Gynogenetic haploid plants analysis for agronomic and enzymatic markers in maize (Zea mays L.)

Summary Experiments were conducted to investigate whether selection occurs during the processes involved in the production of doubled haploids. Haploid plants produced from two hybrids, each heterozygous for isozyme markers, were subjected to genetic analysis. The distributions of doubled haploid lines and pedigree lines derived from the hybrid C123 x Oh7 were compared with regard to agronomic character. The results suggest that the populations of haploid plants obtained by in vivo gynogenesis represent a random gametic array. Thus, in order to introduce haploid plants into breeding programmes in maize, maternal haploidy seems to be a very attractive method.     

Morphological and molecular evidences for DNA introgression in haploid induction via a high oil inducer CAUHOI in maize

The phenomenon of maternal haploid induction in maize was first described many years ago, but the underlying mechanism is still unclear. In this study, the Stock-6-derived, haploid-inducing line CAUHOI with high kernel oil content (KOC), was used as the pollinator to produce maternal haploids from the maize hybrid ZD958 with low KOC. CAUHOI is homozygous for the dominant marker gene R1-nj. Haploids were identified by morphological and cytological investigations. The frequency of haploid induction from this cross was 2.21%. Unexpectedly, many haploid kernels had weakly pigmented purple color on the embryo, and some haploid kernels had high KOC. Simple sequence repeat (SSR) analysis showed that 43.18% of the haploids carried segments from CAUHOI, and a small proportion (average 1.84%) of the genome of CAUHOI was introgressed into haploids. Haploid kernels with high KOC had a higher frequency of segment introgression from CAUHOI (2.92%) than that in haploid kernels with low KOC (1.79%), showing that the marker gene R1-nj and high-oil genes from CAUHOI were expressed during the development of some haploid embryos, and confirmed that the DNA introgression from the inducer parent occurred during maternal haploid induction. Together, these results suggested that the chromosome elimination was probably responsible for haploid induction in maize, and late somatic elimination might occur. Several possible mechanisms underlying haploid formation are discussed. Liang Li and Xiaowei Xu contributed equally to this work.     

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.     

Production of haploids from anther culture of banana [<Emphasis Type="Italic">Musa balbisiana</Emphasis> (BB)]

We report here, for the first time, the production of haploid plants of banana Musa balbisiana (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype Pisang klutuk, 12 from Pisang batu, seven from Pisang klutuk wulung and four from Tani. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important material for the improvement of banana through breeding programmes.     

Use of matroclinous haploid plants in recurrent selection in corn]

Two cycles of recurrent selection were performed in maize with the use of matroclinous haploids. Two synthetic populations, SP and SA, were improved. Each cycle consisted of two stages: (1) isolation of haploids from the synthetic populations and (2) growth of the haploids, pollination with pollen from diploid plants, and selection. The selection was performed for ear size in haploid plants. The mean gain in productivity in the synthetic populations SP and SA the per cycle was 16.48 and 20.98%, respectively. It is suggested that the high value of this index is related to the fact that haploid plants reveal useful genes with additive and epistatic effects. Natural selection may have played a part, too. The combination of artificial and natural selection in haploids resulted in a considerable gain in productivity in the synthetic populations to be improved.     

The Use of Matroclinous Maize Haploids for Recurrent Selection

Two cycles of recurrent selection were performed in maize with the use of matroclinous haploids. Two synthetic populations, SP and SA, were improved. Each cycle consisted of two stages: (1) isolation of haploids from the synthetic populations and (2) growth of the haploids, pollination with pollen from diploid plants, and selection. The selection was performed for ear size in haploid plants. The mean gain in productivity in the synthetic populations SP and SA the per cycle was 16.48 and 20.98%, respectively. It is suggested that the high value of this index is related to the fact that haploid plants reveal useful genes with additive and epistatic effects. Natural selection may have played a part, too. The combination of artificial and natural selection in haploids resulted in a considerable gain in productivity in the synthetic populations to be improved.     

