Method for efficiently breeding actinidia chinensis

Abstract

The application provides a method for efficiently breeding Actinidia chinensis, comprising the following steps: taking leaves or stem segments of Actinidia chinensis as explants, and performing culture treatment including induction culture on the explants to obtain Actinidia seedlings; wherein an induction culture medium used in the induction culture comprises an MS culture medium and an additive, the additive is selected from at least two of KT, 2,4-D, 6-BA, NAA, IBA and IAA; and in the culture medium, the content of the KT is 0.05-0.1 mg / L, the content of the 2,4-D is 0.5-1.0 mg / L, the content of the 6-BA is 0.1-0.5 mg / L, the content of the NAA is 0.5-1.0 mg / L, the content of the IBA is 0.01-0.05 mg / L, and the content of the IAA is 0.5-1.0 mg / L. The method provided by the application overcomes the influence of environmental factors on the breeding of Actinidia chinensis, and helps the protection of biodiversity.

Description

Technical Field

[0001] This invention relates to a method for the efficient propagation of Chinese kiwifruit, belonging to the field of biotechnology. Background Technology

[0002] Chinese kiwifruit (Actinidia chinensis), also known as kiwi, vine pear, sheep peach vine, sheep peach, yangtao, kiwi, and Jinggangshan kiwifruit, belongs to the genus Actinidia in the family Actinidiaceae. It grows in mountain forests at altitudes of 200-600 meters, typically in tall grass thickets, shrublands, or secondary sparse forests. It prefers humus-rich, well-drained soil. Kiwifruit distributed in higher latitudes thrives in warm, humid, sheltered, and sunny environments. Analysis shows that the fruit generally contains 100-200 mg of vitamins per 100 grams of fresh sample, with some reaching 400 mg, approximately 5-10 times that of citrus fruits. It contains 8-14% sugars, 1.4-2.0% acids, and 12 kinds of amino acids, including tyrosine. Chinese kiwifruit is highly valued for its economic and nutritional value and is widely loved.

[0003] Currently, due to the low natural reproductive capacity of *Actinidia chinensis*, the number of wild individuals is decreasing year by year, necessitating urgent conservation efforts. *Actinidia chinensis*, *Actinidia arguta*, *Actinidia arguta*, *Actinidia chinensis* var. *chinensis*, and *Actinidia arguta* var. *chinensis* are all listed as Class II National Key Protected Wild Plants. Clearly, protecting rare plant resources is of paramount importance.

[0004] Generally, the propagation methods for Chinese kiwifruit mainly include sowing, cutting, and grafting. For sowing, select large, ripe fruits, allow them to ripen for six to seven days after harvesting, extract the seeds, wash them with water, dry them, wrap them in gauze, and then bury them in slightly moist sand for 1-2 months to improve germination vigor and rate. For cutting, select well-drained, loose, permeable sandy soil with good water retention. Mix an appropriate amount of river sand into the top 15 cm of soil to promote rooting. For grafting, select healthy, robust one-year-old branches with plump buds as scions. Leave 1-2 strong buds on each scion. The long cut of the scion should be 3-4 cm, and the short cut 0.5-1.0 cm. Insert the long cut of the scion into the cut on the rootstock, ensuring that the cambium layers align on one side after grafting.

[0005] However, the above-mentioned breeding methods are traditional breeding methods, which are affected to varying degrees by environmental factors such as season, weather, and temperature, severely limiting the breeding of Chinese kiwifruit. Summary of the Invention

[0006] This invention provides a method for the efficient propagation of Chinese kiwifruit. The method uses Chinese kiwifruit stem segments or leaves as explants, and obtains kiwifruit seedlings through induction culture. It is not affected by environmental factors and can achieve efficient propagation of Chinese kiwifruit.

[0007] This invention provides a method for efficiently propagating Chinese kiwifruit, comprising the following steps:

[0008] Kiwifruit leaves or stem segments were used as explants, and the explants were subjected to culture treatments including induction culture to obtain kiwifruit seedlings.

[0009] The induction culture medium used includes MS medium and additives, wherein the additives are selected from at least two of KT, 2,4-D, 6-BA, NAA, IBA, and IAA.

[0010] Furthermore, in the induction medium, the content of KT is 0.05-0.1 mg / L, the content of 2,4-D is 0.5-1.0 mg / L, the content of 6-BA is 0.1-0.5 mg / L, the content of NAA is 0.5-1.0 mg / L, the content of IBA is 0.01-0.05 mg / L, and the content of IAA is 0.5-1.0 mg / L.

