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.     

Fertilization and Embryo Development in Wheat x Maize Crosses

The fertilization and embryo development in crosses of hexaploid wheat “Kangxuan 9” X maize “SS 7700” were studied. Of 180 florets fi,ced after pollination 34(18.9%) had embryo and endosperm, 46(25.6%) had only embryo and 12(6.7%) had only endosperm. Percentages of single or double fertilization were higher than that in control (“Chinese Spring” X maize). The hybrid embryos and endosperms obtained were karyotypically unstable and characterized by rapid elimination of the maize chromosomes to produce haploid wheat embryos. The potentials for wheat haploid production and transfer of DNA segments, including transposable elements, from maize to wheat is discussed.     

人工合成冬性六倍体小麦的研究

以冬性四倍体硬粒小麦(Triticum durum,2n=28,AABB)为母本与粗山羊草(Aegilops tauschii,2n=14,DD)杂交,得到的单倍体幼胚(n=21,ABD)经组织培养拯救,获得的幼苗经染色体加倍而成为合成小麦(AABBDD)。从中鉴定、筛选出冬性的合成小麦。幼胚仅在1/2 MS培养基上培养,成苗率为75.81%;根据幼胚的发育状态,将发育较完善的幼胚直接接种在1/2 MS培养基上,将发育不良的幼胚先接种于1/2 MS+2 mg/L 2,4-D培养基上进一步养育幼胚,之后视幼胚发育状况再将其转入1/2 MS培养基中培养成苗,此方法的成苗率为92.44%,较前者的成苗率提高了16.63%。染色体加倍在冬季塑膜拱棚内用0.05%秋水仙素进行半根法处理,较容易获得健壮苗,并且分蘖多。     

Differences among auxin treatments on haploid production in durum wheat × maize crosses

Three doubled haploid lines of durum wheat [Triticum turgidum ssp. durum (Desf.) Husn.] were crossed with maize (Zea mays L.), and five hormone treatments were applied to test their effect on the production of caryopses, embryos and haploid plants. The auxin treatments consisted of 100 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mg/l or 50 mg/l dicamba and two combination mixtures of 95/5 mg/l and 50/50 mg/l 2,4-D plus dicamba, respectively. Hormones were added to the culture medium of the detached tillers. Differences were not observed among the four hormone treatments that contained dicamba, nevertheless, these treatments significantly increased the production of caryopses, embryos and haploid plants. On average, 8.9 caryopses, 2.6 embryos and 1.3 haploid plants per spike were obtained following the treatment with 100 mg/l 2,4-D, and 15.0 caryopses, 6.0 embryos and 3.0 haploid plants per spike were obtained following the various treatments with dicamba. We propose the application of dicamba alone, or dicamba plus 2,4-D, as a means for improving the yield of haploid plants of durum wheat through crosses with maize.     

Haploid regeneration from tetraploid wheat using maize pollen.

Crosses were made between 21 tetraploid wheat genotypes (6 parents, 15 F1 hybrids) and a single F1 hybrid of maize that was used as the male parent. Plants were grown under controlled greenhouse conditions (daylength, 16 h; temperature, 25 degrees C days and 15 degrees C nights). To enhance embryo survival, 2,4-D (10 mg/L) was applied to spikes 24 h after pollination with maize. Embryos were recovered from the tetraploid wheat genotypes at a rate of 2.34-14.14/100 developed ovaries. Sixty-nine haploid plants were obtained from 3 parents and 12 F1, hybrids. Fifty-six of these were successfully doubled. General combining ability was significant for the two traits studied, indicating that additive genetic control is important for the number of developed ovaries and haploid embryo production in tetraploid wheat x maize crosses. In this report, we demonstrate the potential of using maize pollen to produce haploid plants from tetraploid wheat genotypes.     

Chlorophyllian durum wheat plants obtained by isolated microspores culture: importance of the pre-treatments

In Triticum turgidum subsp. durum (Desf.) Husn., the utilization of in vitro anther culture is hampered by the very high frequency of albinism of the regenerated plants reaching in most cases 100%. Only in vitro ovary culture or intergeneric crosses with maize produce gynogenetic green haploid and doubled haploid plants. This paper is concerned with another very interesting method of androgenetic doubled haploid plant production, the in vitro isolated microspore culture. It is shown that this method, associated with cold alone or cold plus mannitol pre-treatments, of the spikes kept within their sheath leaves, during different times, have significant positive effects, not only on embryo production, but also on chlorophyllian plant regeneration. All pre-treatments and control taken together, a total of 16 490 embryos was obtained from 17.4 x 10(6) microspores of two T. durum varieties, among which 9320 embryos were transferred to regeneration medium and developed 150 chlorophyllian plants. Thus a long-term (five weeks) 4 degrees C cold pre-treatment of the microspores could be promising for green regeneration in durum wheat.     

