 0 . 25 5 . 7'Jc C () r✓ 10 70 0 . 44: 4 . 9±0 . 40 ,, r, (f' b ■ G;c' D 3 10 60 0 . 47 5 . 2±0 . 35 6 . 7% E 5 10 60 0 . 50 5 . 1±0 . 31 6 . 1 ~10 V 7_--'-• n ·~_ (. ti l i t :' 1 ,.... Av . F e: ·i ni tj_,i,:L ce nt l e11 ~;: t 1:-1 j_ ( ! rn r;1 . 23. The rel o,ti vely low degre e of v n,riation i n the b i oas s ay , Fhich was perf ormed unde:r c on trolled c onc1.i t i on s, is s h own by t h e coefficients of var iability , which r a nged from 5 . 7 t o 8 .2 p er ce nt . of varisbility. Ot her tests s h owed si~il~r magn itµ des Wit~ a kn owled~e of t h ese st a ti s tics f or a pair af un correl a ted me a ns one c ould ca lcula te t he standar d ~ error 0f t c-rn c"J.iffe}:-ence of t he r:1 eR.ns , usi ng the f orm ula : 2 Tt e ab so ~u t e di ff erence w~ ic h was necess ary f or s igni fi ca nce bet~een t he me ans c ou ld t~en be ob t a ined, u si ng t h e equa ti on : Differe nc e = 11 t 11 va lue x S. E. diff . 11 Acc ordi nF: : to Fis he r ' s t 11 t 1-)_b le, t he 11 t 11 v a_lue for p :: 0.05 is 2 . 09 an d f or p = 0 . 01 is 2 .54 for 1 9 de grees of freedo m. Thr ough the use ~f these e ou ~tions the differences necess~y f or si gn ific an ce between the aver a ge r e l a tive growth of the emb ryos cultured in t he presence an d in t h e abs ence of embryo f2,ctor were c omputed an d lis t ed in t ab le 11. Tci,b le 1 1 . Di ff er e nces ne c essc•_r y f or Si (:n ific a n ce 'c etwee n t he av er ~~e re l a tiv e browths of pairs of □ e a n s wit h diff er en t st on dur d er ro rs . s . E . 1 s. E.0 (._. Diff ere r. c ,::;c ,-.ece ss .0 _::.~? - 0 . 05 0 - ~ ·~ l,, 0 s i re:nif i CE,.nce = 0 . 01 -- 0 . 2. 0.2 0.6 0.7 0. 2 0.3 0.7 0.9 0.2 0.4 0.9 1.1 1--- - - -- ·- · -- · -- 29 . From the statistic a l da t a p resented in t a ble 11, it see ms t ta t 0.9 ~ay be c on side red a si gnificant diff e r en ce be t ·ween t h e relo.tive 6rov/cns of 2 sets of embryos c ul-ture d for 3 da ys in different n edi a . Th e t hr es ho ld difference f or si gnificance between 2 me an s will necessarily dep end upon t l".e len gth of ti ,~ e of cult ure. Tl1e :·1e cu1S 1vi tl"J t h eir re sp ective s t a ndard er~ors f or Sets A and D of t ab le 10 have b een c a lcul a te d f or growt h a f t er 1, 2 , 3, an d 4 da ys and are given in t ab le 12. Also incl ud ed i n t he tab le a re t he differ en ces necessary f or si cn ificance between set A a nd set D a ft er differe ~t len g t h s of ti me of c ulture. The me a ns f or set A and set D wit h the resp e ctive differences neces sar y f or si gn ific an ce between the t wo at t h e p = 0 . 05 level a re plo tt ed i n fi gure 5. From the gr aph s one c a n re a dily observe t h e signi fic 2n t di ff s rences i n grow t h b et we e n the e mb r y os suppl i ed wit h embryo f ~ctor and t h os e no t su)p lied ~1 t h e mbr yo f a c tor. T~e di ffer 2n c e c&n be noted ns e arly a s af t er 2 days of c u lt ure. Th is crit eri on of si g- nific c.nce, r, s expressed. in t 1-"1 e l e ~;t 2 rows of t s_bl e 18 , -vril1 b e followe d t hr cushou t t h e subse q~en t parts of t ~is t hesis . at Av. relative growth a fter C '1 ..c. . 0 D: Knbryo f.s.ctor 0.51 0 . 29 0 .18 5 .1 0 . 38 3-~ 0 . 03 5 . 0 e.. o 5. 2 0.70 0 .5 2 0.35 6 . 7 0 . 24 o. os 3 . 3 4.0 var. var. 0 . 22 2.7 0 . 07 4 . 4 var. va r. 1" 0 c,.42 6 . 8 0.95 0 .70 ·--·-· .0 ,__, . 4 . 8- 0 . 25 5 . 2 ------- l day 2 days 3 day s 4 days Av. S.E. Coef. Av. s. E-. Coef. Av. s.E. Coef. Av. S.E. Coef. 1. 6 P=0.05 Di ff . ne cess. for si gnif. P=0.01 n1.1mJero/ Va ri a tia~ i n e Llbr yo growth a fter diff e r ent v days of culture. A: Control Set Tab le 12. 0 ~ . 31 . . t-~t i ½ t -+- t ·t---+-'-• -+ t I +• .,I.. ·: .t -+• + ·I t: :+ ?,· .:::: :t-t-t .. f ·j-+- 4-,.. dI I-+ +· 1-i '1 "tr :+ + -tr +-t +L H- ,. ~ ··- t +· I ·+-i a ·++ •+ ---h- .. .. } :r.;: 1 -H f+ .L. .l ~! r + + tt + ... :~t -~ ;- + ti -t L+ -L • 't t + H- H- --;:: .:r ·+ + H- 1 + -+- .,. --i--f ·- ·•• - t:• _; it tt: , ,_-+ + ..:! .. :t:: .! ·+ ~ - . •+· -1-+ - -+-+ --n:± : • f ·-+ -+-+ TT +--1; j.:: f ...... C ..... L• + T f i --t-r + fl I t-r ·t- I ~t r~ .+-~ : j l,. ;'. t1 ::.:: / + .I- + + £ ~ ....t .! + H t ti .ti .F .; ;-- .. ti: +: + H + t -~ :1 ·•· t I EE .... H- t:t- 1+ :-t ... .. + -+-~ .... ~ t ·t :;: • 1+ .t ti M -~ ·:--' -:;: r:t.::I I-+ +.-:-- :rt t--+- ---t ·- .,- I+ :!:) + ·• ' ~1 H 1 --·t- £" ~... I+ H- t+t1:ttttt1:tttEi 32. a) Tem oera ture. E~bryos, which were 0.25 to 0.65 ~~. i n l~ngth, were grown in a 2 per cent ~cx~r os e ~ edl~m (2 per cent dextrose table 1) with and without t he ~\ddition of e~bryo factor. The growth st a tus of the embryos grown at different tern9 er a. t ur e s f or d j_ff ere ti t 1 e :1f ; t i-_s ') f ti -,~e are shaw n in t a.b 1 e 13 Qnd plotted in fisure 6. Table te r:11.>cre~tu1 e s . 1 -----=--,,--,,...,....--y---.,,--•---..-·-·--- - - - - - - - - - - - - - - - - · - - - Rel a tive Per cone. of cent .Av. relative gr oHth enbr yo viaf ecto r bility 23 hr. 42 hr. 91 hr.- 117 J-..--.•-~l---------,1-----_;;_,-+-----~· ------- 2st1°c 0 40 1 60 1.4 1. 9 2.7 1.3 1.4 1.8 2.6 co 1.2. 1.4 0 70 1.3 1.8 1 80 1.4 1.6 70 0 60 1 S.1 1.6 2.3 2.0 3.5 3.0 2 90 2.0 0 40 1.6 1 70 n 70 (...., l.fi n {_,. 1 -1.. 2.8 3.9 4. 8 4.0 7. 6 12.5 10.2 . 0 ~ 7.3 5 .7 9.7 ,1 --...: 5.1 H" . ., -, + ti: + . ·+ .... t ' ·~ ++ ~ ~ !: J :.! \ • ~ -t- [F.:;:; -'- I-' _c ,.; -ti H" ::.+ -~ H" . w T ,, r-t-+- ~-•N ~ .,. .~ M 1, .... ~ '-H~-l-H 1 ;: : H ,-i tT++-t+ttt-rt l . HlfHFt - -i. 1 I i ·;-+ l y with in c re a s i ng t e m; e r ~t uree t imum ~~c re n ched. u) t o 32° C. , whe re an 00- At 17 ° C. t he gr owt h w&s e ~~all~ ~oor i n bo t h t t e µ rese n ce and t he ab sence Df embryo f a ctor . op ti ma l embryo jrow t h . A I t mus t be pointed o~t, ~owever , t :1c. t t he emb ryo s used i n t r. i s t e st Ceve l oped i n t ;-ie dextrose medi:.En r,. l one . Tc·.i s is E>.n e xcef ti on t o t >e u s ua l f ailure of e~br~os t o ~r o~ in t he dextrose c on trol ~ed i urn a nd will be tre a ted i n more det a il in t h e Discussi on se cti on of t Lis t he sis. b) £1:i of me dium. Preli mi nar y t es t s on t he eff e c t ,::,f t h e di ur;i ) H of th e i:1e- on t he t YOt,th of _"Qa t ur§: e :nb170s i n vitr o were cono.uc t e,:;_. Dur ing: t he e ::,rly e:>:v eri men ts t }.10 f ollowi nr:; b '.J_f fe rs were u.sec1 in 0.01 mo l a r c once n t ra ti on s: _ _ u.-,, c1·trnt . ___ e. buf~ier _ far p H 3 to 4, an d ·1 ) ~oa"i 2) mon obasic- di ba sic po t a ssium phosph c\.te buffer for pH 5 to 8 . In the cultures buffared with the above mixtures only a few of the e mbryo s a.e ve lop ed. Tll.e gr ow t h was ver y ·uns;.c tis- f a c t,ory even in t h e prese n ce of embryo f a ctor. It wa s found l ater , as shown by t h e fi gures of table 14, tha t a 0.01 molar dipot a ssiura- non osodi um phospha te b uffer did no t affect the growth of embryos adversely . 35. T.::;_ble 1 4 . .E ffec t of I~HP0 4 -NaH2P0 4 buffer on the growth of Da t ~ e mbryos . I!I ediur.i. pH No . of embryos 'Pe r · c ent v:l a bility ' 5.4 15 60 2. 4 15 40 2 .4 Av . re lat i ~ g r owth .s_f ter 2. days -- s u crose --t buffer - buffer ,, sucrose + 3 drops of c o conut milk oer v ial 2 (/o + buffer 5.7 15 67 4.8 - b u ffer 5 .5 15 60 4.3 Thr ough t~e use of t h is buffer t~e followi ng series o f tests on the eff ect of pH on embrro grov1th was con ducted. Embry o s ab ou t 0 . 2 to 0 . 6 mm. in length were grown in a ~-?. per· cent S 1J.crose in g from 5 t o 9 . rn edi un wh i ch wa s b uff e red a t p H's rang- T:1e da t a v1 ere c om9 l l ed fr om e xperimen t s exte nd in g over a p eriod of seve r al mo~ths . The ~H val~es g iven i n t h e t r b le re p re s ent t h e p H 1 s of t h e ~~d ia a t t h e ti:ne of t h e f in o. l ,:1 e ~_s "J.re men ts, wh ic h v s_ried bet vveen 36 t o 48 hours of cult ure a t 32t 0 . 5 ° C. The pe rcent a ~e vi a bility vel o)ed a t t h e v::..,_rl ous 9 H'' s , are presented in t 2_b le 15 a nd fi g ur e 7. "7 C 00. Tab le 15. Effect of pH on t he growt h of eobryos in 2 per cent sucros e ~edi urn . inTti al leng th of e mbr •ro s i n !7C1 • ::...:L.:::..::,- Per cent vi a b ilit ,r 421123 0 . 31 0 421 207 0 .41 40 421208 0 , 31 0 421208 0 . 34 0 4211 2, 3 0 . 38 0 4 2 1207 0 . 44 30 5. 2 421103 0 . 37 0 5. 3 421119 0 . 33 40 1. 5 421123 0 .39 20 1. 6 421130 0 . 4 2, 20 5. 6 421116 o. 30 40 1. 9 5. 7 42 1 22 3 0 . 23 60 2.0 5. 8 4~:11 19 0 . 36 1 00 1. 9 5.9 421 203 0 . 3 :3 60 2.2 42 1 ::~03 0 . 35 70 6. 0 430112 0 . 24 60 6 .1 421130 0 . 37 60 2 .6 6. 4 421 207 0 .40 70 2 .3 6. 5 421116 0 . 28 60 6. 6 421212 0 o L1'J 80 421212 0 . 40 80 ,421 2 1~2 0 . 34 100 42 1217 0 .33 90 Av. pH ~-- - Tes t no . . 5. 0 5 .1 (C on ti nued on nex t ~;,!..- '"8 e"=-'' 8) .:....I -~ Av. relativ e g rmvth --- . ..., 1 c, 2. 