Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Allotetraploid hybrids produced by protoplast fusion for seedless triploid <Emphasis Type="BoldItalic">Citrus</Emphasis> breeding

In connection with crop improvement strategies for marginal regions it has been proposed to increase the outcrossing rate in barley which would presuppose high and persistent pollen viability to ensure successful cross fertilisation. The present study was designed to investigate the viability of mature pollen from extruded and non-extruded anthers of cultivated and wild barley (Hordeum vulgare ssp vulgare and ssp spontaneum, respectively) in comparison with the closely related but obligatory outcrossing species H. bulbosum. Pollen viability (PV) was assessed employing the p-phenylenediamine-peroxidase-test on pollen derived from spikes or anthers immediately after collection and after treatment at 20, 30 and 40 ^∘C for 1, 2, 4, 8 and 16 h. The latter treatment was interposed after 8 h by a 10 h period of darkness at 12 ^∘C and thus called 26 h treatment, consecutively. Initial PV was high with 98% across all genotypes and even at 40 ^∘C did not fall below 80% after 8 h. After the 26 h treatment, PV of two H. vulgare genotypes originating from semi-arid regions and of H. bulbosum fell below 60% while the other genotypes retained a PV of > 80%. Viability of pollen of extruded anthers in H. vulgare ssp. spontaneum was on average slightly but significantly lower than PV of non-extruded anthers but still remained above 90%, even after the 26 h treatment at 40 ^∘C. Pollen viability of the outcrossing species H. bulbosum ranged on a very similar level as PV of H. vulgare. Results indicate that pollen of H. vulgare retains a sufficiently high level of viability to ensure successful cross-fertilisation over a period of at least 26 h even at high temperatures of up to 40 ^∘C.     

Hybrid Tea-rose pollen. I. Germination and storage

Summary Germination and storage trials were carried out with pollen of several rose varieties. The pollen grains germinated well in a 15% sucrose solution with 40 ppm boric acid. Staining the pollen with a 0.1% tetrazolium solution and standardizing the degree of colour at which the pollen grains are counted as viable, provided a good viability estimate, simpler to carry out than in vitro germination. Germination capacity and staining ability of the pollen were greatly impeded-about halved-by dehydration during storage in desiccators at low humidity. This effect could be corrected by humidifying the pollen beforehand for about one hour, though this pre-treatment increased the percentage of germinated pollen grains more than the percentage stained. There was no difference between the two percentages in fresh or in deep-frozen pollen.Pollen stored at 1°C and high relative humidity soon lost its germination capacity: between 0 and 20% humidity a considerable proportion of the pollen remained viable for 9 months and longer. Storage for the same period in vacuum-sealed glass tubes at –24°C maintained viability as well or better and would probably prolong it further. Some of the cold-stored pollen induced a reasonable seed set after one year, a low seed set was obtained even after two years of storage at 1°C and low humidity.     

Storage of avocado pollen

Summary Avocado pollen was stored at a range of temperatures and relative humidities (RH) for up to one year and the pollen was tested for viability in vivo.Pollen stored for one month was capable of germination on the stigma and penetrating the ovule when stored at 4°C with <1,23,55 and 75% r.h. and at -196°C with 0% r.h. Most pollen samples stored at 25 and -15°C at a range of r.h. would germinate on the stigma but none would penetrate the ovule.After one year of storage, pollen at 4°C and <1 and 23% r.h. would germinate on the stigma but would not penetrate the ovule. There was no germination of pollen stored at 4°C and 55 and 75% r.h. Only pollen stored at -196°C and 0% r.h. would penetrate the ovule, but thawing and refreezing once during the year destroyed the viability.     