小麦×高粱的受精率和小麦单倍体的诱导(简报)

远缘杂交无论在植物遗传学理论的建立还是在新品种的培育上都是一项十分有用的技术,特别在小麦上成绩更为突出。近来发现小麦不但和其亲缘属(如山羊草属、偃麦草属,赖草属等)植物可以杂交,而且和亲缘关系较远的植物种属如玉米、珍珠栗、大刍草、摩擦禾等杂交也有很高的成胚率,并且通过杂种胚发育过程中父本染色体的消失,可以获得小麦单倍体。Laurie和Bennett以高粱属的     

Production of durum wheat substitution haploids from durum x maize crosses and their cytological characterization.

The objective of this study was to investigate the effect of individual durum wheat (Triticum turgidum L.) chromosomes on crossability with maize (Zea mays L.) and to cytologically characterize the haploids recovered. Fourteen 'Langdon' (LDN) D-genome disomic substitution lines, a LDN Ph mutant (Ph1b ph1b), and normal 'Langdon' were pollinated with maize pollen. After pollination, hormonal treatment was given daily for up to 14 days. Haploid embryos were obtained from all lines and were aseptically cultured. From a total of 55,358 pollinated florets, 895 embryos were obtained. Only 14 of the embryos germinated and developed into healthy plants. Different substitution lines showed varying degrees of success. The most successful was the substitution 5D(5B) for both embryo formation and haploid plantlet production. These results indicate that the substitution of 5D for 5B confers on durum wheat a greater ability to produce haploids. Fluorescent genomic in situ hybridization (GISH) showed that the substitution haploids consisted of 7 A-genome chromosomes, 6 B-genome chromosomes, and 1 D-genome chromosome. Triticum urartu Turn. genomic DNA was efficient in probing the 7 A-genome chromosomes, although the D-genome chromosome also showed intermediate hybridization. This shows a close affinity between the A genome and D genome. We also elucidated the evolutionary translocation involving the chromosomes 4A and 7B that occurred at the time of evolution of durum wheat. We found that the distal segment translocated from chromosome 7B constitutes about 24% of the long arm of 4A.     

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.     

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).     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

Ageing of isolated embryos and endosperms of durum wheat: An analysis of chromosome damage

Embryos (EM) and endosperms (END) were isolated from durum wheat seeds and aged separately for 3 years. Analysis of chromosome damage was carried out on root tip meristems of transplants in which aged (a) embryos and/or endosperms were used. The aim of the experiments was to investigate the ageing process when embryos and endosperms age separately and to evaluate the possible influence of the endosperm during storage of the seed.The results obtained indicate that (i) both aged embryo and endosperm produce mutagenic substances capable of inducing nuclear damage in the radicle meristem and (ii) age-induced damage in the embryo is not the consequence of endosperm ageing. The relationships between chromosome damage produced by aged embryos and endosperms in the transplants are discussed.     

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.     

禾木科植物染色体消除型远缘杂交的研究进展

植物远缘杂交是作物育种中广泛应用的技术。除了核型稳定的种间杂交可以获得杂种以外,还可以利用核型不稳定的种间杂交后父本染色体消除的现象,通过胚培养和染色体加倍处理获得加倍单倍体(DH)植株。然而从小麦×玉米杂交获得的DH后代与其理论上应完全同质的遗传表现却不相符,总有2～5％的DH植株发生了形态学变异。最近的研究证明。通过小麦×玉米的受精作用,一些玉米特异DNA可以被转移到小麦DH后代的基因组中。     

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。     

Production of wheat doubled haploids by pollination with Job's tears (Coix lachryma-jobi. L.)

