Tissue culture method of round-leaf grapes

Abstract

The invention provides a method for tissue culture of round-leaf grapes. The tissue culture seedling of the round-leaf grape prepared by using the existing method shows that lateral buds cannot be generated, the lateral buds are few, the round-leaf grape does not extend and the like, the culture success rate is low, and batch propagation cannot be realized. Through deep research, the inventor finds that the non-ideal propagation effect in tissue culture is determined by two factors, namely a basic culture medium and hormone content, and after it is tried that propagation is difficult to realize in different hormone contents, the inventor selects to improve the basic culture medium, including adjusting the amount of ammonium nitrate and adding casein hydrolysate; therefore, efficient tissue culture of the round-leaf grapes is realized.

Description

Technical Field

[0001] The invention belongs to the field of biology and biotechnology, and more particularly, relates to a tissue culture propagation method for muscadine grapes. Background Art

[0002] Muscadine grapes (Vitis rotundifolia Michx.) belong to the subgenus Muscadine of the genus Vitis in the family Vitaceae. Unlike common grape varieties (subgenus Vitis, with 19 pairs of chromosomes), Muscadine grapes possess 20 pairs of chromosomes. Muscadine grapes are characterized by their resilient bark, the absence of transverse diaphragms at the nodes of branches and vines when longitudinally sectioned, and unbranched tendrils. Muscadine grapes originated in the southeastern United States and are a relatively unique type of American grape. Pests and diseases are one of the major factors hindering the healthy development of the grape industry. Muscadine grapes possess excellent disease and insect resistance, showing high resistance or even immunity to various pests such as phylloxera, Xiphinema, and root-knot nematodes, as well as to powdery mildew, black pox, and Pierce's disease, and are virtually immune to downy mildew. Furthermore, Muscadine grapes' high resistance and immunity to pests and diseases make them a valuable breeding material, making them a valuable germplasm resource.

[0003] Compared to other common Vitis varieties, muscadine grapes possess a unique shape that resists humidity and heat, making them suitable for cultivation in southern my country, where grape cultivation has traditionally been unsuitable and reliant on greenhouses. Muscadine grapes come in a wide variety of varieties, suitable for both fresh consumption and winemaking, with some also suitable for both fresh consumption and processing. Muscadine grapes have large berries with a unique aroma and flavor, and their skins produce antioxidants. Muscadine wines are rich in polyphenols and antioxidants, with significantly higher levels of resveratrol than more common Eurasian wines. They are also the only high-end wine to contain citric acid (a substance with anti-cancer properties), giving them a unique flavor. Therefore, the introduction of this variety will provide consumers with fresh-eating varieties and wines with unique flavors, enrich my country's existing grape resources, provide excellent stress-resistance genes for grape breeding, and potentially open up new open-air grape production areas in southern China.

[0004] Muscadine grape tissue culture seedlings is the solution of the limitation of breaking through the traditional seedling raising method, the rapid batch breeding of improved varieties seedlings. Although there is the tissue culture seedling system of other grape varieties (eugrape subgenus) at present, but Muscadine grape is because belonging to different grape subgenus, and there is the obvious difference on chromosome and external character with the kind of eugrape subgenus, the tissue culture seedling technology of Muscadine grape is also not reported at present, and the scientific research field feedback generally exists the bottleneck problem that is difficult to increase.

[0005] The invention utilizes tissue culture technology to carry out tissue culture seedling cultivation on muscadine grapes, which can not only shorten the seedling cultivation time but also realize batch seedling cultivation, and has important significance for the development of the muscadine grape industry in southern my country. Summary of the Invention

[0006] The present invention aims to provide a tissue culture propagation method for muscadine grapes.

[0007] In a first aspect of the present invention, a tissue culture method for muscadine grapes (Vitis rotundifolia Michx.) is provided, comprising: primary culture, proliferation culture, rooting culture and transplanting acclimation; the proliferation culture adopts a modified MS culture medium to which 6-benzyladenine, naphthaleneacetic acid and vitamin B2 are added; wherein the modified MS comprises: setting ammonium nitrate to a final concentration of 900±80 mg / L (preferably 900±60 mg / L; more preferably 900±40 mg / L; more preferably 900±20 mg / L; more preferably 900±10 mg / L), and adding hydrolyzed casein to a final concentration of 200±50 mg / L (preferably 200±40 mg / L; more preferably 200±30 mg / L; more preferably 200±20 mg / L; more preferably 200±10 mg / L).

[0008] In one or more embodiments, the method comprises: using muscadine grape branches as explants, obtaining stem segments with buds, and inducing the production of sprouts using a primary culture medium; the primary culture medium is MS medium, to which 6-benzyladenine (6-BA) and naphthaleneacetic acid (NAA) are added.

