CN112586350A - Strawberry virus-free seedling culture method - Google Patents
Strawberry virus-free seedling culture method Download PDFInfo
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- CN112586350A CN112586350A CN202011414054.4A CN202011414054A CN112586350A CN 112586350 A CN112586350 A CN 112586350A CN 202011414054 A CN202011414054 A CN 202011414054A CN 112586350 A CN112586350 A CN 112586350A
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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
- A01H4/001—Culture apparatus for tissue culture
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- Biotechnology (AREA)
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Abstract
The invention discloses a strawberry virus-free seedling raising method. The preparation method comprises the following steps: s1, selecting an explant; s2, sterilizing the explants; s3, inoculating and culturing explants; s4, differentiating to form virus-free seedlings; s5, acclimatization and transplantation of seedlings. The preparation method of the invention has the advantages of good detoxification effect, fast seedling propagation, high transplanting survival rate, and improved yield and quality of strawberries, and the cultivated strawberry detoxification seedlings have strong growth vigor.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a strawberry virus-free seedling raising method.
Background
Strawberry is perennial evergreen herbaceous plant of Rosaceae strawberry, belongs to polyploid, and is a crop asexually propagated by stolons, and the traditional seedling cultivation method adopts asexual propagation mode of stolons propagation, so that the efficiency is low, the popularization of good varieties is not facilitated, and the virus infection is very easy. Four types of strawberry viruses identified clearly in China, namely strawberry mottle virus (SMOV), Strawberry Mild Yellow Edge Virus (SMYEV), strawberry shrinkage virus (SCRV) and Strawberry Vein Banding Virus (SVBV). The common virus infection of four seedlings is a bottleneck problem in strawberry production, and strawberries infected with viruses have weaker growth vigor, poor resistance, obviously reduced yield and deteriorated fruit quality and commodity characters compared with strawberries not infected with viruses. For virus diseases, no medicament can be used for treating at present, so that the cultivation of virus-free maternal seedlings and the cultivation of virus-free nursery stocks are fundamental measures for preventing and treating the virus diseases of the strawberries.
At present, virus-free maternal seedlings are mainly obtained by virus pathogen removal through strawberry virus-free tissue culture technology, namely, anther tissue culture, micro-stem tip tissue culture and other technologies to remove virus pathogens, so that the excellent properties of the breeds and breeds of strawberry seedlings are restored, and the purposes of high quality and high yield are achieved. However, the cultivation of the virus-free maternal seedling of the strawberry is not easy, the explant for tissue culture has defects, the condition control in the culture process is insufficient, and the like, so that the problems of poor detoxification effect, pollution, vitrification, low breeding coefficient, low transplanting survival rate and the like can be caused, and great loss is caused to production.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the strawberry detoxification seedling method which has the advantages of good detoxification effect, fast seedling propagation, high transplanting survival rate, and improvement of the yield and quality of strawberries, and the cultured strawberry detoxification seedlings have strong plant growth vigor.
In order to achieve the purpose, the invention provides the following technical scheme: a strawberry virus-free seedling raising method comprises the following steps:
s1, selection of explants: selecting strong strawberry plants with good growth vigor, no diseases and pests and no deformity, taking stolon segments of the strong strawberry plants, and selecting stolons with four undeveloped leaves at the top ends; storing the selected explant at 6 deg.C for 10-18 h;
s2, disinfection and sterilization of explants: naturally heating the stolons processed in the step S1 from a low-temperature state to room temperature, shearing 6-8cm stolons between the second undeveloped leaf and the third undeveloped leaf on the top, placing the stolons in 75% alcohol for soaking for 30-40S, then processing for 1min by using 0.15-0.2% mercuric chloride solution, finally placing the stolons in 75% alcohol again for soaking for 30-40S, and repeatedly washing for three times by using sterile distilled water after processing;
s3, inoculating and culturing explants: inoculating the explant treated in the step S2 on a stem tip induction culture medium, culturing at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 2000Lux and the illumination time of 14h/d until the remaining conditions are unchanged, the light intensity is enhanced to 3000Lux after six weeks, continuously culturing until the light intensity is weakened to 2000Lux after eight weeks until a regeneration plant bud is induced from the stem tip, and culturing for 2-3 months at this stage to finish primary culture;
s4, differentiating to form detoxified seedlings: cutting the plant sprouts treated in the step S3 into small pieces, inoculating the small pieces on a multiplication culture medium, and culturing the small pieces at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 2000 and 3000Lux and the illumination time of 14h/d until the small pieces grow into 5-8cm long seedlings;
s5, domestication and transplantation of seedlings: and (5) transplanting the seedlings in the step S4 into sterilized nutrient soil, taking out the seedlings from an artificial climate incubator, culturing the seedlings on a light culture rack, transferring the seedlings to an outdoor sunshade net environment for growth, transplanting the seedlings into soil after the seedlings grow for 2-3 weeks, periodically watering and fertilizing, and paying attention to the growth state of plants.
