Low relative humidity increases haploid production in durum wheat X maize crosses

Haploidization is a useful tool for genetic analysis and plant breeding, but a consistent and satisfactory protocol for haploid production has been difficult to achieve in durum wheat. The objective of this study was to analyze the influence of the relative humidity of the environment, when culturing detached tillers during the production of haploids plants in durum wheat by the maize method. Thirty‐eight F3 lines from eight crosses of durum wheat were pollinated with bulked pollen from three commercial maize hybrids. A mixture of 2‐4D and dicamba was used as a hormone treatment. The numbers of caryopses, embryos and haploids plants were scored. When 65‐85% (light‐dark) humidity was substituted for 55‐65% the number of haploids per spike increased notably. This increased frequency was largely attributed to a rise in the production of generated caryopses. On average, 15.2 vs. 9.3 caryopses, 5.0 vs. 2.8 embryos, and 3.1 vs. 0.6 haploid plants, per spike, were produced under low and high humidity regimes, respectively.     

Factors affecting oat haploid production following oat × maize hybridization

Doubled haploids (DHs) are becoming increasingly important in crop breeding programmes but methods for producing oat DHs remain inefficient. In this study haploid and DH oat plants were produced using the oat × maize hybridization method. Factors influencing the rate of caryopsis and haploid embryo production including genotype, post‐pollination plant growth regulator application and temperature were investigated. The four growth regulators tested showed significant differences in their capacity to induce caryopsis formation with dicamba producing the highest numbers of caryopses, followed by picloram, 2,4‐dichlorophenoxyacetic acid (2,4‐D) and gibberellic acid (GA₃). No significant differences were observed between these growth regulators for their effect on embryo production. The concentration of dicamba was also important and was found to influence caryopsis but not embryo production, with 50 and 100 mg/l dicamba producing significantly more caryopses than 25 or 5 mg/l. Temperature had a significant impact on both caryopsis and embryo production with the magnitude and direction of response depending on genotype. Rates of haploid embryo production observed were between 0.8% and 6.7% of the pollinated florets. The proportion of haploids, which survived and were successfully doubled with colchicine following transfer to soil was between 72% and 81%.     

Improved Techniques for Haploid Production in Wheat using Chromosome Elimination

Wheat × maize and wheat × pearl millet crosses have proved efficient for haploid production using various genotypes of wheat; 22 and 27 % of florets produced embryos. In favourable conditions 6—9 haploid plants per spike were produced. The following simplifications or improvements in technique are recommended: 1. Only a single treatment with an aqueous solution of dicamba or 2,4-D (50–100 ppm) for embryo stimulation in vivo; 2. Application by spraying or dipping the spikes; 3. Application time two to four days after pollination; 4. Embryo rescue 15 to 18 days after pollination; 5. Crosses without emasculation are possible if pollination occurs 1–2 days before anthesis. More than 450 haploids and some doubled haploid (DH) lines (after colchicine treatment in vitro) were produced using these methods. No hybrid plants, chromosome additions or substitutions were found.     

Effect of pollinator on haploid production in durum wheat crossed with maize and pearl millet

The aim of this work was to analyse the influence of the male parent on the production of embryos and haploid plants in durum wheat crossed with maize and pearl millet, to find a proper trait to identify the most efficient pollinators and to evaluate the mixtures of pollen. Two genotypes of durum wheat, low and high responding, were crossed with eight pollen samples: (i) three maize hybrids, (ii) three pearl millet inbred lines, (iii) a mixture of maize pollen and (iv) another mixture of pearl millet pollen. No significant differences on embryos and haploid plant production were observed among the four samples of maize pollen, but there were clear genotypic differences for the production of haploids between genotypes of pearl millet. The best pearl millet genotype produced significantly more haploid plants than the other two and the mixture of pollen. There was no correlation between the production of embryos and haploid plants. Therefore, the production of haploid plants must be the criterion to identify superior pollinators. In addition, a mixture of pollen is inappropriate except when using genotypes previously identified as good pollinators.     

Effect of mannitol pretreatment to improve green plant regeneration on isolated microspore culture in Triticum turgidum ssp. durum cv. 'Jennah Khetifa'

The use of doubled haploids improves the efficiency of cultivar development in many crops and can be helpful in genetic and molecular studies. The major problem with this approach is the low efficiency of green plant regeneration. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat cv. ‘Jennah Khetifa’. Tillers from donor plants were pretreated in 0.3 m mannitol and were stored at 4°C at various times: 3, 5, 6, 7, 8, 10 and 12 days. Our results showed clearly that the novel pretreatment combined mannitol 0.3 m and cold for 7 days had a strong effect on the number of embryos produced and regenerated green plants. Under this condition 13 475 mature embryos were produced from 2 693 500 microspores. Moreover, 85 green plants were obtained. High green plants regeneration frequency was recorded. As an average 11.55 green plants were produced per 100 000 microspores (about the equivalent of six plants per spike). Therefore, this study showed clearly that our results are the best ones published until now in durum wheat.     