[0009] In one or more embodiments, the method comprises: cutting the axillary buds induced by the primary culture into small segments, inoculating them into a proliferation medium for culture, and obtaining proliferated seedlings; the proliferation medium is a modified MS medium to which 6-benzyladenine, naphthaleneacetic acid and vitamin B2 are added.

[0010] In one or more embodiments, in the method, a single bud of the terminal bud of the proliferated seedling is obtained, inoculated into a rooting medium for culture, and rooted seedlings are obtained; the rooting medium is 1 / 2 MS medium, to which naphthaleneacetic acid is added.

[0011] In one or more embodiments, in the method, the cultured rooted seedlings are transplanted for acclimatization and then cultivated in the field.

[0012] In one or more embodiments, before the primary culture, the method further comprises sterilizing the muscadine explants; preferably, the explants are soaked in alcohol and then sterilized with sodium hypochlorite.

[0013] In one or more embodiments, the muscadine grape branches are cut into small segments with 1 to 2 axillary buds; preferably, the length of the small segments is 1 to 4 cm, such as 2 or 3 cm.

[0014] In one or more embodiments, after each solution is sterilized, it is rinsed with sterile water 1 to 5 times (e.g., 2, 3, 4 times) or for 1 to 15 minutes (e.g., 2, 3, 5, 6, 8, 9, 10, 12 minutes).

[0015] In one or more embodiments, the alcohol solution is 75% alcohol; preferably, the alcohol is immersed for 20 to 120 seconds, such as 30, 50, 60, 70, 80, 90, 100 seconds.

[0016] In one or more embodiments, the sodium hypochlorite is 10% sodium hypochlorite; preferably, the disinfection is performed for 5 to 20 minutes, such as 6, 8, 10, 12, 15, or 18 minutes.

[0017] In one or more embodiments, in the primary culture, the primary culture medium comprises: 0.2-0.7 mg / L 6-benzyladenine, 0.05-0.5 mg / L naphthaleneacetic acid; preferably comprises: 0.3-0.6 mg / L 6-benzyladenine, 0.1-0.3 mg / L naphthaleneacetic acid.

[0018] In one or more embodiments, in the primary culture, the time for inducing the production of sprouts using the primary culture medium is 15 to 35 days (eg, 20, 25, or 30 days), preferably 20 to 30 days.

[0019] In one or more embodiments, during primary culture, the light intensity is 2000-3500 LUX (preferably 2500-3000 LUX), the light duration is 16±2 hours / day, and the temperature is 25±2°C.

[0020] In one or more embodiments, in the proliferation culture, the proliferation culture medium includes: 0.2-1 mg / L of 6-benzyladenine, 0.08-0.5 mg / L of naphthaleneacetic acid, and 0.5-3 mg / L of vitamin B2; preferably includes: 0.3-0.8 mg / L of 6-benzyladenine, 0.1-0.3 mg / L of naphthaleneacetic acid, and 1-2 mg / L of vitamin B2.

[0021] In one or more embodiments, during proliferation culture, subculture is performed every 20 to 35 days (e.g., 22, 24, 25, 26, 28, 30, 32 days), preferably every 22 to 32 days.

[0022] In one or more embodiments, in the rooting culture, the rooting medium comprises: 0.05-0.8 mg / L (such as 0.08, 0.1, 0.2, 0.3, 0.4, 0.6, 0.7 mg / L) of naphthaleneacetic acid; preferably 0.1-0.5 mg / L.

[0023] In one or more embodiments, during the rooting culture, the light intensity is 2000-3500 LUX (preferably 2500-3000 LUX), the light duration is 16±2 hours / day, and the temperature is 25±2°C.

[0024] In one or more embodiments, in the rooting culture, the rooting culture time is 25 to 45 days (such as 28, 30, 32, 35, 38, 40, 42 days); preferably 30 to 40 days.

[0025] In one or more embodiments, during transplanting and acclimation, the seedling substrate for transplanting and acclimation includes peat soil, perlite, vermiculite or a combination thereof; preferably, it is a substrate mixed with peat soil, perlite and vermiculite in a ratio of 2:1:1; preferably, its humidity is greater than 80%.

[0026] In one or more embodiments, in the primary culture, proliferation culture, and rooting culture, the culture medium further comprises: sucrose and / or agar (for fixing tissue culture).

[0027] In one or more embodiments, the culture medium contains sucrose at a concentration of 15-25 mg / L; preferably 18-22 mg / L, such as 20 mg / L.

