Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars

Different pretreatments were given to anthers of barley before culturing, and their effects assessed on the frequency of embryos and green doubled haploid plants produced. Mannitol pretreatment was better than cold pretreatment for some low responding cultivars. Optimal concentration of mannitol for pretreatment depended on cultivar. Low responding genotypes needed a higher concentration of mannitol than responsive ones. The addition of Ficoll to liquid medium increased the number of embryos and green plants. The influence of the growth regulators 2,4-D and TIBA was assayed using ten cultivars of barley grown in Spain. The anti-auxin TIBA gave good embryo production with some of the low responding cultivars. Two row-type cultivars always produced higher number of embryos and green plantlets than six row-type. The application of these modifications to 10 F₁ hybrids with potential agronomic value, allowed the production of almost 1000 doubled haploid plants from only 3500 anthers. Up to two doubled haploid plants per flower were produced from the cross Monlon × Sonja. This revised version was published online in June 2006 with corrections to the Cover Date.     

Improved embryogenesis from anther culture and plant regeneration in timothy

Timothy (Phleum pratense L.) is an important forage grass grown in northern temperate areas. Development of haploid cell culture techniques for timothy has been limited due to the recalcitrance of timothy in tissue culture. In this study, timothy anther culture techniques were established. Liquid PG-96 (Pulli and Guo, 1996) induction medium significantly promoted embryo yield; the best result was 800–1000 embryos (calli) per 100 anthers. Genotype was an important factor in androgenetic embryogenesis of timothy. Embryos were obtained from 16 genotypes out of the 28 genotypes tested. The optimum stage for microspore development was between the very late uninucleate stage and the binucleate stage. Cold pretreatment applied to the donor plants (spikes) increased embryo yield. Despite a high embryo induction rate, green plant regeneration rate was relatively low. The frequency of albinos was reduced by use of low light intensity conditions during regeneration. Over 300 green plants were recovered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anther Culture in Nicotiana tabacum: The Role of the Culture Vessel Atmosphere in Pollen Embryo Induction and Growth

A comparison of four volumes of culture vessel atmosphere revealedthat this variable greatly influenced the induction and growthof pollen embryos from anthers of Nicotiana tabacum. The optimumfrequency of anthers producing embryos and plantlets was foundwith a culture atmosphere of 15 ml per anther, whereas the optimumnumber of plantlets was found with 5.5 ml per anther. A smallvolume (0.5 ml per anther) almost completely suppressed embryoinduction. Removal of specific components of the culture atmosphere(ethylene, carbon dioxide, oxygen) influenced the response ofthe anthers but did not produce a satisfactory explanation ofthe inhibition of pollen embryogenesis by the small cultureatmosphere volume. In particular, the influence of ethyleneabsorption on embryo induction and growth depended both on theculture atmosphere volume and on the stage of development ofthe pollen at the start of culture. Using anthers containingpollen at a stage after the first pollen grain mitosis. ethyleneabsorption was found to increase the survival of induced embryos.Treatment of anthers for 3 d with silver nitrate, a known antagonistof ethylene action, was not an efficient means of increasingthe yield of pollen plantlets.     

Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Effect of plant growth conditions, plating density, and genotype on the anther culture response of soft white spring wheat hybrids

Ten soft white spring wheat (Triticum aestivum L.) F₁ hybrids were grown under three temperature regimes, and anthers were cultured at two plating densities to investigate the effect of plant growth conditions, plating density, and genotype on embryo induction and plant regeneration. Anthers from plants grown at high temperature (25 °/18 °C) or from plants transferred from low (15 °/12 °C) to high temperature generally produced more embryos and green shoots, with a lower frequency of albinos, than did anthers from plants grown at low temperature. However, plating densities of 10 versus 20 anthers per milliliter, had little effect on anther response. Four of the five hybrids with `Fielder' as the female parent produced more embryos and green shoots than did hybrids with `AC Reed' as the female parent. Received: 12 July 1996 / Revision received: 1 April 1997 / Accepted: 30 April 1997     