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.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Maternal haploids of Petunia axillaris (Lam.) B.S.P. via culture of placenta attached ovules

Summary Hybridization of Petunia axillaris and P. parodii with Nicotiana tabacum was attempted using the method of in vitro pollination and fertilization. Seedlings were produced when the Petunia species and N. tabacum were used as the maternal parents; however, most of these had the identical somatic chromosome complement of the maternal parent. With crosses involving P. axillaris as the maternal parent, a low frequency of haploids was also produced. Due to the potential of haploids in basic and applied genetic research, additional experiments were carried out to determine whether in vitro pollination was necessary to stimulate haploid production and to more closely define the optimal time for ovule excision and culture. Four treatments were applied to accomplish these objectives. They were: placentas cultured prior to the time of anthesis, with and without pollination, and placentas cultured after the time of anthesis, with and without pollination. In vitro pollination had no effect on the frequency of haploids produced. Placenta attached ovules cultured prior to the time of anthesis produced significantly more haploids than those cultured after anthesis. The preanthesis treatment produced a frequency of 6.5 haploids per 100 ovaries cultured. The culture of placenta attached ovules provides an alternative to anther culture as a means for haploid production.The investigations reported herein were supported by USDA/SEA/CRGO Project 59-2213-1-1-613-0 and the paper (No. 84-3-36) is published with the approval of the Director of the Kentucky Agricultural Experiment StationThe authors are Graduate Research Assistant and Professor, respectively, Department of Agronomy, University of Kentucky, Lexington 40546-0091. The research reported in this paper is in partial fulfillment of the PhD requirements for the senior author     

Should maize doubled haploids be induced among F<Subscript>1</Subscript> or F<Subscript>2</Subscript> plants?

Maize (Zea mays L.) doubled haploid lines are typically produced from F₁ plants. Studies have suggested that the low frequency of recombinants in doubled haploids may reduce the response to selection. My objective was to determine if, for sustaining long-term response, doubled haploids should be induced in F₁ or F₂ plants during maize inbred development. In simulation experiments, I examined the response to multiple cycles of testcross selection among doubled haploid lines derived from F₁ plants (denoted by DH), doubled haploid lines derived from F₂ plants (DH_F2), and recombinant inbred (RI) lines derived by single-seed descent. For a trait controlled by 100 or more quantitative trait loci (QTL), the cumulative responses to selection were up to 4–6% larger among DH_F2 lines than among DH lines. The cumulative responses were up to 5–8% larger among RI lines than among DH lines. The QTL become unlinked as the number of QTL in a finite genome decreases, and the responses among RI, DH, and DH_F2 lines were equal or nearly equal when only 20 QTL controlled the trait. Metabolic-flux epistasis reduced the differences in the response among RI, DH, and DH_F2 lines. Overall, the results indicated that doubled haploids should be induced from F₂ plants rather than from F₁ plants. If year-round nurseries are used and new F₁ crosses for inbred development are initially created on a speculative basis, the development of doubled haploids from F₂ rather than F₁ plants should not cause a delay in inbred development.     

The RUBY reporter enables efficient haploid identification in maize and tomato

In vivo haploid induction has been extended from maize to monocotyledonous plants like rice, wheat, millet and dicotyledonous plants such as tomato, rapeseed, tobacco and cabbage. Accurate identification of haploids is a crucial step of doubled haploid technology, where a useful identification marker is very pivotal. R1-nj is an extensively used visual marker for haploid identification in maize. RFP and eGFP have been shown to be feasible in identifying haploid. However, these methods are either limited to specific species, or require specific equipment. It still lacks an efficient visual marker that is practical across different crop species. In this study, we introduced the RUBY reporter, a betalain biosynthesis system, into maize and tomato haploid inducers as a new marker for haploid identification. Results showed that expression of RUBY could result in deep betalain pigmentation in maize embryos as early as 10 days after pollination, and enabled 100% accuracy of immature haploid embryo identification. Further investigation in tomato revealed that the new marker led to deep red pigmentation in radicles and haploids can be identified easily and accurately. The results demonstrated that the RUBY reporter is a background-independent and efficient marker for haploid identification and would be promising in doubled haploid breeding across different crop species.     

