CN107853181B - Tobacco explant anti-browning method based on activated carbon - Google Patents
Tobacco explant anti-browning method based on activated carbon Download PDFInfo
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- CN107853181B CN107853181B CN201711329629.0A CN201711329629A CN107853181B CN 107853181 B CN107853181 B CN 107853181B CN 201711329629 A CN201711329629 A CN 201711329629A CN 107853181 B CN107853181 B CN 107853181B
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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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention relates to an activated carbon-based tobacco explant anti-browning method, which comprises the following steps: step (1), disinfecting seeds; step (2), seed culture; step (3), culturing tobacco explants; step (4), performing anti-browning culture; step (5), rooting culture; and (6) hardening and transplanting seedlings. The invention not only can effectively relieve the browning phenomenon of the tobacco explant, but also can improve the integral differentiation state of the explant. The root system not only has accelerated growth, but also has flexible texture and is not easy to break, and the transplanting survival rate of the rooted seedlings is high. Convenient use, low cost and good effect.
Description
Technical Field
The invention relates to a tobacco explant anti-browning method, in particular to an activated carbon-based tobacco explant anti-browning method, and belongs to the field of plant tissue culture.
Background
Browning of explants during plant tissue culture is an important factor affecting tissue culture. The research shows that the browning of the explant is caused by that polyphenol oxidase in the tissue is activated, and phenolic substances are oxidized to generate quinone substances which inhibit the activity of other enzymes and poison the whole explant tissue.
Current studies indicate that the causes of explant browning in tissue culture are: the genotype of the plant, the explant itself, the culture conditions, the culture medium, etc. Although the factors causing browning are large, selection of an appropriate explant and selection of an appropriate medium and culture conditions are effective in preventing browning. However, prolonged low light culture reduces the viability of the explants and even causes death.
Tobacco (A)Nicotiana tabacum) Not only important commercial crops, but also model plants for gene function research. The browning problem of tobacco in the tissue culture process is also frequent, and the browning can cause that the tobacco explants in the culture medium can not be continuously cultured and even die, so the browning prevention is the problem to be solved to the utmost extent in the tobacco tissue culture.
In addition, the tobacco root system is fragile and slow to grow due to improper use of the browning agent or improper selection of a culture medium and culture conditions, and further how to strengthen the toughness of the tobacco root system while solving the browning problem is a problem which needs to be solved urgently at present.
Disclosure of Invention
In order to solve the problems, the invention aims to provide an activated carbon-based tobacco explant anti-browning method, which can effectively relieve the browning problem in the tobacco tissue culture process, improve the explant survival rate of tobacco tissue culture and improve the toughness of a root system.
The specific scheme of the invention is as follows:
an activated carbon-based tobacco explant anti-browning method comprises the following steps:
step (1), disinfecting seeds;
step (2), seed culture: inoculating the seeds obtained in the step (1) into an MS culture medium for culture, and germinating the seeds to grow into seedlings; the seed culture conditions are as follows: the culture temperature is 25 + -1 deg.C, and the illumination intensity is 30-50 μmol/(m)2S) culturing for 60d under the condition of 16h/d of illumination time;
step (a)3) Culturing tobacco explants: punching the seedlings obtained in the step (2) by using a puncher to obtain a tobacco leaf disc, and culturing the tobacco leaf disc in an MS (Mass Spectrometry) differentiation culture medium under the culture conditions that: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 5d to obtain explants;
step (4), anti-browning culture: culturing the explant obtained in the step (3) in MS anti-browning culture medium, and culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing for 40d under the condition of dark culture at 25 ℃ for 8h/d, wherein the MS anti-browning culture medium is an MS basic culture medium added with 0.5-5g/L of active carbon, 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (5), rooting culture: culturing the explant obtained in the step (4) in an MS rooting culture medium, and culturing the explant at 28 ℃ for 16h/d under illumination with the illumination intensity of 30-50 mu mol/(m)2S), culturing at 25 ℃ in the dark for 8h/d, and rooting the tobacco when culturing for 10 d; the MS rooting culture medium is prepared by adding 0.5-5g/L of active carbon, 30g/L of sucrose and 4g/L of agar into an MS basic culture medium, and the pH value of the culture medium is 5.7.
