Intra- and interspecific transmission of androgenetic competence in diploid potato species

Summary In an attempt to determine the transmission of androgenetic competence, 10 families resulting from intra- and interspecific hybrids including three reciprocal hybrids were examined in anther culture. Hybrid families were generated between competent clones of Solanum phureja and incompetent clones of S. phureja, S. microdontum and S. berthaultii. S. phureja clones PP5 and A95 (derived by androgenesis of a 2n microspore of PP5) were found to be consistently competent for androgenesis. Androgenetic competence was observed to segregate in all hybrid families with some highly responsive and some unresponsive genotypes in all families. A total of 9,465 cultured anthers have yielded 936 embryoids and 91 plants, including 29 monoploids. The cytoplasm of species lacking competence appeared to have greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. Expression of androgenesis varied among half-sib hybrid families indicating that competence for androgenesis was influenced by the parents lacking competence. The anther culture data on a backcross between a highly responsive hybrid and its unresponsive parent indicated that competence may be under control of a single dominant gene.     

High frequency spontaneous production of doubled haploid plants in microspore cultures of Brassica rapa ssp. chinensis

Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosome doubling of mature haploid Brussels sprout plants by colchicine treatment

Summary The effect of colchicine concentration, and the mode and time of colchicine application in doubling the chromosomes of haploid Brussels sprout plants was assessed by scoring treated plants for the presence of diploid flowers and seed set after self-pollination.When colchicine treatments were applied after the plants had been vernalised, using 2 dose rates and 3 methods of application, only 38.1% became doubled and only 13.8% produced seed. Treatment with 0.1% colchicine with or without the addition of 2% dimethyl sulphoxide gave doubling rates similar to those reached with 0.05% colchicine, but resulted in more damage to the apices.When 0.05% colchicine solution was injected into the plants' apices at varying times during vernalisation, the rate of doubling was 71.2% on average and 50.7% of plants gave seed on selfing.Overall doubling rates of plants where the apex was easily accessible were 79.0%, while for plants with dense terminal heads they were 35.0% and for plants with visible buds 31.4%.     

Effect of genotype,environment and carbohydrate on anther culture response in head cabbage (Brassica oleracea L. convar. capitata (L.) Alef.)

Summary The effect of genotype, growing conditions for donor plants and type and concentration of carbohydrate in the culture medium was investigated for anther culture of head cabbage (white cabbage, savoy cabbage, pointed-headed cabbage). Strong genotypic effects on embryo formation from the cultured anthers were shown as well as superior embryo formation from anthers of field grown donor plants compared to plants grown in the greenhouse. When comparing 7, 10 and 13% sucrose in the medium, embryo response increased with increasing sucrose concentration. With maltose, which was generally inferior to sucrose as carbohydrate source for anther culture, the embryo response did not increase with maltose concentration above 10 per cent.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

Somatic embryogenesis in pea (Pisum sativum L. and Pisum arvense L.): Diallel analysis and genetic control

Summary Six lines of Pisum were tested in vitro for their ability to produce somatic embryos from apices. Significant quantitative variation was observed. Inheritance of the ability to form somatic embryos was studied using a diallel cross among six different lines. About 80% of the observed genotypic variation was due to additive effects. There is a tendency for the favourable genes to be recessive. It appears that there are two genetic systems involved. Analysis of the distribution of F₃ families means from a cross among two extreme lines seems to indicate the presence of a few major genes in the control of somatic embryogenesis of pea.     

Selection for fast germination in rapeseed (Brassica napus L. and B. Campestris L.)

Summary Eleven populations of Brassica napus L. and twelve populations of B. campestris L. were subjected to three cycles of selection for fast germination at 2°C and at 25°C. The seeds from the selected populations, and unselected control populations grown in the same environment as the selected populations, were examined for germination behaviour at 2°C and 25°C, and for growth behaviour at 10°C. The populations in both species responded differently in terms of germination behaviour to selection for fast germination. In most of the populations that did respond positively to selection, selection practised at 2°C was superior to selection at 25°C in improving percent germination at 2°C, and was as good as the selection at 25°C in improving germination rate at the higher temperature. Selection for fast germination had no effect on growth characteristics of B. napus and B. campestris populations grown at 10°C. Thus, selection for fast germination at one low temperature may lead to improvement in germination characteristics over a range of temperatures, but will not necessarily lead to improved growth performance of the selected populations.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Maternal induced haploids in rye (Secale cereale L.)

