CN109329026B - Method for obtaining sweet potato virus-free seedlings through high-temperature and variable-temperature treatment - Google Patents
Method for obtaining sweet potato virus-free seedlings through high-temperature and variable-temperature treatment Download PDFInfo
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- CN109329026B CN109329026B CN201811453894.4A CN201811453894A CN109329026B CN 109329026 B CN109329026 B CN 109329026B CN 201811453894 A CN201811453894 A CN 201811453894A CN 109329026 B CN109329026 B CN 109329026B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of 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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- 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
Abstract
The invention discloses a method for obtaining sweet potato virus-free seedlings by high-temperature-variable-temperature treatment, and belongs to the technical field of virus-free sweet potato seedling cultivation. The method for obtaining the sweet potato virus-free seedlings by high-temperature and variable-temperature treatment comprises the steps of carrying out high-temperature treatment on the rooted sweet potato seedlings for 5 hours at 40-45 ℃ every day, then carrying out high-temperature and variable-temperature treatment at 28 ℃, and continuously treating for 30-40 days; the method for obtaining the virus-free sweet potato seedlings through high-temperature-variable-temperature treatment has good virus passivation effect, the stripped stem tips are long, the stem tips are easy to strip, the efficiency is high, and the adventitious buds grow fast; the regenerated seedling has good rooting quality, stout seedling and high survival rate.
Description
Technical Field
The invention belongs to the technical field of cultivation of detoxified sweet potato seedlings, and particularly relates to a method for obtaining detoxified sweet potato seedlings by high-temperature-variable-temperature treatment.
Background
Sweet potatoes are important crops processed from grains, feeds and industrial raw materials, and play an important role in agriculture and national economy. The sweet potato is rich in nutrition, and is more and more favored by people in recent years along with the improvement of health care consciousness of people. Sweet potatoes belong to root crops, and are propagated by utilizing a nutrient body in the production process, so that the sweet potatoes are easily infected by viruses, and the virus density is higher and higher along with the prolonging of the cultivation life of the bred variety. More than 20 kinds of sweet potato infecting viruses have been reported at present. The main Chinese diseases are Sweet Potato Feathery Mottle Virus (SPFMV), Sweet Potato Latent Virus (SPLV), Sweet Potato Vein Mosaic Virus (SPVMV), sweet potato mild mottle mosaic virus (SPMMV), sweet potato leaf roll virus (SPLCV), Cucumber Mosaic Virus (CMV) and Tobacco Mosaic Virus (TMV), and the more serious diseases are SPFMMV, SPLV, SPLCV and the like. Sweet potato virus can be transmitted through root tuber, insect vector, grafting and machinery. The virus infection can cause weak growth vigor of plant seedlings, slow seedling return, less caking, small potato blocks, light and unsmooth color of potato skins, brown crack of the potato blocks and the like, so that the yield is reduced, the quality is deteriorated, and even the commodity value is lost. Particularly, in recent years, virus diseases affect the sweet potato production in China, and the sweet potato has a tendency of being more and more intense, and can cause more than 90% of yield loss in severe cases, even the sweet potato is not harvested. At present, SPVD occurs in China, Guangdong, Jiangsu, Sichuan, Anhui, Fujian, Shandong and the like.
The main method for overcoming the harm of virus diseases is to use stem tip culture to produce virus-free seedlings, because the distribution of viruses in plants is uneven, the farther away from the meristem, the higher the virus density, and vice versa, and the meristem generally has no virus. However, the stem tip meristem is not always easy to survive when being stripped for culture. Thus, typically, shoot tips with 1-2 leaf primordia are stripped for culture, but this reduces the detoxification effect. Overcomes the contradiction between the two, improves the regeneration rate and the detoxification rate, and becomes the bottleneck of the production of the detoxified seedling of the sweet potato.
The cultivation of the stem tip of the sweet potato to produce the virus-free seedling has been studied and reported, but the stem tip is stripped from the material taken from the stem and vine which is cultivated in the field or greenhouse in the past; the stem tip is also stripped from potato block material and cultured, but the germination is also accelerated under normal temperature condition. The method is adopted to grow and culture under conventional conditions, and because the material for detoxification has slow growth and weak metabolism, the stem tip stripping culture has poor detoxification effect, the stem tip is not easy to strip, and the regeneration rate is low. In addition, the detoxification effect of the conventional detoxification method is not ideal for some old varieties with serious virus diseases.