[0028] In one or more embodiments, the culture medium contains 3-9 mg / L agar; preferably 5-7 or 5.5-8 mg / L, such as 6 mg / L.

[0029] In another aspect of the present invention, a tissue culture kit for culturing muscadine grapes is provided, comprising: a proliferation medium comprising a modified MS medium supplemented with 0.2-1 mg / L 6-benzyladenine, 0.08-0.5 mg / L naphthaleneacetic acid, and 0.5-3 mg / L vitamin B2; preferably, 0.3-0.8 mg / L 6-benzyladenine, 0.1-0.3 mg / L naphthaleneacetic acid, and 1-2 mg / L vitamin B2.

[0030] In one or more embodiments, the tissue culture kit further includes a primary culture medium, which includes MS culture medium supplemented with 0.2-0.7 mg / L 6-benzyladenine and 0.05-0.5 mg / L naphthaleneacetic acid; preferably includes: 0.3-0.6 mg / L 6-benzyladenine and 0.1-0.3 mg / L naphthaleneacetic acid.

[0031] In one or more embodiments, the tissue culture kit further comprises a rooting medium, which comprises 1 / 2 MS medium, to which 0.05-0.8 mg / L of naphthaleneacetic acid is added; preferably 0.1-0.5 mg / L is added.

[0032] In one or more embodiments, the tissue culture kit further comprises sucrose.

[0033] In one or more embodiments, the tissue culture kit further comprises agar.

[0034] In another aspect of the present invention, there is provided a use of the tissue culture kit for tissue culture of muscadine grapes.

[0035] Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 , axillary buds germinated on the explants 15 days after primary culture.

[0037] Figure 2 , axillary buds germinated on the explants 30 days after primary culture.

[0038] Figure 3 , bottle seedlings grown on proliferation medium for 30 days.

[0039] Figure 4 , single buds grown on proliferation medium for 30 days.

[0040] Figure 5 , rooted seedlings with single buds in rooting medium for 30 days.

[0041] Figure 6 , transplant the tissue culture rooted seedlings into the substrate for 20 days to acclimate the seedlings.

[0042] Figure 7 , transplant the tissue culture rooted seedlings into the substrate for 40 days to acclimate the seedlings. DETAILED DESCRIPTION

[0043] The present invention discloses a tissue culture method for muscadine grapes (Vitis muscadinea). This method, by fully leveraging the totipotency of plant tissue cells and plant tissue culture technology, optimizes and improves the composition of the basic culture medium to achieve a highly efficient tissue culture system for muscadine grapes. This method, through the steps of primary explant culture, proliferation culture, and rooting culture, combined with process optimization, achieves highly efficient tissue culture of muscadine grapes, ultimately yielding tissue-cultured muscadine grape plants with a high rooting rate and transplant survival rate.

[0044] Muscadine grapes (Vitis muscadinea) belong to a different subgenus than other common grape varieties (Vitis euphylla), with significant differences in chromosome structure and external fruit characteristics. However, existing tissue culture methods are all targeted at Euphylla.

[0045] In practice, the inventors found that using existing methods to grow muscadine grape tissue culture seedlings resulted in the inability to produce lateral buds / few lateral buds / no elongation, resulting in a low success rate for cultivation and the inability to achieve batch propagation. After in-depth research, it was found that the unsatisfactory propagation effect in tissue culture was determined by two factors: the basal culture medium (MS) and the hormone content during the proliferation culture phase. After trying different hormone contents and still finding it difficult to propagate, the basal culture medium (MS) during the proliferation culture phase was modified, including adjusting the amount of ammonium nitrate to 900±80mg / L and adding 200±50mg / L hydrolyzed casein. In addition, vitamin B2 was added to the proliferation and expansion culture medium to help produce lateral buds.

[0046] As used in the present invention, the "stem segment with buds" is a stem segment with axillary buds on the branches of the plant; preferably, the branches of the plant are branches grown in the current year.

[0047] As used in the present invention, the "light culture" or "normal light conditions" or "normal light culture" can be used interchangeably, which means 16±2 hours, preferably 16±1 hours of light culture and 8±1 hours of culture in a dark environment in 24 hours a day, wherein the light intensity is 2000-3500LUX; preferably 2500-3000LUX.

[0048] As used in the present invention, the natural environment includes but is not limited to: planting environments such as nurseries, fields, and forests.

[0049] As used herein, "comprising," "including," or "comprising" means that various components can be used together in the mixture, composition, or culture medium of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the terms "comprising," "including," or "comprising."

[0050] As used in the present invention, the tissue culture kit refers to a package / box that is convenient for commercial-scale production and use, which usually contains a container / small package, and the culture medium, solution, single component or component of the present invention can be placed in the container / small package.

