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.     

Factors affecting seed set in the crosses between <Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> (Ramat.) Kitamura and its wild species

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.     

Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

Modelling rice competition with <Emphasis Type="Italic">Echinochloa crus-galli</Emphasis> and <Emphasis Type="Italic">Eleocharis kuroguwai</Emphasis> in transplanted rice cultivation

Field experiments were conducted to investigate rice — Echinochloa crus-galli and rice — Eleocharis kuroguwai competition under transplanted rice cultivation in four major rice production areas; Suwon, Daejeon, Iksan, and Naju in Korea. Rice yield data were used to predict rice yield as a function of plant densities of E. crus-galli and E. kuroguwai using a rectangular hyperbola and to determine economic threshold (ET) levels of the weeds. Both weed species significantly reduced number of tillers at early rice growth stage, resulting in significant reduction in number of spikes, and the other yield components such as number of grains, maturity and 1,000-grain weight at later growth stage. The weed competitivity represented by parameter ranged from 0.0145 to 0.0346 for E. crus-galli and from 0.0037 to 0.0187 for E. kuroguwai, indicating that the competition effect of E. crus-galli on rice yield was slightly greater than that of E. kuroguwai. The ET values of E. crus-galli were between 0.298 and 1.078 plants m⁻², while those of E. kuroguwai were between 0.848 and 5.298 plants m⁻², depending on weed competitivity and herbicide price. Therefore, our results can be used to support decision-making on herbicide application for E. crus-galli and E. kuroguwai management in transplanted rice cultivation.     

Occurrence of viable unreduced pollen in a <Emphasis Type="Italic">Begonia</Emphasis> collection

Polyploidy is widespread in plants and has played a major role in the evolution and diversification of the plant kingdom. Unreduced (2n) gametes are an interesting tool for polyploidisation and the creation of genetic variation in plant breeding. Especially in ornamentals, polyploidisation can broaden attractive features within a species. A Begonia collection was screened on the occurrence of 2n pollen with the aid of four different techniques: pollen size measurement, flow cytometric analysis of nuclei isolated from germinated and non germinated pollen, investigation of the microsporogenesis and analysis of progeny. In ten of the 70 screened genotypes (B. dregei, B. pearcei, B. ‘Anna Christina’, B. ‘Bubbles’, B. ‘Florence Rita’, B. ‘Orococo’, B. ‘Rubaiyat’, B. ‘Spatflacier’, B. ‘Tamo’ and B276), large pollen were observed with a rather spherical than normal ellipsoidal shape. Flow cytometric data proved that these aberrantly shaped pollen were associated with 2n ploidy levels, although they were not always viable. Meiotic aberrations in these large pollen producers resulted mainly in dyads although also monads, triads and polyads were observed. Successful crosses were obtained with B. dregei, B. ‘Orococo’, and B276 as pollinators; DNA content had increased in all or a part of the progeny. The results show that the occurrence of 2n pollen is not a rare phenomenon in Begonia.     

Pollen production in hexaploid triticale

Summary Comparative studies were made of the pollen characteristics of triticale, wheat, and rye. Measurements were made of the anther length, width, and percent extrusion; pollen viability; size and number of pollen grains per anther; and dispersal on 10-mm² slide area pollen traps. Triticale anthers were intermediate in length between and significantly different from both wheat and rye. Rye pollen grains per anther were four and two times greater in number than those of wheat and triticale, respectively. Pollen viability was not significantly different between species. Rye pollen grains were smaller than those of wheat and of some triticale cultivars. Simple correlations between anther length and anther width, pollen grains per anther, pollen grain trapped per 10 mm² slide area, and plant height were significantly positive.     

