Segregation distortion in homozygous lines obtained via anther culture and maize doubled haploid methods in comparison to single seed descent in wheat (Triticum aestivum L.)

Background: The quality of wheat grain depends on several characteristics, among which the composition of high molecular weight glutenin subunits, encoded by Glu-1 loci, are the most important. Application of biotechnological tools to accelerate the attainment of homozygous lines may influence the proportion of segregated genotypes. The objective was to determine, whether the selection pressure generated by the methods based on in vitro cultures, may cause a loss of genotypes with desirable Glu-1 alleles. Results: Homozygous lineswere derived from six winter wheat crosses by pollination with maize (DH-MP), anther culture (DH-AC) and single seed descent (SSD) technique. Androgenetically-derived plants that originated from the same callus were examined before chromosome doubling using allele-specific and microsatellite markers. It was found that segregation distortion in SSD and DH-MP populations occurred only in one case, whereas in anther-derived lines they were observed in five out of six analyzed combinations. Conclusions: Segregation distortion in DH-AC populations was caused by the development of more than one plant of the same genotype from one callus. This distortion was minimized if only one plant per callus was included in the population. Selection of haploid wheat plants before chromosome doubling based on allele-specific markers allows us to choose genotypes that possess desirable Glu-1 alleles and to reduce the number of plants in the next steps of DH production. The SSD technique appeared to be themost advantageous in terms of Mendelian segregation, thus the occurrence of residual heterozygosity can be minimized by continuous selfing beyond the F6 generation

Doubled haploid production of winter and spring triticale hybrids using colchicine in anther cultures

Celem pracy było podwyższenie efektywności otrzymywania podwojonych haploidów w kultu¬rach pylnikowych 6 form mieszańcowych pszenżyta ozimego i 6 form jarych poprzez zastosowanie kolchicyny w pożywce indukującej androgenezę. Modyfikowano stężenie kolchicyny i czas traktowania (0,1 mg/l lub 1,0 mg/l kolchicyny odpowiednio przez 24, 48 i 72 godziny) w płynnej pożywce C17. Stwierdzono, że optymalnymi parametrami zastosowania kolchicyny były stężenia 1,0 mg/l przez 24 godziny oraz 0,1 mg/l przez 48 godzin. Na podstawie analiz cytometrycznych, w tych warunkach stwierdzono zwiększenie liczby roślin o podwojonej liczbie chromosomów. Wynik ten obserwowano u 6 form ozimych (średnio 66,2%) oraz u 2 form jarych (średnio 52,7%), w porów¬naniu z warunkami kontrolnymi na pożywce bez kolchicyny, w których uzyskano około 30% podwojonych haploidów.The aim of this work was to increase the efficiency of doubled haploids production in anther cultures of 6 winter and 6 spring triticale hybrids. The colchicine concentration and the time of its use (0.1 mg/l or 1.0 mg/l colchicine, for 24, 48 and 72 h, respectively) in androgenesis inducing C17 liquid medium were modified. It was found that the optimal parameters of colchicine treatment were 1.0 mg/l for 24 hours, and 0.1 mg/l for 48 hours. Based on flow cytometry analysis, in those conditions an increase in a number of doubled haploids was observed. The increase was observed in 6 forms of winter (average 66.2%) and 2 forms of spring triticale (average of 52.7%), as compared to control conditions (the C17 medium without colchicine), producing of about 30.0% of doubled haploid

Production of spontaneous and induced doubled-haploid lines of winter triticale obtained through anther culture