[0051] The present invention provides a method for promoting the proliferation and rooting of muscadine grapes. This method, developed after years of observation and experimentation, fully considers the characteristics of the muscadine species and its differences from other grapes. The method primarily includes the steps of primary culture, proliferation culture, rooting culture, and transplanting and acclimation. Furthermore, prior to the primary culture, a disinfection step is preferably included; during the transplanting of test tube seedlings, a hardening and acclimation process is preferably also included.

[0052] In a preferred embodiment, during the primary culture, the inventors obtained muscadine grape branches, preferably branches that emerged in the current year, as explants, obtained stem segments with buds, and cultured them with an initial induction medium to induce axillary buds.

[0053] During primary culture, the culture medium is a basic MS culture medium, and 6-BA and NAA are further added to the primary culture medium.

[0054] The sprouts that have undergone primary culture are further subjected to proliferation culture. During the proliferation stage of culture, the sprouts that have undergone primary culture are cut into small buds, inoculated into a proliferation medium for culture, and proliferated sprouts are obtained. The proliferation medium is a modified MS medium, in which the dosage of the components is optimized, specifically, the amount of ammonium nitrate is adjusted (for example, reduced), and hydrolyzed casein is added to a relatively high amount; more preferably, ammonium nitrate is adjusted to a final concentration of 900±80 mg / L, and hydrolyzed casein is added to a final concentration of 200±50 mg / L. In addition, Vb2, 6-BA and NAA are further added to the proliferation medium.

[0055] After the aforementioned proliferation culture, the inventors further carried out rooting culture of muscadine grape seedlings, comprising: inoculating the proliferated apical buds of the seedlings into a rooting medium for culture to obtain rooted seedlings; the rooting medium is 1 / 2MS medium to which naphthaleneacetic acid is added.

[0056] After obtaining the rooted test tube seedlings as described above, the inventors further transplanted the test tube seedlings into a seedling culture medium for hardening and acclimation; thereafter, the acclimated seedlings were transplanted into a natural environment.

[0057] As a preferred embodiment of the present invention, during the seedling hardening and acclimation process, the seedling culture medium includes peat soil, perlite, vermiculite or a combination thereof.

[0058] In the technical solution of the present invention, the branches of muscadine grapes are used as explants, so before the initial culture, a step of sterilizing the branches is also included.

[0059] The number of disinfection cycles has a certain relationship with the contamination rate during subsequent incubation. Insufficient disinfection may lead to an increase in the contamination rate. Therefore, as a preferred method of the present invention, it is preferable to soak in alcohol followed by disinfection with sodium hypochlorite. In a preferred embodiment, after disinfection with each solution, rinse with sterile water 1-3 times.

[0060] Based on the tissue culture scheme of muscadine grapes provided by the present invention, a tissue culture kit for culturing muscadine grapes is also provided, which includes: a primary culture medium, a proliferation culture medium, and a rooting culture medium.

[0061] As a preferred embodiment of the present invention, the tissue culture kit further comprises sucrose and agar, which are added to the culture medium.

[0062] As a preferred embodiment of the present invention, the tissue culture kit further includes some basic culture media for plant cultivation, including: MS culture medium, 1 / 2MS culture medium, or culture media formed by appropriate changes based on them.

[0063] As a preferred embodiment of the present invention, the tissue culture kit also includes instructions for use, which describe the use of each culture medium, or also describe the treatment method for plants or plant tissues before or after using the culture medium, so as to help guide those skilled in the art to use the tissue culture kit in an appropriate manner.

[0064] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0065] Materials and methods

[0066] MS medium: purchased from CMS, product number M519.

[0067] 1 / 2MS medium: Based on MS medium (Cimeijie product number M519), the content of macroelements is reduced by half.

[0068] MS modified medium: MS medium (Cimeijie product number M519) is prepared based on Chinese medicine chemicals, with ammonium nitrate adjusted to a final concentration of 900 mg / L, and hydrolyzed casein added to a final concentration of 200 mg / L.

[0069] The range of primary culture medium formula values and example values are shown in Table 1.

[0070] Table 1

[0071]

[0072] The formula of proliferation medium is shown in Table 2.

[0073] Table 2

[0074]

[0075] The rooting medium formula is shown in Table 3.

[0076] Table 3

[0077]

[0078] All media were sterilized by autoclaving at 121°C for 20 minutes.

[0079] Unless otherwise specified, cells were cultured in a light-sensitive environment with a 16-hour / 8-hour (day / night) illumination cycle and a light intensity of 2000-3000 Lux. If dark-sensitive cultures were specified, cells were cultured in a dark environment for 24 hours.

