A reproducible protocol for the production of gynogenic haploids of mulberry, Morus alba L.

Gynogenic haploids of a female clone of mulberry (Morus alba L. Cv. K-2) were produced by unpollinated ovary culture. Segments from in vitro developed inflorescences were cultured on MS+ BAP (8.5 μM)+2,4-D (4.5 μM) and after three weeks individual ovaries were transferred to MS+2,4-D (4.5 μm)+glycine (6660 μM)+proline (1738 μM). In this treatment sixteen per cent of the ovaries developed a gynogenic seedling. The gynogenic plants have been established in soil. Out of twenty gynogenic plants examined cytologically, twelve showed the haploid number of chromosomes (2n=x=14) and the other eight were aneuploids, with 13–17 chromosomes in their root tip cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Spontaneous and induced chromosome doubling in gynogenic lines of onion (Allium cepa L.)

The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and 6-benzyladenine (6—BA) in the medium is required to induce an acceptable yield of gynogenic embryos from unfertilized ovary and flower cultures in onion. Four different exposure times of ovary and flower cultures to exogenous growth regulators (15, 30, and 45 days, and the entire culture period) were assayed. The objective was to ascertain the effect of these substances and of their period of application on the formation of gynogenic embryos and on the yield of haploids. An exposure of 15 days was sufficient for ovaries and flowers to be stimulated towards the gynogenic response, whereas, for the remaining period of 30—80 days, the pro-embryos could easily grow on a growth-regulator-free medium. In the gynogenic material obtained, phenotypic differences were visible between genetically independent lines but not between plants within each line, even when they had a different ploidy level (n or 2n). Almost all lines obtained by gynogenesis were sterile. Only a small percentage (1%) became fertile through spontaneous chromosome doubling, and these produced 2—20 seeds each, giving normal plants. The recovery of fertility occurred more often during the generations of bulbils. To exploit this natural propensity towards diploidization in this phase, different amounts and numbers of applications of colchicine were evaluated in two experiments. The treatments corresponding to 10 and 100 mg/1 of colchicine applied for 24h gave the highest number of diploid cells in root tips but no diploidization occurred in the shoot apices. Three days of colchicine treatment at 10 mg/1 produced 46% of plants completely diploid in the shoot apex. The flower fertility of these doubled haploid plants is being evaluated.     

Dicamba and growth condition effects on doubled haploid production in durum wheat crossed with maize

Doubled haploid plants are useful in genetic studies and plant breeding, but a consistent and satisfactory frequency of production has been difficult to achieve in durum wheat. Triticum turgidum L., using the maize pollen method. The objective of this study was to develop an objective method of producing doubled haploids in durum wheat. Plant growing and handling conditions, aspects of hormone treatments, wheat genotype and pollen source were considered. The number of caryopses, embryos, haploids, doubled plants and doubled plants that set seed were measured. Although growth conditions, pollen source, method of handling plants and wheat genotype are important considerations, the type of hormone was found to be most significant in the production of doubled haploid plants. When 50mg/l dicamba was substituted for 100 mg/l 2,4‐D the number of doubled haploids per spike increased from 0.2 for the best 2,4‐D treatment to 1.3 for the dicamba treatment. This increased frequency was largely attributed to an increase in the number of caryopses generated for each spike emasculated and from an increased frequency of germination of embryos to haploid plantlets. The best production of caryopses was 0.41 caryopses per florest with 2,4‐D. The best production of haploids per 100 florets was 12 with dicamba and 1.65 with 2,4‐D. The frequency of one doubled haploid per emasculated spike through the use of dicamba is a practical level for generating populations for genetic studies.     

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.)

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.     

Second cycle gynogenesis in onion, Allium cepa L., and genetic analysis of the plants

