Preparation method of Calibrachoa protoplast

A technology of petunia florida and protoplasts, which is applied in the direction of plant cells, can solve the problems of large differences in protoplast separation, long separation time, and low efficiency of petunia floret protoplast separation, achieving high yield and good growth status Effect

Active Publication Date: 2015-11-18
SHANGHAI JIAO TONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

The isolation of protoplasts of different varieties of the same species of different species is quite differe...

Method used

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  • Preparation method of Calibrachoa protoplast
  • Preparation method of Calibrachoa protoplast
  • Preparation method of Calibrachoa protoplast

Examples

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preparation example Construction

[0031] The present invention relates to a kind of preparation method of petunia florida protoplast, described method comprises the following steps:

[0032] A, under dark conditions, the young leaves of Petunia florida are pretreated;

[0033] B. Use the enzymolysis solution to enzymolyze the leaves treated in step A; the solvent of the enzymolysis solution is 0.25M phosphate solution, and the solute is the following final concentration: 2wt% cellulase, 0.8wt% eluent , 0.2wt% pectinase, 0.11wt% anhydrous calcium chloride, 0.10wt% bovine serum albumin; the pH of the enzymolysis solution is 5.6-5.7;

[0034] C. Add CPW washing solution equal to the volume of the enzymolysis solution to the solution after the enzymolysis in step B, shake the enzyme solution, filter through a 300-mesh cell sieve and centrifuge at 1100r / min for 2min, and suck off the supernatant; The solvent in the CPW lotion is CPW salt solution, the solute is mannitol with a final concentration of 0.25M, and the...

Embodiment

[0069] This example relates to the preparation of petunia floret mesophyll protoplasts. The variety of petunia florida is Calibrachoahybrid 'lindurayellow' with yellow flower color, purchased from Fides company. Specifically include the following steps:

[0070] 1. Preparation materials: Take the 1st to 2nd young leaves under the terminal buds of Petunia florida, and weigh 0.33g.

[0071] 2. Separation of plasmodium: pretreatment of leaves, under dark conditions, collect young leaves and place them in clear water at 3-5°C for 20-25 hours of pretreatment.

[0072] 3. Enzymatic hydrolysis: Add 5ml of enzymatic hydrolysis solution to a 6cm small petri dish, cut the pretreated leaves into 0.1cm×0.2cm strips and lay them flat in the enzyme solution so that they do not overlap each other. Static hydrolysis at 26±1°C for 5 hours in the dark, during which the culture dish was gently shaken every 1 hour to fully contact the two phases. The enzymolysis solution is 0.25M phosphate solu...

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Abstract

The invention discloses a preparation method of a Calibrachoa protoplast. The method comprises the following steps: preprocessing tender leaves in 3-5DEG C clear water under dark conditions for 20-25h; carrying out enzymatic hydrolysis on the preprocessed blades by using an enzymatic hydrolysis solution; adding a CPW washing solution having a same volume with the enzymatic hydrolysis solution, shaking up, filtering by using a 300 mesh cell sieve, centrifuging at a rotating speed of 1100r/min for 2min, and absorbing the obtained supernatant; and adding a CPW washing solution to the obtained precipitate to wash a protoplast, centrifuging at a rotating speed of 1100r/min for 2min, collecting the obtained precipitate, adding a suspension, and suspending to obtain the protoplast with the output of (4.7-5.3)*10<6>/g and the activity of 85%, wherein the activity of 80% of the protoplast is maintained 9h later. The protoplast with high activity, high output and good state is obtained through a separation technology, protoplast culturing and plant regeneration conditions are met, and an experiment material is provided for plant protoplast fusion cultivation of new kinds.

Description

technical field [0001] The invention relates to a preparation method of petunia floret protoplast. Background technique [0002] Calibrachoahybrid, also known as Wuchunhua, is a herbaceous flower of the genus Calibrachoa in the family Solanaceae. Because of its rich color, beautiful flower shape, long flowering period and large amount of flowers, the plant shape is compact, and it has a good effect on medium and high pH values. Features such as tolerance are widely used in urban landscaping, and the potential for commercial development is huge. At the same time, it can also be used as a breeding resource to carry out protoplast fusion with other plants to cultivate new varieties, especially to open up a new way for the cultivation of yellow petunias. [0003] The type and proportion of enzyme solution, osmotic pressure, enzymatic hydrolysis time, pretreatment method and purification method and other factors affect the separation of plant protoplasts. At present, there are ...

Claims

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Application Information

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IPC IPC(8): C12N5/04
Inventor 曾丽刘国锋王鹏刘晓丛彭勇政赵子刚林登贵王梦茹秦泽冠周嘉桢
Owner SHANGHAI JIAO TONG UNIV
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