Unilateral cross compatibility in Vaccinium elliottii × V. arboreum, an intersectional blueberry hybrid

Vaccinium elliottii Chapmn., a diploid blueberry in Vacciniumsection Cyanococcus, was crossed with V. arboreum Marsh, a diploid blueberry insection Batodendron. The goal was toproduce hybrids that could be used toincorporate traits from these species intotetraploid southern highbush cultivars. The crosses were made reciprocally usingtwo clones of each species. A recessiveseedling marker gene for anthocyanin-freecotyledons in the V. elliottii clonesused as female parents permitted thetesting of mentor pollination. For thementor pollinations, V. elliottiiplants that were homozygous for therecessive marker allele were pollinatedwith a small amount of viable V.elliottii pollen carrying the recessivemarker mixed with a large amount of V.arboreum pollen carrying the dominantmarker. Thousands of intersectional hybridseedlings were obtained when V.elliottii was the seed parent, both withand without mentor pollination, but nohybrids were obtained from 2735 flowerspollinated in the reciprocal crosses. Theseeds that gave rise to hybrid embryos weresmaller than normal V. elliottiiseeds, but germinated well. Hybridseedlings grew slowly at first, buteventually produced some vigorous plants inthe field, although many plants remainedmuch smaller than normal seedlings of thetwo parental species. Some of the hybridsproduced numerous flowers during the 6years they were observed in the field, buteven with many diploid, tetraploid, andhexaploid section Cyanococcus blueberryplants growing nearby that could haveprovided pollen for their flowers, thehybrids produced no viable seed.     

Use of Vaccinium octoploids to facilitate 4x-6x gene transfers

The use of an 8x Vaccinium produced through doubling a tetraploid hybrid with colchicine was studied as a means of facilitating gene exchange between V. corymbosum L. and V. ashei Reade. Analysis included meiotic observations and crossability studies of the 8x plant, as well as attempting to develop 6x breeding lines. Meiotic analysis revealed the presence of micronuclei in Telphase II products and polyspory in sporads. In the crossability studies five 4x highbush plants were pollinated with 8x o2 4x pollen. Data gathered included: % fruit set, average weight/fruit, average number of seeds/fruit, average number of seedlings/pollination, and number of 6x seedlings. Octoploid pollinations were significantly lower than 4x pollinations in all parameters. One 6x plant was produced from the 4x-8x crosses but was found to be mitotically unstable, having somatic cells with chromosome numbers ranging from 48 to 168.     

Unreduced pollen frequencies versus hybrid production in diploid-tetraploid Vaccinium crosses

Summary Reciprocal crosses were made between highbush Vaccinium corymbosum L. cultivars (4X) and high, medium and low frequency diplandroid pollen producing diploid clones of V. elliotti (Chapm.) and diploid pubescent forms of V. corymbosum. Pollination of 3,342 flowers produced 15 hybrids-11 tetraploids and 4 triploids. All V. elliottii hybrids were tetraploid while each successful diploid-tetraploid V. corymbosum cross produced at least one triploid. The ratio of tetraploid hybrids to 100 pollinations (H: 100P) was considered to be the best criterion for assessing crossability. Vaccinium elliottii produced a total of 8 4X hybrids while 2X V. corymbosum only produced 3. Vaccinium elliottii was more successful as a female parent and diploid V. corymbosum only produced hybrids as male parents. High frequency diplandroid clones showed a lower mean crossability as pollen parents (H: 100P=0.2) than medium frequency diplandroid clones (H: 100P=0.8). As predicted, 0% diplandroid clones wer unsuccessful as pollen parents. Unreduced pollen production was essential for tetraploid hybrid production in 4X×2X crosses. There appeared to be no consistent, inherent pattern between 2n pollen and 2n egg production in a clone nor was a tetraploid genotype effect on crossability consistently observed.Paper No. 10781 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC., 27695-7601     

The performance of double or mixed pollinations with compatible and self-incompatible or incongruous pollen of pear and apple

