Overcoming the self-incompatibility of Raphanus sativus by application of plant hormones,amino acids and vitamines,and by temperature treatment of pollen

Summary Overcoming self-incompatibility by application of three kinds of plant hormones, sucrose, 3 kinds of amino acids and 2 kinds of vitamines was tested in cvs. Honbashi-taibyo Minowase (H-Mino) and Minowase (Mino) of Raphanus sativus. Effects differed between the cultivars. In H-Mino, BA (100 mg/l) and glutamic acid, folic acid and nicotinic acid (500 mg/l) resulted in higher fruit set and higher number of seeds per pollinated flower. In Mino, BA and NAA (100 mg/l) and glutamic acid and glycine (500 mg/l) induced a high number of seeds per pollinated flower. These chemicals, however, induced parthenocarpic fruit set, especially GA3. From the observation of pollen on stigmas washed with glutamic acid, it appeared that the pollen-tube penetrated into a papilla cell after 1 hour and openings of papillae and detached pollen grains and tubes were found after 2 hours as the result of successful pollentube penetration of papillae. Pollen was heated at 50°C for 30, 45 or 60 minutes, at 60°C for 15, 30 or 45 minutes and at 70°C for 10, 20 or 30 minutes prior to self-pollination. In H-Mino, 60 and 70°C were effective, and expecially 60°C for 15 or 30 minutes resulted a higher percentage fruit set and more seeds per fruit. In Mino, although 50–70°C were effective, the mean number of seeds per pollinated flower was lower than in H-Mino.     

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.     

Levels of self-incompatibility in wallflower,Cheiranthus cheiri L.

Summary Approximately 1000 plants from 30 wallflower cultivars were selfpollinated in the open flower and bud stage. Only a very small number of plants failed to set seed and the averages ranged from 3.3 to 11.0 seeds per siliqua. Cultivars with mixed colours appeared to show most self-incompatibility and it is suggested that they form un-evolved populations in terms of the incompatibility system by comparison with their related single-colour cultivars.S1 progenies tended to remain at the same self-incompatibility level as their parents although some segregation occurred with approximately the same number of plants from low to high self-incompatibility and vice-versa.S1 progenies showed no apparent inbreeding depression, as is the case also for their descendants, but there was a tendency for progeny heterogeneity to be associated with high parental self-incompatibility while homogeneous inbreds were most often derived from more self-compatible parents.     

Identification of self-incompatibility alleles and pollen incompatibility groups in sweet cherry by PCR based s-allele typing and controlled pollination

A total of 17 pollen incompatibility groups in sweet cherry (Prunusavium L.) were identified among 46 accessions by PCR based S-alleletyping analysis and by controlled test pollinations. Two putativeS-alleles different from S ₁ to S ₆,S _z and S _y were identified. Five S-genotypes, S ₁ S ₅, S ₁ S ₆,S ₂ S ₆, S ₄ S ₆, andS ₅ S ₆, combinations of S ₁ toS ₆ alleles that had not previously been identified from cultivars in NYSAES, were positively confirmed by PCR based S-genotyping analysis. Also, the S-genotypes of cultivars in some pollen incompatibility groups that had previously been incorrectly reported have been clarified. Several popular cultivars, which were previously used as testers for S-allele typing analysis, were found to have been inaccurately genotyped. In addition, the S-genotypes and self-incompatibility groups of some relatively recentlyintroduced cultivars were identified. The molecular typing system ofS-genotypes based on PCR is a useful and rapid method for identifying newS-alleles and incompatibility groups in sweet cherry.     

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.     

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.     