Haploid induction in onion via gynogenesis provides inbred lines that can be used in production of hybrid cultivars. Despite several successful reports, the gynogenic induction rate is still relatively low, ranging mainly from 0.5 to six embryos per 100 cultured flowers. In the present study, gynogenesis was induced in selfed progeny of three genetically homozygous and stable fertile doubled haploid lines previously obtained through in vitro gynogenesis. The aims were to acquire information on whether the gynogenic responsiveness of a particular heterozygous cultivar is correlated with higher or lower gynogenic capacity in the second cycle of gynogenesis, to evaluate the non-genetic component of the variability, and to monitor by random amplified polymorphic DNA (RAPD) analysis the onset of eventual gametoclonal variation induced during the gynogenic processes. The results from the gynogenic induction of 9708 flowers showed significant differences among three lines, one of them being extremely responsive, showing a mean yield of 118.3 haploid embryos per 100 cultured flowers (118.3%). The high embryo yield of this line was determined by the line's particular genotype. However, the induction rate of gynogenic embryos obtained within 24 genetically uniform donor plants of this responsive line varied from 67 to 196% and this variation can be attributed to uncontrolled environmental conditions. A total of 157 plants sampled from two lines and obtained through a first (RI) and a second (SCG) cycle of gynogenesis, was analysed by RAPDs. The single band profiles were compared either between or within the RI and SCG plants of each line. A total number of 4600 RAPD fragments were scored and no variation was found in the highly responsive line, while in the less responsive one, minor changes were detected for the presence/absence of two RAPD bands. These results indicate high genetic stability of onion homozygous lines passed through two cycles of gynogenesis.     

Methods for the Selection of Low-Temperature Tolerant Mutants of Chrysanthemum morifolium Ramat. by Using Irradiated Cell Suspension Cultures

In order to improve mutant selection procedures, 5000 plantlets were regenerated in three different harvests (flush A, B and C) from X-irradiated (15 and 20 Gy) and non-irradiated cell suspensions of Chrysanthemum morifolium cv. ‘Parliament’. After screening under suboptimal energy condition (12°C greenhouse temperature, winter low-light), a total of 95 early flowering plants were selected. These plants flowered at least as early as a low-temperature tolerant (LTT) mutant E₂, used as a reference. The percentage of early flowering variants was highest among plants obtained from calli, X-irradiated with 15 Gy and regenerating relatively late (flush B). It appeared that the applied selection method (e.q. harvest) had a large impact on the number of early plants obtained.     

Transfer of cytoplasmic male sterility by spontaneous androgenesis in tobacco (Nicotiana tabacum L.)

Summary Five cytoplasmic male sterile lines of Nicotiana tabacum homozygous for a dominant rootless mutation [Rac^-] were used as female parents in crosses with three male fertile varieties. Androgenetic haploids were selected by their ability to form plantlets with a normal root system, whereas hybrid plantlets failed to grow. High frequencies of androgenetic haploid plants were obtained with N. debneyi cytoplasm, and with one paternal genotype. Chromosome doubling was performed using three different methods (pollination, in vitro culture, acenaphthen) and cytoplasmic male sterile doubled haploid plants have been obtained. The results demonstrate the efficiency of this method for a rapid cytoplasm transfer in Nicotiana tabacum; the integrity of the cytoplasm is conserved. For the first time, we possess a counter selectable marker universally usable for Nicotiana tabacum and closely related species.     

Quantity production of anther-derived haploids from a multiple disease resistant tobacco hybrid. I. Frequency of plants with resistance or susceptibility to tobacco mosaic virus (TMV), potato virus Y (PVY), and root knot (RK)

Summary Methods are described for producing large numbers of haploid plantlets from anthers of a flue-cured tobacco hybrid with monogenic resistance to tobacco mosaic virus, (TMV), potato virus Y (PVY) and root knot (RK), respectively. Additional details are given on colchicine treatment for converting haploids to doubled haploids (DH's) and on the frequency of spontaneous DH's among untreated plantlets. Disparate genetic ratios of TMV-resistant to TMV-susceptible plants were obtained among colchicine-treated haploid plantlets, induced DH's and untreated haploids when compared with F₂ and BC₁ progenies. Haploids (gametes) with the gene for TMV resistance occurred more frequently than expected and plantlets with the gene for RK resistance occurred less frequenctly than expected. Transmission of the gene for PVY resistance differed only slightly from Mendelian expectations. These unexpected ratios, in addition to the frequent occurrence of plastid chimeras among anther-derived plantlets, strengthened our conviction that haploidy is somehow associated with mutation.Joint contribution from the Departments of Genetics, Crop Science and Plant Pathology, North Carolina State University, and the Tobacco Research Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Oxford, North Carolina. Paper No. 5576 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Variation among androgenic and gynogenic doubled haploids of tobacco (Nicotiana Tabacum L.)

