A method for inducing homozygous tetraploid plants by chicory protoplasts
A technology of protoplasts and tetraploids, which is applied in 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
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Embodiment 1
[0089] (1) Material: Take 7-14 days old, diploid chicory seeds of sterile seedling leaves, or vigorously growing tissue culture seedling leaves, and cut them into thin strips of 2-3 mm lengthwise, removing the central main vein and edge of the leaves , As a culture material.
[0090] (2) Enzymatic hydrolysis: Put the culture material into a petri dish containing enzymatic hydrolysis solution, the mass volume ratio of the leaves to the enzyme treatment solution is 1 g: 10 ml, the petri dish is sealed with Parafilm; the petri dish is placed Enzymatic hydrolysis at 32°C under dark conditions for 5.5 hours to obtain enzymatic hydrolysis products.
[0091] The above enzymatic hydrolysis solution includes: cellulase R-10 with a concentration of 0.25% by mass, pectinase with a concentration of 0.2% by mass, and 10mM CaCl 2 .2H 2 O, 0.7mM KH 2 PO 4 , Mannitol with a concentration of 10% by mass and MES (2-(N-morpholine)ethanesulfonic acid) with a concentration of 0.1% by mass are filtered ...
Embodiment 2
[0112] (1) Obtaining materials: refer to step (1) in Example 1 to obtain culture materials;
[0113] (2) Enzymatic hydrolysis: Put the culture material into a petri dish containing enzymatic hydrolysis solution, the mass-volume ratio of the leaves to the enzyme treatment solution is 1 g: 10 ml, the petri dish is sealed with Parafilm; the petri dish is placed Enzymatic hydrolysis at 32°C under dark conditions for 5 hours to obtain enzymatic hydrolysis products.
[0114] The above enzymatic hydrolysis solution includes: cellulase R-10 with a concentration of 0.25% by mass, pectinase with a concentration of 0.2% by mass, and 10mM CaCl 2 .2H 2 O, 0.7mM KH 2 PO 4 , Mannitol with a concentration of 10% by mass and MES (2-(N-morpholine)ethanesulfonic acid) with a concentration of 0.1% by mass are filtered and sterilized with a 0.22μm microporous membrane.
[0115] During the enzymolysis process, every 20 minutes, manually shake the petri dish for 10 seconds to speed up the enzymolysis react...
Embodiment 3
[0127] (1) Obtaining materials: refer to step (1) in Example 1 to obtain culture materials;
[0128] (2) Enzymatic hydrolysis: Put the culture material into a petri dish containing enzymatic hydrolysis solution, the mass-volume ratio of the leaves to the enzyme treatment solution is 1 g: 10 ml, the petri dish is sealed with Parafilm; the petri dish is placed Enzymatic hydrolysis was carried out at 32°C under dark conditions for 5.5 hours to obtain enzymatic hydrolysis products.
[0129] The above-mentioned enzymolysis solution includes: cellulase R-10 with a concentration of 0.25% by mass, pectinase with a concentration of 0.25% by mass, and 10mM CaCl 2 .2H 2 O, 0.7mM KH 2 PO 4 , Mannitol with a concentration of 10% by mass and MES (2-(N-morpholine)ethanesulfonic acid) with a concentration of 0.1% by mass are filtered and sterilized with a 0.22μm microporous membrane.
[0130] During the enzymolysis process, every 20 minutes, manually shake the petri dish for 10 seconds to speed up t...
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