CN113692970A - Method for improving rooting rate and root growth effect of tissue culture seedlings of apples - Google Patents
Method for improving rooting rate and root growth effect of tissue culture seedlings of apples Download PDFInfo
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- CN113692970A CN113692970A CN202111091464.4A CN202111091464A CN113692970A CN 113692970 A CN113692970 A CN 113692970A CN 202111091464 A CN202111091464 A CN 202111091464A CN 113692970 A CN113692970 A CN 113692970A
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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
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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
- A01H4/002—Culture media for tissue culture
Abstract
The invention discloses a method for improving rooting rate and growth effect of tissue culture seedlings of apples, which comprises the following steps: 1) and (3) inducing axillary buds: inoculating axillary buds emitted in spring to an axillary bud induction culture medium for culture at 24 +/-1 ℃ for 12h/d for 40-45 days; 2) and (3) differentiation and proliferation culture of seedlings: transferring the induced axillary buds to a proliferation culture medium, irradiating for 12h/d, cutting off seedlings with the growth height of more than 1cm every 35-40 days for propagation, wherein the propagation times are not more than 8 generations; 3) rooting culture of seedlings: cutting off the tissue culture seedling with the growth height of more than 2cm, inoculating the cut tissue culture seedling on a rooting culture medium, and placing the cut tissue culture seedling at 24 +/-1 ℃ for 12h/d for illumination; compared with the conventional culture medium, the method shortens the root germination time by about 13 days and the culture time by about 20 days.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a method for improving rooting rate and root growth effect of tissue culture seedlings of apples.
Background
The apple is a deciduous tree and is used as a cross-pollinating plant, most varieties of self-flowers cannot fruit, and the propagation method generally adopts cutting, grafting and tissue culture modes. The plant tissue culture is a vegetative propagation technology developed in recent decades, and is a technology for inducing adventitious buds, adventitious roots and callus under the conditions of sterile and proper artificial culture medium, temperature and illumination light by utilizing isolated organs, tissues or cells and protoplasts of a plant body, and finally forming a complete plant through propagation, proliferation and rooting, so that the plant can be multiplied in a certain time, and a large amount of research time and precious plants can be saved.
The tissue culture of herbaceous plants is relatively easy, the tissue culture of woody plants takes longer time, the induction and proliferation stages are relatively stable, and the rooting stage culture is a key and is relatively difficult for woody plants. At present, the in-vitro rooting technology such as matrix culture, water culture, aerosol culture, filter paper method and the like is widely applied to tissue culture of flowers and precious nursery stocks for rooting in test tubes and rooting outside test tubes.
The whole process of the conventional tissue culture of the apples is generally carried out in a culture medium, axillary buds of the apples are treated in spring and then inoculated in an induction culture medium, the pH of all the culture medium is 5.8, the sucrose is 30g/L, the agar is 5.5g/L, the sterilization is carried out for 20min at the temperature of 121 ℃, the induction culture medium is MS + BA1.0mg/ml + IAA0.2mg/ml, the temperature is 24 +/-1 ℃, the illumination is carried out for 12h/d, and the culture is carried out for about 40 days. Transferring the induced axillary buds to a multiplication culture medium, wherein the multiplication culture medium is MS +6-BA2.5mg/L + NAA0.1mg/L, 24 +/-1 ℃, 12h/d illumination, cutting off seedlings with the growth height of more than 1cm for propagation every 35-40 days, and the propagation times are not more than 8 generations. Cutting off the seedling growing over 2cm, inoculating the cut seedling on a rooting culture medium, 1/2MS + NAA0.5mg/L + IAA1.0mg/L, placing the cut seedling at 24 +/-1 ℃ under 12h/d illumination, starting callus to appear about 20 days after culture, starting root bud growth on the callus at 25-30 days, culturing for 50-55 days, wherein the rooting rate is about 65-75%, and carrying out acclimatization and transplantation after culturing for 65 days. The total time is about 370 days, and the tissue culture seedlings of the apples can be induced and cultured in batches.
