Comparative responses of tetraploid wheats pollinated with Zea mays L. and Hordeum bulbosum L.

Ten different tetraploid wheat (Triticum turgidum) genotypes were pollinated with maize (Zea mays). Fertilization was achieved in all ten genotypes and no significant difference in fertilization frequency between the tetraploid wheat genotypes was detected. A mean of 41.1% of pollinated ovaries contained an embryo. All these crosses were characterized by the elimination of the maize chromosomes, and the resulting embryos were haploids. Six of the tetraploid wheat genotypes were also pollinated with Hordeum bulbosum. Fertilization frequencies with H. bulbosum were much lower (mean=13.4%), and significant differences between the tetraploid wheat genotypes were detected. Observation of pollen tube growth revealed that part of the incompatibility reaction between tetraploid wheats and H. bulbosum was due to an effect similar to that of the Kr genes, namely pollen tube growth inhibition. These results indicate that pollinations with maize may have potential as a broad spectrum haploid production system for tetraploid wheats. Present address: Agriculture Canada, Research Branch, Central Experimental Farm, Bldg 50, Ohawa, Ontario, Canada K1A OC6     

Induction of Haploid Durum Wheat Plants Through Pollination of Maize Pollen

When tetraploid wheat (Triticum durum Desf. ) variety DR147 was crossed with maize (Zea mays L. ) variety suppersweet ss 7700, pollen readily germinated on the stigma and one or more pollen tubes reached the embryo sac in 83.4% of wheat florets. The frequency of fertilization and embryo formation was 44.5% and 42. 6% respectively. The hybrids were karyotypically unstable and the maize chromosomes were eliminated early in the development. Thus haploid wheat embryos were form. Although the double fertilization frequency of durum wheat X maize was high (32.7%) to form embryos and endosperms, yet the endosperms were highly abnormal. It was very difficult to produce viable mature seeds from the mother durum wheat plants. The survival of hybrid embryos produced by durum wheat X maize could be improved or prolonged by treatment with 100 ppm 2, 4-D (either by dipping inflorescences in solution or injecting 0.3 to 0.5 mL 2, 4-D solution into the uppermost internodes of the wheat stem). 9 to 13 days after pollination, caryopsis were excised from the pollinated spikes and surface sterilized for peeling of the embryos in different developing stages. The embryos were plated on MS solid medium containing 3% sucrose, 200 mg/L casein hydrolysate for embryo rescue. The experimental results revealed that the well developed embryos (larger than 0. 5 mm with scutellum structure) were easy to produce calli by callus induction or produce haploid wheat plants by embryo rescue, whereas the poorly developed embryos (globular, pear or torpedo-shaped embryos smaller than 0.3 mm) responsed very poorly. The germination frequencies of well and poorly developed embryos were 83.3 % and 12.5 %, respectively. Chromosome counts of root tip cells of the rescued plants proved their haploid nature (2n= 2x= 14).     

Haploid Wheat Induction by Crosses Between Different Ploidy Wheat and Different Populations of Maize

Haploid embryo and plant producing frequencies were studied by crossesing diploid, tetraploid and hexaploid wheat with landraces,hybrids and inbred lines of maize. It showed significant differences among the wheat and maize populations. The tetraploid and hexaploid wheat were better than the diploid. High frequencies were obtained by using tetraploid wheat of Triticum turgidum cv. TG14 and maize landraces cv. Xiaoyumi and Xiao Huangmaya. The highest haploid plant producing frequency (6.95 %) was obtained in the TG14 × Xiaoyumi.     

Production of double haploids in oat (Avena sativa L.) by pollination with maize (Zea mays L.)

The aim of the study was to optimize the method of oat haploid production by pollination with maize. Seventeen oat genotypes were used in the experiment. Various factors influencing the growth and development of ovaries and embryo production were investigated: genotype, time of pollination, growth regulators and time of their application. Emasculated before anthesis, oat florets were pollinated with maize pollen after 0, 1 or 2 days. Next, one of two auxins analogues (2,4-D or dicamba) were applied to oat pistils. These auxins had no significant influence on the number of enlarged ovaries and embryos. The time of application of these growth regulators had a significant influence on embryo production. Haploid embryos were obtained from all used genotypes, although the frequency of enlarged ovaries and obtained embryos did not differ markedly between the genotypes. On average, 85% of ovaries were enlarged and 11.7% of them produced haploid embryos. Depending on the regeneration medium, 24–41% of embryos were germinated, of which 12% had developed into green plants. A strong significant difference in the number of germinating embryos and haploid plants was observed between the kind of regenerating medium used. There were no albino plants and all the obtained plants were haploid.     