Chromosome pairing in haploid plants of radish derived from alien monosomic addition lines

Six haploid plants of radish were obtained via alien monosomic addition lines (2n = 19). One plant was derived from anther culture of an Raphanus sativus‐Brassica oleracea addition line, one plant from the selected smaller seed of an R. sativus‐Sinapis arvensis addition line and four plants from an R. sativus‐B. rapa addition line. During metaphase I of pollen mother cells, two plants exhibited the chromosome pairing of (0‐3)II+ (3‐9)I, three showed (0‐1)III + (0‐3)II + (3‐9)I and the remaining plant (0‐2)II + (5‐9)I. Trivalents seemed to be formed by the pairing between two larger chromosomes and the smaller one with somewhat loose pairing. All haploid plants were inferior to the radish cv. ‘Shogoin’ (2n = 18) with respect to vegetative growth. Their flowers were smaller, with sterile pollen grains, but a few normal flowers with fertile pollen sporadically developed and then produced a few seeds. It is suggested that the radish genome (R, n = 9) might comprise three pairs of homoeologous chromosomes, with the remaining three chromosomes carrying the homologous region(s) that results in a trivalent formation.     

New insights into genotypic thermodependency of cytoplasmic male sterility for hybrid barley breeding

The cytoplasmic male sterility (CMS) system msm1 in barley is known to be thermosensitive, sometimes resulting in spontaneous fertility restoration in the absence of the corresponding restorer gene Rfm1. Here, we investigated genotypic differences concerning temperature sensitivity and the plant developmental stage at which elevated temperature induces spontaneous fertility restoration in three CMS mother lines. While one line stayed completely male sterile, a significantly higher fertility was observed in two lines after treatment from growth stage DC 41 until maturation. Microscopic analysis revealed that sterile anthers contained neither intact pollen, nor remains of aborted pollen grains, whereas pollen was visible in anthers of potentially fertile plants. We conclude that the barley CMS system affects anther and pollen development prior to meiosis. Elevated temperature during heading and flowering can lead to a spontaneous fertility restoration by reactivating pollen growth. Nevertheless, genotypic variation exists enabling the selection for stable CMS mother lines and the development of F₁ hybrids with high hybridity. As spontaneous fertility restoration due to environmental effects is difficult to phenotype, further investigations will focus on the development of molecular markers for marker‐assisted selection.     

Use of ovule culture to cross between Delphinium species of different ploidy

The applicability of ovule culture was examined in reciprocal crosses between the diploid species D. grandiflorum L. and the tetraploid cultivar D. hybridum ‘Galahad’. An interspecific hybrid plant of D. hybridum ‘Galahad’ × D. grandiflorum was obtained,and hybridity was confirmed by chromosome counting. The hybrid was different from the parents in flower color and size but intermediate between the parents in leaf shape. The hybrid showed low pollen fertility and failed to produce viable seeds by either self- or backcross-pollination using fertile pollen grains from its parents. The applicability and efficiency of the ovule culture technique for genetic improvement of the genus Delphinium through interspecific hybridization between different ploidies is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reproductive barriers in the intergeneric hybridization between <Emphasis Type="Italic">Chrysanthemum grandiflorum</Emphasis> (Ramat.) Kitam. and <Emphasis Type="Italic">Ajania przewalskii</Emphasis> Poljak. (Asteraceae)

In order to reveal the causes of low seed set in the intergeneric cross between Chrysanthemum grandiflorum (Ramat.) Kitam. ‘Zhongshanjingui’ (female parent) and Ajania przewalskii Poljak. (male parent), we systematically investigated megasporogenesis and megagametogenesis of female parents, pollen germinability of male parents, pollen–pistil interaction and hybrid embryo development using paraffin section and light, fluorescence and scanning electron microscopy. The results indicate that development of megagametophyte of female parent belongs to Polygonum type and 84.6% ovules developed to normal 8-nucleate embryo sac at the stage of pollination; the pollen germinability in vitro of male parent was 39.3%; a large number of pollen grains germinated normally and no callose deposited on the female stigmas within 24 h after pollination. Furthermore, about 75% ovules were fertilized, whereas only 17.2% ovules developed to normal maturing embryos at 15 days after pollination due to abortion at various developmental stages. Taken together, these results suggest that sterile ovules of female parent, pollen viability and pollen–pistil interaction have no significant influence on seed set in the cross. A high percentage of embryo abortion or degeneration, i.e. post-fertilization barrier, may be the main factor for low seed yield.     