4 2.4 2 .5 37 . Table 15 . pH Tes t no. (Co ntinuet fron p revious page) . Av . initial len o· th of e mbryos i n mrr. . 0 - ---- Per cen t Av . re l at i ve vi ag rowth bil i ty ,_ ___ , ~ . . -- - __ 6. 6 42 12 17 0 . 33 70 2 .7 6.7 42 1126 0 . 35 60 2.2 421210 0 . 35 90 2.3 42 1 210 '7 I; 0 • vv 50 3. 0 421210 0 . 44 60 2. 4 421210 0 . 43 70 2.6 430104 0 . 47 70 2.4 421 130 0 . 38 80 2.6 421 2.03 0 . 34 40 2.8 421203 0 . 33 80 3.2 42 1 210 0 . 37 50 2.9 42123 1 0 , 32 90 3.4 42123 1 0 . 38 90 3.1 421207 0 . 31 50 3.0 430 106 · 0 . 54 60 2.0 4301 19 0 .51 70 2.3 430119 0 . 26 50 2.3 421 215 0 . 34 60 2. 6 430 111 0 . 46 50 2. 3 430113 0 . 36 90 2.8 430 118 0 . 43 50 2.4 7.2 421130 0 . 34 60 2. 5 7 .3 42 1~~ 10 0 . 38 60 2.4· 8. 3 4212 15 0 . 32 20 2.2 3. 9 421110 0 . 27 0 --- 6.8 6. 9 7.0 -- 58 . Tn"'--M""T"M"I ~ ft ~ ,_. ttif -t- ..... + ,,... ..... ... ' f :t ,t H- + .j... + + -· + ! J ,-+ I-++,--, -t '1H+i---++++++<t.......+++->-++4 i-l + +-+ .; + ' H-+<-+++++H -+ -~ 1 + 3 3. Fr o~ t ~e da t a , it is ap ~e ren t t ha t t he highest p erce n t a ~e vi ab ilit y Hn d t h e f as te st r a t e of ~~ owt h of Da tura embr yo s aurin g t he first 2 day s of cul t ure i n vitro i n t he 2 p er cent sucrose medium oc cu~: r'ed bet 11.i een p H 6 to 7. • Wt.e t h- er o~ not the s ame optimum app lied to l a rger emb~yos an d whe t he r it ap) l ied t o emb ryos grown i n the p res en ce of embryo fac t or was de t er min ed b y the f o: l owin g t es t. abou t O. ~~ n" ~ l O mr1 \..-'V <.,.;l..L l v~ • !. ! o • i " l e· n~th . l l .. ,;._ , 0 E6bryo s r e sp ectively , we re grown in a 2 pe r ce n t s~crose 7 edium, which was buffered a t 2 pH ' s, with a nd wi t hou t the a dd iti on of e~bryo f a ctor. The fin a l· me a sureme nt s we re ~ade a ft er 2 day s and are given in t ab le 16. Table 16 . oH -· 5. G 6. 5 - 5.7 6.7 Growth of embr yo s of di ffer Pn t sizes a t 2 pH's. Aver uge l ength of e:nb ry os in m~,1. ··- - Rel D.tiv e c one . of e,:.br:.ro f D. ct or - -·~----·- ,,. Per ce nt - v ~ Ct- - l~ il i t·v __ ,. Av . reL._tiv e '. r ow t}~_ -' 0 . 35 0 · 30 0 . 36 1 40 3. 2 0 . 39 .:; u 50 4.1 0 . 36 0 50 3 .1 0 . 35 1 50 3.5 0 . 35 5 70 4. 6 1. 2 0 70 1. 9 1.1 1 70 2. 0 1.0 5 70 . ,::, • 0 0 . 92 0 100 2.8 1. 1 1 100 2.9 Q. 93 5 40 3.4 2. 6 n ,, 40 . The da t a cle a rl y s how t ha t t he l a r ge ; a s well as t he s ma lle~ embry os resp onded to p H differences in t h e sam e way . Bot h gr o~p s of e~bryo s greK be tt ef a t p H 6 . 5 t han 5 . 6 . Fur- th er more , t h e sam e p H respo ns e existed i n t ~e pr es e nce an d in t he ubsc:;n ce of embr yo f :?cct or. All of t h e previ ou s t e sts on th e effec t of pH on embr yo gr owt ~ were t~rmina ted a ft er 2 da ys . Th e auest i6n as t o t he ef f ect o f t h e p H of t he :1e di um ·on tl-:.e c onti.n·t1co. fc:r·ov,t h of t he .enbr :;ro s wa s i nve s ti fsD. t e d in t he f oll owin g expe:::, i ment. Emb r yo s , which wer e 0 . 25 t o .J . 5 m!:-: . in l eni;: th , wer e ~rov,-n i n t he 2 p er c en t suc r ose ::i e cUum with D.nd wi t "'.1 out t he r; dc\i ti on of 3 dr ops of c oc on~ t Lli l k per vi a l . we re cul t ur ed ~t 3 2 t 0 . 5° C. Tab l e 17. ..., Medi um pH f--;:,;-;;r- 27Q s ucro se + 0 a.r ap s of coc onu t ;ri i l l~ • 7 p e:e V l8-.~ <",)01 ,-.J 1'-' SU- + 3 drop s of c oc om,1. t milk p er vi a l C:".' 0 88 T~e ~e dium wa s buff er ed ~ e a s ur eme~ ts of t he l e ng t h of G·Y-ov;t h of e2br?0 s D. t dif f er en t P er ce n t vi a bility )H 1 s . Av. reL: Lti ve gr owth l d~CJ. . 2 d.G, . 3 d D, . 4 da . 5 clD, . Av. wet in !1 ~-; . :::Ste r 11 O" "> VS v: t. ~- 5.1 33 1. 4 2.0 3. 0 3 .7 4.3 2. 0 6 ~9 73 2 .1 2. 9 Z; . 6 •L l 5. 5 0 .7 7.5 67 2. 0 2. 5 3. 4 4 .4 5 .2 0.5 r: 0 73 2.0 3.8 6. 8 9. 8 1 6 .l 13 . 0 6. 9 81 2. 7 5.0 7 .1 8 .3 9 .1 1.7 7.5 S-4 2 .7 4 ,7 7.5 8 .4 9.8 2.0 ;._j . r 41 -I+ ++ _..., H e+ --f • I!" -l .I +·· !! = J~ -'-4 ➔ :i: -i- +!--1--1- I± ti.pt tt ~:j I+: ij !it ·t-• t H -1 r' ++ + UilJ.il.Ul"1" • j.; 1 .................,........... ..l+ I-+ 42. As in the case of t he p revi ou s t ~s t s , the optimum pH duri:':t_; '::1-:.e f i:?'."'~t 2 days of growth was a r ound p H 6. 9. How- ever, af ter 4 days of growth the pH optimum changed and t he embryos 11:hich ori g inally grew slowly a t pH 5. 5 fin e lly ou tgrew t he embryos cultured a t the more alka li ne pH 1 de spite t he 1: : etter sk., rt of t he l a ttet. T:te po or growth of t he em- bryos c,t pH 5.1 wa s expected fro r1 t he resul ts of e arli er e:~eriments shown i n fi gure 7. The po ssibility t ha t the decreased growt h a t the hi gh& pH was due t o t~e Giffere nce i n the r a ti o between t he sodium and the p otassium ions, which were introduce d with t h e buffer, was elimin a t ed by t he res ults of t':rn foll'.)-win g experi~ent. Embryos 0 . 2 t o 0 .4 mm . in l ength were cult ured in 2 per cent sucrose media, which were bu ffered a t p H 5 and 7 with the soluti on s listed in t G,1)le 1 8 i n J.01 !Ilol ar con- centr a ti ons. It is to b e noted t ~& t 2 buf fers were used for e a ch pH; on e had a hi gh sodiuffi : po t a ssiu~ ra t io , 0hil e t he ot~er h~C c low sodi~m : po t a ssium r a ti o. c oc onu t ~ilk wa s ~dde6 cult ured ~t 3 2fG.5 ° C. 3 drop s of i.-.nd. t \e T~e results ar e s hown i n t ab ie 1 3 It c on be c ,:H~ c1ude d , t ,-:. er ef ore, t !.'"l8,t t >e p H of t h e □ ediu ra 2nd e r::b r yo s. no t t ~e ionic rati o be t wee n s odiun ~ ~d ~o t ~ss i tm T:1e r/:i.!Jtd i ni tie.l r u te of 1;:r o1r:th 2,t pH 6. 3 v:a s f oI.loi.,e d by a sl cvvi ng dovm , wl:i.lle tLe sl ov,;- i::11 tL:::;l grov:t h a t pH wa s f ollo~ed by a s ub se c\uen t This •w&s observed whe th e r t h e ~ed i u□ wa s buff er ed ~1th a solutlo~ with ~ low or one ~1th a h i gh sodium: pot a s s l ~m r a tio . Tab le 18 . Effect of different buffer soluti ons on t h e growt h of Da t ~ru e mbryos . Buf- Cor1:J osi tion fer No . of pH embryos Per c en t Av . rel a tive grov,th viability 2 dD~ · 3 cJ.c.1 . 5 de~. 7 da . A 3 c c . I\TN HP O~ 5 . 5 50cc . h'l KH2P04 13 32 2 .5 4.0 8.8 15.2 B 10cc . IiiK2HP04 6 . 9 3c c. I!'.N D.H2P0 4 13 77 4.4 6.2 7.6 9.0 C 10cc . MNa2HP04 6 . 7 0 . 6cc . MIC-.,HP0 t.., 4 13 62 2. 3 3. 7 7.4 13.5 D 50cc. L'.Na 2 HP0 4 6 . 8 12 cc . LdCH 2P O4 15 60 4.4 5.8 -7 . 2 8 .1 OJ(..) ~ "]: - -... :I -t-l .... ++ '1 ir J I-.\. a....] -, H :? :' ,'I-+ -4 1/- f ~ ,-:-:tr:i r- ... '-'- + 1T •'-4 LllJ..J 1-t+ 7::i H - TI -z:1 1n M I w. l:t:t i,.: n :: . ' :!~ r:: t-t-tt-t-t+,--H-H+t_;+I + I I- --" I-+ I-'- -1--! ' -j- :±l:l:1rt-Ht:t:::t'.t±--'-:±lli-'t±lli ft + -+ --f t ,+r -tL....... L [J_J It -t l H •• p -±5 . Cr:apter 5 Effect of chemic a l subst an ces on _gr oi·Jt h '.J f D::it .'.r ,:i er/b r •.ro s 1 1 Duri n6 t h e c ourse of t hi s ~nv e s t ic0 ti 07 t ½e effect of diffe ren t s ubs t &nces on the s rowt h 0f e ~bry os was teste~ Of t h e s e, on l y s ucrose Rnd ~uxin were f oun d t b be b eneficia ~ e s \•; il l ·c e disc ussed in ch ap t er 7 . Other s ubst 2nces , wh ich are listed in table 19 , showed very l ittle or no e ffects i n elicitir. g f a v or ab le gr owt ~ resp onse s i n the e mbr yos cult .1rcd in vitr o . 1 Ts.ble 1 9 . Subst a nces in~ctive in 0ron otin g t h~ growth of Da tura embry os in vi~3:.2 · _______________...._ u_, onc . oer cu lt ure vi· a _ ---,'""'" um S·, ·1.bs_,.t-a-.,.-:~-_c-e,tt,-·---.--...,..,,.,. Medi l, . KCl - C . P. - C.P. Ho 2.;i; L .nd I s :1 i nor ele ..1e r. ts 1 0& 0 ,J dext!'ose O . OOl~vi t o O . 03LI 2~~-~ de:;:t:-eose 0 . OOll.i: t o O. 031.: 2c,; sucrose 0 . 1 to l OOx ori ginal C) (J1 0 . 0 1 to 0 . l ~'o I ,_. i ~' • S v. CI'OS8 «1 .1 0 'ia C.ext:t o se Lk :. l tose o c1_ ; •,./ c.1 -L u.ct ose 0-~ dext r ose dextrose I .'1 0 ~Q de x trose l1ie 111 bi ose Oc'. ,- dextr ose Xy lose 05~ cJ~e:(tr' ose c1- .itr c.bi no se O~:;A dextr ose LJxose . 0 (,1 , :J 6.ext:?.~ose Dextrose -t l- rham nose + a my~::dnlin (C ont in ued on ne xt page) 0 . 2'7'1 of ec.c h 46 . Tab le 1 9 . (C on t i n~ed from p r evious page ). Subst a nce Med i uu d- :1Icrn noce + d- 2.r Etb in ose + arb ut in .._, lo Mal t os e+ 1- sorbose 1- sal icj_ n G '1 0 r;; d s v.cro s e 0. 27~{, of e a ch n ---1 s u cr o se 0 . .0r7,<l . . , , o of d- Gl uc os am in e + d- g~ l ~c t ose + esc u l ine 2% s u c r o s e d-La c t ose + d-fructose + -·..n~ '_n_lor 1' u"z1" n °~ r ce ,~, o s t·c~ ,. ,J._; d- iir abin ose + c(.- :1 et h.y l g l uc osi de + o<.- me t hy l• ;:, ma nnos11.,, _e 0 :fl ,-.., , o ~ Co n c. oe r c ultur e v i a l ea ch O. 27% of e c1.ch .1. Phy tic e.c i d s u crose. a.ex L ,rose • ~ .,I_. 0 . 0 00 1 t o 0 . 05M tr:i ;:t ure o f: · a.1- rae t h i on i ne g lut a;n i c c'.c id l - ,9_s oart ic 2-c i d 1- (- )- le uc i !