Genotype,plant, bud size and media factors affecting anther culture of cauliflowers (Brassica oleracea var. botrytis)

Summary Eleven F₁ hybrid cultivars of cauliflower, representing a range of maturity types, were examined for their responsiveness to anther culture. Embryos were produced from each of the cultivars tested, and the mean embryo yield varied from 82.2 embryos per 100 anthers cultured for cv Dova to 0.6 embryos for cv Serrano. Variation between genotypes and between plants within a genotype was significant, both in terms of embryo yield and percentage responsive anthers. Autumn and winter maturing cauliflowers were generally more responsive than summer types. Embryo yields were enhanced by culturing anthers on solid rather than on liquid media. An increase in concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D) from 0.1 to 0.3 mg/l also increased embryo yield. Embryo yield was doubled when anthers were cultured on solid media containing 0.3 mg/l 2,4-D compared to liquid media containing 0.1 mg/l 2,4-D. Although bud size alone did not have a significant effect on embryo production, genotype x bud size and plant x bud size (within genotype) interactions were significant. Estimation of the variance components demonstrated that, apart from the residual plate-to-plate variation, variation between plants was the largest source of variation, accounting for approximately 30% of total variance. Plant x bud size (within genotype) interaction accounted for 18% of total variance and genotypic differences for approximately 8%.     

Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture

Summary Inheritance of the ability to respond in wheat anther culture was studied from 6×2 reciprocal crosses between six varieties with high and two varieties with low capacity for green plant formation and their parents, replicated in two environments. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 78.4% and 85.4% of total variation, respectively, while smaller genetic effects were indicated for regeneration. Nuclear genes could explain almost all the genotype effects in this material. Embryo formation showed heterosis over high parent for 5 of the 12 hybrids, while percentages of green plants from the hybrids were intermediate to the parents. General Combining Ability (GCA) could explain 78.8% of the variation for embryo formation among the hybrids, whereas differences in percentage of green plants were dominated by Specific Combining Ability (SCA), accounting for 67.9% of hybrid variation. A positive correlation (r=0.81^**) was observed between the genetic capacity for regeneration and green plant formation. Analysis of covariance indicated that effects causing GCA for green plant formation were mainly responsible for this correlation. A regression model with two parallel lines divided the six parent lines with high green plant formation into three groups with respect to their reactions with the two testers. The results are discussed with regard to possible involvement of two sets of nuclear genes affecting the percentage of green plants obtained in wheat anther culture: one set consisting of mainly additive effects affecting green plant percentage through an initial effect on regeneration ability, and another set of two or a few more major genes with dominance or epistatic effects uncorrelated with regeneration.     

Enhancement of embryogenesis and plant regeneration from barley anther culture by low doses of gamma irradiation

Summary The effects of 0,5 and 10 Gy doses of gamma irradiation on the enhancement of embryogenesis and plant regeneration efficiency of three barley (Hordeum vulgare L.) genotypes, Igri, Arabi Abiad and AECS 76, were evaluated. Embryo yields at 5 and 10 Gy doses were significantly higher than those of the control (OGy). This effect was genotype-dependent. The most responsive genotype was Igri, with 592.8 embryos 32 anthers exposed to 10 Gy. However, despite a high embryo induction rate, the green plant regeneration rate was low. Arbi Abiad had a higher ability to generate green plants produced from, with 28. 13 plantlets obtained from 32 anthers at 10 Gy; irradiation had no significant effect on regeneration of Igri and AECS 76 genotypes. In general, the 10 Gy dose produced a much higher embryo yield than the 5 Gy dose. The root-tip chromosome number and the fertility of 298 regenerating green plants of cv. Igri revealed that 64% of the tested plants were spontaneously doubled haploids (DHs) and fertile.     