Differential cold sensitivity of pollen grain germination in two Prunus species

Summary Pollen grains from selected cutivars of almond [Prunus dulcis (Mill.) D. A. Webb] and peach (Prunus persica Batsch L.) were exposed to a range of temperatures between 1°C and 34°C on a thermogradient plate. Pollen germination at temperatures below 9°C was conspicuously greater in almond than peach. Miximal germination percentages were attained at about 16°C (almond) and 23°C (peach). The two species did not differ in their capacity for pollen tube elongation over a broad range of temperatures. Maximal pollen tube elongation occurred at temperatures 5°C to 8°C higher than maximal pollen germination. Species affiliation appeared to be of much greater consequence than chilling requirement or bloom date of the sporophyte in predicting gametophytic response to temperature.     

In vitro pollen germination and tube growth of tomato (Lycopersicon esculentum Mill.) and its relation with plant growth

Summary In vitro pollen germination at 10°C, 14°C and 22°C of four groups of two pure line tomato varieties was compared with their plant growth at 19°C D/10°C N under controlled environmental conditions. Generally, pollen germination was slow at 10°C but after 6 h the percentage of germination was similar to that at 22°C. Maximum germination was obtained at 14°C already after 4 h. The longest pollen tubes occurred at 22°C. The two varieties within each group differed significantly from each other for percentage of pollen germination. For one group, this difference was greater at 10°C than at 22°C. The varieties in two groups also differed significantly for pollen tube growth. These differences in pollen tube growth rate were greater at 22°C than at 10°C. There was no clear relationship between pollen germination, pollen tube growth and plant growth in any of the four pairs of varieties. The results are discussed with regard to the possibility of pollen selection at low temperature in order to improve the efficiency of breeding for growth at low temperature.     

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.     

Introgression of unique characteristics of floral initiation under 24 hour day-length of Fragaria chiloensis ‘CHI-24-1’ into F. × ananassa

A unique flower initiation of the wild Junebearing strawberry strain ‘CHI-24-1’ in Fragaria chiloensis occurs under 24 h DL and high temperature conditions. To introduce the floral initiation characteristics of ‘CHI-24-1’ into cultivated strawberries of F. × ananassa, a cross pollination was conducted between ‘CHI-24-1’ and the Japanese short-day type strawberry cultivar ‘Nyoho’. The floral initiation of ‘CHI-24-1’ was induced in both parent and daughter plants linked with runners under a 24 h DL and 23/20 ^∘C, but not 8 and 16 h DLs at the same temperatures. Of the 21 F1 hybrids grown under the 24 h DL, 12 showed flower truss production in the parent and/or daughter plants linked with runners. Among 64 F1 hybrids, 26 exposed to 8, 16 and 24 h DLs for 30 days produced no flower trusses. However, 32, one and five F1 hybrids produced flower trusses under the 24 h DL alone, 8 h DL alone and both the 8 and 24 h DLs, respectively. The results of the experiments indicated that none of the F1 hybrids were day-neutral plants, but approximately 60% had the characteristics of floral initiation under 24 h DL, which was inherited from the pollen parent of ‘CHI-24-1’. The importance of the unique floral initiation characteristics under 24 h DL with high temperature for strawberry breeding was discussed.     

Pollen and pollination experiments. V. An empirical basis for a mentor pollen effect observed on the growth of incompatible pollen tubes in pear

Summary The self-incompatible pear cultivar Doyenne du Comice was selfed with the aid of the mentor pollen technique (self pollen mixed 1:1 with compatible pollen) and the pioneer pollen method (compatible pollen applied 14 h in advance of the self pollen). Observations on tube growth in the style showed that inviable methylated pollen was ineffective either as mentor or pioncer pollen, having no effect on the performance of the self pollen which stopped growing at about one quarter of the style from the stigma. Calculations made on the basis of the obtained data indicated that the viable untreated or irradiated pioneer and mentor pollen, the former somewhat better than the latter, aided the self pollen tubes to reach the base of the style.     

