Spontaneous Diploidization Research Articles

Spontaneous diploidization of the maternal chromosome set in Nile tilapia (Oreochromis niloticus L.) eggs

Spontaneous diploidization of the maternal chromosome set (SDM) in Oreochromis niloticus is described here for the first time. The SDM phenomenon was observed in progeny of only one XY neofemale out of 11 such neofemales from which eggs had been fertilized with UV-irradiated sperm: this treatment produced only the expected haploid embryos from the other females. SDM progeny were produced from three different batches of eggs from this female. No significant differences in survival at different stages of embryonic development were observed between the SDM and the diploid control group. The maternal inheritance of SDM progeny was verified using multilocus DNA fingerprinting. Only diploid karyotypes were observed in these fish and diploid control groups. In both the SDM and control groups, sex ratios were significantly skewed towards males (93.3% and 65.0% males respectively). The actual mechanism of SDM in eggs from this particular female remains unknown.

Production of gynogenic plants from hybrids of Allium cepa L. and A. roylei Stearn

Allium roylei is a non-bulbing wild relative of common onion ( Allium cepa) with several desirable traits, including resistance to Botrytis leaf blight (BLB) and downy mildew. We obtained gynogenic plants by culturing flower buds from two A. cepa lines and several generations of plants derived from crosses of A. cepa and A. roylei. The donor plants used showed substantial differences in response to induction of gynogenesis. We obtained 59 plantlets from responsive A. roylei-derived plants. Backcross generations of A. roylei-derived plants (BC 1F 1 and BC 1F 2) provided the majority of the gynogenic plantlets; plants from earlier generations (F 1 and F 2), showed either very low or no gynogenic response. About half of the gynogenic plantlets continued to grow into plants. Flow cytometry revealed that most gynogenic A. cepa plants (86%) were haploids, while 9% were spontaneous diploids. In contrast, 77% of the gynogenic plants from A. roylei-derived plants were spontaneous diploids. Two gynogenic plants obtained from a BC 1F 1 and one from a BC 1F 2 plant produced small or medium size bulbs that dried down like A. cepa material; the remaining gynogenic plants produced non-dormant bulb-like structures. The possible contributions of doubled haploid A. roylei-derived plants to onion breeding are discussed.

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.

IDENTIFICATION OF CULTIVARS AND HYBRIDS IN SYRINGA AND CUCURBITA BY DNA FINGERPRINTING

We have tested the applicability of AFLP analysis in varietal identification in Syringa and parentage identification after hybrid breeding in Cucurbita in a practical micropropagation situation. Both in Syringa as well as in Cucurbita, AFLP fingerprints were highly reproducible and produced similar band patterns irrespective of the starting material being either tissue culture-derived or greenhouse material. A single primer combination yielded enough polymorphic bands to unequivocally discriminate among 13 closely related lilac cultivars. Following hybrid production after wide crosses between Cucurbita pepo x C. maxima lines AFLP fingerprints clearly identified the parental genomes in regenerating hybrid plants and could be used to discriminate between true hybrids and gynogenic haploids or spontaneous diploids.

Associations between anther-culture response and molecular markers on chromosomes 2H, 3H and 4H of barley (Hordeum vulgare L.)

Segregation distortion is a common phenomenon among anther-culture-derived plants and it has been suggested that the distorted areas may contain genes affecting survival in anther-culture. Segregation of 111 markers was checked in an androgenetic barley (Hordeum vulgare L.) progeny and a linkage map was constructed. Thirty one progeny lines were tested for their anther-culture ability and associations between molecular markers and anther-culture traits were established. Two regions on chromosomes 2H and 4H were associated with anther response, three on 2H (two areas) and 3H with plant regeneration rate, and one on 4H with spontaneous diploidization. The chromosomal regions controlling anther-culture response and the regions where distorted segregation was found were not always the same.

Polyploidization in embryogenic microspore cultures ofBrassica napus L. cv. Topas enables the generation of doubled haploid clones by somatic embryogenesis

Embryogenic microspore and pollen culture followed by subculture of microspore-derived plantlets enabled the production of clones ofBrassica napus cv. Topas. Flow-cytometric analysis revealed that most microspore- and pollen-derived embryos (pEMs) were haploid initially. Spontaneous diploidization occurred at the globular stage of the pEMs, and was expressed as the relative increase of the 2C and 4C nuclear DNA content. Diploidization occurred throughout various organs of the pEMs and resulted in the formation of haploid and doubled haploid chimerics. In some embryos, nearly all cells were doubled haploid. From early cotyledon stage onward, pure haploid embryos were not observed anymore. At late cotyledon and germination stages, pure doubled haploid embryos and plantlets increased in number. Tetraploid pEMs were found occasionally. A culture regime was established to induce somatic embryos on the pEM-derived young plantlets. The ploidy of the somatic embryos varied highly and tended to be the same as that of the tissue at the initiation site on the pEM-plant. The results show that during the embryogenic development ofB. napus microspores, spontaneous diploidization occurs at globular stage, and increases progressively, resulting in the formation of chimerical haploid and doubled haploid plants as well as pure doubled haploid plants; ploidy neither affects pEM development at embryo developmental stages nor somatic embryogenesis, that starts on young pEM-derived plantlets; doubled haploid somatic embryos can be cloned from single pEM-derived plantlets; and doubled haploid embryos develop to fertile plants.