W kulturach pylnikowych 15 form mieszańcowych pszenżyta ozimego badano częstotliwość uzyskiwania androgenicznych roślin. Ze wszystkich genotypów otrzymano zielone rośliny, przy czym częstotliwość wahała się od 0,4 do 15,2/100 pylników w poszczególnych genotypach. Dla regeneracji roślin zastosowano pożywkę 190-2 z dwoma stężeniami kinetyny (0,5 lub 1,5 mg/l). Na pożywce zawierającej 0,5 mg/l kinetyny otrzymano więcej zielonych roślin, tj. od 2,5 do 46,7/100 androgenicznych struktur. Poziom ploidalności oznaczono cytometrycznie u 350 roślin i w zależności od genotypu stwierdzono od 17,6 do 80,0% spontanicznie podwojonych haploidów. Ogółem uzyskano 80,3% linii DH pszenżyta ozimego, wśród których było 38,0% spontanicznie podwojonych haploidów, natomiast w wyniku kolchicynowania 209 roślin haploidalnych otrzymano 70,8% linii DH.We investigated the rate of obtaining androgenic plants using anther culture of 15 winter triticale hybrids. Green plants were obtained from all genotypes, with the frequency ranging from 0.4 to 15.2/100 anthers. The medium 190-2, with two different concentrations of kinetin (0.5 and 1.5 mg/l), was used for plant regeneration. Higher number of green plants was observed in the case of the medium with 0.5 mg/l of kinetin (2.5–46.7/ 100 androgenic structures). Ploidy level was determined by flow cytometry in 350 plants and showed from 17.6 to 80.0% spontaneously doubled haploids, depending on the genotype. A total of 80.3% of DH lines was obtained, among which 38.0% were spontaneously doubled haploids, whereas 70.8% of DH lines was obtained from colchicine treatment of 209 haploid plants

Regeneration of oat androgenic plants in relation to induction media and culture conditions of embryo-like structures

The effect of C17 and W14 induction media on the formation of embryo-like structures (ELS) from F3 generation of nine hexaploid oat hybrids was investigated in the study. In all genotypes, the highest number of ELS (0.6 - 12.1/100 anthers) was obtained on C17 medium. The efficiency of plant regeneration on medium 190-2 was tested, in relation to different ELS culture conditions. The highest rate of green plants per 100 ELS (3.3 - 42.4) was produced by incubation at 22oC in the dark for the first two weeks. Among 36 green regenerants, 28 (77.8%) were haploid and 8 (22.2%) were spontaneous doubled haploids, fully fertile. After colchicine treatment of haploid plants, 19 were partially fertile and set from 1 to 15 seed per panicle

Pollen dimorphism and androgenesis in Hordeum vulgare

Dimorphism of binucleate pollen grains of Hordeum vulgare has been confirmed. It is considered, however, in contrast to the accepted opinions, that some of the large pollen grains with dense cytoplasm lying close to the tapetum are the outset forms for embryoids, and not the small pollen grains with scarce cytoplasm lying in the pollen sac centre

Segregation distortion in homozygous lines obtained via anther culture and maize doubled haploid methods in comparison to single seed descent in wheat (Triticum aestivum L.)

Background: The quality of wheat grain depends on several characteristics, among which the composition of high molecular weight glutenin subunits, encoded by Glu-1 loci, are the most important. Application of biotechnological tools to accelerate the attainment of homozygous lines may influence the proportion of segregated genotypes. The objective was to determine, whether the selection pressure generated by the methods based on in vitro cultures, may cause a loss of genotypes with desirable Glu-1 alleles. Results: Homozygous lines were derived from six winter wheat crosses by pollination with maize (DH-MP), anther culture (DH-AC) and single seed descent (SSD) technique. Androgenetically-derived plants that originated from the same callus were examined before chromosome doubling using allele-specific and microsatellite markers. It was found that segregation distortion in SSD and DH-MP populations occurred only in one case, whereas in anther-derived lines they were observed in five out of six analyzed combinations. Conclusions: Segregation distortion in DH-AC populations was caused by the development of more than one plant of the same genotype from one callus. This distortion was minimized if only one plant per callus was included in the population. Selection of haploid wheat plants before chromosome doubling based on allele-specific markers allows us to choose genotypes that possess desirable Glu-1 alleles and to reduce the number of plants in the next steps of DH production. The SSD technique appeared to be the most advantageous in terms of Mendelian segregation, thus the occurrence of residual heterozygosity can be minimized by continuous selfing beyond the F6 generation