Summary Six pear and five apple trials were carried out to ascertain the outcome of combinations of compatible pollen (C) with self (S) or incongruous pollen (I) as to the pollination index (PI=seeds/pollinated flower). The PI of the mixture C+I (1:5) was consistently depressed as compared to that of the control C. The results of the double pollinations S/C and I/C were affected by the temperature at pollination; their PI's at <15°C were twice as high as those at >15°C, being well above and below the PI of C in the former and latter case respectively. The opposite was true for the C/S combination, the PI of which increased with the pollination temperature; the PI of C/I did not differ much from the PI of C, irrespective of temperature. The conclusion was reached that the interaction previously and presently found between compatible and self-incompatible pollen also exists to a fair extent between compatible and incongruous pollen. However, in pear neither the mentor nor the pioneer pollen technique proved to aid its hybridization with apple, the formation of self seed was not observed either. In apple the production of apple × pear hybrids was likewise doubtful, but the double pollinations S/C and C/S formed 4–10% self seed.     

Restrictions of the efficiency of haploid plant production in apple cultivar Idared,through parthenogenesisin situ

Summary For haploid plant production in Idared apple, parthenogenesisin situ was induced by irradiated pollen. The most important restrictions were seed set, embryo germination and green plant recovery from germinated embryos. The efficiency of green plant production was strongly influenced by irradiation dose, picking time and quality of the irradiated pollen. 250 Gy pollen was more efficient for green plant production than 500 Gy pollen. Using 250 Gy pollen with reduced germination capacity resulted in a five-fold lower green plant yield. Delaying picking of the fruits (140 instead of 70 days after pollination) had a significant positive effect. Up to 20 plants per 1000 pollinated flowers, showing no expression of a marker gene present in the pollen parent, were obtained in some treatments. Induction efficiency was high when seed weight was high. This offers the possibility of selecting seeds with the best chances for haploid plant production. Heavy seeds generally contained visual normal developed embryos, which germinated better than those from lighter seeds and gave normal plant development. Delayed picking increased the proportion of large seeds, suggesting that pollination with irradiated pollen retards embryo development. From treatments with irradiated pollen of poor quality more small seeds were harvested.     

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     

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.     

Germplasm conservation of oca (Oxalis tuberosa Mol.) through botanical seed. Seed formation under a system of polymorphic incompatibility

Seven clonal accessions representing all three stylar morphs of tristylous oca were intercrossed and selfed in a complete diallelic crossing design. The four traits total capsule production, total number of seeds/capsule, total number of seeds containing an embryo/capsule, and percent seeds containing an embryo of all seeds per capsule were recorded. Both capsule and seed production were highest in the legitimate pollinations (in which styles are pollinated with pollen of the corresponding anther level), but also in illegitimate pollinations of mid-styled seed parents with long-level anther pollen of short-styled accessions. All 18 types of pollination, consisting of 6 legitimate, 6 illegitimate intermorph, and 6 illegitimate intramorph pollinations, expressed gradually different levels of capsule and seed production. Intramorph pollinations and selfs of mid-styled seed parents were less incompatible than intramorph pollinations and selfs of the long- and short-styled morphs. All accessions studied expressed an intact system of heteromorphic stylar incompatibility despite their exclusive clonal propagation for millennia. A maximum of 16,3 seeds/capsule containing an embryo was obtained in an individual legitimate cross of two accessions, although an average of 25 ovules are present in a flower of O. tuberosa. The results are discussed in relation to the establishment of a genebank based on botanical seed of this clonally propagated, valuable Andean tuber crop. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Reproductive capacity of F1 hybrids from Rosa rugosa and Chinensis cultivars

Summary Reciprocal crosses were carried out for 6 combinations of 4 R. rugosa with 3 R. chinensis cultivars. The parental combinations were evaluated for successful pollinations, seed germination and flowering and seed setting seedlings in populations of 3 year old plants. Seeds and viable seedlings were obtained from each of the 12 seedling populations. Only 3 populations produced female fertile offssprings. The average of seed setting seedlings was 1.5%. The percentage of flowering seedlings was significantly reduced in the offspring from certain parents. The success of cross pollination, seed germination and the ability to initiate flowers were influenced by cytoplasmic factors.The differences in fertility between the present hybrids and the only other rugosa x chinensis hybrid described, R. calocarpa (Andrê) Willmott, are discussed.Contribution No. 73/2.     

