Popelka Juan Carlos, Xu Jianping, Altpeter Fredy
Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben, Corrensstrasse 3, 06466 Gatersleben, Germany.
Transgenic Res. 2003 Oct;12(5):587-96. doi: 10.1023/a:1025822731661.
Rye is extremely recalcitrant to tissue culture and genetic transformation. We describe the efficient and reproducible production of stably expressing transgenic rye plants after biolistic gene transfer to callus tissue derived from immature embryos. Key factors in the production of transgenic rye plants include the identification of biolistic gene transfer parameters and a selection protocol, which does not affect its regeneration ability. The bar gene was used as a selectable marker and selection was performed by spraying the regenerated shoots with 0.05% Basta solution without any previous selection of tissue cultures. Based on Southern blot analysis, a total of 21 transgenic rye plants with independent transgene integration patterns were produced. A low transgene copy number was observed in most transgenic plants and 40% of the plants had a single transgene copy insert. The high frequency of single transgene copy inserts might be a consequence of the selection system, which is based on the identification of stably expressing transgenic plantlets rather than stably expressing tissue cultures. All transgenic rye lines with single transgene inserts showed stable transgene expression in sexual progenies, but indications of transcriptional and post-transcriptional gene silencing were observed in few transgenic lines with multiple transgene inserts. Tissue culture-based selection was not necessary for the generation of transgenic rye. The identification of 17 transgenic rye plants without using any selectable marker gene by PCR amplification of transgene sequences is also demonstrated. Instant generation of selectable marker-free transgenic rye avoids a negative impact of selective agents on the transgenic tissue cultures, responds to public concerns on the safety of selectable markers and will support multiple transformation cycles for transgene pyramiding.
黑麦对组织培养和遗传转化极具抗性。我们描述了通过将基因枪法介导的基因转移至源自未成熟胚的愈伤组织后，高效且可重复地产生稳定表达的转基因黑麦植株的方法。产生转基因黑麦植株的关键因素包括确定基因枪法介导的基因转移参数和一种不影响其再生能力的选择方案。使用bar基因作为选择标记，通过向再生芽喷洒0.05%的草丁膦溶液进行选择，而无需对组织培养物进行任何预先选择。基于Southern杂交分析，共产生了21株具有独立转基因整合模式的转基因黑麦植株。在大多数转基因植株中观察到低转基因拷贝数，40%的植株有单个转基因拷贝插入。单个转基因拷贝插入的高频率可能是选择系统的结果，该系统基于对稳定表达的转基因植株而非稳定表达的组织培养物的鉴定。所有具有单个转基因插入的转基因黑麦品系在有性后代中均表现出稳定的转基因表达，但在少数具有多个转基因插入的转基因品系中观察到转录和转录后基因沉默的迹象。基于组织培养的选择对于转基因黑麦的产生并非必要。还展示了通过对转基因序列进行PCR扩增，在不使用任何选择标记基因的情况下鉴定出17株转基因黑麦植株。即时产生无选择标记的转基因黑麦可避免选择剂对转基因组织培养物的负面影响，回应公众对选择标记安全性的担忧，并将支持用于转基因聚合的多次转化循环。
