Manipulation of in vivo pollination techniques to improve the fertilization efficiency of interspecies crosses in the genus Phaseolus

Phaseolus angustissimus A. Gray contains genes for traits of interest for dry bean (P. vulgaris) breeders. F₁ hybrids can be produced but introgression through backcrossing has been a problem. One of the main impediments is the time required between pollination and fertilization when F₁ hybrids of P. vulgaris/P. angustissimus are backcrossed with P. vulgaris. In an attempt to reduce this time, the effect of alternative pollination techniques was studied. The rate and the time of fertilization were ascertained using three different pollen types (pollen germinated in vitro, fresh pollen (FP) mixed in pollen‐germinating media and FP), and two methods of pollination (cut‐style and stigmatic pollinations). An optimal in vitro pollen germination medium for Phaseolus pollen was developed. Low temperatures (6–9°C) were demonstrated to be necessary for Phaseolus pollen germination. Pollination using a cut‐style technique coupled with pregerminated pollen reduced the time for fertilization in the backcrosses of interspecies hybrids by approximately 28 h. This technique improved the success rate of fertilization in wide crosses of Phaseolus designed to introgress genes from wild relatives.     

Widening the gene pool of cultivated lentils through introgression of alien chromatin from wild Lens subspecies

Wild Lens species/subspecies are a potential source for increasing genetic diversity in cultivated lentil. Four intraspecific crosses were attempted between cultivated and wild lentils. Viable hybrids were produced between L. culinaris ssp. culinaris × L. culinaris ssp. orientalis and L. culinaris ssp. culinaris × L. culinaris ssp. odomensis. Normal meiosis and pollen fertility were observed in the first set of crosses, whereas chromosomal abnormalities and reduced pollen fertility were observed in the second set of crosses. These crosses were also studied for some quantitative traits. The range, mean and coefficient of variation were calculated in parents, F₁, F₂ and BC₁ generations to determine the extent of variability generated in the cultivated lentil through introgression of genes from wild lentil. The cultivated lentil × L. culinaris ssp. orientalis crosses showed substantially higher variability for all the traits than crosses involving cultivated lentil ×L. culinaris ssp. odomensis. The results of the present study indicated that these wild subspecies can be exploited for breeding purposes and their variation can easily be utilized to widen the genetic base of the cultivated lentil.     

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.     

Prezygotic interspecific hybridization barriers between kenaf (Hibiscus cannabinus L.) and four wild relatives

Use of interspecific hybridization in genetic improvement of kenaf (Hibiscus cannabinus), a multipurpose crop, is limited by low cross‐compatibility. Previous hybridization studies suggest that part of incompatibility barrier is prezygotic. Pollen germination, stigma receptivity and growth of alien pollen on kenaf pistil were studied using wild relatives of kenaf. Four wild and semi‐domesticated species, H. surattensis, H. acetosella, H. radiatus and H. vitifolius, were used as pollen donors in the present experiment. H. radiatus exhibited highest pollen germination (65.89%) on H. cannabinus stigma. For closely related species (H. surattensis, H. acetosella, H. radiatus), stylar incompatibility was noticed, while for distantly related species H. vitifolius, the incompatibility was stigmatic. Number of pollen tubes entering ovule of H. cannabinus pistil was higher for H. radiatus (1.30). Alien pollen growth and penetration of pollen tube through ovule were higher in species sharing same genome of H. cannabinus. High callose deposition was observed in incompatible crosses, suggesting a general mechanism for prezygotic incompatibility in Hibiscus section Furcaria. Callose deposition in incompatible crosses increased with time.     

Effect of mentor pollen on pistil-pollen incongruities among species of Cucumis L.

Summary The merits of mentor pollen in certain interspecific hybridizations in the genus Cucumis L. were evaluated. African wild cucumber species Cucumis metuliferus Naud. and C. africanus L. and Asian species C. sativus var. hardwickii Alef and var. sikkimensis Hook, were reciprocally crossed. Pollen tube growth was arrested halfway down the style in all combinations except in C. sativus × C. africanus. Mentor pollen irradiated with 100 and 200 krad grew through the style and into the ovules in all species. Fruitset was obtained in all cases when mentor pollen was used either alone or in mixtures. Fruits set after self pollination with mentor pollen yielded only seeds without embryos.In several crosses, aided by mentor pollen, a number of relatively large seeds (with enlarged embryosac) were obtained. A sample of these embryosacs contained a globular structure like an embryo. Embryosacs with embryo-like structures were explanted on several different media but no development was obtained.     