Conservation and storage of a haploid cucumber (Cucumis sativus L.) collection under in vitro conditions

 Over 3 consecutive years (1992–1994), a collection of cucumber haploids was obtained from three different lines and one hybrid. Attempts were made to maintain and store a subcollection of these haploids for 3 years. Cucumber haploids appeared to be stable when cultured in vitro. There were no instances of spontaneous doubling and only one morphologically changed plant. During the first year of storage, between 30% and 80% the clones were lost, due to disturbances in plant development, increased levels of endogenous bacteria, and physiological changes resulting in continuous flowering. After 2 years of storage haploids showed reduced vigour. Therefore, plants were regenerated directly from primordial leaf microexplants. Haploid plants were obtained from nearly all of the previous haploid plants. The rejuvenated haploids possessed the same ploidy level and morphological traits as the old collection. The only new characteristic was faster vegetative growth. Received: 17 March 1998 / Revision received: 14 April 1999 / Accepted: 10 May 1999     

Genetic control of maternal haploidy in maize (Zea mays L.) and selection of haploid inducing lines

Summary The effect of genotype on maternal haploid plant production in maize was studied. The frequency of gynogenetic plants when Stock 6 was used as pollinator varied according to the female parent genotype. No simple relation was observed between genotypic aptitudes for gynogenetic and androgenetic development, which occured after pollination of W23 plant carrying the indeterminate gametophyte gene. Furthermore, the population NS, a favorably responsive genotype to anther culture, does not exhibit exceptional ability for in vivo gynogenesis. The effect of inbreeding and the influence of maternal haploid origin suggest that specific genes control maternal haploid initiation and development. However, gynogenetic development is not limited to a particular genotype. The frequency of maternal haploids may be increased by using specific pollen parents. Attempts were made to select for a high haploidyinducing trait and the present study reports the successful development of lines that can be utilized as pollen parents to induce haploids for experimental purposes and breeding programmes. When an inbred line WS14, derived from the cross W23 x Stock 6, was used as pollen parent, 2%–5% maternal haploids were obtained according to the female parent genotype. A high haploidy-inducing potential is a heritable trait and may be controlled by a limited number of genes. Genetic determination of the haploidy-inducing character was examined in relation to the efficiency of the selecting method and the mechanisms involved in the origin of maternal haploids.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation of maize germ cells

A transfer DNA (T-DNA) carrying the marker gene nptII was detected in the genomes of diploid and haploid maize plants obtained after the treatment of pistil filaments with a suspension of Agrobacterium during artificial pollination. PCR analysis of total DNA isolated from 155 canamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 111 plants (32.7% of the total number of analyzed seeds). The example of matroclinal haploids was used to demonstrate that T-DNA may be transported to the egg cell by the growing pollen tube (PT). Twelve out of 16 analyzed haploid plants contained the T-DNA insertion. The possible mechanism of the transfer of the Agrobacterium T-DNA to the maize genome during pollination is discussed.     

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.     

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.     

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

Key message

We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified.

Abstract

Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Chromosome elimination and in vivo haploid production induced by Stock 6-derived inducer line in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

In vivo haploid production induced by inducer lines derived from Stock 6 is widely used in breeding program of maize (Zea mays L.), but the mechanisms behind have not yet been fully understood. In this study, average frequency of haploid induction in four inbred lines by Stock 6-derived inducer line HZI1 was above 10%. About 0.2% kernels from the cross Hua24 x HZI1 had mosaic endosperm showing yellow shrunken parts from Hua24 to normal parts with purple aleurone from HZI1. Individual lagged chromosomes and micronuclei were observed in mitotic cells of ovules pollinated by HZI1. Above 56.4% of the radicles from the kernels with purple aleurone and colorless embryos were mixoploid (2n = 9-21), and more than 45.22% cells were haploid cells (2n = 10) in three crosses. More than 62.5% of the radicles from the kernels with purple aleurone and purple embryos were mixoploid (2n = 9-21) having 54.27% cells with 2n = 20. SSR analysis showed that all haploids from the cross Hua24 x HZI1 shared the same genomic compositions as Hua24 except for plants Nos. 862 and 857 with some polymorphic DNA bands. The results revealed that chromosome elimination after fertilization caused the haploid production in maize.     

Effect of the 1B/1R translocation on anther culture ability in wheat (Triticum aestivum L.)

Using two varieties, their reciprocal hybrids, F₈ lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two independent traits: regeneration ability and the frequency of albinos. F₈ lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they only regenerated green plants. Based upon cytological examination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the 1BL-1RS translocated chromosome from Aurora.Abbreviations DH doubled haploids - MS Murashige and Skoog - MPG multicellular pollen grains