Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.     

The production of homozygous diploid plants of Solanum verrucosum by tissue culture techniques

Summary Monohaploid plants of S. verrucosum (2n=x=12) were induced in anther culture. Axillary buds from these plants were treated with colchicine in shoot tip culture for 48 hours and then transferred to a colchicine free medium. The resulting plantlets were scored for diploidy by stomatal chloroplast counts and root tip cytology and it was found that doubling of the chromosome number had occurred.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

Androgenesis in a Lolium multiflorum × Festuca arundinacea hybrid to generate genotypic variation for drought resistance

The pentaploid hybrid of Lolium multiflorum and Festuca arundinacea (2n = 5x = 35) combines the high growth rate of L. multiforum with the drought resistance and freezing-tolerance of F. arundinacea. Unfortunately, it also displays the deleterious traits associated with Festuca, namely those associated with high leaf fibre content giving rise to poor palatability and digestibility. To access different combinations of these characters, anther cultures were initiated and regenerated into single embryo derived plants. The anther culture method was very productive since out of a total of 2349 androgenic plants derived from the same parent plant, 57% were green plantlets, although only 507 (22%) subsequently established into plants following transfer to soil. Chromosome counts of randomly selected lines showed that plants with euploid chromosome numbers (14, 21, and 28) would appear to have selective advantage during regeneration. There was wide variation between mature androgenic plants grown under field conditions in plant height, leaf length, leaf width, tiller number and herbage dry matter. The variation between genotypes in response to drought stress was assessed by placing replicate clones under rain-out shelters or under irrigated control conditions in the field. Herbage dry matter under drought was higher in a number of androgenic lines than either parents, but not higher than the pentaploid hybrid. Androgenesis was shown to be a highly effective procedure to expose diverse phenotypic variation all derived from the same Lolium × Festuca hybrid genotype. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient application of in vitro anther culture for different European winter wheat (Triticum aestivum L.) breeding programmes

The efficiency of our anther culture protocol was tested with high‐ and low‐responding genotypes, ‘Svilena’ and ‘Berengar’, and 93 F₁ winter wheat crosses in 2010 and 2011. Based on data for these genotypes, the effect of genotype influenced the number of embryo‐like structures, regenerated plantlets and green plantlets, while the number of albino plantlets was affected by genotype, year and environmental factors. Although genotype also influenced the production of green plantlets from breeding crosses, with green plantlets per 100 anthers ranging from 0.04 to 28.67, the average regeneration rate over all crosses was 5.3 green plantlets/100 anthers, which resulted in a total of 11 416 well‐rooted green plantlets. The survival rate of green plantlets following acclimatization was 97.21% in 2010 and 96.34% in 2011. In this study, the phenomenon of albinism and genotype dependency did not hinder the production of more than five thousand green plantlets each year. In our experiments, anther culture proved to be an efficient method in winter wheat breeding programmes with lower costs than alternative technologies.     

Experiments on anther culture in barley. Influence of culture methods on cell proliferation and organ differentiation

Summary Several experiments have been performed in order to induce cell proliferation and plant differentiation from pollen grains by anther culture in barley. Some modifications of the culture media, pretreatments and transfer of the anthers increased notably the frequency of cell division, but the low proportion of normal green plantlets differentiating from microspores and calluses remains the major obstacle preventing the practical use of anther culture in barley breeding.     

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。     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Chromosome numbers of Saintpaulia ionantha plantlets regenerated from leaves cultured in vitro with caffeine and colchicine

Summary Parts of Saintpaulia ionantha leaves were cultured on a medium supplemented with different concentrations of colchicine or caffeine. The explants were transferred to a control medium without these chemicals after different periods of time. The chromosome number of the regenerated plantlets was counted. In all the experiments, polyploid plantlets were found, but the percentage of them was higher with colchicine. There were no differences in frequency of polyploid plantlets among treatments with the same chemical. In both cases a high percentage of cytochimeric plantlets was observed and possible explanations of their occurrence are given.     