New self-incompatibility alleles in apricot (Prunus armeniaca L.) revealed by stylar ribonuclease assay and S-PCR analysis

Apricot (Prunus armeniaca L.) shows gametophytic self-incompatibility controlled by a single locus with several allelic variants. An allele for self-compatibility (S_C) and seven alleles for self-incompatibility (S₁–S₇) were described previously. Our experiments were carried out to ascertain whether the number of allelic variants of apricot S-locus was indeed so small. Twenty-seven apricot accessions were analysed for stylar ribonucleases by non-equilibrium pH gradient electrofocusing (NEpHGE) to determine their S-genotype. To validate the results of electrofocusing, the applicability of the S-gene-specific consensus PCR primers designed from sweet cherry sequences was tested. NEpHGE revealed 12 bands associated with distinct S-alleles in newly genotyped cultivars. Cherry consensus primers amplified 11 alleles out from 16 ones, which indicated that these primers could also recognize most of the S-RNase sequences in apricot, and provided an efficient tool to confirm or reject NEpHGE results. By combining the protein and DNA-based methods, complete or partial S-genotyping was achieved for 23 apricot accessions and nine putatively new alleles (provisionally labelled S₈–S₁₆) were found. Their identity needs to be confirmed by pollination tests or S-allele sequencing. This study provides evidence that similarly to other Prunus species, the S-locus of apricot is more variable than previously believed.     

外来入侵植物五爪金龙对5种蔬菜种子萌发和幼苗生长的化感效应

五爪金龙是一种外来入侵植物,为了解五爪金龙对其它植物的影响,采用种子萌发试验法测定了五爪金龙提取物对萝卜、小白菜、芥菜、莴苣和生菜5种蔬菜种子萌发和幼苗生长的化感活性。结果表明,五爪金龙甲醇提取物对小白菜和芥菜种子的萌发有明显的抑制作用,特别是对芥菜种子发芽的影响较大,处理后第2天、3天和4天的发芽率分别降低了44.66%、43.34%和42.00%,化感指数分别为-0.5630,-0.5372和-0.5122;对莴苣和生菜种子能延缓其发芽;对萝卜种子的发芽无影响。五爪金龙提取物对5种蔬菜幼苗根生长有明显的抑制作用,并且随着处理浓度的增加抑制作用加强,表现为根明显缩短变粗,根毛减少或无根毛,颜色变成褐色或枯死。对萝卜、芥菜、莴苣和生菜4种蔬菜芽的生长也有明显的抑制作用,但对小白菜芽的生长有一定的促进作用,而且抑制或促进作用的大小与处理的浓度有关。五爪金龙化感物质在茎和叶中均有分布,而且分布的量也相似。     

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.     

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.     

‘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.     

A comparison of methods for hybrid seed production using self-incompatibility in swedes (Brassica napus ssp. napobrassica)

Procedures for producing seed of hybrid swedes using self-incompatibility were examined. Single-cross, double-cross and modified double-cross hybrids were compared in isolation plots using natural pollinators and in polythene tunnels using blow-flies. With good coincidence of flowering and the same flower colour, nearly 100% hybrid seed was produced by natural pollinators with the single-crosses, the double-cross and one of the two modified double-cross hybrids; the other modified double-cross hybrid produced 87%hybrid seed. With poor coincidence of flowering and different flower colours the proportion of hybrids dropped to 61%. Using different flower colours and blow-flies as pollinators in polythene tunnels, higher levels of outcrossing were produced than in isolation plots with natural pollinators; the opposite result was obtained when the same flower colour was used. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Identification of self-incompatibility (S) alleles in Latvian and Swedish sweet cherry genetic resources collections by PCR based typing

