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.     

Haploidy breeding and mutagenesis for drought tolerance in wheat

Several intraspecific crosses between known drought tolerant wheat varieties and stable high yielding recombinants were made with the objective to develop improved cultivars for the moisture stressed rainfed areas of Pakistan. Five of these crosses were selected for further creation of useful mutations through the application of low doses of gamma rays and development of doubled haploids through anther culture. Anther culture response of the selected irradiated F₁ generations was studied on liquid and solid induction media. The highest number of calli among almost all crosses was produced on Potato-2induction medium. All the crosses varied greatly in response to callus induction and maximum calli (75%) were obtained from Lyl-73/vee’s’ cross. Similarly, genotypic differences were found for green vs. albino regenerants. The highest number of green plantlets (12.1%) was recorded for Lu-26/3062. From the developed doubled haploid population 25 DH-mutants were initially selected and nine lines were finally included in multi-locational field tests. Two DH-mutants (i.e. DHML-50 and DHML-9) have potential for better grain yield, earliness, disease resistance and moisture stress tolerance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparative evaluation of exotic and adapted germplasm of spring wheat for floral characteristics in the Indo-Gangetic Plains of northern India

Four hundred spring wheat genotypes, comprising exotic and indigenous germplasm and adapted cultivars for commercial cultivation, were investigated during four crop seasons for six floral characteristics related to outcrossing behaviour. Genetic variability and heritability were investigated along with phenotypic correlation coefficients among these characteristics. A significant quantitative variation existed among the characteristics in all three groups. High heritability for stigma length, openness of florets, anther extrusion and duration of floral opening indicated that selection for these characteristics may be effective. The adapted cultivars were found more promising than the exotic and indigenous germplasm. Most of the characteristics showed significantly positive correlation with each other and high correlation was observed between anther and stigma length, and anther extrusion and duration of floral opening. Superiority of varietal mixtures and segregating generations (F₃ and F₄) of two crosses over parental lines suggested that heterozygosity and heterogeneity can provide yield advantages. Germplasm lines possessing superior expression of floral characteristics may be used to improve yielding ability through enhanced heterozygosity or the development of hybrids.     

Repeatability of regression stability indexes for grain yield of oats (Avena sativa L.)

Summary Two lines of descent were established from an F₃ bulk lot of oats (Avena sativa L.) initiated by mixing seeds from approximately 250 crosses. For one line of descent, seeds were radiated with thermal neutrons or X-rays from F₃ through F₆, followed by five generations of bulk propagation. The second was propagated for 10 generations. No artificial selection was practiced in either line of descent. Grain yield data from 20 random strains from each of four generations from the radiated (F₇, F₈, F₉, and F₁₁) and five from the nonradiated (F₃, F₆, F₇, F₈, and F₁₂) line of descent and 20 check cultivars tested in 14 environments were used for estimating regression stability indexes of oat strains.The 14 environments were assigned randomly to two sets of seven, and regression stability indexes were computed for the 180 experimental oat strains for both sets. Intrageneration correlations between regression stability indexes from the two sets of environments ranged from –0.35 to 0.64 (18 d.f.), and only one of nine was significant, indicating poor repeatability for estimates of this statistic computed from different sets of environments.Correlations between regression stability indexes from two sets of environments, one in which the environments varied by soil nitrogen levels and a second in which they varied by soil phosphorus levels, ranged from –0.01 to 0.28, none of which was significant.The relative magnitudes and ranking of the regression stability index values for the oat genotypes were nearly identical when environmental productivity indexes were assessed with any number of check cultivars from 2 to 20.Journal paper No. J-8080 of the Iowa Agriculture and Home Economics Experiment Station. Ames. Iowa. USA 50010. Project 1752.     

Enhanced anther culture efficiency of indica rice (Oryza sativa L.) through modification of the culture media