[0080] Example 1. Initial culture analysis

[0081] There are two common tissue culture methods for grape varieties of the subgenus Vitis, which are most similar to Muscadine grapes: one-step tissue culture (directly using rooting medium for rooting and sprouting at the same time, and continuing to use the same rooting medium for rooting after cutting a seedling into 1.5 cm stem segments) and two-step tissue culture (including proliferation and rooting).

[0082] During the development of the invention, the inventors attempted to cultivate muscadine grapes using several commonly used tissue culture protocols for grape varieties of the subgenus Vitis. Some of these protocols were patented for tissue culture of other euvitis grape varieties, while others were publicly available and commonly used protocols. Despite repeated cultivation and analysis, the inventors discovered that these protocols were not suitable for muscadine grapes.

[0083] 1. Comparison of the effects of one-step tissue culture on muscadine grapes and common euryphaloid varieties

[0084] Stem segments of two muscadine grape varieties, GV and Carlos, and several common Vitis vinifera varieties (seedless white, cabernet sauvignon, and double red) were cultured on two rooting media:

[0085] The first type: MS+0.2mg / L NAA+0.1mg / L IAA (see patent CN201310126991.3);

[0086] The second type: 1 / 2MS+0.3mg / L NAA+20mg / L sucrose.

[0087] Analyzing the rooting rate, there is no significant difference between the rooting rate of Muscadine grape and other grape varieties of the subgenus Vitis, and the rooting rate is basically above 90%.

[0088] Observations show that lateral buds readily germinate after rooting in cultivars of the subgenus Euphorbia. After 30 days, individual plants exceeding 5 cm in height can be obtained. Cutting the seedlings into stem segments with a single axillary bud and placing them in the two rooting media mentioned above repeatedly allows for subculture and proliferation, meaning that a single culture medium can achieve both proliferation and rooting. However, lateral buds in muscadine varieties are difficult to germinate after rooting, with only 10%-20% of them germinating. Furthermore, these buds do not grow as vigorously as in other cultivars, producing only one or two leaves and a height of 1.5 cm after 30-40 days, indicating failure to achieve proliferation (Table 4).

[0089] Table 4

[0090]

[0091] Therefore, the one-step tissue culture method applicable to traditional Vitis euryale varieties is not suitable for tissue culture of Muscadine varieties.

[0092] 2. Comparison of the effects of one-step tissue culture on muscadine grapes and common eurypho-Vitis subgenus varieties

[0093] Muscadine grapes were cultured using the existing patented rooting medium B5+0.1 mg / L IAA+50 mg / L penicillin (see patent 201710231766.4).

[0094] After 30 days of cultivation, 90% of the seedlings took root, but less than 5% of the stem segments produced side buds, and after 20 days the stem segments turned yellow and withered.

[0095] From the results, it can be seen that muscadine grapes are not suitable for this culture medium. They can only take root, but it is not easy to induce lateral buds.

[0096] Example 2: Optimized tissue culture method for muscadine grapes

[0097] 1. Explant disinfection

[0098] Collect the tender branches of the current year, wash them with water, and then cut them into small segments with 1-2 axillary buds and a length of about 1.5-2.5 cm. Soak them in 75% alcohol for 60 seconds on a clean bench, then disinfect them with 10% sodium hypochlorite for 10-15 minutes, and rinse them with sterile water 4 times.

[0099] 2. Primary Culture

[0100] The disinfected and treated stem segments with buds were inoculated into primary culture medium (MS+0.5 mg / L 6-BA+0.25 mg / L NAA+20 mg / L sucrose+6 mg / L agar) for bud induction culture, and sterile seedlings were obtained after 20-30 days of culture.

[0101] The culture conditions for tissue culture seedlings are light intensity 2500-3000LUX, light duration 16h / d, and temperature 24-26℃.

[0102] Axillary buds germinated from explants after 15 days of primary culture Figure 1 , it can be seen that the axillary buds are thick, bright green in color and growing well.

[0103] 3. Proliferation culture

[0104] The axillary buds induced in the primary culture medium were cut into small segments of about 1.5 cm and inoculated into a proliferation medium (MS modified + 0.5 mg / L 6-BA + 0.25 mg / L NAA + 1 mg / L Vb2 + 20 mg / L sugar cane + 6 mg / L agar). Ten stem segments were accessed in each bottle of culture medium and subcultured once every 25-30 days. Wherein, ammonium nitrate was set to 900 mg / L in the MS modified culture medium, and 200 mg / L casein hydrolyzate was set.