The Latvian and the Swedish sweet cherry (Prunus avium L.) genetic resources collections comprise valuable material for breeding. The collections represent local Latvian and Scandinavian genetic resources: semi-wild samples, landraces, and cultivars developed in local breeding programmes, as well as diverse germplasm from the northern temperate zone. The objective of this investigation was to determine which S ₁ –S ₆ alleles are most important in the sweet cherry genetic resources collections and to compare the identified allelic and genotypic frequencies in material of different origin. Accessions in the two collections were screened for the presence of the self-incompatibility (S) S ₁ to S ₆ alleles, using PCR based typing. Significant differences (P < 0.05) between screened collections were found in frequencies of S ₄ and S ₅ alleles. Analysis of allele combinations identified the high occurrence of selections with the S-genotype S ₃ S ₆ in both collections. Compared to the S-allele frequencies published for over 250 sweet cherry cultivars from Western and Southern Europe, the Latvian and Swedish germplasm appeared to have a high frequency of the S ₆ allele in both collections, and a relatively high frequency of the S ₅ allele in Latvian germplasm. This study represents the first comprehensive S-allele screening for the sweet cherry genetic resources collections in Latvia and Sweden. Both sweet cherry collections contain high proportion of accessions adapted to north central European growing conditions, not typical for the majority of the documented sweet cherry genetic resources, which explains differences in certain S-allele occurrence.     

The effect of alternating temperatures on the self-incompatibility of some clones of Brussels sprouts (Brassica oleracea L. var. Gemmifera (DC.) Schulz)

Summary The influence of temperature on self-incompatibility of six S-homozygous clones of Brussels sprouts was studied. The clones were treated with constant temperatures of 14°, 17° and 20°C and alternating day and night temperatures of 17/14°, 20/14°, 23/14° and 26/14°C. To determine the degree of incompatibility the mean number of pollen tubes per style after selfing was calculated.The clone with the weak S-allele S-5 was less self-incompatible in the 23/14° and 26/14°C treatments than in the other treatments. The other clones with the weak S-alleles S-15 and S-45 remained sufficiently self-incompatible during all treatments. The clone with the rather strong S-39 showed the highest level of self-incompatibility at 26/14°C.In the two weakly self-incompatible clones with S-5 and S-39 a clear difference in self-incompatibility was found between young and old flowers. The strongly SI clones with S-39 showed no difference and in the two clones with S-15 the differences were small and significant in one case only.     

Occurrence of self-compatibility, self-incompatibility and unilateral incompatibility after crossing diploid S. tuberosum (SI) with S. verrucosum (SC): I. Expression and inheritance of self-compatibility

Diploid Solanum tuberosum (tbr), 2n=2x=24,can be crossed with S. verrucosum (ver) only when the latter is used as a pistillate parent but not reciprocally. This conforms to the phenomenon of unilateral incompatibility (UI) where a self-compatible species, like ver (SC) cannot be used as a male parent to cross with a self-incompatible (SI) parent like tbr. Even if ver × tbr hybrids are made, the F1 hybrids possess cytoplasmic male sterility and thus hinder genetic analysis of crossing barriers. Exceptionally, however, some diploid genotypes of tbr (SI) can be used as pistillate parents to cross with ver, and such exceptional tbr clones are called `acceptors'. Repeated backcrossing of acceptors to ver have resulted in male fertile genotypes that possess tbr cytoplasm and ver nucleus. These genotypes were used for the genetic analysis of `acceptance' and UI in thse experiments. It was found that acceptance of ver-pollen by tbr-pistils is based on a dominant gene A that expresses only in the absence of an inhibitor I. In the F₁ hybrids, only the S-allele of tbr was expressedbut not that of ver. Concomitant with this observation, it was shown that ver does not produce style-specific S-glycoproteins that are responsible for self-incompatible reaction in diploid potato. Although the the F₁ populations were SC, they segregated into SC and SI genotypes giving skewed segregation ratios for this trait. Because of this as well as the disappearance and re-appearance of SC trait in the offspring generations, it was necessary to postulate a more complex interaction between A and I. Models are presented in order to explain acceptance, non-acceptance and the expression of UI. It is concluded that at least four different loci are involved in the expression of UI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Self-incompatibility interactions in Brassica napus

Summary The interactions of nine S-gene lines of Brassica napus were examined. Seven different S-genes were involved in the crosses, with three lines of different origin having the same S-gene. In general, the reactions on selfing the heterozygotes were as expected from the results of test crosses with the homozygous parent lines. All types of dominance relationships were found, including reversal of dominance in pollen and pistil. Weakening of activity of one or both genes was found in several cases. Background genotype was found have to some effect and reciprocal differences in reactions were also observed.     