However, in the rooting culture stage of woody plant tissue culture, 80% of the roots of the tissue culture seedlings are planted on calluses growing at the bottom of the seedlings in the culture medium, and the calluses are easy to fall off during hardening and cleaning, so that rootless seedlings are caused, transplanting cannot grow healthily, the transplanting survival rate of the tissue culture seedlings is reduced, and the rooting culture and hardening and transplanting stages take longer about 65 days, so that the input cost of woody tissue culture is high.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the rooting rate of an apple tissue culture seedling, shortening the culture time and having a good growth effect on a seedling root system by using different sterilization culture mediums in the rooting process of the apple tissue culture seedling, so as to solve the defects caused in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for improving rooting rate and growth effect of tissue culture seedlings of apples comprises the following steps:
1) and (3) inducing axillary buds: inoculating axillary buds emitted in spring to an axillary bud induction culture medium for culture at 24 +/-1 ℃ for 12h/d for 40-45 days;
2) and (3) differentiation and proliferation culture of seedlings: transferring the induced axillary buds to a proliferation culture medium, irradiating for 12h/d, cutting off seedlings with the growth height of more than 1cm every 35-40 days for propagation, wherein the propagation times are not more than 8 generations;
3) rooting culture of seedlings: cutting off the tissue culture seedling with the growth height of more than 2cm, inoculating the cut tissue culture seedling on a rooting culture medium, and placing the cut tissue culture seedling at 24 +/-1 ℃ for 12h/d illumination.
Preferably, the axillary bud induction medium: MS + BA1.0-1.5mg/ml + IAA0.2-0.5mg/ml, pH5.8, sucrose 30g/L, agar 5.5g/L, sterilizing at 121 deg.C for 20 min.
Preferably, the propagation medium: MS +6-BA2.5-3.0mg/L + NAA0.1-0.3mg/L, pH5.8, sucrose 30g/L, agar 5.5g/L, sterilizing at 121 deg.C for 20 min.
Preferably, the rooting culture medium:
1)20g of coconut coir and 20ml of rooting culture solution;
2)8g of coconut coir, 2g of perlite and 20ml of rooting culture solution;
3) 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, pH5.7-5.8, sucrose 30g/L, agar 5.5g/L as control.
Preferably, all substrates and media are sterilized at 121 ℃ for 20 min.
Preferably, the rooting culture solution: 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, sucrose 30 g/L.
The beneficial effect of adopting above technical scheme is: the method provided by the invention can improve the rooting rate of the tissue culture seedlings of the apples, the roots of the seedlings begin to sprout at the bottom of the seedlings at the 7 th day of the rooting stage, 60% of the roots at the bottom of the seedlings grow at the 14 th day, the rooting rate reaches 95% at the 30 th day of the cultivation, the roots grow well, the number of the roots is more than 6, and the height of the seedlings is more than 4cm, so that the hardening transplantation can be carried out. Compared with the conventional culture medium, the method shortens the root germination time by about 13 days and the culture time by about 20 days.
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FIG. 120 g coconut coir +20ml culture medium cultured for 30 days;
FIG. 28g coconut husk +2g perlite +20ml culture broth cultured for 30 days;
FIG. 3 the medium was cultured for 30 days.
Detailed Description
Preferred embodiments of the present invention are described in detail below.
The method provided by the invention is a method for rapidly culturing the root system germination and growth of the tissue culture seedling of the apple by adopting 20g of coconut husk and 20ml of culture solution, 8g of coconut husk and 2g of perlite and 20ml of culture solution and a culture medium (contrast) as a culture medium in the rooting stage of the tissue culture seedling of the apple, taking tissue culture of a rootstock of Lijiang chastetree fruit of the apple as an example, and comprises the following specific processes:
and (3) inducing axillary buds: cleaning axillary buds sent in spring, cleaning in an ultra-clean workbench, cleaning with alcohol for 30 seconds, cleaning with sterile water for 2 times, shaking and sterilizing with 0.1% mercuric chloride for 10-12 minutes, cleaning with sterile water for 3-4 times, cutting into stem segments with 1-2 axillary buds, inoculating 3-4 axillary buds in each bottle, inoculating in an axillary bud induction culture medium MS + BA1.0-1.5mg/ml + IAA0.2-0.5mg/ml, sterilizing at pH5.8, agar 5.5g/L, sucrose 30g/L and 121 ℃ for 20 minutes, placing in a tissue culture rack for culturing at the temperature of 24 +/-1 ℃, 12h/d illumination, culturing for 40-45 days, observing the growth of the axillary buds, cutting and inoculating in a differentiation and proliferation culture medium for continuous culture when the length of the axillary buds exceeds 1 cm;
and (3) differentiation and proliferation culture of seedlings: inoculating the induced axillary buds into a proliferation culture medium for continuous culture, and sterilizing for 20min at 121 ℃ by MS +6-BA2.5-3.0mg/L + NAA0.1-0.3mg/L, pH5.8, agar 5.5g/L and sucrose 30 g/L. Placing the seedlings on a tissue culture frame for continuous culture at the temperature of 24 +/-1 ℃ under the illumination of 12h/d, cutting off seedlings with the growth height of more than 1cm every 35-40 days for propagation, wherein the propagation times are not more than 8 generations.