In vitro androgenesis and segregation distortion inBrassica napus L.: spontaneous versus colchicine-doubled lines.

A total of 750 plantlets were regenerated from 1,400 embryos produced through microspore cultures from one F1 plant of the cross 'Darmor-bzh' x 'Yudal'. Fifty-three percent of the regenerants were evaluated by flow cytometric analysis, which revealed that 31% were spontaneous diploid (SD), 63% were still haploid and the remaining 6% plants had other ploidy levels. Available segregation data (266 markers) produced on this androgenic progeny were used to study the interference between segregation distortion and the mode of chromosome doubling of androgenc lines. On the basis of the present results it is not possible to conclude that distortions are peculiar to one type of regenerated plant; only a difference in the intensity of the bias might be assumed.

Variation of gynogenesis ability in onion (Allium cepa L.)

Onion varieties from Northern-Europe, Eastern-Europe, Southern-Europe and the U.S. including populations, inbreds, synthetics and clones were tested for their gynogenesis ability for three years: 1993-1995. Embryos were induced in Petri dishes using in vitro culture of flower buds. Embryo rate, regeneration rate, survival rate, embryo quality and ploidy level of regenerants are described. Important genotype and variety effects are pointed out; embryo production and plant regeneration ranged from 0 to 17% and from 0 to 11% of inoculated flowers, respectively. Highest results were obtained with inbreds and synthetics for embryo production as well as for plant regeneration. Populations expressed various responses which were generally low. Geographic origin and year effects were covered by the effects of genetic structure of varieties and specific genotype of donor plant. Among regenerants, 80% were haploid and 13% were spontaneous diploid. No relation has been found between general quality and ploidy level of regenerants. The choice of plant material for successful gynogenesis in onion is discussed.

Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis

Induced parthenogenesis as a possible method of haploidization in sunflower (H. annuus L.) was tested in previous investigations (Todorova et al., 1994) and conditions for reproducible regeneration of gynogenic doubled haploids were established by the present work. Forty eight treatments were studied, involving four pollen donors and four recipient hybrids. Pollen was irradiated with doses of 300 Gy, 600 Gy and 900 Gy. In total, 2279 embryos were cultivated in vitro of which 1107 plants were obtained and 582 of them produced seeds after selfing. The ploidy level of the regenerants was evaluated at the two – three leaf stage and 296 of the plantlets obtained were haploids. Some of them underwent spontaneous diploidization, the others were treated with colchicine solution for chromosome doubling. The diploid plantlets were checked for their gynogenic origin by genetical and biochemical methods. The effectiveness of the method expressed as the number of agronomically useful DH lines to the number obtained that were fertile and resistant to downy mildew, branched and unbranched plants was 8,6% on average.

Spontaneous diploidization of maternal chromosome set in ornamental (Koi) common carp ( Cyprinus carpio L.)

Spontaneous diploidization of maternal chromosome set in ornamental (Koi) common carp ( Cyprinus carpio L.)

Evidence for the heritable nature of spontaneous diploidization in common carp, Cyprinus carpio L., eggs

Evidence for the heritable nature of spontaneous diploidization in common carp, Cyprinus carpio L., eggs

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1-9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3-13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.

Induced diploid gynogenesis and polyploidy in crucian carp, Carassius auratus gibelio (Bloch), x common carp, Cyprinus carpio L., hybrids.

. Results of a 16 year (1977–1992) study of hybrids obtained from crossing two cyprinid fish, crucian carp, Carassius auratus gibelio (Bloch) (bisexual form), and common carp, Cyprinus carpio L., are presented. The ability to produce diploid eggs in F1 females (resulting from chromosome endoreduplication in early oogenesis) was responsible for the high yield of spontaneous diploids in gynogenetic progenies of F1 females and for triploidy in hybrids from back-crosses of F1 females with males of the parent species. Different types of hybrids, including diploid and triploid gynogenetic, tetraploid (amphidiploid) and androgenetic progenies, were obtained. Diploid and some triploid hybrids (females only) were reproduced inter se by induced gynogenesis in several subsequent gynogenetic generations. Amphidiploid hybrids were obtained by crossing triploid F8 females with diploid common carp males or by crossing diploid gynogenetic females with diploid inverted hybrid males producing diploid sperm. Results of cytogenetic investigations and fish culture properties in hybrids are presented. The scientific and practical significance of the data obtained are discussed.