By adopting the technical scheme, the stolons with undeveloped leaves are adopted as explants, and the pollution rate of the undeveloped leaves is low, so that the obtained seedlings have good detoxification effect and low pollution rate; the stolons are cultivated and propagated into nutrition propagation, so that the strawberry stolons are put in a culture medium containing comprehensive nutrient components to cultivate seedlings basically identical to the original stolons under the condition of disinfection and sterilization, and the characteristics of high propagation speed, small environmental influence and excellent detoxification effect are achieved; the explant is subjected to secondary disinfection treatment by alcohol, mercuric chloride and alcohol, and can be subjected to stem tip induction culture and substance induction by adopting light irradiation in different periods in the culture process, so that the germination rate of the stem bud of the explant can be remarkably improved; the plant plumule is cut into small blocks and inoculated into a multiplication culture medium, the plant plumule can be well preserved and induced to multiply, the exponential increase of the number of the plant plumule is realized while the plant plumule is preserved, the multiplication coefficient of the plant plumule is improved, and the plant plumule cut into small blocks is induced to form a new bud and a new root under the conditions of specific temperature, humidity and light intensity; the induced seedlings are taken to a light culture frame for culture and domestication, then the domesticated seedlings are transplanted to an outdoor sunshade net for growth for 2-3 weeks, then the seedlings which grow robustly are transplanted to soil, and through continuous domestication treatment, the survival rate of aseptic seedling transplantation is greatly improved, meanwhile, infection of rhizopus fungi is avoided, the yield and quality of strawberries are greatly improved, and the cultured strawberry virus-free seedlings have the advantage of strong plant growth vigor.
Further, the stolon collection length of the step S1 is 12-16 cm.
By adopting the technical scheme, the creeping stems of undeveloped leaves have low pollution rate and low toxicity, and the collection length of the creeping stems is 12-16cm, so that the explants are prevented from being polluted by external pollution sources, the toxicity of the explants is further reduced, and the detoxification rate of seedlings is improved.
Further, 1 to 3 drops of tween 20 are added dropwise to the mercuric chloride solution in the step S2.
By adopting the technical scheme, because the Tween 20 is the nonionic surfactant, the nonspecific combination is reduced, the surface tension of the explant is reduced, a better disinfection effect is achieved, the surface sterilization effect of the explant is improved, and the pollution rate of seedling culture is reduced.
Further, a step S2.1 is added in steps S2 and S3, where in step S2.1, the stolons processed in step S2 are placed on a clean bench, the stem skin of the outer wrapping is sequentially stripped from outside to inside under a dissecting mirror, a growth point is obtained, and a tissue on the upper portion of the growth point is picked by a dissecting needle to serve as an explant.
By adopting the technical scheme, the stolons are placed on an ultra-clean super-clean platform, so that the pollution of endophytes to the explants is further reduced in the culture process, and the pollution rate of generated seedlings is reduced; meanwhile, a growth point is selected, namely a growth cone or a meristem of the strawberry stolon, the cell division activity is vigorous, the differentiation degree is low, and the toxicity in the plant is greatly reduced and even is non-toxic, so that the period of cultivating the seedling by the plant is shortened, the toxicity of the seedling is reduced, and the detoxification rate is improved.
Further, the length of the explant left is 0.4mm ± 0.05 mm.
