Cytogenetic studies in hexaploid Helianthus species and their F1 hybrids with cultivated sunflower, H. annum

Hexaploid sunflower species provide potential sources of desirable genes in breeding for resistance to pathogens as well as high protein and oleic acid content. The possibility of using them in sunflower breeding was studied by testing the crossability of hexaploid species to cultivated sunflower, by the analysis of meiosis and pollen viability in the F₁ interspecific hybrids. The hybrids were obtained from 16 combinations, with 1–18 F₁ plants per combination. The F₁ hybrids in general exhibited the dominant phenotype of the wild species. Sterility ranged from 0 to 87.5% per F₁ combination, while pollen viability ranged from 43.0 to 70.2%. Meiosis was almost normal in the species analysed but irregular in the F₁ hybrids. Most of the meiocytes showed predominantly bivalents (86.79–90.22%), but univalents (0.00–4.99%) and the multivalents (8.23–10.09%) occurred as well. At anaphase I, chromosome bridges were detected in 6.88–8.48% of meiocytes. Fast chromosomes in meta-phase I and lagging chromosomes in anaphase I and telophase II were observed in many meiocytes.     

Meiosis and pollen grain viability in Helianthus mollis,Helianthus salicifolius,Helianthus maximiliani and their F1 hybrids with cultivated sunflower

Three diploid perennial sunflower species are useful for variety improvement: Helianthus mollis, because of sessile leaves, H. salicifolius, because of a high oil concentration, and H. maximiliani, a potential source of resistance to Sclerotinia sclerotiorum. The crossability of these species to cultivated sunflower was examined.Hybrids were obtained from eight combinations, with 3–15 F₁ plants per combination. The F₁'s exhibited the dominant phenotype of the wild species. Pollen viability varied between 32.1 and 69.9%. Meiosis was irregular in the F₁ hybrids. At diakinesis, bivalents (62.7–97.9% of meiocytes), univalents (0–31.23%), and multivalents (3.84–7.68%) were detected. At anaphase I, chromosome bridges were detected in 6.77 to 11.44% of meiocytes. Fast chromosomes in metaphase I, and lagging chromosomes in anaphase I and telophase II were evidenced in a high percentage of meiocytes.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

Cytology and pollen grain fertility in creeping bentgrass interspecific and intergeneric hybrids

Creeping bentgrass (Agrostis stolonifera L. or A. palustris Huds.) is a highly outcrossing allotetraploid species. It can form hybrids with a number of other Agrostis species and Polypogon genus. However, cytology and pollen grain fertility of the creeping bentgrass interspecific and intergeneric hybrids are not well known. In this research, chromosome pairing behaviors during meiosis I in F₁ and pollen viability of F₁ hybrids, as well as seed set rate and seed germination rate of backcrosses were studied in hybrids between creeping bentgrass, and other bentgrass species and three species of Polypogon genus. Abnormal chromosome pairing, laggard chromosomes, and premature segregation in F₁ hybrids were found. Pollen viability ranged from 1.6 to 48.5% amongst F₁ hybrids, significantly lower than that of the parents (85.5–94.1%). Some hybrids produced pollens of different sizes within the same anther. Seed set following backcrosses using F₁ hybrids as the male parent and creeping bentgrass as the recurrent parent was significantly lower than their parents. The study of chromosome paring behaviors and progeny fecundity are important in utilizing the alien genes to improve bio-stress and abio-stress resistance, and in assessing the potential transgene risks of creeping bentgrass.     

