Improvement of anther cultures conditions using the Taguchi method in three cereal crops

Background: Plant tissue cultures have the potential to reprogram the development of microspores from normal gametophytic to sporophytic pathway resulting in the formation of androgenic embryos. The efficiency of this process depends on the genotype, media composition and external conditions. However, this process frequently results in the regeneration of albino instead of green plants. Successful regeneration of green plants is affected by the concentration of copper sulfate (CuSO4) and silver nitrate (AgNO3) and the length of induction step. In this study, we aimed at concurrent optimization of these three factors in barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and triticale (x Triticosecale spp. Wittmack ex A. Camus 1927) using the Taguchi method. We evaluated uniform donor plants under varying experimental conditions of in vitro anther culture using the Taguchi approach, and verified the optimized conditions. Results: Optimization of the regeneration conditions resulted in an increase in the number of green regenerants compared with the control. Statistic Taguchi method for optimization of the in vitro tissue culture plant regeneration via anther cultures allowed reduction of the number of experimental designs from 27 needed if full factorial analysis is used to 9. With the increase in the number of green regenerants, the number of spontaneous doubled haploids decreased. Moreover, in barley and triticale, the number of albino regenerants was reduced. Conclusion: The statistic Taguchi approach could be successfully used for various factors (here components of induction media, time of incubation on induction media) at a one time, that may impact on cereals anther cultures to improve the regeneration efficiency

Exploring factors affecting anther culture in rice (Oryza sativa L.)

Anther culture is an important in vitro culture technique for the production of double haploids. It is largely speciesand genotype specific. Asian-cultivated rice (Oryza sativa, ssp. indica) is recalcitrant to anther culture which limits itspractical application in rice breeding. Several researchers tried to optimize the medium recipes and culture techniquesfor callus induction and plantlet regeneration. Negligible response to callus induction and recovery of a higherfrequency of albino plants are the major hindrance for the use of the technique in crop improvement. Shortening theculture period and sexual hybridization of rice subgroups (japonica/indica) may be adopted to improve green plantregeneration from anther-derived callus in indica rice. However, genetic transformation technology using individualgenes related to different aspects of anther culture could be a more direct approach. Besides, the role of genotypes,physiological status of donor plant, developmental stage of pollen, pre-treatment, culture media, phytohormones, andculture conditions for successful anther culture have been discussed.

In vitro pollen germination and anther culture of Senna obtusifolia / 中草药

Objective: To observe the morphology, measure the activity of pollen of Senna obtusifolia, determine the optimum conditions for pollen germination, induce the formation of callus from its anthers as well as identify its ploidy levels, and thus to lay a basis for its haploid breeding. Methods: Microscopic observation of the anther squashed method was used to reveal the morphology of pollen; The pollen activity was investigated using I2-KI staining and the size of the flower buds with the highest pollen activity was determined; Liquid culture and microscopic observation were performed to determine the optimum culture medium, pH value, and temperature for pollen germination; Microscopic observation of the acetocarmine-stained pollen was performed to determine microspore development stages; The optimum conditions for callus formation were studied by in vitro anther culture; The phenol fuchsin staining was improved for the identification of callus ploidy levels. Results: Pollen of S. obtusifolia is ellipsoid with three germinal furrows, and mostly two germination holes showed germination; The highest pollen activity was found in buds with vertical diameters about 1.0-1.1 cm; The optimum culture medium for pollen germination was determined to be 10% sucrose + 1% boric acid, with maximum germination cultured for 1-3 h at 25 ℃; The uni-nucleate microspore stage was in buds with vertical diameters about 0.9-1.1 cm; Calluses were successfully obtained by culturing the uni-nucleate stage anthers in an inducing medium MS + 1.0 mg/L 6-BA + 2.0 mg/L NAA + 3% sucrose + 0.6% agar and callus enrichment culture was done in MS + 1.0 mg/L 6-BA + 1.0 mg/L IAA + 1.0 mg/L 2,4-D + 1.0 mg/LGA3 + 3% sucrose; There was ploidy separation in callus, with the haploid and diploid cells coexisted. Conclusion: The optimum culture medium for pollen germination is 10% sucrose + 1% boric acid; The optimum anthers for in vitro culture is within flower buds with a vertical diameter of 0.9-1.1 cm; The chimeric calluses obtained from anthers of S. obtusifolia lay a solid foundation for the further induction of haploid plants from its pollen for breeding.

Androgenesis in chickpea: Anther culture and expressed sequence tags derived annotation.