By adopting the technical scheme, the upper tissues of the growing points with the length of 0.4mm +/-0.05 mm are selected from the explants, so that the growth rate of the stem buds is improved, the growth time of seedlings is further shortened, and the propagation rate is greatly improved.
Further, step S2.2 is added to step S2.1, and step S2.2 is to immerse the explant processed in step S2.1 in an anti-browning medium for 10-15 min.
By adopting the technical scheme, the generation of explant browning is effectively prevented, and the survival rate of the explant is ensured to be more than 90%.
Further, the anti-browning culture medium consists of 15000-25000mg/L sodium thiosulfate, 200-400mg/L vitamin C, 1-5mg activated carbon, 20-30g/L sucrose and 6g/L agar, and the pH value of the anti-browning culture medium is between 5.4 and 5.8.
By adopting the technical scheme, the browning phenomenon is easy to occur due to the fact that the browning resistant culture medium solves the problem that the rapid propagation of the strawberry explant is easy to occur, and the survival rate and the quality of seedlings are improved.
Further, the anti-browning culture medium is also added with 1-2g/L PVP.
By adopting the technical scheme, the browning effect of the explant is further inhibited, and the browning resistance rate is improved.
Further, the stem tip induction culture medium in the step S3 is agar with the MS culture medium as a basic culture medium, and MS +6-BA, NAA, a carbon source and gel are added into the MS culture medium, wherein the concentration of the MS +6-BA is 0.3mg/L-0.5mg/L, the concentration of the NAA is 0.05-0.1mg/L, the concentration of the carbon source is 20-30g/L sucrose, and the concentration of the gel is 6 g/L.
By adopting the technical scheme, MS +6-BA is cytokinin, NAA is one of naphthylacetic acid as auxin, so that the growth of explants is promoted, the differentiation of roots and buds is facilitated, and the growth is increased to culture when the plant plumule is suitable for plant plumule, so that the generation of callus is facilitated, and the differentiation of buds is promoted; sucrose provides a carbon source for explant tissues and regulates osmotic pressure; the agar has supporting and fixing functions on the stem tip induction culture medium, so that the germination rate and rooting rate of the explant stem buds can be obviously improved.
Further, the concentration of MS +6-BA is preferably 0.4mg/L, and the concentration of NAA is preferably 0.08 mg/L.
By adopting the technical scheme, the germination rate and rooting rate of the shoot buds of the explants are further improved, the proliferation effect on strawberry seedlings is remarkably improved, and the proliferation rate is improved.
In conclusion, the invention has the following beneficial effects:
firstly, because the stolons among the four undeveloped leaves and the seedling culture conditions are adopted, the virus-free seedlings are obtained, the seedlings with low pollution rate are obtained, the yield and the quality of the strawberries are improved, and the cultured virus-free strawberry seedlings have the effect of strong plant growth vigor.
Secondly, the strawberry virus-free seedling culture method improves the virus-free mode of the stolon tips of the exophyte, adopts the stolon cultured plants between the second undeveloped leaves and the third undeveloped leaves of the stolon to peel off and detoxify the stem buds, greatly reduces the pollution rate and the browning rate, greatly improves the virus-free rate and the survival rate of the meristem of the stem tips, effectively improves the working efficiency and greatly reduces the cost.
Thirdly, the stem tip of the plant obtained by the method is stored under the conditions of proper temperature, humidity and illumination, so that the excellent characters and genetic stability of the variety can be maintained, the period from detoxification to mass production is effectively shortened, and the production cost and labor cost are effectively reduced.
Detailed Description
The present invention will be described in further detail with reference to examples. The test time of each of the following examples is 2018 to 2020, and the test site is the Unionidae offshore pearl agriculture science and technology development Co.