玉米与摩擦禾、薏苡的杂交不亲和性

采用荧光显微技术, 对摩擦禾、薏苡花粉在玉米柱头上的萌发和生长过程进行了观察。摩擦禾花粉粒在玉米柱头上均能萌发, 花粉管在柱头中伸长并到达花柱基部, 且可将雄配子送入胚囊内, 玉米果穗顶端有受精结实痕迹, 说明摩擦禾与玉米的杂交障碍不是杂交不亲和, 而是胚囊不亲和或杂种衰亡。薏苡花粉粒在玉米柱头也能萌发, 花粉管能伸入花柱, 但玉米与薏苡杂交生殖隔离较摩擦禾严格, 杂交极其困难, 杂交障碍为胚囊不亲和或花柱不亲和。玉米与薏苡杂交时, 薏米花粉管能到达玉米花柱基部, 而川谷花粉管却在花柱中停止生长, 杂交障碍与薏苡种类有关。玉米与薏苡杂交的花粉管异常率高于玉米与摩擦禾杂交花粉管异常率, 反映了玉米与摩擦禾的亲缘关系较与薏苡近。     

Effect of Abscisic Acid on Pollen Germination and Tube Growth of Maize Genotypes1

As a preliminary step to using gametophytic selection to obtain genotypes of maize, Zea mays L., differing for abscisic acid (ABA) content and/or sensitivity, the influence of ABA on the in vitro germination and tube growth of maize pollen and the presence of genetic variability for these traits was investigated. In a first experiment, pollen samples of three genotypes were germinated on substrates containing increasing amounts of ABA. Low ABA concentration (0.5 νM) significantly increased pollen germination and tube growth, while 500 νM ABA significantly decreased both traits, thus indicating that ABA can influence the growth of maize pollen in vitro. In a second experiment, eleven inbred lines were screened for the in vitro reactivity of their microgametophytes to ABA. The lines differed significantly for the response (% of control) of tube length, but not of pollen germination to ABA.     

Variation of prezygotic barriers in the interspecific hybridization between oilseed rape and wild radish

Escape of transgenes from genetically modified oilseed rape, Brassica napus, into wild radish, Raphanus raphanistrum, depends on sexual compatibility. The variation in prezygotic barriers of two different cultivars for interspecific hybridization with a population of wild radish was investigated by hand crossing and fluorescence microscopy of pistils. Significant differences were observed between oil seed rape cultivars in their ability to accept wild radish pollen germinating onto their stigma and the rate of fertilization of ovules. Some differences among the pollen donor plants were also detected. These results suggest that the rate of interspecific hybridization in the field would depend upon the oilseed rape cultivar and the genotype composition of the local wild populations. The implication of S-related genes, as revealed through identification by pistil tissue prints of class I and II S-types of SLG (S-Locus Glycoprotein) and SLR1 (S-Locus Related),and immuno-IEF, was not significant. This revised version was published online in August 2006 with corrections to the Cover Date.     

A reproductive calendar of Lilium longiflorum Thunb. cv. Gelria

Summary The development and timing of different processes in the floral biology and sexual reproduction of Lilium longiflorum were studied and brought together into a chronological reproductive calendar. Care must be taken when emasculating flowers later than 4 days before anthesis (DBA), because the stomium of the anther was open from 8 DBA and the pollen grains appear to be mature from 3–2 DBA. The receptivity period of the pistil lasts for a rather long period, but before wilting of an unpollinated flower as determined by a change of the colour of the perianth from white into brown, the flower had already lost its receptivity. Because L. longiflorum pollen grains can easily be stored, the pistil receptivity should thus determine the pollination schedule. The relationships between pollen tubes and pistil are discussed with respect to plant breeding.Abbreviations DAA Days After Anthesis - DAP Days After Pollination - DBA Days Before Anthesis     

Assessment of Cold and Heat Tolerance of Winter-grown Canola (Brassica napus L.) Cultivars by Pollen-based Parameters

Winter‐grown canola (Brassica napus L.) production is limited mostly by frost and winter kill in the southern canola‐growing regions of the United States. Tolerance to cold and heat were assessed by studying percentage of pollen viability (PV), in vitro pollen germination (PG) and pollen tube length (PTL) for 12 field‐grown cultivars. Freshly collected pollen from all cultivars were incubated on artificial solid growth media at a constant temperature ranging from 10 to 35 °C at 5 °C interval for 30 h to determine PG and PTL. A modified bilinear model best described the temperature response functions of PG and PTL. Canola cultivars showed significant variability (P < 0.001) for PV (61.3 % to 89.7 %), PG (29.0 % to 48.2 %) and PTL (463 to 931 μm). The average cardinal temperatures, T_min, T_opt and T_max, for PG and PTL were 6.4, 24.3 and 33.7 °C, respectively. Principal component analysis revealed that maximum PG, PTL, T_min and T_opt of both PG and PTL were the most important factors in determining cold tolerance, whereas T_max of PG and PTL, and maximum PG and PTL were more responsible in separating the cultivars for heat tolerance. The canola cultivar, KS3077, was the most cold tolerant with the lowest T_min and the widest temperature adaptability range, and the cultivar Kadore was the most heat tolerant with the highest T_max for the PG. The identified cold‐ and heat‐tolerant cultivars may be useful in canola‐breeding programmes to develop cultivars suitable for a niche environment.     