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Summary Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.Abbreviations BAP Benzyl Amino Purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - MS Murashige & Skoog medium - IAA Indole Acetic Acid     

Cytogenetic study of Helianthus laevigatus and its F1 and BC1F1 hybrids with cultivated sunflower, Helianthus annuus

Although the wild sunflower species Helianthus laevigatus has not been extensively studied it may be considered for sunflower breeding as a potential source of desirable genes for Sclerotinia stalk rot resistance and high contents of proteins and linoleic acid in the seed. A set of six H. laevigatus populations was crossed to cultivated sun~ower lines and produced nine F₁ (2-14 plants) and 66 BC₁F₁ hybrid combinations (1-13 plants). Male sterility occurred in F₁ and BC₁F₁ hybrid combinations and pollen viability was lower in the progenies than in the parents (51.6-77.2%in F₁ and in F₁ and 4.8-34.0% in BC₁F₁). Meiosis was normal in the H. laevigatus populations It was found that this tetraploid species also occurred in a hexaploid form Numerous irregularities were observed in the meiosis of the F₁ interspecific hybrids During diakinesis, quadrivalents and hexavalents were recorded in addition to bivalents Dislocated chromosomes and chromosome bridges were present in the other phases The chromosome number in F₁ was 68 (tetraploid). Irregularities in chromosome pairing were observed in the interspecific hybrids at BC₁F₁. There were many univalents, and trivalents quadrivalents and hexavalents were also present The chromosome number in the BC₁F₁ generation ranged from 34 to 60. The occurrence of meiotic irregularities in the F₁ and BC₁F₁ interspecific hybrids indicates that H. laevigatus and the cultivated sunflower differ in genome constitution.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Moricandia arvensis cytoplasm based system of cytoplasmic male sterility in Brassica juncea: Reappraisal of fertility restoration and agronomic potential

Cytoplasmic male sterility (CMS) system based on the cytoplasm from Moricandia arvensis (mori) was investigated for fertility restoration and agronomic potential. Fertility restorer gene for mori CMS was introgressed from cytoplasm donor species as all the evaluated Brassica juncea genotypes (155) acted as sterility maintainers. The allosyndetic pairing between M^a and the A/B genome chromosomes in the monosomic addition plants (2n= 18II + 1M^a) facilitated the gene introgression. Partial fertility restoration (43–52% pollen grain stainability) in F₁ hybrids and absence of segregation for male sterility in F₂ progenies suggested gametophytic control of fertility restoration. The pollen fertility in the F₁ hybrids was, however, sufficient to ensure complete seed set upon bag selfing. Introgression from M. arvensis also helped in correction of chlorosis associated with mori cytoplasm in CMS and fertile alloplasmic B. juncea plants. Yield evaluation of thirty F₁ hybrids having the same nuclear genotype but varied male sterilizing cytoplasms (mori, oxy, lyr, refined ogu), in comparison to respective euplasmic hand bred control hybrids, allowed an estimate of yield penalty associated with different CMS systems. It ranged from 1.8% to 61.6%. Hybrids based on cytoplasmically refined ogu were most productive followed by those based on cytoplasmically refined mori CMS. The male sterility systems emanating from somatic hybridization were found superior than those developed from sexual hybridization.     

Intergeneric hybrids and an amphiploid between Elymus canadensis and Psathyrostachys juncea

Summary Fifty four hybrid plants between Elymus canadensis and Psathyrostachys juncea were obtained by handpollination and embryo culture. The average cross compatibility between both species was 31.2 percent. One amphiploid plant was induced by colchicine treatment. The hybrid and amphiploid plants resembled P. juncea in appearance but showed a higher plant height and dry matter yield than the parents. The hybrids showed extremely low pollen stainability and were completely sterile. With the exception of one plant (2n=3x+1=22), all hybrid plants were allotriploids (SHN, 2n=3x=21). The amphiploid plant (SSHHNN, 2n=6x=42) showed 58.9% pollen stainability and 11.6% seed fertility.Mean chromosome associations of the hybrids and amphiploid at metaphase I were 0.02IV+0.06III+2.03II+16.91I and 0.07III+18.00II+5.85I, respectively. Lagging chromosomes, chromosome bridges, abnormal cytokinesis, and micronuclei were occasionally observed at the anaphase, telophase, or tetrad stage.     

