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Preparation method of lily petal protoplast

A protoplast and lily technology is applied in the field of preparation of lily petal protoplasts to achieve the effect of overcoming low separation efficiency

Active Publication Date: 2022-03-04
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On this basis, in recent years, there have been some reports on the separation and purification of protoplasts from oriental lily, wild lily, edible lily, etc., but the materials used are mostly concentrated in callus or suspension cells. There are few studies on plants, and reports directly taken from petals are even rarer

Method used

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  • Preparation method of lily petal protoplast
  • Preparation method of lily petal protoplast
  • Preparation method of lily petal protoplast

Examples

Experimental program
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Effect test

Embodiment 1 100

[0039] The preparation method of embodiment 1 lily petal protoplast

[0040] The present embodiment provides the method for lily petal protoplast isolation and purification, described method comprises the following steps:

[0041] 1. Dark pretreatment: Select the flower buds (not open) of Oriental lily 'Sorbonne' in good condition and free from diseases and insect pests, cut them off and put them in vases, and carry out dark treatment at room temperature. In the present embodiment, the dark treatment time is 72h.

[0042] 2, material processing: with step 1 gained first open inner round perianth piece (being inner round petal, see figure 1 ) soaked in soapy water, rinsed with running water, soaked in 75% alcohol for 5 minutes, rinsed with sterile water, blotted dry, and set aside.

[0043]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 hy...

Embodiment 2

[0050] The influence of embodiment 2 dark treatment on lily petal protoplast preparation

[0051] 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 petals should be darkened for 72 hours (Table 1).

[0052] Table 1 Yield and vigor of lily protoplasts after dark treatment

[0053]

[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, dark treatment has a very significant impact on the yield of petal protoplasts of lily, and as the treatment time prolongs, the yield of protoplasts increases significantly ( Figure 4 ), the protoplast production of petals reached the maximum after 72 hours of treatment 4.82×10 5 individual / g, at this time...

Embodiment 3

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

[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 petal enzymatic hydrolysis time gradient is 8h, 10h, 12h; Petals in bloom and full bloom ( figure 1 ). Enzymolysis was carried out according to the test conditions of each group, and the yield and activity of protoplasts were counted.

[0059] From the R values ​​in Table 2, we can see that the order of the influence of four factors on the yield of petal protoplasts is: cellulase concentration>time of sampling>enzymolysis time>concentration of isolated enzyme. Among them, the concentration of cellulase has a very significa...

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Abstract

The invention discloses a preparation method of lily petal protoplasts, which comprises the steps of: (1) taking a lily plant with unopened flower buds and performing dark treatment; (2) taking the inner round petals of the plant, cleaning and disinfecting them; Add enzymolysis solution to the petal material, vacuum extract in the dark, and then stand in the dark for several hours to obtain the enzymolysis solution containing petal protoplasts; (4) The obtained enzymolysis solution is diluted with W5 solution, filtered through a mesh screen, The obtained filtrate was centrifuged at low speed, the supernatant was discarded, the protoplast precipitate was resuspended with W5 solution, the protoplast pellet was resuspended with MMG solution, the protoplast pellet was resuspended, and the supernatant was discarded. The invention overcomes the problem of low separation efficiency of the lily petal protoplast, and effectively establishes a method for preparing the lily petal protoplast.

Description

technical field [0001] The invention relates to plant protoplast preparation technology, in particular to a preparation method of lily petal protoplast. Background technique [0002] Lily (Lilium spp.) is a perennial bulbous flower of the genus Lilium in the family Liliaceae, and is a world-renowned ornamental flower. 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. As a basic technical means of genetic engineering, the isolation of protoplasts is of great significance for the study of lily-related gene functions and genetic breeding. [0003] As a highly heterozygous monocotyledonous plant, lily is difficult to isolate and cultivate protoplasts. At present, there are few research reports on the preparation of lily protoplasts at home and abroad. Horita first successfully cultivated somatic cell hybrid regenerated plants of Lilium orientalis in 2002 by isolati...

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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