The production of microspore-derived green plants from anther culture of indica rice is generally very low compared with japonica cultivars. A modified anther culture medium, consisting of a higher KNO₃ content (31 mm ) and casein hydrolysate (CH, 500 mg/1) but without ammonium salts, was tested in comparison with a medium consisting of the widely-used N6 medium nitrogen background, using four indica × indica F₁ hybrids as test materials. Green plant regeneration frequency was at least three-fold higher in the microspore-calli derived from the former medium than in those derived from the modified N6 medium. More than 700 microspore-derived plants were raised in the field. Another study was carried out using indica × japonica and indica × javanica F₁ hybrids. The results indicated that a medium with higher (3.5 mm ) ammonium sulphate may induce a higher frequency of anthers with microspore-calli but not necessarily lead to a larger number of green-plant regenerating calli. Subsequently, using the indica cv. ‘IR-43’ as the test material, use of a lower level (1.75 mm ) of (NH₄)₂SO₄, in addition to KNO₃ (31 mm ), was found to be better than CH (500 mg/l) for anther-response as well as green plant regenerability of the derived microspore-calli. Nitrate-nitrogen or ammonium-nitrogen alone elicited poor response. Twenty-five media involving combinations of KNO₃ (20–34 mm ) and (NH₄)₂SO₄ (1–3 mm ) were tested for their effects on anther response. Combinations involving KNO₃ (31–34 mM) and (NH₄)₂SO₄ (2.0–2.5 mm ) were found superior not only for achieving greater anther response but also, for subsequent green-plant regeneration. This contrasts with the 28 mm of KNO₃ and 3.5 mm of (NH₄)₂SO₄ in the widely-used N6 medium developed for japonica rice. Other than potassium nitrate and ammonium sulphate, and potassium phosphate to some extent, the levels of other inorganic salts tested did not make any significant difference to the process of anther response. Based on these results, modified media with three levels of ammonium sulphate were tested for anther culture efficiency of indica × japonica and indica × javanica derivatives (F₃s). Microspore-calli derived from a medium of a lower (1.75 mm ) level of (NH₄)₂SO₄ showed a higher regeneration potential overall than those derived from a higher (3.5 mm ) level. A revised medium has been suggested, on the basis of these results, for the realization of improved anther culture efficiency and, consequently, improved feasibility of using doubled haploids in genetic and breeding research with indica rice.     

Mapping of QTLs underlying flowering time in sorghum [Sorghum bicolor (L.) Moench]

Due to its critical importance in crop yield, the photoperiodic regulation of flowering time is considered an important trait in sorghum breeding programs. In this study, quantitative trait loci for flowering time were detected using an F₂ population derived from a cross between Kikuchi Zairai, a late-flowering cultivar originating from Japan and SC112, an early-flowering cultivar originating from Ethiopia. F₂ plants were grown with their parents under a natural day length and a 12 h day length. Two linkage maps were constructed using 213 simple sequence repeats markers. Nine quantitative trait loci controlling flowering time were identified in F₂ plants grown under a natural day length, whereas 7 QTLs were identified under a 12 h day length. Five QTLs controlling flowering time were shared under both of the day length conditions.     

A genetic study of growth characters and yield characters of oilseed rape

Summary Analysis of single plants in parental, F₁ and F₂ populations of three crosses of oilseed rape indicated cytoplasmic effects in F₁ or F₂ generations for most of the observed characters. There was evidence of dominance for early flowering and early maturity. Heterosis was exhibited for yield and yield components. The heritabilities for the characters under study were generally very low.     

The inheritance of nitrate content in lettuce (Lactuca sativa L.)

Summary The inheritance of nitrate content in lettuce was analysed using 16 F₂ populations and three F₃ populations. Frequency distributions of nitrate content in F₂ and F₃ populations were unimodal and symmetrical, indicating a quantitative inheritance. Both significant positive and negative deviations of the F₂ mean from the mid-parent value were found, indicating dominance or epistasis. Deviations towards low nitrate content were more frequent than deviations in the other direction. Estimates of heritabilities for nitrate content in the F₂ populations ranged from 18% to 69% and were in most cases above 50%. Crosses between low nitrate cultivars did not have lower estimates of heritability in the F₂ than crosses between cultivars with larger differences in nitrate content. In one case a genotype x experiment interaction for nitrate content of parental cultivars was found.Three F₃ populations of crosses between cultivars with low nitrate content were analysed. Estimates of heritabilities for F₃ line means ranged from 78% to 91% and estimates of the genetic standard deviation of nitrate content in unselected advanced generations ranged from 0.24–0.33 g kg^–1. The estimates of heritabilities and of genetic variation in advanced generations offer good prospects of selection of low nitrate genotypes in lettuce. A comparison of efficiency of selection in the F₂ generation and F₃ line selection is made.     

Inheritance of flowering time in six pea cultivars (Pisum sativum L.)

Summary The dates of flowering of six pea cultivars and their F₁ and F₂ generations grown under a moderate level of top-yellows virus infection were analysed by both Griffing's and Jinks' methods to assess the stability of the polygenic control of flowering period. Flowering period was shown to be under the control of an additive system with dominance for the late flowering characteristic. Some gene interaction was indicated in both analyses, mainly between the later-flowering cultivars, although tests for apparent over-dominance were insignificant. It was considered that the degree of virus infection within the experiment had little, if any, effect upon flowering times and that selections for flowering date in these circumstances should be almost as reliable as those made in a disease-free environment.Slightly increased earliness over the early parent was evident in five F₂ progenies and it appears possible to make further slight gains in earliness by selection in the present material.     

