Pigna Gaia, Dhillon Taniya, Dlugosz Elizabeth M, Yuan Joshua S, Gorman Connor, Morandini Piero, Lenaghan Scott C, Stewart C Neal
Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee, USA.
Department of Biosciences, University of Milan, Milano, Italy.
Biotechnol J. 2016 Dec;11(12):1657-1666. doi: 10.1002/biot.201600486. Epub 2016 Nov 28.
Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.
芦竹是地中海地区一种很有前景的生物燃料原料。尽管人们对其遗传改良颇感兴趣，但芦竹的组织培养和转化仍然很困难。作者开发了芦竹细胞和组织培养以及基因工程的方法。进行了培养基筛选，并利用茎腋芽外植体诱导的愈伤组织建立了悬浮培养。发现含有9 µM 2,4-D和4.4 µM BAP的DBAP培养基是测试的诱导细胞悬浮培养的最有效培养基，在14天内组织质量增加了五倍。相比之下，含有13 µM 2,4-D的CIM培养基在同一时期内质量仅增加了1.4倍。优化后的悬浮培养在增加组织质量方面优于先前描述的基于固化培养基的愈伤组织培养方法。悬浮培养被证明对随后的原生质体分离非常有效。原生质体电穿孔产生了3.3±1.5%的转化效率。一个双荧光报告基因载体能够在芦竹的单细胞中直接比较花椰菜花叶病毒35S启动子和柳枝稷ubi2启动子。与35S启动子在芦竹中赋予的表达相比，柳枝稷ubi2启动子导致报告基因表达明显更高。