Summary An inbred line of rye named P8 was found to possess haploid plants in their diploid stock. Phenomenon of spontaneous haploidy was examined in this line. Fifty families derived from this inbred line were screened to determine their ploidy level by mitotic chromosome count taking root tips. Seven families were found to contain haploid plants. Among these seven families, plants from two families which were diploid were crossed reciprocally with the standard out breeding cultivar Conrah. As all of the tested plants of P8 were found aa allele for 6-Phosphogluconic dehydrogenase (6-PGD) isozyme, therefore, only those Conrah plants which have bb alleles for the same isozyme were used in crossing programme.F1 seedlings were screened with the same isozyme marker and out of 1741 F1 seedlings tested, 51 were found to be non-hybrid phenotype for 6-PGD enzyme. Among these, only five plants were found haploid and the rest were diploid. Haploid plants were obtained only from those crosses where P8 was female which indicates that haploids in this line was maternally induced.     

Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp. oleifera (DC.) Metzger

The influence of temperature/photoperiod treatment and gibberellic acid concentration (0, 0.1 or 1.0 mg/l) on direct conversion of microspore-derived embryos (MDEs) to plantlets of winter oilseed rape was investigated. Physiologically mature, 21-day-old MDEs were transferred to a solid B5 medium supplemented with gibberellic acid, and cultured at 24 ^°C, 4 ^°C or 1 ^°C for 14 days, and then at 24 ^°C for the next 21 days. Low temperature was linked with short photoperiod (8 h light/16 h dark), and high temperature was linked with long photoperiod (16 h light/8 h dark). The highest embryo conversion rate was at 1 ^°C with over 70%, compared to<20% at 4 ^°C. Two-way analysis of variance confirmed the significance of the effect of temperature/photoperiod treatment. By contrast, gibberellic acid concentration had no significant effect on stimulation of shoot development from apical meristems of MDEs. Roots developed from apical root meristems of MDEs very easily. The best obtained conversion rate of MDEs induced with cold treatment at1 ^°C for 14 days was 86.5%. Observations on morphological development of MDEs showed clear differences in reaction at various temperature/photoperiod treatments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Green haploid plants from unpollinated ovary culture in tetraploid wheat (Triticum durum Defs.)

In Triticum durum Defs., gynogenesis was investigated on six genotypes, Cocorit, Isly, Jori, Oued Zénati, Sarif, issued from Morocco,and Cham1 from Syria. The experiments described here were made during two seasons (spring and summer) in which batches of 1,036 and 3,750 unpollinated ovaries were used. A 4 ^°C pretreatment was applied, during 7, 11 or 15 days and the A (modified Jähne's et al.,1991) and B (modifed San N?um's, 1976) induction media were compared. The spikes were harvested when microspores were at bi- or trinucleate stage. After the 4 ^°C cold pretreatment, they were sterilized, and the excised ovaries underwent the following phases and sequence of successive media. The A or B ‘induction’ media, induced cell divisions in the female gametophytes, and after 4 to 9 weeks in dark conditions, the swelling ovaries burst. The growing calluses were excised and transferred to 16 h. light onto a differentiation medium (dif); there generated shoots were then placed on a development (dev), followed by a rooting medium (r). The comparison of the two experiments showed that summer was the best season, for in vitro unpollinated ovary culture; this period corresponds to a growth of mother-plants during spring, while those developing in winter gave rise to lower scores. In Experiment 1 the best level of regenerated plants per 100 ovaries were obtained for Isly with 14.3% and for Oued Zénati with 10.1%. In Experiment 2, the best values were those of Isly with 21.8%, after a 7 day cold pretreatment and use of induction medium B, and of Cocorit with 18.1%. Jori, exhibited good results, in three define situations, with 17.1%, 16%, and 9.3%.Only Cham1 and Sarif showed very low scores or no plants. Jori remaining aside, for each genotype, the best values rose up from a specific treatment combination, as a very strong interaction was expressed between genotype, induction medium and cold pretreatment duration. After this ‘first phase’ of regeneration by ovary culture 150 haploid plants were obtained. When combining these results with other attempts, a total number of 191 green plants was regenerated, 90 for Isly, 74 for Jori, 19 for Cocorit, 7 for Oued Zénati and 1 for Cham1. In a ‘second phase’, permanent regenerating callus lines were obtained through subculture on dif medium. Depending on genotype, 2 to 9 transfers (up to one year for Jori) were performed, giving 507 regenerated plants. From direct regeneration and subcultures, a total of 698 plants were produced, all green and mostly haploid. Fertile doubled haploid plants were obtained, either spontaneously or by colchicine treatment, for all these genotypes. These results are of interest for breeders, who need pure lines for genotypes evaluation and for the creation of improved homozygous varieties.     

