Efficient adventitious bud regeneration method for new sakura variety Himalaya

Abstract

The invention relates to an efficient adventitious bud regeneration method of a new sakura variety 'Himalaya', which comprises the following steps: by taking leaves of an evergreen new variety 'Himalaya' sakura as explants, performing adventitious bud regeneration induction, cluster bud proliferation and rooting culture to obtain rooting tissue culture seedlings, and performing seedling hardening and domestication to obtain sakura seedlings. According to the method, 6-BA and NAA with a certain concentration are added into an induction culture medium, adventitious buds are induced from leaf explants through a direct organ regeneration way, the adventitious buds can form cluster buds and proliferation can be achieved by adding 6-BA, IBA and GA3 into a proliferation culture medium, the proliferation coefficient reaches about 5.5, and adventitious roots are formed at the stem bases by adding IBA and NAA into a rooting culture medium. The method is used for tissue culture and rapid propagation of a new variety of oriental cherry, the seedling propagation time is shortened, the seedling production efficiency is improved, a oriental cherry direct organ regeneration technology is established, and the oriental cherry can be used as a receptor of oriental cherry genetic transformation.

Description

Technical Field

[0001] The invention belongs to the field of explant induced regeneration, and specifically relates to a high-efficiency adventitious bud regeneration method for a new cherry variety "Himalaya". Background Art

[0002] The Himalayan cherry is an evergreen early cherry variety bred by the Shanghai Academy of Agricultural Sciences suitable for planting in Shanghai. It obtained the forest tree variety certificate from the Shanghai Forestry Bureau in November 2023. The flowering period in Shanghai is from November to December. It has strong heat resistance and can survive the winter normally in occasional low temperatures above -8°C. Low temperatures below -9°C and lasting for more than 1 week will cause frost damage. It has strong disease resistance. Leaf shrinkage, perforated brown spot disease, and cherry root cancer disease were found on Japanese "Somei Yoshino" cherry trees cultivated in the same area, but not on Himalayan cherry trees.

[0003] As a precious ornamental plant, the Himalayan cherry blossoms are favored by garden enthusiasts in Shanghai for their pink flowers in winter. However, their slow natural reproduction process and the great influence of environmental factors limit their application in landscaping. In the prior art, the reproduction of Himalayan cherry blossoms mainly relies on seed propagation and traditional cutting methods, which have the problems of low reproduction efficiency, large genetic variation, and difficulty in large-scale production. Summary of the invention

[0004] The purpose of the present invention is to provide a method for in vitro regeneration using "Himalayan" cherry leaves, to establish a technology for direct organogenesis of "Himalayan" cherry, to effectively improve the proliferation efficiency, and to shorten the seedling propagation cycle. This regeneration method can be used for the receptor system of genetic transformation, which is helpful for future genetic improvement.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for efficiently regenerating adventitious buds of a new cherry variety "Himalaya", comprising the following steps:

[0007] 1) Explant pretreatment

[0008] Select the leaves of the tissue culture seedlings of Sakura "Himalaya" as explants, and cut them into small cubes with dissecting scissors;

[0009] 2) Induce adventitious bud regeneration

[0010] The cut leaf cubes were inoculated into the induction medium and cultured for 50 to 60 days to induce adventitious bud regeneration;

[0011] The induction medium is based on modified MS as the basic medium, and is supplemented with 2.0-3.0 mg / L 6-benzyladenine, 0.01-0.05 mg / L naphthaleneacetic acid, and 0.4-0.5 g / L hydrolyzed casein; wherein the modified MS is NH 4 NO 3 、KNO 3 , CaCl 2 ·2H 2 O.

[0012] MgSO 4 7H 2 O and KH 2 PO 4 MS medium with half the content;

[0013] 3) Proliferation culture

[0014] The adventitious buds are transferred to a proliferation medium and cultured under light for 30 to 45 days to form clustered buds;

[0016] The light culture conditions are: temperature 25-28°C, light duration 14-16 hours, light

[0017] The illumination intensity is 2000-3000 lux;

[0018] The proliferation medium is based on WPM as the basic medium, and is supplemented with 0.2-0.5 mg / L 6-benzyladenine, 0.05-0.1 mg / L indolebutyric acid, and 0.4-0.6 mg / L gibberellin.