[0011] The additives in the method described above include at least one of a first additive combination, a second additive combination, a third additive combination, and a fourth additive combination;

[0012] The first additive combination includes KT, 2,4-D, and NAA; the second additive combination includes 6-BA and IAA; the third additive combination includes KT and NAA; and the fourth additive combination includes KT, 6-BA, 2,4-D, IBA, and NAA.

[0013] As described above, the leaves of the Chinese kiwifruit are 5-10 days old and have a length × width of 1-2 cm × 1-2 cm.

[0014] The method described above further includes adventitious shoot proliferation culture after the induction culture.

[0015] The adventitious bud proliferation culture medium used in the adventitious bud proliferation culture includes MS medium and proliferation additives, wherein the proliferation additives include at least two of KT, 2,4-D, 6-BA, and NAA;

[0016] Furthermore, in the adventitious bud proliferation medium, the content of KT is 0.05-0.2 mg / L, the content of 2,4-D is 0.5-1.0 mg / L, the content of 6-BA is 0.1-1.0 mg / L, and the content of NAA is 0.1-1.0 mg / L.

[0017] The adventitious bud proliferation medium, as described above, comprises KT, 6-BA, and NAA.

[0018] Furthermore, in the adventitious bud proliferation medium, the content of KT is 0.05-0.1 mg / L, the content of 6-BA is 0.5-1.0 mg / L, and the content of NAA is 0.5-1.0 mg / L.

[0019] As described above, after the adventitious bud proliferation culture, the method further includes seedling strengthening culture, and the seedling strengthening culture medium used includes MS medium, 0.08 mg / L KT, 0.25 mg / L 6-BA, and 0.8 mg / L NAA.

[0020] As described above, after the seedling culture, the method further includes rooting culture, and the rooting culture medium used includes MS medium, 0.5 ml / L IBA, and 0.1 g / L Ac.

[0021] As described above, after the rooting culture, the method also includes transplanting culture, and the substrate used for the transplanting culture includes garden soil, perlite, peat, and vermiculite in a volume ratio of 3:1:2:1.

[0022] The method described above further includes a sterilization process before the induction culture, which includes the following steps:

[0023] The explants were cleaned, then soaked in 75% medical alcohol (by volume), rinsed, and then soaked in 0.1 wt% mercuric chloride solution with Tween 80 added. After rinsing, the explants were disinfected.

[0024] As described above, during the cultivation process, the illumination time is 12 h / d, the light intensity is 2500-3000 lx, and the cultivation temperature is 23-27℃.

[0025] The present invention provides a method for the efficient propagation of Actinidia chinensis, which uses stem segments or leaves of Actinidia chinensis as explants and induces the explants with a specific induction culture medium to obtain kiwi seedlings. The method of the present invention can not only overcome the influence of environmental factors on the propagation of Actinidia chinensis, but also achieve the increase in the number of propagated plants, thus contributing to the preservation of germplasm resources and the protection of biodiversity of Actinidia chinensis. Attached Figure Description

[0026] Figure 1 This is a photograph of the leaf wrinkling and dedifferentiation beginning in Example 1;

[0027] Figure 2 Here are detailed photographs of the leaf callus tissue in Example 1;

[0028] Figure 3 Photographs of adventitious shoots induced from callus tissue in Example 1;

[0029] Figure 4 Photographs of adventitious bud proliferation in Example 1;

[0030] Figure 5 These are photos showing the results of adventitious bud rooting culture after 10 days in Example 1;

[0031] Figure 6 These are photos showing the results of adventitious bud rooting culture after 20 days in Example 1;

[0032] Figure 7 This is a photo of the outdoor growth scene of Chinese kiwifruit in Example 1. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] This invention provides a method for efficiently propagating Chinese kiwifruit, comprising the following steps:

[0035] Kiwifruit leaves or stem segments were used as explants, and culture treatments, including induction culture, were performed on the explants to obtain kiwifruit seedlings.

[0036] The induction culture medium used includes MS medium and additives, and the additives are selected from at least two of KT, 2,4-D, 6-BA, NAA, IBA and IAA.

[0037] Furthermore, the induction medium contains 0.05-0.1 mg / L of KT, 0.5-1.0 mg / L of 2,4-D, 0.1-0.5 mg / L of 6-BA, 0.5-1.0 mg / L of NAA, 0.01-0.05 mg / L of IBA, and 0.5-1.0 mg / L of IAA.

[0038] The method provided by this invention establishes a highly efficient propagation system that uses leaves or stem segments of Chinese kiwifruit as explants to induce callus formation, and then obtains adventitious buds from the callus to cultivate kiwifruit seedlings. In this system, the adventitious bud induction culture is achieved through a specific induction culture medium, which can reduce the influence of external environmental factors and shorten the seedling cycle.