[0105] The seedlings grown on the proliferation medium for 25 days were Figure 3 , it can be seen that the seedlings are growing vigorously and the leaves are bright green.

[0106] The expansion and proliferation can reach more than 3 times.

[0107] 4. Rooting culture

[0108] Select the terminal bud that has grown to about 2 cm or more, cut a single bud of 1.5-2 cm, and inoculate it into rooting medium (1 / 2MS + NAA 0.4 mg / L + sucrose 20 mg / L + agar 6 mg / L) for culture under light conditions.

[0109] Culture conditions: light intensity 2500-3000LUX, light duration 16h / d, temperature 24-26℃.

[0110] It will start to take root in about 7-10 days, with a rooting rate of 100%, and can be transplanted after about 30-40 days.

[0111] Rooting seedlings with single buds in rooting medium for 30 days Figure 5 , it can be seen that the root phenotype is thick, the growth is healthy, and it can provide good nutrition for the plant.

[0112] 5. Transplantation and domestication

[0113] For the seedlings that have been rooting for about 35 days, wash off the agar on the roots with water and transplant them into a matrix mixed with peat soil, perlite and vermiculite in a ratio of 2:1:1. Maintain the humidity above 80% for the first 10 days.

[0114] The rooted seedlings were transplanted into the substrate for 20 days. Figure 6 It can be seen that the seedlings were successfully transplanted and domesticated, and the leaves were thick and healthy, showing a dark green color.

[0115] The results showed that the seeds could be transplanted into the field after 30 days.

[0116] Analysis of the transplant survival rate showed that the survival rate reached 100%.

[0117] Example 3: Optimized tissue culture method for muscadine grapes

[0118] 1. Explant disinfection

[0119] Collect the tender branches of the current year, wash them with water, and then cut them into small segments with 1-2 axillary buds and a length of about 1.5-2.5 cm. Soak them in 75% alcohol for 60 seconds on a clean bench, then disinfect them with 10% sodium hypochlorite for 10-15 minutes, and rinse them with sterile water 4 times.

[0120] 2. Primary Culture

[0121] The disinfected and treated stem segments with buds were inoculated into primary culture medium (MS+6-BA 0.4 mg / L+NAA 0.1 mg / L+sucrose 20 mg / L+6 mg / L agar) for bud induction culture, and sterile seedlings were obtained after 20-30 days of culture.

[0122] The culture conditions for tissue culture seedlings are light intensity 2500-3000LUX, light duration 16h / d, and temperature 24-26℃.

[0123] The axillary buds that germinated on the explants after 30 days of primary culture were as follows: Figure 2 Compared with the axillary buds cultured for 15 days, the axillary buds cultured for 30 days grew significantly, with small leaves appearing, the color was emerald green, and the growth was very good.

[0124] 3. Proliferation culture

[0125] The axillary buds induced in the primary culture medium were cut into small segments of about 1.5 cm and inoculated into a proliferation medium (MS modified + 0.7 mg / L 6-BA + 0.15 mg / L NAA + 2 mg / L Vb2 + 20 mg / L sucrose + 6 mg / L agar). Ten stem segments were accessed in each bottle of culture medium and subcultured once every 25-30 days. Wherein, ammonium nitrate in the MS modified culture medium was set to a final concentration of 900 mg / L, and casein hydrolyzate was set to 200 mg / L.

[0126] The bud clusters of single buds grown on the proliferation medium for 30 days are as follows Figure 4 , you can see that the buds are green and growing very well.

[0127] The expansion and multiplication can reach more than 4 times.

[0128] 4. Rooting culture

[0129] Select the terminal bud that has grown to about 2 cm or more, cut a single bud of 1.5-2 cm, and inoculate it into rooting medium (1 / 2MS+0.15 mg / L NAA+20 mg / L sucrose+6 mg / L agar) for culture under light conditions.

[0130] The culture conditions are light intensity 2500-3000LUX, light duration 16h / d, and temperature 24-26℃.

[0131] Rooting begins in about 7-10 days, with a rooting rate of 99%. Transplanting can be done after about 30-40 days.

[0132] 5. Transplantation and domestication

[0133] For the seedlings that have been rooting for about 35 days, wash off the agar on the roots with water and transplant them into a matrix mixed with peat soil, perlite and vermiculite in a ratio of 2:1:1. Maintain the humidity above 80% for the first 10 days.

[0134] The results showed that the crops could be transplanted into the field in 30 days or later.

[0135] The rooted seedlings were transplanted into the substrate for 40 days. Figure 7 It can be seen that the seedlings were successfully transplanted and domesticated, and the leaves were thick and healthy, showing a dark green color.

