OVERCOMING INCOMPATIBILITY IN WIDE CgOSSES

0. C. SASTRI and M. MALLIKARJUNA

Wild rpacier of crop plrntr rttrrct w c h rttention rr r valurble gene pool. A few ruccrrrer in improving crop plrntr have rerulted from crorring vild trxr with cultivrted onar. Several other trxr are not crorrrble vith thrir cultivrted ralrtLver and rrr therefore unrvailrble for rexurl gene tranrfarr. Hethodr for breaking there barrier8 to interrpecific hybridirrtioa and hybrid production hrve been drveloprd.

The literature on exotic gelaplrsn rboundr with exrmpler of genetic introgrersion from wild tax. conventionrlly crorred vith their cultivrted relativer. But there ir r wealth of ~ e m p l r a that cannot be croraed with cultivatrd trxr.

The barrirrr to hybridizrtion were knovn evrn beforr chr present range of germplaam became avrilrble. During the lrrt five decades, intereet in incomprtibility hrr incrrrred to encompeee phylogenetic-taxonomic purporer and the genetic improvement of crop planto. While emerging somattc u t h o d r rhou promise, eexual aanlpulation is still the firut choice, and eexurl rnethodo have contributed significrntly to loproving cropr.

The geraplaem amenable to sexual manipulation ir dif'f icult to quantify. I t may be an insipnlficant proportion of axlrting germplasm. Evcn in the well-worked genue N i c o t l a ~ , of vhich about 65 species are knovn, only a little more than 300 hybrid8 have been realized. About 902 ot the croeeee in thir ~ e n u r have not. The situation i n ~ i m i l a r for moat other crops. A number of review artlclea and booke on tncomprtibility end method8 to break it are available (9, 15, 16, 1 7 , 10, 41, 49, 51).

CAUSES OF SEED FAII.URE IN INCOMPATItlLE CROSSES

The characteristic prefcrtilizat ion b d r r lera r nd pobtfertilizat ion breakdovrl oC zygote or embryo i n lncoapat ible crob;ae6 have been exterlsively described. Int~l bl t ion of pollen emin in at ion on rit1g.s and pollen tube growth through the rtylr,

Cytoqvwrt~c~st and rewatch asoc~ale. lnterwtwal Crops A w c h Imtrtutr f~ UU !hfnt+hd T ~ o p ~ a (ICRISATI, P a t a n m u P. 0.502 324, A. P.. I~dls.

failure to fertilize, and zygote growth cessation at 8-C' ecage of development are cotmaon to many failing crosses (41, 49, 56). Unfortunately, their molecular causes are little understood. Even 80, the fragmentary information available has led to development of method8 for breaking croesability barriers in a few taxa.

METHODS

Among the several methods used are recognition pollen, hormones, i~unoeuppreseants, and temperature. Early surgical methods are 8180 important (28, 37, 41). Delayed and bud pollinations have overcome both sporophytic r a i c spp.) and gametophytic (Petunia epp,) self-incompatibilities in a few taxa, but not any inter8pecif ic incompat ibil i ty. Thei r success depends on the stigma's receptivenera, i.e., buds of Arachis hypogaea even a day before antheeis are not receptive even to compatible pollen; so bud pollination does not work in A. hypogaea (43).

The discovery of growth hormones in plants inspired investigations on their potential in overcoming incompatibility, which is not mentioned in books and conferences on plant growth horwnee. The influence of exogenous and endogenous hormones on developing fruit has been 1nvestigat.ed in n o m l compatible (self) crosses but rarely in incompatible crosses. The efafects of different growth hormones on free pollen grains in in vitro cultures have been extensively studied (25, 26, 51). The effect^ of hormonee on compatible and self-incompatible pollen tube growth in pjstils of Lilium lon~iflorum (14) and a few other rpecies (9, 41) have been reported but the studies are too few tor conclueione on self-incompatible taxa, let alone interspecif ic ones. Hormone appl icat ions have, however, been found to enhance a few incompatible fruit and seed set crosses.

