Pollen pistil interactions and interspecific incompatibility in Pennisetum

Summary The causes of low crossability between Pennisetum species were studied. In the compatible cross P. typhoides x P. violaceum, the pollen tube growth was normal and there was good seed set. In P. typhoides x P. cenchroides, pollen tubes reached the ovary but no seed set was obtained indicating ovarian incompatibility. The stylar incompatibility was found in the crosses of P. typhoides with P. schweinfurthii, P. hohenackeri, P. orientale and P. squamulatum. In crosses P. typhoides x P. polystachyon and P. typhoides x P. pedicellatum, mostly pollen tubes did not grow beyond stigmatic surface and only few reached the style. Stigmatic as well as stylar incompatibilities seem to be the reasons for no seed set in these crosses.     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

Interspecific hybridization between rice bean (Vigna umbellata) and its wild relative (V. minima): Fertility-sterility relationships

Summary The pre-and post-fertilization barriers in the interspecific crosses between Vigna umbellata and V. minima were investigated. In the reciprocal crosses (V. minima as the parent) the entry of pollen tubes into the ovary was delayed by about 4 h, and no seed set was observed. However, no pre-fertilization barriers were encountered in crosses involving V. minima as the parent and V. umbellata as the parent (normal cross). The delay/absence of divisions in the endosperm and the failure of embryo to divide were the post-fertilization barriers responsible for somatoplastic sterility in normal crosses which yielded a few hybrid seeds. The hybrid seeds showed poor germinability. The F₁ hybrids were intermediate between the parents in most morphological characters, and are completely sterile for pollen. Backcrossing of F₁ hybrid with both the parents did not restore fertility in the progenies. V. minima is considered as the tertiary gene pool of the rice bean.     

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

Summary Germination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatum Lad. and C. cuneatum Rich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F₁ plants.     

The reproductive biology of okra. 1. Study of the breeding system in four Abelmoschus species

Summary The breeding system of Abelmoschus species is not well known. Although being self-compatibles allogamy reaches 63%. The aim of the study was to place four Abelmoschus species on the log P/O scale devised by Cruden (1977). On this scale P = pollen grains per anther × anthers per flower and O = ovules production. Pollens and ovules productions were therefore assessed for 68 accessions of two cultivated species, A. esculentus and A. caillei, and two wild, A. manihot and A. moschatus. Extreme log P/O values ranged from 1.69 for the variety Clemson Spineliss (A. esculentus) to 2.94 for ORS 278 (A. manihot). Averages of wild species A. moschatus (2.17) and A. manihot (2.19) indicate facultative autogamy. Averages for cultivated species A. esculentus (2.00) and A. caillei (2.05) suggest more autogamy.     

Elucidating the behaviour of pollen tubes in intra-and interspecific pollinations of Phaseolus vulgaris L. and P. coccineus Lam.

Summary The behaviour of pollen tubes in intra-and interspecific pollinations of P. vulgaris and P. coccineus has been examined using a combination of phase-contrast, and UV fluorescence microscopy. The results indicate that P. coccineus is self-compatible in terms of pollen tube growth, and that pollen tube inhibition is not responsible for the reciprocal differences in seed set which have been observed in crosses between these species.     

Effect of selection for pollen grain size on various traits in common bean

Summary Selection among microgametophytes usually exploits variation in pollen grain germination. Studies of variation in pollen grain size in common bean (Phaseolus vulgaris L.) suggested that selection for size might lead to changes in sporophytic traits. To determine whether microgametophytic selection based on size would affect pollen grain size in subsequent generations or sporophytic traits that were correlated with pollen grain size, pollen grains from three crosses were separated into two size categories by sieving and then used to pollinate cv. Diacol Calima. Selection resulted in changes in pollen grain diameter for pollen from F₁, F₂ and F₃ plants for all crosses. In vitro germination indicated no differences between vigor of large and small grains, but extraction and sieving reduced germinability. F₁ seed from two of the crosses with size-selected pollen varied in weight according to pollen grain size, but in subsequent generations, the effect disappeared. Both size categories of selected pollen resulted in F₂ progeny with reduced numbers of seeds per pod as compared to controls, suggesting that the size selection process may have resulted in indirect selection for traits reducing seed set. The overall results suggested that genes determining pollen grain size in bean have little or no effect on sporophytic traits such as seed size and seed yield.     

