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[IUIIKWMIL 11111111511111.1112 ' H E515 This is to certify that the thesis entitled RECOVERY OF INTERSPECIFIC VIGNA HYBRIDS VIA EMBRYO CULTURE presented by James Francis Parrot has been accepted towards fulfillment of the requirements for M.S. degree in Horticulture we é Major profegoflrf Xf Date September 10, 1981 0-7639 'rv1531.) BEIURNING MATERIAL§z Place in book drop to LJBRARJES remove this checkout from lelzgzle. your record. FINES will be charged if book is returned after the date stamped below. !__ RECOVERY OF INTERSPECIFIC VIGNA HYBRIDS VIA EMBRYO CULTURE By James Francis Parrot A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1981 .K/f/ // /, ~v -. _1 ABSTRACT RECOVERY OF INTERSPECIFIC ylgflA_HYBRIDS VIA EMBRYO CULTURE By James Francis Parrot Embryo culture was used for 28 interspecific crosses representing 18 species combinations of various accessions of Vigna angularis (Willd.) Ohwi & Ohashi, 1. glabrescens Maréchal, Mascherpa, & Stainier, 1. mungo (L.) Hepper, y, radiata (L.) Wilcz., and y, umbellata (Thunb.) Ohwi & Ohashi. Mature hybrid plants were recovered fron ten interspecific combinations including three that are apparently new: ‘3, glabrescens x ‘1. mungo,'!. glabrescens x.!. radiata, and Cl. radiata xii. umbellata) amphidiploid x.!. glabrescens. Although some hybrids were partially fertile, few produced seed. The ease of pr0pagating and maintaining each hybrid depended upon its indeterminate vegetative habit. Problems with germination, quiescence, callus, deformity, leaf expansion, root development, and acclimation of plants from culture were encountered. A cutting procedure to overcome one type of deformity and an aseptic potting system to facilitate acclimation were developed. ACKNOWLEDGEMENTS I take this Opportunity to express my appreciation to my former major professor, Dr. L. R. Baker, for extending me this opportunity for graduate study and for his guidance, assistance, encouragement, and advice throughout this project and in preparation of the manuscript. I thank Dr. P. S. Carlson for granting me full use of his laboratory, advising and encouraging me in my work, and reviewing the manuscript. I am grateful to my major professor, Dr. J. F. Kelly, and to Dr. S. Honma for their assistance and review of the manuscript. For the facilities, time, and efforts they provided, I thank Drs. J. Fobes and w. Tai. A special thanks goes to my colleague, Dr. N. C. Chen, whose work provided the hybrid embryos for this study, and to my brother Jerome for his invaluable assistance in data processing and producing quality prints from my mediocre slides. I thank Mrs. M. Machado and Mr. G. R. Bauchan for performing the cytogenetics reported herein. For encouragement, suggestions, and help in the execution of my work, I thank Mrs. B. Floyd, Mrs. G. Zabala, Drs. S. McCormick, R. Griesbach, G. E. Lester, R. Malmberg, and Messrs. J. Hunsperger, T. Jacobs, and T. Kumashiro. Too numerous to list, I thank all my colleagues, the staff of this University, and the taxpayers of Michigan and the United States. To my mother, Patricia, I offer my very special thanks for moral support and help in proof-reading various drafts of the manuscript. ii Finally, for less direct but very important support, I thank my father, Joseph, my late aunts Catherine and Estelle, my late uncle Frank, the Most Rev. J. S. Sullivan, D.D., the Very Rev. B. Panchuk, 0.S.B.M., and the Rev. w. Hodde. iii TABLE OF CONTENTS LIST OF TMLES 0.0...00......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... LIST OF FIGURES .0OOOOOOOOOOOOOOOOOOOOO0.0.0..OOOOOOOOOOOOOOOOOOOOO. I'qTRODUCTION 00......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO REVIEUJ OF LITERATURE 0.0.00.0...OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.000... MATERIALS AND METHODS .............................................. P1ant materials 000......O...00.000.00.00...OOOOOOOOOOOOOOOOOO. CUItura] conditions 0.00.00.00.00...0....OOOOOOOOOOOOOOOOOOOOOO crOSSing MethOds 0......0.0.0.0000...00......OOOOOOOOOOOOOOOOOO leritro MethOdS OOOOOOOOOOOOOOO0.0.0.0......OOOOOOOOOOOIOOOOOO Iber-id Characteristics .0.00.0.00...OIOOOOOOOOOOOOO0.0.0.000... StatiStica] Analy51$ 0.0.0.0.0....OOOCOOOOOOOOOOOOOOOOOOOO0.... RESULTS AND DISCUSSION COCOOOCOOOOOOOOOOOOOCOOOOOOOOOOOIOOOO ...... .0 |< O radiataXVO umbe11ata OOOOOOOOOOOOOOOOOOOOO0.00000000000000 umbellata x V. an ularis ................................... angularis x V} umbellata ................................... an u aris x . radiata ..................................... mungo x.V. afigularis ....................................... mungo x V. radiata ......................................... mungo le. umbellata ....................................... g abrescens x‘V. umbellata ................................. glabrescens x V. radiata ................................... radiata x V, gTabrescens ................................... glabrescens x-V. mung ..................................... radiata x V. umbellata) amphidiploid X.V. umbellata ....... (V. radiata x V. umbellata) amphidiploid x'V. radiata ......... V. radiata x (V. radiata x V, umbellata) amphidiploi ......... '(V. radiata x V, umbellata) amphidiploid x V. radiata, BCZ .... (V. radiata x V. umbellata) amphidiploid x‘V} glabrescens ..... V. radiata x (VI umbellata x V. angularis) .................... TV, umbellata i V. an ularisY—x V. mungo ...................... General Considerations an Sumnary <|<|<|<|<|<|< O <4fdwo can sucmomom o_nmummm> capm< uzu we mema52: copmmmuu<~ ~m<= -- A~<< x ma: co fiav _L Am_ce_=m=e a» x mum—.652: .»¢ Emmoaamm -- A~m==mm x Neaaaav Amazam -- xeakaa x mamv u_o_m+e_;asc .i «masgm -- xeA~== x ogav Amen—.695: .> x mu~_uac .>v uo>> mmokow ~a -- mummmmuampm a» H~== -- A~== x Na: co “av Na: fismmwm Ha -- ma: can _z -- m== «Noe -- Na: mmoe .Hm-m. .. fin: mace .xz. camp m6_m man—_mae= .> ”Nam -- Ham x Nam co Haw mfiam -- mag x Hum co ac max comecw Ha -- egg cemekm Ha -- max “mad -- Nam MANN .m-az. .. "ax "flaw .H ‘ =~=_~». cmmnm==z mu~_umc .> E: : 32 x a: .6 5 22 wow 2 -- i #2: mfifim .m-h. saga xue_m mamma..> ~<< Nwfim .oHN * mg. .. H<< ¢-m .=a_;m =a_=u. camp _x=~u< m.L~_=a=~ .> muou ~Lmnsac A:o_um>_cmuv so use: :oEEou mw_uwam :o_mmmuu< .msa: Lm>_u—=u. .co_umNpu_cn»: u_»puaumcmu:_ Lo» mucmgma mo wow: mcm>_u_=u ecu mmvumam camp) .fi m_am» in 20 cm pots containing either a commercial potting mix (Metro Mix 200 or Redi-Earth, N. R. Grace and Co., Cambridge, Massachusetts) or a mixture of soil:sand:peat (1:1:1). Nutrient levels were maintained with bi-weekly applications of 20N-9P-17K (39/pot). Crossing Methods All the hybrid embryos for this investigation were supplied from a simultaneous study by Chen (1980) who followed the pollination techniques of Boling et al. (1961) and Buishand (1956). In Vitro Methods Embryo cultures were prepared from 28 parental combinations representing 18 different interspecific hybrid crosses and backcrosses (Table 2). Pods were collected 7-25 days after pollination either at abscision, at early signs of degeneration (yellowing, bloating, loss of turgor), or at a predetermined number of days after pollination just prior to the anticipated onset of degeneration. Pods were surface disinfected in a laminar-airflow hood by dipping in 95% ethanol and flaming briefly. Ovules were excised and placed on moist sterile filter paper for embryo excision under a dissecting microscope. Much time and effort was spent excising numerous selfed embryos in order to develop, practice, and gain dexterity in the technique so that hybrid embryos could be excised with a minimum of damage. Embryos were measured using an ocular micrometer. Embryos had one to four cotyledons, so the cotyledon lengths were summed for each embryo and divided by two. Unless noted otherwise, the hypocotyl measurement is the length from the cotyledonary node to, but not including, the suspensor which often remained attached to the hypocotyl. Occasionally, 10 mmummsz z en a H~== x “ms: was: _ co m was x fizz “~22 _ ea H “2: x Hz: fins: 3 ea c H2D.x z: i. camp—mas: .> x 0 use .> Ham: ~ ea N “mm.x Hz: i. mumpcas .> x amass .> - x c :25 .> m~m< o ea c max x H<< Hfim< o co «H i.~ma x H<< .I muoPumc .> x m_Lc—=mcm .> -=< 3 co mg |.~== x ~<< i. mum—pone: .> x mwgm—amcm .> ~m<= o No .fio w H<< x ma: -<= zwm No .fio ma i.~<< x as: .I m_L~—:mcm .> x mum—_mnsa .> fifizm mzs .ozm a mu .Nm .fim .mo .Ho ea ms: x "am .i mum_—mnE: .> x mum_umc .> muou xmucmEummcu hucw5=oc_>cm ~Es_ume umumpa mmocu u_»_umamgmuc_ e_ta»= coum_»uou a=_ue_a _Q_SF=~ moxenEm .ocu_> :. meaupao L_m;u we m=o_uwucou use mu_caxz acm_> o_wpumgmgmu:~ .m m—nm» 11 Emma”. 2.3 S .8 cm :2 x was. =+HNiaomm u co N NNmu x HNmmam N8 .333. six 3.333% Acva—mas: .> x mum_cmg .>V Nwfiamm N no .No .fio m Nwsnam x Ham .i u_o—q_uw;a5w Anus—_mnss .> x mum_uoc .Mu x muo_emc a» Hmwmzz N no .No .Ho NN “mm x Nmaamz NNmmsm zam no .No .fio NH Nag x Nmzaaa HNmmam N no .No .Ho Ho flux x Nozzaa SEE .>Ix Boastame Amum_—wns= .> x campus; .>V HNoznm J em .mm .NN .HN mm Hm: x Nmsamm mp~_eec .>r« ewe—aec_;ase 52.85: .> x 32%.. .a fizu> Hzm .Nzw e co HM i. #2: x wo>> .i omens .> x mcmummcnmpm .> we: a E 2 8% x 5.”. i memommcnm—m .> x mum_vuc .> mg; in .03 m E N me. x 8; Hao> NzN .Hzm .ozmH >a=am ea .u ON i. “mm x co>> .i mumwvmc .> x mcmommgampm .> m=o> u u N .i was x wu>> i. wumppmnss .> x mcmummcampm .> «ecu xmucmsummcu aucm5:og_>=u ~e=_ume vows—a mmocu u_ppumamcmu=_ e_cn»: coua_»uou m=_pe_a _e_pw=~ mosses“ vmzepucou N m_nmh 12 .=pueu:_ pee— meeeepaueu o ;p_3 mexgeEe N: mmuee_ee_ =o3N= .meeu .