-ie c.-v a l ine c.U- serine 1-(-)- oroli ne cJ.1- l y s in e 0 . 0000 3 to 0 . 2 5o/o of e a c h .1- h i st i cJ j_ r1 e Ci s t i ne d- or nit h i ne f- 1::.lan i ne dl- c y ste i n e ,a.1- i so l euc i ne dl- nor leuc i ri. e dl- p:-,e ny Ls. L:::..n i ne 1~(-)- hydro xypro li ne X2.nt h ine 0 cfl Hy,o oxD. nt h in e n .1 G /a -I~ , -...J,• s ucros e 0 . 0 05 t o 0 . 05 rng:. s ucr ose 0 . 001 t o 0 . 01 rn(; . Th e an t r.or i. s L1te b tc c3 t o Dr . Cr:rl l ~ie r:w nn f or t c·ie s ug &rs 8nd J ly c osi de s , vh ic h i: ar e use d in t h i s se r ie s of BX!J eri rr: ents . 1 47. Embry o factor in natural extracts A c a sua l i nv esti ga t io n wa s c onduct e d on t h e promotin g effects of various natur a l ~xtracts on the growth of Da_t~r s. embryos 1!2 ,Yl_tro. table 20. The active extracts are listed in They were obt a ined in the followin g way: 1) Coconut □ eat extract: 30 g . of fresh coconu t meat was ground in a Warin g blendor a nd s haken mechanically with 400 cc. of 50 per cent eth yl al coho l for 2 days . to l extra ct wc~s ev 3_p orc;, ted to dry nec:s at vacuo ------wG.ter. ~;~:-i cJ~ r r-, OU Th e alco- in to 60° C 0' ) wets tc.ken UiJ in 125 cc. of tl'Je residue (1. 0 o• 0;:-1e drop of t 11e filtrate was plo.ced in eac h culture vi a l for the te s t. 2) Datura ovule extract: 16 g. of fresh Datura ovules fro m 9 capsules about 14 days ~ft e r pollination wa s macerate d in a mo rt a r with 100 cc. of water a nd left overnight in t h e refri ge r ~tor. The filtr a t e was concentr a ted a t 50 to 60° C. in vacuo to 1/4 of its original vol u~ e. One c1r op of t h is concentrate wa s p l ace d in ea ch cu lture vial. 3) D£_t~ ov1-,tle wo,ter di ffus a. te: 8. 8 g . of fresh D:, t urc1: ovules D.tout 14 dny s '- '. :fter ool lin a tion wc~s placeo. i !·i o, f l e E~-;:. ~,. nd cover e d 1:i th 56 cc. of VJ E:.ter and \ e) t i n t he -for ref::.,i ve r e.tor~=tbout 4 de.ys. 306 cc. cohol WP.S 8.dc3 ed to ti~e c3.iff JE: . te 1 c.'.. of 95 cc. of ethyl a l- nd the mixture filtered. The filtr a te wa s ev29orated t o dryness at 50 to 60° C. in vacuo 2,nd tr..e residue t aken up in :?2 cc. of water. 5 drops of t h is solution was added to e a ch vi a l for t h e test. 48. 4) Datura ovule alcohol diffusate: 37 g. of fresh Datura ovules was 9laced in a flask and·cov2red with 125 cc. The filtrate-was evn)or a ted to Gryness a t 5~ to 60° C in vacuo and t h e res idue t '~ce n F) in 37 cc. of 1 D., w2ter. T~e solution was shaken 3 ti me2 ~1th 100 cc. par- ti on s of )urified dj_ethyl ether.· The ~c ter fracti on was tte resi- 30 g . unc1 C'.)nv erE'e Cc , •J f Qf c erm ( Horto11 Los An ge les) w,.,. s soa':- ed in 10 0 cc.· of distilled water for 4 days. 55J cc. of 95 p er cent ethyl alcohol was add e d to t h e filtrate, re sul tin ~ in the for~ mation of a 9re ci ~ita te. T~e )re cipit 2te was dried in v2cuo . a nd t he dried residue t aken uo in 40 cc. of wo ter. 5 dro~ of ttis s olution was 2d6ed to each cult ur e vial. Penic k and Co. of Ne w Yor\ City) was soaked in 200 cc. of distilled ~~ ter a nd left in t~e refri ge rstor for 2 J 2ys. One (ro) ~r tte filtr a te was added t~ e2cb test vial. 7) Yeast e xtract: sus p ended in t: . ,cc_:-.1.·e Dtfco ye:1, st ext i_-'r.ct powder w;:, s a liqu ot a f:l.n :::, l c u:we!1tL,tio.n :,f ye~ •. st extract in tte medium All of the above extracts s~owed ac tivity i~ ; ~o~ot- ing t ~e growt~ of D&t ur a e~bryos L1 vitr o . Little 01° no ~J t ~toes, l enon pulp , c orn me a l, le mon Jee l, urine , a nd c a s ei:1 hydro l :r sate. :.;e t :t.o d I ·i,c:; t e r a lc oho l tlff u s a te :..... ):) 2% wa.ter extra ct wuer eJ~tra ct Alr:wnd Ye e.st extr a ct ,~0 q,,o· s ucrose s·0. crose dextr ose ext r~. ct meD. l l r:',o \iD.tGl1 sucrose 0 :11 G/0 b,ext rose l~ ,,,·I ts. e j:t !.'·o s e l '-I'.~ s ·J.crose 0:1 Whe o. t g erni I Da t u r B. ov·c.les ovules water diffus e. te Da t ur a ,1).. U .J.....,.,., 9,-.,...,., ~ c+- .·, \ r ·L,L,l~c v ~ t::: U U 8xL1. 2·. 1..,t Dat ura ,,,eclt I ·:·:~~e~ Co: o;"J. t D2..ys 2 r, C:., r, t., 2 2 5 7- u - ·- 0 . 33 0 . 44 0 . 34 0 . 35 0 . 34 0 . 47 0 . 26 ':)0 0 . 22 0 . 38 0 . 43 ~ V e s-.JV 0 . 43 0 . 50 o. '.:'.6 __@ _ '2:'.1 t~:~.~[~_x tr c.'<. C -~ mm. in 8 !71.bryo l eng th of -1:v. ini ti D.l c,-.-o 80 0 70 0 10 70 ron Ov 50 70 50 90 80 70 l C0!1 t ro 11 E xtr P ~r ce n t vi a b ility 8, C I 3. 0 2. 4 3.4 1.5 2. 3 4.0 3.4 L S 5. 3 1. 7 4.0 4.0 t :c on tr O 1 1Ex!r 8. c} Av. reL~. ti ve growt h E~br yo grow t~ stimu l a ti on by natura l extracts . of c u lt u.re - f --- - t - - -•- Tes tin g Sc)Urce of med i um e xtr ac ti on - - -~----, Tab le 20 . 0 Ul . Cho.p ter 7 Chemica l studies on embry o f a ctor i n c oc onut Ti1ilk ---a ) Preli mina.ry frD.C ti on9_ti on of c oc onu t -- - ;i-: i l.k. Th e me di um us ed in t h e t es t ing f or emb r yo f nct or acti v it y in the fr a cti on s ob t a ined durin g t ~e s e 9relimina r y fr ~cti on a ti ons wa s the l per cent de xtr ose med i um, given i n t able 1 C 11 \A lt1'-' r e c•i t ?,5°+_ 1° C. ~ AS , c}lOWD o. t·ne_ a· a t a l• D t 8.0' 1 e 21 , t·.t18 'OJ embry o f nctor, as tested on l p er ce nt dextrose me di um, wa s bu t :.'1 c:tlcoJ: ol , 2.cet one , ethyl .'. "J cetctG , _'u c, nzene , ce.r bon te t r o.chlor i de , r:.nd :) :'J i CL 1e , bu t wr:.s solub le 0 c ent ethyl al c ohol . ~ L1 wnt er ~:1d 80 pe r t- 1. V e art t , Ll '.)yd I s reG,:,:en t (E l i LLl.l y ;:-,_nd Co. ) , 6 0 -100 !!le sh fl orisil (F loridin Co ., Warren, P a .), 4 0 t o 60 mesh dec~lso (Air Conditi •min,1: Co ., Los Ani; eles), e.nd ,,lu;-:iin a (Ee ff or d 8.cti v, · t e6. D. l um ina ) . Tab l e 21 . Fr a cti ona t i on of c oc onu t 7 ilk , a s tested on 1 1_~e1') ce 11t de x t i') o t~e 1.~e'-~~i -~;.r1 . ----·--·-·- -- - -- ~ - - - - - - - - ~"°% V l a b l li t y Original soln. Aci d ether extra cti on E tl1er Ba sic e t h er extro.ction Ether 90 2.3 0 0 _ _ _ _ _ _ _ __ _ _ _ ...,, -✓ --- - ---- ,,· - --~----~------·---,- 52. Tab le 21. (Co n ti nued fr o~;; p revio'J.S [Jag e ). Che r~i c :1.l t r e 2. t r.1e n t Fr a.cti ons dJO vi a bi lit y . .. ·- Av . r e l e.ti ve growth ------ - Ac e tone extre. cti on Aceto n e 0 --- EtOAc e .xtre,ct ion Ac etccte 0 --- Be :1 ze ne extract i on Be nzen e 0 --- CC1 4 e :;:trciCti or.. CCl-4 0 Pyridi n e e::traction P y ri d i ne 0 ----- 8 0 -'{) EtOH I Alco ho l 80 2 . ~2 e xtrac ti on ,....,_, ·- ------~-·Or i g i na l s o l n . 90 2.3 No rit A Filtra te 100 2 .0 Fuller' s e a rt h Fil tr::1_ te so 2.0 Ll oyd 's reu .s_:e n t Filtra te 80 2 .4 ..... ·- Dec ::i. lso Ori gi nal soln . C h r Oi71.'.'t t O i_!)'.' D.m • 80 - ··- ~- ,. 7 Fil tr ::i.te 90 2.0 Fl o1° i sil c }_1r omD,t o ;; r 2Ji1 Fi 1 t ':;1. te 90 1.9 Al um i na c):1r otna t o :;ram Filtra te 60 1. 0 . .. b ) Su cioose . I n vie 1.,v ( ) f t h e C'Jr r el ;- ti or, in fr :i. c t i 0n 2. t io n b e h o.vior e f fec t of s n cr o ::c on t t e "T'OHt '} ,;f e mb r yos na t ur a ll y sug- I n t >1e first exp er L1 e n t e1,,bryo s b ei:1-.: een 0 . 35 and. 0 . 45 mm . in le n;;: t h were cu Lt ur•ed i n ., 1_ 2 pe r c e :,t e.n d a n 8 pe::."' ce n t dextr o se <",;. nd s 1w rose ,,1edi r-,_, res ,:; 8ct ivel y . T 11 e sup- 53. ple!nen t a r y v~ t am i ns , each case. ana. i notgani c s a lts wer e added in D.g:. r, Final measurements of the growth of the embr yos were made &ft er 2 da ys with t h e f oll owing re su l ts: Tc.b le G-r ow t h r::ieo. i a . •-- -) f e,:1br yo s in dex tr ose EU1d s u.cr ose - Fer ce:1t vi ab ility Av. rela tlve t:·rowth ·· (11_ ,~ dextrose 20 1.3 21 s ucr os e 60 1.6 8'1i dextr ose I 0 8'1o sucrose 90 Medium 2 I I 0 1.9 The second exp erimen t which <5. emon strated t he growthp ro ~o ting effects of sucrose co nsist ed of t h e growing of embryos , wh ic''I ,:ere 0.3 t o 0.5 mm . in len gth, in a 2 .p er cent 6.extrose :: nd o. 2 p er ce nt sucrose media, resp ectively, ea c~ c on t a i ~i ~g 4 dr op s of coco~ut 7 ilk ~ er culture vi a l. T:::.b le 2o . Eff e ct of sucrose 0:1 t}-;. e ;s_:r outh ,::; f e:nbryos . Fe r ce n t vi a Av. rel a tive ~rowth 1--- - - - - - - - - --·- - -- ---i:.--·_c: __; __l_i~y 1 d.c:.y 2 d._L~:·s 4 c.2ys r, ,,.._ : • 0 t 1· e 0.. cs od ~grovt h 2~ d ~e ~rl y t wic e a s ~igh a ) ercen t ~se vi Lb ility vio.s seen both i n t r. e presen ce anC:~ t ".e .: ~bse:--:-:;e ;:; f c oc cmu. t mil .k . To de t er ~ i ne the res90:1 se cf e mbryos t o di ff er en t 1. 8 , 2 . 5 , 3.5, 3.9, and 7.7 p er ce~t Bt 1 cr ose, re s;c ct iv el y, of 5 . 2 a.nd 7 . 0 , r e specTi.ve l r , 1,-: i t'-} s u lf u.rtc .:::. cia r,no. po t as- t he medis ~ere me ~sure d wit n a Beckm2n n pH ~e ter ~~ d t h e Acc a rdi~ ~ t~ t ~e fig ures ci v8n 1~ t~e t a ble, t he op- ti :'lu:n cot1centr a t.ion of sucr ose L-:ccl _1d ec:t 1 t:ide r a nge be t ween l and 5 r;er c e:1 t at p H tai ne d •. it:~. sX<' c cn ce ntr:Jt:'..an s f sucrose. of sucr ose durin g tte ~irs t 2 c ?./ S •) f c·"" l t,."