Pollen germination and pollen tube growth following self-pollination and intra- and interspecific pollination of Medicago species

Summary Pollen germination and pollen tube growth after self- and intra- and interspecific pollinations of annual [M. scutellata (L.) Mill., M. disciformis DC., and M. rigidula (L.) All.] and perennial (M. sativa L.) Medicago species were studied using a Carl Zeiss microscope with a fluorescence filter attachment.Self-pollination of self-fertile annual and intraspecific pollination of cross-fertile perennials were characterized by high pollen germination and rapid normal pollen tube growth. In contrast pollen germination percentages were very low and many pollen tube growth abnormalities occurred following interspecific pollination and self-pollination of self-sterile plants. The time period from pollination to fertilization for interspecific crosses was about double that following self-pollination of the annual species. However, fertilization occurred frequently following interspecific pollinations. Much of the sterility was due to factor(s) operating after fertilization.Cooperative investigations of the Kansas Agricultural Experiment Station, Dept. of Agronomy and the U.S. Department of Agriculture, Agricultural Research Service. Contribution No. 82-582-J- Mention of a trademark name or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture, and does not imply its approval over other products that also may be suitable.     

Influence of timing of cross-versus self-pollination on self-incompatibility in Brussels sprout inbreds

Summary The impact of cross pollination 30 h, 4 h, and immediately prior to self-pollination, and self-pollination 2, 4, 8, 12, 24 and 30 h prior to cross-pollination was assessed for pollen tube number per style, seed number per siliqua, and proportion of self-seed (sibs) per siliqua, in two inbred lines of brussels sprout. Pollination procedure had a marked effect on the amount of sib-and hybrid seed produced. Cross pollination 30 h prior to self-pollination produced significantly greater numbers of sib progeny, as did self-pollination 8–12 h before cross pollination. Prior application of self pollen reduces the number of pollen tubes in the style. The results of this experiment suggest that self-incompatibility in brussels sprout could better be assessed by self-pollination with a subsequent cross pollination 8 h later, and the determination of amounts of sib and F₁ hybrid seed per siliqua.     

Control pollination and pollen management in Sesbania sesban (L.) Merr.

Sesbania sesban was amiable to controlled cross pollination when the emasculation and pollination operations were performed in the morning. A medium of 10% sucrose solution was found to be optimal for in vitro germination of the pollen grains. The pollen is quite tolerant of orthodox storage conditions enabling controlled crosses to be performed among accessions that flower in differing seasons of the year.     

Reproductive biology of three commercially valuable <Emphasis Type="Italic">Santalum</Emphasis> species: development of flowers and inflorescences,breeding systems,and interspecific crossability

Santalum (sandalwood) spp. are hemi-parasitic trees, the heartwood of which produces valuable aromatic oil. There appears to be a significant commercial opportunity for establishment of a planted sandalwood resource. However, lack of basic biological knowledge is one constraint on such development. The study reported here addresses one such constraint. Controlled pollination using 13 genotypes of Santalum lanceolatum was undertaken to elucidate (i) self-incompatibility (ii) intraspecific cross-compatibility in the species, and (iii) interspecific cross-compatibility with S. album and S. austrocaledonicum. S. lanceolatum may be considered to have a facultative allogamous (incomplete outbreeding) breeding system. This study found variation between genotypes in the level of putative self-incompatibility: some (20%) were found to set seed following self-pollination, while the remaining 80% had no seed development with such pollinations. However, a significantly greater proportion of genotypes developed seed following intraspecific cross-pollination (62%) compared with self-pollination (20%). While total geographic isolation and significant morphological divergence exists between S. lanceolatum with each of S. album and S. austrocaledonicum this study found no indication of reproductive barrier(s) between them, indicating potential for use of interspecific hybridization in genetic improvement, but also suggesting the potential of undesirable gene flow between native and introduced species.     

The reproductive biology of three perennial Sesbania species (Leguminosae)

Summary The reproductive biology of Sesbania sesban, S. goetzei and S. keniensis (Leguminosae; Papilionoideae) was studied. Fifty percent flowering was observed at 102 to 153 days after germination for S. sesban accessions; 96 to 146 days for S. goetzei and 131 to 176 days for S. keniensis accessions. Flowers opened in the afternoon and remained fresh for 2–3 days. Bee species including Xylocopa sp., Apis mellifera, Megachile bituberculata and Chalicodoma sp. visited the flowers. Hand pollination experiments showed that all three species were self and cross compatible. The percentage of fully developed seeds was greater in pods formed after cross pollination compared to self pollination. No evidence was found for stigmatic or stylar self incompatibility. Outcrossing is probably the common method of reproduction under natural conditions, although in isolated trees substantial seed set by selfing might occur. Pod production under natural conditions was 34% for S. sesban; 49% for S. goetzei and 39% for S. keniensis. Considerable variation was found in pod production under open pollination between accessions of the same species. Selective abortion was observed within pods, with more mature seeds formed at the distal end of the pod.     