Doubled haploids of wheat from wheat x maize crosses:genotypic influence, fertility and inheritance of the 1BL-1RS chromosome

The wheat x maize cross as a technique for haploid induction in wheat was evaluated in a replicated block design comprising 18 wheat F₁ hybrids and five Zea mays L. parents. Haploid plants were regenerated at an average of 9.1 (4.4–14.7) plants per 100 florets processed. Genotypic differences for haploid production efficiency were recorded for both wheat and Zea mays L. Interaction between parents was significant for number of plants/100 florets. All 610 of the 1,703 regenerated plantlets that were analyzed by flow cytometry were haploid. At maturity, 70% (60–81 %) of the colchicinetreated haploid plants were fertile, but the frequency of fertile and sterile plants was not consistent over the wheat hybrids from which they were derived. Flow cytometry performed using the first tiller which arose following colchicine treatment enabled prediction of fertility. The 1BL-1RS chromosome was found at the expected ratios in the F₂ and in the haploid progenies produced through the wheat x maize cross but deviated from the 11 ratio in the haploid progenies produced by anther culture.     

Pseudogamous Apomixis in Maize and Sorghum in Diploid-Tetraploid Crosses

Apomictic seed development is a complex process including formation of unreduced embryo sac, parthenogenetic embryo development from the egg cell, and endosperm formation either autonomously, or due to fertilization of polar nuclei by the sperm (under pseudogamous form of apomixis). In the latter case, an obstacle to the normal endosperm development is disturbance of maternal (m) -to-paternal (p) genomic ratio 2m: 1p that occurs in the cases of pollination of unreduced embryo sac with haploid sperms. Usage of tetraploid pollinators can overcome this problem because in such crosses maternal-to-paternal genomic ratio is 4m: 2p that provides formation of kernels with plump endosperm. Using tetraploid lines as pollen parents we observed formation of plump kernels on the ears and panicles of diploid maize and sorghum accessions. These kernels had hybrid endosperm and diploid maternaltype embryo or hybrid embryo with different ploidy level (2n, 3n, 4n). The frequencies of plump kernels on the ear ranged from 0.2-0.3% to 5.7-6.2% counting from the number of ovaries. Maternal-type plants were found in two maize lines, their frequency varying from 10.7 to 37.5% of the progeny plants. In CMS-lines of sorghum pollinated with tetraploid sorghum accessions, the frequency of plump kernels ranged from 0.6 to 14.0% counting from the number of ovaries; the frequency of maternal-type plants varied from 33.0 up to 96.1%. The hybrid nature of endosperm of the kernels that gave rise to maternal-type plants has been proved by marker gene expression and by SDS-electrophoresis of endosperm proteins. These data testify to variable modes of seed formation under diploid × tetraploid crosses in maize and sorghum both by amphi- and by apomixis. Therefore, usage of tetraploid pollinators might be a promising approach for isolation of apomixis in maize and sorghum accessions.     

The Crossability of Triticum aestivum with Tetraploid Hordeum bulbosum

In order to study the crossability of wheat with H. bulbosum a series of wheat varieties from various sources and their F1 hybrids as well as tetraploid H. bulbosum from different countries were used as parents in this experiment. The main results of the experiment are showed as follows: 1. Twenty-one wheat varieties from Europe, West Asia, America, China, Australia etc. 11 countries and regions respectively as famele parents were crossed with four tetraploid H. bulbosum from Hungary, USSR, Canada and Germany. The seed set percentages in the intergeneric cross combinations ranged from 0.00% to 49.93%. Statistical analysis revealed that there were significant differences between the seed set percentages of wheat varieties. The crossability, with H. bulbosum might differed in different varieties of wheat from same country. 2. Various F1 hybrids resulting from 13 and 26 intervarietal cross combinations of wheat and their parents were crossed with H. bulbosum from Hungary and USSR respectively in different years. The mean seed set percentages of F1 hybrids, their male and female parents were 26.53%, 15.38%, 20.30% and 39.1%, 34.8%, 26.7% respectively. The results indicated that when some wheat varieties having poor-crossability with H. bulbosum were hybridized with other varieties especially with those varieties having high- crossability, the crossability of their F, hybrids probably had higher crossability than their parents having poor-crossability. Six F1 hybrids of wheat obtained from six com- binations of reciprocal crosses, in which the completely non-crossable varieties Hope, Xiao- Bai-Mang and high-crossable varieties Chinese Spring, Fortunate were used as male or female parent alternately, were crossed as female parents with H. bulbosum. All of 6 F1 hybrids were crossable and gave the percentage of seed set from 7.00% to 42.57%, although they ought to carry the dominant Kr genes responsible for non-crossability, which were passed on to F1 hybrids by non-crossable varieties, the parent Hope or Xiao-Bai- Mang. Clearly that is due to the gene interaction between female and male parents. Be- sides Kr loci in wheat, probably other genes can influence the crossability. 3. There were significant differences between clones of H. bulbosum in the crossa- bilities with wheat. In conclusion F1 hybrids of wheat have higher crossability with H. bulbosum than their parents having poor-crossability. This could be advantageous for exploitation of this technique. By selecting even more efficient clones of H. bulbosum and improving embryo culture techniques, the H. bulbosum technique will be used in wheat breeding program- mes probably.     