[0039] In detail, leaves or stem segments of Chinese kiwifruit are cut using sterile instruments as explants. The explants are then inoculated onto an induction medium for induction culture. During the induction culture process, callus tissue, which is bright green or slightly reddish, gradually appears on the explants. As the culture time extends, adventitious buds appear in different parts of the callus tissue. The adventitious buds are then treated to obtain kiwifruit seedlings.

[0040] This invention does not limit the specific growth time of leaves and stem segments of Chinese kiwifruit. Leaves and stem segments can be selected according to actual needs. In one specific embodiment, leaves and stem segments with a growth period of 5-10 days are selected as explants.

[0041] This invention does not limit the specific length of the stem segment as the explant. The stem segment of appropriate length can be cut according to the actual situation, such as a tender stem segment with a length of 1-2 cm.

[0042] The MS medium formulation for the induction medium of this invention is disclosed. The MS medium includes inorganic salts, organic substances, vitamins, etc., which can effectively promote the division and growth of plant cells. In this invention, KT refers to kinetin, NAA refers to naphthaleneacetic acid, 2,4-D refers to dichlorophenoxyacetic acid, 6-BA refers to 6-benzylaminopurine, IAA refers to indoleacetic acid, and IBA refers to indolebutyric acid.

[0043] To ensure sufficient carbon source for each induction medium and to improve the support of the medium, the medium of the present invention may also include sucrose and agar. The present invention does not limit the specific content of sucrose and agar, and the content of sucrose and agar can be selected according to actual needs.

[0044] In one specific embodiment, the induction culture medium contains 30g of sucrose and 7g of agar.

[0045] This invention does not limit the pH value of the induction culture medium; a suitable pH range can be selected according to the actual situation, such as pH 5.5-6.0.

[0046] The present invention does not limit the method of adjusting the pH of the culture medium. For example, before the culture medium is completely dissolved and dispensed, the initial pH value of the culture medium is first measured with pH test paper or pH meter. Then, according to the test value, 1 mol / L HCl or NaOH is added dropwise to the culture medium to adjust the pH value of the culture medium to 5.5-6.0.

[0047] This invention does not limit the specific time of induction culture. It is understood that as the induced callus tissue continues to grow, the nutrients and auxins in the induction culture medium are gradually consumed. At this time, the same type of induction culture medium can be replaced to continue culturing the explants.

[0048] It is understood that the cultivation treatment also includes seedling strengthening, rooting, and transplanting, until kiwi seedlings are obtained. This invention does not limit the specific steps of seedling strengthening, rooting, and transplanting; methods commonly used in the art can be selected to perform seedling strengthening, rooting, and transplanting treatments on adventitious bud clusters.

[0049] The method provided by this invention uses stem segments or leaves of Actinidia chinensis as explants and induces adventitious buds through a specific culture medium, thereby achieving efficient propagation of Actinidia chinensis, shortening the propagation cycle of Actinidia chinensis, and overcoming the influence of the external environment on the propagation of Actinidia chinensis.

[0050] Furthermore, in one specific embodiment of the present invention, the additive includes at least one of a first additive combination, a second additive combination, a third additive combination, and a fourth additive combination;

[0051] The first additive combination includes KT, 2,4-D, and NAA; the second additive combination includes 6-BA and IAA; the third additive combination includes KT and NAA; and the fourth additive combination includes KT, 6-BA, 2,4-D, IBA, and NAA.

[0052] In detail, the induction medium consisting of the first additive combination includes MS medium, 0.05-0.1 mg / L KT, 0.5-1.0 mg / L 2,4-D, 0.5-0.85 mg / L NAA, 30 g sucrose, and 7 g agar.

[0053] The induction medium consisting of the second additive combination includes MS medium, 0.1–0.5 mg / L 6-BA, 0.5–1.0 mg / L IAA, 30 g sucrose, and 7 g agar.

[0054] The third additive combination constitutes the induction medium, which includes MS medium, 0.05–0.1 mg / L KT, 0.5–1.0 mg / L NAA, 30 g sucrose, and 7 g agar.

[0055] The fourth additive combination constitutes the induction medium, which includes MS medium, 0.05-0.1 mg / L KT, 0.1-0.5 mg / L 6-BA, 0.5-1.0 mg / L 2,4-D, 0.01-0.05 mg / L IBA, 0.15-0.35 mg / L NAA, 30 g sucrose, and 7 g agar.

[0056] In one specific embodiment, the induction culture medium composed of the fourth additive combination includes MS medium, 0.1 mg / L KT, 0.25 mg / L 6-BA, 0.8 mg / L 2,4-D, 0.02 mg / L IBA, 0.3 mg / L NAA, 30 g sucrose, and 7 g agar.