Meiotic restitution mechanisms and 2n pollen formation in a Solanum tuberosum dihaploid and in dihaploid × wild species hybrids*

A Solatium tuberosum dihaploid that proved to be a 2n pollen producer, exhibited two types of meiotic abnormality involved in 2n pollen formation: premature cytokinesis at prophase II and parallel/tripolar spindles in meiosis II. For this reason such a dihaploid was preferentially used as a seed parent in crosses with the diploid species S. tarijense, S. sanctae-rosae, and S. phureja. Diploid interspecific hybrids were evaluated for male fertility, for 2n pollen production and stability over three years and for meiotic nuclear restitution mechanisms. Pollen stainability ranged from 6 to 85% and 2n pollen from 5 to 35% among hybrids. The meiotic mechanism generating 2n pollen was that of spindle abnormality in meiosis II and consequent dyad/triad formation. High correlation was found between fused/parallel spindles and dyads.     

Interspecific hybrid of Helianthus annuus × H. simulans: Characterization and utilization in improvement of cultivated sunflower (H. annuus L.)

The first success at interspecific hybridization between cultivated sunflower(H. annuus) and a diploid perennial species, H. simulans is reported. The F₁s from both direct and reciprocal directions exhibited dominance of the wild species phenotype and were pollen sterile. Meiosis was irregular in the F₁ hybrids and both univalents and multivalents were observed. Multiplication of the interspecific hybrids was achieved through in vitro culture of nodal sections on Murashige & Skoog medium supplemented with 0.5 mg l^-1 benzyladenine. Fertility of the interspecific hybrids was improved by subjecting the in vitro proliferating shoots to 0.001% colchicine incorporated in the shoot multiplication medium. The amphiploids serve as fertile bridges and facilitate interspecific gene transfer through conventional breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytogenetic analysis of interspecific hybrids in genus Citrullus (Cucurbitaceae)

Investigations on interspecific crossability in two species of the genus Citrullus were studied based on their morphology, chromosome pairing and pollen fertility in their hybrids resulting from 12 combinations(including reciprocals). These have been utilized for tracing the phylogenetic relationship between the taxa and factors responsible for their differentiation. A collective evaluation of data on cross compatibility,morphology, chromosome association and pollen fertility indicated a close phylogenetic relationship between the species C. colocynthis (wild2n = 22) and C. vulgaris (cultivated 2n = 22). Significant differences for crossability and pollen fertility were observed between hybrids of C. colocynthis × C. vulgaris and reciprocals. Meiotic analysis of F₁ hybrids revealed that these two species are freely crossable and provide normal chromosome association, involving an average of 9 to maximum 11 pairs of chromosomes. This indicated the possibility of high recombination and thereby genetic introgression of desirable traits. As both the species are of economic significance, breeding strategies can be developed for the genetic improvement of these species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Gene flow between cultivated sunflower and <Emphasis Type="Italic">Helianthus petiolaris</Emphasis> (Asteraceae)

Helianthus petiolaris (Asteraceae) native to North America has naturalized in Argentina. The extensive overlapping with sunflower crop regions, the coincidence of life cycles and the common pollinators facilitate interspecific crosses with sunflower, H. annuus var. macrocarpus. To estimate the occurrence of crop-to-wild and wild-to-crop gene flow, off-type plants in progenies of pure stands of both species flowering in coincidence and the presence of crop alleles in H. petiolaris populations were investigated in 26 wild populations and nine crop fields. Morphological traits and RAPD markers were used to attempt hybrid characterization. Off-type individuals were found in frequencies of 0.5 and 0.3% among crop progenies and wild populations, respectively. Off-type plants showed intermediate values for metric morphological traits and low fertility traits. Some off-type plants proved to carry crop alleles based on molecular analysis. The average frequency of cultivar-marker introgression across the wild populations was very low (0.02). Although observed hybridization rates seem to be low, the extension of crop-wild overlapping in Argentina make hybrid formation a noticeable process. Therefore, the new sunflower varieties and eventually GM varieties would transfer their traits through pollen flow and they would persist in H. petiolaris populations.     

