A method for rapidly extracting epidermal cell protoplasts from young and complete leaves of plants
A protoplast and epidermal cell technology, applied in the field of plant cell engineering, can solve problems such as inability to cut
Active Publication Date: 2022-03-25
SHANDONG NORMAL UNIV
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Problems solved by technology
In the prior art, in the process of extracting protoplasts, tools such as scalpels are used to first scrape off the epidermal cells of the upper layer of leaves, and then cut into filaments for enzymatic hydrolysis, etc., and there is no research on the extraction of protoplasts from leaf epidermal cells. Especially for the small true leaves in the early stage of development, the scalpel cannot cut the operation. The invention provides a 1 μm glass needle cutting method, which can be widely applied to other plants
Method used
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Embodiment 1
[0059] 1. Sow the sterilized Limonium bicolor seeds evenly on the MS medium, let it grow under normal conditions, and cultivate sterile seedlings.
[0060] 2. When the first true leaf of the plant has just grown (5-7 days after sowing), if figure 1 As shown in A, ready for sampling figure 1 b.
[0061] 3. Place the plant under a dissecting microscope and take out the first true leaf with fine-tipped tweezers, and use a microelectrode puller to pull out a 1 μm fine glass needle ( figure 1 C) Cutting of true leaves ( figure 1 D. figure 2 ), make wounds, cut 2-3 sections of a true leaf, cut them off completely, each section is 1-2 mm long, put the cut leaves immediately into a centrifuge tube containing 20 ml of enzymatic hydrolysis solution, and hydrolyze Keep the liquid away from light.
[0062] The formula of enzymatic hydrolysis solution (ready to use) is shown in Table 2.
[0063] Table 2
[0064]
[0065] 4. Enrich 200 leaves in the enzymatic hydrolysis solutio...
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Abstract
The invention belongs to the field of plant cell engineering, and in particular relates to a method for rapidly extracting epidermal cell protoplasts by using young and complete leaves of plants as materials. The method is realized through the following steps: when the seeds are cultivated to the length of the first true leaf of 3-5 mm, the first true leaf is taken out, cut with a glass needle, and the cut leaves are enzymatically hydrolyzed; after the enzymatic hydrolysis, Shake from the horizontal direction to the vertical direction; stop the enzymatic hydrolysis reaction, shake from the horizontal direction to the vertical direction, filter and centrifuge, wash the white precipitated protoplasts, and centrifuge; resuspend after natural sedimentation. For the first time, the present invention uses intact leaves as materials to extract protoplasts, which can achieve a purity of more than 95% and a live rate of more than 95%. It is of great significance to study epidermal cell differentiation, and secondly, it can also be used for subcellular localization research and protoplast transformation. And other downstream experiments, such as gene function verification, subcellular localization, cell sequencing, etc.
Description
technical field [0001] The invention belongs to the field of plant cell engineering, and in particular relates to a method for rapidly extracting epidermal cell protoplasts by using young and complete leaves of plants as materials. Background technique [0002] Two-color Limonium ( Limonium bicolor ( bag. ) Kuntze) Limonaceae, Limonium is a perennial herbaceous plant, also known as prairie dry branch plum, Fushi pine, wild distribution in Mongolian Plateau, Inner Mongolia, Xinjiang, Siberia, colorful, natural shape, is a characteristic dried flower Resource plants with good ornamental value. [0003] At present, there are many methods for extracting plant protoplasts, usually the method of enzymatic hydrolysis after cutting leaves (Sun, Lang et al.2013, Shen, Fu et al.2014), such as Arabidopsis, tobacco, corn, etc., but the extraction All come out are mesophyll cells, but there is no research on the extraction method of plant epidermal cell protoplasts. The solution pro...
Claims
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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 袁芳赵博庆鲁朝霞王宝山陈敏
Owner SHANDONG NORMAL UNIV