Crane and Harke ( 7 ) and Brock ( 5 ) first used hormones to delay floral abacission in incompatibly pollinated flowers with short life. Interest in use of hormones in incompatible crosses mubaequently grew. The latest hormone-aided hybridizations are inter8pecific crorses in Arachis, In a few cases application of gibberellic acid to the bases of incompatibly pollinated flowers stimulates the initiation and geotropic elongation of the peg, the aerial phase of Arachis fruit (Table 1; 43, 44, 45). Subaequent application of IAA, N U , or kinetin increases the number of pods (42) and cultured ovules and embryos (29 ) .

Over 20 intersp~cific or intergeneric hybrids have resulted from exogenour application of natural or synthetic plant growth h o m n e a (41). We have yet to understand how hormonee stimulate hybrid development from incompatible crosses. A comparieon of hormonal balances in ovaries between compatibly and incompatibly pollinated flowers is called for and specific effects of exouenoue hormones in successful cases need study. Nessling and brrir (32) found that endoeperm from an interspecific Phaeeolus cro.8 doe. not develop normally end has much less cytokinin than

Ssctlon Aruhrs x Soctton Rhrromarow A. hypowa cv Robut 33 -1 /Ar~ l1 ts sp. Cdl. No. 8648 A. hypogua cv Robut 33-1lArachrs sp. Cdl. No. 9787 A hyp- cv Robut 33. I/Arrchis sp. Cdl . No. 8808 A. hypo- cv TMV2/Arxhrs sp PI 276233 A. hypog.sr cv TMV2IArachn sp. Coll. No. 9849 A. hypoma cv MU 374/Arxhn sp. PI 276233 A. hypogmo cv MK 374lArachrs sp. Coil. No 8849 A h y w r cv M 131Aruchrs sp. PI 276233 A. h y w cv Ch~coIArach~s sp. PI 276233 A. hypog.sa cv ChicolArrchrs sp. Coll. No. 8649

that from selfed pollinetions. Such etudies not only help elucidate hormonal regulation of fruit morphogenenis, but a180 assist in formulating strategy for further work with incompatible crosses.

Recognition pollen

Since Tsinger and Pctrovskaya-Bananovd ( 6 2 ) showed "pollen grain walls [to be] physiologically active suhstuncetl," Interest in poetpollination changes in pollen grain^ and p i ~ r 1 1 )tau grown. I t is now be1 ieved that ext reccl lular (will 1 ) I I proteins coaununicate with extracellular (hurtace) s t l ~ r n d ~ pnpillae proteins: pol 1c.n grainu a r c thereby rrcoKrj1ztacl .in el ther compatible or irlcompatible ( I h , 17, 18). 'The. exlntrnce of pollen grain willls (16) and stigma tiurfilces 1 , ? ) with a protein component has been demonstrated in n number of taxa, and in a few, even stigmas of immature flowers are lined with proteins ( 5 2 , 5 1 , 50).

Knox et a1 (27) exploited this information to produce hybrids from an interspecific cross in Populus, tarller Stettler (57) had achieved interspecif ic hybriditat ion in l'opulue by mixing live incompatible pollen grains with gamma-irradiated (killed) compatible pollen grains called recognition or aentor pollen (27). Mentor pollen stlmlated incompatible pollen grain8 to produce fruit and seed.

Subsequently this method was tested in other taxa and in other systems of self-incompatibility and interepecific incompatibility. Sporophytic self-incompatibility vae overcow in Coswa bipinnatue ( 2 1 ) . Brassica oleraces (40), and Raphanur sativus (48). but not in Brassica campestris (48). Cametophytic self-incompatibility vae overcome in apple (81, Petunia hybrid.

(47, 48) end Nicotiana alata ( 5 5 ) , but not 1;-'Oenothera organensis; ( 5 5 ) . Recognition pollen did not work in other interspecific crosses in Sefiamum ( 4 6 ) , Arachis (43). Ipomoea (13), Trifolium (581, and Cucumis (10).