Analysis of pollen tube growth in situ to investigate self-incompatibility in the wild potato Solanum commersonii

Summary Pollen tube growth was investigated in a diallelic crossing design with seven genotypes of the diploid wild potato species Solanum commersonii, accession O/S UR-9, CIP 762459. Pollen tube growth in the style was recorded using a combined quantitative and qualitative evaluation scale. Clear-cut differences in pollen tube growth behavior in compatible and in partially or completely incompatible crosses were detected. Diallelic crossing of the seven randomly chosen genotypes, intercrossing within two progeny families, and backcrossing of two progeny populations to the parents revealed the existence of a one-locus gametophytic system of stylar incompatibility. The S-allele status of all genotypes investigated was determined.     

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.     

The genetics of hard seed coat in the genus Lens

Summary Seeds of the cultivated lentil are capable of germinating shortly after maturation. The seed dormancy of wild lentil species is due to a hard seed coat. In crosses between the cultivated species L. culinaris and its wild progenitor L. orientalis the hard seed coat of the wild species was controlled by a single recessive gene in homozygous condition. In a cross between the wild species L. ervoides and L. culinaris the hard seed coat of L. ervoides was controlled by a single dominant gene. The significance of the genetics of seed coat hardness in the domestication of lentil is briefly discussed.     

Soybean Pollen Anatomy,Viability and Pod Set under High Temperature Stress

High temperature (HT) stress is one of the major environmental factors influencing yield of soybean (Glycine max L. Merr.) in the semi‐arid regions. Experiments were conducted in controlled environments to study the effects of HT stress on anatomical changes of pollen and their relationship to pollen function in soybean genotype K 03‐2897. Objectives of this study were to (a) quantify the effect of HT stress during flowering on pollen function and pod set and (b) observe the anatomical changes in pollen grains of soybean plants grown under HT stress. Plants were exposed to HT (38/28 °C) or optimum temperature (OT, 28/18 °C) for 14 days at flowering stage. HT stress significantly decreased in vitro pollen germination by 22.7 % compared to OT. Pollen from HT stress was deformed; it had a thicker exine wall and a disintegrated tapetum layer. HT stress decreased pod set percentage (35.2 %) compared to OT. This study showed that decreases in pollen in vitro germination by HT stress were caused by anatomical changes in pollen, leading to decreased pod set percentage under HT stress.     

Genetic diversity and taxonomic relationships within the genus Lens as revealed by allozyme polymorphism

Summary A survey of allozyme polymorphism at 11 loci was carried out on 439 accessions from the genus Lens. This comprised 153 Lens culinaris subsp. orientalis, 35 L. odemensis, 117 L. ervoides, 32 L. nigricans, 2 of a differentiated cytotype of L. nigricans and 100 landrace accessions of the cultivated lentil (L. culinaris subsp. culinaris), from 10 different countries. The aim of the survey was to determine intra-specific genetic diversity and species relationships, based on phylogenetic and phenetic analyses, particularly regarding the position of L. odemensis and the differentiated cytotype of L. nigricans. Diversity was described by three statistics. The level of diversity in the cultivated taxon was lower than in any of the wild species according to two of these statistics, the percentage of polymorphic loci and mean number of alleles per locus. For the third measure (Nei's mean genetic diversity) it was only greater than L. ervoides. Genetic diversity statistics of the wild species indicated differences in the nature of between-population genetic diversity within the different taxa. Phylogenetic analysis suggests that L. odemensis and L. ervoides evolved from a common ancestor, and L. culinaris subsp. orientalis subsequently evolved from L. odemensis. Phenetic analysis, however, places L. odemensis closer to L. culinaris subsp. orientalis than to L. ervoides. Nei's mean genetic distance of L. odemensis from both L. culinaris subsp. culinaris (0.204) and L. culinaris subsp. orientalis (0.110) was greater than the distance between them (0.062). This evidence is not conclusive in determining whether L. odemensis should retain its specific status. Further crossability studies should be carried out on a range of genotypes to assess the potential for gene flow. The evidence presented shows the differentiated cytotype of L. nigricans to be quite distinct from other L. nigricans accessions, both phenetically and phylogenetically. This indicates that the differentiated cytotype of L. nigricans may constitute a new taxon. Discriminant function analysis reveals that isozymes may be useful in validating species classification.     