Rooting culture of seedlings: cutting off the tissue culture seedling with the growth of more than 2cm, inoculating the cut tissue culture seedling to rooting culture mediums I, II and III, and placing the tissue culture seedlings at 24 +/-1 ℃ for 12h/d illumination. 20g of coconut coir and 20ml of rooting culture solution; ② 8g of coconut coir, 2g of perlite and 20ml of rooting culture solution; (rooting culture solution 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, pH5.7-5.8, sucrose 30g/L) 3 Mi culture medium 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, pH5.8, sucrose 30g/L, agar 5.5g/L as control. All the substrates and culture media were sterilized at 121 ℃ for 20 min. And (3) taking root growth of the root system of the Lijiang mountain vitex rotundifolia of the apple as a control, treating about 100 seedlings with the length of 2cm in each group, observing the root growth conditions from the 7 th day, the 14 th day, the 30 th day, the 45 th day and the 55 th day, and recording the root growth conditions of the seedlings in each treatment group. From the table 1, it can be seen that from the 7 th day, the root system germination appears at the bottom of the tissue culture seedlings; processing the bottom of the tissue culture seedling to sprout; and the contrast treatment group has no change. On 14 th day, firstly, treating 60% of seedlings in the group to grow roots; ② 58% of the seedlings in the treatment group grow; and thirdly, callus appears at the bottom of the seedlings of the control treatment group. On the 30 th day, firstly, 95% of the roots of the seedlings in the treatment group grow, the height of the seedlings is more than 4cm, and the roots grow well; secondly, 95% of the roots of the seedlings in the treatment group grow, the height of the seedlings is more than 4cm, the roots grow well, and more fibrous roots exist; and thirdly, the control treatment group sends out root systems on the callus tissues at the bottom of 30 percent of the seedlings. On 45 th day, processing 95% of seedlings with root systems to be hardened; ② 95 percent of the seedlings in the treatment group can be hardened; and thirdly, giving out root systems on 60 percent of calluses at the bottoms of the seedlings of the control treatment group. On the 55 th day, the transplanting survival rate of the treatment group reaches 100 percent; ② the transplanting survival rate of the treatment group reaches 100 percent; thirdly, 70% of callus at the bottom of the seedling of the control treatment group sends out roots, hardening seedling transplantation can be carried out after 65 days, hardening seedling transplantation time is 7 days, during transplantation, the roots fall off along with the callus, no-root seedlings are caused, the transplantation loss is about 20% -35%, and the survival rate after transplantation is 65%.
TABLE 1 rooting substrate and medium for tissue culture of malus baccata, rooting stage registration form
FIG. 1 shows that 20g of coconut coir plus 20ml of culture medium are cultured for 30 days; FIG. 28g coconut coir +2g perlite +20ml culture solution for 30 days, FIG. 3 culture medium for 30 days, and it can be seen from FIG. 1, FIG. 2 and FIG. 3 that the induction and growth time of the root system of the tissue culture seedling can be shortened by the method provided by the present invention.