Intra species variation in Atylosia scarabaeoides (L.) Benth., a wild relative of pigeonpea (Cajanus cajan (L.) Millsp.)

Summary Atylosia scarabaeoides (L.) Benth., a wild relative of pigeonpea, possesses several useful genes which can be utilized for pigeonpea improvement. In the present study, 33 accessions of A. scarabaeoides were evaluated at ICRISAT Center during the 1987 rainy season for variation in some useful traits to identify parents for inter-generic hybridization. A large variation was observed for leaf components, seed size, pod length, seeds/pod, days to flowering, seed protein, sulphur amino acids, resistance to cyst nematode, phytophthora blight, sterility mosaic, fusarium wilt, pod borer, pod fly, and pod wasp. Only four accessions were found to have more than 28% protein content. Methionine and cystine contents were marginally higher than in pigeonpea but the variation was not large enough to utilize them in the breeding program. In A. scarabaeoides. accessions resistant to fusarium wilt, phytophthora blight, sterility mosaic, and cyst nematode were detected. Compared to pigeonpea, the A. scarabaeoides accessions were less susceptible to lepidopteran borer and were immune to pod fly damage. Accessions ICPW 89 and ICPW 111 in short- (100–120 days), and ICPW 94 and ICPW 118 in medium-duration (140–180 days) were identified as potential parents for use in inter-generic hybridization.ICRISAT Journal Article No. 967     

The transfer of characters from Brassica campestris L. to Brassica napus L.: Production of clubroot-resistant oil-seed rape (B. napus ssp oleifera)

Summary Four oil-seed rape lines were crossed with a clubroot resistant Brassica campestris line from the European Clubroot Differential sct. The allotriploid hybrids were backcrossed to the rape lines to introgress clubroot resistance into oil-seed rape. Using a combination of screening for disease resistance and chromosome number, a high proportion of 38-chromosome, clubroot resistant selections were obtained.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. I. Production of F1 hybrids through embryo rescue

Summary Triazine resistant Brassica napus ssp. oleifera and ssp. rapifera were hybridized to cultivars of B. oleracea ssp. italica, ssp. botrytis, ssp. capitata and ssp. fimbriata. The interspecific embryos did not survive in vivo but could be rescued in vitro using a culture medium developed by Monnier (1973). The embryos did not grow directly into normal plants but were successfully regenerated using the protocol developed by Keller (1984). Hybridization efficiency ranged from 0 to 2.64 hybrids per pollination. Interspecific embryo abortion may be related to abnormal endosperm development.     

Properties of maternal haploid maize plants and potential application to maize breeding

Summary Presented are the results of a two-year study of haploid maize plants in the field. The haploids were produced with the aid of inducer line ZMS. In total, 604 and 1030 haploids were obtained and studied in the first and second years, respectively. Tassels of haploid plants were found to be almost completley sterile. Fertility of ears was studied by pollinating them with the pollen from diploid inbred lines, the cross resulting in almost all of the haploid ears carrying kernels. On average 27.4 kernels per ear of haploid plant were obtained in the first year of study and 26.3 in the second. These gave rise to normal diploid plants. This property allows genotypes selected at the level of haploid plants to be involved in breeding process. Unusual plants were found among haploids, phenotypically resembling homozygous lines. It was assumed that the plants had resulted from spontaneous chromosome doubling in haploids. The results of comparative studies of progenies of unusual plants and inbred lines derived from the same synthetic population are presented.     