Double haploid technique is not routinely used in legume breeding programs, though recent publications report haploid plants via anther culture in chickpea (Cicer arietinum L.). The focus of this study was to develop an efficient and reproducible protocol for the production of double haploids with the application of multiple stress pre-treatments such as centrifugation and osmotic shock for genotypes of interest in chickpea for their direct use in breeding programs. Four genotypes, ICC 4958, WR315, ICCV 95423 and Arearti were tested for anther culture experiments. The yield was shown to be consistent with 3-5 nucleate microspores and 2-7 celled structures with no further growth. To gain a further insight into the molecular mechanism underlying the switch from microsporogenesis to androgenesis, bioinformatics tools were employed. The challenges on the roles of such genes were reviewed while an attempt was made to find putative candidates for androgenesis using Expressed Sequenced Tags (EST) and interolog based protein interaction analyses.

Nuevos genotipos de arroz resistentes a la Piriculariosis obtenidos por cultivo de anteras / New blast resistant rice genotypes obtained by and anthers culture

En la Estación Experimental del Arroz Los Palacios, perteneciente al Instituto Nacional de Ciencias Agrícolas de Cuba (INCA), se efectuaron cruzamientos entre cuatro cultivares resistentes a la Piriculariosis y seis de buen comportamiento agronómico y las anteras de las plantas F2 fueron cultivadas in vitro para evaluar la formación de callos en tres medios líquidos: N6-1, N6-m y NL, así como la regeneración de plantas verdes y albinas, en el medio MS. Las dos primeras generaciones de las nuevas líneas obtenidas fueron evaluadas para caracteres agronómicos y la segunda generación, además, para resistencia frente a la Piriculariosis. Las líneas que combinaron resistencia a la Piriculariosis y buenos caracteres agronómicos fueron evaluadas en condiciones de infección natural, con alta presión del patógeno. La utilización de la técnica del cultivo de anteras mostró alta dependencia del genotipo y el medio de cultivo. Con el medio NL se lograron los valores más altos para la formación de callos. Se obtuvieron nuevos genotipos resistentes a la Piriculariosis y de alto rendimiento agrícola.

Crosses were made between four blast resistant and six rice varieties of good agronomic performance, at the Los Palacios Rice Research Station of the National Agricultural Sciences Institute of Cuba (INCA) and the anthers from F2 plants were in vitro culture using three liquid media: N6-1, N6m, and NL, for callus formation and after plants regenerations using MS medium. The first two generations of breeding lines were evaluated for agronomic characters and the second generations, also, for Blast resistant. The lines that combined resistance to Blast and good agronomic performance were evaluated under high pressure of natural Blast infection conditions. The success rate of anther culture was highly dependent on the genotype and culture media used. NL medium led to the highest callus formation values. In the process, new blast resistant and high yielding genotypes were obtained.

Desenvolvimento de populações duplo-haplóides de cevada cervejeira associadas à atividade das enzimas (1-3, 1-4)-β-glucanases / Development of doubled-haploids populations in malting barley associated to activity of enzymes (1-3, 1-4)-β-glucanases

Populações duplo-haplóides apresentam especial vantagem para análises genéticas, uma vez que a informação que elas oferecem pode ser maximizada, devido ao fato que todos os locos encontram-se em homozigose. O objetivo deste trabalho foi o desenvolvimento de duas populações duplo-haplóides (DHs) de cevada (Hordeum vulgare ssp. vulgare L.) segregantes para a atividade das enzimas (1-3, 1-4)-β-glucanases, através da técnica de cultura de anteras. Foram realizados dois cruzamentos com cultivares contrastantes para esta característica. As cultivares parentais selecionadas foram 'MN 698' e 'CEV 97047', para o desenvolvimento da população "malte verde" (MV), e 'Embrapa 127' e 'CEV 96025' para o desenvolvimento da população "malte seco" (MS). Foram cultivadas 10.734 anteras da população MS e 4.139 anteras da população MV. A população MV produziu 50 por cento mais plântulas verdes quando comparada à população MS, refletindo a importância do genótipo na resposta à cultura de anteras e na regeneração. A maioria das plantas adultas duplo-haplóides foi obtida através da duplicação espontânea in vitro do genoma haplóide, ocorrendo em 66 por cento das plantas da população MS e 76 por cento das plantas da população MV. Também foram observadas, em menor frequência, plantas haplóides, triplóides e tetraplóides. Através da técnica de cultura de anteras, foram desenvolvidas 204 linhagens duplo-haplóides, das quais 72 linhagens são da população "malte seco" e 132 linhagens são da população "malte verde". Este material constitui um importante germoplasma para o melhoramento genético da cevada.