The effects of bud self-pollination and open flower self-pollination on the field characteristics of broccoli (Brassica oleracea var. Italica)

Summary Broccoli (Brassica oleracea L. var. italica) progenies grown from seed obtained by greenhouse budpollination (BP), open-flower self-pollination (OS), open-flower self-pollination with ether treatment of the stigma (OSE), and cross.pollination (CP) were compared in the field. For plant height, plant weight, head diameter, and head weight, BP resulted in the lowest values, OS and OSE were intermediate, and CP gave highest values. BP was highest, OS and OSE intermediate, and CP lowest for days to head emergence and days to harvest. Differences between OS and OSE were slight and all were non-significant for the 7 lines with complete data, but were significant for plant weight and head diameter when 20 lines were analyzed by the unweighted means method. The comparison BP versus OS-OSE was non-significant for plant height in both analyses and for head weight in the 7-line analysis only. In a second experiment using only BP and OS, and omitting days to head emergence, trends were similar but only the effects on head diameter were statistically significant. In the greenhouse, BP produced much more seed than either OS or OSE, and CP produced more than BP. Treatment of the stigma of open flowers with ether to overcome self-incompatibility did not result in greater seed production. Weight/100 seeds was higher in CP but did not differ among the three self-pollination methods.Technical Paper 5089, Oregon Agricultural Expriment Station.     

Genetic relationships among cold, salt and drought tolerance during seed germination in an interspecific cross of tomato

Seed of BC₁ progeny of an interspecific cross between a slow germinating Lycopersicon esculentum breeding line(NC84173; maternal and recurrent parent) and a fast germinating L.pimpinellifolium accession (LA722) were evaluated for germination under cold stress, salt stress and drought stress, and in each treatment the most rapidly germinating seeds (first 2%) were selected. Selected individuals were grown to maturity and self-pollinated to produce BC₁S₁ progeny families. The selected BC₁S₁ progeny from each experiment were evaluated for germination rate in each of a non stress (control),cold-, salt- and drought-stress treatment, and their performances were compared with those of a non selected BC₁S₁population in the same treatments. Results indicated that selection for rapid seed germination in each of the three stress treatments was effective and significantly improved progeny germination rate under all three stress conditions. The results support the suggestion that same genes might control the rate of seed germination under cold, salt and drought stress. Furthermore, selection in each of the three stress treatments resulted in improved progeny seed germination rate under nonstress conditions, suggesting that genetic mechanisms that facilitate rapid seed germination under stress conditions might also contribute to rapid germination under nonstress conditions. In practice, therefore, selection for rapid seed germination under a single stress environment may result in progeny with improved seed germination under a wide range of environmental conditions. Furthermore, to improve germination rate under nonstress conditions, it might be more efficient to make selections under stress conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Failure in an interspecific cross between Solanum khasianum Clarke and Solanum mammosum L.

Summary Attempts to obtain a successful interspecific cross between Solanum khasianum Clarke and Solanum mammosum L. were unsuccessful, and the probable reasons for this failure were investigated. A reduced number of viable pollen grains and low receptivity of the stigmas of S. mammosum L. and heterostyly in both species are the probable reasons for the failure of the attempted interspecific crosses between S. khasianum Clarke and S. mammosum L.     

Interspecific hybrids from cross incompatible relatives of sweetpotato

Summary Hybrids were obtained from Ipomoea interspecific crosses through ovule culture. The hybridity of the progeny obtained from I. triloba × IitI. trifida and (I. triloba × I. lacunosa) × I. batatas (4x) crosses was established by comparisons of floral morphology and analyses of peroxidase and esterase isozymes. The hybrids displayed the inflorescence type and sepal shape and texture of their male parents, while corolla size and anther and nectary color tended to be intermediate to their parents. The isozyme banding patterns of the hybrids contained bands present in the patterns of each of their parents. Pollen grain viability, measured by aceto-carmine stainability, was 44.1%, 92.3% and 82.4%, respectively, for the I. triloba × I. trifida hybrid and the (I. triloba × I. lacunosa) × I. batatas (4x) hybrids, H₁ and H₂. A controlled pollination study revealed that the I. triloba × I. trifida, and the (I. triloba × I. lacunosa) × I. batatas (4x) hybrids, H₁ and H₂ were partially self fertile with 6%, 70% and 13%, respectively, of the pollinated flowers producing viable seed. Success in backcrossing and sib-mating varied with the cross combination.     