Vigor and variation expressed by anther-derived doubled haploids of burley tobacco (Nicotiana tabacum L.). I. Comparison of sexual and doubled-haploid populations

Summary The genetic consequences of anther culture and chromosome-doubling techniques on burley tobacco (Nicotiana tabacum L.) were examined in this study. Three diploid populations, obtained from a burley tobacco inbred by conventional and anther-culture techniques, were compared. The first population consisted of 50 conventionally-selfed lines; the second population was made up of 35 doubled-haploid lines obtained from individual haploid plants by in vitro techniques (IVDH); and the third population consisted of 20 doubled-haploid lines whose chromosome complements had been doubled with colchicine (CDH). Comparisons of doubled-haploid lines with sexually-derived lines revealed significant differences for yield, maturity, leaf length, and alkaloid content. Yield reductions in the doubled-haploid populations averaged 8.5%. Significant differences observed between the IVDH and CDH populations indicate that the reported deleterious effects of colchicine contributed to the vigor reduction of doubled haploids. The anther derived lines in this study exhibited greater variation than did the sexual materials. This variation could provide useful variation for a breeding program. Variation exhibited by the sexual progeny of the highly inbred line, Kentucky 16, suggests that the differences among anther-derived materials are at least partially due to natural phenomena.Contribution from the department of Agronomy, University of Kentucky, Lexington, KY 40546-0091. This research was supported, in part, by a grant from R. J. Reynolds Tobacco Co. This paper (no 85-3-5) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salt-tolerance in Brassica juncea L. II. Salt-stress induced changes in polypeptide pattern of in vitro selected NaCl-tolerant plants

Summary The effect of salt-stress was studied on SDS-PAGE pattern of polypeptides in seedling, leaf and siliqua tissues of four genetically stable lines (SR2P1-2, SR3P2-1, SR3P6-1 and SR3P6-2) of in vitro selected NaCl-tolerant plants, a non-selected somaclone (CP5-2) and parent variety Prakash of Brassica juncea L. Seedlings were raised in 0, 50, 100 and 150 meq/l NaCl solutions and plants were irrigated with nutrient solution with 0, 30, 60 & 90 meq/l NaCl. Salinity induced distinct genotype specific changes in polypeptide pattern of leaf and siliqua tissues, while it had no effect on the polypeptide pattern of seedlings, radicle or hypocotyl tissues in any of the lines. In leaves, at vegetative stage, a high molecular weight protein of 53.2 kD while disappered at 60 mM and higher NaCl level in cv. Prakash and SR3P2-1, it appeared in SR2P1-2 and CP5-2 only at these higher salt levels and in SR3P6 lines it was present irrespective of stress conditions. Differences were also observed for a 93.8 kD protein which appeared anew under stress in cv. Prakash, CP5-2 and SR2P1-2, while it was absent in SR-3 lines. Intensity of the 57.3 kD protein decreased in cv. Prakash, increased in SR-2 and CP-5 lines whereas remained unchanged in SR-3 lines under salt-stress. In siliquae, salt stress induced the expression of four new polypeptides (56.1–70.8 kD) at 60 mM NaCl in cv. Prakash, and at 30 mM in SR2P1-2, SR3P2-1 and SR3P6-1 lines, while these were present in CP5-2 and SR3P6-2 even in the absence of NaCl stress.     

Possibility of genetic improvement of pigeonpea (Cajanus cajan (L.)Millsp.) utilizing wild gene sources

Summary Various wild relatives of pigeonpea,Cajanus cajan, namely some species ofAtylosia andRhynchosia, possess desirable characteristics that could be utilized for effecting genetic improvement of this crop. In total 73 cross combinations among two cultivars ofC. cajan and one accession each of eightAtylosia species and one ofRhynchosia were attempted. Twelve hybrids were obtained. Seven of these were analysed for F₁ fertility and their utility for agronomic improvement of theC. cajan. Fertility behaviour of the different F₁ hybrids varied and indicated that potential of gene transfer between the two genera,Atylosia andCajanus, was as good as within the genusAtylosia. From F₂ and F₃ families ofC. cajan × A. scarabaeoides andC. cajan × A. albicans, plants were selected with greater physiological efficiency and agronomic superiority. The prospects of transferring pod borer resistance and higher seed protein content from someAtylosia species to pigeonpea are discussed.