Effects of Temperature on Pollen Tube Growth and Fruit Set in Reciprocal Crosses Between Cucumis sativus and C. metuliferus

Interspecific hybridization between Cucumis sativus and C. metuliferus may be used to introduce disease resistance into the cultivated cucumber. Hybrid plant development, however, is restricted by strong barriers to crossing. The present study was undertaken to examine temperature effects on pollen tube growth and fruit set in reciprocal crosses between C. sativus and C. metuliferus. When crossing C. sativus×C. metuliferus, at 20°C, pollen tube growth was arrested in the stylar region of the pistil and no fruits containing embryos were found. However, at 23° and 26°C, C. metuliferus pollen tubes penetrated into the C. sativus ovules. The enhanced pollen tube penetration was correlated with improved embryo development. Although pre-fertilization barriers were overcome, post-fertilization barriers caused abortion of hybrid embryo development at the globular stage. No effects of higher temperatures were found in the cross C. metuliferus×C. sativus; Pollen tubes were generally arrested in the stylar region of the pistil.     

Pollen–pistil interaction in the inter-specific crosses of Sesamum sp

The wild species of Sesamum have long been recognized as an important source of many valuable pest and disease resistance genes and other novel traits such as male sterility and plant type for the cultivated taxon. Utilization of these species in crop improvement is marginal because of their inherent low crossability with the cultigen. An understanding of the biological nature of incompatibility systems that impede hybridization offers tools for successful introgressions into cultivated sesame. The objective of this investigation was to observe pollen germination and pollen tube growth in cross pollinated S. indicum pistils as an indicator of pre-zygotic barriers operating in wide crosses involving S. indicum. Three wild species: S. alatum, S. radiatum and S. laciniatum were used as pollen donors in hybridization with S. indicum. In the cross S. indicum × S. alatum 85.23% of pollen grains germinated and pollen tubes reached ovaries within 8 h after pollination. In other two crosses, S. indicum × S. laciniatum and S. indicum × S. radiatum the level of incompatibility was high as evidenced by a reduced pollen germination and slow rate of pollen tube growth. Measures to circumvent incompatibility in these crosses to produce interspecific hybrids are discussed.     

Introgression of resistance to Columbia and Northern root-knot nematodes fromSolanum bulbocastanum into cultivated potato

Summary Resistance toMeliodogyne chitwoodi races 1 (MC1) and 2 (MC2) andM. hapla (MH) derived fromSolanum bulbocastanum was introduced into the cultivated potato gene pool through somatic fusion. The initial F₁ hybrids showed resistance to the three nematodes. Resistance to reproduction on roots by MC1 was accompanied by resistance to tuber damage in F₁ clones. Tuber damage sometimes occurred, however, in hybrids of BC₁ progeny resistant to reproduction on roots when MC2 and MH were the challenging nematodes. Resistance to reproduction was transferred into BC₁ individuals, but a greater proportion of BC₁ progeny was resistant to MC1 than to MC2 or MH. Resistance to MC1 appears to be dominant and discretely inherited. F₁ and BC₁ progeny were pollen sterile, but seed were produced from crosses using cultivated tetraploid pollen sources. Approximately 11 and 33 per cent of pollinations produced berries on F₁ and BC₁ pistillate parents, respectively. Seed yield increased fourfold overall in crosses with F₁ compared to BC₁ individuals.Abbreviations MC1 Meloidogyne chitwoodi race 1 - MC2 Meloidogyne chitwoodi race 2 - MH Meloidogyne hapla - Rf Reproductive factor     

Investigation on pre-zygotic barriers in the interspecific crosses involving Gossypium barbadense and four diploid wild species

Interspecific hybridisation has played significant role in the improvement of economically useful traits such as productivity, earliness, fibre quality and resistance to pests and diseases in cotton. However, wide crosses are often limited by the operation of either pre or/and post-fertilization barriers. The present study on pollen tube behaviour of four diploid wild species viz., G. triphyllum, G. davidsonii, G. thurberi and G. armourianum in the pistils of G. barbadense was taken up to determine the extent of pre-zygotic barriers operating in these crosses. High rate of pollen germination (∼80%) and normal growth of pollen tubes were observed upon selfing of both G. barbadense and four wild species. Pollen tubes reached the pistils and fertilization was accomplished within 8 HAP in all the five parental species. In the interspecific crosses, pollen germination was normal in the cross involving G. armourianum and drastically inhibited in the crosses involving G. triphyllum, G. davidsonii and G. thurberi. Even though delayed pollen tube growth was a general feature in all the four crosses, successful fertilization was observed only in the cross involving G. armourianum. In crosses involving G. triphyllum, G. davidsonii and G. thurberi, pollen tubes failed to reach the ovary even at 24 HAP due to the presence of strong stylar and ovarian incompatibility. Abnormalities in pollen tubes such as swelling, twisting, intense callose plug formation, breakage, branching and reverse orientation were frequently observed among the incompatible crosses.     