Wheat (Triticum aestivum L.) haploids were produced by crossing with Job's tears (Coix lachryma-jobi L.) as the pollen parent. Pollination was followed by 2,4-D treatment, detached tiller culture, and embryo culture, as described for maize pollination. The frequency of embryo formation was similar to that obtained by crossing wheat with maize pollen. Job's tears is a perennial plant which forms several stalks and its pollen can be collected throughout the year when the plant is maintained in a controlled environment. Our results indicate that Job's tears can be used as the pollen parent for wheat crosses for haploid production without requiring synchronization of flowering dates.     

Production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum durum</Emphasis> Desf.) through culture of unpollinated ovaries

The objective of this work was to produce doubled haploid plants from durum wheat through gynogenesis using unpollinated ovary culture of three local Tunisian genotypes (Jenah Khotifa, Hmira, Azizi) and three improved cultivars (Karim, Khiar, Razzek). A total of 12,000 unpollinated ovaries were cultured in this study. Spikes were either pretreated at 4°C for 14 days or at 4°C in a mannitol solution (0.3 M) for 7 days. Induction was performed using two media. We showed that ovary development, callus and plantlet regeneration was influenced significantly by genotype and growth conditions. The highest regeneration frequency was obtained when the microspore population was in the late mononucleate to binucleate stage. Our results suggested that the cold pretreatment for 14 days was more efficient than the cold treatment in a mannitol solution. Furthermore, the addition of 2,4-D, vitamins and glutamine, and the use of maltose as sugar source in media improved the ovary culture. When the unpollinated ovaries were cultured under the conditions found to be optimal in the present study, a total of 84 plants were produced, all green and haploid. The best levels for regenerated plants were obtained with the cultivars Khiar (3.5%), Hmira (3.1%) and Karim (1.5%). Fertile doubled haploid plants were obtained by colchicine treatment. This result represents a modern tool for breeders to produce durum wheat homozygous lines in a few months.     

Durum wheat haploid production using maize wide-crossing

While anther culture or pollinations with Hordeum bulbosum have provided suitable methods for haploid production in bread wheat, they have been largely unsuccessful in durum wheat. Pollinations with maize were used in an attempt to produce haploid seedlings and, from these, fertile doubled haploids of durum wheats. Moreover, the effect of various concentrations and combinations of a synthetic auxin, 2, 4-dichlorophenoxyacetic acid (2,4-D), kinetin, and an ethylene inhibitor, silver nitrate (AgNO₃), on embryo recovery were also investigated. Haploid seedlings were recovered from Triticum turgidum ssp. turgidum cv Rampton Rivet pollinated with maize following in-vivo treatment of ovaries with 2,4-D for 2 weeks and subsequent embryo culture. The recovery of haploid seedlings from T. turgidum ssp. durum cv. Wakona pollinated with maize necessitated the addition of AgNO₃, to the 2,4-D treatment. Overall, haploid seedlings were produced in 1.7% and 3.3% of pollinated florets for Rampton Rivet and Wakona respectively. The success of the present work represents a significant breakthrough for haploid production in durum wheats. Wide hybridization with maize followed by in-vivo treatment of ovaries with 2,4-D alone, or in combination with AgNO₃, may provide a widely-applicable method of haploid production in tetraploid wheats.     

Analysis of stunter1, a maize mutant with reduced gametophyte size and maternal effects on seed development

Many higher eukaryotes have evolved strategies for the maternal control of growth and development of their offspring. In higher plants this is achieved in part by postmeiotic gene activity controlling the development of the haploid female gametophyte. stunter1 (stt1) is a novel, recessive, maternal effect mutant in maize that displays viable, miniature kernels. Maternal inheritance of stt1 results in seeds with reduced but otherwise normal endosperms and embryos. The stt1 mutation displays reduced transmission through the male and female parents and causes significant changes in the sizes of both male and female gametophytes. stt1 pollen grains are smaller than wild type, have reduced germination efficiency, and reduced pollen tube growth. stt1 embryo sacs have smaller central cells and abnormal antipodal cells that are larger, more vacuolated, and fewer in number than wild type. Embryos and endosperms produced by fertilization of stt1 embryo sacs develop and grow more slowly than wild type. The data suggest that the morphology of mutant embryo sacs influences endosperm development, leading to the production of miniature kernels in stt1. Analysis of seeds carrying a mutant maternal allele of stt1 over a deletion of the paternal allele demonstrates that both parental alleles are active after fertilization in both the endosperm and embryo. This analysis also indicates that embryo development until the globular stage in maize can proceed without endosperm development and is likely supported directly by the diploid mother plant.