Testing the Efficiency of Pollination Bag Materials

Crossing bags manufactured from three grades of polyester and three types of paper bags were tested for their permeability to grass pollen. Bags were placed on L. perenne plants of known PGI isozyme genotype. Any pollen passing through the crossing bag and pollinating the plant was detected by assaying its progeny for PGI isozymes. The results obtained allowed differentiation of the various materials for their suitability for pollen proofing.     

The site of action of the crossability genes (Kr 1, Kr 2) between Triticum and Secale. I. Pollen germination,pollen tube growth,and number of pollen tubes

Summary Seven genotypes of common wheat (Triticum aestivum L.) were crossed with rye (Secale cereale L.) in order to find the site or sites of action of the crossability genes, Kr ₁ and Kr ₂, of wheat. The data obtained, by fluorescence microscopy, were compared to the controls (wheat x wheat). The results indicate that the crossability genes have little effect on pollen germination and on the time taken for the pollen tubes to reach the microphyle, irrespective of their crossabilities with rye. The number of pollen tubes reaching the microphyle is, however, affected by the Kr-genes, as high crossable genotypes have more pollen tubes than the low crossable ones. There was a high correlation between the mean number of pollen tubes at the micropyle with seed set, which also reflects the crossability. The Kr-genes seem to manifest themselves in the retardation and inhibition of pollen tube growth between the style base and the top of the embryo sac, where the effect is most distinct in the low crossable genotypes.     

Interspecific hybridization in Cucumis L. II. The crossability of species,an investigation of in vivo pollen tube growth and seed set

Summary The crossability of 12 Cucumis species of African and Asiatic origin was studied in a diallel cross, in order to find ways to realise the cross between the common cucumber (C. sativus L.) and its wild relatives which carry resistances against diseases and pests.Self-pollinations and cross-pollinations within species gave normal pollen tube growth and seet set. The different accessions within a species, as a rule reacted alike in interspecific crosses. In crosses between African species different crossing patterns were found, viz. bilateral congruity, bilateral incongruity and unilateral incongruity. Within C. sativus all accessions intercrossed freely, except one, which displayed unilateral incongruity.Good seed was harvested from several crosses and in some cases embryo culture was needed for further development of seeds. No good seeds were obtained from any cross between a species of the African group and C. sativus L.     

Reaction of wild species of the geneus Lens to drought

Summary Susceptibility to drought stress is a key factor in dry land lentil (Lens culinaris Medikus) production in the Mediterranean region of West Asia and North Africa. This study examined the response to drought stress of 121 accessions representing all subspecies of the genus Lens; cultivated, and the wild L. culinaris ssp. orientalis (Boiss.) Ponert, L. culinaris ssp. odemensis (Ladiz.), L. nigricans M.B. Godr. ssp. nigricans Godr. and L. nigricans ssp. ervoides (Brign.) Ladiz. for their potential use in breeding for dry land conditions. Accessions were grown under two moisture regimes (dry land and dry land plus supplemental irrigation) at Breda, Syria during the 1990–91 and 1991–92 seasons. The cultivated lentil had markedly superior seed and straw production than did the wild Lens species. Time to flowering accounted for less than 10% of the variation in yield of wild accessions under rainfed conditions in the two seasons, showing that, in contrast to the cultivated germplasm, drought escape was relatively unimportant in wild lentil. Performance under drought in wild lentil, measured in terms of dry land seed yield or drought susceptibility index (S), was randomly distributed among collection locations with little relation to collection site aridity. Direct selection of wild lentil germplasm for biomass yield under dry conditions is of little value and an evaluation of wild accessions in hybrid combination is needed.     

Floral Traits and Seed Production of Sorghum A-, B- and R-lines under Chilling Field Temperatures

In A-lines of sorghum ( Sorghum bicolor L. Moench), seed production under field conditions with manual pollination is generally lower than that in self-pollinated B-lines. This may be associated with floral differences. Six pairs of A/B-lines and four R-lines were evaluated during 2005 and 2006 at Montecillo, State of México (2240 m altitude). Rachis length, number of primary branches and fertile flowers per panicle, 100-seed weight, seed number, seed yield and seed set per panicle were evaluated. In the A-lines, the pistil characteristics were also measured and in the male-fertile lines, the size of anthers and the amount and size of viable pollen were recorded. Compared with the A- and B-lines, the R-lines exhibited significantly higher (P ≤ 0.05) numbers of flowers, seed set and seed yield per panicle and they also produced more pollen grains of larger size and with greater viability during their longer flowering period (FP). Between A- and B-lines, there were differences (P ≤ 0.05) in most of the yield traits, which also interacted with year. The proportion of viable pollen in B- and R-lines (75 %) was not considered to be a factor that might account for their low seed production. Chilling temperatures (3.5–8.4 °C) during the FP could have affected stigma receptivity in the three different line types and thus may have reduced seed set in the male-fertile lines.