[0136] Analysis of the transplant survival rate showed that the survival rate reached 100%.

[0137] Example 4: Comparison of the effects of culturing muscadine grapes using the prior art three-step method

[0138] A stem segment of a muscadine grape variety (GV, Geweil) with 1-2 axillary buds was cultured on the following proliferation media:

[0139] MS+0.6 mg / L 6-BA+0.1 mg / L NAA (self-designed solution before basal medium improvement);

[0140] MS + GA3 0.3 mg / L + 6-BA 0.3 mg / L + IBA 0.5 mg / L (refer to CN 201510762618.6 and Example 1, but the grape variety is Muscadine Vitis vinifera, and the three-step method includes primary culture, expansion culture, and rooting culture);

[0141] 1 / 2MS+NAA 0.05 mg / L+IBA 0.5 mg / L (refer to the corresponding concentration group in CN 201811112855.8, and the grape variety is Muscadine Vitis vinifera).

[0142] The culture results are shown in Table 5.

[0143] Table 5

[0144]

[0145] Based on the above, the growth conditions of the muscadine grape variety GV on the three culture media were not ideal.

[0146] Example 5: Comparison of the effects of culturing muscadine grapes and common euvitis subgenus varieties using the three-step tissue culture method and the conventional grape tissue culture protocol with unmodified basal medium

[0147] The three-step tissue culture process included primary culture, expansion, and rooting. The primary culture and rooting steps were the same as those in Example 2. In the proliferation culture, stem segments with one to two axillary buds from a muscadine grape variety (GV, Gwiel) and several common euvice grape varieties (seedless white, Cabernet Sauvignon, and Double Red) were placed in a proliferation medium containing MS + 0.6 mg / L 6-BA + 0.1 mg / L NAA (a conventional grape tissue culture protocol using unmodified basal medium).

[0148] Significant differences were observed between the Muscadine Vitis genus and the other three varieties. The other three Vitis genus varieties grew vigorously, with 4-6 leaves and could reach heights exceeding 3 cm, achieving a multiplication rate of more than 3 times. However, the Muscadine Vitis genus only produced a single leaf per plant, and its vitrification and weak growth, only about 1-1.5 cm in height, failed to achieve the desired multiplication rate (Table 6).

[0149] Table 6

[0150] variety Explant (strain) Number of lateral buds (plants) Height (cm) Muscadine Grape GV 8 9 1-1.5 Seedless White 8 30 3-4.5 Cabernet Sauvignon 8 32 3-5 Double Red 8 40 3-4.5

[0151] The above results indicate that the tissue culture scheme adapted to the genus Euclid cannot achieve good proliferation of Muscadine grapes.

[0152] Example 6 Comparison of culturing muscadine grapes using the modified MS proliferation medium of the present invention In this example, the culturing effects of the optimized technical methods of Examples 2 and 3 were verified.

[0153] A three-step tissue culture process was performed, including primary culture, expansion, and rooting. The primary culture and rooting steps were the same as in Example 2. During the proliferation culture, stem segments of a muscadine grape variety with one to two axillary buds were placed in a MS-modified proliferation medium (MS-modified + 0.6 mg / L 6-BA + 0.1 mg / L NAA + 2 mg / L Vb2). The ammonium nitrate in the MS-modified medium was adjusted to a final concentration of 900 mg / L, and the hydrolyzed casein concentration was set at 200 mg / L.

[0154] The results showed that the two varieties of muscadine grapes grew vigorously and robustly, with 4-5 side buds and a height of about 3 cm in 30 days (Table 7).

[0155] Table 7

[0156] variety Explant (strain) Number of lateral buds (plants) 30-day height (cm) Muscadine Grape GV 8 35 3.1-4.5 Carlos Muscadine 8 32 3.3-4.1

[0157] In summary, muscadine grapes require a special culture scheme that is different from other varieties, and the optimized scheme of the present invention is very suitable for tissue culture of muscadine grapes.

[0158] The above-described embodiments merely illustrate several embodiments of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of the present invention. Therefore, the scope of the present invention shall be determined by the appended claims. All references mentioned herein are incorporated by reference in this application, just as if each reference were incorporated by reference individually.

Claims

1. A tissue culture method for muscadine grapes (Vitis rotundifolia Michx.), comprising: Primary culture, proliferation culture, rooting culture and transplanting acclimatization; The proliferation culture medium used in the proliferation culture is a modified MS medium, to which 6-benzyladenine, naphthaleneacetic acid and vitamin B2 are added; The MS improvement includes: setting ammonium nitrate to a final concentration of 900±80 mg / L and adding hydrolyzed casein to a final concentration of 200±50 mg / L.