Summary Agronomic field performance traits and leaf chemical characteristics of gynogenic doubled haploids (GDH) and androgenic double haploids (ADH) were compared with selfed lines of a burley tobacco cultivar to determine which type of doubled haploids was superior. Three sets of ADH and GDH lines performed similar to their selfed lines, but one set each of ADH and GDH lines had reduced yield, plant height, leaf number, stalk diameter, and leaf length in comparison to the respective selfed lines of KY 17.Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. This paper (No. 87-3-287) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Properties of maternal haploid maize plants and potential application to maize breeding

Summary Presented are the results of a two-year study of haploid maize plants in the field. The haploids were produced with the aid of inducer line ZMS. In total, 604 and 1030 haploids were obtained and studied in the first and second years, respectively. Tassels of haploid plants were found to be almost completley sterile. Fertility of ears was studied by pollinating them with the pollen from diploid inbred lines, the cross resulting in almost all of the haploid ears carrying kernels. On average 27.4 kernels per ear of haploid plant were obtained in the first year of study and 26.3 in the second. These gave rise to normal diploid plants. This property allows genotypes selected at the level of haploid plants to be involved in breeding process. Unusual plants were found among haploids, phenotypically resembling homozygous lines. It was assumed that the plants had resulted from spontaneous chromosome doubling in haploids. The results of comparative studies of progenies of unusual plants and inbred lines derived from the same synthetic population are presented.     

Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

Culture of unpollinated ovules,ovaries, and flower buds in some species of the genus Allium and haploid induction via gynogenesis in onion (Allium cepa L.)

Summary Induction of haploid plants is of great importance for breeding purposes because of the possibility to obtain from haploids homozygous material by artificial chromosome doubling in relatively short times. The present study reports the first evidence of successfull haploid induction in onion. Isolated ovules, ovaries, or whole flower buds of different Allium species were cultured on BDS agar medium. Testa browning in the ovules and an extensive growth of the latter were observed. In cultures of ovaries and flower buds, development of callus and subsequent regeneration of plantlets from the region of the nectaries were observed. In leek, sometimes supernumerary flower organs like ovules were formed in this callus. In onion (Allium cepa L.), plantlets developed from the ovules in all culture methods. Chromosome numbers of these plantlets were counted in root tip squash preparations. They were found to be haploid. Haploid plants were significantly smaller than diploid ones. They were transferred to soil and developed until bulb formation. Because of their importance for breeding, haploid plants obtained by gynogenesis are further stored in vitro.     

Induction of haploids and aneuhaploids in colchicine-induced tetraploid Solanum chacoense Bitt.

Summary The species Solanum chacoense BITT. (2n=2x=24) is a tuber-bearing, self-incompatible species which is important both for breeding and for genetic research. It crosses readily with most other tuber-bearing Solanum species including the common potato S. tuberosum (2n=4x=48). Gametophytic incompatibility hampers research in and utilization of this species. Doubling the chromosome number by colchicine makes it a self-compatible autotetraploid. By crossing selfed progeny of 4x-S. chacoense with a number of haploid-inducing diploid Solanum species a high yield of different dihaploid S. chacoense individuals (2n=24) could be obtained from one originally diploid clone: S. chacoense CPC 1153. More than 160 haploids showing a large variability were identified. The average haploid frequency was 53.7 per 100 berries. Most hybrid plants (70–100%) from four 4x × 2x crosses studied were tetraploid. The frequency of triploid hybrids was low (0–10%). Haploid-inducing capacity of fifteen male parents used in this study varied from 0–141 haploids per 100 berries. Careful examination of 156 haploids revealed 15 viable aneuhaploids (2n=25, 26, and 27), i.e. 9.4%. The potential value of these aneuhaploids is discussed.About 50% of the haploids were sufficiently male fertile to use them in crossing. A few of them set berries after selfing. Five aneuhaploids including the one with 27 chromosomes were successfully crossed as females with a diploid hybrid clone.     