Breakdown of self-incompatibility in perennial ryegrass at high temperature and its uses in breeding

Summary The maximum temperature (34°C) at which perennial ryegrass plants from one breeding population successfully shed pollen was determined and the seed set of ten clones taken from the same population and self-pollinated both in an unheated glasshouse and at a constant 34°C during anthesis was compared. Seed set by selfing of all ten clones was markedly increased by heat treatment, from a mean of 2.3% to 30.7%. Heat treatment of anthers, but not stigmas, greatly enhanced pollen tube growth on stigmas from the same genotype. Germination of progeny was not affected by the heat treatment. Heat treatment of ten clones from a breeding population with contrasting morphology and growth rate was not so successful. Only one clone shed pollen freely at 34°C and showed a marked improvement in subsequent seed set. The advantages to the breeder of routinely producing full-sib families of elite perennial ryegrass parents by selfing are outlined.     

Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Effect of incubation temperature regimes and culture medium on broccoli microspore culture embryogenesis

Conditions for reliable induction of embryogenesis from isolated microspores were studied in ten genotypes of broccoli. Embryo yields were significantly increased in almost all of the broccoli genotypes by the incubation at 32.5 ^°C for 1 day, than when the standard incubation at 30 ^°C for 2 days was used. Treatments of 48 hours at 32.5 ^°C produced less than optimal results suggesting that broccoli microspores are more sensitive to high temperatures than those of B. napus. The use of the ¹/₂ NLN-13 medium yielded greater number of embryos than the standard NLN-13. The magnitude of the response to the redution of the concentration of major salts by half in the NLN medium varied with the different genotypes. High embryogenic broccoli cultivars, such as ‘Shogun’, ‘SDB9’, and ‘Green Valiant’, presented a better response to the reduction of the concentration of major salts by half in NLN-13. Reduction never produced a detrimental effect on embryo yield and seems not to have any effect in the subsequent development of embryos in plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Overcoming the self-incompatibility of Lilium longiflorum Thunb. by application of flower-organ extract or temperature treatment of pollen

Summary The application of flower organ extracts to stigmas and the temperature treatment of pollen were tried to overcome self-incompatibility of Lilium longiflorum cv. Georgia.Substances in stigma, style, ovary and anther were extracted with ethanol and fractionated with ethylacetate into the acidic, basic and aqueous fractions. The extracs melted in a small volume of distilled water were applied to stigmas prior to self-pollination. Hinomoto stigma extract, self-pollinated and cross-pollinated Georgia stigma extracts of high concentrations and Georgia anther extract of high concentration were effective in overcoming the self-incompatibility and resulted in a high percentage of fruit set and many normal seeds. Extracts from Hinomoto ovary, style and anther were ineffective, except a basic fraction, which was very slightly effective.Pollen was treated with 40°C for 60 or 90 minutes and 50°C for 30 or 60 minutes, and a half of each lot was followed by –20°C for 24 h, prior to self-pollination. All treatments were effective, especially at 40°C for 60 minutes or 50°C for 30 minutes, and 40°C for 90 minutes or 50°C for 60 minutes followed by –20°C for 24 h.     

In vitro interspecific fertilization,embryo development and formation of hybrid seedlings between Gossypium hirsutum and G. arboreum

Summary In vitro hybridization between Gossypium hirsutum, and G. arboreum was carried out. Hybrid seedlings were obtained after successive use of the following five kinds of media: 1) pollen grain germination medium, 2) double-fertilization medium, 3) embryo development medium, 4) seedlings formation medium, 5) green seedlings growth medium. The factors affecting in vitro interspecific fertilization, embryogenesis and seedling formation were studied. The key factors were temperature and relative humidity (RH). The optimum RH for in vitro interspecific fertilization was 65%, and a suitable temperature range was 26–30°C. Pollen grain germination ratio decreased rapidly at a RH of 80%. When the temperature and the RH were higher than 32°C and 80%, respectively, the fertilization rate decreased to zero.Effects of petals and calyces of the maternal flowers on in vitro interspecific fertilization and ovule growth were identified. Even though the petals or calyces were excised, hybrid plantlets were also obtained after media were improved and a shake culture was used. In addition, the process of double-fertilization and embryo development were studied cytologically. The developmental characteristics of the hybrid embryos derived from in vitro interspecific fertilization were a later occurrence of the double-fertilization process and a much lesser division of endosperm cells. But the embryo development was not affected by these characteristics, and young hybrid embryos can develop to their cotyledon stage finally on artificial media.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Pollen longevity and artificial cross-pollination in Coffea arabica L