Overcoming unilateral breeding barriers between Lycopersicon peruvianum and cultivated tomato,Lycopersicon esculentum

Summary Treatment of Lycopersicon peruvianum stigmas with an artificial medium analogous to stigmatic exudate allowed pollen germination and growth on immature pistils. Growth of Lycopersicon esculentum pollen tubes to L. peruvianum ovules, an otherwise incompatible cross, was achieved following such treated bud-pollinations. No plantlets were recovered, although a few embryos from this cross at the globularity heart stage of development were excised at 22 days after pollination, indicating the presence of crossing failures as severe as in the reciprocal cross. Hybrid plants were obtained from the reciprocal cross, using as pollen parent an L. peruvianum line selected for congruity with L. esculentum. Bud pollinations to L. peruvianum, using these interspecific F₁ hybrids as the pollen parent, allowed viable embryo development and plantlet recovery. Resulting backcross plants, which possess approximately 1/4 L. esculentum genome in a L. peruvianum cytoplasm, may facilitate further introgression of the L. esculentum nuclear genes into in foreign cytoplasm.     

Cytogenetics of interspecific hybrids in the genus Capsicum L.

Summary Crosses between a wild species C. chacoense and three cultivated species of chili pepper viz. C. annuum, C. frutescens and C. chinense yielded hybrids when C. chacoense was the seed parent but the reciprocal crosses were unsuccessful. C. chacoense × C. annuum F₁ hybrids were partly fertile and therefore an F₂ population could be raised; the other two F₁ hybrids were totally sterile. Chromosome pairing in the F₁ plants resulted largely in bivalents and a few multivalents and univalents. The genomes of the four species share large homologies and the role of chromosome structural changes in genome differentiation is suggested. Hybrid sterility is the major reproductive isolation mechanism.     

Hybridization between cotton and Malvaceae species as a tool for production of partial interspecific aneuploid cotton plants

Cotton although is an autogamous species could be cross pollinated under favorable climate conditions and/or in the presence of pollinators. The coexistence of cotton with Malvaceae species raises questions on the possibility pollen to be exchanged among Malvaceae species and on the resulted consequences. The present work was undertaken to evaluate the in situ response of cotton flowers (G.hirsutum L, G. barbadense L.) and their F₁ interspecific hybrids when are artificially pollinated with Malva sylvestris L., Hibiscus syriacus L. and Abelmoschus esculentus Moench. Furthermore, an in vitro protocol was attempted to support embryos’ growth in order to produce viable progenies originating from crosses between cotton and the aforementioned Malvaceae species. The obtained results gave evidence that pollen from the above Malvaceae species stimulated cotton ovaries without successful hybridization. The interaction between pollen and cotton’s stigmas was higher at early stages when M. sylvestris was used as pollinator but in crosses with H. syriacus and A. esculentus more ovules were activated as revealed by the percentage of carpodesis and life-time of bolls onto maternal plants. Only crosses between cotton with okra produced cotton seeds under in situ conditions. The in vitro embryo-ovule culture protocol, used, increased the number of regenerated cotton plants, especially in crosses among F₁ interspecific cotton hybrids and A. esculentus. In this case, regenerated plants were recombinant aneuploids, combining traits from both cotton species. This novel cotton germplasm possessing unique chromosome rearrangements, at aneuploid level could be proved useful after cytogenetic, molecular or QTL genetic analysis referring to important agronomic traits.     

Overcoming interspecific barriers in Sorghum and molecular characterization of the derivatives

Crop improvement in sorghum has progressed extensively availing the variation available in the primary gene pool of S. bicolor. Crossing further, the yield plateau can be accomplished by bringing in the genes from tertiary gene pool into the cultivated gene pool. Earlier efforts for wide hybridization in sorghum with tertiary gene pool were not successful due to inhibition of wild pollen germination on the stigma of cultivated species. In the current study, elite lines of cultivated Sorghum species were pollinated with wild species using different floral treatments. The results showed that spray with the synthetic auxin, 2,4-dichlorophenoxyacetic acid, has promoted the seed set. Overall, 76% crosses showed seed set, but many seeds were underdeveloped and deformed. F₁ progeny showed traits of both cultivated and wild parents. Hybrids were confirmed through PCR analysis. Many (80%) F₁ plants were sterile, and 20% of F₁s were advanced to F₂ generation. Wide variation was observed in F₂ progeny, and the phenotype selections were made and are being evaluated in the field.     

