CN108184675B - Rapid germination culture medium for primary culture of sequoia zhongshanensis - Google Patents
Rapid germination culture medium for primary culture of sequoia zhongshanensis Download PDFInfo
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- CN108184675B CN108184675B CN201810215028.5A CN201810215028A CN108184675B CN 108184675 B CN108184675 B CN 108184675B CN 201810215028 A CN201810215028 A CN 201810215028A CN 108184675 B CN108184675 B CN 108184675B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention relates to a rapid germination culture medium for the primary culture of sequoia intermedia, which comprises an induction culture medium and a strong seedling culture medium; the culture medium provided by the invention is rich in nutrient components, is suitable for proliferation and rapid germination of the sequoia zhongshanensis, greatly shortens the culture period of the sequoia zhongshanensis, and reduces the production cost; the germination rate of the explant obtained by primary culture of the sequoia zhongshanensis exceeds 98%, the average germination time is less than 18d, and the germination rate is high; can effectively reduce the browning rate of the explant during tissue culture, improve the survival rate of the taxus chinensis explant and improve the propagation efficiency.
Description
Technical Field
The invention relates to the technical field of tissue culture of sequoia intermedia, in particular to a rapid germination culture medium for primary culture of sequoia intermedia.
Background
The sequoia intermedia is a semi-evergreen tall tree with tall and straight trunk and beautiful tree shape, is a superior interspecific hybrid of 3 species of sequoia sempervirens, sequoia mexicana and sequoia fortunei, and is bred by a plant research institute of Chinese academy of sciences of Jiangsu province after years of experimental research. Has the characteristics of long green period, fast growth, water and humidity resistance, salt and alkali resistance, strong wind resistance, less plant diseases and insect pests, wide adaptability, fast growth, excellent material quality and the like which are better than those of parents. Not only is a good tree species for farmland forest networks and beach forestation, but also has wide development prospect in the aspects of urban landscaping, water conservation, water and soil conservation, green landscape channels, ecological construction and the like.
At present, the main means for breeding the seedlings of the taxus chinensis is cuttage, which can ensure that the seedlings keep the excellent characteristics of mother trees, but the lignification of the taxus chinensis seeds is earlier, the cuttage survival rate is low, the number of the seeds is limited, the speed is low, the seedling yield is low, and the large-area popularization and planting of the taxus chinensis is restricted. Therefore, the tissue culture propagation is the first choice for solving the problem of the new excellent clone propagation of the sequoia intermedia, and the research on a culture medium capable of improving the primary culture germination rate of the sequoia intermedia is the urgent priority for improving the rapid propagation of the sequoia intermedia.
Disclosure of Invention
In order to solve the technical problems, the rapid germination medium for the primary culture of the sequoia intermedia is provided, which can improve the germination rate of the sequoia intermedia, shorten the average germination time and reduce the browning rate.
The invention provides the following technical scheme:
a rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium; the induction culture medium can effectively induce the generation of adventitious buds, promote the formation of adventitious roots, reduce the germination time and improve the germination rate of the sequoia intermedia; the strong seedling culture medium can effectively enhance the proliferation effect of the sequoia intermedia and improve the survival rate of the tissue culture of the sequoia intermedia;
wherein the formula of the induction culture medium is 2/3MS culture medium, 3-5 mg/L2, 4-D, 5-7mg/L NAA, 5-10mg/L banana puree, 0.01-0.05mg/L TDZ, 2-4 mg/L6-BA, 10-20g/L agar, 0.02-0.06 organic matter, 4-6g sucrose and 2-3mg/L camphor oil; the 2/3 culture medium is comprehensive in nutrition, wherein TDZ has strong cell division activity, regeneration and propagation of plant buds are promoted, eyes of the buds are broken, the cluster bud induction time can be obviously shortened by matching with 6-BA, the explant browning rate can be reduced by matching with organic matters and camphor oil, and the matching between other components can provide a carbon source and a growth platform for the Chinese fir explant, so that the production cost is reduced, the germination rate of primary culture is improved, and the germination time is shortened.
The formula of the strong seedling culture medium is 1/2MS culture medium, 10-30mg/L compound sodium nitrophenolate, 10-30mg/L ascorbic acid, 5-10mg/L GA, 6-10mg/L IAA, 0.03-0.06mg/L PVP, 10-20mg/L active carbon powder, 2-4g/L sucrose and 10-20g/L agar; wherein the ascorbic acid is a reducing substance, has no side effect on the plant body of the tissue culture, can reduce the oxidized quinine into a phenolic substance, prevents the quinone substance from further spontaneously polymerizing to form a pigment substance, and the combination of the PVP and the activated carbon powder can effectively reduce the browning rate of the seedlings and improve the survival rate of the seedlings; the combination of the compound sodium nitrophenolate and other components can improve the height of the seedling of the taxus chinensis, the number of the leaves, the fresh weight of the leaves, the quality of the roots and the length of the roots, improve the survival rate of the taxus chinensis when being transplanted and improve the economic benefit.
