QTL mapping and associated marker selection for the efficacy of green plant regeneration in anther culture of rice

Anther culturability of rice is a quantitative trait controlled by nuclear‐encoded genes. The identification of quantitative trait loci (QTL) and associated marker selection for anther culturability is important for increasing the efficiency of green plant regeneration from microspores. QTL associated with the capacity for green plant regeneration in anther culture of rice were mapped on chromosomes 3 and 10 using 164 recombinant inbred (RI) lines from a cross between ‘Milyang 23’ and ‘Gihobyeo’. The quantitative trait locus located on chromosome 10 was detected repeatedly when three anther culture methods were applied and was tightly linked to the markers, RG323, RG241 and RZ400. Associations between these markers and the efficacy of green plant regeneration in 43 rice cultivars and two F₂ populations, ‘MG RI036’/‘Milyang 23’, and ‘MG RI036’;/‘IR 36’ were analysed. One of these markers, RZ400, was able to identify effectively genotypes with good (> 10.0%) and poor (< 3.0%) regenerability, based on the marker genotypes in the cultivars and two F₂ populations. This marker enables the screening of rice germplasm for anther culturability and introgression into elite lines in breeding programmes.     

Adaptation of quinoa (Chenopodium quinoa) to Northern European agriculture: studies on developmental pattern

The inheritance of resistance to clubroot, caused by Plasmodiophora brassicae in Brassica oleracea was studied in the F₁, F₂and backcross progenies of three crosses between resistant and susceptible doubled haploid lines. Fresh weights of affected (CW) and healthy (HRW) parts of the root system were measured and R = ln((HRW+0.5)/(CW+0.5)) was used as a measure of resistance. R was shown to have a normally distributed error term with a constant standard deviation over the entire scale. Analysis of means indicated the presence of two complementary resistance genes in one of the crosses. In the other two crosses analysis of means also indicated two-locus interactions, in these cases reducing the cumulative effects of loci homozygous for resistance or for susceptibility alleles. The numbers of segregating resistance genes in these two crosses could not be determined. The presence of epistatic interactions precluded analysis of variance; therefore in this case the measurements were not more informative than the symptom grades analyzed in a previous study.R showed a high rank correlation (-0.85) with symptom grades assigned to the same plants. The results of genetic analyses of R were generally in agreement with those obtained earlier using symptom grades. Symptom grades can therefore be regarded as a reliable measure of symptom development, despite their more subjective nature.     

Effects of D-genome chromosomes on crossability of hexaploid triticale (X Triticosecale Wittmack) with maize

With the aim of producing polyhaploids of hexaploid triticale, 20 genotypes from a CIMMYT breeding programme and eight D-genome chromosome substitution lines of ‘Rhino’ were crossed with maize. In crosses between 20 triticale genotypes and maize, 15 lines produced embryos. Frequencies of embryo formation ranged from 0.0 to 5.4%, with an average of 1.1%. From a total of 200 pollinated spikes, 62 plants were regenerated. Most regenerated plants were polyhaploids with 21 chromosomes, and few aneuhaploids with 22 chromosomes were found. In crosses of triticale substitution lines with maize, all the lines produced embryos, while ‘Rhino’ produced no embryos at all. Higher frequencies of embryo formation were obtained in substitution lines with chromosomes 2D and 4D. These results suggest that D-genome chromosomes in a triticale genetic background have the effect of increasing the frequency of polyhaploid production in triticale x maize crosses.     

Wide mapping of a T-DNA insertion site in oilseed rape using bulk segregant analysis and comparative mapping

Doubled haploid oilseed rape lines segregating for a transgene inducing herbicide resistance (bar gene) were investigated for the wide mapping of the T-DNA insertion site. Bulk segregant analysis using presence/absence and intensity polymorphisms between the bulks, as well as comparative mapping with a linkage group deriving from another cross, led to the identification of 11 random amplified polymorphic DNA (RAPD) markers tightly or loosely linked to the bar gene. Ten RAPD loci out of 11 were located on the same side of the bar locus, strongly suggesting that the T-DNA integrated in a telomeric or subtelomeric position. The eleventh RAPD marker exhibited a strong segregation distortion, which could be the result of a heteroduplex formation. Comparison of the linkage groups obtained from the two crosses showed different recombination rates between markers, possibly reflecting differences in parental genetic backgrounds. Consequences and potential applications in transgene dispersal safety assessment studies are discussed.     