In addition, seedlings grow on a solid MS culture medium before domestication and transplantation of the existing sweet potatoes, but the culture medium is difficult to clean during transplantation and is susceptible to bacteria. The roots are easy to be damaged during transplanting, and the virus-free seedlings are slow to recover.
Disclosure of Invention
Aiming at the problems in the prior detoxification technology, the invention aims to provide a method for obtaining sweet potato detoxified seedlings by high-temperature variable-temperature treatment.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for obtaining sweet potato virus-free seedlings by high-temperature-variable-temperature treatment comprises the following steps:
(1) selecting sweet potato blocks which are not damaged by worms and damaged, and insolating for 2-3 days under the sun;
(2) cleaning potato blocks with clear water, covering sand on the potato blocks, and putting the potato blocks into an incubator at 28 ℃ for accelerating germination;
(3) when the bud seedling grows to be more than 10cm, taking the top 2-3cm long (with young leaves) of the seedling, cutting off leaves visible to naked eyes, washing with clear water, then carrying out surface disinfection, and inserting the disinfected seedling into an MS culture medium for culture;
(4) after the seedlings take roots, carrying out high-temperature treatment at 40-45 ℃ for 5 hours every day in an incubator, then carrying out high-temperature-variable-temperature treatment at 28 ℃, and continuously treating for 30-40 days;
(5) stem tips with leaf primordium length of 0.5-0.8mm are stripped under a stereoscopic dissecting mirror, inoculated on an MS culture medium added with 0.1-0.2mg/LNAA and 1.0-2.0mg/L BAP, cultured at 25-27 ℃ for 13 hours per day under illumination of 2000 and 3000lx, cultured for 3-4 weeks, and differentiation of adventitious buds is induced;
(6) when the regeneration plantlet grows to be more than 2cm, cutting off the regeneration plantlet from the base, and transferring the regeneration plantlet to a culture medium without hormone addition to obtain a complete detoxified plantlet;
(7) before acclimatization and transplantation, transferring the sterile seedlings into an MS liquid culture medium for culture;
(8) after hardening off the test-tube plantlets, cleaning a culture medium, directly planting the test-tube plantlets in soil of a plastic greenhouse in a way that the distance between plants is 20-25cm, and adding insect-proof nets at two ends of the plastic greenhouse; the humidity in the greenhouse was kept above 80% for the first 1 week.
The technical scheme of the invention has the advantages that:
the method for obtaining the virus-free sweet potato seedlings through high-temperature-variable-temperature treatment has good virus passivation effect, the stripped stem tips are long, the stem tips are easy to strip, the efficiency is high, and the adventitious buds grow fast; the regenerated seedling has good rooting quality, stout seedling and high survival rate.
Compared with the prior sweet potato virus-free seedling method (patent number 201410291953.8 of invention' a preparation method of sweet potato virus-free seedling),
firstly, the high-temperature treatment (35-37 ℃) is carried out during potato block culture in the prior method, the cut seedling buds are transferred to a culture medium after being sterilized, then the high-temperature treatment at 40-45 ℃ is carried out, if the seedlings are not transferred to the culture medium, the high-temperature treatment at 40-45 ℃ is directly carried out, and the seedlings are difficult to survive in sandy soil; the virus inactivation effect is better after the temperature is raised;
secondly, after the cut sprouts are treated by high-temperature change, the virus passivation effect is better, so that larger stem tips (which can be increased to 0.5-0.8m) with leaf primordium can be stripped under a stereoscopic dissection mirror. The large stem tip is easy to strip, the efficiency is higher, the time for growing adventitious buds is short, the existing method has large stripping difficulty, difficult survival and slow growth;
the regenerated seedling has better rooting quality in liquid culture medium than solid MS culture medium, is sturdy, easy to wash, and not easy to damage the root during transplanting, and the seedling is easy to survive.
The high-temperature and variable-temperature treatment of the invention has good virus passivation effect and good stem tip detoxification effect, and the stem tip is easy to strip due to the elongation of the leaf primordial internodes (the meristem is easy to see under a dissecting mirror due to the elongation of the leaf primordial internodes, the stem tip is easy to strip), and the regeneration rate is high (the growth and the metabolism are vigorous due to the higher temperature). Experiments show that after the sweet potato is continuously treated for 1 month in a culture room of an incubator at 40-45 ℃ for 5 hours at high temperature every day, the growth of seedlings of most sweet potato varieties is not obviously influenced.