Further, the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1. The glutathione reductase can scavenge free radicals and active oxygen, and can also be used as antioxidant to inhibit oxidation of phenols. Cysteine can inhibit enzyme activity by irreversibly combining with copper ions of PPO active sites, or replace histidine residues of PPO active sites, reduce the combination of phenols at the cell disruption parts of Taxus chinensis explants and polyphenol oxidase to generate brown quinone, and reduce browning rate.
Furthermore, the preparation method of the rapid germination culture medium for the primary culture of the sequoia intermedia comprises the following steps:
s1, cutting bananas into cube blocks with the length of 1-1.5cm, adding water according to the formula amount, and stirring to obtain banana puree for later use;
s2, preparing an induction culture solution from the banana puree obtained in the step S1 and other elements in the formula according to the formula amount, and adjusting the pH value of the induction culture solution to 6.0-6.5;
s3, preparing strong seedling culture solution according to the formula amount, and adjusting the pH value of the strong seedling culture solution to 6.0-6.5;
s4, subpackaging the induction culture solution and the strong seedling culture solution obtained in the steps S2 and S3 respectively, and sterilizing at 110-120 ℃ for 2 h;
s5, cooling the separately-packaged induction culture solution and strong seedling culture solution obtained in the step S4 to solidify, and obtaining a finished product.
Further, in the steps S2 and S3, 1mol/L acetic acid solution and 1.5mol/L NaOH solution are used for adjusting the pH value. Wherein the pH value is adjusted by using an acetic acid solution, and the acetic acid solution is weak acid and does not influence active ingredients in other components in the culture solution.
The invention has the beneficial effects that:
(1) through the mutual synergistic effect of TDZ and 6-BA of the induction culture medium, the induction effect is good, the germination rate of the explant obtained by primary culture of the sequoia intermedia exceeds 98 percent, the average germination time is less than 18 days, and the germination rate is high;
(2) through the mutual synergistic effect of ascorbic acid and Gibberellin (GA) in the strong seedling culture medium, the nutrient composition is rich, the culture medium is suitable for the proliferation and rooting of the taxus chinensis, the culture period of the taxus chinensis is greatly shortened, and the production cost is reduced;
(3) the culture medium can effectively reduce the browning rate of the explant during tissue culture, improve the survival rate of the taxus chinensis explant and improve the economic benefit.
Detailed Description
Example 1
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 3 mg/L2, 4-D, 5mg/L NAA, 5mg/L banana puree, 0.01mg/L TDZ, 2 mg/L6-BA, 10g/L agar, 0.02 organic matter, 4g cane sugar and 2mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 10mg/L compound sodium nitrophenolate, 10mg/L ascorbic acid, 5mg/L GA, 6mg/L IAA, 0.03mg/L PVP, 10mg/L activated carbon powder, 2g/L sucrose and 10g/L agar.
Example 2
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 5 mg/L2, 4-D, 7mg/L NAA, 10mg/L banana puree, 0.05mg/L TDZ, 4 mg/L6-BA, 20g/L agar, 0.06 organic matter, 6g cane sugar and 3mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 30mg/L compound sodium nitrophenolate, 30mg/L ascorbic acid, 10mg/L GA, 10mg/L IAA, 0.06mg/L PVP, 20mg/L activated carbon powder, 4g/L sucrose and 20g/L agar.
Comparative example 1
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 3 mg/L2, 4-D, 5mg/L NAA, 5mg/L banana puree, 0.01mg/L TDZ, 10g/L agar, 0.02 organic matter, 4g sucrose and 2mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 10mg/L compound sodium nitrophenolate, 10mg/L ascorbic acid, 5mg/L GA, 6mg/L IAA, 0.03mg/L PVP, 10mg/L activated carbon powder, 2g/L sucrose and 10g/L agar.
Comparative example 2
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 3 mg/L2, 4-D, 5mg/L NAA, 5mg/L banana puree, 2 mg/L6-BA, 10g/L agar, 0.02 organic matter, 4g cane sugar and 2mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 10mg/L compound sodium nitrophenolate, 10mg/L ascorbic acid, 5mg/L GA, 6mg/L IAA, 0.03mg/L PVP, 10mg/L activated carbon powder, 2g/L sucrose and 10g/L agar.
Comparative example 3
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 5 mg/L2, 4-D, 7mg/L NAA, 10mg/L banana puree, 0.05mg/L TDZ, 4 mg/L6-BA, 20g/L agar, 0.06 organic matter, 6g cane sugar and 3mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 30mg/L compound sodium nitrophenolate, 10mg/L GA, 10mg/L IAA, 0.06mg/L PVP, 20mg/L activated carbon powder, 4g/L sucrose and 20g/L agar.
Comparative example 4
A rapid germination culture medium for primary culture of Sequoia intermedia comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 5 mg/L2, 4-D, 7mg/L NAA, 10mg/L banana puree, 0.05mg/L TDZ, 4 mg/L6-BA, 20g/L agar, 0.06 organic matter, 6g cane sugar and 3mg/L camphor oil; the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1.