[0057] Through extensive creative experiments, the inventors discovered that when the induction culture medium includes the above-mentioned components, callus tissue can appear on the 10th-15th day of induction culture, and adventitious buds can appear after 30-40 days of continued culture, which greatly shortens the induction culture cycle. The reason is that the tissue cells of young leaves of Chinese kiwifruit undergo dedifferentiation and redifferentiation in a relatively short period of time under the synergistic effect of the various components of the induction culture medium, thereby completing the transformation from leaves to callus tissue and then to adventitious buds.

[0058] Furthermore, in one specific embodiment of the present invention, the leaves of the Chinese kiwifruit are 5-10 days old and have a length × width of 1-2cm × 1-2cm.

[0059] Specifically, the age of the leaves of *Actinidia chinensis* used as explants is 5-10 days. For example, the age range includes, but is not limited to, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or any combination thereof.

[0060] It is understood that the shape of the leaves of the Chinese kiwifruit is not a perfectly regular rectangle. In this invention, the length of the Chinese kiwifruit leaf refers to the longest dimension of the Chinese kiwifruit leaf, which is usually the straight-line distance from the base of the leaf to the tip.

[0061] The width of the leaves of the Chinese kiwifruit refers to the straight-line distance at the widest point of the leaf, usually the distance from the left to the right side of the leaf.

[0062] The leaves of *Actinidia chinensis* used as explants are 1-2 cm long and 1-2 cm wide. For example, the length includes, but is not limited to, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2.0 cm, or any combination thereof; the width includes, but is not limited to, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2.0 cm, or any combination thereof.

[0063] When the explants were selected from the leaves of *Actinidia chinensis*, the adventitious bud clusters obtained after induction culture at the same induction time were larger in area and the adventitious buds grew vigorously. This is because the young *Actinidia chinensis* leaves of the aforementioned age and size have strong cell activity, which is conducive to the formation of callus tissue. The callus tissue formed is more suitable for adventitious bud induction, and the induced adventitious buds have a strong ability to absorb nutrients and grow vigorously.

[0064] Furthermore, in one specific embodiment of the present invention, the induction culture further includes adventitious shoot proliferation culture.

[0065] The adventitious shoot proliferation culture medium used includes MS medium and proliferation additives, and the proliferation additives include at least two of KT, 2,4-D, 6-BA and NAA.

[0066] Furthermore, the adventitious bud proliferation medium contained 0.05-0.2 mg / L of KT, 0.5-1.0 mg / L of 2,4-D, 0.1-1.0 mg / L of 6-BA, and 0.1-1.0 mg / L of NAA.

[0067] In detail, in one specific embodiment, the callus tissue with adventitious buds obtained by induction is taken out of the culture bottle, the part of the explant that has not been induced into callus tissue is removed using sterile instruments, and the callus tissue with adventitious buds of appropriate size is inoculated onto the adventitious bud proliferation medium for adventitious bud proliferation culture to obtain proliferated adventitious bud clusters.

[0068] The present invention does not limit the specific size of the callus tissue with adventitious buds before adventitious bud proliferation culture. A suitable callus tissue can be selected according to the size of the culture device, for example, a callus tissue with a length of 1 cm and a width of 1 cm.

[0069] The present invention does not limit the number of adventitious buds in the callus tissue with adventitious buds. The number of adventitious buds can be selected according to the actual situation, for example, 1-5 adventitious buds.

[0070] This invention does not limit the time of adventitious bud proliferation culture. It is understood that, with the extension of time, under the condition of sufficient nutrients and auxin, the proliferation coefficient of adventitious bud clusters will gradually increase.

[0071] In this invention, the proliferation coefficient refers to the volume ratio of the callus tissue before and after proliferation, that is, the volume of the callus tissue after proliferation divided by the volume of the callus tissue before proliferation.

[0072] In one specific embodiment, callus tissue with a length of 1 cm, a width of 1 cm, and 1-3 adventitious buds was cultured for 45 days, and the proliferation coefficient reached 6.5.

[0073] By using the above-mentioned adventitious bud proliferation medium to culture adventitious buds, the synergistic effect of various proliferation additives promoted cell division and differentiation in the adventitious bud clusters, achieving rapid expansion of the number of adventitious buds and improving the propagation efficiency of Chinese kiwifruit.

[0074] Furthermore, in one specific embodiment of the present invention, the adventitious bud proliferation culture medium includes KT, 6-BA, and NAA;

[0075] Furthermore, in the adventitious bud proliferation medium, the content of KT is 0.05-0.1 mg / L, the content of 6-BA is 0.5-1.0 mg / L, and the content of NAA is 0.5-1.0 mg / L.