!T'e l t w2.s found t hnt other subst2:1c es b e si~es s~cros e were needed in the basic r:ie di urJ f or t1-rn c or. ti c;ueC: c rowt 1: .-.r t}--ce er1bryos . Un- li ~e t ~e emb r :os supp li ed ~.ith c 0c onut mi l~ th ose dultured i n the 2 9e r cent sucr os e ~edium a l one s oo n c eased gro~th, as i 11 us tr ;:., ted in t c,.'.::-le 25 , u1d f igu.re 1 0 . I n t"' i s experlmen t 55 . e mbryo s at ou t 0.35 mm. i n leng t h were c ul t ur e d in s edi a con t a ining different c oncentr a tions of s ucr ose with and wit hout t h e add it i on 0f a n a ctive s ol u tion (stoc k D). I n this respect, t~e graph in fi g ure 11 is a lso p ertine n t. Here, t he a verage fresh wei gh t of t h e embr yo s c ul- tured f or 11 da ys a t 32°±0.5° Cin a 2 pe r ce n t sucr os e medi um a t pH 6.~ is p lotted agains t the rel a ti ve concentr a ti on of embryo f a ct or add ed. It is t o b e noted t ha t t h e wei g~ t Furtherm ore, t h e v e r~ low eve n o t t h e op t i mum c sn centr a ti o~ of s uc rose . Tab le r: /1 r._, -..t • Eff e c t Jf d iff e re~t c o~ c c ntrat io~ s of sucrose on t 1']8 s rowt h of e~b r y os a t p H 5 . 2 a nd 6 .4. -Per ce n t s ucro se Av . re l o_-ti ve f,.~ ro wt h Per ce n t vi abi lit y pH o.o 6. 4 0 0 C :, • JO 6.5 1. 3 10 1 . c.J 1. 5 :J ,..... ;·, t._; • C 7.7 lO C .':' • r:~) . 0 { i ~ 2.. 2. 10 6.4 ..._ ______________.........___________. , 56 . Tab le :?. 5 . Gr owt :'."; -; f e::;b r y0 s in S"t.1 c ro s e :o~e dii..rn in t h e pr e sen ce an d t h e ~t sence of s toc ~ D. ----------------- --------~-p~~e--r-c_e_n~t ---r-A-::-v-.-r-e~~-a-.~t-~i-v_e_ ~----r -0-w~t~t-, 1 .___ _ _ _ _ _ IJi'_e-'o ;;;..i·_1-'__;t_m_ __ _ _ _ _ _..._v_i_a_b__i_l_,i_·...._t., ,__y__~ 2 da ys 4 day s 0 . 0% s u cros e , no stoc k D 0 0 . 31 s uc r ose , no s t oc i D 40 1.5 3. 0 2 .4% s ucro se , no s t oc k D 40 1.5 2.8 4 . 8% s u cr os e , n o s t oc k D 100 1. 9 3.0 0 . 0% s ucr ose, stock D 0 0 . 8~~ suc ro se , st ock D 70 2.5 6. 0 2 . 4% s uc ro se , st oc k D 90 3.0 8. 2 4. 8% s ucrose, s t ock D 100 2 .7 7 .1 Durin g t h e c our s e of these i nvesti ga tions, it wa s found t hat t h e respon s e of t h e excised e mbr yo s t p s ucrose could b e decre a se d to zero by ~e ep i ng t h e c ap s u J.es off t ~e ~l a n t for sever a l d a ys before the iso lati on a nd t h e culturing of t h e emb ryos. A typ ica l inv e sti gation is presented b e lov; . A ca.p sule WEt S o ic ~rn c1 fr om a ,_) Lrn t in t}1. e f iel d c.:n.d t h e ov u l e s were re moved an d ~ l a ced in a s t e rile Pet r i dis h in a moist ch2mb e r a t 23 ° C. Ovules wer e t ~ken r ~nd oml y fro m t h e dish e;,ft er diff er en t len ~:t ':1 s of ti ::, e 2 :r1d t h e e rr1br yo s wer e iso l a te d ~nd c ~lt ured i n a 5 pe r ce nt s u cr oce □ e d i u□ Fr o~ t h e fi gur es i n t ob le 26 it c 2n be s e r n t ha t t h e +-+ ·t+ ·r~ .... t .~ 8 ~I . .: t ... •.~lt-7 t H- +l ·.,_, :j: + - ++ rtl: ... ;--r+ I' . . . . j..j. H+ tttt11-ttt- ' .... : + ,~. + .. t l:t It ' 11 I' .L t:t µ. t+ ... + .1-: - SB . ·•·I ± ± f+ +-i.; i1 HI '-" i +- If :t: ! l:l i :;: t ii x'f O :t: +- I ,- ..-ot a ·+-- l;h4 .:.,"' t:t ~ ~ ..... ~ t. ·- t :11 ~ "-+ -/7.S I } t l ~ + -1- "1 :t.. ~ ~ ti ,., - l ~ -I: .-..· ~! :t.~ I+ ........ ., ,, v.£ :t ~~ + nft Ii H - .µ + + .... + -~ t j- ....... r J. ·• , h i . ,. ..,. ;· . H ll [ t ,- ; tr ct -.. r: l"t I, ,l f ++ C [:J ~ .:r, +-+ '-'-" ~ ·t ~++++> .:, . u'j- ;. I + :H: ·14- +· -:tl l--l-+-l---l-+4-+-l--l-+--l-+--1-14-+-1-+-1---1-+-++H-l-+-I---Cl--l-++-HHSH-+++__,. .14 ·tt T .. :r. ij t-+ ' :!: 58 . c ons i de r ab le growt h i n t h e s u c rose ~edium ev en in t he abs ence of K- 2 dur i ng t h e first 4 day s of culture. Aft e r ag- in g for a si ng le da y the gr ow th of t h e embr y os in the sucros e ,n eo.i um wi t h:Yu. t K- 2 c1rorJ1) ed t o a very ~ow r ,.te . N'.J gr ow t h wa s ob t a ine d whe n t h e e mbr yo s we r e aged f or 4 day s or l onger a nd gr oKn in .t h e 5 p er cent s u c ro s e medium without t h e e.d di ti on of K- 2. Si:ni l e.r e r:1br ~ro s , howeve r, 2:re w v e r y nicel y in t }:"e su cro s e :ue di "J:7 v~'h en K- 2 WEl.S add e d . Tab le 26 . Growt h 0f ~g e d e~br yo s i n 5~ s ucro s e medi ura . I Le ng t :i of a g i ng Li e di um Per ce r: t v i a.b ill t y 0 O.D.y no K- 2 ., .L dB.·::/ 4 o.e.y s 6 dci.ys .- Av . rel c: :. ti ve - ~;rowt h 2 -~Dy s 3 day s dny s 60 2 .1 --- 3.4 K- 2 50 2. • L!:.t --- ~:1: . 3 n o K- 2 40 --- 1.3 --- K- 2 80 --- 3. 9 --~ no K- 2 0 --- --- --- K - <,) 70 1. ~ ., --- --- no K- 2 0 --- --- --- K- 2 70 2.. 0 --- --- 4 -- c) Growt ~ i nh i b it or . One of t he c~ i ef d iffi c ~l ti eE An c aun t e r ed in t te use of t ~e bio- s s say de scr i be d i n t he ~re vtous os.ges is t he se nsit iv it y ~f D&t ilra embr yo s i n vitro t o f r ow t h i n~ i b it a r s . Of t en, a s ol u t ion wt ic h did n ~t 0r o~o te t b e growt h ~f t ~e e,7'.bryos did so a ft e r c ert a in c h emic &l tre ;•t rri e nt s , whereby t h e gr owt h i nh i b iti ng s ubs t an c es had b e en re moved . Th e 60. • presence of a growth inhibitor w~s i ndic a ted fro m t h~ experi'Jents on t he eff sc t ,Jf sucrose on the r;:r01-rtl-: cf Datur a embrycs i ri _::'.:itl"'_'.2 • S1J.c.ros e (C hemi c c=d Co ., C. P. ? r a o.e) wa s recryet o llized f r ~m u 50 a er ce nt eth y l a lcoh ol soluti on . The ori s inal ana t he rec~ y s t itl li ze d sucrose were &60e6, re0 - ·ec .l. ..L' v""C 1-L. ,..;· , u _c-1 1· l··i , t ~t le 1. , l···"e of t ~e dextrose in t h e ~e di uo give n in _L...- c , 1... • E □ br y os , wh ic~ were 0 . 2 5 to U. 5 5 c7 . i n len g th , vver e u s eo. u~e J rese~te d in Ta ble ~7 . Gro~t ~ of ecb ry o s in r ecryst &llized sucr oce . Per c ent vi c:tbility Con e . of sucrose 0 . oocri s ucrose I - Av . rel a tivE g r □ v{ t h 0 --- 1. 0% C. P. sucr ose 0 --- 2,. 3~t C. P. s:~_c1 o se 1 40 1.3 ,, 401 • /'..l C. P . s1..1.crose 70 2.0 5li". C. P. 8UC:10SO 50 1.3 10 1 . on "-_i: • q I_,• • l • 0 ,-:1 -,·o r e C }_, : ,r S • sucr oEe ~ ,ff recr::s . S'_1cr ose 40 1.4 'i; . 4~10 A recrys . sucrose 80 1. 8 C 0". ~. V • I., recrys . sucrose 90 2.0 r, G • 0/0 ·-- - - ----- a sharp drop in the rel2tive ~ro~ t~ j f t h e e~bry a s cultured in t h e h i ghest sucr ose c oncentr ution of 9 . G p er ce~t . deo~eaE 8 in t ~e percentage Qf A v i able embr y os p2r n lleled t h is 61. decPeas e i n r e l ::, . tive ~:roi.vt h . An i c7.en tiC c\l 6.r c) wns no t ob t ~i~ ed in t~ e eillb r yos , whi c~ were gro wn i~ med i a c on t a i ni ng recrystalli zec1 S:..,1 Crose . Th is, o f c our se , c ou ld b e ex- p l ained on the b a sis 0f t he remov a l 0f a gr owth inh i bitor I n- fro~ the sucrose t hrough t~e recr yst a llizat ion process. as. _,..rnu.c h as no i nhi b it ory effect was obse rv ed in t :1e low s ucrose c on ce n tr a ti ons , the C.P. sucrose was used for t h e c u lture me dia of subse ou e n t bi o- as s ay s. A s i □ ilar in h ibit ory effec t was a l so fo und i n pre- vio u sly 2c tiv e ext rac ts, which had been a u t oclaved or hea te d c'. t 60° C f or a fe w ho1J.r s . Dr!_t a f or a t~rp ic s.l exper i r,1 e nt of t h is n~ture a re s hown i n t abl e 28 and fi gure 12. An a ctive extra ct of DRt J r a ovules wa s added to a 2 per ce nt s u cr ose ;:1edi'..1.m . were Ha l f of t he cul t ure vL,.ls ~ ,~_ut oclc..ved [ind the 1 were o t he r l-:1:1, lf 1.'::::t:::S n ,:-. t c:,'.1 t oc l av ed of t er t:~e ad. di ti on c, f t h e extr Qct . E7bryos , wh ich were 0 . 25 t o J . 6 mm . i ~ l en g t ~ , ., o-,.:\,,__._ e 62 . Ta.b le 28 . Growt h of emb r ::ns i n s ut'.} clav e d 2,_ n d nonau t oclave d ovule extra ct. Dr op s per vi a l Contr ols (2% sucrose) Av. rel a ti v e g ro wth vi a bility 70 2 day s 3 _da;l~ A 60 2.2 3.5 B 58 2 .4 3.4 o. 25➔~ 70 2 .7 4.4 . o. 5 60 2 .7 4.7 1.0 80 3 .0 '-'. 1.5 90 3. 2 5.0 2. 0 60 3.1 5. 0 0 . 25 40 2.0 3.6 0.5 70 2 .6 4.6 1.0 50 2.8 4.2 1.5 80 2.3 3.8 2.0 50 1.5 1. 8 Not autoclaved i; -l Aut oclaved ·- The e mbr y o grow th in the vials c on t a ini ~g 2 dr op s of au tocla ved extr ac t was far le ss than even tha t of t he c ontrols . S ome of t ~ e inhib itor y sub st a nces c ou l d b e re mo ved by extr a cti on wit h et hy l a lc oho l a nd bu t yl ~ lco ho l an d by p r ecip it a t i on wit h le a d a cet a t e . Su 9por ti n g da t a f or t h is st a te ment :~.i"e incl ud ed i n t ab le 29 . I n the fir s t exp eri ment 100 c c . of a c o c onu t mil k c oncentr ~t e (c), wh ich was obt a i ned we s O. 25 drop 11 per vi al me an s : Th e extr1:1.ct was diluted 4 ti~es and 1 drop of t he d ilute d s o l u ti on wa s added t o e a ch cult ur e vi a l. "~ 11 65. ..j I !"