The inheritance of quantitative traits in Brassica napus ssp. rapifera (swedes): Augmented triple test cross analyses of production characters

Two F₂ triple test crosses, augmented with F₃s, produced from crosses between different inbred lines of swedes (Brassica napus ssp.rapifera L.) were assessed in field trials at Dundee in 1988 and 1989,respectively. This paper reports the analyses of resistance to powdery mildew, neck length, growth cracks, sugar content and hardness; analyses of yield have been published previously. Additive genetical variation was found for all traits while non-additive variation was less important, the highest degree of dominance being 0.44 for hardness. There was evidence of additive × dominance and dominance × dominance epistasis for mildew and additive × additive epistasis for neck length and hardness. Significant,consistent reciprocal differences were found and these were particularly large for neck length and growth cracks. Sugar determination was carried out on the basic generations of the second cross, the parental lines of which showed large differences in concentration of glucose, fructose and sucrose. Directional dominance was found for high glucose but not for fructose or sucrose. The implications of these results for swede breeding are discussed and it is proposed that inbred cultivars would be a more practical option than F₁ hybrids. This revised version was published online in August 2006 with corrections to the Cover Date.     

A Survey of the Anther Culture Response of Brassica oleracea L. Cultivars Grown Under Field Conditions

Anther culture studies were conducted on a wide variety of different vegetable Brassica oleracea L. cultivars grown under field conditions. Representative selections were included from different varieties including: broccoli, var. italica; heading broccoli, var. botrytis; Brussels sprouts, var. gemnifera; Chinese Kale, var. alboglabra; Kales, vars. acephala, medullosa, ramosa, selensia; cauliflower, var. botrytis; kohlrabi, var. gongylodes; and cabbage, vars. capitata and sabanda. At least some microspore derived embryos were recovered from the majority of cultivars tested; however, rates of embryo formation were sometimes lower than those obtained under controlled environmental conditions. In general the anther culture response was greatest for cabbage and other vegetables that required vernalization for flowering. Results were lower for materials exposed to high temperature stresses before culture. Nevertheless, it is clear that the use of anther culture procedures in breeding programs is possible without access to extensive controlled environmental facilities.     

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F₂ plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F₂ segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.     

Response to selection, combining ability and heritability of coleoptile length in winter wheat

Selection of winter wheat cultivars with long coleoptiles is an important component of improving emergence, weed suppression and grain yield in low rainfall regions of the world. Seven winter wheat cultivars were hybridized in a Griffing’s half-diallel mating design, method 2 (reciprocals excluded, parents included), model 1 (fixed), and the progeny analyzed for coleoptile length in the F₁ through F₄ for response to selection, general combining ability (GCA), specific combining ability (SCA) and heritability. Highly significant differences existed for GCA among progeny in each generation. SCA was highly significant in the F₁, F₂ and F₄, although SCA estimates were inconsistent among generations in each progeny population. The SCA/GCA ratio was 0.15, 0.11, 0.06 and 0.04 in the F₁ through the F₄, respectively, indicating that additive effects increased with homozygosity. Broad-sense heritability for coleoptile length was estimated at 0.86, 0.76, 0.60 and 0.78 for the F₁ through the F₄, respectively. Realized heritabilities ranged from −0.16 to 0.85 with a mean of 0.32 in the F₃ and ranged from −0.68 to 0.68 with a mean of 0.13 in the F₄. Spearman’s rank correlation coefficients (R_S) were significant for all generation comparisons except the F₁:F₂, indicating that changes in rank for coleoptile length were insignificant. Cultivars with long coleoptiles and high GCA were the best parents for improving coleoptile length. Sustained selection over generations for coleoptile length was more effective than one generation of selection for genotypes both with and without reduced height genes.     

Genic analysis of hybrid sterility caused by anther indehiscence between distantly related rice varieties

Hybrid sterility of the cross between Silewah, an Indonesian native variety and Hayakogane, a Hokkaido rice variety in Japan was confirmed to be caused by anther indehiscence, based on a lot of spikelets with fewer numbers than 3 of dehiscent anthers and fewer numbers of pollens poured onto stigma per spikelet in Silewah/Hayakogane F1. In Silewah/Hayakogane//Hayakogane BC₁F₁, spikelet fertility was correlated with mean number of dehiscent anthers per spikelet. So, genic analyses for hybrid sterility by anther indehiscence were conducted by scoring spikelet sterility in F₂, BC₁F₁s and triple cross. As a result, it was concluded that the hybrid sterility was controlled by complementary action of three dominant genes. Silewah has one of the three genes and Hayakogane has the other two. It was presumed that the two genes which Hayakogane possesses were derived from those of Aikoku, an old native variety in Japan. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rye doubled haploids as a research and breeding tool – a practical point of view

Doubled haploid (DH) plants were produced using anther culture from out‐crossing rye, including breeders’ lines, cultivars and F₁ plants with DH parents, to examine the feasibility of using the DH technique for breeding and specifically for developing mapping populations. Only 10–36% of green regenerants produced via anther culture were suitable for research or breeding purposes because of low survival rate or low fertility. Spontaneously arising DH regenerants were more often fertile compared with the colchicine‐treated ones. The fertility of spontaneous DHs varied from sterile to half that found in a normal rye population, which has implications for the design of a crossing scheme and subsequent anther culture. In the reciprocal crosses within one DH population, fertility was the lowest observed, probably because of self‐incompatibility factors, whereas in the DH crosses with normal heterozygous cultivars fertility was the highest. Two mapping populations using DHs were established, the first for out‐crossing rye it would seem. These populations will be used for mapping two important traits, the semi‐dwarf growth habit and preharvest sprouting resistance in rye.     