Step (6), hardening and transplanting seedlings: and (3) opening the sealing film after the root system of the rooting plant obtained in the step (5) is developed for 25 days, pouring a proper amount of water into the culture bottle, placing the culture bottle in a greenhouse, placing the culture bottle in a shade place for growth for 1-2 days, placing the culture bottle in slightly dry natural soil together with the culture medium, sleeving a pricked transparent plastic bag, culturing for 7 days, and moving the culture bottle to the outside.
Further, in the step (3), the MS differentiation medium is MS minimal medium added with 0.5mg/L NAA, 2 mg/L6-benzyladenine 6-BA, 30g/L sucrose and 4g/L agar, and the pH value of the medium is 5.7.
Further, in the step (1), the specific steps of seed disinfection are as follows: taking plump tobacco seeds, transferring to a clean bench, soaking the seeds in 75% alcohol for 30s, washing with sterile water for 2 times, and adding 1% AgNO3Sterilizing for 10min, soaking and cleaning with sterile water for 5 times, and drying explant surface water with sterile filter paperInoculating, wherein the sterile water is ultrapure water sterilized by high pressure.
Further, in the step (2), the MS culture medium is MS minimal medium added with 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7.
Further, in the step (4), the MS anti-browning culture medium is prepared by the following method:
mixing 4-5g MS minimal medium, 10-40g sucrose and 1L ultrapure water, adjusting pH of the medium to 5.7, adding 0.5-5g activated carbon and 3-6g agar, and sterilizing at 121 deg.C for 15 min.
Compared with the prior art, the invention has the beneficial effects that:
1. after a proper amount of activated carbon is added into a culture medium, the browning phenomenon of the tobacco explant can be effectively relieved, and the integral differentiation state of the explant can be improved, wherein the browning rate is 17.8% -24.8%.
2. The root system of the tissue culture seedling added with proper amount of active carbon not only has accelerated growth, but also has flexible texture and is not easy to break, and the root system and the texture of the contrast are easy to be crisp. Finally, statistics shows that the transplanting survival rate of the rooted seedlings cultured by adding the activated carbon is 77.1-84.2%, which are all higher than that of a control.
3. The method of the invention has the advantages of convenient use, low cost and good effect.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.
Example 1
The tobacco explant anti-browning method based on activated carbon comprises the following steps:
step (1), disinfecting seeds: taking full tobacco seeds, and then moving the tobacco seeds to an ultraclean workbenchSoaking the seeds in 75% alcohol for 30s, washing with sterile water for 2 times, and adding 1% AgNO3Sterilizing for 10min, soaking and cleaning with sterile water for 5 times, and inoculating after drying water on explant surface with sterile filter paper, wherein the sterile water is ultrapure water sterilized under high pressure.
Step (2), seed culture: inoculating the seeds obtained in the step (1) into an MS culture medium, and culturing at the temperature of 25 +/-1 ℃ under the illumination intensity of 30 mu mol/(m)2S) culturing for 60 days under the condition of illumination time of 16h/d, and germinating and growing the seeds into seedlings, wherein the seed MS culture medium is an MS minimal medium added with 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (3), culturing tobacco explants: punching the seedlings obtained in the step (2) by using a puncher to obtain a tobacco leaf disc, and culturing the tobacco leaf disc in an MS (Mass Spectrometry) differentiation culture medium under the culture conditions that: culturing at 28 deg.C for 16h/d with illumination intensity of 30 μmol/(m)2S) culturing at 25 ℃ for 8h/d in the dark and for 5d to obtain an explant; the leaf disc MS differentiation culture medium is an MS basic culture medium added with 0.5mg/L of NAA, 2mg/L of 6-benzyladenine 6-BA, 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (4), anti-browning culture: placing the explant obtained in the step (3) in an MS anti-browning culture medium, and culturing the explant at 28 ℃ for 16h/d under illumination with the illumination intensity of 30 mu mol/(m)2S) and culturing for 40d under the condition of dark culture at 25 ℃ for 8h/d, wherein the MS anti-browning culture medium is an MS basic culture medium added with 1.5g/L of active carbon, 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7.