Example 1
The strawberry virus-free seedling culture method comprises the following operation steps:
selection of strawberry plants
Selecting healthy strawberry plants with good growth, no diseases and insect pests and no deformity, placing the healthy strawberry plants into a culture room, treating the healthy strawberry plants under the conditions of 36 ℃ and 2100Lux illumination for 16h every day, culturing the healthy strawberry plants under the conditions of 30 ℃ and dark for the rest 8h, and continuously carrying out circulating temperature change treatment for 4 weeks;
2 selection and Sterilization of explants
2.1 selection of explants: selecting strawberry plants cultured in the step (1), rubbing stolon stem sections of the strawberry plants with washing liquid for 3 times, washing with running water for 10min, and cutting stolons with four undeveloped leaves at the top ends, wherein the stolons are 14cm in collection length; storing the selected stolons at a low temperature of 6 ℃ for 14 h;
2.2 Disinfection and sterilization of explants: naturally heating the stolons treated in the step (2.1) to room temperature from a low-temperature state, transferring the stolons of the stem segments to an ultra-clean bench, cutting 7cm stolons between the second undeveloped leaves and the third undeveloped leaves from the top, placing the cut stolons in 75% alcohol for 35s of soaking, then sterilizing the stolons with 0.2% mercuric chloride solution and 2 drops of Tween 20 as bactericide for 1min, placing the stolons in 75% alcohol again for 35s of soaking, finally repeatedly washing the stolons subjected to sterilization and sterilization treatment with sterile distilled water for three times, and sucking surface moisture with sterile filter paper;
2.2.1 placing the stolons processed in the step (2.2) on a clean bench, sequentially stripping the wrapped stem skin from outside to inside under a dissecting mirror to obtain a growth point, picking the upper tissue of the growth point by using a sterilized dissecting needle to serve as an explant, reserving small particles with the length of 0.4mm +/-0.05 mm at the growth point of the explant, operating in an aseptic environment, and draining water on the surface of the growth point of the explant;
2.2.2 placing the explant growth point treated in the step (2.2.1) in an anti-browning culture medium for soaking for 12min, taking out the explant growth point, repeatedly washing the explant growth point with sterile distilled water for three times, and then sucking surface water by sterile filter paper; wherein the anti-browning culture medium comprises sodium thiosulfate with the concentration of 20000mg/L, 300mg/L vitamin C, 3mg of activated carbon, 30g/L sucrose, 6g/L agar and 1.5g/L PVP, and the pH value of the anti-browning culture medium is between 5.4 and 5.8;
3 inoculation culture of explants
Inoculating the explant growth point subjected to anti-browning treatment in the step (2.2.2) to a stem tip induction culture medium, culturing at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 2000Lux and the illumination time of 14h/d until the remaining conditions are unchanged, the light intensity is increased to 3000Lux after six weeks, continuously culturing until the light intensity is reduced to 2000Lux after eight weeks until a regeneration plant bud is induced from the stem tip, and culturing for 2-3 months at this stage to complete primary culture; wherein the stem tip induction culture medium takes an MS culture medium as a basic culture medium, MS +6-BA, NAA, a carbon source and gel are added into the MS culture medium, wherein the concentration of the MS +6-BA is 0.4mg/L, the concentration of the NAA is 0.08mg/L, the carbon source is 30g/L sucrose, the concentration of the gel is 6g/L agar, and the pH value of the stem tip induction culture medium is between 5.4 and 5.8; 4 differentiation to form detoxified seedling
Repeatedly washing plant plumule obtained after the stem tip induction culture medium treatment in the step (3) with sterile distilled water for three times, and then sucking surface water by sterile filter paper; placing the treated plant plumule on a super clean bench, cutting into small pieces, inoculating the cut small pieces into a proliferation culture medium, and culturing at 25 + -3 deg.C, humidity of 75 deg.C, light intensity of 2500Lux, and illumination time of 14h/d until the plant plumule grows into 5-8cm long seedling; wherein the enrichment culture medium is MSM minimal medium and basic culture medium, 0.5mg/L6-BA, 200mg/L glutamine, 30g/L sucrose and 6g/L agar are added into the MS culture medium, and the PH value of the enrichment culture medium is between 5.4 and 5.8;
5 domestication and transplantation of seedlings
And (4) transplanting the long seedlings obtained by the treatment in the step (4) into sterilized nutrient soil, taking out the seedlings from an artificial climate incubator, culturing the seedlings on a light culture frame, transferring the seedlings to an outdoor sunshade net environment for growth, transplanting the seedlings into soil after 3 weeks of growth at the stage, periodically watering and fertilizing, and paying attention to the growth state of plants.
