The Taguchi method and metAFLP analysis were used to optimise barley regenerants towards maximum and minimum levels of tissue culture-induced variation. The subtle effects of symmetric and asymmetric methylation changes in regenerants were identified. Plant tissue cultures (PTCs) provide researchers with unique materials that accelerate the development of new breeding cultivars and facilitate studies on off-type regenerants. The emerging variability of regenerants derived from PTCs may have both genetic and epigenetic origins, and may be desirable or degrade the value of regenerated plants. Thus, it is crucial to determine how the PTC variation level can be controlled. The easiest way to manipulate total tissue culture-induced variation (TTCIV) is to utilise appropriate stress factors and suitable medium components. This study describes the optimisation of in vitro tissue culture-induced variation in plant regenerants derived from barley anther culture, and maximizes and minimizes regenerant variation compared with the source explants. The approach relied on methylation amplified fragment length polymorphism (metAFLP)-derived TTCIV characteristics, which were evaluated in regenerants derived under distinct tissue culture conditions and analysed via Taguchi statistics. The factors that may trigger TTCIV included CuSO4, AgNO3 and the total time spent on the induction medium. The donor plants prepared for regeneration purposes had 5.75% and 2.01% polymorphic metAFLP loci with methylation and sequence changes, respectively. The level of TTCIV (as the sum of all metAFLP characteristics analyzed) identified in optimisation and verification experiments reached 7.51 and 10.46%, respectively. In the trial designed to produce a minimum number of differences between donor and regenerant plants, CuSO4 and AgNO3 were more crucial than time, which was not a significant factor. In the trial designed to produce a maximum number of differences between donor and regenerant plants, all factors had comparable impact on variation. The Taguchi method reduced the time required for experimental trials compared with a grid method and suggested that medium modifications were required to control regenerant variation. Finally, the effects of symmetric and asymmetric methylation changes on regenerants were identified using novel aspects of the metAFLP method developed for this analysis.

中文翻译：

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大麦体外再生方案优化过程中组织培养诱导变异的精确评估。


使用田口方法和metAFLP分析来优化大麦再生剂，以实现组织培养诱导变异的最大和最小水平。鉴定了再生体中对称和不对称甲基化变化的微妙影响。植物组织培养 (PTC) 为研究人员提供了独特的材料，可加速新育种品种的开发并促进异型再生体的研究。源自 PTC 的再生体的新出现的变异性可能具有遗传和表观遗传起源，并且可能是理想的或降低再生植物的价值。因此，确定如何控制 PTC 变化水平至关重要。操纵总组织培养诱导变异 (TTCIV) 的最简单方法是利用适当的应激因子和适当的培养基成分。本研究描述了大麦花药培养物中体外组织培养诱导的变异的优化，并与源外植体相比最大化和最小化再生变异。该方法依赖于甲基化扩增片段长度多态性 (metAFLP) 衍生的 TTCIV 特征，这些特征在不同组织培养条件下衍生的再生体中进行评估，并通过田口统计进行分析。可能触发 TTCIV 的因素包括 CuSO4、AgNO3 和在诱导培养基上花费的总时间。用于再生目的的供体植物分别具有 5.75% 和 2.01% 的多态性 metAFLP 位点，其中甲基化和序列发生变化。优化和验证实验中确定的 TTCIV 水平（作为分析的所有 metAFLP 特征的总和）分别达到 7.51 和 10.46%。 在旨在使供体植物和再生植物之间的差异最小化的试验中，CuSO4 和 AgNO3 比时间更重要，而时间并不是一个重要因素。在旨在产生供体植物和再生植物之间最大数量差异的试验中，所有因素对变异都有类似的影响。与网格方法相比，田口方法减少了实验所需的时间，并表明需要对培养基进行修改来控制再生体变异。最后，使用为此分析开发的metAFLP方法的新颖方面，确定了对称和不对称甲基化变化对再生体的影响。