[0076] In detail, the adventitious bud proliferation medium includes MS medium, 0.05-0.1 mg / L KT, 0.5-1.0 mg / L 6-BA, 0.5-1.0 mg / L NAA, 30 g sucrose, and 7 g agar.

[0077] In one specific embodiment, the adventitious bud proliferation medium includes MS medium, 0.08 mg / L KT, 0.5 mg / L 6-BA, 0.6 mg / L NAA, 30 g sucrose, and 7 g agar.

[0078] When the adventitious bud proliferation medium includes the above components, the synergistic effect of various plant growth regulators is further optimized, the adventitious bud proliferation rate can reach 548.6%, and the proliferation coefficient can reach 6.49, which further improves the propagation efficiency, accelerates the cultivation of Chinese kiwifruit, and realizes the large-scale propagation of Chinese kiwifruit.

[0079] Furthermore, in a specific embodiment of the present invention, after the adventitious bud proliferation culture, the process further includes seedling strengthening culture, wherein the seedling strengthening culture medium used includes MS medium, 0.08 mg / L KT, 0.25 mg / L 6-BA, and 0.8 mg / L NAA.

[0080] In detail, the proliferated adventitious bud clusters are removed using sterile instruments, the adventitious buds are separated from the base, and the individual adventitious buds are inoculated into a seedling culture medium for seedling cultivation to obtain robust adventitious bud seedlings.

[0081] The medium included MS medium for seedling growth, 0.08 mg / L KT, 0.25 mg / L 6-BA, 0.8 mg / L NAA, 30 g sucrose, and 7 g agar.

[0082] This invention does not limit the specific growth state of the adventitious buds participating in the seedling cultivation, and suitable adventitious buds can be selected according to the actual situation.

[0083] In one specific implementation, the adventitious buds participating in the seedling cultivation have a bud length of not less than 1 cm.

[0084] Understandably, the pH of the seedling culture medium can be adjusted as needed, for example, to 5.5-6.0.

[0085] This invention does not limit the specific time for cultivating strong seedlings. With sufficient nutrients and growth hormones, as the cultivation time for strong seedlings is extended, the bud length and number of leaves of adventitious seedlings further increase.

[0086] In one specific implementation, the seedling cultivation can be terminated when the length of the adventitious buds in the seedling cultivation is not less than 2cm.

[0087] The above-mentioned seedling culture medium provides appropriate nutrients and growth regulators for adventitious buds, promoting their growth and development and laying the foundation for subsequent experiments.

[0088] Furthermore, in one specific embodiment of the present invention, after the seedling cultivation, rooting culture is also included. The rooting culture medium used for the rooting culture includes MS medium, 0.5 ml / L IBA, and 0.1 g / L Ac.

[0089] In detail, when the adventitious buds grown from the seedlings reach 2-3 cm in length, they are transferred to a rooting medium using sterile equipment to undergo rooting culture, thus obtaining rooted seedlings.

[0090] Specifically, the rooting medium includes MS medium, 0.5 ml / L IBA, 0.1 g / L AC, 30 g sucrose, and 7 g agar. IBA refers to indolebutyric acid, which is a type of auxin hormone. AC refers to activated carbon. The pH of the rooting medium is 5.5-6.0.

[0091] In one specific embodiment, 2-3 cm adventitious buds are transferred to a rooting medium using sterile instruments for rooting culture. Roots develop at the base of the adventitious buds on days 5-10. By day 20-30, roots longer than 5 cm grow at the base of the adventitious buds, with more than 10 roots.

[0092] Using the above-mentioned culture medium to cultivate the roots of adventitious buds, the synergistic effect of each component promotes the formation of a healthy root system, providing a good foundation for subsequent transplanting and growth.

[0093] Furthermore, in a specific embodiment of the present invention, after rooting culture, transplanting culture is also included. The substrate used for transplanting culture includes garden soil, perlite, peat, and vermiculite in a volume ratio of 3:1:2:1.

[0094] In detail, the rooted seedlings are transplanted into seedling trays filled with substrate for cultivation. After 20 to 30 days of cultivation, transplanted seedlings with root length greater than 5 cm and more than 10 roots are obtained.

[0095] In one specific embodiment, rooted seedlings with stem segments reaching 1 cm in length and 4-6 leaves are selected and placed in clean water. The rooting culture medium attached to the seedlings is gently washed off with a brush to prevent contamination from sucrose and agar, which would affect the survival rate. After washing, the seedlings are first soaked in a 0.3% carbendazim solution for 1 hour, then removed and placed on newspapers laid on the ground for 1-2 hours to absorb the moisture from the leaves and stems. Finally, the seedlings are transplanted into a substrate consisting of garden soil, perlite, peat moss, and vermiculite in a volume ratio of 3:1:2:1 for cultivation.