Responsiveness to anther culture in cultivars and F1 crosses of spring wheat

Anther culturability of 43 cultivars and 6 F1 crosses representing different quality classes of spring wheat was studied using a glucose-containing, modified N6 medium (HNG). Generally high pollen embryoid formation (up to 111 embryoids per 100 anthers) was associated with lower green plant regeneration (up to 9.1 green plantlets per 100 embryoids) frequencies and a high proportion (63% on average) of albino plants. Anther response was found to be strongly affected by the genotype of the donor plants. Seven of the screened cultivars yielded more than one green regenerated plant per 100 anthers. The most responsive cultivars were Veery #2 (6.2), ST 6 (4.0), and Leader (3.6). No ability to regenerate green plantlets was shown by 10 of the genotypes. Anther responsiveness of F1 progenies as compared with the parental cultivars were different in each cross. Differences found between reciprocal crosses suggest that the cytoplasm of a donor plant may affect anther response. Haploid plants constituted 45% of the anther derived regenerants, while the remaining part was divided into 29% of spontaneous diploids and 26% of plants with abnormal chromosome numbers. Key words: Triticum aestivum, anther culture, doubled haploids, wheat

Production of Spontaneous Diploid Lines from Isolated Microspores Following Cryopreservation in Spring Rapeseed (Brassica napus L.)

AbstractCryopreservation of microspores provides a potentially powerful means for increasing percentage of spontaneous diploids in microspore‐derived populations in rapeseed (Brassica napus L.). This study was to investigate the production of spontaneous diploid lines in relation to steps of cryopreserving micro‐spores of two spring rapeseed breeding lines G‐231 and M3‐124. Microspores of both genotypes were isolated and subjected to Cryopreservation. The step of cooling process (before liquid nitrogen immersion) in a Cryopreservation protocol resulted in high percentages of spontaneous diploids in lines regenerated from frozen microspores in both genotypes. Liquid nitrogen immersion and subsequent thawing process had no influence on the percentage of spontaneous diploids. A linear relationship between percentage of spontaneous diploids and cooling temperatures was detected. The lowest cooling temperature of −40 °C yielded the highest percentages of spontaneous diploids (51.1 % in G‐231 and 47.7 % in M3‐124, respectively), which were more than doubled in comparison to those in the corresponding unfrozen controls. The current study indicated that cold treatment may be utilized to greatly enhance the percentage of spontaneous diploids in rapeseed breeding programs.

Spontaneous diploidization of maternal chromosome set in ornamental (koi) carp, Cyprinus carpio L.

An unusually high frequency of spontaneous diploidization in a maternal chromosome set (SDM) was discovered in one ornamental carp (koi) female in an experiment on these fish for induced gynogenesis. Spontaneous appearance of diploid embryos in the gynogenetic offspring (intact eggs × irradiated sperm) and appearance of triploids among control fish (intact eggs × intact sperm) obtained from koi female and males of edible carp indicated spontaneous diploidization of maternal chromosomes (SDM). Possible cytological processes causing this phenomenon are discussed.

A comparison of anther and microspore culture as a breeding tool in Brassica napus

A direct comparison of microspore culture and anther culture was made in Brassica napus using F1 crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 2∶1 for the population obtained from the Regent x Golden crosses but 1∶1 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

AbstractThe use of anther culture in breeding winter wheat.II. Production of new doubled haploid lines of winter wheat with 1AL—1RS‐translocationA total of 4472 anhers was cultured from 8 F1 populations of winter wheat with the 1AL—1RS wheat‐rye translocation cultivar ‘Amigo’ as one of the crossing arents. When averaged over all populations a frequency of embruoid formation of 10% and of regeneration efficiency of green plants of 1% were observed. In addition to the 45 green regenerated plants, 93 albinos were obtained. 44% of the green plantlets had 21 chromosome in root tips and 29% were spontaneous diploids. Multiple peroxidases were used a biochemical markers in the subsequent characterization of the homogeneous breeding material. The electrophoretic patterns showed that 16 doubled haploids without rye chromosome segments were produced. In addition the features of dh0‐ plants showed, that several of the new 1AL—1RS‐translocation lines were awnless.

Inheritance patterns of erucic acid content in populations of Brassica napus microspore-derived spontaneous diploids

The levels of erucic acid and other fatty acids in seeds of microspore-derived spontaneous diploid plants from crosses between low and high erucic acid parents were examined. The analysis confirmed that erucic acid is simply inherited and is determined by two genes that act in an additive manner. The effects of the genes for erucic acid on the levels of the other fatty acids was also determined and many significant correlations were found. In particular, erucic acid levels were negatively correlated with oleic acid and linoleic acid levels. The study also illustrates several advantages of using haploidy to analyze the inheritance of agronomically important traits. In particular, the number of phenotypic classes is smaller in androgenic populations and differences between classes are greater than in an F2 population.

Occurrence of aneuploidy in radiation gynogenesis of carp, Cyprinus carpio L.

DNA content and chromosome numbers were studied in gynogenetic and normal carp embryos. Gynogenetic embryos showed the haploid chromosome number; the occurrence of spontaneous diploidization was rare. Early cold shock treatment of the gynogenetic zygotes resulted in the retention of the second polar body producing a much higher proportion of normally developing viable larvae. The appearance of large numbers of aneuploid embryos in the cold‐treated gynogenetic group is explained by the loss or breakdown of chromosomes that are caused in their turn by the absorbtion of the second polar body through the cold treatment.