Characterization of an S-allele associated protein in Japanese pear

Summary This paper describes some characteristics of a stylar protein associated with the S₂ self-incompatibility allele (S₂-protein) in Japanese pear reported earlier (Hiratsuka, 1992). The term style refers to style plus stigma in this paper unless indicated otherwise.The S₂-protein, which is a relatively major component of styles with a pI of 6.5, was present only in the style, and the stigmatic zone involved the protein much higher in quantity than upper half of the style, followed by lower half. Molecular weight was assumed to be 24,000 judged from migration distance in sodium dodecyl sulfate (SDS) polyacrylamide gel. Immature styles from 8 to 4 days before anthesis also contained the protein though the amount was relatively small. Neither the heat treatment of prepollinated styles nor the pollination (compatible or incompatible) altered the pI value and staining concentration of S₂-protein in the gel. The protein did not have so strong ribonuclease (RNase) activity as reported in S-proteins of Nicotiana alata (McClure et al., 1989) and the RNase activities of extractable stylar proteins from self-incompatible strains were almost the same as those from self-compatible strains.Abbreviations CBB Coomassie Brilliant Blue - IEF-PAGE isoelectricfocusing polyacrylamide gel electrophoresis - RNase ribonuclease - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Detection and inheritance of a stylar protein associated with a self-incompatibility genotype of Japanese pear

Summary Stylar proteins in 6 cultivars of Japanese pear (Pyrus serotina Rehd.) with different self-incompatibility alleles were examined by isoelectricfocusing polyacrylamide gel electrophoresis (IEF-PAGE). Cultivars used involved 1 self-compatible mutant Osa-Nijisseiki obtained from Nijisseiki (S₂S₄). Banding patterns of proteins were almost the same among the 6 cultivars except for 3 bands; one of which associated clearly with self-incompatibility S₂-allele (hereafter the protein referred to as S₂-protein) and was easily detectable because of its large amount and its pI value differed from many other proteins. Two other bands were seemingly associated with S₃- and S₄-alleles, respectively. The S₂-protein was apparently present in the styles of Osa-Nijisseiki.The inheritance of S₂-protein was studied by using 15 selfed plants of Osa-Nijisseiki. Eight out of 15 plants possessed the S₂-protein as a major component of the stylar proteins and 3 plants as a minor component. The remainder 4 plants did not have the protein. This result may support that the S₂-protein is the actual S₂-gene product.Abbreviations IEF-PAGE isoelectrifocusing polyacrylamide gel electrophoresis - CBB Coomassie Brilliant Blue     

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.     

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.     

Sequence of the S 9-RNase cDNA and PCR-RFLP system for discriminating S 1- to S 9-allele in Japanese pear

A new S ₉-allele was discovered in 6 Japanese pear cultivars, ‘Shinkou’, ‘Shinsei’, ‘Niitaka’, ‘Amanogawa’, ‘Nangetsu’ and ‘Nansui’. cDNA encoding S ₉-RNase, a stylar product of S ₉-allele, was cloned from pistils of ‘Shinkou’ and ‘Shinsei’ by 3' and 5' RACE. The S ₉-RNase gene had an open reading frame of 684 nucleotides encoding 228 amino acid residues. S ₉-RNase had a hypervariable (HV) region different from S ₁- to S ₈-RNase and shared higher similarity (95.2%) with apple S ₃-RNase than with 8 Japanese pear S-RNases (from 61.0% to 70.7%). Genomic PCR with primers ‘FTQQYQ’ and ‘anti-(I/T) IWPNV’ provided S ₁- to S ₉-amplicon (product), but could not discriminate the S ₂ from the S ₉ of ca. 1.3 kb. The S ₂ and S ₉ were distinguished by digestion with AflII and BstBI, respectively. The digestion with nine S-allele-specific restriction endonucleases, SfcI, AflII, PpuMI, NdeI,AlwNI, HincII, AccII, NruI and BstBI, distinguished S ₁ to S ₉, establishing that this PCR-RFLP system is useful for S-genotype assignments in Japanese pear harboring S ₁- to S ₉-allele. ‘Shinkou’, ‘Shinsei’, ‘Nangetsu’ and ‘Nansui’ assigned as S ₄ S ₉ were determined to be cross incompatible. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inter-varietal pollen tube growth and ovule penetration in the avocado

Summary Results are presented showing number of pollen tubes in the pistil, and ovule penetrations in crosses involving the avocado varieties Edranol, Ryan, Hass, Reed, Talbot, Jalna, Fuerte, Bacon and Sharwil. Between 5% and 100% of the ovules in each cross were penetrated by a pollen tube. The female parent exerted more control than the male on numbers of tubes and ovule penetrations and the results for type A varieties were more consistent than those for type B varieties. Penetration of an ovule by two tubes was observed in all varieties except Reed and Fuerte and penetration of an ovule by three tubes was observed in the variety Edranol only. With the possible exception of the variety Bacon as female parent there was no difference between pollination with pollen from type A or B varieties.     