Example 2
The operation method is the same as that of example 1, wherein the scheme that the outer wrapping stem skin of the stolons is stripped in the step (2.2.1) is cancelled, and the upper tissue of the growing point is obtained.
Example 3
The procedure is as in example 1, with the elimination of the anti-browning medium protocol in step (2.2.2).
Example 4
The procedure is as in example 1, wherein 1.5g/L PVP in the browning resistant medium in step (2.2.2) is changed to an equivalent amount of 20000mg/L sodium thiosulfate.
Comparative example 1
The operation method is the same as that of example 1, wherein the method for (2.1) selecting the explant and (2.2) sterilizing the explant in the step (2) is changed into the following method:
2.1 selection of explants: selecting strawberry plants cultured in the step (1), rubbing stolon stem sections of the strawberry plants with washing liquid for 3 times, washing with running water for 10min, and cutting stolons with four spread leaves at the top ends, wherein the collection length of the stolons is 14 cm; storing the selected stolons at a low temperature of 6 ℃ for 14 h;
2.2 Disinfection and sterilization of explants: naturally heating the stolons treated in the step (2.1) from a low-temperature state to room temperature, transferring the stolons of the stem segments to a clean bench, cutting 7cm stolons between the second and third unfolding leaves from the top, placing the cut stolons in 75% alcohol for 35s, then guessing the sterilizing treatment of mercuric chloride solution with the mass fraction of 0.2% and 2 drops of Tween 20 as a bactericide for 1min, placing the stolons in 75% alcohol again for 35s, finally repeatedly washing the stolons subjected to the sterilizing treatment with sterile distilled water for three times, and sucking surface moisture with sterile filter paper.
Comparative example 2
The operation method is the same as that of example 1, wherein the method for (2.1) selecting the explant and (2.2) sterilizing the explant in the step (2) is changed into the following method:
2.1 selection of explants: selecting strawberry plants cultured in the step (1), selecting stolon stem sections of the strawberry plants, rubbing the surfaces of stolons for 3 times by using a washing solution, washing the surfaces of the stolons for 10min by using running water, and cutting stolons at the top ends, wherein the collection length of the stolons is 14 cm; storing the selected stolons at a low temperature of 6 ℃ for 14 h;
2.2 Disinfection and sterilization of explants: naturally heating the stolons treated in the step (2.1) from a low-temperature state to room temperature, transferring the stolons of the stem segments to a clean bench, cutting 7cm stolons counted from the top, soaking the cut stolons in 75% alcohol for 35s, then guessing the sterilizing treatment of mercuric chloride solution with the mass fraction of 0.2% and 2 drops of Tween 20 as a bactericide for 1min, soaking the stolons in 75% alcohol for 35s again, and finally repeatedly washing the sterilized stolons with sterile distilled water for three times and aseptically sucking surface moisture.
Comparative example 3
The operation method is the same as that of example 1, wherein the inoculation culture method of the explant in the step (3) is changed into the following method:
3 inoculation culture of explants
Inoculating the explant growth point subjected to the anti-browning treatment in the step (2.2.2) to a stem tip induction culture medium, and culturing at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 3000Lux and the illumination time of 14h/d for 5-6 months at the stage so as to induce a regeneration plant bud from the stem tip, thereby completing primary culture; the stem tip induction culture medium takes an MS culture medium as a basic culture medium, MS +6-BA, NAA, a carbon source and gel are added into the MS culture medium, wherein the concentration of the MS +6-BA is 0.4mg/L, the concentration of the NAA is 0.08mg/L, the carbon source is 30g/L of cane sugar, the concentration of the gel is 6g/L of agar, and the pH value of the stem tip induction culture medium is between 5.4 and 5.8.
And (4) statistics of test results:
comparison of detoxification effects: taking 70 explant samples from each group, respectively adopting the method of the embodiment 1-4 to carry out operation, statistically counting the stem tip inoculation survival rate and the pollution rate in the culture process, carrying out virus detection and counting the detoxification rate after obtaining the virus-free seedling, the transplanting survival rate, the height of the main stem after transplanting and the plant growth situation.