Effect of floral morphology on pollination in Brassica rapa L

Summary Flower structure, especially the anther–stigma separation (ASS), is well known to affect pollination efficiency, and thus to potentially increase or decrease seed production in crops. Therefore, investigating the relationship between flower characteristics and pollination ability is crucial to a full understanding of mechanisms to improve F₁ seed production in Brassica rapa. We used image analysis to measure three flower characteristics: short stamen height (SSH); long stamen height (LSH); and pistil height (PH) in seven cultivars. We calculated the ratio of PH to LSH as an index of anther–stigma separation (ASS). We investigated the number of pollen grains (NPG) deposited on the stigma and the seed-set percentage (SSP) under open-pollination and self-pollination conditions (with and without insects, respectively). Nested ANOVA indicated significant differences between the seven cultivars in the floral characteristics except for PH. Moreover, much larger variation was observed in NPG and SSP than in floral characteristics. Although stepwise multiple regression analysis indicated that plants with relatively high PHs produced more seed under self-pollination, the number of seeds that resulted from self-pollination did not affect seed production because of incompatibility. Therefore, the effect of the spatial position of pistils on the F₁ seed production was low. Possibly other factors such as the total pollen production and visiting times of pollinators were important factors in the low yields observed in some cultivars     

Self and cross fertility in cranberry

Summary Eight varieties of cranberry (Vaccinium macrocarpon Ait.) were crossed in a complete diallel experimental design to examine self and cross fertility. Significant differences were detected for total, developed, and percent developed seed set among the varieties as females. Female fertility of Howes and Wilcox was reduced between 30 and 50% as compared to other varieties. Developed and percent developed seed set were significantly reduced for all varieties upon self-pollination. Reduction in developed seed set appeared to be the result of an increased level of abortion in self-pollinated flowers. Significant differences were found among varieties for ovule number. A positive correlation between berry weight and developed seed/berry was observed for most varieties. Genotypic variability in fertility and self-fertility could be exploited in developing varieties with increased productivity.     

Flatpea crossing methods and germplasm screening for reduced 2,4-diaminobutyric acid composition

Summary Flatpea (Lathyrus sylvestris L.) is a potentially valuable forage legume, but it contains high levels of 2,4-diaminobutyric acid (A₂bu), a compound that can have adverse effects on some animals, including rats and poultry. This study evaluated intercrossed pollination methodology needed for cultivar improvement and characterized intercrossed progeny for A₂bu concentration. Pollen germination on an artificial medium, using pollen collected during different stages of flower development, was used to identify appropriate stages for flower emasculation. Pollen dehiscence occurred at an early-bud stage when petal color was first evident. Slightly more mature buds contained pollen with substantially higher in vitro germination than pollen from early-bud stage flowers. Lines that produced high numbers of seeds per pollination were identified from crosses between flatpea accessions. Cross-pollination resulted in significantly higher seed set than selfing. Pollination using caged honeybees was the most efficient method of hybridization, although the degree of inbreeding in bee-pollinated plants could not be determined. Intercrossed progeny produced by honeybee pollination contained a lower mean concentration of foliar A₂bu than the mean for flatpea accessions.Abbreviations HPLC high performance liquid chromatography - OPA ortho-phthalaldehyde     

‘Thermally aided pollination’: A new method of breaking self-incompatibility in Brassica oleracea L.

Summary Application of heat during self-pollination of open flowers by means of an electric mini soldering iron resulted in seed set in self-incompatible Brassica oleracea varieties. Temperatures of 60, 70 and 80°C were tested. Compared with bud-pollination, in some cases the so-called thermally aided pollination (TAP) method, gave a considerably higher seed yield at 70 and 80°C. In view of the ease and rapidity of TAP it should be ascertained how far this method can replace bud-pollination in maintaining inbred lines. The possible mechanism behind the TAP response is briefly discussed.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

Brassilexin accumulation and resistance to Leptosphaeria maculans in Brassica spp. and progeny of an interspecific cross B. juncea × B. napus