Identification of self-incompatibility alleles in pear cultivars (Pyrus communis L.)

Self and cross-incompatibility determination by means of fruit and seed set experiments or pollen tube growth observations in the style has been frequently reported to be unclear in pear (Pyrus communis L.). Thus,in order to develop a reliable in vivo method to test pollen-pistil incompatibility in pear, pollen tube performance has been studied along the pistil following self and cross-pollinations. Results show that, while pollen tube growth in the style may be an unclear test, ovule observation at the microscope for the presence of pollen tube in the nucellus is a proper method to test incompatibility in this crop. With this analysis we could identify S-alleles of ‘Williams’ (S₁S₂) and ‘Coscia’(S₃S₄), and three of the four possible S-genotypes resulting from the ‘Williams’ × ‘Coscia’ cross, as represented by ‘Butirra Precoz Morettini’ (S₁S₃), ‘Santa Maria Morettini’ (S₂S₃)and ‘Tosca’ (S₁S₄). This result demonstrates that ‘Williams’ and ‘Coscia’ cultivars do not share any allele in common. We also established two new inter-incompatibility groups in pear. Furthermore, the presence of a common allele between ‘Williams’ and ‘Agua de Aranjuez’,and ‘Coscia’ and ‘Agua de Aranjuez’, three apparently unrelated old cultivars, may indicate a narrower genetic base than expected for European pear. This finding together with the fact that 40% of new released cultivars have direct or indirect parental relationship with the cultivars ‘Coscia’ and/or ‘Williams’, anticipates the possibility of new cases of cross-incompatibility for this crop in the future. Both the method described and the determination of the S-genotypes will facilitate the characterisation of self and cross-incompatibility relationships in this species. This revised version was published online in August 2006 with corrections to the Cover Date.     

The interaction between compatible and self-incompatible pollen of apple and pear as influcenced by their ratio in the pollen cloud

Summary The production of self-seed was investigated in apple and pear with the aid of viable and compatible mentor/ pioneer pollen in relation to the proportion of self-imcompatible pollen present in the pollen cloud.Treatments consisted of mixtures of compatible and self-incompatible pollen at ratios of 1:1, 1:2 and 1:9 applied once or twice or followed by self-pollination. Selfing only, whether once or twice, produced virtually no fruits or seeds, while mixed pollinations did. Generally, fruit set tended to decrease and the self-seed to increase with increasing amounts of self pollen in the pollinations.The pioncer-pollen method (compatible in advance of self pollen) appeared more effective than the mentor pollen method (compatible and self pollen mixed). The use of viable instead of dead mentor/pioneer pollen causes competition for the ovules with the self pollen, but has the advantage that, in addition to some self-seed, other seeds are formed which, e.g. in apple, are necessary to keep the fruits on the tree until harvest.Fruit set was moderately reduced at ratios of compatible pollen to self pollen not exceeding 1:9 with pear and 1:5 with apple which constitute maximal ratios in practice as regards pollinator trees: trees of the main cultivar. In fruit orchards and probably in nature with other species, the interaction between compatible and self pollen may result in more self-seed, and so suggests that inbreeding played a greater part in the evolution of self-incompatible species, than the meager results of articial self-pollination imply.     

A molecular technique for selection of self-compatible varieties of Japanese pear (Pyrus pyrifolia Nakai)

The pear cultivar ‘Osa-Nijisseiki’ (S₂S^sm ₄; sm = stylar-part mutant) has been used as a parent to breed self-compatible cultivars that produce excellent fruits. However, determination of the self-compatibility of ‘Osa-Nijisseiki’ offspring requires a lot of time, 6 years or more, by conventional cross breeding. We have designed a rapid reliable method for the identification of self-compatible varieties of ‘Osa-Nijisseiki’ offspring based on the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with S-allele specific restriction endonucleases. By using this method, 8 self-compatible varieties were selected among 16 selections resulting from a cross between the self-compatible cultivar ‘Osa-Nijisseiki’ (S₂S^sm ₄) and the self-incompatible cultivars ‘Niitaka’ (S₃S₉), ‘Whasan’ (S₃S₅), ‘Chuwhangbae’ (S₄S₆). The S-genotypes of 16 ‘Osa-Nijisseiki’ offsprings were also determined. This revised version was published online in August 2006 with corrections to the Cover Date.     