Production of a desirable Brassica oleracea CMS line using an alloplasmic B. rapa CMS line carrying Diplotaxis erucoides cytoplasm as a bridge plant

In Brassica oleracea, production of F₁ hybrid seeds mainly makes use of the improved Ogura cytoplasmic male sterile (CMS) line. However, reliance on one particular line is a risk, and it would be advantageous to develop other CMS lines. In this study, we transferred Diplotaxis erucoides cytoplasm to B. oleracea cultivars using an alloplasmic B. rapaCMS line as a bridge plant to avoid incompatibility between donor and recipient plants. The new B. oleraceaCMS lines, which were derived by four generations of backcrossing, had small rudimentary anthers with no pollen grain and showed complete male sterility. There was no functional defect in other floral organs, and the ability to receive normal pollen did not appear to be impaired. Moreover, the B. oleraceaCMS lines carrying D. erucoides cytoplasm had larger leaf areas and a normal plastochron. As a consequence, the B. oleraceaCMS lines carrying D. erucoides cytoplasm have the potential to be valuable alternatives for use in commercial B. oleracea hybrid seed production.     

Wheat pollen dispersal under semiarid field conditions: potential outcrossing with Triticum aestivum and Triticum turgidum

Isolation distance is the main barrier to crop-to-crop gene-flow. A 3-year study assessed the maximum potential outcrossing under field conditions between two wheat cultivars (Triticum aestivum L.) and between wheat and durum wheat (Triticum turgidum L. var. durum). Outcrossing was measured by seed set on emasculated recipient plants placed at four sides with different distances from a 3 m × 3 m T. aestivum (cultivar Chinese Spring) pollen source. Frequencies of seed set at 0 m distance were 45% (37–56%) for T. aestivum cultivars and 18% (5–30%) with T. turgidum. These values agree with hybridization in non-limiting pollen conditions measured by manual crosses in greenhouse. The number of pollen grains and the outcrossing frequencies decreased at increasing distances influenced by the prevailing wind direction. Under semiarid conditions of this assay, viable pollen was found 14 m from the pollen source, with a maximum distance of 8 m at which cross-pollination decreases below 1%. Ambient conditions affect pollen viability, hybridization and pollen dispersal. Data presented in this paper emphasize the major role played by environmental conditions in outcrossing. Data obtained in one area may therefore not coincide with the prevailing situation in different locations and climates.     

Performance Studies in F6 Lines of Autotetraploid Fenugreek

Hybridization followed by pedigree selection was carried out between two autotetraploid strains of fenugreek (Trigonella foenum-graecum L.). Morphological and cytological investigations were carried out in two autotetraploid parents, their F₁ hybrids and F₆ lines derived from F₁ after selfing. Cytomorphological data of F₆ lines were compared with those of F₁, hybrids and of their parents. The hybrid plants were intermediate with respect to different vegetative traits, however, they showed increased seed set as compared to their autotetraploid parents. The mean pod number per plant in F₆ was one and half times as high as one of the high yielding tetraploid parents and approximately twice as high as that of F₁ hybrid. Meiosis in F₆ was more regular than in F, and the parents. Thus, hybridization followed by pedigree selection is helpful not only in regularizing the meiotic behavior, but vigour and seed fertility have also improved in autotetraploid fenugreek.     

Yield, yield components and plant architecture in the F3 generation of common bean (Phaseolus vulgaris L.) derived from a cross between the determinate cultivar ‘Prelude’ and an indeterminate landrace