=me_pe—e we uueuep “we. mceee—xuee me geese: ;u_: mexceee Ne Leases: moaeuvecp eeeu ceuoecegu e co m seem .mexgesw esem sec» ee>esec ace: meeempaeee seen so eeox .mucwECOLw>:m chm 90.0....» cw twacwucou mucwECOLw>cm Lmsuo 0:8 cw mexgese m__=: .w aceeeec_>ew ea eeccowmeecp ego: o smeeceu 2 use N gmeeccu m muemsceLP>=e cw m=_:ega mexcese .mpmmme> cemcep ea cewmeecp we es_e we» u< .m—mmme> comcep e» gmemeecu p_uee mepuepe Eecw aeeuwm e? mew: mucmseecw>ce omega .mepemec we» cw eeee: we unmexm .mueee=ecw>ee meewce> use we mee_u_eeeu see c e—eea mum» .uxm» use =_ emeevuees »_—ee_mwueem we uneexm mewuuee _ppee mewuepe see» eeme Seems m:_ee—e —ewu_:~ .eweee m=e_ce> use we cepu_meeseu co» m epeeu wemu mrmmzumm a»: an N . «2: x ~m<= .i e :23 .>Ix Amwcepemee um_x eueppmeE: .>V .mm-me<=-m a ea m ~m<= x am .i Am_ce_e an AP x mum—Fees: .»v x euepeec .> MMnmmu>=m e co m eu>>ix NNmeeexz mceumwgeenm .> x e_e—e_e_;e5e Amen—pens: .w.x e»e_eec .Mq meeu xmucmEuemcu xuemscec_>em ~Ee_eee emuepe mmece u_wwuwemgmue~ e_cn»= cone—xeoe acpee_a _~_e_=~ usages“ eeee_u=ee N «_eep 13 a clear delineation betwen hypocotyl and cotyledon could not be made, so total length of each embryo was divided into hypocotyl and cotyledon length according to the hypocotyl:cotyledon ratio of the other embryos of the same cross. Each embryo was plated onto agar-solidified medium consisting of Murashige and Skoog (1962) mineral salts with supplements (Table 3) prepared in advance and dispensed into Petri dishes. Care was taken to place the radicle in contact with the medium. A channel, gradually increasing in depth, was cut into the agar from the embryo toward an Open area of the agar surface so as to prevent beading of excess moisture around the embryo. Petri dishes were sealed with Parafilm to retard moisture loss from the medium during incubation (Figure 1A). Plates were placed on shelves in a room equipped with unshielded fluorescent tubes (cool white or cool whitezwarm white as indicated in Tables 2 and 4). Irradiation just above the plates was measured (under cool white regimes only) with a foot-candle meter (Type 214 Light Meter, General Electric, Fairfield, Connecticut). This meter was later calibrated with a Li-Cor Radiometer LI-185A (LI-COR, Inc., Lincoln, Nebraska) over a spectrum of 400 to 700 nm. Because of the spectrum changes characteristic of fluorescent tubes, both measurements are given but should be regarded as approximations. The irradiation changes from daytime (16 hr) to nighttime (8 hr) and vice versa were made abruptly. Several different environments for embryo culture were used during this study (Table 4) and the environments used for each of the particular hybrids are listed in Table 2. Embryos were transferred to large sterilized jars for further deveTOpment when the shoot reached an average length of 18 mm and had, 14 99 99999999 99 99999 .E=_ems zoom 99999 9999 99 999999 some so» emceuee geese: esp :99 999: 99999 999 999 9.9 99 99999999 99: =9 999 .Ee_ewe mecmie=PceEEe m>99 9» N9 5999 eeuu_se mozezz ——<: .9:_>e—eeu=e 99999 eeeee Aee~99pwuegeeev 9999: useeueu ee~___cwum Leuppux eu:_ eeueceegeuep mapem me eczeEe meg mpeeam meexm 999 99999999: 99 eeueuPee9 xu_uce=e egg mwe_u .ANmm9v aeexm e99 wmwsmegaz we owes» me 0599 use use mu_em pegmevxa .uoHN9 we 9:_>9_eeeee 9:9 .9999 .wmeguem .99999 Lee 99999 99 eemmocaxe mueeeeeeeu _—<~ 9.9 9.9 9.9 9.9 9.99 9.9 9.99 9.9 9.9 9.9 9.9 9.9 9.9 99 x9999: 9999999 9.9 9.9 9.9 9.9 9.99 9.99 9.99 9.99 9.9 9.9 9.9 9.9 9.9 9 9999 99-99 9.99 9.9 9.9 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9 9999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.99 9.9 9.9 9.9 99 999999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 9999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 99: 999x999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 9999 9.9999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 99: 99999999 9.999 9.99 9.9 9.9 9.999 9.999 9.999 9.999 9.999 9.999 9.999 9.999 9.99 99 9999999_-999 9.9 9.9 99.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9- 999999 9999999 99 99 N9 99 99 99 N9 99 99 .99 N9 99 9 9999 9999999999 9999999i999 99999 999999 mewuueq._wuee 99999—9 sec» emu: upeee 999999999 999< ~99eee —999e:999 99 oceupeo 999950 Le; emu: ewees me ce_u_meeseu .m open» Figure 1. 15 Stages of the embryo culture system. A. Stage 1 embryo germinating on petri plate. 8. Stage 2 embryo transferred to larger vessel for expansion of roots, stem, and leaves. Note bottom half of Petri dish inside. C. Stage 3 plantlets. At left is a plantlet recently potted aseptically for gradual acclimatization. Note wire support frame that clamps firmly to the pot. At right is a plantlet nearly ready for transplanting to the greenhouse. Note deep lid and its supports that allow gradual decrease of humidity toward the end of stage 3. 0. Stage 4 plantlet fully acclimated and ready to transplant to the greenhouse. .9999» 999;: 999: :99: : geeseec9>ee me 9999» acmemege99w 999:3 9eee one me 999; wee 999999999 99 eeueeec9 99: e pauseec9>em .9 unusee99>ce 9e» emcemeee we: 99: 9999909999 .999959999 999 99999995 9e: 99: z ecoscecp>em ge>e 9 pewsee99>eo we 9099999999 9999999999 .e9999 999 :9 90999 uemumegee—9 9995: 599399999: peeu meueu9999 zzuzu .9999» uemummcee9m 999:: _eeu meume9999 3o .99999 e: 9m99u_e:_ o~ : 9:959eg_>=e ems» .mgueee m 999 9 pewsee99>eo 99;» .9999: e Lem z acmseec9>ee : e geesee99>em cage .mxmwz m so; : newaeeg9>em emgu .9999: 9 go; o aeoE=eL9>em > e ucmseeg9>ee cog» .9999: m 9e» : «emseec_>ee ems» .mxeez m-N so» u acesceg_>ee : I geesee99>ee case .9999 9 new 9 aceseec9>ee 9 e 99e59999>ee cusp .mxmm: n so» : uemsee99>em m a. acmgogzcm case .3029 H .5..— : ”Em—99:99:95 a 9 weasee99>eo cage .mxmm: 9 new : acmsee99>cm c 9 ueosceg9>ee even .9999 N9 gem z ucmaceg9>em a m u:m=.co.9.—>:w :93 .UmucmEou .3395 p.595 < acmEcosgcm o o ”Em—590.3290 cmzu .Umucmfizou :HZOLG 75:: < ”Ewe—5.92:0 z 7 m wavegoggcw :09.» .vmucméou 59930.5 :29: < u:mE:o.9_>:m 2 9.9.9.9 999-99 9.9-9.9 999-99 39 N999 9 9 9 -- -- 3:939 9999 99 9.9.9.9 99-99 9.9-9.9 999-999 :9 9999 99 9 9 9.9.9.9 999-999 39 9999 I 9 9 9.9.9.9 999-999 39 9999 9 9 9 9.9.9.9 999-99 39 9999 9 9 9 9.9.9.9 999-999 :9 9999 9 9 9 9.9.9.9 999-99 39 9999 9 9 9 9.9.9.9 999-999 :9 9999 9 9 9 9.9-9.9 999-99 :9 9999 9 9 9 9 9 9 9+9N 9 9-999-999 9-99 9-999-999 9-99 99999 9. 99999999999 999999 99 99992 999999 999 999 99999 9999 99999999999 99999999 e99 »w_uee:u unm99 .9999999 99999 9999999 9999999 9999 9999999999 99 9999999999 .9 99999 18 generally, at least one expanded leaf. This transfer was usually accomplished by removing the Parafilm from the Petri plate, cleaning the bottom half of the plate (including its sides) with a pad saturated with 95% ethanol, discarding the t0p of the plate, and placing the lower half with the embryo into the larger jar (Figure 18). A few dr0ps of sterile water had been added to the larger jar to assure adequate humidity at the time of transfer. Occasionally, however, the embryo was transferred to a larger vessel into which fresh medium had been dispensed. In some cases these jars were of non-borosilicate glass and later observation indicated leaching of ions into the medium. These media (Table 5) are identified as "defective" by a "d” prefixed to the medium designation. Once in larger jars, cultures growing in environments B through F and M through 0 were transferred to environment 0, while embryos in other environments continued in those same environments. This general technique was supplemented in some cases where abnormal growth occurred. Some embryos which callused heavily were transferred to a cytokinin-containing medium (51, 32, $3, or S4; see Table 5) in an effort to obtain shoots (Skoog and Miller, 1957). Also, shoot tips of embryos which produced multiple shoots or grossly elongated and spindly shoots were excised (about 10 mm in length) and the cut end of each was pushed about 2-4 mm into fresh 04 medium for rooting and possible development into normal plantlets. The rest of the plantlet was either discarded or saved to produce new shoots. Plantlets were potted after adequate deveIOpment (avg. of 51 mm, but minimum of 8 mm shoot length with at least one expanded leaf). The first few hybrid plantlets (all RR x UU and one RRUU x RR plantlets) were pot- ted in perlite or a commercial potting mixture and gradually acclimated 19 .599995 9999 99:9 999999999999 99999 99 999959 999 999999 99999 999 99999999: 99 999999999 99999999 999 99599 9999» 999 99999999: 99 99999 99 9599 999 999 99999 9999:9zx 9999 :9 599995 9999 999 9999999 99959: 999 999999 99999999 999 99995 999 9999 99999 999 5999 99999999 9999 999999>9 99: 99999 .999999 99999 .999999 9999999 999 .99999 99999—99-9999 9999 99999 99 9995 999999> 9999999 9999 999999999 999: 9999 99995 99999999 A9>99999999 :9: 9993 999999999 999999999999 9999z9 .90999 99 999>99uou99 999 .9999 .9999999 .9999— 999 99999 :9 9999999x9 9999999599 99<9 99 99999999 99 99999 :99 999: 99999 999 999 9.9 99 99999999 99: =9 999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9 9999 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9.99 9 9999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 999999 9.9 9.9 9.99 9.99 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 9999999 99999999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9; 9999999999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 9999 999999-9-999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 99: 9999999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 9999 999999999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 99 99: 99999999 9.999 9.999 9.999 9.999 9.99 9.999 9.99 9.999 9.999 