Genetics of fertility restoration in A2-based cytoplasmic genetic male sterility system in pigeonpea (Cajanus cajan)

The three short duration cytoplasmic genetic male sterility (CGMS) hybrids developed using A₂ (Cajanus scarabeoides) cytoplasm-based male sterile lines (CORG 990047A, CORG 990052A and CORG 7A) and the restorer inbred AK 261322 and their segregating populations (F₂ and BC₁F₁) were subjected to the study of inheritance of fertility restoration in pigeonpea. The fertility restoration was studied based on three different criteria, namely, anther colour, pollen grain fertility and pollen grain morphology and staining. The F₂ and BC₁F₁ populations of the three crosses, namely, CORG 990047A × AK 261322, CORG 990052A × AK 261322 and CORG 7A × AK 261322, segregated in the ratio of 3:1 and 1:1, for anther colour (yellow:pale yellow), pollen grain fertility (fertile:sterile) and for pollen grain morphology and staining. The above study confirmed that the trait fertility restoration was controlled by single dominant gene. This finding can be utilized for the identification of potential restorers, which can be further used in the development of CGMS-based hybrids in pigeonpea.     

三系杂交棉花粉育性对高温和低温胁迫的反应

为研究在高温和低温胁迫条件下的三系杂交棉花粉育性稳定性问题,利用棉花细胞质雄性不育系和恢复系,配制2个三系杂交棉组合,抗A×浙大强恢(记作：强恢F₁)和抗A×DES-HMF277(记作：弱恢F₁),并以保持系(抗B)为对照,分别进行温室控温试验和田间自然温度试验,分析三系杂交棉对高温和低温胁迫的反应和花粉育性转换(可育至不育)的临界温度。试验表明,一般三系杂交棉的花粉育性对胁迫温度的反应比保持系敏感,常常花粉散粉少和花粉活力较低。不同三系杂交棉组合对胁迫温度的抗性存在明显差异,强恢F₁明显高于弱恢F₁,与保持系的育性相似,可育花粉率和自交结铃率较高,不孕籽率较低。经可育花粉率(Y)与温度(T)的回归分析,花粉育性转换的临界温度符合Y = a (T–T_opt)²+b模型。强恢F₁育性转换的上限和下限温度分别为38.0℃和13.0℃,弱恢F₁为36.0℃和14.0℃,保持系为38.5℃和10.0℃。与低温胁迫比较,高温胁迫在我国大部分棉区更普遍,持续时间更长,对产量影响更大。提高三系杂交棉在胁迫气温条件下的花粉育性的稳定性是近期育种的重要目标。     

Characterization of interspecific hybrids and first backcross generations from crosses between two cultivated eggplants (Solanum melongena and S. aethiopicum Kumba group) and implications for eggplant breeding

Common (Solanum melongena L.) and scarlet (S. aethiopicum L.) eggplants are cultivated for their fruits and form part of the same genepool. We have studied plant and fruit characteristics, pollen viability and seed set, phenolics content, and fruit flesh browning in accessions of S. melongena and S. aethiopicum Kumba group, as well as interspecific hybrids between these species and first backcross generations to each parental species. Respective genotypes were also characterized with seven polymorphic SSR markers. The results demonstrate that many differences exist for plant and fruit morphology among S. melongena, S. aethiopicum and the interspecific hybrids. The latter are very vigorous and generally intermediate between the two parents, except for fruit size which is smaller (and parthenocarpic due to a high pollen sterility) than those of any of the parents. Backcross progenies also exhibited morphological variation with moderate heritability values for the attributes evaluated. Variation for fruit size was present in the backcross generations but fruits were small resulting in little variation for fruit shape. Backcross plants with moderate fertility produced seeded fruits. Primary hybrids had fruit phenolics content similar to that of S. aethiopicum, the parent with lowest phenolics concentration, and were heterotic for fruit flesh browning. Backcross progenies were quite variable for both traits. SSR markers did not reveal segregation distortion in the backcross generations for these interspecific hybrids. The results demonstrate that generations derived from sexual interspecific hybridization can be a powerful tool for S. melongena and S. aethiopicum Kumba group breeding.     