Growth trials were conducted outdoors in the UK to determine the yield, yield components and plant architectural differences between determinate and indeterminate Phaseolus vulgaris bean plants. F3 lines derived from crosses between ‘Prelude’, a determinate cultivar and ‘V8’, an indeterminate landrace, were grown together with the parents and ‘Carioca’, an indeterminate landrace from Brazil. Data were recorded on flowering date, number of nodes on main stem at flowering, plant height at maturity, number of pods/plant, number of seeds/pod, number of seeds/plant, 100-seed weight, seed mass/plant, percentages of diseased and healthy seeds/plant. Determinate F3 lines had significantly lower (P < 0.05) seed mass/plant, fewer pods/plant, fewer seeds/pod, fewer seeds/plant, lower harvest index, shorter stems, earlier date of flowering, fewer nodes at flowering and fewer healthy seeds/plant than indeterminate F3 lines. However, determinate genotypes had significantly larger (P < 0.05) pods to accommodate their larger seeds compared with indeterminate lines. A similar situation was found when the yield and yield components of ‘V8’, ‘Prelude’ and ‘Carioca’ were evaluated; the highest seed mass/plant was given by ‘V8’ and the lowest by ‘Prelude’. Furthermore, some indeterminate genotypes with Type IV growth habit yielded significantly higher (P < 0.05) than the high-yielding parent, ‘V8’, indicating a positive influence on seed yield by crossing different genotypes.     

Pollen storage at low temperature as a procedure for the improvement of cold tolerance in spring rape, Brassica napus L.

The possibility of selecting spring rape for cold tolerance at the mature pollen grain stage was studied by investigating the effects of pollen storage at low temperatures on the quality of pollen grains and on the cold tolerance of the plants generated from them. Pollen treatments of F¹ hybrids affected fertilization ability much more than viability and even after 10 days storage at 3 or 10°C the pollen germination percentage was reasonably high. Pollen storage for 7 or 10 days at 3 or 10°C significantly increased the cold tolerance of F² seed germination, with 3°C being more effective. Pollen storage for a shorter time had no effect upon the number of resulting genotypes tolerant to low temperature. This approach may be successfully applied in plant breeding to enrich segregating plant populations with cold-tolerant genotypes.     

Induction of 2n pollen in tulips by arresting the meiotic process with nitrous oxide gas

Triploid tulips have agronomically desirable traits such as vigorous growth and large flower size, but only a portion of all cultivated tulips is triploid. To apply 2n pollen to polyploid breeding of tulips, the polyploidizing agent, nitrous oxide gas (N₂O), was applied to bulbs. In tulips, meiosis in anthers occurs inside the bulbs from mid- to late-October. When meiosis in anthers (excised from bulbs) reached metaphase I, we treated other bulbs of the same clones with N₂O for 24–48 h. Most of the treated plants produced pollen grains with a wide-ranging or bimodal size distribution, indicating a mixture of n, 2n and aneuploid pollen grains. The use of pollen containing a relatively high proportion of giant pollen grains tended to yield larger numbers of triploids in the progeny. The number of giant pollen grains could be increased when N₂O-treated pollen grains were suspended in 10% sucrose and then sieved through a nylon mesh. Very few polyploids were observed in some cross combinations, even those involving pollen with a relatively high proportion of giant grains. Even so, this low polyploid yield most likely is due to a triploid block, because the capsules obtained in the crosses of the diploid×N₂O-treated plants contained some abnormal seeds, which were mostly triploid. Embryo culture was useful in rescuing abnormal embryos. The present study reveals that 2n pollen can be produced at high frequency using N₂O during tulip breeding.     

Pollen production in soybeans with respect to genotype,environment, and stamen position

Summary Our objective was to determine the average numbers of pollen grains from fertile plants (Ms ₁) and the average numbers of coenocytic microspores from genetic male sterile plants (ms ₁ ms ₁) in soybeans, Glycine max L. Merr. Comparisons were made between the average numbers of pollen grains and the average numbers of coenocytic microspores with respect to environment where plants were grown and to stamen position in the flower. Five male sterile lines were used. They included the North Carolina ms ₁ mutant, the cultivar Hark with the ms ₁ gene, and mutants identified as the Urbana, Tonica, and Ames male steriles. Three environments used were the Agronomy Farm, University of Georgia, Athens, Georgia; the Agronomy and Agricultural Engineering Research Center, Iowa State University, Ames, Iowa; and the Agronomy Greenhouse, Iowa State University, Ames, Iowa.Pollen production from fertile plants varied from 374 to 760 pollen grains per anther among genetic lines and environments. This variation may be an important consideration in selecting a male parent to use as a pollinator for hybrid seed production.Among fertile plants, the average numbers of pollen grains per anther of the separate stamen and and of the lower whorl of stamens were significantly different only in greenhouse-grown plants. Among male sterile plants, the average numbers of coenocytic microspores per anther of the separate stamen and of the lower whorl of stamens were significantly different in three genotype x environment combinations. These three exceptions did not conform to any genetic or environmental pattern. Deviations from the expected ratio of 4 pollen grains from fertile plants: 1 coenocytic microspore from sterile plants were attributed to initial differences in the average number of microspore mother cells between the two genotypes.Joint contribution: Agricultural Research Service, USDA, North Central Region, and Journal Paper No. J-8910 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Cytological mechanism of 2n pollen formation and unilateral sexual polyploidization in Lolium