Efficient production of haploid wheat (Triticum aestivum) through crosses between Japanese wheat and maize (Zea mays)

Summary Four Japanese wheat varieties, three crossable and one non-crossable with Hordeum bulbosum, were pollinated with maize pollen of 5 genotypes. By the application of 2,4-dichlorophenoxyacetic acid after pollination, embryos kept developing on wheat plants until 14 days after pollination. The frequency of embryo formation was significantly different among the maize genotypes, varying from 18.0% to 31.9%, but not among the wheat varieties. By bagging spikes with flag leaves the frequency of embryo formation was increased by about 7%. Ten- to twelve-day-old embryos gave higher frequencies of plant formation (83.6%) than 14-day-old embryos(50.0%). All 6 regenerated plants investigated cytologically were found to be haploid. Twelve of the 14 colchicine-treated plants produced florets setting seeds. The overall efficiency of our procedure is considered to be higher than that reported by Laurie and Bennett (1988).     

Effect of zearalenone treatment on the production of wheat haploids via the maize pollination system

The ability of zearalenone (ZEN) to stimulate the growth of haploid wheat embryos formed following the pollination of wheat spikes with maize pollen was tested. The maize pollination system was used as a model to compare the activity of ZEN with that of auxin analogues. Three solutions, each with a different concentration of ZEN (6.0, 3.0 or 1.5 microM), and a solution of 2,4-dichlorophenoxyacetic acid (control) were tested for their effect on ovary swelling, frequency of embryo formation and the ability to regenerate plants. In total, 3,105 florets of 282 spikes from five different cultivars of hexaploid winter wheat ( Triticum aestivum L.) were pollinated with maize ( Zea mays L. cv. Gama) pollen and treated with the ZEN solutions. The highest concentration of ZEN (6.0 microM) was the most effective in inducing ovary swelling (84 swollen ovaries/100 pollinated florets) and increasing the frequency of embryo induction (18.9 embryos/100 pollinated florets), but these embryos were severely deformed. They had low capability to germinate in vitro, while callus was easily formed and indirect regeneration of plants was possible. The lowest ZEN concentration (1.5 micrroM) induced ovary swelling in 42.8/100 pollinated florets and embryo growth in 3.3 out of 100 pollinated florets. The embryos were regular in shape, and almost half of them germinated in vitro while callus induction from them failed. The concentration of 3 microM ZEN had an intermediate effect. The type of response of the various wheat genotypes was similar, while frequencies were different, with cv. Izolda being the most responsive. The results show that ZEN has some of the properties of an auxin analogue, while other effects of its action are unique. It can be used in the maize pollination system of doubled haploid production to replace auxin analogues when indirect regeneration of plants via callus tissue is planned.     

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.     

水稻84-15细胞胚胎学的研究

粳稻品种８４＿１５是非洲长花药野生稻（Ｏ．ｌｏｎｇｉｓｔａｍｉｎａｔａ）和栽培稻８０＿６１９５杂交,在杂种第三代出现的不分离粳稻品系。它们的杂种Ｆ１０代农艺性状保持整齐一致（赵世绪,１９９０;陈建三,１９９５）。它比水稻品种郑粳１０７增产２２．２％。用粳稻８４＿１５与水稻喜峰杂交,在杂种第二代选育出中新一号比秋光增产１１％,已种植Ｆ１０代农艺性状保持一致。为了证实粳稻８４＿１５固定杂种优势的原因,我们进行了细胞胚胎学的研究。通过研究发现了粳稻８４＿１５珠心胚生殖现象（陈建三,１９８８;１９９０;１９９１）。本文旨在进一步研究此种现象。     

Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat. Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

小麦与玉米杂交及单倍体的产生

应用普通小麦不同品种(品系)与苏玉4号杂交,喷洒100ppm的2,4—D一至二次,授粉后子房膨大,其膨大率因品种不同而异。在部分膨大子房中剥取到发育幼胚,经组织培养后,获得单倍体小麦植株,植株未结实。     