Pollination requirements for seed set in ulluco (Ullucus tuberosus)

Summary Pollination experiments were carried out on ulluco (Ullucus tuberosus), known as a very poor seed producer. All the clones used were diploids. The effect of self-vs. cross-pollination was studied in Experiment 1. Caged selfpollinated flowers produced no seeds. Different clones produced seeds in 0–8% of caged cross-pollinated flowers and in 5–19% of cross-pollinated non-caged flowers. Ulluco is thus basically an outbreeder.Experiment 2 studied whether seed set is limited by pollen availability. Total seed number per inflorescence number did not differ between plants treated with supplemental pollen and control plants; pollen availability thus did not limit seed set. The treated plants, however, produced more seeds in cross-pollinated than in other flowers on the plant, and the control flowers on treated plants produced fewer seeds than those on the control plants. In the treated plants, resources may have been allocated to artificially cross-pollinated flowers at the expense of other flowers. In some clones, seed set after artificial cross-pollination was at a level practicable for breeding work.     

Compatibility and incompatibility in witloof chicory (Cichorium intybus L.). 4. Formation of self-seeds on a self-incompatible and a moderately self-compatible genotype after double and triple pollinations

Summary After double or triple pollinations including selfing and the use of one-day-old compatible foreign pollen a severely self-incompatible clone could be forced to develop a reasonable amount of self-seeds per flower head. This number of self-seeds increased when the foreign pollination was delayed for e.g. 240 minutes in the double pollination experiment. Even more self seeds were obtained in the triple pollination experiment with a delayed second selfing.The influence of the old foreign pollen may be caused by a mentor pollen or a pioneer pollen effect.     

Variation in pollen performance among plants of Fagopyrum esculentum

Summary In genetically-heterogeneous outcrossing species, there is the opportunity for selection based on the male gamete. Buckwheat (Fagopyrum esculentum Moench) is self-incompatible with one ovule per flower, so pollen competition at each ovule can be studied. The occurrence of selection among pollen parents was determined, as well as the relative importance of prezygotic and post-zygotic selection. Mixed pollinations from two donors produced nonrandom paternity, with one of the donors being favored about 2:1 on several females. Individual plants showed significant variation in the speed of pollen-tube growth. Therefore, prezygotic selection is likely to have occurred based on the speed of pollen tube growth. In single-donor pollinations, donors had equal success as expected in the absence of post-zygotic selection among donors. However, a significant male x female interaction was found, consistent with postzygotic selection against particular parental combinations. To test whether male fitness is reduced by increased allocation to seed filling during pollen production, large- and small-seeded lines were compared, both as pollen donor and as pollen recipient. The large-seeded line was better in both roles, thus there was no evidence that greater allocation to seeds reduced the quality of the pollen.     

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape. This revised version was published online in August 2006 with corrections to the Cover Date.     

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).     

Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple

Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established.     

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     

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.     

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). II. Fertilization and early post-fertilization developments

Summary Fertilization and early seed development was studied in the variety Chinese Spring of common wheat (Triticum aestivum L.) after pollination with rye (Secale cereale L.) and selfing, and in the common wheat variety Hope after selfing. In all three combinations the first pollen tube reached the micropyle in about 40 min after pollination. When pollinated with rye the migration of the sperm nuclei to the egg cell and the polar nuclei was delayed by about an hour. In the subsequent development until 72 hours after pollination the average cellular and nuclear doubling times of embryo and endosperm were about 16 and 8 hours for the interspecific combination. 18 and 9 hours for Chinese Spring selfed and 20 and 12 hours for Hope selfed.