Summary Plants of Lolium perenne, L. multiflorum and L. hybridum (331, 51 and 18, respectively) were screened for 2n pollen production. The screening was based on the size differences that are expected to be found between n and 2n pollen of a plant. It was found that 28 plants of perennial ryegrass-belonging to 13 cultivars-produced produced pollen grains of heterogenous size (big and small). The estimated frequency of big pollen grain production was higher than 10% in ten out of 28 plants and in three of them it reached a value of 100%. Eight plants obtained from two 4x × 2x crosses, in which the male parent had been previously identified as producing pollen grains of heterogenous size, was tetraploid. The cytological mechanism of big pollen grain formation observed in three plants consists in the lack of spindle formation and, consequently, of chromosome migration in anaphase II. From these breeding and cytological results, it was concluded that the big pollen grains observed were viable 2n pollen grains. The nuclei of the 2n pollen grains produced by this mechanism are genetically equivalent to those formed by the restitution of the second meiotic division. The value of these gametophytes in a breeding program of Lolium is discussed.     

Creation of novel interspecific-interploid Hylocereus hybrids (Cactaceae) via embryo rescue

In this study we developed a technique for rescuing embryos at a very early developmental stage following interspecific-interploid crosses between Hylocereus sp. Controlled hand pollinations were performed between the tetraploid H. megalanthus (Vaup.) Bauer as the female parent and either diploid H. monacanthus (Lem.) Britton et Rose or H. undatus (Haw.) Britton et Rose as the male parent. The fertilized ovules were excised from ovaries 10 or 30 days after pollination (DAP). Pollinated ovules containing the funiculi and placental tissue and immature embryos were placed on half-strength basal Murashige and Skoog (MS) medium containing 680 μM glutamine, 0.54 μM α-naphthaleneacetic acid, and 0.45 μM thidiazuron and supplemented with 0.00, 0.09, 0.17 or 0.26 M sucrose concentrations. The best ovule response was recorded at 30 DAP, and the most prolific callus formation was observed at 10 DAP. Callus formation was observed in most of the treatments using whole ovules but not in the isolated immature embryos. The calli were mucilaginous or compact, transparent and friable, but they did not form embryogenic structures. Embryo development was significantly affected by the sucrose concentration, and the best results were obtained with 0.17 M sucrose. More than 70 % of the obtained hybrids were successfully hardened off and transplanted in soil where they grew normally. Ploidy level analyses of 77 putative hybrids exposed diploid, triploid, tetraploid, and higher than tetraploid levels. Among those hybrids studied, 22 progenies were randomly chosen for amplified fragment length polymorphism analysis, and all were identified as genuine hybrids. The technology described here is an additional stage in the breeding program for Hylocereus species resulting in novel, interspecific hybrids obtained using the embryo rescue technique.     

同源四倍体高粱不育系和保持系的生物学及生理特性研究

以二倍体高粱不育系及其保持系和由它们诱变成的同源四倍体选系为材料, 研究 了同源四倍体不育系和保持系的生物学特征及生理特性, 结果发现, 四倍体的株高一般为 二倍体的80.6％～90.1％, 单株总叶片数减少1～2片; 保持系加倍后, 顶部三叶面 积增 加4.6％～9.4％; 不育系的花药由二倍体时的白色变为四倍体的淡黄色,