Broadening the genetic base of lentil cultivars through inter‐sub‐specific and interspecific crosses of Lens taxa

Wild Lens taxa are invaluable sources of useful traits for broadening genetic base of cultivated lentil. Nine inter‐sub‐specific and interspecific crosses were made successfully between cultivated (Lens culinaris ssp. culinaris) and wild lentils (L. culinaris ssp. orientalis, odemensis, lamottei and ervoides). The effect of species groups, day length and temperature on crossability in lentils was evident under normal winter sowing in New Delhi and in summer Himalayan nursery at Sangla in Himachal Pradesh, India, although pollen fertility assessed in all the cross‐combinations showed no significant variation. True hybridity of nine inter‐sub‐specific and interspecific crosses was confirmed through morphological and molecular (ISSR) markers, in which three of 120 primers could confirm the hybridity of all the crosses. All cross‐combinations were also studied for important quantitative traits related to yield. The range, mean and coefficient of variation were estimated in parental lines, F₁ and F₂ generations to determine the extent of variability generated in cultivated lentils through the introgression of genes from wild L. taxa. A high level of heterosis was observed in F₁ crosses for important traits studied. Substantially higher variations for seed yield and its attributing traits were exhibited in F₂ generations indicating transgressive segregation. The results of the present investigation revealed that wild L. taxa can be successfully exploited for lentil improvement programmes, and the variations generated could be easily utilized for broadening the genetic base of cultivated lentil gene pool for improving the yield as well as wider adaptation.     

Pollen germination,pollen tube growth and fertilization following self and interspecific pollination of Paspalum species

Summary Crossability between most Paspalum species is very low. This study was undertaken to identify the impediments to hybridization. Accessions of P. intermedium Munro. ex Morong, P. jurgensii Hackel and P. dilatatum Poir were self-pollinated and crossed with one anther. Paspalum intermedium is essentially self-sterile but P. jurgensii and P. dilatatum are highly self-fertile. Following pollination, pollen germination and tube growth were studied by observing the pollinated pistils with fluorescent microscopy. Examination of self-pollinated pistils revealed that the pollen germinated shortly after contacting the stigmas. Germination was over 80% for the P. intermedium and P. dilatatum accessions but only 57% for P. jurgensii. Pollen tubes grew to the micropyle within 45 minutes after pollination in P. dilatatum and 1 hour and 15 minutes in P. jurgensii. However, in the P. intermedium accessions most tubes did not grow beyond the stigma and very few penetrated the style and ovary. Apparently stylar-incompatibility is the reason for the low selfed seed set. In the cross-pollinations, pollen germinated shortly after pollination and germination ranged from 57 to 88% for the different crosses. In all crosses the pollen tubes grew to the micropyle within 30 minutes to 2 hours after pollination indicating that a cross-incompatibility system is not the cause for low crossability among these species. By examining embryo sacs from P. intermedium × P. dilatatum, its reciprocal and P. intermedium × P. urvillei crosses, it was determined that gametes failed to unite in some crosses and this is a major reason for low crossability.     

Factors affecting seed set in the crosses between <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> (Ramat.) Kitamura and its wild species

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.     

Inheritance of seed resistance to bruchids in cultivated mungbean (Vigna radiata, L. Wilczek)

Bruchid beetles or seed weevils are the most devastating stored pests of grain legumes causing considerable loss to mungbean (Vigna radiata (L.) Wilczek). Breeding for bruchid resistance is a major goal in mungbean improvement. Few sources of resistance in cultivated genepool were identified and characterized, however, there has been no study on the genetic control of the resistance. In this study, we investigated the inheritance of seed resistance to Callosobruchus chinensis (L.) and C. maculatus (F.) in two landrace mungbean accessions, V2709BG and V2802BG. The F₁, F₂ and BC generations were developed from crosses between the resistant and susceptible accessions and evaluated for resistance to the insects. It was found that resistance to bruchids in seeds is controlled by maternal plant genotype. All F₁ plants derived from both direct and reciprocal crosses exhibited resistance to the bruchids. Segregation pattern of reaction to the beetles in the F₂ and backcross populations showed that the resistance is controlled by a major gene, with resistance is dominant at varying degrees of expressivity. Although the presence of modifiers was also observed. The gene is likely the same locus in both V2709BG and V2802BG. The resistant gene is considered very useful in breeding for seed resistance to bruchids in mungbean.