1L of browning prevention culture medium can be prepared at one time, 4.43g of MS basic culture medium, 30g of cane sugar and 1L of ultrapure water are mixed, the pH value of the culture medium is adjusted to be 5.7, then 1.5g of activated carbon and 4g of agar are added, sterilization is carried out for 15min at 121 ℃, after sterilization, the culture medium is shaken up to prevent the activated carbon from easily sinking to the bottom, 30 MS browning prevention culture medium 1L can be poured into 100mm culture dishes, 10 culture dishes 150mm and 15 culture bottles 300 mL;
step (5), rooting culture: placing the explant obtained in the step (4) in an MS rooting culture medium, and culturing at 28 ℃ in the light16h/d, the illumination intensity is 30-50 mu mol/(m)2S), culturing at 25 ℃ in the dark for 8h/d, wherein the tobacco roots when cultured for 10d, the MS rooting culture medium is MS basic culture medium added with 1.0g/L of active carbon, 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (6), hardening and transplanting seedlings: and (3) opening the sealing film after the root system of the rooting plant obtained in the step (5) is developed for 25 days, pouring a proper amount of water into the culture bottle, placing the culture bottle in a greenhouse, placing the culture bottle in a shade place for 1 day of growth, placing the culture bottle in slightly dry natural soil together with the culture medium, covering the culture bottle with a pricked transparent plastic bag, culturing for about 7 days, and moving the culture bottle to the outside.
In this example, the explant browning rate was 17.8%, and the statistical transplant survival rate was 78.7%.
The root system growth is accelerated, and the texture is flexible and not easy to break. The growth needs about 7 days, and the texture cannot be expressed by strength values
Example 2
The activated carbon-based tobacco explant anti-browning method of the present embodiment, wherein:
steps (1) to (3) are the same as in example 1;
step (4), anti-browning culture: placing the explant obtained in the step (3) in an MS anti-browning culture medium, and culturing for 16h/d at 28 ℃ under illumination with the illumination intensity of 30-50 mu mol/(m)2And s), culturing for 40d respectively under the condition of dark culture at 25 ℃ for 8h/d, wherein the MS anti-browning culture medium is an MS basic culture medium added with 2.5g/L of active carbon, 30g/L of cane sugar and 4g/L of agar, and the pH value of the culture medium is 5.7.
Step (5), rooting culture: placing the explant obtained in the step (4) in an MS rooting culture medium, and culturing for 16h/d at 28 ℃ under illumination with the illumination intensity of 45 mu mol/(m)2S), culturing at 25 ℃ in the dark for 8h/d, wherein tobacco roots are generated when 5d is cultured, the MS rooting culture medium is an MS basic culture medium added with 2.5g/L of active carbon, 30g/L of sucrose and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (6) was the same as in example 1.
In this example, the explant browning rate was 23.6%, and the statistical transplant survival rate was 84.2%.
Example 3
The tobacco explant anti-browning method based on activated carbon comprises the following steps:
in the steps (2) to (3), the illumination intensity is 50 [ mu ] mol/(m)2·s)；
Step (4), anti-browning culture: placing the explant obtained in the step (3) in an MS anti-browning culture medium, and culturing for 16h/d at 28 ℃ under illumination with illumination intensity of 50 mu mol/(m)2And s), culturing for 40d respectively under the condition of dark culture at 25 ℃ for 8h/d, wherein the MS anti-browning culture medium is an MS basic culture medium added with 5.0g/L of active carbon, 35g/L of sucrose and 6g/L of agar, and the pH value of the culture medium is 5.7.
Step (5), rooting culture: placing the explant obtained in the step (4) in an MS rooting culture medium, and culturing for 16h/d at 28 ℃ under illumination with illumination intensity of 50 mu mol/(m)2S), culturing at 25 ℃ in the dark for 8h/d, wherein tobacco roots are generated when culturing for 9d, the MS rooting culture medium is an MS basic culture medium added with 5.0g/L of active carbon, 30g/L of cane sugar and 4g/L of agar, and the pH value of the culture medium is 5.7;
step (6), hardening and transplanting seedlings: and (3) opening the sealing film after the root system of the rooting plant obtained in the step (5) is developed for 25 days, pouring a proper amount of water into the culture bottle, placing the culture bottle in a greenhouse, placing the culture bottle in a shade place for 2 days, placing the culture bottle in slightly dry natural soil together with the culture medium, covering the culture bottle with a pricked transparent plastic bag, culturing for about 7 days, and moving the culture bottle to the outside.
In this example, the explant browning rate was 24.8%, and the statistical transplantation survival rate was 77.1%.
The rest is the same as in example 1.