9.999 9.99 9.999 99 99999999-999 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9.9 9- 999999 9999999 99 99 99 99 999 999 999 99- -99 9999 9999999999 99995 999999999 99999 99999999599 99999 9999 99995 999999—99 99m< 999995 999995999999 999 9999999 999959 999 9999 99995 99 99999999599 .m 99999 20 to greenhouse conditions by use of high humidity and/or misting chambers. An aseptic potting procedure was developed due to the high mortality of this preliminary method. A clay pot (6 cm in diameter) was fitted with an aluminum wire frame to support the plantlet and facilitate subsequent aseptic manipulation of the pot. The pot with its frame was placed inside a 946 ml glass jar and autoclaved. A potting medium of either pure sand or a mixture (ca. 1:1 v/v) of perlite and commercial potting mixture was put into another container, moistened, autoclaved, then flushed with an autoclaved nutrient solution. Four nutrient solutions (L1, L2, L3, and L4; see Table 3) were used at the beginning of this study, but L1 was soon ad0pted as the preferred solution. The plantlet was loosened from the agar in the culture vessel and placed into sterile water to wash away any agar still clinging to the roots. The water that clung to the plantlet after this soaking afforded the plantlet some protection from dessication during the actual potting procedure, but as an added precaution, a small area of the laminar air flow was blocked to provide a guarded area. The flushed, aseptic potting medium was spooned into the pot around the hybrid plantlet. The potted plantlet was then put into an autoclaved 946 ml jar with a glass lid, sealed with Parafilm, and incubated with irradiance of 5.6 nEs-lcm-2 (33o ft-c) 16 hr/day at 21:}°C for acclimation (Figure 1C). The plantlet was allowed to acclimate over 10-79 days based on expansion of a new leaf as a signal of adequate acclimation. For prolonged adaptation periods, the pot was flushed occasionally with fresh nutrient solution and transferred to a new jar. Near the end of the adaptation phase, the pot was flushed and transferred to an autoclaved jar with a deep lid (Figure 10) which was gradually opened over a five to ten day period. After the plant had 21 adapted to ambient laboratory conditions (Figure 10), it was moved to the greenhouse on a cloudy day, and transplanted to a larger pot inoculated with a mixed Rhizobium preparation (courtesy of J. C. Burton, Nitragin Co., Milwaukee, Wisconsin) primarily to provide a non-pathogenic microbial population around the young plant. A four-point evaluation system to score the development of each embryo was based on the main stages of the culture procedure. Thus, the various points of the scoring system indicate, respectively, 1) that the plated embryo had initiated growth, 2) that the embryo had, or had nearly, developed a basic root-stem-leaf structure and was transferred to a larger culture vessel, 3) that the plantlet had developed enough photosynthetic area to move to a pot, and 4) that the plantlet had fully acclimated and was ready to be transplanted to the greenhouse. Hybrid Characteristics The hybrid plants, or hybrid plants grown from cuttings, were compared with the parental species in greenhouses for morphological traits with emphasis on the patroclinous characters of the hybrid. Pollen fertility was estimated by use of a saturated Iz-KI solution, counting plump, evenly stained pollen grains as fertile. Statistical Analysis When several hybrid embryos were obtained from the same parental combination (cultivar by cultivar) they were sometimes given different treatments with respect to cotyledon removal, medium, and/or incubation environment (Table 2). Means of different treatment effects within each hybrid combination were compared by use of the t-test, but because the data often did not meet all the assumptions for this test, observed 22 differences are termed "apparent" rather than "significant". Differences in the embryos that were beyond experimental control (ovule condition, presence of suspensor, embryo size, etc.) were also noted for their effects and analyzed by t-tests or linear or curvilinear regression for many, but not all, of the crosses. Again, as above, interpretation of the results is qualified. Some hybrid characteristics (3.3., leaf measurements) were also compared by t-test to those of the maternal parent. The chi-square test was used to determine goodness of fit of observed phenotypic ratios to a proposed genetic model. RESULTS AND DISCUSSION The 28 crosses representing 18 species combinations responded to embryo culture with different degrees of success (Tables 6, 7, 8, 9). Compositing the 28 crosses into their 18 species combinations, in only one case was there a total failure of embryos to grow (RR x RRUU). In seven of the crosses growth commenced, but mature (flowering) plants were not obtained (AA x RR, MM x AA, MM x RR, MM x UU, RRUU x UU, RR x UA, and UA x MM). Mature hybrid plants were recovered from ten different species crosses; viz.: three diploid x diploid crosses (RR x UU, UU x AA, AA x UU), one amphidiploid x amphidiploid cross (RRUU x VVGG), one diploid x amphidiploid cross (RR x VVGG), four amphidiploid x diploid crosses (RRUU x RR, VVGG x UU, VVGG x RR, VVGG x MM), and one allotriploid x diploid backcross (RUR x RR). The initial culture medium did not seem to have much effect on the final degree of success. Only one cross (RRUU62 x UU1) gave any indication of an apparent difference. Medium E1 appeared to be better than either E2 or E4 for these embryos, but this was confounded with age in that all of the embryos at the extremes of the age spectrum (11 and 17 days after pollination) were plated on E2 and E4. If the youngest and oldest embryos were dropped from consideration, there was no longer a detectable difference between these media effects. 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E385. mcmumecaepm .wrw ewe_ewewsamw “camp—ease .> x avowemg .>v meeu eeunee< gee ew new ow guzeco wxueueew: cove—xueu e—=>o chwuepq eeuewn mmecu uwwwueamcoucu ewes»: (pi m N H, :ewmwuxe an Asav erm com: um emu mexcasu hmmeuuam we magmas eagnsu .meweeam muggy mcw>we>cw mewgnaz ocmw> uwwwumemceacw we menu—so eagnsm .o e—nmw 28 score, but were also confounded. In one cross (UU1 x AAI) embryos incubated in the light did not perform as well as those initially incubated in darkness. (None of these light-incubated embryos grew.) However, this possible light effect was confounded with age in that all of the embryos initially incubated in the light were older (15-16 days) than those incubated in the dark (12-14 days). The germination of other interSpecific hybrids in the present investigation, including a cross of the same two species but a different cultivar as the maternal parent, was not inhibited by light. Probably, the older embryos were simply excised after irreversible degeneration of the embryo had begun, as was reported for embryos of wheat x rye crosses (Taira and Larter, 1978). No other differential effects of initial incubation conditions were detected for other hybrid combinations. Cotyledon removal treatments (Table 2) did not appear to be different in their effect on the final scores attained by the embryos. V. radiata x V. umbellata From the cross of RR1 x UU1, 12 abscised pods at 7-25 days after pollination provided 14 embryos for culture. Embryos were normal in appearance except that one had no cotyledons; it could not be determined whether they had never developed or were lost at excision. Ten embryos grew normally, two failed to develop epicotyls, one produced callus, and one did not grow at all. In four weeks, eight of the ten normal plantlets were transferred to larger vessels containing fresh 05 medium. The other two had poor leaf eXpansion and ceased growth in stage one. Of the eight plantlets that were transferred, one was lost to contamination, 29 two failed to devel0p adequately, and five were eventually potted. These embryos were not potted aseptically, and only one of them survived. It took about ten weeks from excision to greenhouse readiness. Sawa (1973) also used embryo culture to obtain four mature plants from 23 embryos of this hybrid combination. Other workers obtained mature plants from viable seeds without the use of embryo culture (Ahn, 1976; Ahn and Hartmann, 1978c; Asian Vegetable Research and Development Center, 1974, 1975, 1976; Chen, 1980; Chen et al., 1978; Dana, 1966c). The hybrid obtained by embryo culture in the present work need not be described again as it is the same as that obtained from viable seed by Chen (1980). Fertility of the F1 of this combination is low and no diploid F2 p0pulation has been reported. However, amphidiploids have been derived by treatment with colchicine (Ahn, 1976; Ahn and Hartmann, 1978c; Chen, 1980; Dana, 1966c; Sawa, 1974) and spontaneously (Asian Vegetable Research and DevelOpment Center, 1976a, 1976b) which produced viable progeny. V. umbellata x V. angularis Two different V. umbellata accessions, UU1 and UU3 were used as the maternal parent in crosses with AA1. From the first of these crosses, UU1 x AAl, nine pods ranging form 12-16 days after pollination provided 19 ovules that contained normal embryos and 2 ovules with small or collapsed embryos. The 19 normal embryos were plated but only six embryos grew. Four of these ceased growth when the shoot reached 6 mm or less in length. Two embryos proceeded to the second stage and were ultimately tranSplanted to the greenhouse after an average 187 days from excision. 