CN108552059B - Plant tissue culture method for promoting strong roots of potato seedlings - Google Patents
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Classifications
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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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
-
- 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
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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the field of plant tissue culture, in particular to a tissue culture method for promoting strong roots of potato seedlings. The tissue culture method of the present invention comprises the steps of: (1) collecting and sterilizing materials; (2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia; (3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; (4) rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; the tissue culture method of the invention utilizes the isolated culture of the stem tip, thus overcoming the problem of large using amount of the seed potato; the invention optimizes the culture medium and culture conditions of primary culture and rooting culture, not only can make the potatoes have more roots and fast rooting, but also can make the tissue culture seedlings of the potatoes robust and be easier to transplant and survive.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a plant tissue culture method for promoting strong roots of potato seedlings.
Background
The plant tissue culture technology is developed in the beginning of the 20 th century, and refers to a process of in vitro culture of in vitro plant organs, tissues, cells, protoplasts and the like under a proper condition under an aseptic condition, induction generation of callus, adventitious buds and the like, and then formation of a complete plant. The tissue culture technology has the advantages of controlling the growth and differentiation of cells by utilizing various artificial culture conditions, and the like, powerfully promotes the cross development of plant physiology, pathology, pharmacy, genetics, breeding, biochemistry and other disciplines, is widely applied to agriculture, forestry, gardening, industry, pharmaceutical industry and other industries, generates huge economic benefit and social benefit, and has the following aspects: 1. in-vitro rapid propagation of the nursery stock; 2. cultivating virus-free seedlings; 3. the method is applied to plant breeding such as haploid breeding, protoplast fusion, culture medium transgenic breeding of embryo and endosperm and the like; 4. producing secondary metabolites such as Chinese medicinal components; 5. artificial seed and germplasm preservation; 6. gene function studies, etc.
The potato is a high-yield economic crop which is wide in distribution, strong in adaptability, high in yield, rich in nutrition, suitable for grains, vegetables, feeds and industrial raw materials, and is also one of ten kinds of nutritional foods in the world. The potato contains protein, carbohydrate, fat, calcium, phosphorus, zinc, iron, thiamine, nucleotide, vitamins, etc. And the amino acid-rich food contains rich amino acid, and is well appreciated by the development of modern nutritional foods. In addition, potatoes contain nutrients such as carbohydrates, fats, sugars, crude fibers, ash, and various vitamins, which are indispensable to the human body. At present, regeneration plant culture is carried out by using various explants of potato leaves, stem segments, petioles, roots and the like, and multi-step seedling culture is adopted. However, the existing potato field cultivation mainly adopts tuber propagation, and the tuber propagation has many advantages, but has obvious disadvantages of large potato seed consumption, low propagation coefficient and easy accumulation of virus, which leads to serious quality degradation.
And the test-tube plantlets obtained by tissue culture are often weak in growth vigor, undeveloped in root system and low in survival rate after transplantation, and the culture efficiency and further application effect of the test-tube plantlets are seriously influenced. Therefore, it is very important to establish a tissue culture method for promoting the growth of the roots of the potato seedlings.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a tissue culture method for promoting the strong root of potato seedlings, which adopts the isolated culture of the stem tip to overcome the problem of large using amount of seed potatoes; the invention optimizes the culture medium and culture conditions of primary culture and rooting culture, not only can make the potatoes have more roots and fast rooting, but also can make the tissue culture seedlings of the potatoes robust and be easier to transplant and survive.
The invention is realized by the following technical scheme:
a plant tissue culture method for promoting strong roots of potato seedlings comprises the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.2-2.5mg/L, the final concentration of ZT is 0.08-0.18mg/L, the final concentration of ethephon is 0.2-0.4mg/L, and the final concentration of uniconazole is 0.1-0.2 mg/L;
(4) rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; the rooting medium comprises the following components: MS culture medium, 0.5-3 mg/L of hymexazol, 25-30 g/L of cane sugar, 0.5-1.0 mg/L of cytokinin and 4.5-5.5 g/L of agar, and the pH value is 5.5-6.0.
Further, tween 80 is dropwise added into the mercuric chloride solution, and the final concentration of the tween 80 is 0.02%.
Further, the conditions of the primary culture are as follows: the culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
Further, the rooting culture conditions are as follows: the illumination is carried out for 12-14 h every day, the illumination intensity is 1500-2000 LX, and the culture temperature is 23-27 ℃.
