Establishment of transient and stable gene transformation systems in medicinal woody plant Acanthopanax senticosus

IF 5.2 2区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Chemical and Biological Technologies in Agriculture Pub Date : 2024-09-19 DOI:10.1186/s40538-024-00669-8

Huan Liu, Panpan Sun, Yaqian Tong, Xinglei Gao, Zhonghua Tang, Guizhi Fan

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引用次数: 0

Abstract

Background

Transient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plants in vitro.

Results

Embryogenic callus protoplast isolation and transient transformation system were successfully established. The highest yield of protoplasts was approximately 1.88 × 10⁶ cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% macerozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng μL⁻¹, and 25 min heat shock at 45 °C. In addition, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions.

Conclusion

This study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research on A. senticosus.

Graphical Abstract

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建立药用木本植物刺五加的瞬时和稳定基因转化系统

背景瞬时和稳定的基因转化系统在阐明基因功能和推动植物遗传改良方面发挥着至关重要的作用。结果成功建立了冻存胼胝体原生质体分离和瞬时转化系统。在 1.5% 纤维素酶和 0.2% 大环酶的作用下，原生质体的最高产量约为每克 1.88 × 106 个细胞，存活率为 90%，酶解过程在黑暗中进行 6 小时，然后在 400×g 转速下离心 5 分钟。在 PEG 4000 浓度为 40%、转染时间为 40 分钟、黑暗培养 16 小时、质粒浓度为 1.5 ng μL-1、45℃热休克 25 分钟的条件下，原生质体的瞬时转染率达到 45.56%。结论本研究成功地建立了瞬时和稳定的遗传转化系统，为今后的仙客来分子生物学研究铺平了道路。

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来源期刊

Chemical and Biological Technologies in Agriculture Biochemistry, Genetics and Molecular Biology-Biotechnology

CiteScore

6.80

自引率

3.00%

发文量

审稿时长

15 weeks

期刊介绍： Chemical and Biological Technologies in Agriculture is an international, interdisciplinary, peer-reviewed forum for the advancement and application to all fields of agriculture of modern chemical, biochemical and molecular technologies. The scope of this journal includes chemical and biochemical processes aimed to increase sustainable agricultural and food production, the evaluation of quality and origin of raw primary products and their transformation into foods and chemicals, as well as environmental monitoring and remediation. Of special interest are the effects of chemical and biochemical technologies, also at the nano and supramolecular scale, on the relationships between soil, plants, microorganisms and their environment, with the help of modern bioinformatics. Another special focus is the use of modern bioorganic and biological chemistry to develop new technologies for plant nutrition and bio-stimulation, advancement of biorefineries from biomasses, safe and traceable food products, carbon storage in soil and plants and restoration of contaminated soils to agriculture. This journal presents the first opportunity to bring together researchers from a wide number of disciplines within the agricultural chemical and biological sciences, from both industry and academia. The principle aim of Chemical and Biological Technologies in Agriculture is to allow the exchange of the most advanced chemical and biochemical knowledge to develop technologies which address one of the most pressing challenges of our times - sustaining a growing world population. Chemical and Biological Technologies in Agriculture publishes original research articles, short letters and invited reviews. Articles from scientists in industry, academia as well as private research institutes, non-governmental and environmental organizations are encouraged.