Genetic transformation of the oilseed crop camelina using immature zygotic embryos.

IF 4.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
Barno Ruzimurodovna Rezaeva, Ingrid Otto, Götz Hensel, Pouneh Pouramini, Anton Peterson, Jochen Kumlehn
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Abstract

Background: Camelina is an oilseed crop with particularly useful fatty acid and amino acid profiles of its seeds, high resilience to abiotic and biotic stresses, and a short life cycle. Previous genetic engineering approaches in camelina have largely relied on the floral dip method which is, however, associated with genotype-dependent efficiency and incompatibility with methods of direct biomolecule delivery.

Results: Here, we established a novel method of transgenesis for camelina, taking advantage of the high regenerative capacity of immature embryos. Various culture conditions and treatments were experimentally validated, which included the duration of explant pre-cultivation, wounding of explants and its time of application, Agrobacterium strain and density of inoculum, acetosyringone concentration, duration of explant-Agrobacterium co-cultivation, as well as application time and concentration of the selective agent hygromycin. We provide convergent evidence of stable transgenicity and transgene inheritance by (1) selection for resistance to hygromycin, (2) PCR, (3) detection of the transgene product GFP, and (4) DNA gel blot analysis involving primary transgenic plants and segregating progeny. Primary transgenics examined in detail featured one to three T-DNA integration loci, with one to seven T-DNA copies being integrated in total per plant. The established method proved efficient across all three tested accessions including two current cultivars, whereby transformation efficiencies, determined as PCR-positive primary transgenic plants related to agro-inoculated explants, of between 13 and 17% were obtained.

Conclusion: With this method, we provide a viable platform for the functional validation of genes-of-interest and for biotechnological improvements of plant performance and quality features in camelina.

利用未成熟合子胚胎对油料作物亚麻荠进行遗传转化。
背景:亚麻荠是一种油籽作物,其种子具有特别有用的脂肪酸和氨基酸谱,对非生物和生物胁迫具有很高的抗逆性,并且生命周期短。以前的亚麻荠基因工程方法主要依赖于花浸提法,然而,这种方法具有基因型依赖的效率,并且与直接生物分子传递方法不相容。结果:利用未成熟胚的高再生能力,建立了一种新的亚麻荠转基因方法。实验验证了外植体预培养时间、外植体损伤及施用时间、农杆菌菌种及接种密度、乙酰丁香酮浓度、外植体-农杆菌共培养时间、选择性剂潮霉素的施用时间及浓度等培养条件和处理方法。我们通过(1)对潮霉素抗性的选择,(2)PCR,(3)转基因产物GFP的检测,以及(4)对原代转基因植物和分离后代的DNA凝胶印迹分析,提供了稳定的转基因性和转基因遗传的趋同证据。详细检查的初级转基因具有1至3个T-DNA整合位点,每株植物总共整合1至7个T-DNA拷贝。所建立的方法被证明在所有三个被测试的品种中都是有效的,其中包括两个现有的品种,由此获得了与农业接种外植体相关的pcr阳性原代转基因植株的转化效率,在13%到17%之间。结论:该方法为相关基因的功能验证和亚麻荠植物性能和品质特征的生物技术改良提供了可行的平台。
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来源期刊
Plant Methods
Plant Methods 生物-植物科学
CiteScore
9.20
自引率
3.90%
发文量
121
审稿时长
2 months
期刊介绍: Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences. There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics. Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.
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