甘薯和巴西牵牛18S rRNA基因的克隆和序列分析

【研究目的】为甘薯和侵染甘薯病毒的基因表达研究提供内参基因序列信息。【方法】分别以‘商薯19’、‘北京553’两甘薯品种和巴西牵牛的基因组DNA为模板,利用PCR方法克隆甘薯和巴西牵牛18S rRNA基因序列。【结果】测序结果表明,获得的供试两甘薯品种和巴西牵牛的18S rRNA基因序列长度均为1630 bp;序列比对结果表明,甘薯和巴西牵牛与裂叶牵牛、烟草等双子叶植物的18S rRNA基因相应序列的一致性均达98%以上,与单子叶植物Lilium superbum的18S rRNA基因序列也有较高的一致性。【结论】从两甘薯品种和巴西牵牛的基因组中克隆出了18S rRNA基因部分序列,研究结果不仅为利用18S rRNA基因作为内参基因分析甘薯和侵染甘薯病毒基因的表达研究提供了序列依据,而且可为甘薯和巴西牵牛的分子系统学研究提供序列参考。     

Molecular mechanism of the S-RNase-based gametophytic self-incompatibility in fruit trees of Rosaceae

Self-incompatibility (SI) is a major obstacle for stable fruit production in fruit trees of Rosaceae. SI of Rosaceae is controlled by the S locus on which at least two genes, pistil S and pollen S, are located. The product of the pistil S gene is a polymorphic and extracellular ribonuclease, called S-RNase, while that of the pollen S gene is a protein containing the F-box motif, SFB (S haplotype-specific F-box protein)/SFBB (S locus F-box brothers). Recent studies suggested that SI of Rosaceae includes two different systems, i.e., Prunus of tribe Amygdaleae exhibits a self-recognition system in which its SFB recognizes self-S-RNase, while tribe Pyreae (Pyrus and Malus) shows a non-self-recognition system in which many SFBB proteins are involved in SI, each recognizing subset of non-self-S-RNases. Further biochemical and biological characterization of the S locus genes, as well as other genes required for SI not located at the S locus, will help our understanding of the molecular mechanisms, origin, and evolution of SI of Rosaceae, and may provide the basis for breeding of self-compatible fruit tree cultivars.     

Identification of self-incompatibility alleles (S) by PCR-RFLP in radish (Raphanus sativus L.)

From 16 inbred lines of cultivated radishes (Raphanus sativus L.), 6 S-alleles tentatively named S²⁰¹ to S²⁰⁶ were identified, and their dominance relationships were examined. Among the S-alleles, S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴ were found to be co-dominant. These 4 S-alleles showed dominance with S²⁰⁵ in pollen and with S²⁰⁶ in both pollen and stigma, while S²⁰⁵ and S²⁰⁶ were co-dominant. Polymerase chain reaction (PCR) was performed using the radish inbred lines randomly selected from the 6 S-allele groups. The primers were based on the highly conserved sequences of the S-locus specific glycoprotein (SLG) genes in Brassica oleracea. As a result of the PCR, a single DNA fragment of about 1.16kb was amplified as expected from the original sequence of B.oleracea. The S-allele specific pattern in the restriction fragments of the PCR products (PCR-RFLP) was confirmed for the first group of S-alleles (S²⁰¹, S²⁰², S²⁰³ and S²⁰⁴). However, for the second group of the S-alleles (S²⁰⁵ and S²⁰⁶), no PCR products were obtained. The usefulness of the PCR-RFLP in a radish breeding program is described. This revised version was published online in July 2006 with corrections to the Cover Date.