开发实用的染色体加倍体系构建成烟草DH群体

通过对4种染色体加倍方法(烟草浸花药法、浸花培苗法,叶片再生法及浸腋芽法等)的比较研究表明：以4g／L浓度的秋水仙碱浸泡花药法加倍率最高达47．5％～75％,其次为叶片再生法(30％～36．67％)和浸苗法(16．67％～32．35％),而浸腋芽法较低(17．78％)。前两种方法加倍率虽高,但有较高畸形苗比率,叶片再生法工序较繁琐。作者认为烟草加倍单倍体产生,应以采用浸苗法为主。在使用秋水仙碱浸苗加倍时,添加DMSO可明显促进秋水仙碱的加倍效率,且促进作用随时间延长而提高：从高效、快捷和节约等原则考虑,我们开发了烟草有效的染色体加倍体系,以4g／L的秋水仙碱 20g／LDMSO溶液浸苗48h的效果最好。对两个组合烟草单倍体苗浸12h以上,其染色体加倍效率达到对照的2．30-2．93倍。本试验用较低浓度秋水仙碱添加DMSO有利于节约实验费用。通过完善染色体加倍技术程序已构建成2个DH群体,供基因定位研究。     

Somaclonal and chromosomal effects of genotype, ploidy and culture duration in Asparagus officinalis L.

Chromosome and morphological variations of embryogenic calli-derived plants of gynogenic haploid, diploid, triploid and tetraploid asparagus (Asparagus officinalis L.) were investigated. Artificial tetraploids were produced using colchicine treatment of seeds of diploid cultivar, ‘Poultom’. ‘Haidel’ (2X) was crossed with the artificial tetraploids, from which one gynogenic haploid, one diploid, 6 triploid, 3 mixoploids were obtained. Embryogenic calli were first obtained from crown buds, subsequently induced to form somatic embryos, and after 30 days, induced to germinate. Chromosome variation in embryogenic calli-derived plants increased with increasing duration of subculture, particularly when low ploidy levels of plants such as haploid and diploid were used as explants. Approximately 80% of haploid-derived plants showed morphological variations such as dwarfness and abnormal morphological characteristics, although no differences were observed in cladodes and stem characteristics between other polyploid-derived plants and their parents. The data presented here would supply important fundamental information for commercial mass-propagation using somatic embryogenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple

Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established.     

Main Factors Affecting Cucumber (Cucumis sativus L.) Haploid Embryo Development and Haploid Plant Characteristics

This report gives the first detailed analysis of cucumber haploid plants and the first evaluation of genotype and season effects on haploid embryo development. Female flowers of four F1 varieties and two inbred lines were pollinated with 300 Gy-treated pollen and three-week old embryos were cultured in vitro on E20A medium. The variety ‘Polan’ produced the greatest number of embryos (1.34 per 100 seeds), 51 % of which were capable of further development. The number of embryos able to develop was four times higher in summer than in spring. All the plants obtained, except one, were haploids or aneuhaploids. Haploid plants grew slowly and were sterile, although they occasionally set seedless fruits. The length of the stems, leaves and guard cells was smaller than those of diploids.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

The use of anther culture in breeding winter wheat. II. Production of new doubled haploid lines of winter wheat with 1AL—1RS-translocation A total of 4472 anhers was cultured from 8 F¹ populations of winter wheat with the 1AL—1RS wheat-rye translocation cultivar ‘Amigo’ as one of the crossing arents. When averaged over all populations a frequency of embruoid formation of 10% and of regeneration efficiency of green plants of 1% were observed. In addition to the 45 green regenerated plants, 93 albinos were obtained. 44% of the green plantlets had 21 chromosome in root tips and 29% were spontaneous diploids. Multiple peroxidases were used a biochemical markers in the subsequent characterization of the homogeneous breeding material. The electrophoretic patterns showed that 16 doubled haploids without rye chromosome segments were produced. In addition the features of dh₀- plants showed, that several of the new 1AL—1RS-translocation lines were awnless.     

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.     

Anther Culture Derived Homozygous Lines in Hordeum bulbosum

The production of homozygous doubled haploids via anther culture was investigated in the self-incompatible diploid species Hordeum bulbosum. Anthers from three accessions (GBC77, GBC752 and GBC753) were cultured on FHG media using IAA or three levels (1, 50 and 100 mg/l) of the auxin phenylacetic acid (PAA). Four green plants and 63 albino plants were obtained from a total of 1620 plated anthers (540/accession). The best mean anther response, number of embryogenic calli and regenerated plants were obtained with 50 mg/1 PAA. Three of the four green plants survived to maturity and, based on root-tip squashes stained with feulgen, all had 14 chromosomes like the anther donor parent. These anther culture-derived plants contained only some of the parental DNA bands, as observed by PCR analysis, indicating that they are of gametic origin rather than arising from somatic parental tissue. This is the first report of homozygous lines produced from this self-incompatible species.