Review of doubled haploid production in durum and common wheat through wheat × maize hybridization

Production of doubled haploids (DHs) is an important methodology to speed the process of breeding and development of mapping populations in crops. The procedure for DH production includes two major steps: haploid induction and chromosome doubling. In recent years, wide hybridization between wheat and maize has become a main approach for haploid production in wheat. In this method, the maize chromosomes are completely eliminated during the early development of the hybrid seeds after wheat spikes were pollinated with maize pollen. Numerous wheat cultivars and mapping populations have been developed using wheat–maize hybridization. In this study, we review the procedures of DH production of durum and common wheat via wide hybridization with maize, the factors which affect the efficiency of DH production, and the mechanism of selective elimination of the maize genome during the early development of the hybrid embryos. We also report a highly efficient protocol for DH production in durum and common wheat, which was established based on the optimal conditions for each of the factors that affect the efficiency of DH production.     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

Production of doubled haploids in triticale (×Triticosecale Wittm.) by means of crosses with maize (Zea mays L.) using picloram and dicamba

The aim of the present study of triticale × maize crosses was to find an appropriate growth regulator treatment to improve the yield of triticale haploids and the subsequent production of doubled haploids. The growth effect in unpollinated ovaries of triticale was examined after treatment with 1000 mg/1 indole-3-acetic acid (IAA) or 100 mg/1 solutions of the following auxin analogues: 2,4-dichlorophenoxyacetic acid (2,4-D), 3,6-dichloro-o-anisic acid (dicamba), 4-chloro-o-tolyloxyacetic acid (MCPA), phenylacetic acid (PAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4,5-trichlorophenoxyacetic acid (2,4,5- T), respectively. Dicamba stimulated growth of the ovaries significantly more than picloram and both stimulated more growth than the other growth regulators tested. Neither dicamba nor picloram induced embryo development in unpollinated pistils. Dicamba and picloram solutions, at concentrations of 25, 50, 75 and 100 mg/1, were subsequently applied to pistils of triticale pollinated with maize. On average, between 17.1 and 21.5 embryos/100 fiorets were excised after treatment with 75 or 100 mg/1 solutions of picloram or dicamba but the concentrations of 20 and 50 mg were less effective. The frequencies of excised embryos did not differ between genotypes. Seventy-six green haploids were obtained from 100 embryos rescued in vitro on the 190–2 and modified B5 media, the first medium being superior. The plants were subjected to colchicine treatment at the 3–4 tiller stage. Out of 68 plants brought to maturity, 25 exhibited fertile sectors. In comparison with previous studies, picloram and dicamba significantly improved the efficiency of the triticale × maize crossing. The low dependence on the mother germplasm makes triticale × maize crossing an efficient alternative to the androgenetic methods of doubled haploid production in triticale.     

小麦与玉米杂交产生小麦单倍体与双单倍体的稳定性

小麦与玉米杂交是诱导小麦单倍体最有效的途径之一, 但单倍体和双单倍体产生频率不稳定影响了该技术的应用。选用13个小麦杂种F₁代单交组合与玉米杂交, 研究了不同小麦生长环境、生长素处理、培养基和壮苗处理对单倍体及双单倍体产生频率的影响。小麦生长在大田, 去雄后割穗培养与玉米杂交平均得胚率为23.9%, 每个杂交穗平均得胚数6.8个, 均是返青后从大田移回冷温室盆栽的3倍以上;不同小麦杂交组合间胚产生频率存在明显差异。生长素Dicamba蘸穗处理平均得胚率是21.5%, 与2,4-D处理得胚率(21.1%)无显著差异, 但不同杂交组合间差异显著。B5培养基幼胚萌发率为70.9%~88.3%, 平均82.0%;1/2 MS培养基胚萌发率为70.0%~86.0%, 平均76.6%;两种培养基平均胚萌发率无显著差异。试管苗经壮苗培养基壮苗处理与试管苗经移栽壮苗处理后加倍效率分别是67.6%和8.6%。移栽壮苗处理的苗分蘖少, 生长较弱, 加倍处理后存活率低和加倍率低是其单倍体加倍效率低的原因。     

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.     