小麦与玉米杂交及单倍体的产生

应用普通小麦不同品种（品系）与苏玉４号杂交,喷洒１１０ｐｐｍ的２,４－Ｄ一至二次,授粉后子房膨大,其膨大率因品种不同而异。在部分膨大子房中剥取到发育幼胚,经组织培养后,获得单倍体小植株,植株未结实。     

水稻84-15细胞胚胎学的研究

粳稻品种84-15是非洲长花药野生稻(O．longistaminata)和栽培稻80-6195杂交,在杂种第三代出现的不分离粳稻品系。它们的杂种F10代农艺性状保持整齐—致(赵世绪,1990;陈建三,1995)。它比水稻品种郑粳107增产 22.2％。用粳稻84-15与水稻喜峰杂交,在杂种第二代选育出中新一号比秋光增产11％,已种植F10代农艺性状保持—致。为了证实粳稻84-15固定杂种优势的原因,我们进行了细胞胚胎学的研究。通过研究发现了粳稻84-15珠心胚生殖现象(陈建三,1988;1990;1991)。本文旨在进一步研究此种现象。     

The production of haploid wheat plants from wheat x maize crosses

Summary Hybrid embryos from hexaploid wheat x maize crosses rapidly lose the maize chromosomes to produce haploid wheat embryos. Such embryos almost always aborted when left to develop on the plant, and only 1 was recovered from 2440 florets (0.17% of the expected number). Embryos had greater viability in spikelet culture, 47 (26.5% of the expected number) being recovered from 706 ovaries. Thirty-two of these embryos germinated to give green plants, 31 of which were haploid (21 wheat chromosomes) and 1 of which was euploid (42 wheat chromosomes). Spikelet culture enabled 17.1% of the expected number of embryos to be recovered as haploid plants, a 100-fold improvement on allowing embryos to develop in vivo. Ten haploid plants of Chinese Spring (kr1, kr2), 13 plants of Chinese Spring (Hope 5A) (kr1, Kr2), and 8 of Hope (Kr1, Kr2) were recovered. The potential of wheat x maize crosses for wheat haploid production and for gene transfer from maize to wheat is discussed.     

The Production of Haploid Wheat Plants via Wheat X Maize Hybridization

The intergeneric cross between wheat and maize are characterized by a high frequency of formation of hybrid embryos, but maize chromosomes are rapidly eliminated in the first few cell division cycles to produce haploid wheat embryos. If left the embryos on the plants they will soon abort, as a result of the absence or poor development of the endosperm. Embryo rescue techniques should not enable these embryos to grow to plants because of the embryos were extremely young for embryo culture. Viable embryos were obtained at much higher frequenc, if spikes containing cross-pollinated florets were dipped in 100 ppm 2,4-D solution 4 hours after pollination. Of the 382 florets treated, 64(16.8%) embryos were obtained 10 days after treatment, and 47 plants recovered on the culture medium. In control (2,4-D not applied) only 1 (0.96%) embryo and 1 plant was obtained from 104 florets. This simplified technique should enable haploid wheat plants production through wheat - maize to apply to practical breeding.     

Implementation of extensive citrus triploid breeding programs based on 4x?×?2x sexual hybridisations

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x?×?2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x?×?2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3?%), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x?×?2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations.     

Relationships between four measures of genetic distance and breeding behavior in spring wheat

We estimated the genetic distances among 10 spring wheat genotypes based on pedigree data, morphological traits and AFLP markers, used individually and combined with morphological traits, to find the best predictors of general- and specific-combining abilities among parental genotypes. Ten wheat parents were crossed in a diallel form, disregarding reciprocal hybrids, totaling 45 combinations. The F(1) hybrids, F(2) populations and parents were evaluated in the field in 2007. The experimental plots consisted of 20 plants for F(1) hybrids and 40 plants for parental and F(2) populations. All methods (pedigree data, AFLP markers and morphological traits, used individually and combined) were found to be useful for the assessment of genetic diversity. The significant coefficient correlations ranged from low (0.45) to moderate (0.67) between the distance measures and hybrid performance. There was significant agreement between the distance measures based on AFLP markers vs morphological traits + AFLP markers (r = 0.47) and between pedigree data vs morphological traits + AFLP markers (r = 0.43). The pedigree distance was positively associated with traits 100-kernel weight and grain yield per plant in F(1) (correlations of 0.67 and 0.62, respectively) and F(2) (correlations of 0.62 and 0.59, respectively) generations. These correlation values indicate that the genetic distance, based on pedigree data, could replace diallel crosses for the selection of parents with higher combining ability and with moderate reliability.