Summary Various aspects of pollen longevity, in vitro germination of pollen and controlled pollination in Coffea arabica were investigated. High pollen viability could be maintained for more than two years, much longer than previously reported, by storing it under vacuum at 18°C. The most satisfactory method of in vitro germination of pollen was the hanging drop in a Van Tieghem cell, with a 10°, sucrose solution. For artificial cross-pollination it is necessary to carry out emasculation and bagging at the latest one day before anthesis. The stigmas of unpollinated flowers remained receptive for at least nine days.This paper is published with permission of the Director, Coffee Research Station, Ruiru, Kenya     

Incompatible pollen tube growth and protein composition in styles of Japanese pear following high temperature treatments

Summary Immersing the excised-prepollinated styles of self-incompatible Japanese pear, Pyrus serotina Rehd., into distilled water at 45°C for 1.5–2.5 min promoted the growth of self-pollen tubes; both the number of styles penetrating the pollen tubes through styles to cut ends and of pollen tubes protruding from cut ends were increased. The subsequent growth of the protruded pollen tubes on agar media was also enhanced by the treatments. Treatments at 40°C did not promote the growth of self-pollen tubes and those at 50°C led the styles to necrosis. In the stylar proteins analyzed by isoelectricfocusing polyacrylamide gel electrophoresis, several changes in banding pattern were found following the heat treatment; one of the bands was a glycoprotein that increases in quantity in developing styles as self-incompatibility of the style becomes stronger (Hiratsuka et al., 1986). This protein band was stained more weakly by Coomassie Brilliant Blue-G250 dye. Antigenic substances in the styles examined by immunodiffusion seemed not to be denatured by high temperature treatments.Abbreviations IEF-PAGE isoelectricfocusing polyacrylamide gel electrophoresis - CBB Coomassie Brilliant Blue     

A new method for selecting cytochimeras by the maximum number of nucleoli per cell in Allium wakegi Araki and A. fistulosum L.

Summary A new method for determining ploidy levels in cytochimeral plants was developed by examining the maximum number of nucleoli per cell. Colchicine-treated plants of Allium wakegi Araki and A. fistulosum L., which have different ploidy levels in the first (LI), the second (LII), and the third (LIII) germ layer, were used together with untreated 2-2-2 plants of the same species. Nucleoli in guard cells for LI and in mesophyll cells for LII were stained in a 50% AgNO₃ aqueous solution at 55° C for three hours under dark, humid conditions. In both species the ploidy levels of LI determined by the maximum number of nucleoli per guard cell accorded well with those determined by guard cell length. In A. fistulosum the ploidy levels of LII determined by the maximum number of nucleoli per mesophyll cell clearly agreed with those determined by pollen size. This method provided more precise and efficient identification for LI and LII ploidy than the conventional methods of using guard cell length for LI and pollen size for LII.     

The genomes of Arachis hypogaea. 1. Cytogenetic studies of putative genome donors

Summary Cytological studies of wild diploid Arachis species in the same section of the genus (sect. Arachis) as the cultivated peanut A. hypogaea L. show, with one exception, a karyotype characterized by the presence of 9 pairs of larger chromosomes and one pair of small (A) chromosomes. The exceptional species A. batozocoi Krap. et Greg. has a more uniform karyotype. Interspecific hybrids between diploid species of similar karyotype have moderate to high pollen stainability, those involving A. batizocoi have zero pollen stainability and a very irregular PMC meiosis. Such infertile hybrids are the most likely to produce fertile, stable amphidiploids on doubling the chromosome complement. It is suggested that the cultivated peanut could have originated from such a sterile interspecific hybrid and on morphological and phytogeographic grounds the most likely genome donors are A. cardenasii (nomen nudum) and A. batizocoi of the species within section Arachis, which have been collected up to the present time.Paper number 5560 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27650