Relative efficiency of maize and Imperata cylindrica for haploid induction in Triticum durum following chromosome elimination‐mediated approach of doubled haploid breeding

Intergeneric hybridization in seven diverse durum wheat genotypes was carried out using two composite varieties of Himalayan maize, viz., Bajaura Makka and Early Composite, and a wild grass, Imperata cylindrica, as pollen sources. Observations related to various haploid induction parameters put forth I. cylindrica as significantly better pollen source for haploid induction in durum wheat over maize in terms of pseudoseed formation (46.93%), embryo formation (38.06%), haploid regeneration (40.42%) and haploid formation efficiency (7.44%). The line x tester analysis revealed that both male and female genotypes had significant effects on all haploid induction parameters except haploid formation frequency in later. Among the pollen sources, I. cylindrica emerged as best combiner based on GCA values when compared with the two Himalayan maize composites. Durum wheat genotype, A‐9‐30‐1 was recognized as the best general combiner followed by PDW 314. The present investigation proposed durum wheat × I. cylindrica as a superior technique over maize‐mediated system, and its large‐scale use can open a new horizon in the sphere of durum wheat doubled haploidy breeding programme.     

Use of asynchrony in flowering for easy and economical polyhaploid induction in wheat following Imperata cylindrica‐mediated chromosome elimination approach

Wheat × Imperata cylindrica‐mediated approach of doubled haploidy breeding requires hand emasculation followed by pollination with I. cylindrica pollen. The pace of this endeavour can be enhanced by utilizing asynchronous flowering of wheat spikes by direct pollination without emasculation followed by morphological marker–assisted screening of selfed and crossed seeds. The emasculated and un‐emasculated spikes of 13 spring and six winter wheat genotypes and two triticale × wheat derivatives were pollinated with I. cylindrica pollen. The response of different genotypes for production of crossed and selfed seeds with direct pollination varied significantly within and between groups for spring and winter wheats, whereas triticale × wheat derivatives responded similarly to each other but significantly different from spring and winter wheats. Although, the proportion of pseudoseed formation was lower in case of direct pollination, yet in some genotypes, it was comparable to that of pollination after emasculation. Moreover, the response for haploid embryo induction frequency was similar in both the cases. The method of direct pollination can be utilized for easy and economical induction of haploids.     

Durum wheat × maize crosses for haploid wheat production: Influence of parental genotypes and various experimental factors

To improve haploid plant production in durum wheat, the haplomethod involving intergeneric crossing with maize followed by embryo rescue was used. The influence of parental genotypes and various experimental factors were studied. Ten cultivars of Triticum turgidum ssp. durum (female parent) were crossed with eight genotypes of Zea mays. After pollination, plant stems were either maintained in situ or cut near the base and kept in a 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐sucrose solution. Ten to 18 days after pollination, embryos were excised from developed ovaries and cultured on one of MS, MS/2, or B5 media. Haploid embryos and plants were obtained (78 green haploid plants regenerated in 0 year). The wheat genotype was significant for ovary development, embryo and plant formation, whereas the maize genotype was significant only for embryo formation. Detailed results of all crosses showed the best crossing partner for each wheat genotype. Cutting the plant stems after pollination gave better results than maintaining them in situ. The optimal stage for embryo rescue was 14 days and B5 and MS/2 media were more efficient than MS for embryo culture.     

Doubled haploid production in winter wheat and triticale genotypes,using the Hordeum bulbosum system

Summary An attempt was made to produce doubled haploids on 16 winter wheat and six spring and winter triticale genotypes thought to carry genes for interspecific incompatibility. The potential for haploid production was maximized by the use of Hordeum bulbosum genotypes selected for high crossability on crossable wheat genotypes, the use of two post-pollination applications of gibberellic acid and by the pollination of immature florets.A low frequency of seed was set on both the wheat and the triticale genotypes, having mean seed sets of 0.20 per cent and 0.27 per cent respectively. Although the frequency of embryos (seed quality) was high, doubled haploid production was further limited by poor embryo differentiation and regeneration. Haploid plantlets were obtained from the wheat cultivars Moulin and Renard, although successful chromosome doubling and doubled haploid production was achieved in Moulin only.     