[0096] It is understandable that the rooted seedlings can be hardened off before transplanting. This invention does not limit the specific process of hardening off, as long as it enables the rooted seedlings to better adapt to adverse environments.

[0097] Furthermore, in one specific embodiment of the present invention, a sterilization process is included before induction culture, and the sterilization process includes the following steps:

[0098] The explants were cleaned, then soaked in 75% medical alcohol, rinsed, and then soaked in 0.1 wt% mercuric chloride solution with Tween 80 added. After rinsing, the explants were disinfected.

[0099] In detail, the collected leaves or stem segments of *Actinidia chinensis* used as explants were wrapped in gauze and soaked in a beaker containing laundry detergent for 5 minutes, followed by rinsing with running water for 45 minutes. Then, the explants were placed on a clean bench and soaked in 75% medical alcohol for 30 seconds. After discarding the alcohol solution, they were immediately rinsed once with sterile water. Next, they were soaked in 0.1 wt% mercuric chloride solution with 3-5 drops of Tween 80 for 5-10 minutes, followed by rinsing with sterile water 3-5 times. Finally, the explants were blotted dry with sterile filter paper to obtain sterilized explants.

[0100] The soaking time of the present invention is 5-10 min. For example, the soaking time includes, but is not limited to, a range of 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any combination thereof.

[0101] It is understandable that the reagents used in the disinfection process may damage the incision site of the explant, causing the incision tissue to become damaged and discolored. This damaged and discolored tissue may cause secondary contamination of the explant. Therefore, in order to reduce the risk of secondary contamination and improve the survival rate of the explant, the damaged and discolored part of the explant caused by disinfection can be removed.

[0102] The present invention does not limit the specific operation of removing the damaged and discolored part; the damaged and discolored part of the explant can be removed using a sterile excision operation method commonly used in the art.

[0103] The above disinfection process ensures that the explants undergoing induction culture are in a sterile state, effectively preventing contamination by bacteria or other microorganisms during the induction culture process, and improving the survival rate and induction rate of explant callus induction culture.

[0104] Furthermore, in a specific embodiment of the present invention, during the cultivation process, the illumination time is 12h / d, the light intensity is 2500-3000lx, and the cultivation temperature is 23-27℃.

[0105] In detail, the light intensity is 2500-3000 lx. For example, the light intensity includes, but is not limited to, 2500 lx, 2600 lx, 2700 lx, 2800 lx, 2900 lx, 3000 lx, or any combination thereof.

[0106] The incubation temperature is 23-27℃. For example, the incubation temperature includes, but is not limited to, 23℃, 23.5℃, 24℃, 24.5℃, 25℃, 25.5℃, 26℃, 26.5℃, 27℃, or any combination thereof.

[0107] It is understandable that the environmental humidity needs to be controlled during the cultivation process. In this invention, the environmental humidity can be controlled to 100% before transplanting and to 50-70% during transplanting.

[0108] When the above-mentioned culture conditions are used in the cultivation process, the induction treatment, proliferation treatment, seedling strengthening treatment, and rooting treatment can be effectively promoted, thereby improving the growth efficiency of seedlings and further accelerating the propagation of Chinese kiwifruit.

[0109] The following detailed description of the efficient method for propagating Chinese kiwifruit provided by the present invention is illustrated through specific embodiments.

[0110] The kiwifruit parent plant used in this embodiment of the invention was taken from the planting resource nursery of the Yangtze Rare Plant Research Institute of China Three Gorges Corporation, located in Yichang City, Hubei Province, where the Three Gorges Dam is situated.

[0111] Example 1

[0112] 1. Explant Pretreatment: In the greenhouse of the planting resource nursery, leaves of *Actinidia chinensis* aged 5-10 days, leaves of *Actinidia chinensis* aged over 60 days, and young stem segments of current-year *Actinidia chinensis* were selected as explants. They were cut from the base, wrapped in gauze, and soaked in a beaker containing laundry detergent for 5 minutes, followed by rinsing with running water for 45 minutes. After outdoor pretreatment, the explants were placed on a clean bench and soaked in 75% medical alcohol for 30 seconds. After discarding the alcohol solution, they were immediately rinsed once with sterile water. Then, they were soaked in 0.1% mercuric chloride solution with 5 drops of Tween 80 for 5-10 minutes, followed by rinsing with sterile water 3-5 times. Finally, the leaves were blotted dry with sterile filter paper. Since sterilization and rinsing damage the cut tissue of the explants, damaged or discolored areas were removed before inoculation. Each treatment consisted of 30 bottles, replicated 3 times.