The formula of the strong seedling culture medium is 1/2MS culture medium, 30mg/L compound sodium nitrophenolate, 30mg/L ascorbic acid, 10mg/L GA, 10mg/L IAA, 4g/L sucrose and 20g/L agar.
Examples 1-2 and comparative examples 1-4 were prepared as follows:
s1, cutting bananas into cube blocks with the length of 1-1.5cm, adding water according to the formula amount, and stirring to obtain banana puree for later use;
s2, preparing an induction culture solution from the banana puree obtained in the step S1 and other elements in the formula according to the formula amount, and adjusting the pH value of the induction culture solution to 6.0-6.5;
s3, preparing strong seedling culture solution according to the formula amount, and adjusting the pH value of the strong seedling culture solution to 6.0-6.5;
s4, subpackaging the induction culture solution and the strong seedling culture solution obtained in the steps S2 and S3 respectively, and sterilizing at 110-120 ℃ for 2 h;
s5, cooling the separately-packaged induction culture solution and strong seedling culture solution obtained in the step S4 to solidify, and obtaining a finished product.
In an aseptic environment, the branches of the taxus chinensis with terminal buds and axillary buds are respectively inoculated in the culture media prepared in the examples 1-2 and the comparative examples 1-4, and then are cultured under the conditions of 12h light/12 h dark cycle, the light intensity of 000lux and the temperature of 25 +/-1 ℃. The germination rate, germination time and browning rate of the Taxus chinensis explants were recorded after 30 days of culture, and the results are shown in the table below.
| Experimental group | Germination Rate (%) | Average time to germination (d) | Browning rate (%) |
| Example 1 | 98.31 | 17.58 | 6.78 |
| Example 2 | 99.54 | 16.54 | 7.34 |
| Comparative example 1 | 74.81 | 20.33 | 7.16 |
| Comparative example 2 | 87.11 | 19.61 | 6.54 |
| Comparative example 3 | 97.69 | 17.31 | 28.65 |
| Comparative example 4 | 98.01 | 16.14 | 31.07 |
As can be seen from the experimental data of the example 1 and the comparative examples 1-2 in the table above, the TDZ and the 6-BA are matched with each other to promote the regeneration and the propagation of plant buds, break the eyes of the buds and obviously shorten the cluster bud induction time; the experimental data of example 2 and comparative examples 3-4 show that ascorbic acid can reduce oxidized quinone into phenolic substances, prevent quinone substances from further spontaneous polymerization to form pigment substances, and the combination of PVP and activated carbon powder can effectively reduce the browning rate of seedlings and improve the survival rate of the seedlings. The culture medium provided by the invention has rich nutrient components, is suitable for the proliferation and rooting of the sequoia intermedia, greatly shortens the culture period of the sequoia intermedia, and reduces the production cost; the germination rate of the explant obtained by primary culture of the sequoia zhongshanensis exceeds 98%, the average germination time is less than 18d, and the germination rate is high; can effectively reduce the browning rate of the explant during tissue culture, improve the survival rate of the taxus chinensis explant and improve the economic benefit.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (1)
1. A rapid germination culture medium for primary culture of sequoia zhongshanensis is characterized in that: comprises an induction culture medium and a strong seedling culture medium;
the formula of the induction culture medium is 2/3MS culture medium, 3-5 mg/L2, 4-D, 5-7mg/L NAA, 5-10mg/L banana puree, 0.01-0.05mg/L TDZ, 2-4 mg/L6-BA, 10-20g/L agar, 0.02-0.06 mg/L organic matter, 4-6 g/L sucrose and 2-3mg/L camphor oil;
the formula of the strong seedling culture medium is 1/2MS culture medium, 10-30mg/L compound sodium nitrophenolate, 10-30mg/L ascorbic acid, 5-10mg/L GA, 6-10mg/L IAA, 0.03-0.06mg/L PVP, 10-20mg/L active carbon powder, 2-4g/L sucrose and 10-20g/L agar;
the organic matter is a mixture of glutathione reductase and cysteine, and the mass ratio of the glutathione reductase to the cysteine is 2: 1;
the preparation method of the rapid germination culture medium for the primary culture of the sequoia intermedia comprises the following steps:
s1, cutting bananas into cube blocks with the length of 1-1.5cm, adding water according to the formula amount, and stirring to obtain banana puree for later use;
s2, preparing an induction culture solution from the banana puree obtained in the step S1 and other elements in the formula according to the formula amount, and adjusting the pH value of the induction culture solution to 6.0-6.5;
s3, preparing a strong seedling culture solution according to the formula amount, and adjusting the pH value of the strong seedling culture solution to 6.0-6.5;
s4, subpackaging the induction culture solution and the strong seedling culture solution obtained in the steps S2 and S3 respectively, and sterilizing at 110-120 ℃ for 2 hours;
s5, cooling the separately-packaged induction culture solution and strong seedling culture solution obtained in the step S4 to solidify to obtain a finished product;
in the steps S2 and S3, 1mol/L acetic acid solution and 1.5mol/L NaOH solution are adopted to adjust the pH value.
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