Induction of microspore embryos in a CMS line of Brassica juncea and formation of the androgenic plantlets

Anther culture of two wide hybrids (Diplotaxis erucoides × Brassica campestris) × B. juncea and (D. berthautii × B. campestris) × B. juncea, their CMS lines and the parent species elicited a range of responses highlighting the importance of the genotype. Androgenesis was expressed in cultured anthers of CMS (D. erucoides) B. juncea (22.8%), in restored pollen fertile plants of this CMS line (1.66%), and in the parent, B. juncea cv Pusa Bold (13.02%). AgNO₃ was essential for androgenic response in the CMS lines, and it markedly increased the frequency of androgenesis in the cultivated species. Multiple crops of microspore embryos were obtained from responsive anthers of CMS plants in anther recultures. As high as92% microspore embryos of the CMS line germinated on basal B₅medium and formed normal plantlets. This revised version was published online in July 2006 with corrections to the Cover Date.     

Haploidy breeding and mutagenesis for drought tolerance in wheat

Several intraspecific crosses between known drought tolerant wheat varieties and stable high yielding recombinants were made with the objective to develop improved cultivars for the moisture stressed rainfed areas of Pakistan. Five of these crosses were selected for further creation of useful mutations through the application of low doses of gamma rays and development of doubled haploids through anther culture. Anther culture response of the selected irradiated F₁ generations was studied on liquid and solid induction media. The highest number of calli among almost all crosses was produced on Potato-2induction medium. All the crosses varied greatly in response to callus induction and maximum calli (75%) were obtained from Lyl-73/vee’s’ cross. Similarly, genotypic differences were found for green vs. albino regenerants. The highest number of green plantlets (12.1%) was recorded for Lu-26/3062. From the developed doubled haploid population 25 DH-mutants were initially selected and nine lines were finally included in multi-locational field tests. Two DH-mutants (i.e. DHML-50 and DHML-9) have potential for better grain yield, earliness, disease resistance and moisture stress tolerance. This revised version was published online in August 2006 with corrections to the Cover Date.     

Risk assessment of outcrossing of transgenic rapessed to related species:

Summary The risk for a gene dispersal is reported for reciprocal crosses between a transgenic rapeseed variety resistant to the herbicide phosphinotricin and five related species. The first stages after pollination were cytologically observed and fertilized ovaries were established in in vitro culture for the production of interspecific hybrids. A similar classification was observed for the index of pollination compatibility and embryo yield. From the 243 embryos produced, 109 plantlets were obtained in a greenhouse. All the interspecific combinations tested were able to produce hybrid plants. A higher number of hybrids was obtained when rapeseed was used as the female parent. The hybrids had the expected triploid structure except for two amphidiploid, B. napus × B. oleracea, and one amphidiploid, B. napus × S. arvensis, plants with 56 chromosomes. The triploid hybrids were sterile or partially fertile but two of the amphidiploid plants, B. napus × B. oleracea, were fully fertile. The cytoplasm source did not seem to affect the fertility of the hybrids.     

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.     

Pollen mother cell and anther wall development in a photoperiod-insensitive male-sterile mutant of pigeon pea (Cajanus cajan (L.) Millsp.)

Summary The development of the pollen mother cells (PMC's) and anther wall of a male-sterile mutant was microscopically compared with that of fertile sib plants in pigeon pea (Cajanus cajan). Male-sterility was complete and caused by breakdown of microsporogenesis at prophase I. Delayed and incomplete development of the anther wall appeared to be responsible for PMC degeneration. The sterile mutant also differed from the fertile plants in the size and number of the PMC's.On leave from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

不同世代太谷核不育小麦对花药成株的影响

通过对太谷核不育小麦杂交一代可育株和不育株,及二、三、四代可育株花药的离体培养,结果表明:1.不育株花药培养能够获得植株再生;2.杂交一代的可育株花药出愈率最高,绿苗分化率亦最高;3.基因型对花药出愈率和绿苗分化率有明显的影响.     

Plant Regeneration Through Anther Culture of Three Wild Species of Hordeum (H. murinum, H. marinum and H. bulbosum)

Plant regeneration was achieved through anther culture of three wild species of Hordeum (H. murinum, H. marinum and H, bulbosum). Calli or embryoids were formed from microspores in anthers cultured on a medium containing 6-benzylammopurine (BAP) and ficoll. These calli or embryoids regenerated green or albino shoots and roots after transfer to regeneration media. Green plantlets which developed on regeneration media were transferred to soil where they showed further growth.     