In general, the smaller the stem tip stripped from the sweet potato, the higher the death rate of the sweet potato, the lower the seedling rate and the higher the detoxification rate; on the contrary, the death rate is greatly reduced, but the detoxification rate is lower or the aim of detoxification cannot be achieved. The ideal stem tip stripping length is 0.2-0.4mm when the virus-free seedlings are produced at present, but the virus-free seedlings have low seedling rate and slow growth. The invention increases the length of stem tip of the seedling after high temperature treatment to 0.5-0.8mm under a stereoscope, and then transfers the seedling to a culture medium for callus induction, adventitious bud regeneration and seedling regeneration, so that the cultured adventitious bud grows faster and can achieve the aim of detoxification.
The sweet potato sterile seedlings are transferred to an MS liquid culture medium and cultured for 15 d. Compared with a solid MS culture medium, the method has the advantages of good rooting quality, sturdy seedlings, easy washing of the culture medium, difficult damage to roots during transplanting and easier survival of the seedlings.
Drawings
FIG. 1 sweet potato seedlings infected with virus (left: Beijing 553; right: Nicotiana tabacum 25);
FIG. 2 shows the seedlings after high temperature-variable temperature treatment;
FIG. 3 shows adventitious buds growing from callus formed by stem tip culture (left side: seedling is subjected to high temperature swing treatment and adventitious buds growing from 0.5-0.8 mm-sized stem tip are peeled off; right side: seedling is not subjected to high temperature swing treatment and adventitious buds growing from less than 0.4 mm-sized stem tip are peeled off);
FIG. 4 rooting of detoxified shoots in solid and liquid medium (left: rooting of detoxified shoots in solid medium; right: rooting of detoxified shoots in liquid medium);
FIG. 5 shows indirect ELISA detection of virus-free seedlings (1: positive control, 2: infected seedlings, 3: negative control, 4-8: virus-free seedlings generated by peeling stem tips with a size of 0.5-0.8mm after high temperature variable temperature treatment).
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
The method for preparing the sweet potato virus-free seedlings by high-temperature and variable-temperature treatment specifically comprises the following steps:
1. treatment before germination acceleration of sweet potato blocks
Selecting sweet potato varieties Beijing 553 and Nicotiana tabacum 25 with serious virus disease incidence in the field seedling stage, harvesting in autumn, taking the potato blocks without worm damage and injury, and insolating under the sun for 2-3 days.
2. Accelerating germination
Cleaning the potato blocks with clear water, putting into sand, covering the potato blocks with sand with the thickness of 1-2cm, and putting into an incubator at 28 ℃ for accelerating germination. Culturing in dark for 1 week before germination, and illuminating for 13h every day at 1000-.
3. Stem tip stripping and culturing
(1) And (3) disinfection of materials: when the bud grows to be more than 10cm, the virus disease symptom is very obvious (figure 1), 2-3cm long (with young leaves) at the top end of the seedling is taken, leaves visible to naked eyes are cut off, the leaves are washed by clear water, then the leaves are soaked in 70% alcohol for 10-20 seconds in a hyperstatic workbench, then sodium hypochlorite solution with the mass percent of 2% is used for soaking for 5-7min for surface disinfection, and finally the leaves are rinsed for 3-5 times by sterile water and inserted into an MS culture medium.
(2) After the seedlings are rooted for 5-7 days, the seedlings are treated at high temperature for 5 hours every day in a culture room at the temperature of 40-45 ℃, and then are treated at high temperature and variable temperature at the temperature of 28 ℃ for 30-40 days continuously (figure 2).
(3) 0.5-0.8mm of stem tip with leaf primordium is stripped under a stereoscopic dissecting mirror, and inoculated on an MS culture medium for culture. The shoot tips began to form callus after about 1 week of culture, and adventitious buds began to form from the callus wounds 3-4 weeks later (FIG. 3). As can be seen from FIG. 3, the adventitious buds growing on the callus formed by the shoot tips with the size of 0.5-0.8mm grow faster than the shoot tips with the size of less than 0.4 mm. After culturing for 1 month on MS culture medium added with 0.2mg/LNAA and 2.0mg/LBAP, when the regeneration plantlet grows to be more than 2cm, the regeneration plantlet is cut from the base and transferred to MS culture medium without hormone to grow into complete detoxified plantlet.