[0113] 2. Induction culture

[0114] 1) The effect of different tissues on induction culture

[0115] Sterilized leaves and stem segments were inoculated onto induction medium for induction culture. The induction status of stem segments and leaves was statistically analyzed using a visual statistical method, as shown in Table 1. The induction number refers to the number of explants that showed callus tissue on the leaves on the 30th day of culture.

[0116] The induction medium included MS medium, 0.1 mg / L KT, 0.25 mg / L 6-BA, 0.8 mg / L 2,4-D, 0.02 mg / L IBA, 0.3 mg / L NAA, 30 g sucrose, and 7 g agar. The pH of the induction medium was 6. The environmental changes during the induction culture of leaves as explants were consistent with those during the induction culture of stem segments as explants. The light intensity was 2500-3000 lx, the culture temperature was 23-27℃, and the photoperiod was 12 h / d.

[0117] Table 1

[0118]

[0119] 2) Effects of different culture media on induction culture

[0120] Young leaves aged 5-10 days, after being treated according to the above explant pretreatment steps, were inoculated onto 15 groups of induction culture media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3. A total of 30 samples were treated in each group of induction culture media.

[0121] The specific composition of the induction medium is shown in Table 2. The induction medium also includes MS medium, 30g sucrose, and 7g agar, with a pH of 6.0. During the culture process, the environmental changes of each induction medium were consistent, with a light intensity of 2500-3000 lx, a culture temperature of 23-27℃, and a photoperiod of 12h / d.

[0122] After 10-15 days of cultivation, the leaves begin to grow wrinkled callus tissue, forming a bright green or slightly reddish color. Figure 1 , Figure 2 As shown, with the extension of the culture time, adventitious buds gradually grow from different locations on the callus tissue, such as... Figure 3 As shown in Table 2, the callus formation was statistically analyzed after 30 days of continuous culture. The induction rate refers to the ratio between the number of leaves that induced callus and the number of leaves initially, while the adventitious bud induction rate refers to the ratio between the number of adventitious buds and the number of leaves initially.

[0123] On day 20, the induction medium was replaced with an induction medium of the same composition to ensure the supply of nutrients and auxin.

[0124] Table 2

[0125]

[0126]

[0127] 3. Adventitious bud proliferation culture

[0128] The adventitious buds obtained by the above induction culture were used for proliferation culture. The explants with adventitious buds were removed from the induction culture medium using sterile instruments. Callus tissue with 1-3 adventitious buds and a length of 1 cm and a width of 1 cm was cut and inoculated onto 15 groups of proliferation culture media numbered 1-1, 1-2, 1-3, 2-1, 2-2, 2-3, 3-1, 3-2, 3-3, 4-1, 4-2, 4-3, 5-1, 5-2, and 5-3 for proliferation culture. A total of 30 samples were processed in each group of proliferation culture media.

[0129] The specific composition of the proliferation medium is shown in Table 3. The proliferation medium also includes MS medium, 30g sucrose, and 7g agar, with a pH of 6.0. During the culture process, the environmental changes of each proliferation medium were consistent, with a light intensity of 2500-3000 lx, a culture temperature of 23-27℃, and a photoperiod of 12h / d.

[0130] After 45 days of cultivation, the proliferation of adventitious buds is as follows: Figure 4 As shown in Table 3, the adventitious bud proliferation rate is calculated as follows: (Number of proliferating adventitious buds - Number of initial adventitious buds) / Number of initial adventitious buds × 100%.

[0131] Table 3

[0132]

[0133] 4. Cultivation of strong seedlings

[0134] The adventitious bud clusters formed after proliferation were removed, and adventitious buds were separated from the base. 180 adventitious buds with a length of not less than 1 cm were selected and inoculated onto three groups of seedling culture media numbered A, B, and C for seedling cultivation.

[0135] The seedling strengthening culture media all included MS medium, 30g sucrose and 7g agar. The seedling strengthening culture media of group A also included 0.05mg / L KT, 0.1mg / L 6-BA and 0.5mg / L NAA; the seedling strengthening culture media of group B also included 0.06mg / L KT, 0.2mg / L 6-BA and 1.0mg / L NAA; the seedling strengthening culture media of group C also included 0.1mg / L KT, 0.5mg / L 6-BA and 1.0mg / L NAA. The pH of the seedling strengthening culture media was 6.

[0136] During the cultivation process, the environment of each seedling culture medium changed in a consistent manner, with a light intensity of 2500-3000 lx, a cultivation temperature of 23-27℃, and a light duration of 12 h / d.

[0137] After 30 days of cultivation, the growth rate of adventitious buds and seedlings was statistically analyzed. It was found that the growth rate of adventitious buds in group A was 79.6%, that in group B was 99.8%, and that in group C was 87.5%. Moreover, the adventitious buds in group B grew vigorously and the leaves were tender and green.