Control experiment:
the control experiment was conducted in the same manner as in example 1 except that no activated carbon was added, and the results are shown in Table 1:
TABLE 1 comparison of the effects of the control and examples 1-3
As can be seen from Table 1, the addition of activated carbon not only improves the browning rate, but also improves the survival rate of transplantation to some extent, and it makes the texture flexible and not easy to break, and the growth only needs about 7 days, which is not expected before.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
1. An activated carbon-based tobacco explant anti-browning method is characterized by comprising the following steps: the method comprises the following steps:
step (1), disinfecting seeds;
step (2), seed culture: inoculating the seeds obtained in the step (1) into an MS culture medium for culture, and germinating the seeds to grow into seedlings; the seed culture conditions are as follows: the culture temperature is 25 + -1 deg.C, and the illumination intensity is 30-50 μmol/(m)2S) culturing for 60d under the condition of 16h/d of illumination time;
step (3), culturing tobacco explants: punching the seedlings obtained in the step (2) by using a puncher to obtain a tobacco leaf disc, and culturing the tobacco leaf disc in an MS (Mass Spectrometry) differentiation culture medium under the culture conditions that: culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S), culturing at 25 ℃ for 8h/d in the dark, and culturing for 5d to obtain explants;
step (4), anti-browning culture: culturing the explant obtained in the step (3) in MS anti-browning culture medium, and culturing at 28 deg.C for 16h/d with illumination intensity of 30-50 μmol/(m)2S) culturing at 25 deg.C in dark for 8h/d for 40d, adding 0.5-5g/L activated carbon, 30g/L sucrose and 4g/L agar into MS minimal medium, and culturing in dark for 40dThe pH value of (1) is 5.7;
step (5), rooting culture: culturing the explant obtained in the step (4) in an MS rooting culture medium, and culturing the explant at 28 ℃ for 16h/d under illumination with the illumination intensity of 30-50 mu mol/(m)2S), culturing at 25 ℃ in the dark for 8h/d, and rooting the tobacco when culturing for 10 d; the MS rooting culture medium is prepared by adding 0.5-5g/L of active carbon, 30g/L of sucrose and 4g/L of agar into an MS basic culture medium, and the pH value of the culture medium is 5.7;
step (6), hardening and transplanting seedlings: and (3) opening the sealing film after the root system of the rooting plant obtained in the step (5) is developed for 25 days, pouring a proper amount of water into the culture bottle, placing the culture bottle in a greenhouse, placing the culture bottle in a shade place for growth for 1-2 days, placing the culture bottle in slightly dry natural soil together with the culture medium, sleeving a pricked transparent plastic bag, culturing for 7 days, and moving the culture bottle to the outside.
2. The activated carbon-based tobacco explant anti-browning method of claim 1, wherein: in the step (3), the MS differentiation medium is an MS minimal medium added with 0.5mg/L of NAA, 2mg/L of 6-benzyladenine 6-BA, 30g/L of sucrose and 4g/L of agar, and the pH value of the medium is 5.7.
3. The activated carbon-based tobacco explant anti-browning method of claim 1, wherein: in the step (1), the specific steps of seed disinfection are as follows: taking plump tobacco seeds, transferring to a clean bench, soaking the seeds in 75% alcohol for 30s, washing with sterile water for 2 times, and adding 1% AgNO3Sterilizing for 10min, soaking and cleaning with sterile water for 5 times, and inoculating after drying water on explant surface with sterile filter paper, wherein the sterile water is ultrapure water sterilized under high pressure.
4. The activated carbon-based tobacco explant anti-browning method of claim 1, wherein: in the step (2), the MS culture medium is MS minimal medium added with 30g/L sucrose and 4g/L agar, and the pH value of the culture medium is 5.7.
5. The activated carbon-based tobacco explant anti-browning method of claim 1, wherein: in the step (4), the preparation method of the MS anti-browning culture medium comprises the following steps:
mixing 4-5g MS minimal medium, 10-40g sucrose and 1L ultrapure water, adjusting pH of the medium to 5.7, adding 0.5-5g activated carbon and 3-6g agar, and sterilizing at 121 deg.C for 15 min.
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| CN115968787A (en) * | 2023-02-20 | 2023-04-18 | 广西农业职业技术大学 | Method for reducing browning of kidney bean in-vitro regeneration system |
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Non-Patent Citations (2)
| Title |
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| 不同防褐剂对烟草愈伤组织培养褐化现象的抑制效应;王颖等;《贵州农业科学》;20121231;第40卷(第1期);第26-27页，尤其是第26页摘要，第1.1和1.2节 * |
| 烟草组织培养的技术探讨;邹永梅等;《江苏教育学院学报（自然科学版）》;20070930;第24卷(第3期);第28-29页，尤其是第29页第1.3，2.1-2.1节 * |
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