Improved doubled haploid production protocol for Brassica napus using microspore colchicine treatment in vitro and ploidy determination by flow cytometry

A rapid and efficient microspore culture protocol was applied to produce homozygous progeny of crosses between low erucic canola and high erucic resynthesized rapeseed (Brassica napus L.). Microspores of Canadian cultivars ‘Excel’ and ‘Profit’ as well as three F₁ hybrids with the resynthetic line ‘RS239’ were treated with colchicine immediately after isolation. Flow cytometry was applied for early identification of doubled haploid (DH) regenerants. The diploidization rate was subsequently verified by scoring flower morphology. In vitro colchicine treatment had a positive effect on induced diploidization, and was associated with the frequency of preliminary spontaneous diploidization which was, however, determined by the genotype. In addition, the effects of colchicine treatment on embryoid formation and regeneration have been evaluated. The method presented is feasible for commercial large‐scale production of DHs in rapeseed as the genotype‐specific diploidization can be efficiently balanced by in vitro colchicine treatment. In addition, the use of flow cytometry immediately after in vitro culture allows efficient selection for DHs, thus saving labour and cost and in the laboratory and subsequent greenhouse phase.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of anther orientation on microspore-derived plant production in barley (Hordeum vulgare L.)

Summary The effect of anther orientation on culture response has been examined in a range of barley cultivars of agronomic importance. Anther orientation did not significantly affect the percentage of anthers responding. However, orientation of the anthers in the up position with one lobe in contact with the medium significantly increased the number of green plants produced when compared to anthers cultured in the flat position. Green plantlet production was observed directly from embryoids i.e. without an intermediate callus phase. Cytological examination of the regenerants indicated that the majority of the plantlets possessed the normal barley karyotype. The implications of these results for Barley breeding are discussed.     

The evaluation of superior Hordeum bulbosum L. genotypes for use in a doubled haploid barley breeding programme

Summary Eight Hordeum bulbosum selections were produced from a cross between Cb 2920/4 and Cb 2929/1, two genotypes widely used in doubled haploid breeding programmes. The selections were hybridized with barley to evaluate their ability to produce high proportions of well-differentiated haploid embryos compared with Cb 2929/1 as control. We report here an initial small-scale investigation followed by a larger-scale test in two different environments to assess seed setting, haploid embryo differentiation rates and VB hybrid formation. These VB embryos contain both parental sets of chromosomes and occur more frequently in the glasshouse during the winter. Two of the eight selections were identified as combining the desirable characteristics of both parents, namely high seed setting on cv. Vada which is partially incompatible with H. bulbosum, large numbers of well-differentiated haploid embryos and a low incidence of VB hybrids. The selections are available for release to interested research workers and plant breedersAbbreviations DH doubled haploid - VB a hybrid from H. vulgare x H. bulbosum which contains both parental sets of chromosomes     

小孢子培养获得松花型花椰菜DH再生植株

对5个松花型花椰菜(Brassica oleraceavar.botrytisL.)杂种一代的小孢子培养研究表明,小孢子胎胚发生主要依赖于基因型,庆农65天的每花蕾胚状体产量最高,平均达15.5个。松花菜的胚状体萌发率一般在30%左右,并有效地获得了大量的DH再生植株。冷激预处理能显著地影响花椰菜小孢子的胚胎发生,但供体材料间存在不同的结果。结球期和开花结角期再生植株的生育期与育性出现较大分离,可育且能正常结角的比例约占全部小孢子再生植株的50%以上,因而不再需要加倍处理。     

K型小麦雄性不育系诱导单倍体胚的胚胎学观察

从胚胎学方面研究了粘果山羊草细胞质的小麦雄性不育系K83(21)35A与保持系83(21)35B杂交的双受精过程,胚和胚乳的发育特点,以及单倍体胚和孪生胚发生的规律。结果表明:双受精过程、胚和胚乳的发育与普通小麦自交相似。n型单倍体胚来自助细胞的孤雌生殖;n-n型双胚来自助细胞和卵细胞的孤雌生殖;2n-n型双胚中的单倍体胚来自助细胞,二倍体胚来自受精卵。适当延迟授粉,能提高单倍体发生的频率。