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Method for inducing homozygous tetraploid plant from protoplast of Cichorium intybus L.

A technology of protoplasts and tetraploids, applied in the fields of botany equipment and methods, plant regeneration, horticultural methods, etc., can solve the problems of time-consuming, low frequency of homozygous polyploid induction, low frequency of tetraploid induction, etc. problem, to achieve the effect of high ploidy controllability

Inactive Publication Date: 2015-12-02
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The domestic routine polyploid induction method of chicory is usually to use colchicine and other chemical agents to treat the organs, tissues, callus or multicellular mass of chicory, and the obtained polyploid plants are often accompanied by mixed ploidy and chimerism, It takes repeated subcultures and isolation cultures to screen out homozygous polyploid plants. The process is not only labor-intensive and time-consuming, but also the induction frequency of homozygous polyploids is not high.
Foreign countries have used protoplast fusion technology to induce chicory tetraploids, but the induction frequency of tetraploids is low (Rambaud et al., 1996)

Method used

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  • Method for inducing homozygous tetraploid plant from protoplast of Cichorium intybus L.
  • Method for inducing homozygous tetraploid plant from protoplast of Cichorium intybus L.
  • Method for inducing homozygous tetraploid plant from protoplast of Cichorium intybus L.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] (1) Material collection: take 7-14 day old, diploid chicory seedling leaves of aseptic seedlings, or vigorously growing tissue culture seedling leaves, cut longitudinally into thin strips of 2-3 mm, and remove the central main vein and edges of the leaves , as a culture material.

[0090] (2) Enzymolysis: put the culture material into a petri dish equipped with enzymolysis solution, the mass volume ratio of leaves and enzyme treatment solution is 1 g: 10 ml, and the petri dish is sealed with Parafilm film; place the petri dish in Enzymolysis was carried out at 32° C. for 5.5 hours in the dark to obtain an enzymolysis product.

[0091] The above-mentioned enzymolysis solution includes: cellulase R-10 with a mass percent concentration of 0.25%, pectinase with a mass percent concentration of 0.2%, and 10 mM CaCl 2 .2H 2 O, 0.7 mM KH 2 PO 4 10% mannitol in mass percent concentration, 0.1% MES (2-(N-morpholine) ethanesulfonic acid) in mass percent concentration, and ster...

Embodiment 2

[0112] (1) Material collection: operate with reference to the method of step (1) in Example 1 to obtain culture materials;

[0113] (2) Enzymolysis: put the culture material into the petri dish that enzymolysis solution is housed again, the mass volume ratio of leaf and enzyme treatment solution is 1 gram: 10 milliliters, and petri dish is sealed with Parafilm film; Place this petri dish Enzymolysis was carried out at 32° C. for 5 hours in the dark to obtain an enzymatic hydrolysis product.

[0114] The above-mentioned enzymolysis solution includes: cellulase R-10 with a mass percent concentration of 0.25%, pectinase with a mass percent concentration of 0.2%, and 10 mM CaCl 2 .2H 2 O, 0.7 mM KH 2 PO 4 10% mannitol in mass percent concentration, 0.1% MES (2-(N-morpholine) ethanesulfonic acid) in mass percent concentration, and sterilized by filtration through a 0.22 μm microporous membrane.

[0115] During the enzymolysis process, every 20 minutes, the petri dish was shaken...

Embodiment 3

[0127] (1) Material collection: operate with reference to the method of step (1) in Example 1 to obtain culture materials;

[0128] (2) Enzymolysis: put the culture material into the petri dish that enzymolysis solution is housed again, the mass volume ratio of leaf and enzyme treatment solution is 1 gram: 10 milliliters, and petri dish is sealed with Parafilm film; Place this petri dish Enzymolysis was carried out at 32° C. for 5.5 hours under dark conditions to obtain an enzymolysis product.

[0129] The above-mentioned enzymolysis solution includes: cellulase R-10 with a mass percent concentration of 0.25%, pectinase with a mass percent concentration of 0.25%, and 10 mM CaCl 2 .2H 2 O, 0.7 mM KH 2 PO 4 10% mannitol in mass percent concentration, 0.1% MES (2-(N-morpholine) ethanesulfonic acid) in mass percent concentration, and sterilized by filtration through a 0.22 μm microporous membrane.

[0130] During the enzymolysis process, every 20 minutes, the petri dish was sh...

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Abstract

The invention belongs to the field of culture of plant protoplasts, and particularly relates to a method for inducing a homozygous tetraploid plant from a protoplast of Cichorium intybus L. According to the method, colchicineis used for inducing the protoplast of Cichorium intybus L. According to the method, a diploid culture material of Cichorium intybus L. is subjected to enzymolysis, purification, induction, cell cluster and small callus culture, callus reproduction and embryoid induction and differentiation so as to obtain regeneration buds. According to the invention, colchicineis used for inducing the protoplast of Cichorium intybus L., all the steps and all parameters have synergistic effect, and thus the regeneration rate of the tetraploid plant is high, and is extremely higher than that of a tetraploid plant induced by a protoplast fusion method.

Description

technical field [0001] The invention belongs to the field of plant protoplast culture, and in particular relates to a method for inducing homozygous tetraploid plants by chicory protoplasts. The method uses colchicine to induce the chicory protoplasts to obtain tetraploid regenerated plants. Background technique [0002] Chicory (Cichorium intybus L.) belongs to Compositae. It is a perennial herbaceous plant. It is a three-purpose crop for food, medicine and feed. Its roots are rich in inulin, oligomeric and super high fructose, which is very beneficial to human health. It can also be processed into low Caloric health food and coffee substitute; softened chicory can be used as fresh edible high-grade vegetables; in addition, chicory is also a high-yield and high-quality forage. [0003] Chicory originates in Europe and was introduced to China in the early 1980s. Large-scale trial planting and rapid promotion have begun, and it has good development potential. However, the br...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/08A01H4/00
Inventor 张秀海陈绪清杜运鹏王璐邢礼军李宏潮
Owner BEIJING AGRO BIOTECH RES CENT
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