2,4-D浓度对玉米花粉诱导小麦产生单倍体幼胚得胚率的影响

在玉米花粉诱导普通小麦产生单倍体技术中,2,4-D浓度是影响幼胚得胚率的重要因素之一.为了确定2,4-D的适宜浓度,利用9个玉米材料和16个小麦材料进行了研究.在小麦散粉前和散粉后,授玉米花粉后12h和36 h分别用150、200、250、300、350 mg/L的2,4-D蘸穗2次,调查数据显示,散粉前处理得胚率平均值分别为6.74%、7.19%、8.44%、7.53%和6.61%;散粉后分别为2.53%、3.11%、4.70%、2.81%和2.67%.结果表明,小麦散粉前授玉米花粉后用250 mg/L的2,4-D处理得胚率最高.     

AFLP analysis indicates no introgression of maize DNA in wheat x maize crosses

Amplified fragment length polymorphisms (AFLPs) were used to follow the possible introgression of maize DNA into haploids of wheat as a side‐effect of exploiting wheat x maize hybridization for haploid production. AFLPs were generated with 64 MseI/ EcoRI and 64 MseI/ PstI primer combinations, and the AFLP profiles of haploids were tested against those of maize and of the regular wheat varieties involved in the crosses. On average, 45.1 and 110.7 fragments were produced per assay with the MseI/EcoRI and MseI/PstI combinations, respectively. Different numbers of fragments were produced for wheat and maize: an average of 81 in the haploid, 80 in the wheat parent, and only 67.1 in maize. No evidence was found for introgression of maize into the wheat genome. Three unique AFLP fragments were detected in haploids, which were not present in the parental wheat genotypes. These ‘novel’ AFLP bands in the haploids could be caused by nucleo‐cytoplasmic interaction in the hybrid zygote. Such instability in the wheat genome is defined as temporal, as it was not detected in further generations when colchicine‐doubled progeny of the haploids was tested for the presence of polymorphic fragments.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

The effects of parthenogenesis on wheat embryo formation and haploid production with and without maize pollination

Doubled haploid (DH) lines are important in wheat (Triticum aestivum L.) breeding, and haploids produced via maize pollination precede DH line development. Although maize pollination has proven reliable and broadly applicable to wheat, its success is determined by the wheat and maize genotypes employed. A wheat genotype consisting of nuclear and cytoplasm components predisposing it to parthenogenesis was compared with three other genotypes, each possessing only one or neither component necessary for parthenogenesis. In a glasshouse experiment, each genotype was pollinated with maize and subsequently treated with a2,4-Dichlorophenoxyacetic Acid (2,4-D) solution to determine if parthenogenesis affected embryo formation frequency (EFF)and haploid formation efficiency (HFE). Wheat genotypes were also treated with the2,4-D solution alone to determine if embryos and haploid plants could be produced in vivo without maize pollination. ‘Salmon(K)’, a parthenogenetic genotype consisting of a Salmon 1BL.1RS nucleus in a Ae. kotschyii cytoplasm, had a mean EFF of 32%; whereas, the non-parthenogenetic genotypes had mean EFF calculations ranging from 7 to 21%. Mean HFE for Salmon(K) was not significantly different than the mean HFE for non-parthenogenetic Salmon; however, EFF and HFE calculations for Salmon(K) and Salmon, each with a 1BL.1RS translocation, were generally higher than calculations for genotypes without the translocation. Salmon(K) was the only genotype to produce a 3% or higher EFF and HFE after treatment with 2,4-D alone. Parthenogenesis significantly affected the frequency at which embryos were produced after pollination with maize and the frequency at which embryos and haploid plants were produced after treatment with 2,4-D alone. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of fertility between the F1, F2 and anther derived lines in the crosses of indica/japonica and japonica/indica in rice (Oryza sativa L.)

Summary Response of anthers in in vitro culture was examined in the indica-japonica hybrids of rice (Oryza sativa L.). Significant genotypic differences were observed for callus induction and regeneration among the different interracial hybrids of indica-japonica races. Induction frequency of haploids ranged from 57.7 to 72.9 per cent and doubled haploid androgenic lines ranged from 27.1 to 42.3 per cent in the anther culture of the different hybrids. The indica-japonica hybrids recorded partial pollen grain and spikelet fertility in F1 (29.9 to 41.5% and 19.4 to 48.7% respectively) as well as in F2 (42.7 to 50.6% and 37.1 to 54.4% respectively). In contrast, the androgenic doubled haploid lines recorded significant increase and the pollen grain and spikelet fertility was 76.3 and 78.6 per cent respecitively. The results suggested that the sterility barriers for realising genetic recombinants and fixation of fertile homozygous lines in indica-japonica hybridization programme could be overcome through F1 anther culture technique.Abbreviations BAP Benzyl Amino Purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - MS Murashige & Skoog medium - IAA Indole Acetic Acid     

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike. This revised version was published online in July 2006 with corrections to the Cover Date.