Further, the primary medium consists of: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.5-1.8mg/L, the final concentration of ZT is 0.12-0.15mg/L, the final concentration of ethephon is 0.25-0.3mg/L, and the final concentration of uniconazole is 0.16-0.18 mg/L.
Further, the rooting medium consists of: MS culture medium, 1.5-2 mg/L hymexazol, 25-30 g/L cane sugar, 0.7-0.8 mg/L cytokinin and 5-5.2 g/L agar, and the pH value is 5.5-6.0.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a tissue culture method for promoting strong roots of potato seedlings, which uses stem tips to culture in vitro and overcomes the problem of large using amount of potato seeds;
(2) the pretreatment and the sterilization treatment adopted by the invention are combined, and the terminal buds of the potato seedlings can be effectively sterilized, so that the pollution rate of the explants is effectively reduced, and the explants cannot be damaged.
(3) The primary culture medium meets the requirements of the potato on the culture medium in the stages of bud section induction, subculture value increase, test tube miniature potato induction, rooting and seedling strengthening and the like, and has remarkable promoting effects on the induction rate of the potato bud explants, the differentiation rate of callus tissues, the germination rate and the rooting effect.
(4) The rooting culture medium can ensure that the potatoes have more roots and fast rooting, and can also ensure that the tissue culture seedlings of the potatoes are robust and are easier to transplant and survive.
Detailed Description
The following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention.
Example 1:
a plant tissue culture method for promoting strong roots of potato seedlings comprises the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; wherein the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.2mg/L, the final concentration of ZT is 0.08mg/L, the final concentration of ethephon is 0.2mg/L, and the final concentration of uniconazole is 0.1 mg/L; the primary culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
(4) Rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; wherein the rooting medium comprises the following components: MS culture medium, 0.5mg/L hymexazol, 25g/L cane sugar, 0.5mg/L cytokinin and 5g/L agar, and the pH value is 5.5-6.0. The rooting culture conditions are as follows: the illumination is carried out for 12 hours every day, the illumination intensity is 1500-2000 LX, and the culture temperature is 23 ℃.
Example 2:
a plant tissue culture method for promoting strong roots of potato seedlings comprises the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use; wherein 2 drops of Tween 80 are added into the mercuric chloride solution;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; wherein the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.5mg/L, the final concentration of ZT is 0.12mg/L, the final concentration of ethephon is 0.25mg/L, and the final concentration of uniconazole is 0.16 mg/L; the primary culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
(4) Rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; wherein the rooting medium comprises the following components: MS culture medium +1.5mg/L hymexazol +25g/L cane sugar +0.7mg/L cytokinin +5g/L agar, pH 5.8. The rooting culture conditions are as follows: the illumination is carried out for 12 hours every day, the illumination intensity is 1500-2000 LX, and the culture temperature is 25 ℃.
Example 3:
a plant tissue culture method for promoting strong roots of potato seedlings comprises the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; wherein the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.8mg/L, the final concentration of ZT is 0.15mg/L, the final concentration of ethephon is 0.3mg/L, and the final concentration of uniconazole is 0.18 mg/L; the primary culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
(4) Rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; wherein the rooting medium comprises the following components: MS culture medium +2mg/L hymexazol +30g/L cane sugar +0.8mg/L cytokinin +5.2g/L agar, pH 5.8. The rooting culture conditions are as follows: the culture medium is irradiated by light 123 every day, the illumination intensity is 1500-2000 LX, and the culture temperature is 24 ℃.
Example 4:
a plant tissue culture method for promoting strong roots of potato seedlings comprises the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use; wherein 2 drops of Tween 80 are added into the mercuric chloride solution;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; wherein the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 2.5mg/L, the final concentration of ZT is 0.18mg/L, the final concentration of ethephon is 0.4mg/L, and the final concentration of uniconazole is 0.20 mg/L; the primary culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
(4) Rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; wherein the rooting medium comprises the following components: MS culture medium +3mg/L hymexazol +30g/L cane sugar +1.0mg/L cytokinin +5.2g/L agar, pH 5.8. The rooting culture conditions are as follows: the illumination is carried out for 14 hours every day, the illumination intensity is 1500-2000 LX, and the culture temperature is 27 ℃.
Effect verification
1. Rate of differentiation into shoots
The primary culture differentiation into buds of the explants of examples 1, 2, 3 and 4 was observed and counted, and the results are shown in Table 1.