Inheritance of resistance to angular leaf spot in Nicotiana tabacum L. cultivars burley 21 and kentucky 14

Summary The genetics of resistance to angular leaf spot caused by Pseudomonas syringae pv. tabaci in Nicotiana tabacum cultivars Burley 21 and Kentucky 14 was investigated by studying disease reactions to three isolates of parental, F₁, F₂ and backcross generations derived from crosses between the resistant cultivars and the susceptible cultivar Judy's Pride. Studies were conducted in the greenhouse and in field plant beds. Chi-square values were computed to determine whether the observed ratios for disease reactions deviated from expected Mendelian ratios for a single, dominant gene controlling resistance to angular leaf spot in tobacco. Based on the resistance of the F₁ and the backcross generation to the resistant parent (BC-R), a 3 resistant: 1 susceptible segregation ratio in the F₂, and a 1 resistant: 1 susceptible segregation ratio in the backcross generation to the susceptible parent (BC-S), it was concluded that resistance to three isolates of Pseudomonas syringae pv. tabaci is governed by a single, dominant gene.     

Production of salt tolerant rice breeding line via doubled haploid

Summary A haploid breeding program was initiated to develop doubled haploid salt tolerant rice breeding line via anther culture. Two sensitive breeding lines BR4608-R₁-R₂ and BR4909-R₁-R₂ were crossed with a salt tolerant line IR13146-13-3-3 to transfer its salt tolerant character to the doubled haploids.Anther from confirmed F₁s of the two crosses were cultured in defined medium for callus induction and eventual plant regeneration. Fifteen doubled haploid (DH) lines were obtained from two crosses. Test for salt tolerance were done in vitro. Five out of 15 lines were found tolerant at the level of 8–10 decisiemens/m (ds/m) while the rests were sensitive to that level of salinity.Field experiment was conducted to evaluate the doubled haploids under saline and non saline soil. Five salt tolerant lines produced comparable yield with the resistant control (BR 23) under saline condition, whereas these lines yielded even higher in non saline soil under irrigated condition when evaluated with other 10 sensitive DH linesAbbreviations LSD Least Significant Difference - NAA Napthalene Acetic Acid     

Potential use of doubled haploid lines for the screening of resistance to yellow rust (Puccinia striiformis) in hexaploid wheat

Doubled haploid lines derived from anther culture of two Iranian spring wheat genotypes‘Ghods’susceptible and‘9106’resistant to yellow rust in Iranian field conditions, and their F₁ hybrids were used in this study. Seedlings of 36 doubled haploid lines, selected out of 96 according to their agronomic traits and the two parental genotypes were inoculated with eight races of yellow rust. The parental genotypes (‘Ghods’and‘9106’) were segregating for some of the races but their doubled haploid lines were either resistant or susceptible to them.‘Ghods’was susceptible to three of the races studied but three doubled haploid lines derived from it were resistant to them. Five selected doubled haploids from the‘9106’genotype and six from F₁ hybrid plants were resistant to all eight races tested. After further investigations in Iranian field conditions it was found that some of these lines can be used as donor genotypes for resistance to yellow rust in wheat breeding programmes. Use of these genotypes should be possible if the French yellow rust races used for selection also represent the dominant races in Iran. It can be concluded that anther culture provides an efficient method for fixing genes of resistance to yellow rust and desirable doubled haploids from F₁ plants can be derived.     

Large-scale production of wheat and triticale double haploids through the use of a single-anther culture method

A total of 257 parental wheat and 38 triticale lines were used for anther culture. On average, 2.1 green wheat haploids were obtained per spike. This response occurred irrespective of the origin of the material (Germany, France, Sweden or UK) and 5 years of testing. Triticale responded with 5.3 green haploids per spike. Using the criterion that one parental line should give at least one green haploid per spike in the screening experiment, green haploids were produced from 88 out of 91 F₁ wheat breeding combinations and from each of 21 F₁ and F₂ triticale breeding combinations. An average of 4.7 green plants were obtained per spike from the wheat production programme, while the triticale programme gave an average of 6.2 green plants per spike. A single medium supplemented with different hormones for anthers and embryos was used for culture of both crops.