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Preparation method of lily mesophyll protoplast

A technology of protoplasts and lily leaves, applied in the field of preparation of lily mesophyll protoplasts, can solve the problems of less research on mature plants, failure to obtain regenerated plants, etc., and achieve the effect of overcoming low separation efficiency

Active Publication Date: 2022-03-08
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In 2006, Liu Yujing (Liu Yujing. Lily Protoplast Isolation and Culture Research [D]. Master. Northeast Agricultural University, 2006.) used oriental lily 'Bernini', Asiatic lily 'Elite' and Lily lily (L.lancifolium) as materials Preliminary experiments were carried out on the preparation and cultivation of lily protoplasts, but failed to obtain regenerated plants
On this basis, in recent years, there has been some research on the isolation and purification of protoplasts from oriental lily, wild lily, edible lily, etc., but most of the materials used are concentrated in callus or suspension cells. Plants are less studied

Method used

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  • Preparation method of lily mesophyll protoplast
  • Preparation method of lily mesophyll protoplast
  • Preparation method of lily mesophyll protoplast

Examples

Experimental program
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Effect test

Embodiment 1 100

[0040] The preparation method of embodiment 1 lily mesophyll protoplast

[0041] The present embodiment provides the method for the separation and purification of lily mesophyll protoplasts, the method comprising the following steps:

[0042] 1. Dark pretreatment: select oriental lily 'Sorbonne' plants that have grown for about 30 days and are in good condition and free from diseases and insect pests, and perform dark treatment at room temperature for 48 hours.

[0043] 2, material processing: the top new leaf ( figure 1) rinse with clean water, soak in soapy water for disinfection, rinse with running water, soak in 75% alcohol for 5 minutes, rinse with sterile water, dry the water, and set aside.

[0044] 3. Enzymatic hydrolysis treatment: Cut the material obtained in step 2 into strips with a width of 2 mm with a sterilized blade, weigh 1 g of strips, and put them into 10 ml of enzymatic hydrolysis solution with tweezers. Use a vacuum pump to extract in the dark for 30 min...

Embodiment 2

[0051] The influence of embodiment 2 dark treatment on the preparation of lily mesophyll protoplast

[0052] For the dark treatment time in the method of Example 1, the present invention sets 5 time gradients: 0, 24, 36, 48, 72h. The material after dark treatment was subjected to enzymatic hydrolysis and purification according to the method in Example 1. The results showed that the leaves should be treated for 48h (Table 1).

[0053] Table 1 Yield and vigor of lily protoplasts after dark treatment

[0054]

[0055] Note: The same lowercase letters are not included, indicating that the difference is significant at the P<0.05 level.

[0056] As shown in Table 1, the dark treatment had a very significant effect on the yield of protoplasts in the leaves of Lily, and as the treatment time was extended, the yield of protoplasts increased significantly ( Figure 4 ), the protoplast output of leaves reached the maximum 7.30×10 after 48 hours of treatment 5 individual / g, at this...

Embodiment 3

[0057] Example 3 Effects of Enzyme Concentration Ratio, Enzymolysis Time and Material Collection Period on the Preparation of Lily Mesophyll Protoplasts

[0058] For the material collection period, enzyme concentration ratio and enzymolysis time in steps 2 and 4, the present invention sets 4 factors and 3 levels of L 9 (3 4 ) Orthogonal test (Table 2). The cellulase concentration level is 1%, 1.5%, 2%; the isolated enzyme concentration is set to 0.2%, 0.5%, 1%; the leaf enzymatic hydrolysis time gradient is 4h, 7h, 10h; the leaves are taken from plants that have grown for about 30 days The upper, middle and bottom blades ( figure 1 ). Enzymolysis was carried out according to the test conditions of each group, and the yield and activity of protoplasts were counted.

[0059] The results are shown in Table 2. It can be seen from the range (R value) that the order of influence of the four factors on leaf protoplast yield is: cellulase concentration > sampling site > isolated e...

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Abstract

The invention discloses a method for preparing lily mesophyll protoplasts, which comprises the steps of: (1) taking lily plants with unopened flower buds, and performing dark treatment; (2) taking leaves of the plants, cleaning and disinfecting them; (3) feeding the mesophyll materials Add enzymolysis solution in the dark place, vacuum extraction in the dark, and then stand in the dark for several hours to obtain the enzymolysis solution containing mesophyll protoplasts; (4) Dilute the obtained enzymolysis solution with W5 solution, filter through a mesh sieve, and obtain the filtrate After low-speed centrifugation, discard the supernatant, resuspend the protoplast pellet with W5 solution, centrifuge at low speed, discard the supernatant, and repeat washing once; resuspend the protoplast pellet with MMG solution. The invention overcomes the problem of low separation efficiency of the lily mesophyll protoplast, and effectively establishes a method for preparing the lily mesophyll protoplast.

Description

technical field [0001] The invention relates to the technology for preparing plant protoplasts, in particular to a method for preparing lily mesophyll protoplasts. Background technique [0002] Protoplast refers to a naked cell surrounded by a plasma membrane with the cell wall removed. Protoplasts are pluripotent and can regenerate into complete plants. Protoplasts can absorb foreign genes, viruses, plasmids and other substances, and are excellent materials for a series of operations and gene transformation in plant cell engineering. In addition, through protoplast fusion technology, genetic material can also be changed to obtain new varieties. Therefore, the separation and regeneration of protoplasts are of great significance in the fields of plant genetic breeding, trait improvement and genetic transformation. [0003] Lily (Lilium spp.) is a perennial bulbous flower of the genus Lilium in the family Liliaceae, and is a world-renowned ornamental flower. With the expan...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 孙明石雪珺张启翔张腾旬钟剑陈俊通程堂仁潘会堂王佳
Owner BEIJING FORESTRY UNIVERSITY
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