Doubled haploid populations offer special advantages in genetic analyses, since the information they provide may be maximized due to the fact that all loci are homozygous. The aim of this study was to develop two barley (Hordeum vulgare ssp.vulgare L.) doubled-haploid (DHs) populations segregating to (1-3, 1-4)-β-glucanases activity, utilizing the anther culture protocol. Two crosses were performed with contrasting cultivars to (1-3, 1-4)-β-glucanases activity. Parental cultivars used were 'MN 698' and 'CEV 97047' for the development of 'green malt' population (MV) and Embrapa 127 and 'CEV 96025' for the development of "dry malt" population (MS). For the MS and MV populations, 10,734 and 4,139 anthers were cultured, respectively. MV population produced 50 percent more green seedlings as compared to MS population, which reflects the importance of genotype to the culturing of anthers and to regeneration. Most doubled-haploid adult plants were obtained by in vitro spontaneous duplication of the haploid genome, which occurred in 66 percent of plants from the MS population and 76 percent of plants from the MV population. Haploid, triploid and tetraploid individuals were also observed, though at low frequencies. The anther culture protocol afforded to develop 204 double-haploid lineages, of which 72 were generated by the 'dry malt' population and 132 from the "green malt" population. This material should be considered as important germplasm for barley genetic improvement.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense / 中草药·英文版

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D).The highest regeneration rate (34.6%)was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine (BA).The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant (2n = 12)with only one haploid plant(n = 6).Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture orB.chinense.

Morphological markers for microspore developmental stage in maize

The use of maize in anther culture has been limited because only few genotypes presented a high androgenetic potential. Obtaining the proper stage of microspore development at culture initiation is one of the most crucial factors for success in the androgenesis. For Brazilian maize genotypes there are no studies reporting a correlation between cytological features and morphological parameters. In this study, morphological parameters were recorded and associated with cytological specific stages of the the microsporogenesis in two Brazilian maize genotypes that were sowed in different places (field and growing chamber). For both genotypes, the plants of the growing chamber presented a delay in development. Spikelets length and anther length/spikelet length ratio are not good parameters since they can be greatly influenced by the environment. The anther length was the more reliable parameter to correlate with a specific developmental stage. Nevertheless, variations between genotypes and environment were detected.

A utilização do milho (Zea mays) na cultura de anteras é limitada devido ao baixo número de genótipos com alto potencial androgenético. A obtenção de micrósporos no estádio de desenvolvimento apropriado no início da cultura é um dos fatores cruciais para o sucesso do processo androgenético. Em genótipos brasileiros de milho não existem estudos relatando a correlação entre características citológicas e parâmetros morfológicos. Neste estudo, parâmetros morfológicos foram avaliados e associados com estádios específicos da microsporogênese em dois genótipos brasileiros de milho os quais foram semeados em diferentes locais (campo e câmara de crescimento). Para ambos os genótipos, as plantas crescidas na câmara de crescimento apresentaram atraso no desenvolvimento. O comprimento da espigueta e a razão comprimento da antera/comprimento da espigueta não são bons parâmetros uma vez que podem ser muito influenciados pelo ambiente. O comprimento da antera foi o melhor parâmetro para indicar o estádio de desenvolvimento do micrósporo. Todavia, variações entre genótipos e ambiente foram detectadas.

Evaluation of gelling agents on anther culture: response of two soybean cultivars

Anthers of two soybean cultivars were cultured in B5 long basal culture media gelled with agarose or PhytagelTM. Cytological examinations of the anthers were carried out during the first 45 days of culture to assay the viability and developmental stage of microspores. Frequency of callus formation was recorded at 45 days of culture. The analysis of variance of the microspore viability assay showed significant Cultivar X Gelling Agent X Day of Culture interactions. The frequencies of viable microspores decreased significantly with time of culture, within each cultivar and gelling agent tested. The interaction Day X Cultivar was significant for the frequencies of binucleate symmetrical grains and multinucleate/multicellular structures. The effect of gelling agents on the frequency of binucleate symmetrical pollens grains and multinucleate/multicellular structures was not significant. About the frequencies of calli and embryogenic calli formed, a significant difference was detected between the cultivars (IAS5= 14.8 percent and BRS 133=6.6 percent). Gelling agents showed no effect over these frequencies.

Anteras de duas cultivares de soja foram cultivadas em meio de cultura basal B5 longo gelificado com agarose ou Phytagel®. Análises citológicas das anteras foram conduzidas durante os primeiros 45 dias de cultura para avaliar a viabilidade e o estágio de desenvolvimento dos micrósporos. A freqüência de formação de calos foi analisada após 45 dias do início da cultura. A análise da variância da viabilidade do micrósporo mostrou interações significativas de Cultivar X Agente Gelificante X Dias de Cultura. As freqüências de grãos de pólen viáveis diminuíram significativamente com o tempo de cultura, dentro de cada cultivar e agente gelificante testado. A interação Dia X Cultivar foi significante para as freqüências de grãos de pólen binucleados simétricos e estruturas multinucleados/multicelulares. O efeito do agente gelificante na freqüência de grãos de pólen binucleados simétricos e estruturas multinucleados/multicelulares não foi significante. Com relação às freqüências de calos e estruturas embriogênicas formadas, houve diferença significativa entre cultivares (IAS5= 14.8 por cento e BRS 133=6.6 por cento). O agente gelificante não mostrou efeito em tais freqüências.