30 In the second cross involving these two species, that is, UU3 x AA1, eight embryos from one shrivelled, chlorotic, 20-day-old pod were cultured. The embryos were normal in appearance except that two of them had slightly distorted cotyledons. All eight of these embryos grew and developed normally; however, two of them quiesced before attaining 1 mm of shoot growth. The other six plantlets reached the potting stage, but two of these succumbed. Four plantlets were transplanted to the greenhouse in an average of 120 days after excision. Chen (1980) has already described the mature plants of UU1 x AA1. The mature plants of UU3 x AA1 did not differ greatly from those of UU1 x AA1 except that the former tended to have greater leaf pubescence and a little different growth habit than the latter. Whereas hybrid plants of UU1 x AA1 were of medium height, somewhat trailing, and produced juvenile shoots from the base of the plants, those of UU3 x AA1 were taller, more vining, and produced axillary branches at the higher nodes. These differences may have been partly due to different environmental conditions. Pollen stainability was 82% (mean based on 100 grains from each of eight flowers) and numerous fruits and seeds were produced. However, pod length and number of seeds per pod were not determined. The cross of V, umbellata x V, angularis has been reported (Ahn, 1976; Ahn and Hartmann, 1978b, 1978c; Chen, 1980; Sawa, 1973) but only with the aid of embryo culture. Although Evans (1975) reported obtain- ing seeds, no mention was made of F1 plants. Ahn and Hartmann (1978b) reported difficulty in obtaining the hybrid plants as many embryos failed to grow in culture or died as seedlings. Poor germination of the UU1 x AA1 embryos was also observed in the present work, although UU3 x AA1 embryos presented no serious difficulty. Sawa (1973) reported the F1 31 plants he obtained to be sterile, but the hybrid plants reported by Ahn and Hartmann (1978b) and those in the present investigation were fertile. UA11 had 77% stainable pollen (Chen, 1980) and UA31 had 82%; both pro- duced plump seeds. F2 plants were grown out by Ahn and Hartmann (1978b) land in this study with no unusual difficulties. Ahn and Hartmann (1978b) also backcrossed the F1 to both parents, obtaining fertile yorogeny. Thus, gene transfer between these two species is probable. An F2 population of the cross UU1 x AA1 was observed for epicotyl colcn~and the shape of the first foliar leaves. Ahn and Hartmann (1978b) arud Chen (1980) reported continuous variation for both of these traits. hhnnerous leaf forms intermediate between and including cordate and 'lanceolate were observed in the F2 population of the present study, iridicating complex inheritance. However, the observations for epicotyl cxalor (Table 10) fit a 9:3:4 ratio where one dominant gene determines purple pigmentation and a second dominant gene intensifies that pigmen- 'tation to dark purple. This agreed with Kakizaki's (1923) genetic model for stem color in V, angularis, P for purple and I which intensifies P. .V,_angularis x V. umbellata The parent AAZ was crossed with UUZ. One bloated, 10-day-old pod and one normal, 11-day-old pod were harvested. Seventeen ovules were Obtained with one or two from each pod beginning to discolor with a red CH" brown hue. Two embryos were damaged during embryo excision so that ("fly 15 were plated. One of the embryos had three cotyledons. Eight of the! embryos, including those from discolored ovules, grew, but in either a deformed or a callused manner. Leaves were generally under-developed anti several embryos failed to form roots. One of these embryos, however, 32 .om.-om. n a memN. u NXN em o.¢H m.oH m.Hm Nu eaouaaxm No_pmc Aenm av emeuaaxm em we a mm mm AHH<=V e_cnwg u_w_uaamcmp=H m m o o Na afi< venowum aw—ecewmmuue eewucm awwezm: mumwomucmw op mpe>o mcwwweeu op mcwcwza mpmcweceumecw xcwm; awmpmcmeee ecwucm mue>e x—eeecn mmcocmcn mew—weep cpwz pomeminam mpmc_ELeumecw 3:2 :23; mcwpcm ewouwme xwwecowmeuuo mpm>e awemoen xwwezm: mmgucegn ueweeunsm new: uumge mensweeeumecw mecmemmnaq wee; :wmcms pmwmemwn wecwEme maecm um_mee_n _mcweeww “Pee: cuzoem m>wpwummm> opewwwaE: aw acmema wecgmuea even»: mememmcnmpm a» pcmcea Pmccmuez owpmwemuumcmzo II. .Am:e>v e_eewe e_w_eeemeeee_ twee“ use .Amasv weep_eee: .> .Aoo>>v meeemeenemm .w.wo meeem_eepeeeeee .HH e_eew 42 Figure 2. V, glabrescens (VVGG), V, umbellata (UU5), and their F1 interspecific hybrid (VGU5). A. Left to right: Plants of V. glabrescens (VVGG), F1 interspecific hybrid (VGU5), and V. umbellata (UUS). B. Close-up of an older specimen of the F1 hybrid (VGUS) showing the trailing branches. 43 belhia A ,2 I 9 I v“ 44 harvested 11-15 days after pollination. The cotyledons of the five embryos from the oldest pod were deteriorated, so were detached without being measured. The cotyledons of the remaining 15 embryos appeared normal. One or both cotyledons were removed from 13 of these embryos (Table 2). All of the embryos grew vigorously to the transfer stage within six weeks, but growth was not entirely normal. Sixteen of the plantlets had some degree of callus, and 11 had some degree of deformity such as coiling of the shoot and growths on the stem and cotyledons. Certain growths on the cotyledons appeared bud-like, but attempts to regenerate them on shoot-inducing media 51, 52, S3, and S4 (Table 5), failed. One plantlet had three first-foliar leaves. In addition, epicotyls of most of the embryos grew into abnormally long, spindly shoots primarily in the internode above the first foliar leaves. The first foliar leaves were well expanded and green, but the first trifoliolate leaves often remained rudimentary or even concealed by the stipules for an extended period. The cool white light regime (environment H, Table 4) was changed to include one half warm white fluorescent (envrionment J) because more red light might alleviate the problem (Borthwick and Hendricks, 1960), but elongation continued and the Spindly and fragile internodes began to break. Axillary shoots also began to grow in a similar manner. The shoot tips were excised (about 10 mn in length) and the cut end of each was pushed about 2-4 mm into new D4 medium. Eighteen primary and axillary cuttings were taken. These cuttings often continued to elongate such that excision of the tip was required as many as three more times. Eventually ten of the cuttings (from eight embryos) assumed a more normal growth pattern, rooted, and were transplanted to pots. Nine of these were transplanted to the 45 greenhouse over a period of 90-285 days from the original plating of the embryos. It has already been stated that no effect of cotyledon removal was detected for any of the hybrids. Nonetheless, a word about cotyledon removal seems appropriate here. Cotyledon removal has been reported to be helpful for hybrid embryos that have begun to deteriorate (Skirm, 1942). It has also been suggested (P. D. Ascher, personal communication) that cotyledons of some species contain inhibitors and that removal is advantageous for immature embryos 12.!1EEQ- In the present work, however, cotyledons were removed from all the embryos that were obviously deteriorated so that no control group existed. Within the group of embryos that had not begun to show deterioration and were otherwise treated alike, no significant difference in the ultimate rescue of plantlets was detected among cotyledon treatments. In the second cross of these species, using an intraspecific F1 V. radiata pollen parent (RR2 x RR1), seven ovules were obtained from a 14-day-old pod. Cotyledons were normal in appearance. Two of the embryos grew in a very deformed manner and the rest had minor deformities and/or callus growth. Nonetheless, six embryos (the largest six for all measurements) were transferred to larger vessels. All of these displayed abnormal elongation. The cutting procedure was applied to six primary and one axillary shoot tips. One of these was lost to contamination and one failed to root, but five (originally from five different embryos) were transplanted and reached the greenhouse in an average of 114 days from initial embryo excision. In the greenhouse, hybrid plants from these two crosses were phenotypically very similar. They were strongly matroclinous for 46 indeterminate growth, but slightly less so for the bright yellow color of the standard. Leaves of the hybrid plants appeared slightly narrower than those of either parent. For other traits, the hybrid plants were intermediate between the parents. The intermediate characters showing the greatest influence of the paternal parent were the leaf and stem pubescence, the greyish-green cast of the keel, and the extension of the keel beyond the wing petal (Table 12, Figure 3). Root tip squashes performed by G. R. Bauchan revealed a somatic chromosomal number of 33. Although flowering was profuse, these hybrids failed to retain pods for more than a day or two. Some particularly interesting features were observed during the develOpment of this hybrid. Pods of this cross frequently reached maturity with no external signs of degeneration. Dana (1965b) also obtained mature pods with well-developed seeds, but these contained only shrivelled, non-viable embryos. During the course of the present investigation, it was found that the axes of embryos excised 15 or more days after pollination appeared to have elongated and pushed the plumule of the embryo into the seed coat. The lack of mature viable seeds in this cross appeared to be more a case of abnormal growth than arrested development. Even_ln_Vit§9, abnormal excessive develOpment was the problem. Embryos 12.11929 demonstrated high (100%) viability, but the internode just above the first foliar leaves continued to elongate. Other difficulties included callus and growths on the stem and cotyledons, coiling of the shoot, and slow devel0pment or suppressed initiation of the first trifoliolate leaves. These observations suggested a hormonal imbalance in the embryo and young plantlet, possibly an over-supply of gibberellin and/or auxin (Cionini et al., 1976; Mok et 47 .Acewuececem an ecu Eeew mmcwuuze e ece .muce_e Ne w .mecewa He e Eecwv meezoww m :0 women mew mew can some somNN NH.o.H N.e xeem-:mmem saw: 3e_—wx zepwm» ewea mews; acm> was >0 N_eeoee oe e_oe_ee eue:_5eeuee :aHNN xecmuceeca sew: 3ewpex ze_Pe» e;m_ee xcwm; x—mumgmvee eum>e apemeea ea mum>o muecwseepeecw mm.o.u N.H :emcm ugmwwm gpwz :owwe» zop_e» e;m_ee Ne_e; N_e;m_.m cwou_ee xwwmcewmmoue epe>o xweeogn xwwesm: mum:_ELmumecw NAEE cwv cowmceuxm _mmx Lewoe _eex cemeceum we eepeu ewe: eucmumeaae wemA eqegm umpeuepn wecwsgow zuzoem e>wueueme> mueweme um “emcee Feceeuee e_ee»: mceummenQNm a» acmcea peaceuez ewemwemuuecegu .Afiau>v e_eeN; owwwememcmucw ewes“ use .Afimmv .H w cecwmw. eumwemc .