TABLE 1 explant differentiation into shoots Rate for each example
| Group of | Example 1 | Example 2 | Example 3 | Example 4 |
| Percentage of differentiation into shoots (%) | 95.6 | 98.6 | 98.4 | 95.8 |
As can be seen from Table 1, the explants in examples 1-4 all had high differentiation into shoots, indicating that the primary medium of the present invention is effective in promoting explant-induced differentiation. Meanwhile, the differentiation and bud formation rate in the examples 2 and 3 is obviously higher than that in the examples 1 and 4, which shows that the explant induced differentiation effect is better when the final concentration of 6-BA in the composition of the primary culture medium is 1.5-1.8mg/L, the final concentration of ZT is 0.12-0.15mg/L, the final concentration of ethephon is 0.25-0.3mg/L, and the final concentration of uniconazole is 0.16-0.18 mg/L.
2. Rooting rate
The rooting conditions of the sterile seedlings obtained by the subculture of the above examples 1-4 after rooting culture for 7 days were observed and counted, and the results are shown in Table 2.
TABLE 2 rooting conditions for rooting culture of aseptic seedlings in the examples
| Group of | Example 1 | Example 2 | Example 3 | Example 4 |
| Number of roots (%) | 100 | 100 | 100 | 100 |
| Root number (root) | 6.3 | 6.8 | 6.9 | 6.4 |
| Root case | Is relatively thick and strong | Rough and strong | Rough and strong | Is relatively thick and strong |
As can be seen from Table 2, the rooting rate of each group of aseptic seedlings is 100%, and the aseptic seedlings have more and robust roots, so that the rooting medium has a good effect of promoting the rooting of the aseptic seedlings, and is favorable for improving the survival rate and the growth speed of the potatoes in subsequent field planting. Meanwhile, the root shape conditions in the examples 2 and 3 are obviously superior to those in the examples 1 and 4, which shows that the MS culture medium, 1.5-2 mg/L hymexazol, 25-30 g/L cane sugar, 0.7-0.8 mg/L cytokinin and 5-5.2 g/L agar have better effects on promoting the rooting and strengthening the roots of the aseptic seedlings.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A plant tissue culture method for promoting strong roots of potato seedlings is characterized by comprising the following steps:
(1) material collection and disinfection: cutting terminal buds of vigorous potato seedlings in the field in 6 months, cutting off leaves, washing with tap water for 40-50min, sucking up water with sterile filter paper, soaking and washing with 75% alcohol for 25s on a super clean bench, sterilizing with 0.1% mercuric chloride for 3-6min, and finally washing with sterile water for 7-8 times for later use;
(2) stripping the stem tip: under a binocular dissecting mirror, peeling off outer leaves of the sterilized potato terminal buds, and only leaving 1-2 leaf primordia;
(3) primary culture: inoculating 5-6 stem tips with size of 0.1-0.2mm on primary culture medium for culture; the primary culture medium comprises the following components: adding 6-BA, ZT, ethephon and uniconazole into 1/2MS culture medium, wherein the final concentration of 6-BA is 1.5-1.8mg/L, the final concentration of ZT is 0.12-0.15mg/L, the final concentration of ethephon is 0.25-0.3mg/L, and the final concentration of uniconazole is 0.16-0.18 mg/L;
(4) rooting culture: inoculating the aseptic seedling in the step (3) into a rooting culture medium according to a single plant for culturing to obtain a detoxified test-tube seedling; the rooting medium comprises the following components: MS culture medium, 0.5-3 mg/L of hymexazol, 25-30 g/L of cane sugar, 0.5-1.0 mg/L of cytokinin and 4.5-5.5 g/L of agar, and the pH value is 5.5-6.0.
2. The method for promoting potato seedling strong root plant tissue culture according to claim 1, wherein tween 80 is added dropwise to the mercuric chloride solution, and the final concentration of tween 80 is 0.02%.
3. The method for culturing a plant tissue capable of promoting potato seedling strong root according to claim 1, wherein the conditions for the primary culture are as follows: the culture temperature is 25 ℃, the illumination intensity is 2000-3000lx, the illumination time is 12h/d, and the culture lasts 40 days.
4. The method for culturing plant tissues for promoting potato seedling strong root according to claim 1, wherein the rooting culture conditions are as follows: the illumination is carried out for 12-14 hours every day, the illumination intensity is 1500-2000 lx, and the culture temperature is 23-27 ℃.
5. The method for plant tissue culture for promoting potato seedling strong root according to claim 1, wherein the rooting medium consists of: MS culture medium, 1.5-2 mg/L hymexazol, 25-30 g/L cane sugar, 0.7-0.8 mg/L cytokinin and 5-5.2 g/L agar, and the pH value is 5.5-6.0.
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