(4) Before acclimatization and transplantation, the sterile seedlings were transferred to MS liquid medium and after 15d of culture, were promoted to root (FIG. 4). As can be seen from FIG. 4, the seedlings rooted more on the liquid medium and were of higher quality than the solid medium, and the medium was easier to wash than the solid medium before transplanting.
4. Virus detection of detoxified shoots
SPVD virus detection is performed on the detoxified seedlings by adopting an indirect ELISA detection method, and the detection result is shown in figure 5: the detoxification rate of 2 varieties reaches 100 percent.
5. Acclimatization and transplantation of detoxified test-tube plantlets
(1) Opening the bottle mouth of the test-tube plantlet after subculture for domestication, and continuously hardening the plantlet for 2-3 d.
(2) Cleaning the test-tube plantlets with a culture medium, and directly planting the test-tube plantlets in the soil of a plastic greenhouse in a single plant mode, wherein the distance between plants is 20-25 cm. Insect-proof nets are added at two ends of the plastic greenhouse, and the humidity in the greenhouse is kept above 80% in the first 1 week.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (2)
1. A method for obtaining sweet potato virus-free seedlings by high-temperature-variable-temperature treatment is characterized by comprising the following steps: the method comprises the following steps:
(1) selecting sweet potato blocks which are not damaged by worms and damaged, and insolating for 2-3 days under the sun;
(2) cleaning potato blocks with clear water, covering sand on the potato blocks, and putting the potato blocks into an incubator at 28 ℃ for accelerating germination;
(3) when the bud grows to be more than 10cm, taking the top end of the seedling, cutting off visible leaves, washing with clear water, then carrying out surface disinfection, and inserting the disinfected bud into an MS culture medium for culture;
(4) after the seedlings take roots, carrying out high-temperature treatment at 40-45 ℃ for 5 hours every day in an incubator, then carrying out high-temperature-variable-temperature treatment at 28 ℃, and continuously treating for 30-40 days;
(5) stripping the stem tip with the leaf primordium under a stereoscopic dissecting mirror, inoculating the stem tip to an MS culture medium added with 0.1-0.2mg/L NAA and 1.0-2.0mg/L BAP, culturing at 25-27 ℃ for 13h illumination every day at 2000-3000lx for 3-4 weeks, and inducing the differentiation of adventitious buds;
(6) when the regeneration plantlet grows to be more than 2cm, cutting off the regeneration plantlet from the base, and transferring the regeneration plantlet to a culture medium without hormone addition to obtain a complete detoxified plantlet;
(7) before acclimatization and transplantation, transferring the sterile seedlings into a liquid culture medium for culture;
(8) after hardening off the test-tube plantlets, cleaning a culture medium, directly planting the test-tube plantlets in soil of a plastic greenhouse in a way that the distance between plants is 20-25cm, and adding insect-proof nets at two ends of the plastic greenhouse; keeping the humidity in the greenhouse to be more than 80% in the first 1 week;
taking the top end of the seedling in the step (3), wherein the top end of the seedling is 2-3cm long and is provided with young leaves;
in the step (5), the length of the stem tip stripped under a stereoscopic dissecting mirror is 0.5-0.8 mm.
2. The method for obtaining sweet potato virus-free seedlings by high-temperature-variable-temperature treatment according to claim 1, which is characterized in that: the liquid culture medium in the step (7) is MS liquid culture medium.
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| CN111436374A (en) * | 2020-05-08 | 2020-07-24 | 佛山市农业科学研究所(佛山市农业技术推广中心) | Detoxification and rapid propagation method of Gaoming Shuiguan pueraria |
| CN115281089B (en) * | 2022-08-08 | 2023-04-18 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN115644063A (en) * | 2022-11-11 | 2023-01-31 | 青岛农业大学 | Rapid propagation method of virus-free raw seedling of sweet potato |
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| WO2010076954A2 (en) * | 2008-11-10 | 2010-07-08 | (주) 마이크로프랜츠 | Mass production of potato microtubers by tissue culture and method for producing seed potatoes |
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| CN104719164B (en) * | 2015-03-30 | 2017-06-16 | 青岛农业大学 | A kind of rapid propagation method of Sweetpotato Viruses Elimination original silkworm egg potato |
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