> .Aou>>v mcmummcnmwm .m.wo muwumwcmuumcmzu .NH m_new 48 Figure 3. _V. glabrescens (VVGG), V, radiata (RR1), and their F1 interspecific hybrid (VGRl). A. Left to right: Plants 0f.¥° glabrescens (VVGG), F1 interspecific hybrid (VGRl), and V, radiata (RR1). B. Left to right: Flowers in the same order. Note the extension of the keel beyond the wing petal. 49 50 al., 1978; Murashige, 1961; Rappaport et al., 1950; Solacolu and Constantinesco, 1936). If excess gibberellin is the cause of these phenomena, application to the maternal parent of an inhibitor of gibberellin biosynthesis such as AMO-1618 that can be translocated into developing seeds (Marth and Mitchell, 1961) may allow recovery of this hybrid without the need of embryo culture. If excess auxin is the problem, it may be possible to facilitate this cross by use of an auxin antagonist in vitro (Newcomb and Wetherell, 1970). V. radiata x V. glabrescens An intraspecific F1 0f.!- radiata (RR2 x RR1) was also used as the maternal parent in a cross with V. glabrescens. Ten ovules were obtained from a pod harvested 14 days after pollination. The embryos appeared "calcified"; that is, more dry and Opaque than other embryos. Two embryos failed to grow, and the other eight grew as mostly disorganized callus. At five weeks, one shoot tip was excised from the embryo obtained from the largest ovule. It rooted, developed normally, and was transplanted to the greenhouse in 83 days. The other embryos, all heavily callused, were transferred to $1, $2, or $3 medium and moved to continuous light (environment K). The calli were subdivided occasionally (with any brown callus being scraped off at that time) and transferred to fresh medium. After 80 days on the 52 medium, one of the pieces (from an embryo above the median in all measurements) began to produce shoots. Ten of these were excised over the next six weeks, moved to 04 medium, and back to a diurnal light regime (environment J, then H). Nine of these shoots developed into plantlets for potting, but only eight were potted. Six of these were transplanted to the greenhouse after an 51 average of 226 days from initial culture. Mature hybrid plants of this cross were similar to the V. glabrescens x V, radiata plants already described. Average pollen stainability was 11% (mean based on 100 grains from each of six flowers) and the hybrid plants failed to set pods. Other workers have obtained mature viable seeds from this cross, but at low frequency. Chen (unpublished data) reared one hybrid plant to flowering from a mature seed. It was phenotypically similar to the hybrid plants obtained in the present study. Dana (1965b) obtained 22 shrunken seeds from 250 pollination attempts, but only one mature hybrid plant which died soon after flowering. Using an autotetraploid of V. radiata, Krishnan and De (1968) obtained a number of seeds, but only one mature F1 plant. They successfully backcrossed that hybrid to V. glabrescens and to the V, radiata autotetraploid, obtaining a few viable seeds and backcross plants in each case. The observations that most of the hybrid embryos in the present work grew as callus, and that shoots were obtained from one callused embryo after transfer to a medium containing cytokinin, suggested that the endogenous auxin level may have been supra-optimal or that cytokinin was sub-Optimal in the hybrid embryos. Nesling and Morris (1979) suggested that reduced levels of cytokinin in ovules of interspecific Phaseolus hybrids may be causally related to abortion of the hybrid embryos. The addition of cytokinin to the medium in the present study may have optimized the auxin-cytokinin balance to allow formation of shoots (Skoog and Miller, 1957). 52 V. glabrescens x V. mungo From the cross of V. glabrescens x MM1, four normal pods, 15 days after pollination, provided 11 ovules. Growth was weak and spindly in one embryo, but proceeded normally in the other ten which reached the transfer stage within three weeks. Two of these plantlets, transferred to a possibly-defective medium (dD4), succumbed. Of the eight embryos 73h transferred in the standard manner, one ceased growth, another produced a gag ~ spindly shoot, and six were aseptically potted. A total of five plants were tranSplanted to the greenhouse in an average of 129 days from plating. Hybridity was obvious because of the wide divergence of the parents. The hybrid plants were less erect than either of the parents, favored the maternal parent in indeterminate growth and leaf pubescence, were like the paternal parent in that some of the leaflets were acuminate, and intermediate for other phenotypic characters (Table 13, Figure 4). In addition to the gross differences in phenotypes, the difference between hybrid and maternal plants was significant for the leaf characters of length, width and l/w ratio (Table 13). The hybrid plants flowered profusely, but pollen stainability was only 0.3%, compared to more than 95% for the parents. The few pods that were set abscised within three or four days of flowering. Dana (1968) attempted the reciprocal cross of these two species and obtained partially filled but viable seeds. Eight of these plants flowered, but there was no pod set and average pollen stainability was only 0.8%. After treatment of shoots with colchicine, however, Dana obtained a few viable seeds. These progeny reached flowering and had 53% pollen stainability. 53 .meezeww m we gene Eecw meweem ooH we uceee e :e eemee mw come seem» .eeu_e>.u-_eewe>.uw ce emmee meepeegegu m _Pe Lew mewueem _ecceuee use Eeew ucecewwwe umeew we: uezu “cope use we ewes» wee eeueeeee meewe> He ecw .mceueeeecu amaze Lew mace—e weeeeues Eeew acmeewwwe x_uceu_wwcmwm wee: vexeesu mueewe ewce»; seem .meeuuecegu weep ace Lew cezue some Eecw aceeewwwe Ame. u av aweceuwwwcmwm wee: eexuecem mace one we mweeew>wecw .eeuceeeeu we: mcwcezeww Lepwe ucewe zeee we :ewueee weeucee esp Eecw :exeu Awe eeew e .Ne Eecw N .He Eecw ev me>em— ce eemee New mew ecu sees :uewu N e: use .eecemees ewe: eexuecem gene we mweeew>wecw ezw m.me . m.e xewe: xweeeeeeee e» xwugmwwm zcwe: Awugmwpm eH.e.nwe.N me.e “we.e men: Shem e.e + me e.N + we mcwcwzp Le peece acwwweee op eemLeueem m.we Newe; »_eee__m co. w. 0. NNO menacece eoeem-e:m new: eueem wage »e__eeee_eem ee__ee eeceemeeee wee; o_eee eee_z\eeeee_ Ase ewe ;He_z Age.=_e eeeee_ Nee_eee_e _ee_eLew gwne: euecwsceuee euecwsceueecw euecwELeueec_ cezeem e>weepeme> emcee a» ewes»: mceemmeeemmuw uwumweeueecegu eeeeee wecgmeee pcecee _eceeue: .Aeze>v ewes»: uwwwewemeeucw ewes» use .Afizzv .mnw. emcee .M..Aoo>>v mceemmceewm .M.we muwumwceueecegu .mH e_eew 54 Figure 4. .V. glabrescens (VVGG), V, mungg (MM1), and their F1 interspecific hybrid (VGMI). A. Left to right: Plants of V. glabrescens (VVGG), F1 interspecific hybrid (VGMl), and V. 92339 (MM1). B. Left to right: Leaves in the same order. 55 56 (V. radiata x V. umbellata) amphidiploid x V. umbellata The amphidiploid, RRUU62, and the diploid, UU1, were hybridized. Thirty-three embryos were obtained from seven abscised pods 11-17 days after pollination. Cotyledons of these embryos were poorly developed. Twenty-one embryos initiated growth, but seven succumbed to callus and/or deformed growth, and the remainder ceased growth before the shoot 3% exceeded 5 mm in length. (in This backcross would lead to introgression of V. radiata genes into ‘V. umbellata, the less cultivated of the two species. Nonetheless, 1. since V. umbellata is a crop plant in China and India and because it produces fertile hybrids with V. angularis (and thus is a potential bridge from V, radiata to V. angularis), introgression of V. radiata genes into V. umbellata could be valuable. The single most important barrier observed in embryo culture of this hybrid was failure of the embryos to develop beyond a few millimeters in length. These embryos were very small at excision, and van Overbeek et al. (1942) suggested that very young embryos may be unable to synthesize an adequate amount of some endogenous growth regulator(s) or other substance(s) to maintain growth. They found that progressively younger QEEEEE embryos required progressively more complex media to grow to tranSplantable seedlings. Went (1954) suggested that factors needed for growth and differentiation might diffuse into the medium and away from the embryo so that an inadequacy develops even though the embryo might be able to produce sufficient quantities. Skene (1969) found that development of immature embryos (15-18 days after pollination) of .Eflgseolus vulgaris 'Hawkesbury Wonder' was restricted unless GA3 was Supplied in the medium. Although many hybrids were obtained in the 57 present investigation without the use of GA3, this particular hybrid may benefit from its presence in the medium. (V. radiata x V. umbellata) amphidiploid x V. radiata Three different amphidiploid X.