In conclusion, the invention adopts the sterilization culture medium, namely 20g of coconut coir plus 20ml of culture solution and 8g of coconut coir plus 2g of perlite plus 20ml of culture solution as the culture medium for the tissue culture rooting stage of apple seedlings, and can culture a large number of strong and hard tissue culture seedlings which are not easy to fall off and have good root growth vigor. The total culture time of the conventional apple tissue culture is about 370 days, and the culture time of the rooting stage is 50-55 days. The adoption of different sterilization matrixes shortens the rooting induction time by about 10-14 days and the rooting culture and seedling hardening and transplanting time by about 30-35 days.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (6)
1. A method for improving rooting rate and growth effect of tissue culture seedlings of apples is characterized by comprising the following steps:
1) and (3) inducing axillary buds: inoculating axillary buds emitted in spring to an axillary bud induction culture medium for culture at 24 +/-1 ℃ for 12h/d for 40-45 days;
2) and (3) differentiation and proliferation culture of seedlings: transferring the induced axillary buds to a proliferation culture medium, irradiating for 12h/d, cutting off seedlings with the growth height of more than 1cm every 35-40 days for propagation, wherein the propagation times are not more than 8 generations;
3) rooting culture of seedlings: cutting off the tissue culture seedling with the growth height of more than 2cm, inoculating the cut tissue culture seedling on a rooting culture medium, and placing the cut tissue culture seedling at 24 +/-1 ℃ for 12h/d illumination.
2. The method for improving rooting rate and growth effect of tissue culture seedlings of apples according to claim 1, wherein,
the axillary bud induction culture medium: MS + BA1.0-1.5mg/ml + IAA0.2-0.5mg/ml, pH5.8, sucrose 30g/L, agar 5.5g/L, sterilizing at 121 deg.C for 20 min.
3. The method for improving rooting rate and growth effect of tissue culture seedlings of apples according to claim 2, wherein,
the proliferation culture medium: MS +6-BA2.5-3.0mg/L + NAA0.1-0.3mg/L, pH5.8, sucrose 30g/L, agar 5.5g/L, sterilizing at 121 deg.C for 20 min.
4. The method for improving rooting rate and growth effect of tissue culture seedlings of apples according to claim 1, wherein the rooting culture medium comprises:
1)20g of coconut coir and 20ml of rooting culture solution;
2)8g of coconut coir, 2g of perlite and 20ml of rooting culture solution;
3) 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, pH5.7-5.8, sucrose 30g/L, agar 5.5g/L, as control.
5. The method for improving rooting rate and growth effect of tissue culture seedlings of apples according to claim 1, wherein all the substrates and the culture media are sterilized at 121 ℃ for 20 min.
6. The method for improving rooting rate and growth effect of tissue culture seedlings of apples according to claim 4, wherein,
the rooting culture solution comprises: 1/2MS + NAA0.5-1.0mg/L + IAA1.0-1.5mg/L, pH5.8, sucrose 30 g/L.
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Citations (4)
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| JP2005102516A (en) * | 2003-09-26 | 2005-04-21 | Nippon Paper Industries Co Ltd | Method for rooting plant tissue |
| CN108124773A (en) * | 2018-01-29 | 2018-06-08 | 宝鸡松良农业科技有限公司 | A kind of method that tissue cultures are carried out using apple stem section |
| CN108401907A (en) * | 2018-04-25 | 2018-08-17 | 河南省农业科学院 | A kind of Apples Dwarf Stocks M9T337 tissue cultures volume production method |
| KR102200064B1 (en) * | 2019-07-24 | 2021-01-08 | 충청북도 (관리부서:충청북도 농업기술원) | Method of producing virus free M.9 and M.26 dwarf apple tree rootstock using apical meristem culture |
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- 2021-09-17 CN CN202111091464.4A patent/CN113692970A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005102516A (en) * | 2003-09-26 | 2005-04-21 | Nippon Paper Industries Co Ltd | Method for rooting plant tissue |
| CN108124773A (en) * | 2018-01-29 | 2018-06-08 | 宝鸡松良农业科技有限公司 | A kind of method that tissue cultures are carried out using apple stem section |
| CN108401907A (en) * | 2018-04-25 | 2018-08-17 | 河南省农业科学院 | A kind of Apples Dwarf Stocks M9T337 tissue cultures volume production method |
| KR102200064B1 (en) * | 2019-07-24 | 2021-01-08 | 충청북도 (관리부서:충청북도 농업기술원) | Method of producing virus free M.9 and M.26 dwarf apple tree rootstock using apical meristem culture |
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