Overexpression of GRF-GIF genes enhances plant regeneration in cassava (Manihot esculenta)

IF 2.2 3区 生物学 Q4 CELL BIOLOGY
Rosana Segatto, Gecele M. Paggi, Nigel J. Taylor
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Abstract

The GROWTH-REGULATING FACTOR (GRF) and its INTERACTING FACTOR (GIF) have been shown to stimulate regeneration of transgenic plants with studies reporting increased transformation efficiency in multiple species, including wheat, beet, and citrus. The present work evaluated the effects of overexpressing GRF4-GIF1 and GRF5 on the regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Effects of GRF4-GIF1 and GRF5 sequences derived from Vitis vinifera and Arabidopsis thaliana were assessed by cloning expression cassettes under control of strong constitutive promoters. Friable embryogenic callus from cassava varieties 60444 and NASE 13 were transformed with Agrobacterium tumefaciens strains LBA4404 and LBA4404 THY-, and multiple independent transgenic plant lines recovered. Expression of the morphogenic genes did not enhance transformation efficiency, nor efficiency or timing of somatic embryo regeneration or whole plant recovery above the green fluorescent protein (GFP) control. Organogenesis experiments were carried out to observe effects of transgenic expression of these genes on morphogenesis from petiole, leaf-petiole, and stem explants. Results differed between the two genotypes evaluated. Expression of Vitis vinifera GRF4-GIF1 was effective for stimulation of rapid organogenesis and shoot regeneration at 30 to 37% from leaf-petiole explants of cultivar 60444. In contrast, Arabidopsis thaliana GRF5 was superior in stimulating shoot regeneration in cultivar NASE 13 with 40 to 50% of leaf-petiole explants regenerating shoots. In both cultivars, caulogenesis occurred rapidly within 3 to 4 wk culture on medium containing the cytokinin meta-topolin. Effects of overexpression of these morphogenic genes at the whole plant level were accessed by establishing plants in the greenhouse. GRF4-GIF1 overexpression resulted in significantly shorter plants with increased leaf size and total leaf area while AtGRF5 stimulated above average storage root weight.

Abstract Image

过表达 GRF-GIF 基因可提高木薯(Manihot esculenta)的植物再生能力
GROWTH-REGULATING FACTOR(生长调节因子)及其 INTERACTING FACTOR(互作因子)已被证明可刺激转基因植物的再生,有研究报告称它们在小麦、甜菜和柑橘等多个物种中提高了转化效率。本研究评估了过表达 GRF4-GIF1 和 GRF5 对木薯(Manihot esculenta Crantz)转基因植物再生的影响。通过克隆强组成型启动子控制下的表达盒,评估了源自葡萄和拟南芥的 GRF4-GIF1 和 GRF5 序列的影响。用农杆菌 LBA4404 和 LBA4404 THY- 菌株转化木薯品种 60444 和 NASE 13 的可胚化胼胝体,获得了多个独立的转基因株系。与绿色荧光蛋白(GFP)对照相比,表达形态发生基因并没有提高转化效率,也没有提高体细胞胚再生或整株植株恢复的效率或时间。器官发生实验观察了转基因表达这些基因对叶柄、叶柄和茎外植体形态发生的影响。两种基因型的评估结果有所不同。葡萄 GRF4-GIF1 的表达对刺激葡萄 60444 栽培品种叶柄外植体的快速器官发生和嫩枝再生有 30% 至 37% 的效果。相比之下,拟南芥 GRF5 在刺激栽培品种 NASE 13 的嫩枝再生方面更具优势,叶柄外植体有 40% 至 50% 再生出嫩枝。在这两个栽培品种中,在含有细胞分裂素meta-topolin的培养基上培养3至4周后,茎发生迅速。通过在温室中建立植株,研究了这些形态发生基因在整个植株水平上的过表达效果。GRF4-GIF1的过表达导致植株明显变矮,叶片大小和总叶面积增加,而AtGRF5则刺激贮藏根重量超过平均水平。
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来源期刊
CiteScore
5.00
自引率
7.70%
发文量
71
审稿时长
6-12 weeks
期刊介绍: Founded in 1965, In Vitro Cellular & Developmental Biology - Plant is the only journal devoted solely to worldwide coverage of in vitro biology in plants. Its high-caliber original research and reviews make it required reading for anyone who needs comprehensive coverage of the latest developments and state-of-the-art research in plant cell and tissue culture and biotechnology from around the world.
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