¥- radiata combinations were attempted. From the cross of RRUU62 x RR1, sixty-one ovules from 11 pods b that had abscised 11-16 days after pollination were prepared for embryo culture. Minor abnormalities were noted, such as two embryos that had three cotyledons and 16 embryos that had only one cotyledon. Growth 1 1 Eu commenced in 44 embryos, but soon ceased. Four embryos were prematurely transferred to fresh media (dF1, dF2, dF3, or dF4) in larger vessels in an effort to overcome the barrier, but to no avail. Eventually some of the 40 remaining embryos resumed growth and 12 reached the transfer stage. Nine of these were transferred to fresh media (05, dF1, dF2, dF3, or dF4) in larger vessels, and the other three were transferred to larger vessels without renewing the media. Four were eventually transplanted to pots, one was lost to mechanical error, and seven ceased growth a second time and eventually died, possibly due in part to the defective media. The first of the plantlets to be transplanted to a pot died, so the other three were potted aseptically, and survived to flowering. Average time from initial plating to establishment in the greenhouse was 153 days. RR2 was also used to pollinate RRUU62. Four abscised pods provided 18 ovules. Cotyledon abnormalities were again noted, both in number and in size. (Two embryos had only one cotyledon and a third had three; the cotyledons of embryos that had two cotyledons were often very unequal in size.) All 18 embryos began to grow, but most failed to develop roots. About one third of the embryos exhibited a slight degree of callus and/or 58 deformed growth. Four embryos were transferred prematurely to new medium (dF1, dF2, dF3, and dF4) in larger vessels and succumbed. Only two of the 14 remaining embryos reached the necessary size for first transfer. These were eventually transplanted to the greenhouse in an average of 128 days from initial plating. A second amphidiploid, RRUU87, was crossed with RR1. Abscised pods I; 8-14 days after pollination provided 22 small, but normal-appearing T? embryos for culture. Of the 19 embryos that grew, two became slightly deformed and none attained more than 3 mn of shoot length. The five hybrid progeny of RRUU62 that reached the greenhouse had either RR1 or RRZ as the paternal parent. The two groups of hybrid plants were phenotypically similar to each other and were intermediate for nearly every character in which the parents were different (Table 14, Figure 5). The growth habit of the hybrid was more similar to that of the maternal parent, but the leaf and pod pubescence and the floral color and structure of the hybrid showed the influence of the paternal parent. The leaves and pods were more coarsely pubescent in the hybrid than in the maternal parent. The green and grey coloration of the keel, as well as the keel's extension beyond the wing petal, was greater in the hybrid than in the maternal parent. The hybrid was triploid (33 chromosomes) as determined by root tip squashes performed by M. Machado. After about a month of flowering, these backcross plants began to set pods, both by natural self-pollination and by backcrossing again to V, radiata. Many of these pods, especially the first ones to set, abscised within two weeks but did provide some embryos for culture. Some of the pods that set later reached maturity and produced viable seeds. The successful back-crossing of the amphidiploid only to its 59 .meezeww N we gene eeew mcweem cewwee ooH we aceeu e :e cemee mw ewes seem» x .Acewueeecea mmeeexeee ecu seew mmcwuuee e ece .mpcewe Ne e .mucewe He e Eeewv mcezeww m :e venue 1cm mew use gems gueNN xewe; xee> xgwe: xee> ea aweueeeees agwe; zwpgmw—m eeeeemeeee wee e.we e.eN m.~e afiue Newpweeeweem ee__ee Ne.e.w N.e NH.e.H N.m NH.e.H N.N NAee ewe eowmeeexe _eex zmgm1ceeem new: sewwex zeem1ceegm new: zeppex :eeem mpeceees new: zewwez Leweu weeg sewwez zeppex ewee pgmwee xweee: e» wpee zeppex pguwca eeeecepm we Lewee cwez wgwe; xgm> xgwe; zgm> xgwe: xge> e» xczee mesmemeeee wee; ueeee mcwcwzu awepeeeees mewcwzp aweuegeees awee: epecwELepee epecwsgepeeew “enzeEem euecweeeueecw penzesem guzeem e>wueueme> _mmmflmmu a» eweezz Neeemm ewowe_ewee5e ewemweepeeeeee pceeee Feceeuee “sewee weeeepez 1111111. 1. . HNememw eweeae mmewweeee ewes» eee .Afimmv .Hw eeewew. eeeweee .> .ANeeemme mewe_ewewee2e fleeew_eee= .> x epmweee .>VN we mewemweepeeeeee .eH e_eew 60 Figure 5. [(V, radiata X.!- umbellata) amphidiploid] (RRUU62), V, radiata (RR1), and their backcross hybrid (RUR621). A. Left to right: Plants of [(V, radiata x V, umbellata) amphidiploid] (RRUU62), the backcross hybrid (RUR621), and V, radiata (RR1). B. Left to right: Flowers in the same order. Note the extension of the keel beyond the wing petal. 6| .-.. 1 . a ‘1 L?‘. '. L‘ Wl'iqefa..‘ ' <1, 1 71 ‘ 3; TJ91 ‘10-: (V. r x V. u)4x Hybrid V. r 62 maternal parent species suggested a possible cytOplasmic-genic interac- tion. The cytoplasm of the amphidiploid was that of V, radiata. The triploid obtained by backcrossing this amphidiploid to V, radiata, in which two haploid genomes of V. radiata and one haploid genome of V. umbellata are present in V. radiata cytOplasm, was successful. The backcross to V. umbellata, in which two haploid genomes of V, umbellata and only one haploid genome of V. radiata would be present in V. radiata cytoplasm, was not successful. Braak and Kooistra (1975) obtained similar results from backcrosses of their Phaseolus vulgaris xufi. ritensis amphidiploid to the two parental species. Nonetheless, the backcross to V. umbellata might be attainable by employing supplemental techniques, such as addition of GA to the culture mediun as discussed in the previous section. V. radiata x (V. radiata x V. umbellata) amphidiploid One pod was harvested 14 days after pollination fran the cross RR1 x RRUUB7 providing nine ovules for excision. The three smallest ovules appeared deteriorated as evidence by an overall brownish cast and a reddish hilum. The cotyledons were partially fused together in each embryo. No embryo growth was observed. Relatively few embryos of this cross were plated and all were from the same pod, so the cause of germination failure cannot be ascertained. Since some of the ovules appeared deteriorated, it is possible that the embryos had already atr0phied in vivo prior to excision. Taira and Larter (1978) reported that wheat x rye embryos older than 14 days after pollination rapidly atrOphied in vivo and responded poorly in vitro. 63 (V. radiata x V. umbellata) amphidiploid x V. radiata, BC2 It was mentioned in a preceding section that the allotriploid individuals produced by backcrossing RRUU62 to RR1 were backcrossed again to V, radiata and that the first of these pods to set abscised before maturity. Therefore, embryo culture was employed until these allotri- ploids began to produce viable seeds. From a cross using RR21 as the paternal parent, two embryos were excised from one abscised 19-day-old pOd. Neither embryo developed ifl.!i££9- When RR1 was used as the paternal parent, many pods that set began to abscise nine to 14 days after pollination. Fifty-four embryos were obtained from 19 of these pods. The cotyledons of one embryo appeared fused together, and another embryo had only one cotyledon. About one third of the 26 embryos that grew had some degree of callus or deformity, two were slightly spindly, and the rest were normal. Seven develOped sufficiently (in an averge of 22 days) to permit transfer to larger vessels. Two of the seven plantlets were placed on le medium and succumbed. Two of the other five that were transferred reached the potting stage, but only one survived to flowering. Ninety-four days elapsed between initial plating for embryo culture and transplanting to the greenhouse. In the greenhouse, phenotypic characters of V. umbellata (brighter yellow flowers and more indeterminate growth) were still evident in this second-backcross—generation plant. Flowers were abundant, but pods, if set, abscised within a couple of days. A range in morphological characters and fertility levels were observed in plants that were derived via viable seeds fron backcrosses and self-pollination of the same allotriploid (Chen, 1980). M. Machado 64 performed root tip squashes on a number of these plants and found them to be 2n=2x=22 and 2n=2x+1=23 for chromosomal numbers. The jg_Vitrg_recovery rate of these embryos from the triploid was very low. It is likely that many of the embryos in the abscising pods were aneuploids of various sorts, and less vigorous due to chromosomal imbalances. .k ‘ ._ ‘. . 