- 1+ ·~ .... c• .... t .L, ~ :+ .... L, .. . ~ t: ~ ctr ir ~:.;. +· :p: ±i '-+ -~ ,.;: ;_,;. .1. ++ e+-< ... H ....j a+ 4< It L t'! 1-l- 1--+ ➔ -H .. H h + ,.~,-,. 1 - H- i--j- .... t~+-++<4++-~H~H--+++H~-+++H~~~-+++~-H-~+++~~ .j: + + ±. l :ti ~ + r+r:... H- -I+ ...j., t ...... ,·, -H .... 64. s haken with 350 cc. of 95 per cent eth yl a lcohol and t h e supe rn a t a nt l iquid decanted fr om the pa sty residue. Both fr 2.ctions were eva,_,;)Or a ted t o dryness in v a cuo at 55 to 60° C. The dried residues, which were &pproximately e qual in wei gh t, were taken up in e qur,.l v olumes of wa ter f or the teet. Em- bryos, whic h were 0. 5 t o 0 . 8 mm. in len ;?;th, were cult ured i n a 2 pe r c en t s ucrose med ium a t 25Il° C. I n the second e xp e r i rr:e nt 15 cc. of c ocon ut milk conc en tr a te (B) was shaken 2 times with 100 cc. por tions of bu t y l a lc oho l. Tw o p er ce n t of t h e dr ? we i gh t was fou nd in t he a lcoho l frac t i8n . Both alcoho l ~~d Ka t e r fracti ons were e v2porc:.tec\ t o drvn ess i n-vacuo ·- - a t 55 to 60° C c1,nd t he residues were t ake n up in e 0u ~l volumes of wa ter. Embry os 0. 25 t o 0 . 5 ~~ - i n l~ ngt h were cult ured on a l p er cent dextros e ~edium f or t t is test . I n both of t 1:le 8.bove fr2.cti onr; ti;:ins t h e r e sidues , a ft e r be ing separ a ted fr om t h e s olv ent, gave a mu ch h i gher g~ow t h t h a n t he ori gi na l ex t rac ts. In t he f irs t e xper i- me n t t he add ition of t h e alcoho l fr a cti on to t h e control med i um s topp ed t ~e gr ow t h of t h e embr yos co mp letely , which g&v e c ·:msia.er;;.b le ½:r owth i n the sucr ose mec'l i um c::. l one . Th e ass um9 ti on t ha t i ~~ i b it or y s~bst ~nces we re ) re se nt i n t h e 2-.lc o':"sol .fr <'•c t ion is , t r, ere fore, logi c c l. In t h e sec ond ex- pe r i:ne r:. t t h e 1;::::,. ter fn.,_ c ti on rer:,cd n i n:?,: s ft er t h e extrc::ction wit h butyl ~lc oh0 l Qr o~oted n ouch h i ~he r r ~t e of gr owt h t han t he or i g i na l c o~ce n tr a te p r i or ta t h e bu t y l a lc oho l extr a cti on . 65 . Ta.,u~ -le 29'- . R e o1ov a--'-1 o_ f' 12:row t h __ --'~ - nn• i 'oi• t or b y e thy 1 and bu t yl a l coho l. Tre a t me nt ------ !Ethyl a lc oho l Frac t ion , · c one . .e_er vial 70 via- bility Av . re l ative growth --- 100 1. 9 1 drop 0 --- drops 0 --- 1 drop 100 4. 3 c.rops 70 2.5 --- 0 --- drop 50 1.7 2 drops 80 2.4 4 drop s 90 2.3 1 dr op 0 --- 2 drops 0 drops 0 ----- 1 dr op 90 2.. 6 dr o;; s 100 4.2 4 drop s 100 4. 9 Co!1 t ro l ,.:. , ( 2:·:; s u crose ) Alc ohol 3 Re s i due 3 Bu t y l 1:1,lc oho l Con trol 1%dextrose ) Con e . B Alc ohol ., l. 4 Water n :::., tJ-, e ::·, t ~: en t of' c oc o m., t mi 1 '.t c on ce n t r a te 1, j_ t h 9 5 ,_:,e r c en t 66 . \ j,'")(.J ..1...:.. \..,I c i p it a ti on wi th le a d a cet a te (Merc k , re a gent le a d i ons were t h e n re n~v ed t h r ough a serie 2 of ste~s inva lvi ng t h e a dt iti on ~f exce s s s~lfuric a c id a nd t he extr a cti on of the e~bryo f2ctor ~i t ~ 0 8 per ce n t e t ty l al c o- D~tur a e~bryos , w~ ic h we re ini ti a lly 0.1 5 to 0 .4 m□ . i n ·~en gt h , vrnre cu l t rr ed i t 32:tl ° C ii1 a 2. per ce n t sucr ose 1 iTiedi u:n, t o i: .:'h ic l--i dif fe re nt f,_li quo ts o f t : --, e diff er e!1 t fr a.cTh e ~r o0t h of t½e e mbryo s Dfte r 2 is ; iven in t ab le 30 and fi gur e 13. Fro m the gra9h s in figure 13, it c a n be seen tha t the e~bryos which were cultured in medie containing the le ~d gro wth. Fra ctio ns D and E, ~ticb ~e re not ~r ecip it &ted by - ctive • 1 82. d a Cet a te , re . -wee- 1neq ---· C 0. T~ le na_ - .. e p oss1''01·11~t~, _• thg. ca t was t h e t oxic f a ct or inv olved seemed unlikely because sL:-:l l a r toxici t ~· sy mp toms v,·ere not observ ed. in fro.cti ons B, D, an d E, \Jh ic h urre s ubj ected to t h e s a.r1 e tre n tme n ts as fra cti on A. T~ese d~ t a , t h er ef ore, str on~ l y s ugges t t he. t a t le e.s t a p.::.rt of t lrn i nhib itory s ucs t e nces b e yr ecip it a t ed by le ~d RCet ~te. C O'J.l d .-,. t .: ' -t ' ± .... .l- q ' f+ ' tt -~ l ·t rtt .. + . . . t _:: .j_ -• + I ' -~ ~ ~ t --<'+ . . _ 1 ++ ... + .+ .......... ti±titiltH:tti±t:tt:t:tl:ti.~ +t L t + .... H .... -I+ '-+ ... 72. d) Root inhibitor. A growth characteristic of Da tura embryos cultured in media containing fresh coconut milk was the lack of root formation. The cotyledons and the hyp ocotyl enla rged but no roots appeared a s long as the embryos were left in the same medium. Bu t if the embryos, after reachin g a size of several millimeters in length, were transferred to a 1 per cent dextrose medium, which did not contain any coconut milk, root formation occurred (47). Attempts to re move this root inhibitor were successful through the use of solvents, such as ethyl alcohol, butyl alcoh ol, a nd diethyl ether. A typical example of the methods used for the re- moval of the r oot inhibitor is the following: 14.7 liters of fresh coconut milk was concentrated at 55 to 60° C in vacuo for 8 hours to a volume of 3.45 liters. 846 cc. of 95 per cent ethyl alcohol was added slowly with vi g orous stirring to 100 cc. of this concentrate (test solution B). The supernat a nt alcoholic fraction was separated from the pasty residue, which was dissolved in 25 cc. of water. The latter solution was shaken with 100 cc. of butyl alcohol and the 2 layers sep a rated. The water l a yer was shaken with 100 cc. of diethyl ether and dialyzed with a cellophane t ubing in distilled water. Th e dialyzate was then concentra ted at 55 to 60° C in va cuo to a volume of 50 cc. (test solution E). 270 cc. of 95 per cent ethyl alcohol was ~dded to this concentra te in small p ortions with vigorous stirring. A precipitate was formed upon the addition of the alcohol. This precip itate was dried in 73. vacuo at 55 to 60° C and taken up in 50 cc. of water (test solution F). Of all the different fractions tested in a 1 p er cent dextrose medium with embryos, which were 0.35 to 0.6 mm. long, only fractions E and F were active. The dat a for fractions B 7 E, and Fare shown in table 31. Table 31. Removal of root inhibitor. % root- Test . Solution % viability Contr ol ( 1% dextrose) 0 --- --- 0 B 0 --- --- 0 E 100 2 .3 6.9 30 F 100 2 .2 5. 9 80 Av. relative growth ;:::; a a y::, 0 aa:ys ing in 5 days Thus, it can be seen tha t the root inhibitor and a lso the growth inhibitor were re moved by successive extra ctions with ethyl a lcoh ol, butyl alc ohol, diethyl ether, and again with ethyl a lcohol. Although the ori ginal concentrate B gave n6 sti mu lation of growth in the embry os, the final fr a ctions E a nd F gave excellent growth. Very good root for ma tion wa s obs erved in t he embryos which were supp lied with fr a ction F. Because of t he simila rity in t he beha vior of t h e root inhib itor in c oconut milk a nd the a uxins t o s olvent e x tractions, Dr. van Ov erbeek investi ga ted t h e cont e nt of the latt er in coconut milk. Coconut milk wa s subjected to dif- f erent tre a t me nts, such a s ether ex traction, a lkaline hydrolysis, boiling, and autoclaving and the auxin content of these fractions determined by the standard Avena coleoptile test (48). Results of such a test are given in table 32. Table 32. Liberation of auxin from coconut milk by different treatments. -·-·---------------------......,..-----------Treatment Avena deg. curvature i----- - - - ----------------1-_:.(.Comparabl~ata) Agar blocks soaked in coconut milk concentrate. 0.6 Concentrate extracted with ether; residue from ether fraction taken up in volume of water equal to that of the original concentrate. 21.5 Concentrate hydrolyzed at pH 10 for 24 hours at 4o C. 7.9 Hydrolysis followed by ether extraction; residue from ether fraction taken up in volume of water equal to that of the original concentrate. 28.5 Concentrate boiled for 5 minutes. 0 Concentrate autoclaved 3 times at 15 pounds pressure for 20 min. 8.7 Boiling followed by ether extraction; residue from ether fraction taken up in volume of water equal to that of original concentrate. 