Detoxification rate the explant tissue culture seedlings of the embodiments and the comparative examples of the invention were tested according to the indicated plant detection method of "technical procedures for virus detection of detoxified strawberry seedling virus", to determine whether the explant tissue culture seedlings were detoxified, and the detoxification rate was calculated.
TABLE 1 comparison of detoxification Effect
The data in table 1 show that the detoxification method provided by the invention combines various modes of selecting the creeping stems without unfolding leaves, a low-temperature treatment detoxification method, stem tip detoxification, anti-browning culture, light intensity irradiation in different time periods, a stem tip induction culture medium and a propagation culture medium, has good virus removal effect on tissue culture and rapid propagation of strawberry detoxification seedling, detects the virus in the stems of the obtained strawberry detoxification seedlings, has a detoxification rate as high as 99.8%, has high transplanting survival rate and low pollution rate in a later planting process, can be directly transplanted, does not need seedling hardening, has high propagation speed, improves the yield and preparation of strawberries, and has the characteristic of good growth situation of the cultivated strawberry detoxification seedlings.
According to the strawberry detoxification seedling method in the embodiment 1, after the method is adopted by the Anhuo Hakka seal agriculture science and technology development limited company, high-quality strawberries are cultivated; the virus-free seedling culture makes breakthrough progress, large-scale industrial production is realized, and the annual yield of the high-quality strawberry virus-free seedling culture at present reaches more than 4000 ten thousand plants. Compared with the conventional seedling, the virus-free seedling obtained by the virus-free strawberry seedling raising method has obvious growth advantage and yield increase effect. Compared with the conventional seedlings, the strawberry detoxified seedlings of different varieties have the advantages that the plant height is increased by 34%, the leaf area is increased by about 65%, the number of stolons is increased by about 93%, the yield and the quality of the plants are improved, and the yield is increased by 50% -60%.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (10)
1. A strawberry virus-free seedling raising method is characterized by comprising the following steps:
s1, selection of explants: selecting strong strawberry plants with good growth vigor, no diseases and pests and no deformity, taking stolon segments of the strong strawberry plants, and selecting stolons with four undeveloped leaves at the top ends; storing the selected stolons at a low temperature of 6 ℃ for 10-18 h;
s2, disinfection and sterilization of explants: naturally heating the stolons processed in the step S1 from a low-temperature state to room temperature, shearing 6-8cm stolons between the second undeveloped leaf and the third undeveloped leaf on the top, placing the stolons in 75% alcohol for soaking for 30-40S, then processing for 1min by using 0.15-0.2% mercuric chloride solution, finally placing the stolons in 75% alcohol again for soaking for 30-40S, and repeatedly washing for three times by using sterile distilled water after processing;
s3, inoculating and culturing explants: inoculating the explant treated in the step S2 on a stem tip induction culture medium, culturing at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 2000Lux and the illumination time of 14h/d until the remaining conditions are unchanged, the light intensity is enhanced to 3000Lux after six weeks, continuously culturing until the light intensity is weakened to 2000Lux after eight weeks until a regeneration plant bud is induced from the stem tip, and culturing for 2-3 months at this stage to finish primary culture;
s4, differentiating to form detoxified seedlings: cutting the plant sprouts treated in the step S3 into small pieces, inoculating the small pieces on a multiplication culture medium, and culturing the small pieces at the temperature of 25 +/-3 ℃, the humidity of 75 ℃, the light intensity of 2000 and 3000Lux and the illumination time of 14h/d until the small pieces grow into 5-8cm long seedlings;
s5, domestication and transplantation of seedlings: and (5) transplanting the seedlings in the step S4 into sterilized nutrient soil, taking out the seedlings from an artificial climate incubator, culturing the seedlings on a light culture rack, transferring the seedlings to an outdoor sunshade net environment for growth, transplanting the seedlings into soil after the seedlings grow for 2-3 weeks, periodically watering and fertilizing, and paying attention to the growth state of plants.
2. A strawberry detoxification seedling raising method according to claim 1, wherein the stolons collection length of the step S1 is 12-16 cm.