Comparison of media for their aptitude in wheat anther culture

Different media were evaluated with anthers of five spring wheat (Triticum aestivum L.) genotypes for their ability to produce embryos and green plants in anther culture. Our first experiment showed that the addition of a combination of 19 amino acids significantly increased the number of embryos and green plants obtained. The mean number of green plants per 100 anthers for the two genotypes in this experiment, HY320 and B723, went from 28.2 without amino acids in the medium, to 46.7 with addition of amino acids. Our second experiment with the genotypes HY320, Wim and Laval-19 showed that liquid medium with Ficoll is more efficient for anther culture (9.9 green plants/100 anthers) than solid (0 green plants), gelationous media (2.5 green plants/100 anthers) or liquid medium with Membrane Rafts (0 green plants; Hoechst Celanese Corp.). Our third experiment revealed that the effect of replacement of sucrose by maltose varied with the genotype of the donor plant. Maltose partially inhibited the androgenesis of three responsive genotypes, HY320, Wim and Reliance (40.3 green plants/100 anthers instead of 43.9 with sucrose), while maltose significantly increased the androgenesis of the recalcitrant genotype Laval-19 (10.8 green plants/100 anthers instead of 5.4 with sucrose). An amino acid x maltose interaction was also observed. Amino acids without maltose increased androgenesis, but the addition of maltose to the amino acid-enriched medium eliminated this positive effect of the amino acids.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

抗除草剂杂交籼稻亲本的配合力分析

本试验以6份新育成的抗除草剂籼型恢复系为父本,5份生产上广泛应用的不育系为母本,采用不完全双列杂交设计配制了30份杂交组合,对其苗期除草剂抗性和主要农艺性状配合力进行了分析。除草剂抗性鉴定表明,亲本恢复系及三系杂交组合抗性接近完全,两系杂交组合抗性达90%以上。配合力分析表明,不育系除单株有效穗数外其他农艺性状的一般配合力均达到极显著差异;恢复系间一般配合力在所有性状中均达到显著或极显著差异;杂交组合间特殊配合力方差仅在单株产量、结实率、播始历期和千粒重4个性状中达到显著或极显著差异。不育系中,金科1A在单株产量、结实率等7个性状上的一般配合力均为最高,但其特殊配合力方差最小;广占63-4S在千粒重性状上具有最高的一般配合力、最大的特殊配合力方差,在播始历期上具有最高的一般配合力负效应;C815S在株高上的一般配合力负效应最大,同时特殊配合力方差较高。恢复系中,华抗恢101在单株有效穗数上具有最高的一般配合力和特殊配合力方差;华抗恢104在穗长上具有最高的一般配合力,在播始历期上具有最高的一般配合力负效应;华抗恢105在单株产量、结实率等性状上具有最高的一般配合力,在株高上具有最高的一般配合力负效应;华抗恢106在千粒重性状上具有最高的一般配合力和特殊配合力效应方差。利用抗除草剂恢复系配制杂交组合,不仅可以改良其除草剂抗性,也可以通过广泛测配,选择一般配合力强、特殊配合力方差大的亲本配组育成强优势组合。     

A study of factors affecting embryo yields from anther culture of cabbage

In cabbage (Brassica oleracea var. capitata), a thermal shock treatment of 24 h at 35 °C at the start of the culture period resulted in higher embryos per 100 anthers (30.0) compared to a treatment of 48 h. Similarly , a chilling treatment of 24 h at 4 °C resulted in a higher embryo yield (6.0) per 100 anthers compared to a treatment of 48 h. However, the embryo yields were significantly higher (p> 0.01) in thermal shock than chilling treatments in all experiments. Treatments of 6 days at either 35 °C or 4 °C gave no embryos. The most responsive cultivar was the F₁ hybrid , Hercules, in all experiments. Although anther culture was successful in the other genotypes, the open pollinated ones, the highest number of embryo yields per 100 anthers was obtained in the hybrid. High temperature treatment before culture had a beneficial effect on the embryo yields. The responsiveness of anthers to addition of increasing concentration of silver nitrate (AgN0₃) (the ethylene inhibitor) to the culture medium, showed a progressive increase in the embryo yields in all the genotypes. Since embryos were also formed in the absence of silver nitrate, probably, due to a greater genotype × medium interaction, it is noted that the presence of silver nitrate in the medium may not be essential for cabbage anther culture as reported earlier. The findings of this study may be recommended for large production of cabbage embryos in culture.     

Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture

Summary The genetics behind response in barley anther culture was studied with 22 reciprocal and one single: cross between three varieties with high and four varieties with low capacity for green plant formation. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 62 and 76% of total variation, respectively, with almost no genetic effect on the ability to regenerate plants from pollen embryos. Nuclear genes could explain all genotype effects in this plant material, since no reciprocal effects were indicated. The three parents with high and the four parents with low capacity for green plant formation formed two phenotypically homogeneous groups, producing 27–52% and 0–7% green plants, respectively. Genetic variation within hybrids for both embryo and green plant formation could be explained completely by general combining ability (GCA). The results are discussed with respect to a previous similar study in hexaploid wheat and the reported existence of DNA deletions in the plastid genomes in albino plants from anther culture of wheat and barley.     

Yield and yield components in flue-cured tobacco and their genetic analysis

Five cultivars and the half diallel set of 10 F₁ hybrids of flue-cured tobacco (Nicotiana tabacum L.) were grown in two seasons. Highly significant differences were assessed between genotypes as concerns flowering time, plant height, number of leaves, leaf length and width and yield per plot. High to moderate values for heritability in the broad sense were obtained in all cases. Hybrids, in general, flowered earlier, were taller, had fewer but shorter and wider leaves and slightly increased yield when compared with the mean value of all parents. The variance associated with general combining ability (GCA) was highly significant in all characters. The estimates of SCA were significant in most cases. High GCA/SCA ratios which largely exceeded the unity were obtained for most attributes. The negative and positive alleles were unequally distributed in the parents for all the studied traits. A small number of effective genes was obtained for all characters except plant height, where one to two groups of genes were distinguished     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Reciprocal effects in anther cultures of wheat hybrids

This study was conducted to determine the reciprocal effects for anther culture response in wheat (Triticum aestivum L.) using a set of 4 × 4 full diallel crosses. Both reciprocal and nuclear genetic effects were highly significant for anther culture response and useful for selection and breeding purposes. General combining ability (GCA) effects were predominant for all investigated anther culture traits. Also, significant differences for specific combining ability (SCA) effects were detected between reciprocal crosses. Although significant reciprocal differences for responding anther, callus number and green plant regeneration were recorded in some reciprocal crosses, there were no significant reciprocal differences for albino plant regeneration. The use of one parent as male or female could lead to change at the production of green plants from the F₁ hybrids and screening of inbred lines for response to anther culture, without reciprocal effects, could decrease the utilization of breeding material.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.     

Isolated microspore culture of Canadian 6× triticale cultivars

Isolated microspore culture was conducted on nine Canadian triticale cultivars (X Triticosecale Wittmack) using two induction media developed for wheat, with or without 100 g l⁻¹ Ficoll. Significant interactions were observed for the number of embryos and calluses induced, green and albino plantlets regenerated and fertility of green plants. Ficoll was beneficial in both media to increase numbers of embryos and green plants for all cultivars. Overall, medium NPB99 supplemented with ficoll provided the most suitable condition for most cultivars. AC Alta performed slightly better on CHB3 supplemented with Ficoll. Only cv. Wapiti was not amenable to androgenesis. The cultivars AC Certa, AC Copia, AC Alta, Sandro, Ultima, Frank, Pronghorn and Banjo produced respectively, 10, 9, 6, 5, 4, 3, 3 and 1 green plants per Petri dish (35,000 microspores), on their optimum treatment. Twenty-two percent of total lines produced were fertile, and considered doubled haploids. The application of isolated microspore culture to triticale, opens new possibilities in breeding triticale, for the utilization of in vitro selection and genetic engineering.