Embryo development and fruit-set in pear induced by untreated and irradiated pollen

Summary Flowers of the pear cultivars Conference, Doyenné du Comice and Gieser Wildeman were pollinated with untreated and 50-krad-irradiated pollen. Fruitlets were sectioned at regular intervals up to 6 weeks after pollination. Tubes of the irradiated pollen grew slower, but within two weeks nearly as many embryos were initiated as in the case of untreated pollen. Thereafter, the proportion of degenerated embryos increased, although the endosperm sometimes persisted. Six weeks after pollination only about 21% of the seeds induced by the irradiated pollen still contained normal embryos. At or before that time in-vitro culture of the immature seeds will be necessary because few embryos survive until harvest. The irradiated pollen stimulated a rather large parthenocarpic fruit-set, presumably associated with endosperm rather than embryo development during the first 4–6 weeks after pollination. The possibilities of haploid production are discussed.     

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

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

Effects of high temperatures on mature pollen grains in wild and cultivated maize accessions

Summary The effects of high temperature on mature pollen of various maize lines were investigated. Genotypic differences in pollen reaction to high temperature were revealed. Pollen grains resistant to high temperature (35°C, 26°C) were characterized by higher germination capacity and better ability to develop normal pollen tubes. The studies are of interest to evaluate reproductive system tolerance and conduct gamete selection at the mature pollen grain stage in maize.     

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

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

The crossing of common wheat (Triticum aestivum L.) with cultivated rye (Secale cereale L.). I. crossability,pollen grain germination and pollen tube growth

Summary The crossing of common wheat (Triticum aestivum L.) with rye (Secale cereale L.), and especially the action of the crossability genes of wheat, was studied using the readily crossable wheat cv Chinese Spring (genotype kr ₁ kr ₁ kr ₂ kr ₂ >), the poorly crossable wheat cv Hope (genotype Kr ₁ Kr ₁ Kr ₂ Kr ₂ ), as well as the disomic substitution line of chromosome 5B of Hope into Chinese Spring (CS/Hope 5B, genotype Kr ₁ Kr ₁ kr ₂ kr ₂ ). By comparing crossability and actual fertilization, the poor crossability with rye of both cv Hope and the CS/Hope 5B substitution line was shown to result from absence of fertilization. Studies of pollen grain germination and pollen tube growth showed that the dominant alleles of the crossability genes manifested themselves through retardation and eventually inhibition of pollen tube growth at the style base and in the ovary wall. In Hope the growth of all pollen tubes was inhibited, whereas in CS/Hope 5B rarely fertilization was achieved. The recessive alleles of the crossability genes do not seem to have an influence on the growth of rye pollen tubes in wheat pistils.     

Factors affecting haploid production in wheat using the Hordeum bulbosum system. 1. Genotypic and environmental effects on pollen grain germination,pollen tube growth and the frequency of fertilization

Summary A quantitative examination of pollen grain germination and pollen tube growth within the overy wall was made in selected wheat × tetraploid H. bulbosum hybridizations, to investigate the effect of the wheat and the H. bulbosum genotype on these characters. As expected, variation at the known crossability loci had no effect on pollen grain germination. The frequency of pollen tube penetration of the ovary wall was, however, severely reduced when the dominant alleles were present. Pollen tube penetration was nevertheless observed in 3 of the 110 ovaries examined of the non-crossable cultivar Highbury. The H. bulbosum genotype had a much smaller effect on these characters, but significant differences between the clones were observed in the numbers of pollen tubes initially penetrating the ovary wall. Although two H. bulbosum genotypes showed no significant differences in the number of ovaries with pollen tubes at the base of the ovule, significant differences in the frequency of fertilization were observed. The possible cause of this discrepancy is discussed.The frequency of fertilization in crossable wheat × H. bulbosum hybridizations was improved by the application of gibberellic acid within 10 minutes of pollination, and reduced by an increase in the ambient temperature from 20°C to 26°C. Fertilization following the pollination of non-crossable wheat genotypes was not affected by either of these factors.     

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.     

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.     

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.     

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.     

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.