Relationship between genetic divergence and hybrid performance in japonica rice grown in a cold water-irrigated field

The present study was carried out to evaluate genetic divergence among eleven japonica rice cultivars and to assess the relationship between genetic distance and hybrid performance in partial non-reciprocal crosses among them. The 44 F1 hybrids along with the eleven parents were evaluated for five cold tolerance-related traits; discoloration at seedling stage, days to heading, culm length, fertility, and spikelets per panicle in a cold water screening nursery (17 ^°C). The eleven parents were examined for DNA polymorphism using amplified fragment length polymorphisms(AFLPs). A total of 855 polymorphic variants were generated and based on the polymorphism data, genetic distances (GDs) ranged from 0.023 to0.524. Very little heterosis was observed in hybrids for most of the traits,whereas heterosis was high for fertility. The correlation values of GDs with F1 performance were mostly non-significant except for discoloration and fertility. The correlations of GDs with mid-parent and better-parent heterosis were not significant and proved to be of no predictive value. Our results indicate that GDs based on AFLP markers are not useful for predicting heterosis for cold tolerance in japonica hybrids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Enhanced grain yield in rice hybrids through complementation of fertility restoration by Rf3 and Rf4 genes as revealed by multilocation evaluation of tropical japonica derived rice (Oryza sativa) hybrids

Thirty-one tropical japonica derived Rf gene carrying rice hybrids were classified into three classes as Rf3, Rf4 and Rf3 + Rf4 hybrids. These hybrids were tested under three different mega-environments. Between Rf3 class and both the classes of hybrids possessing Rf4 genes, significant variation for spikelet and pollen fertility and grain yield was found. The pollen fertility was five times higher among Rf4 hybrids than that of hybrids carrying Rf3 alone. Likewise, spikelet fertility among Rf4 hybrids was two times higher than that of Rf3 hybrids. Parallel grain yield increase in Rf4 hybrids was 2.4 times than in Rf3 hybrids. However, Rf3 gene was found complementing Rf4 to truncate the range of pollen and spikelet fertility. Hybrids possessing Rf3 alone exhibited partial pollen and spikelet sterility, and significant negative standard heterosis for grain yield. The high yielding hybrid, “HYB36” carrying both the Rf genes was found to be widely adapted. The present study established that Rf4 gene is essential either alone or in combination with Rf3 for fertility restoration to achieve enhanced grain yield in WA-CMS based hybrids.     

Meiotic behavior in Brachiaria humidicola (Poaceae) hybrids

In the genus Brachiaria, genetic variation can be exploited directly from germplasm collections or released using sexual reproduction in normally apomictic polyploids. The discovery of a natural sexual polyploid accession H031 of Brachiaria humidicola collected in Africa, opened new opportunities to exploit the genetic variation in this species. This accession was crossed with an apomictic cultivar BRS Tupi with the same chromosome number (2n = 36) and 361 F₁ hybrids were obtained. Following visual selection for leafiness, vigor, growth habit, and the mode of reproduction, 50 hybrids were selected for further agronomic evaluation. The parents and 45 of the 50 selected hybrids were evaluated for the regularity of meiosis. In the female parent (H031), meiosis was somewhat irregular, with 16.3% of abnormal tetrads, whereas the male (cv. BRS Tupi) meiosis was very regular, with only 3.1% of abnormal tetrads. Among hybrids (sexual and apomictic), the percentage of abnormal tetrads ranged from 15.8 to 98.3%. The abnormalities included irregular chromosome segregation, chromosome stickiness and the absence of cytokinesis. Considering that apomixis in the genus Brachiaria is pseudogamic, and that meiotic aberrations can compromise pollen viability, the results of this study present another parameter to aid selection for more stable microsporogenesis. Apomictic derivatives with stable meiosis are candidates for new cultivars whereas sexual hybrids can be retained in breeding for another round of recombination.     

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.