21.6 The data show that although untreated coconut milk concentrate showed only a very low Aven§: curvature the treated concentrates produced large curvatures. Therefore, a good supply of auxin or precursors was present in coconut milk. 75. Since the presence of a uxin in coconut milk was est a b lished t h e effects of the &.d d i tion of auxin purified extract were studied. wj-/4 riH\ a some wh a t Different concentrations of ind o le-3-acetic acid (Merck) were added to a 5 p er cent sucrose medium with and without a n a ctive extra ct (K- 2 ). K-2 wa s obt a ined fro m coconut milk concentr a te a fter successive tre a t ments with ethyl a lcoh ol, diethyl et h e r , c a lcium oxide, and ethy l alcohol. Embryos, which were O. 2 to O. 4 mm. in len g th, were used. The results of the experiment are shown in t a ble 33. Table 33. Medium - K- 2 + K- 2 Effect of ind ole-3-acetic a cid on the growth of Datura embryos in vitro, Cone. of To via---- -Av. rela tive growth Condition IM/liter bility 2 days 8 days of embryo 0 70 2.1 3.9 transp a rent 10 50 2 .0 3.1 transparent 100 60 1. 9 3.3 tran s parent 750 30 2 .2 --- callus 1000 50 1.7 --- callus 0 60 2.7 34t 2 .0 nor mal; no roots 10 90 2 .7 50± 6 .8 normal; 457t rooting 100 70 2.7 28 callus-like 750 60 2.1 6. 9 callus 1000 80 2.0 3.4 callus ·A si g nific a nt sti mula tion in b oth root a nd cotyledon a nd h yp ocot y l growt h wa s foun d wit h t h e a dd ition of 10 ga mm~ 76. of indole-3-acetic acid per liter of medium in the presence of K-2 only . No benefici a l effects were found in the ab- sence of K- 2 . In concentrati ons above 100 gamme:s per 11 ter ca llus for mation resulted in b oth the a bsence a nd the presence of K- 2 . Although the res ul ts of this particula r e x- p eriment were clear a nd convincing, the stimula ting action of 10 gamma s of in dole- 3- gcetic a cid on embryos supp lied wit h K-2 coul d not be dup lic a ted a month later. However, the changes under gone by K- 2 could n ot be asc ertaine d to e xp l a in t his apparent discre pancy. i:iant ing, Y.ltlGbElitS :f ft1;1; ,,:, tlrnt ,;,d,::l:e,1 eebL.lll ;;i1t2i11 u.019 w:;u:. m•t It 1n.;3c be th;t s1tffi f.,rmeD: ill xeqlbi:t@:I: the l( f; Jli sta11d-- f:r :ptin:ia.l About 6 mont hs later, however, a si milar sti mu- grJut':; lat i on in embryo growth was ob tained t hrough the addition of 10 gammas of nao hthalene acetic a cid to a 2 p er cent sucrose medium, containing a p urified embryo factor prepar a ti on. e) Ash. Coconut meat, mil k , a nd husk were a shed separ a tely in p latinum dishes in a muffle furnace a nd te s ted for t h eir effects on the growth of Datura e mbryos in vitro. No benefici a l effects we re f ound in a ll ca ses. The me d- i a and the concentr a ti ons of t he a sh s a mp le s , which were t es t e d, a re giv en in t h e follo wing t able: 77. Table 34. Concentrations of coconut ash found to be inactive in promoting the growth of embryos. Test no. Ash of: Test medium 420902 Meat 2 01/o dextrose equiv. to 0.04 to 135 mg. of dry meat per culture vial. 420831 Husk 2o/~ dextrose equiv. to 0.04 to 125 mg. of husk per culture vial. 430326 Milk 2o/c, sucrose equiv. to 1 to 6 drops of milk per culture vial. Concentration - .__ Among the many subseauent repititions of these experiments, occasionally a beneficial effect of the ash of the growth of the embryos was observed. In all cases, however, the effect was slight, if significant. f) Fractionation of coconut _!Qilk fo,£ ~~r:zo factor. Based on the facts known from the experiments described in the previous pages of this chapter a fractionation of coconut milk was oerformed. The scheme, which was followed, is diagrammed in fractionation II. Tests for embryo factor was conducted with embryos, which were about 0.25 to 0.6 mm. in length, cultured in 2 per cent sucrose medium. The medium was buffered at pH 6.5 to 7.0 with 0.01 molar dipotassium-monosodium phosphate. The final measure- ments were made after 2 days of culture at 32to.5o O. Most of the active fractions were tested several times in different dilutions. In order to calcule,te the amount of embryo factor 78. present after the various fractionation steps a quantit a tive unit was adopted. The unit was desi gnated as the Datura unit and defined as follows: One Datura unit was the quan- tity of embryo factor which had to be added to e. standard culture medium to give a rela tive growth difference in one Datura embryo of 0.5 over that of the control. The stand- ard medium was the 2 per cent sucrose medium (2 per cent sucrose in place of 1 per cent dextrose in the medium given in table 1). The medium was buffered at pH 6.8 with 0.01 molar dipotassium-monosodium phosphate. 3/4 cc. of this medium was placed in each culture vial, which contained one embryo. The relative growth measured after 2 days of culture at 32±1° C was taken as the ratio: initial length. final len g th/ The initial length of the embryos used varied from 0.2 to 0.5 mm. An example is 9resented to show the app lication of the ab ove definition. Embryo growth in the st a ndard culture medium to which 0.002 g. of a test substance was added: Av. initial length of 10 embryos= 0.40 mm. Av. final length of 10 embryos : 1.20 mm. Relative growth= 1.20/0.40 = 3.00 Embryo growth in the st a ndard culture medium only: Av. initial len g th of 20 embryos= 0,35 mm. Av. final length of 20 embryos = 0.87 mm. Relative growth= 0.87/0.35 ·= 2.49 Therefore, relative growth difference= 3.00 2.49 - 0.51 Hence, 0.002 g. of the test subst an ce contained 1 _!2c1.tu~ unit of embryo factor. 79. It must be re a lized, however, that the Datura units given in fracti onation II are only rough approxima tions of the a ctual quantity of e mbry o f a ctor p resent. In t he first were p l a ce, t h e embr yo s ~ se nsiti ve t o inh i b it ors. Furt he r- a10re, t he pH of t he cul ture rn e di um wa s no t a l wcss maint a ined a t e xactly 6 . 8 and t he f inal measure me nts were not a l w8.ys made after e xactly 48 ho urs. Milk fro m fres h c oconuts (fr om L. A. Nu t House of Los An geles) wa s c on ce ntra t e d in va cuo a t 55 to 60° C in a glass still. After this concentration procedure and stand-rng_ for severa l months in t h e refri ger a tor t he concentra te showed no a ctivit y in p romo tin g t h e growth of embr yos. The p os- sibility of a libera tion of t oxic subst a nces suggest e d the a lcohol ext raction step a s de scrib e d in 9art chapter. 11 c 11 of t h is After t h e alcohol tre a t ment activity was a gain observ ed in the residue fr a ction (C-2 ); t he t oxic sub s tances were t aken up b y t h e a lcohol (C-1). The residue fr a ction was dissolved in wa ter and subjected to a le a d pr ecip it a tion process. To the resulting 80 p er cent alcohol soluti on, a fter the re mova l of t he lead ions, t he embry o f a ctor wa s pr ecipit a ted with su l furic acM. The resultin g p reci p ita te (I- 2 ) was a.ctive in a dilution of 1:1200 , which re presented an enrichm ent of 11 times. The type of growth closely rese mbled that of embryos cultured in a medium containin g coconut milk . formed. No roots were It was t h erefore subj e ct ed to several ethyl alcohol extractions in order to rem ove the root inhibitor. The 83 . i) Proembryo of Datura stramonium, 0.1 mm . in diameter. These do not grow in media lacking embryo factor. 3·) Normally differentiated embryos, 4.7 mm . · in length, which had developed from proembryos, 0 . 17 mm . in diameter, after 7 days of culturing in media containing inorganic salts, 9 per cent sucrose, vitamin supplements, and embryo factor. Plate I. 2) Callus-like bodies, 0.35 mm . in diameter, which developed from heart-sh~ped embryos, 0.35 mm . in length, after 11 days of culturing in media containing l per cent dextrose, inorganic salts, vitamin supplements, and autoclaved coconut milk. 4) Embryos, originally 0.4 mm. in length, cultured for l month in the dark. Upper embryo: 3 mm. in length, was grown in a medium containing 2 per cent sucrose, inorganic salts-, and vitamin supplements. · Lower embryo': 15 mm. long, was grown in the same medium, with the addition of fresh coconut milk . Note the absence of roots, as discussed in Chapter 7. Photographs showing different types of growth in Datura embryos cultured in vitro. (Photographs 1, 2, and 3 presented through courtesy of Dr. J. van Overbee k) 84. DISCUSSION➔~ Ideas suggesting a limit in the size of the embryos which ca_n be cultured in vitro, as proposed by La.Rue (29) and Dietrich (20), were shown by va n Overbee k et al (46, 47), and confirmed in this investigation, to be untenable. In view of van Overbeek 1 s success (unpublished data) in the culturing in vitro of Da tura proembryos as small as 0.06 mm. in diameter (approximately the 64 cell stage), the question is clearly one of nutrition and not one of a vit a listic threshold as suggested by earlier workers. The failure of these investigators to grow very small e~bryos was due merely to the lack of a proper culture medium. The culture of mature embryos, which were shown to be a utotro~ic, did not meet with any great difficulty. They could be maintained on strictly inorganic media (42). In some cases the addition of sucrose or vitamin B1 was necessary (27). For large immature embryos of deciduous trees, Lammerts (27) found t hat suga rs were required in the incrgr~ie medium. 