3. The method for detoxifying and growing strawberry seedlings according to claim 1, wherein 1 to 3 drops of tween 20 are added dropwise to the mercuric chloride solution in the step S2.
4. The strawberry detoxification seedling method according to claim 1, wherein step S2.1 is added in steps S2 and S3, and step S2.1 is that the stolons processed in step S2 are placed on a clean bench, the outer wrapped stem skin is sequentially stripped from outside to inside under a dissecting mirror, a growth point is obtained, and the upper tissue of the growth point is picked by a dissecting needle to be used as an explant.
5. The strawberry detoxification seedling method according to claim 4, wherein the length of the explant is 0.4mm ± 0.05 mm.
6. The method for virus-free seedling raising of strawberries as claimed in claim 4, wherein step S2.2 is added in step S2.1, and step S2.2 is to immerse the explants obtained from step S2.1 in the anti-browning culture medium for 10-15 min.
7. The method as claimed in claim 1, wherein the browning resistant medium comprises 15000-25000mg/L sodium thiosulfate, 200-400mg/L vitamin C, 1-5mg activated carbon, 20-30g/L sucrose and 6g/L agar, and the pH of the browning resistant medium is between 5.4-5.8.
8. The method for detoxifying growing strawberry seedlings according to claim 7, wherein 1-2g/L of PVP is further added to the browning resistant medium.
9. The strawberry detoxification seedling method according to claim 1, wherein the stem tip induction culture medium in the step S3 is an MS culture medium as a basic culture medium, and MS +6-BA, NAA, a carbon source and gel are added to the MS culture medium, wherein the concentration of MS +6-BA is 0.3mg/L-0.5mg/L, the concentration of NAA is 0.05 mg/L-0.1 mg/L, the concentration of the carbon source is 20 g/L-30 g/L sucrose, and the concentration of the gel is 6g/L agar.
10. A strawberry detoxification seedling raising method according to claim 6, wherein the concentration of MS +6-BA is preferably 0.4mg/L, and the concentration of NAA is preferably 0.08 mg/L.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05130816A (en) * | 1991-03-04 | 1993-05-28 | Iseki & Co Ltd | Artificial seeds of strawberry |
| CN101946702A (en) * | 2010-08-13 | 2011-01-19 | 北京金六环农业园 | Special medium for tissue culture of strawberry stem tip and method thereof for producing detoxification seedlings |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
| CN105961201A (en) * | 2016-05-16 | 2016-09-28 | 玉溪市六合农业科技发展有限公司 | Tissue culture method for strawberry transplant seedlings |
| JP2017176142A (en) * | 2016-03-31 | 2017-10-05 | 有限会社バイオ・ユー | Production method of strawberry sound seedling |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
-
2020
- 2020-12-02 CN CN202011414054.4A patent/CN112586350A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05130816A (en) * | 1991-03-04 | 1993-05-28 | Iseki & Co Ltd | Artificial seeds of strawberry |
| CN101946702A (en) * | 2010-08-13 | 2011-01-19 | 北京金六环农业园 | Special medium for tissue culture of strawberry stem tip and method thereof for producing detoxification seedlings |
| CN103283595A (en) * | 2013-05-17 | 2013-09-11 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
| JP2017176142A (en) * | 2016-03-31 | 2017-10-05 | 有限会社バイオ・ユー | Production method of strawberry sound seedling |
| CN105961201A (en) * | 2016-05-16 | 2016-09-28 | 玉溪市六合农业科技发展有限公司 | Tissue culture method for strawberry transplant seedlings |
| CN108464240A (en) * | 2018-04-13 | 2018-08-31 | 河北富硕农业科技发展有限公司 | The method of Snow White's strawberry detoxifying fast breeding |
Non-Patent Citations (3)
| Title |
|---|
| JAIN, S.M. ET AL.: "The Prospects of Tissue Culture and Genetic Engineering for Strawberry Improvement", 《ACTA AGRIC. SCAND., SECT.B,SOIL AND PLANT SCI.》 * |
| 孙永平 等: "红颊草莓组培快繁技术研究", 《江苏农业科学》 * |
| 聂园军 等: "草莓茎尖脱毒技术研究", 《黑龙江农业科学》 * |
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