Sastri and Shivanna ( 4 8 ) have suggested that the method of killing the compatible pollen grains determines success. The recognition pollen can be prepared by storage at normal temperature, repeated freezing and thawing, and gama-ray irradiation until pollen grains lose their ability to germinate. In a few instances the recognition pollen function could be effected by leachates of compatible pollen grains (21, 48).

Pandey (33, 3 4 , 35) found that in Nicotiana gene transfers could be effected with jutit gamma-irradiated compatible pollen graine. Jinke et a1 (22) have confirmed this. Recently Shuzlkudo et a1 (54) combined this method with ovule culture to transfer an incomplete chromosome complement of Nicotiana tabaccum into N. ruetica, two species which are not otherwiee crooueble.

About 10 yr ago Batee and his coworkers began using in wide hybridization experiments animal immunosuppressants including E-aminocaproic acid (EACA), chloramphenicol, acriflavin, ralycylic acid, and gentioic acid. The euccess rate varied, but EACA wee the most effective: with EACA, hybrids were obtained in crosaeo between barley and rye, durum wheat and barley, and bread wheat and barley (3, 4). EACA also stimulated embryo development in crovoee between Triticum turgidurn and Secale cereale (60, 6 1 ) .

In another intervpecific cross, Vigna radiatn/V. umbellata, a foliar epray of FACA (100 ppm) effected twice as many hybrids ue those that resulted from the unsprayed controls ( 1 ) . Baker et a1 ( 2 ) obtained optimum results in the same cross by injecting 250 ppm of EACA. Chen et a1 ( 6 ) observed in two cultivars that foliar EACA spray for 14 d starting no later than the premeiotic stage of flower development delnyed but did not prevent embryo abortion. EACA applicatio~i to florets on 4 consecutive d after pollination reduced embryo recovery from 30 to 19%. but increased the number of ovaries forming embryo and endosperm in Triticum t imopheevi/Secele cercale (31). I t was not effective in Festuca arundinrcea/~ectylie glomernta (30).

OTHER METHODS

Embryo, ovule, and ovary cultures have yielded hybrids in more than 50 cromr combinations with early abortion of embryo or endosperm, or both (see 36, 4 1 ) . According to Johnston et a 1 ( 2 4 1 , when there are ploidy differences between the..parents, an abnormal endosperm ie caused by a deviation of the maternal and paternal genome ratio from 2 to 1 in the endosperm itself. By arrigning a epecific number (endosperm balance number) to the endoopera of each parent irrespective of its ploidy, and by

manipulating I : numbers, Johnston and Hannemn (23) h., succeeded in crossing s o w diploid Solanum species.

Ploidy manipulations are not new and a species with higher ploidy is generally better as a feamle parent. (Even when the parents have the same ploidy, one of them is raised to a higher level through colchicine treatment.) In other eituationr, a third species, itself crossable with each incompatible parent, is used as a bridge. Cultivar crossability differences indicate the need for a range of cultivars. Genes for crossability, ruch as Krl and Kr2 in wheat, should be investigated in other taxa. Hoot - - of these aspects have been reviewed by Reee et a1 08), Thour ( 5 9 ) . Driacoll (11). and Riley et a1 (39).

CONCLUSMNS

Several means for hybridization between lncompstible speciaa have been found. Until the somatic methods (e.~., 1 2 ) can find wider applications, theee methods, with or without modiflcationr, m a t be exploited. In some instances combining methode with ovule or embryo culture is needed. For example, in a few interrpecific crosses of Arachis, applying gibberellin to incompatibly pollinated flowers stimulated normal development of the pegr, and a few pods (Table 1) up to certain stager. Only ovules in tho pods were found to be immature, with poorly differentiated embryo8 ( 2 9 , 43, 44, 45). Ovules and embryos had to be cultured in vitro to yield plantlets ( 2 9 , 43, 44). Similarly, in interspecif ic crosses in Sesamum (46) and ('uc-umls, f 10) mentor pollen effected only part of the desired respon.sc8. For further development, another method must be d d o p t e d .

1ncomp.itlbility must be better u~~tlt.rwtt~oii bc.lorc. i t can be overcome and tt~r vild germpla~m exploited for 1 ur ttrrr improvement of crops.

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