1". '" r 11 (V. radiata x V. umbellata) amphidiploid x V. glabrescens An F1 hybrid plant between the two different V, radiata x.V. umbellata amphidiploids, RRUU62 x RRUU87, was hybridized with V, glabrescens. Pods that set began to shrivel about 15 days after pollination, so one pod was harvested at 13 days which yielded nine ovules. The embryos appeared normal except that the two cotyledons of each embryo seemed to be partially fused together. All nine embryos grew normally at first, but two turned chlorotic so that only seven were transferred to larger jars. Five were transplanted to pots and then to the greenhouse. The average time of this culture period was 127 days. The five hybrid plants grew vigorously in the greenhouse and were intermediate between their parents in general appearance (Figure 6). However, the five plants were phenotypically different from each other. Greater detail is given for one of them in Table 15. This slight variation among individual hybrid progeny can be explained by the fact that the maternal parent was an F1 between two different RRUU amphi— diploids and was heterozygous. One of the hybrid plants exhibited an anomaly in the growth of some of its shoots, resulting in a twisted and sometimes a "sheared-looking" stem. It appeared to be caused by a partial obstruction of the growing point by the petiole of the 65 .mcezeww N we seem seew mcweem sew—ea ooH we uceee e no women mw sees seem: x .Apcepe ewcexg ewwweeemeeucw eco ece .mucepe Ne e .mueewe He e Eeewv mcezeww m :e eemee 1em muw ece cums :eemx .eeeeeume pee wee: mecewe eweexg Lecue Eeew eueo .zpwwweecwepm cewwee Lew weeexe zwce Hw peewe ewee»; Lew wee mama» .cewpeeecmm “u use we was“ mw newzz :ewmceexe wee; peeexe .eewuegecem Nu ecu we peg» mw.wregee _eccewme esp Lew ce>wm :ewueweemeo .acegee Peceepes esp we em>cem newe—ewewgese Aeaewpeese .> x eueweeg .>v exp ewes» cmezpme an esz e.e "Nw eeewe m.em m.eH new agape e.mm zANe Newpweeeweem eepwee Se H me 85 H m; :e H 2 LE .5 85:33 33. pzmwpm eueweeEeeucw eueeeeee museumeeee wee; meeemeeeene a» aeweewz NAeeeemm x message eeemweeeeeeeee uceeee Penceuee uceeee weeeepez .1 .111wmwwmmmeweee eweewe ewwweeemeeeew ewes» new .Aco>>v mceomegeeflw .> .AwNmmzammv Hewewewewcese Aeuewweese .> x eueweeg .>vu we muwumwcepuegegu .mH ownew 66 Figure 6. [(V, radiata x V. umbellata) amphidiploid] (RRUU6827), V. glabrescens (VVGG), and their F1 interspecific hybrid (Ruvogg-gz). 67 68 newest leaf bending back over the shoot tip. It was not a serious problem, however, as the shoot tip usually out-grew the condition after a few nodes. Two of the hybrids were placed under short day conditions and flowered, but failed to set pods. Pollen stainability was 10% and 6% for these two plants. Pollen stainabilities for the paternal parent and the F2 progeny of the maternal parent were 90% and 58% respectively. Dana (1964) found that one genome of V, glabrescens was homologous with the genome of V, umbellata. Although a cytogenetic analysis of the present hybrid plants was not performed, the low fertility suggested that the other genome of V, glabrescens is not homologous with that of V, radiata, unless the low fertility is due to genic rather than chromo- somal factors. Krishnan and De (1968) found a maximum of eight V, radiata chromosomes homologous with those of V, glabrescens. V. radiata x (V. umbellata x V. angularis) The intraspecific F1 hybrid of RR1 x RRS was crossed with the F1 interspecific hybrid of UU3 x AA1. Five ovules were obtained from one 17-day-old abscised pod and one 16-day-old pod near abscission. Four of the embryos grew. One produced a shoot of a few millimeters and then ceased growth. One grew normally, but as the root system deteriorated, the shoot tip was rescued and rooted. The other two grew spindly shoots, so shoot tip cuttings were taken of these also. All 3 shoot tips rooted and were transferred to larger vessels, and one plantlet was transplanted to a pot; however, all plantlets were weak and eventually succumbed. This three-species cross was attempted to overcome the incompati- bility between V, radiata and V, angularis, and to possibly (remotely) produce a fertile diploid incorporating a genome of V, radiata and some 69 chromosomes of V. umbellata. Chen (1980) cited previous successful attempts to overcome interspecific incompatibility by use of third-species bridges; Chen himself successfully used V, radiata var. sublobata as a bridge between V. radiata var. radiata and V. umbellata, obtaining 25 to 45 times more viable seeds per 100 attempts than by crossing V. radiata var. radiata x V, umbellata directly. In the cross of (V, umbellata x V, angularis) x V, radiata, he obtained pod set but no viable seed. The reciprocal of that cross, undertaken here, provided some embryos for culture but none reached maturity. The few embryos cultured in vitro indicated a general rather than any common, specific weakness. Perhaps a greater number of embryos from the same cross, or the use of different parental cultivars would allow successful recovery of mature plants. _(V. umbellata x V. angularis) x V. mungo The F1 interspecific hybrid UA31 was crossed with M83. One attached 15-day-old pod and one abscised 14-day-old pod provided seven ovules. Three embryos failed to grow and the other four ceased after attaining a few millimeters of shoot growth. Two had expanded the first foliar leaves, but none develOped roots. All embryos that grew had a - slight degree of callus. Crossing either V. umbellata or V. angularis in any combination with .!-.EEESQ appears difficult as indicated by the number of unsuccessful reports. Many attempts to produce these combinations (Ahn, 1976; Ahn and Hartmann, 1978c; Biswas and Dana, 1975a; Chen, 1980) have yielded only one mature plant from V, myngg_x V, angularis (Chen, 1980) and 12 individual plants 0f.!-.EEEEQ x_V. umbellata (Biswas and Dana, 1975a). 70 As discussed in the previous section, a three-species cross was considered worthwhile. Although some viable embryos were obtained for culture, none reached maturity. The problems encountered in XIEEQ with the present cross are similar to those that Skene (1969) observed and was able to remedy with 10'4M GA3. General Considerations and Summary The first step in the procedure of embryo culture is the selection of material. One is naturally inclined to allow the interspecific embryo to reach its maximum normal develOpment_ig-V1Vg, yet care must be taken to rescue it before the onset of atr0phy (Taira and Larter, 1978). Although experiments were not performed during the present study to determine the Optimum age for embryo excision, some observations were made that illustrate the relationship between age or embryo condition and ultimate recovery, and these could be helpful to future interSpecific hybridization programs. First of all, it was observed that some hybrid combinations (such as VVGG x RR and RRUU x VVGG) failed at a particular point in time after pollination, and could, therefore, be harvested just before the onset of atrophy according to a calendar schedule. Other crosses (such as RR x UU, RRUU x UU, RRUU x RR, and RUR x RR) had a broader range of time over which the pods began to abscise or show other signs of failure. Frequently, embryos were successfully rescued and grown into mature plants if secured the day the pod abscised or began to show signs of degeneration. However, this criterion of visible pod degeneration was not always a reliable indicator of embryo condition. This was most evident in the cross V. glabrescens x V, radiata in which pods reached 71 maturity with no external signs of degeneration although the embryos began to decline about 15 days after pollination, as could be seen immediately upon embryo excision. Although embryos of many ages were grown in culture, due in large part to confounding with other factors, specific age effects ingiggg were generally not clear. As has already been mentioned, 14- to 16-day-old embryos of RRUU62 x U01 outperformed those of 11 and 17 days, but this difference might have been due to media differences. In the backcross of RUR621 x RR1, the 12-day-old embryos did not perfonn as well as those only nine days old, but no other age effects for nine- through 14-day-old embryos of this cross were apparent. There seemed to be a difference among pods in that the percentage of germination of the embryos from five pods was higher that that of two other pods in the cross RRUU62 x RR1. One of these pods whose embryos performed poorly was older than the others (16 days after pollination vs 11 to 15) suggesting that age might be responsible for part of this pod effect. However, the other pod whose embryos performed poorly abscised 12 days after pollination, while embryos from both 11- and 13-day-old pods performed quite well. So this pod effect, if real, might have been due to conditions of the maternal plants in the greenhouse. In the cross of RUR621 x RR1, a number of ovules were beginning to shrivel at the time of excision. Not surprisingly, the performance of embryos from plump ovules appeared better than that of embryos from shrivelled ovules (.84 vs .14 on the 1 to 4 scale). There appeared to be a positive linear correlation (r=.67) between ovule size and recovery of embryos from the cross of RR x VVGG. It is interesting to note that the ten embryos plated were from the same pod, 72 and represented quite a range in development, as judged by ovule size. (It was not noted whether placement in the pod was correlated with ovule size.) The embryo that grew to maturity only after having been placed on cytokinin-containing medium was from the second largest ovule (actually tied with three others for this rank) and the embryo that grew to maturity without exogenous cytokinin was from the largest ovule. Perhaps the ovules on the next step up in size are those few that are capable of going on to devel0p into viable seeds. Size factors of the embryos themselves also seemed to be linearly correlated with recovery in the cross of AA1 x RR1. The correlation coefficient between cotyledon size and the growth score obtained by the embryo 19.21222 was .95 and the correlation between the total embryo size (cotyledon + hypocotyl) and growth score was .89. However, some factor other than age must have been responsible for the degree of development obtained in XIXQ since no linear correlation between age and these two size factors was apparent. In two other hybrid combinations, embryo size factors and recovery of the embryos in liEEQ appeared curvilinearly related. In the cross of VVGG x MM1, the coefficient of determination was .81 for the quadratic regression of final score on cotyledon size (Figure 7). The coefficient of determination was .59 for the quadratic and .92 for the cubic regressions of final embryo score on total embryo size (cotyledon + hypocotyl) for the cross RRUU x VVGG (Figure 8). The existence of a peak or optimum size for recovery of these two hybrid combinations suggests that time of excision is critical -- too soon and the embryos are insufficiently developed to survive -- too late and the embryos, though larger, have begun to atrOphy. 