Production and genetic characterization of the co-isogenic lines of a common wheat Triticum aestivum CV. S-615 for ten major genes

Summary Co-isogenic lines of a common wheat, Triticum aestivum, cv. S-615 with each of the following ten major genes were produced by repeated backcrosses; The gene C on 2D chromosome for the compactum character, s on 3D for the sphaerococcum character, Hd on 4B, B1 on 5A, and B2 on 6B for awn suppression, Hg on 1A for glume hairiness, Hp on 4A for peduncle hairiness, Ne1 on 5B and Ne2 on 2B for hybrid necrosis, and v1 on 3B for virescence. Seven of them showed the typical mendelian fashion of inheritance, while three others (C, s and v1) were transmitted at lower frequencies than their corresponding normal alleles.The effects of those major genes on 24 characters of cv. S-615 were investigated, which are summarized as follows: C: Increased node diameter, number of spikelets per ear and spike density, but decreased lengths of all ear rachis, awn, anther, empty and outer glumes, and grain, and grain index. s: Increased culm diameter and thickness, and spike density, reduced lengths of all 1st and 2nd internodes, culm, flag leaf, rachis, awn, anther, empty and outer glumes, and grain, and grain index, and accelerated heading. Hd: Increased number of spikelets per ear and anther length, while decreased length of awn, and empty and outer glumes. B1: Increased 1st internode, rachis, and anther lengths, and grain index, but decreased spike density, awn length and grain thickness. B2: Increased rachis length, number of spikelets per ear, empty and outer glume lengths and grain index, but reduced awn length and grain thickness. Hg: No effects. Hp: Increased number of spikelets per ear. Ne1: Accelerated heading, and increased empty glume length. Ne2: Increased node and culm diameters. v1: No effects.Two species-specific genes, C and s, affected a large number of characters expressed in various developmental stages. These genes were found to have pleiotropic effects, namely, they influenced, at least, two groups of interdependent characters. Three awn suppressors influenced a limited number of characters, but no evidence of their pleiotropic effects was obtained.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 422. The work was supported in part by a Grant-in-Aid (No. 148,004) from the Ministry of Education, Japan.     

Inhibitory effect of streptomycin on rice tissue cultures and the selection of resistant callus clones

Summary Callus tissues were initiated from seed, radicle and anther cultures of rice (Oryza sativa L.) in order to study the effect of streptomycin on callus growth. Our results showed that the addition of 250 g/ml or more streptomycin to the culture medium caused a significant inhibition of callus proliferation. The degree of inhibition depended upon the genotype, the drug concentration and the tissue source of callus. Selection of resistant cell lines began with seed and immature embryo cultures grown on various levels of streptomycin. The fastest growing sectors of callus were subcultured for additional selection. In this way, a total of 11 comparatively vigorous callus clones were isolated after 7 or 12 subcultures. Some of these clones exhibited a significant increase of resistance index when compared with unselected starting material. After 5 or 6 selection cycles, 79 plantlets were regenerated from resistant callus, but none grew to maturity because all were white (albino).     

Interspecific hybrids from cross incompatible relatives of sweetpotato

Summary Hybrids were obtained from Ipomoea interspecific crosses through ovule culture. The hybridity of the progeny obtained from I. triloba × IitI. trifida and (I. triloba × I. lacunosa) × I. batatas (4x) crosses was established by comparisons of floral morphology and analyses of peroxidase and esterase isozymes. The hybrids displayed the inflorescence type and sepal shape and texture of their male parents, while corolla size and anther and nectary color tended to be intermediate to their parents. The isozyme banding patterns of the hybrids contained bands present in the patterns of each of their parents. Pollen grain viability, measured by aceto-carmine stainability, was 44.1%, 92.3% and 82.4%, respectively, for the I. triloba × I. trifida hybrid and the (I. triloba × I. lacunosa) × I. batatas (4x) hybrids, H₁ and H₂. A controlled pollination study revealed that the I. triloba × I. trifida, and the (I. triloba × I. lacunosa) × I. batatas (4x) hybrids, H₁ and H₂ were partially self fertile with 6%, 70% and 13%, respectively, of the pollinated flowers producing viable seed. Success in backcrossing and sib-mating varied with the cross combination.