2. The method according to claim 1, wherein In the method: The primary culture comprises: using muscadine grape branches as explants, obtaining stem segments with buds, and inducing the production of sprouts using a primary culture medium; the primary culture medium is an MS medium supplemented with 6-benzyladenine and naphthaleneacetic acid; The proliferation culture comprises: cutting the axillary buds induced by the primary culture into small segments, inoculating them into a proliferation culture medium for cultivation, and obtaining proliferated shoots; the proliferation culture medium is a modified MS medium supplemented with 6-benzyladenine, naphthaleneacetic acid, and vitamin B2; The rooting culture comprises: inoculating a single bud of the terminal bud of the proliferated seedling into a rooting medium for cultivation to obtain a rooted seedling; the rooting medium is a 1 / 2MS medium to which naphthaleneacetic acid is added; Transplanting and domestication include: transplanting and domesticating the seedlings that have been rooted and cultured, and then cultivating them in the field.

3. The method according to claim 2, wherein Before the primary culture, the explants of the muscadine grapes are sterilized; preferably, they are soaked in alcohol and then sterilized with sodium hypochlorite.

4. The method according to claim 2, wherein In the primary culture, the primary culture medium comprises: 0.2-0.7 mg / L 6-benzyladenine, 0.05-0.5 mg / L naphthaleneacetic acid; preferably comprises: 0.3-0.6 mg / L 6-benzyladenine, 0.1-0.3 mg / L naphthaleneacetic acid; or In the primary culture, the time for inducing the production of sprouts using the primary culture medium is 15 to 35 days, preferably 20 to 30 days; or During primary culture, the light intensity is 2000-3500 LUX, the light duration is 16±2 hours / day, and the temperature is 25±2°C.

5. The method according to claim 2, wherein In the proliferation culture, the proliferation culture medium comprises: 6-benzyladenine 0.2-1 mg / L, naphthaleneacetic acid 0.08-0.5 mg / L, vitamin B2 0.5-3 mg / L; preferably comprises: 6-benzyladenine 0.3-0.8 mg / L, naphthaleneacetic acid 0.1-0.3 mg / L, vitamin B2 1-2 mg / L; or During proliferation culture, subculture is performed every 20 to 35 days, preferably every 22 to 32 days.

6. The method according to claim 2, wherein In the rooting culture, the rooting medium comprises: 0.05-0.8 mg / L of naphthaleneacetic acid; preferably 0.1-0.5 mg / L; or During rooting culture, the light intensity is 2000-3500 LUX, the light duration is 16±2 hours / day, and the temperature is 25±2℃; or In the rooting culture, the rooting culture time is 25 to 45 days, preferably 30 to 40 days.

7. The method according to claim 2, wherein The seedling raising substrate for transplanting and acclimation includes peat soil, perlite, vermiculite or a combination thereof; preferably, it is a substrate mixed with peat soil, perlite and vermiculite in a ratio of 2:1:1; preferably, its humidity is greater than 80%.

8. The method according to claim 2, wherein In the primary culture, proliferation culture and rooting culture, the culture medium further comprises: sucrose and / or agar; Preferably, the culture medium contains sucrose 15-25 mg / L; more preferably 18-22 mg / L, such as 20 mg / L; Preferably, the culture medium contains 3-9 mg / L agar; more preferably 5-7 or 5.5-8 mg / L, such as 6 mg / L.

9. A tissue culture kit for culturing muscadine grapes, comprising: A proliferation medium comprising a modified MS medium supplemented with 0.2-1 mg / L 6-benzyladenine, 0.08-0.5 mg / L naphthaleneacetic acid, and 0.5-3 mg / L vitamin B2; preferably, 0.3-0.8 mg / L 6-benzyladenine, 0.1-0.3 mg / L naphthaleneacetic acid, and 1-2 mg / L vitamin B2; wherein ammonium nitrate is set to a final concentration of 900±80 mg / L and hydrolyzed casein is added to a final concentration of 200±50 mg / L; More preferably, the tissue culture kit further comprises components selected from the following group: A primary culture medium comprising MS medium supplemented with 0.2-0.7 mg / L 6-benzyladenine and 0.05-0.5 mg / L naphthaleneacetic acid; preferably comprising 0.3-0.6 mg / L 6-benzyladenine and 0.1-0.3 mg / L naphthaleneacetic acid; A rooting medium comprising 1 / 2 MS medium supplemented with 0.05-0.8 mg / L of naphthaleneacetic acid, preferably 0.1-0.5 mg / L; sucrose; Agar.

10. Use of the tissue culture kit according to claim 9 for tissue culture of muscadine grapes.

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