Summary Resistance to Leptosphaeria maculans was assessed in Brassica napus, B. juncea, B. carinata, B. nigra and progeny issuing from an interspecific cross B. napus × B. juncea, using a cotyledon-inoculation test. In these individual plants, brassilexin accumulation was determined following an abiotic, non-specific, elicitation. All the tested B. napus cultivars were highly susceptible to the parasite and weakly accumulated brassilexin. In contrast, B. juncea, B. carinata, and B. nigra usually displayed a hypersensitive response to the inoculation and accumulated more brassilexin than B. napus. The same correlation between resistance to L. maculans and phytoalexin accumulation was observed in the interspecific hybrid progeny. The cotyledon-inoculation test allowed the discrimination of plants displaying a hypersensitive response to the inoculation from those highly sensitive to the parasite, but intermediate disease severity classes were not usually representative of resistance or susceptibility. In this respect, brassilexin determination allowed differentiation, within a set of plants presenting an intermediate response to the pathogen, of plants with a high (B. juncea-like), and with a weak (B. napus-like) ability to accumulate brassilexin.Abbreviations IHP interspecific hybrid progeny - JR B. juncea-type complete resistance to blackleg (Roy, 1984) - W&D test cotyledon-inoculation test as described by Williams & Delwiche (1979)     

Isolation and characterisation of somatic hybrids of diploid Solanum tuberosum and Solanum brevidens and the use of amylose-free starch mutation for detection of introgression

Summary Somatic hybrids of diploid amylose-free (amf) Solanum tuberosum and diploid S. brevidens were made by Poly-Ethylene-Glycol (PEG) or electrofusion methods. For the isolation of interspecific hybrids the use of selection markers (kanamycin and hygromycin resistance) was useful but not essential. In this 2x+2x interspecific combination 4x and 6x somatic hybrids were obtained. Seed set was the best in 4x×4x (S. tuberosum) backcrosses, but seed germination was the best in 6x×4x combinations, using in vitro germination of unripe seeds harvested 25 days after pollination. A high degree of pollen stainability (30–40%) was observed in 7 tetraploid hybrids and very low in all hexaploids. After iodine staining, the recessive amf marker was expressed by a red colour instead of blue, visible in starch-containing cells like columella cells of root tips, (micro)tubers or microspores. As expected, complementation was observed in starch-containing cells of the fusion hybrids. Segregation of the amf marker was clearly observed in microspores of 4x and 6x hybrids. Segregation ratios in the 4x hybrids showed variable recombination frequencies. In the backcross progeny of hexaploid F12-5 with a tetraploid amf mutant one amylose-free recombinant among 67 plants was found, indicating the occurrence of meiotic recombination in the megaspore mother cells.     

Methods of overcoming interspecific barriers in Trifolium

Summary Barriers to interspecific hybridization in Trifolium were investigated by manipulation of mentor pollen treatments, ploidy levels, and compatibility and male sterility systems. Crosses involving the addition of mentor pollen produced fewer seeds and hybrids than crosses involving normal pollination. Lower seed set with mentor pollen was deduced to result from the use of less viable pollen, approximately half the pollen having been killed by alcohol. Pollinations at the diploid level resulted in more hybrids than at the tetraploid level, perhaps because genes for male sterility produced higher female sterility in the tetraploids. The self-compatible stock produced more seeds, mostly selfs, than the self-incompatible stock, but produced more hybrids only in one cross, T. pratense L. × T. diffusum Ehrh. The use of male-sterile female parents reduced selfing but produced fewer hybrids than male-fertile female parents. Techniques of this study were designed to affect prefertilization barriers, but the lack of effect may indicate that postfertilization barriers in Trifolium are of greater importance.Journal Article No. 98-3-208 of Kentucky Agricultural Experiment Station. Published with approval of the Director.     

Increased atmospheric humidity post pollination: A possible aid to the production of inbred line seed from mature flowers in the Brussels sprout (Brassica oleracea var. Gemmifera)

Summary In two highly self incompatible inbred lines of Brussels sprouts the effect of increased atmospheric humidity post pollination was examined immediately following 1) hand pollination of green buds and open flowers, and 2) blowfly pollination of open flowers. Data were obtained for mean number of seeds set per pollination, mean number of fruits setting seed, and mean number of seeds produced per fruit which set for both varieties. Measured as number of seeds produced per minute spent pollinating, it was clear that open flower pollination followed by high humidity conditions was a much more efficient method of producing inbred line seed (46 seeds/minute) than green bud pollination (27 seeds/minute).     

Procedure for artificial cross pollination in clove (Syzygium aromaticum)

Summary A technique for artificial cross pollination in clove is described. This involves emasculating flowers two days before anthesis, and pollinating four days later. Following this procedure a seed set of approximately 25% can be expected.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.