42). Similar results were found by other workers (29, With s ma ller immature embryos, LaRue (29) and White (51) found tha t t h e hea t-stable factors in yeast or fibrin digest were necessary. Th e addition of organic sup- plements was also found necessary f or the growth of immature * A considerab le oortion of the materi a l in this section has been presented in~ symp osium (44) by Dr. J.van Overbee~ who discussed the results of the investigati6ns on embryo culture carried on at the Institute an d at the Carnegie Institution of Washington at Cold Sp ring Ha rbor, Ne w York. F or t he sake of comp letenes s , however, t h e subject is repe a ted in this sec ti.on. 85. Datura embryos in vitro (47). The medium given in table 1 was.found by van Overbeek et al (47).to be suitable for the culturing of medium-size immature Datura embryos in vitro. Whether of not all of the ingredients were necessary has not been established experimentally. The choice of these substances, however, was not without good reason. Although some ar gument existed as to the beneficial effects of glycine for tissue cultures (33, 53, 54) it was included in the medium. The essentiality of thiamine and pyridoxine for the growth of roots was established more clearly through experiments on root cultures (1, 6, 9, 32) and mature pea embryos (7, 24). In similar experiments (11, 15), ascorbic acid was shown to give a vigorous increase in the growth of some varieties of pea embryos in vitro. No stimulation, however, was observed in those varieties which were capable of synthesizing ascorbic acid in appreciable quantities. Datura roots ill vitro were shown (8) to be completely heterotrophic for thiamine, pyridoxine, and nicotinic acid. The response of isolated tomato roots to nicotinic acid in the presence of thiamine and pyridoxine varied with the different clones and strains. sponded and some did not (9). Some re- With isolated pea roots, however, the absence of nicotinic acid and thiamine resulted in a decrease in growth with each weekly transfer. The inclusion of adenine in the culture medium of Datura embryos was based on the discovery of its leaf growth promoting effects (5). Succinic acid was suggested (37) to 86. be essential as one of the members of the 4-carbon-acid hydrogen-carrier cycle, which was presumed to be coupled with auxin-controlled resp iration. Because of the univer- sal occurrence in living things (56) of pantothenic a cid a nd its sti mulatory activity in some p lants and plant tissues in vitro (10, 31, 57) it wa s also included in the medium. The above medium proved s a tisfactory for the culture of i mmature Datura embryos over 0~5 to 1.0 mm. in length ( table 2~). Smaller embryos were not cap able of grolvth in this medium. Since the embryo is nourished by the endo- - - sperm in the seed it seemed logical to van Overbeek et al (47) that the addition of a na tural endosp e rm may stimulate the growth of smaller Datura embryos in vitro. Indeed, it was found to be so when coconut milk was tried (table 3). From the fact that extracts of coconut milk and me a t, Dat~~ ovules, wheat germ, almond meal, and yeast extract were found to be active in supporting the growth of very s mall immature Datura embryos in vitro (table 20), it seems that little sp ecificity existed among the substances necessary for the growth of very small embryos from different species of pla.nts. The same source which nourished the coconut or the alr:iond or the whe a t embryo was found capable of nourishing the _!2at~ra embryo. This non-specificity of nutritive factors for plant embryos was also suggested from the results of earlier investigators. Arnaudov (3) found that moss sporophytes could be removed from their own gametophytes and transplanted successfully into other 87. gametophytes either of the same or of different species. Similar types of transfer were accomplished in the higher plants. Brown and Morris (13) transferred embryos from one barley grain to another. If proper adhesion was ob- tained between the transplanted embryo and the new endosperm the grafted embryo would develop a,s readily as if it were. dependent on its own endosperm. The contents of the foreign endosperm underwent all of the usual changes attending their absorption and, if molds were excluded, e the resulting growth of the embryo was en tirly normal. Partial development was obtained in the transplantation of barley embryos on wheat endosperm. In this case, howeve~ the differences in the size and the shape of the embryos made proper contact between the transplanted embryo and the new endosperm difficult. Nevertheless, that there was a partial development is significant. Better success was obtained by Colla (16) who was able to transplant and grow wheat embryos on the endosperm of rye, barley and oats. The relative growth curves of embryos cultured in medium containing embryo factor (figures 2, 8) resembled those of autocatalytic reactions. This suggested to van Overbeek (44) that all of the cells of the embryos were involved in the 0rocess of growth of the embryos in media containing the growth factors. He, therefore, concluded that the embryo factor promoted cell proliferation chiefly, as contrasted to the action of auxins, in promoting cell elongation in the Avena coleoptile. To further support 88. this contention he brought out the fact that the curves for the relative growth of s mall and of large embryos (figure 4) were the s ame, while the absolute increase in length wa s much l ar ger in the ca se of t he l ar ger embr y os. Furthermor~, a cy t ologic a l examination by Dr. J a ck Schultz reve a led a ctively dividin g nuclei in the ·e mbry os supp lied with embryo factor, while t he nuclei in e mbryos not supplied with em..:. bryo factor quic kly bec ame pycn otic. Embr yos de prived of e mbr yo f a ct or f or a fe w da ys were not able to resume growth when supplied wit h embryo f a ctor. On the basis of the data in figure 4 it also seems . likely that the concentra tion of embryo factor must re a ch a certain threshold va lue, before its effect can be manifested. Onl y 30 p er cent of the embryos, which were 0.5 mm . in leng t h , develop ed a t t he lowest embry o f a ctor concentrati on. At the s a me level 100 p er cent of the larger e mbry os developed. With inc r e a sing a mounts of embryo fac- t or t he percent age of vi able e mbryos increased in the smaller embryos and re mained a t 100 per cent for the larger ones. This, of c ourse, is in har mony with the increasing a utotrophic na ture of incre a sing l y ma ture embryos, as discussed ab ove. As p ointed. out by va n Ov er b eek ( 44) the forma tion of embryo f a ctor in the parent_ p lant a nd t h e supp ly to the embryo may var y with the conditi ons under wh ich the p l a nts we re grown. Th e e mbr yos who se growt h was pl otted in fi gure 4 were t aken from p l a nts which were gr own during the winter months in a r e lativel y p oorly lig hted greenhouse. In t h is m. case, embryos as l a r ge as 1.2 mm . in len g th were not capable of gro·wth in the ab s e nce of added embryo f a.ct or. On the othe r hand, occasionally embryos, as s mall as 0.5 mm. in length , which were t a.ke n from p l a nts gr own in the Spring in the field, were capable of growth in the 1 per cent dextrose medium alone (fi gure 6). This suggested a sufficiency of embryo factor in the embryo, as a result of increased production by the mo ther p lant in t h e Sprin g . A cha.r a cteristic feature of Da tu_!:§: embryos cultured in media containing fresh coconut milk is the lack of root for mation (plate I.). Root development was markedly sup- pressed even on mature embryos cultured i n medium cont a ining autoclaved coconut milk (47). The f a ct that the hypo- cotyl in the embryo s cultured in c o conut milk was not supp ressed along with the root sugges ted to van Overbeek et al (47) t he possibility t ha t auxin was the age nt concerned. The susp icion was strengthened by his determination of auxin in coconut milk (table 32) an d by the removal of the root inhi b itor from coconut milk thr ough extractions with solvents, in which t h e auxins were soluble (table 31). The st a te in which a uxin is present in coconut milk ca n be settled only with further more conclusive exp eriments. When an agar block was soaked directly in fresh milk coconut?" only a very sli ght Avena e,:,leo·.:itile curvature was detected (table 32). ing or a utocla ving . Some auxin w0s rele ase d by boil- Consider able amounts of auxin were detected when the milk wa s extra cted