A sterilization protocol for field-harvested mature maize seed used for in vitro culture and genetic transformation.
A sterilization protocol for field-harvested mature maize seed used for in vitro culture and genetic transformation.
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用于体外培养和遗传转化的田间收获的成熟玉米种子的灭菌方案。
DOI：
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发表时间：
2009
期刊：
影响因子：
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通讯作者：
K. Wang
中科院分区：
文献类型：
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作者：
J. C. Martinez;K. Wang
体外组织培养技术不仅需要无菌的工作环境，而且需要无污染的起始材料。影响体外培养组织的两种常见污染物是细菌和真菌。用于体外培养的培养基的富集使得外植体对这些微生物敏感（Leifert和Waites，Int. Soc. Plant Tissue Cult. Newsletter 60：2-13，1990）。尽管在应用灭菌技术时要小心，但由于外植体中存在大量微生物，污染仍然可能导致田间生长材料的100%损失。植物遗传转化过程中的第一步是获得无菌植物材料用于体外组织培养。对于玉米，当使用未成熟胚时，存在用于初始材料灭菌的完善的方案（Frame等人，体外细胞开发Plant 36：21-29，2000）。然而，尚无针对成熟玉米种子的有效灭菌方案的报道，尤其是对于通常携带大量空气和土壤传播病原体的田间收获种子。克服成熟种子灭菌中的污染问题的一种策略是简单地从大量种子开始，这增加了获得足够数量的灭菌种子用于体外培养的机会。或者，一种首先防止这些污染物进入实验室的方法将是理想的。在这里，我们报告了一种有效的和可重复的种子灭菌方法，这是专门为成熟的玉米种子收获从田间生长的植物。该方法包括三个主要的消毒阶段。首先将玉米种子表面消毒并浸泡在无菌水中，然后将软化的种子消毒并分离成熟胚。最后，解剖的成熟胚在置于植物培养基上进行培养之前进行消毒。使用这种逐步灭菌方法，获得平均98%的无菌成熟胚，并且植物活力（通过幼苗生长速率测量; ISTA，Seed Sci.技术27（补编）：27-32，1999）与未灭菌对照品（Martinez，2008）相比未受损。材料. 1)成熟的玉米种子收获自田间生长的植物。2)80%乙醇（约300 ml，约200粒种子）。3)50%漂白液：将450 ml市售漂白剂（5.25%次氯酸盐）与450 ml含有2滴表面活性剂Tween-20的Millipore水混合。使用~900毫升约200种子。4)15%漂白剂溶液：将15 ml市售漂白剂（5.25%次氯酸盐）与85 ml含有1滴表面活性剂Tween-20的Millipore水混合。约200个解剖胚胎使用约100 ml。5)微孔水（高压灭菌）。6) ...
In vitro tissue culture techniques require not only an aseptic work environment but also contaminant free starting materials Two common contaminants affecting in vitro-cultured tissues are bacteria and fungi. Enrichment of the media used for in vitro culture makes explants susceptible to these microorganisms (Leifert and Waites, Int. Soc. Plant Tissue Cult. Newsletter 60:2-13, 1990). Despite careful attention while applying sterilization techniques, contamination still may cause 100% loss from field-grown material due to large numbers of microorganisms present in the explants One of the first steps in the process of plant genetic transformation is to acquire sterile plant materials for in vitro tissue culture. For maize, a well-established protocol exists for the sterilization of initial material when immature embryos are used (Frame et al., In Vitro Cell Dev. Biol. Plant 36:21-29, 2000). However, no effective sterilization protocols are reported for mature maize seeds, especially for field-harvested seeds that typically carry large amounts of air-and soil-borne pathogens. One strategy to overcome the contamination problem in mature seed sterilization is simply to start with large quantities of seeds, which increases the chances of obtaining adequate numbers of sterilized seed for in vitro culture. Alternatively, a method that prevents introduction of these contaminants into the laboratory in the first place would be ideal. Here we report an efficient and reproducible seed sterilization method, which was developed specifically for mature maize seeds harvested from field-grown plants. The method includes three major disinfection stages. First, the maize seed surface is disinfected and soaked in sterile water, then the softened seeds are disinfected and the mature embryos isolated. Finally, the dissected mature embryos are disinfected before being placed on plant media for culturing. Using this step-wise sterilization method, an average of 98% sterile mature embryos are obtained, and plant vigor (measured by Seedling Growth Rate; ISTA, Seed Sci. Tech-nol. 27(Suppl.):27-32, 1999) is not compromised compared to the non-sterilized control (Martinez, 2008). Materials. 1) Mature maize seeds harvested from field-grown plants. 2) 80% Ethanol (~300 ml for ~200 seeds). 3) 50% bleach solution: mix 450 ml of commercial bleach (5.25% hypochlorite) with 450 ml of Millipore water containing 2 drops of the surfactant Tween-20. Use ~900 ml for ~200 seeds. 4) 15% bleach solution: mix 15 ml of commercial bleach (5.25% hypochlorite) with 85 ml of Millipore water containing 1 drop of the surfactant Tween-20. Use ~100 ml for ~200 dissected embryos. 5) Millipore water (auto-claved). 6) …