基因型和胚龄对小麦未成熟胚离体培养反应的影响

本文对34种基因型的小麦未成熟胚在离体培养中的反应进行了比较。结果表明,94％的供试基因型愈伤组织诱导率都可达到80％以上,若排除供体植株环境条件的不同和接种过程中的人为因素可能造成的影响,不同基因型的愈伤组织诱导率看来没有根本的差异。愈伤组织分化率因基因型的不同变动在0—60％之间,平均为32.7％。虽然同一基因型的盾片愈伤组织分化率在不同年份中有所不同,但是愈伤组织是否具有再生能力?看来是个稳定的遗传性状。因此小麦未成熟胚对愈伤组织诱导的反应和愈伤组织的再生能力可能具有不同的遗传基础。本文的结果还表明,虽然最适于培养的未成熟胚的大小为1毫米左右,伹小至0.3毫米的未成熱胚仍能以几乎100％的频率形成愈伤组织,60％左右的愈伤组织能分化出再生檀株,只是所需的时间比1毫米左右的胚较长。     

Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums

A procedure was established for the induction of regenerable calli from immature inflorescence segments of high-tannin cultivars of sorghum (Sorghum bicolor (L.) Moench). Murashige & Skoog's medium with several components altered was utilized for inducing, maintaining, and regenerating the cultures. Embryogenic calli formed at a frequency of 8–70% depending on the genotype. During a ten-month period, 3600 plants were regenerated from eight genotypes tested. Among the developmental stages of immature inflorescence tested (from differentiation of secondary branch primordia to floret formation) no critical differences were found in potential for callusing, embryogenesis or regeneration. Genotypic differences were observed in pigment production, embryogenic callus formation, shoot differentiation, and in maintenance of regeneration capacity.Abbreviations 2,4-D dichlorophenoxyacetic acid This is Journal Paper Number 11972 from the Purdue University Agricultural Experiment Station     

Development and maturation of surghum seeds on detached panicles grown in vitro

Summary A procedure for culturing detached panicles of sorghum, Sorghum bicolor (L.) Moench, was developed to achieve flowering, fertilization, and subsequent seed development and maturation in vitro. Sixteen sorghum genotypes (five high and eleven low in tannin) were tested for their ability to develop normally in culture. Panicles collected one to two days before the initiation of anthesis were cultured in flasks containing liquid medium. Contamination and medium darkening were the major obstacles encountered. Up to 55% of the panicles cultured reached physiological maturity in vitro. The frequency of seed set ranged from 30 to 97% depending upon genotype and medium. Seed and glume color were normal. Seed produced in vitro resembled those grown in vivo and germinated well, but were smaller than normal (100 kernel weight reached 50 to 70% of the control). Grain polyphenols were synthesized in the cultured panicles. Seed of high tannin genotypes produced in vitro were lower in total phenols and tannins and higher in flavan-4-ols and the 3-deoxyanthocyanidin pigments than control seed. This technique can be used for harvesting late-maturing stocks and for various sorghum studies.     

大麦×小麦杂种愈伤组织及再生植株的细胞学观察

本文研究了Ant-13大麦×中国春小麦未成熟胚诱导的愈伤组织细胞,再生植株体细胞和花粉母细胞染色体的变化情况。发现在这三个不同分化和发育阶段中都存在着混倍体现象。但随着分化和发育过程的进行,混倍体程度越来越小,正常双单倍体细胞的比例越来越大。从再生植株花粉母细胞减数分裂前的有丝分裂开始到整个减数分裂过程中都可以看到染色体行为的异常现象,从而形成败育花粉,造成杂种不孕。花粉败育发生在单核小孢子时期。     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Enhancement of ethylene biosynthesis and germination with thidiazuron and some selected auxins in Striga asiatica seeds

Witchweed [ Striga asiatica (L.) Kuntze], an economically important parasitic weed on several poaceous crops, is difficult to control. In nature, germination and subsequent morphogenesis of Striga are cued to specific host-derived chemical signals. Seeds (approximately 2.4 mg) treated with thidiazuron (TDZ) or the auxins 2,4-dichlorophenoxy-acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), or 2-(4-chloro- o -tolyloxy) propionic acid (MCPP) produced little ethylene (66-138 nl l⁻¹). Combinations of TDZ with the auxins increased ethylene production by 4- to 18-fold. Ethylene production was strongly inhibited (86–92%) by aminoethoxyvinylglycine (AVG), inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Ethylene evolved from seeds treated with TDZ in combination with 2,4-D increased after a lag period and was promoted by a pretreatment in 2,4-D. TDZ or any of the auxins, at the rates tested, effected negligible to low levels of germination (0 to 16%), whereas mixtures of TDZ with the above auxins stimulated 38 to 84% germination. Test solutions containing TDZ and indole-3-acetic acid (IAA) were, however, less effective. TDZ/auxin-induced germination was inhibited by AVG and the ethylene action inhibitor silver thiosulfate (STS). The inhibitory effect of the former was reversed by treatment with ACC. In vitro studies revealed negligible germination (< 1%) on control medium. Seeds germinating on media containing TDZ alone developed into seedlings with distinct shoots and rudimentary roots. Seeds germinating on media containing 2,4-D, irrespective of TDZ concentration, were induced to form calli. The results are consistent with a model in which both germination and subsequent morphogenesis in Striga are associated with exogenous and endogenous phytohormones.     

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo₃ in the infection medium and adding AgNo₃ in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×10⁹ c.f.u. ml^–1 for bacterium inoculation; 100 mg l^–1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T₀ plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T₁ plants were analyzed and transmission of transgenes to the T₁ generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.     

Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghums

Callus and plant regenertion were induced from shoot portions of mature embryos (dry seeds) of five high tannin Sorghum bicolor (L.) Moench cultivars. The explants were cultured on Murashige and Skoog medium with altered concentrations of 5 salts, supplemented with 150 mg/L L-asparagine, 5mg/L 2,4-Dichlorophenoxyacetic acid and 0.05mg/L kinetin. Calli which were yellow and globular were formed with 70–90% frequencies. The subculture medium which gave best results was MS with 2mg/L 2,4-Dichlorophenoxyacetic acid and 0.5mg/L kinetin. Plants were regenerated on MS medium supplemented with 150mg/L L-asparagine and 0.2mg/L kinetin with regeneration frequencies of 11–48%.Abbreviations 2,4-D dichlorophenoxyacetic acid