73 Figure 7. Relationship between cotyledon size and recoveryfl vitro of hybrid embryos of V. glabrescens x V. mungo (Y = ~351.19 + 192.61X - 26.07X2; r2=0.81) Figure 8. Relationship between total embryo size and recoverylnm of hybrid embryos of (V. radiata x V. umbellata) amphidiploid x V, glabrescens (Quadratic: v = -59.53 + 74.97x - 22.1ox2; r2=o.59. Cubic: v = -677.28 + 1175.7ox - 669.89X2 + 125.87X3; r2=o.92). GROWTH SCORE GROWTH SCORE 74 l l l I J l l j l 3.2 3.4 3.6 3.8 4.0 4.2 COTYLEDON SIZE (mm) L _ l l j J j J I I 1.3 1.5 1.7 1.9 2.1 2.3 TOTAL EMBRYO SIZE (mm) 75 Knowledge of these relationships observed between ovule or embryo condition (plumpness and size) and ultimate recovery in XIEEQ are really not very useful in an interspecific breeding program where few embryos are obtainable, unless a non-destructive 12.!il9 measure to determine ovule and embryo condition exists. While age is a useful measure in determining excision time for hybrids that have a predictable growth rate or a particular time of breakdown, another monitor would be useful for those hybrids that do not have either of these characteristics. Although Savithri et al. (1978) did not mention whether or not there was a significant correlation between pod diameter and seed fresh weight in their study of V. radiata develOpment, their figures indicate that development of these two characters are nearly parallel; thus, measurement of pod diameter might prove to be a good, non-destructive monitoring technique to help determine the Optimum time for excision of hybrid embryos. The presence or absence of the suspensor was not often found to have an effect on recovery. In the cross of RUR621 x RR1, however, embryos with suspensor intact appeared to outperform those without suspensors (1.2 vs 0.5). On the other hand, the suspensor-deprived embryos fran the ten-day-old pod of the cross AA2 x UU2 seemed to outperform those with suspensor intact (0.83 vs 0.0); this relationship did not hold for AA2 x UU2 embryos overall (both ten- and 11-day-old embryos). Although these results appear somewhat inconsistent, and the observed inhibitory effect Of the suspensor seems to be in conflict with other work (Cionini et al., 1976; Yeung and Sussex, 1979); the fact that some inhibitors are indeed present in young bean suspensors (Alpi et al., 1975) offers a possible explanation. It may be that the suspensors of AA2 x U02 produced 76 inhibitors (or transported them from maternal tissues) in the early stages 0f.ifl.!i!9 development, which would normally have been deactivated a little later_ianng. Suspensors of embryos excised at ten days may have been deprived of the Opportunity to deactivate this hypothetical inhibitor while the suspensors of embryos excised at 11 days did have the Opportunity to deactivate it. A study of the possible role of these inhibitors in interspecific hybrids might be profitable. Embryos of a few of the hybrid combinations were cultured in ViEEQ and established in the greenhouse without any particular difficulties. More Often, however, each hybrid combination exhibited one or more characteristic problems_ifl-Vitgg that made the recovery procedure more difficult, decreased the number of plantlets that might otherwise have been obtained, or entirely prevented successful recovery. The most comnonly encountered difficulties included low percentage of germination, cessation of growth shortly after its commencement, deformity or callus, difficulty in initiation or expansion of leaves, weak root systems, and problems in acclimation to a potting mixture. No treatments were applied specifically to the problem of low germination when it was encountered (RR1 x RRUU87, UU1 x AA1, and RUR621 x RR); however, all but the first of these hybrids were recovered by plating a greater number of embryos. In an attempt to overcome the problem of cessation of growth shortly after germination, a few embryos of the cross RRUU62 x RR1 were transferred to fresh media. This treatment was not found to be effective, but it was later discovered that the fresh media to which they had been transferred were probably defective. Consequently, the usefulness of transferring to fresh medium to remedy the problem of 77 cessation of growth needs further investigation. Some causes and other possible remedies for this problem were posed in the discussions of the crosses RRUU x UU, RUR x RR, and 0A x MM. Deformity or callus were common maladies for several combinations (AA x UU, MM x RR, MM x UU, VVGG x RR, RR x VVGG, RRUU x UU, and UA x MM). One embryo of AA x UU eventually outgrew its callus and deformity. A cutting procedure was successfully applied to the abnormal elongation problem of V. glabrescens x.V. radiata. Heavily callused embryos of RR x VVGG were transferred to media containing cytokinin and one of these embryos subsequently began to produce a number of shoots that were then excised, rooted, and transplanted to the greenhouse. The discussion of MM x 00 and RRUU x 00 also include comments about possible causes and remedies for deformity and callus. Some of the hybrid combinations, notably AA x UU, NM x AA, MM x UU, VVGG x RR, and UA x MM, had difficulty in expanding or initiating leaves. The embryos of VVGG x RR were incubated with an increased amount of red light (supplied by warm white fluorescent tubes) in an effort to increase leaf expansion. Many of these did eventually expand leaves, but no conclusion can be drawn from this since a control group of embryos for this hybrid (incubation continued in cool white only) did not exist. It should be added that many other hybrid combinations had no difficulty expanding leaves under cool white light, and even some that had initial difficulty eventually expanded leaves without changing the light regime from all cool white light. The use of 0A3, adenine, or grafting have also been suggested to increase leaf expansion in the discussions of AA x UU and MM x AA. The root system of one embryo from the cross RR x UA developed 78 then deteriorated. By taking a shoot-tip cutting a new root system was established. Three hybrid combinations (AA x UU, RRUU x RR, and UA x MM) exhibited weak root systems_in-Vitgg. This did not seem to be the main limiting factor for any of these hybrids, but GA3 and grafting are mentioned as possible remedies for it in the discussions of MM x AA and UA x MM. The last step in the rescue of hybrid plants by embryo culture is adapting them to the £5.11EE9 environment. A number of individual plants from the cross RR x UU were lost to dessication and one of the plantlets from the cross RRUU x RR succumbed to what was assumed to be damping off. A humid, aseptic potting and acclimation system was developed and proved successful. Nonetheless, plants of AA x RR and RR x UA failed to acclimate under this system. These failures could be attributed to occasional flaws in the system (micro-organisms were isolated from one supposedly-aseptic pot) or possibly to inherent weaknesses of the particular hybrid combination to advance beyond the young seedling stage. All of the interspecific hybrids that survived acclimatization grew vigorously upon transfer to the greenhouse. All of these plants survived long enough to reach flowering but some individuals were maintained at non-inductive photoperiods to maintain them vegetatively for asexual propagation. Because of the low number of individual plants of most combinations obtained, it was necessary to propagate asexually many of the hybrid plants (six of the ten interspecific hybrid combinations obtained) to maintain them for further investigations and to increase them so as to obtain a greater number of progeny from those hybrids that were fertile. Although colchicine doubling was beyond the scope of this 79 project, it is a logical next step in advancing sterile hybrids. Asexual propagation would also be useful to facilitate the colchicine procedure. Asexual propagation by cuttings was routinely and successfully performed for these interspecific hybrid combinations. However, asexually propagated hybrid plants differed in their vigor. If at least one parent of hybrids involving two species was strongly indeterminate, then problems related to vegetative vigor were not encountered in plants grown from cuttings. However, in the backcrosses where the parental determinatezindeterminate ratio was 2:1, both the number of cuttings that could be obtained and the vigor of these cuttings were restricted. Furthermore, the photOperiod sensitivity of V. glabrescens and V, umbellata parents allowed the vegetative growth in some of the hybrids (particularly UA31, VGU, VCR, and RVG) to be conveniently controlled by daylength. 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