with ether and the 90. ether fraction tested. In all cases there was a three- to a four-fold increase in.the Avena curvature. The fact that coconut milk after being forced through a collodion membrane and extracted with ether gave the same auxin curvatures as undialyzed coconut milk suggested that the auxin liberated upon ether extraction cannot be due to a liberation from a protein conmlex as postulated by Skoog and Thima nn (34, 38, 55). On the basis of preliminary experiments, van Overbee k et al (47) tentatively proposed the presence of at le a st 3 factors in coconv.t milk, which produced the typ e of growth characteristic of Datura embryos in a medium containing coconut milk: a) t hermolabile factor causing both growth and differentiation of the Datura embryo, b) heat stable factor causing in some cases a callus-like growth but no differentiation, and c) heat stable root inhibitor. In the light of the resul ts obtained in the present investiga tion, these tentative assumptions must be altered. The failure of autocla.ved coconut milk to support organized embryo growth was probably not solely a result of the heat lability of the growth-prom otin g factor. This was suggest- ed by the fact that autoclaved yeast extract was able to support normal embryonic ~rowth (t able 20). The more · prob- able reason W£,. S the libera tion of growth inhibitors. In- hibitors mi ght have a risen fro m a utoclavin g (figure 12) or fro m a prolon ~ed he a ting of coconut milk at about 60° C and sta nding (fractionation II). Indications of growt h 91. inhibitors in untreated plant material were first found in the work of van Overbeek et al (47). They found that when halved ovules containing the embryos were placed in the nutrient medium, such embryos did not grow whereas similar embryos, when completely isolated, did grow. There was some suggestion that auxin was concerned in the above relationships. The experimental facts which led towards this possibility were the following: There was a similarity in behavior between the root inhibitor in coconut milk and auxin. Both were heat stable and extracted with the same solvents. Autoclaved coconut milk supported a callus type of growth (47), as contrasted to the normal differentiated type of growth supported by non-autoclaved coconut milk. It was found that autoclaving liberated auxin (table 32). Furthermore, it was found that high concentrations of auxin added to the medium containing a somewhat purified embryo factor preparation induced callus growth (table 33). The callus formation induced by high ~nxia concentrations of auxin might have been superimposed upon the growth stimulated by the heat stable substances, resulting in an undifferentiated growth. The results are in agreement with the findings of Sioog (44) that Nicotiana callus failed to differentiate into shoots and roots when supplied with a high auxin medium. Although there were some similarities in properties between the growth inhibitor discussed in chapter 7 and auxin it is not certain whether the two are identical. More experiments are needed before any con- 92. clusions can be drawn in this respect. On one hand, therefore, a high auxin· concentration induced callus formation. On the other hand, the figures in table 33 indicated that there was a stimulation in embryo growth upon the addition of 10 gammas of indole-3-acetic acid per liter of medium containing a somewhat purified embryo factor preparation. Rooting was also stimulated by this low auxin concentration. The implication of the neces- sity of a minimum amount of a substance or substances for root formation was supported further by the experiment illustrated in fi gure 11. Of the 4 sets of embryos in this experiment only those which were given the highest concentration of embry o factor formed any roots after 11 days. No roots developed in the others, despite their good hypocotyl and cotyledonary growth. Of course, much more ex- periments are needed before conclusive data can be presented. However, the work of Stephenson (35) is interesting in this respect. He obtained a marked stimulation in the growth of lettuce embryos with the addition of 0.1 mg. of indole-3-acetic acid or naphthalene acetic acid per liter of medium. With the addition of 1 to 10 mg. of auxin per liter cancerous and nodule-like typ es of growth were obtained. Stephenson's investigations, like those in this thesis, were conducted under sterile conditi ons. Anyway, we see present in t he endosp erm of coconut a g ood source of 8.uxin, which is apparently bound. in some unknown way but ~hich c a n be libera ted by different methods. As suggested by the r e sults of t h is investi ga tion, the auxin may be liberated in s ma ll a mounts to stimula te norma l e mbr yo nic d evelopment. Under ab normal or unf a vorab le con- ditions the liberation of unusually high amoun ts of a uxin may b e the cause in the a bn orm a l t ypes of gr owth s ometimes me t in nature. Of cour s e, it is n a ive t o as sume ~ p riori tha t only one or a f ew sub s t a nces ar e necess a r y for the or ganized growth of embryos. The embryo factor as used in t h is t h e- sis, is a generic term to includ e a numbe r of a s yet unidentified subst a nces. And a s in t h e c as e of a uxin, a n opti mum concentra tion of e a c h of t h ese f a ctors may p robably be necessary. Beyond the optimal r a n g e, a bnormal- ities in growt h or inhib itions of t h e effects of the other sub stances may result. Furthermore, accompanying change s in the p H of t h e endosperm may b e necessary. As s hown in fi gure 9, t h ere is a shift in t h e p H opti mum fr om p H 6.8 to p H 5.5 a fter 4 days of culture. Whethe r this shift actually takes p lace in n a t u re, however, needs to be determined. 94 . SUMivlARY 1) The results of van Overbeek et al (47) on the successful culturing of small immature Datura embryos in vitro on media containing coconut milk were confirmed. 2) The growth of Datura embryos in vitro in the l per cent dextrose medium, as gi ven in t ab le 1, was supported also by extracts of coconut meat , Da tura ovules, wheat germ, almond meal, and yeast. 3) A s a tisfactory bio-assay for the subst an ces necessary for t he growt h of Da tura embryos was developed. This bio-assay was based on the growt h of Da tura embryos cultured asepticall y in vitro in a medium containing the solution to be tested relative to the growth of Datura embryos cultured in the control medium in the absence of the test solution. The quantity of embryo factor was expressed in Datura units. One Datura unit was the quantity of embryo factor which had to be added to the standard medium to give a relative growth difference in one Datura embryo of 0.5 over t ha t of the c on trol. in 4) 2.. Jo.'fs. The op timum tempe r a ture for t he growth of Da tura em- - brvos in vitro was found to be 32° C • ., 5) pH 6.8 was the optimum for the growth of small Datura emb ryos during the first 4 to 5 days of culture in vitro. 6) After 4 to 5 days the op timum s hifted to pH 5.5. Besides t he in gredients in the control medium (vit am in Bi, vitamin B6, ascorb ic a cid, nicotinic acid, aderil.ne, 95. succini6 acid, calcium pantothenate, glycine, inorganic s a lts, and a gar) sucrose ~as f ound to be necess a ry. 7) Other sugars, amino ac ids, purines, a nd inor ian ic s a lts were in a ctive in ~he concentrations tried. 8) A stimulation in embryo growth a nd in root formation was f ound with t h e addition of 10 g amma s of indole3- a cetic a cid or naphtha lene a cetic acid ·per liter of a 2 p er cent sucrose medium ( 2 pe r cent sucrose in - l9/v'en. .,~ .r,r6/e / • place of dextrose in the 1 p er cent dextrose me diu~, containing a some wh a t purified embryo factor preparation. Concentra ti ons of 100 gamma s or over p er liter induced c a llus formation. 9) Fr a ctionations of coc onu t milk for t h e re ma ining growth factors revealed t h eir insolubility in the usua l organic solvents. They were not adsorbed by the usual ad sorbents and not p reci p it a ted by t h e usua l p recipit a nts. 10) Th e r oo t inhi b itor in c oc onu t milk was r emoved t h rough extra ctions with ethyl a lcoho l a nd d iethyl ether. A p rob ab le identity with auxin was sugge sted. 11) A growth inhibitor was shown to be for med up on hea ting coc onut rn ilk a t high te mpera tures or a t low tempe ratures for a p rolon ged p eriod. Th is wa s re move d t h rough ethyl a nd butyl alcohol extractions and p reci p itation with lead acetate. 12 ) Purification of coconut milk yie lded preparations with a de tectable a ctivity in a diluti on of 1:19,000, which rep resents a n enrichment of 170 ti mes. 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