[0138] 5. Rooting culture

[0139] Adventitious buds with a length of 2-3 cm obtained through the above series of culture steps were selected and transferred to four groups of rooting culture media numbered A, B, C, and D for rooting culture.

[0140] The specific composition of the four rooting media is shown in Table 4. Each rooting media also includes MS medium, 30g sucrose, and 7g agar. The pH of each rooting media is 5.8.

[0141] During the cultivation process, the environmental conditions of each rooting medium remained consistent: light intensity of 2500-3000 lx, cultivation temperature of 23-27℃, and light duration of 12 h / d. After 30 days of cultivation, the condition of the rooted seedlings was assessed, as detailed in Table 4. Figure 5 , Figure 6 The photos show the adventitious buds after 10 and 20 days of rooting culture, respectively.

[0142] Table 4

[0143]

[0144] 6. Hardening off seedlings and transplanting

[0145] Select robust, well-rooted seedlings with complete and abundant root systems that meet the requirements for hardening-off transplanting. After 5 days in the hardening-off environment, open the bottle cap and harden off for another 10 days. During this period, spray the inside of the bottle and the leaves of the *Actinidia chinensis* seedlings with a 250ml spray bottle to prevent the seedlings from wilting due to dehydration. On the 16th day, remove the seedlings from the culture medium and place them in clean water. Gently wash off the culture medium attached to the tissue culture seedlings with a brush to prevent contamination from sugar and agar, which could affect the survival rate. After washing, soak the seedlings in a 0.3% carbendazim solution for 1 hour, then remove them and place them on newspapers laid out on the ground for 1-2 hours to absorb moisture from the leaves and stems. Finally, plant the seedlings in the four groups of substrates numbered A, B, C, and D for further cultivation.

[0146] The specific composition of the four substrate groups A, B, C, and D is shown in Table 5. During the cultivation process, the environmental changes of each substrate group were consistent, with a light intensity of 2500-3000 lx, a cultivation temperature of 23-27℃, and a light duration of 12 h / d.

[0147] After 30 days of cultivation, the survival rate and seedling growth under different substrates were recorded, as detailed in Table 5. The seedlings were then transplanted outdoors for ground cultivation. Figure 7 This is a picture of the outdoor growth of Chinese kiwifruit.

[0148] Table 5

[0149]

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for propagating Chinese kiwifruit, characterized in that, Includes the following steps: Kiwifruit leaves were used as explants, and the explants were subjected to culture treatments including induction culture to obtain kiwifruit seedlings. The induction culture is followed by adventitious shoot proliferation culture; the adventitious shoot proliferation culture medium used in the adventitious shoot proliferation culture includes MS medium and plant growth regulators, wherein the plant growth regulators consist of 0.05-0.1 mg / L KT, 0.5-1.0 mg / L 6-BA, and 0.5-1.0 mg / L NAA; The adventitious bud proliferation culture also includes seedling strengthening culture; the seedling strengthening culture medium used in the seedling strengthening culture consists of MS medium, 0.06 mg / L KT, 0.2 mg / L 6-BA, 1.0 mg / L NAA, 30 g sucrose, and 7 g agar; The seedling cultivation process also includes rooting culture; the rooting medium used in the rooting culture consists of MS medium, 0.5 mg / L IBA, 0.1% activated carbon, 30 g sucrose, and 7 g agar. The leaves of the Chinese kiwifruit are 5-10 days old. The induction culture medium used in the induction culture consisted of MS medium, 0.05–0.1 mg / L KT, 0.1–0.5 mg / L 6-BA, 0.5–1.0 mg / L 2,4-D, 0.01–0.05 mg / L IBA, 0.15–0.35 mg / L NAA, 30 g sucrose, and 7 g agar.

2. The method according to claim 1, characterized in that, The leaves of the Chinese kiwifruit are 1-2cm long and 1-2cm wide.

3. The method according to claim 1, characterized in that, After the rooting culture, the transplanting culture is also included. The substrate used for the transplanting culture includes garden soil, perlite, peat, and vermiculite in a volume ratio of 3:1:2:

1.

4. The method according to claim 1, characterized in that, The induction culture process also includes a disinfection treatment, which includes the following steps: The explants were cleaned, then soaked in 75% medical alcohol (by volume), rinsed, and then soaked in 0.1 wt% mercuric chloride solution with Tween 80 added. After rinsing, the explants were disinfected.

5. The method according to claim 1, characterized in that, During the cultivation process, the illumination time was 12 h / d, the light intensity was 2500-3000 lx, and the cultivation temperature was 23-27℃.

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