[0019] mg / L, activated carbon 0.2~0.5g / L;

[0020] 4) Rooting culture

[0021] Cut the clustered buds into individual plants and inoculate them into rooting medium. After 25 to 30 days of cultivation,

[0022] Take root, get rooted shoots;

[0023] The culture conditions are: temperature 25-28°C, light duration 15-16 hours, light intensity

[0024] The degree is 2800-3000 lux;

[0025] The rooting medium is based on MS as the basic medium, and is supplemented with 0.8-1 mg / L indolebutyric acid, 0.2-0.5 mg / L naphthaleneacetic acid, and 0.2-0.5 g / L activated carbon; 5) seedling hardening and acclimatization

[0026] Open the tissue culture bottle cap of the cherry rooting seedlings, harden the seedlings at room temperature for 4 to 7 days, transplant them into the substrate, maintain the temperature at 25 to 30°C, spray twice a day, maintain the air humidity above 85%, cover with a shade net with a shading rate of 50%, and cultivate them for 25 to 30 days to obtain seedlings.

[0027] Preferably, the culture conditions in step 2) are darkness and 22-25°C.

[0028] Furthermore, 25-30 g / L sucrose and 6.5-7 g / L agar powder are added to the induction medium, proliferation medium and rooting medium in steps 2) to 4), respectively, and the pH value of the medium is 5.75-5.85.

[0029] Preferably, the culture conditions in steps 3) and 4) are a temperature of 23-26° C., a lighting time of 15-16 hours, and a light intensity of 2500-3000 lux.

[0030] Furthermore, the matrix in step 5) is: peat soil: perlite = 1:1 (volume ratio).

[0031] Furthermore, in the improved MS medium, NH 4 NO 3 The content is 800~850mg / L, KNO 3 The content is 900~1000mg / L, CaCl 2 ·2H 2 O content is 200-240 mg / L, MgSO 4 7H 2 O content is 170~200mg / L, KH 2 PO 4 The content is 80-90 mg / L.

[0032] In the present invention, the formula of MS medium was published by Murashige T and Skoog F in 1962 (Murashige T and Skoog F, 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-479).

[0033] The WPM (Woody Plant medium) culture medium formula of the present invention was published by Lloyd and McCown in 1980 (Commercially feasible micropropagation of mountain laurel, Kalmialatifolia, by use of shoot-tip culture. Proc Int Plant Prop Soc 30: 420-427).

[0034] In the present invention, cherry blossom leaves are used for tissue culture, the number of leaves is large, and explants are easy to obtain. During the induction culture process, 6-BA and NAA are added to induce a small amount of callus tissue from the leaves, thereby directly inducing adventitious buds. The present invention controls the 6-BA content to 2.0-3.0 mg / L and the NAA content to 0.01-0.05 mg / L. At the same time, hydrolyzed casein is added, and a scheme of reducing MS macroelements is adopted, so that the explants can be prevented from being poisoned and the induction rate of adventitious buds can be improved.

[0035] The invention adds activated carbon to the proliferation medium and the rooting medium to increase the proliferation coefficient, prevent callus formation and promote rooting and stem elongation growth.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention utilizes cherry blossom leaves to directly induce adventitious buds to regenerate plants, effectively improving proliferation efficiency and shortening the breeding cycle of seedlings to four months. In addition, this regeneration method can be used for the receptor system of genetic transformation, which is helpful for genetic improvement.

[0038] The present invention utilizes leaves to directly induce adventitious buds to occur, the induction rate reaches 75%, the proliferation coefficient is greatly improved, the proliferation coefficient is about 5.5, and the production cost of cherry blossom seedlings can be significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the in vitro culture of cherry blossom leaves to induce adventitious buds in Example 1 of the present invention.

[0040] Figure 2 This is the cherry blossom cluster bud proliferation culture in Example 1 of the present invention.

[0041] Figure 3 This is the cherry rooting seedling obtained in Example 1 of the present invention.

[0042] Figure 4 These are the cherry blossom seedlings domesticated in Example 1 of the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments.

[0044] Embodiment A method for efficiently regenerating adventitious buds of a new cherry variety "Himalaya" is provided, and the cultivation steps are as follows:

[0045] 1) Selection of explants: The explants were obtained from the leaves of tissue culture seedlings of a new cherry variety “Himalaya” preserved in the tissue culture center of the Forestry and Fruit Research Institute of Shanghai Academy of Agricultural Sciences.

[0046] 2) Explant preparation: Select new leaves that have been transferred to a new proliferation medium for 15 days as explants, and cut the leaves into pieces of about 0.5 cm using tweezers in a clean bench. 2 of small cubes for later use.

[0047] 3) Adventitious bud induction: Place the leaves with their backs facing downwards in the adventitious bud induction medium and culture them in the dark at 23°C for 50 days to induce adventitious buds. Figure 1 On average, each leaf square can induce 3 adventitious buds.

[0048] The adventitious bud induction medium uses the improved MS medium as the basic medium, and is supplemented with 2.0 mg / L 6-benzyladenine (6-BA), 0.05 mg / L naphthaleneacetic acid (NAA), 0.4 g / L hydrolyzed casein, 30 g / L sucrose and 7 g / L agar, and the pH value of the medium is 5.7-5.8.

[0049] 4) Adventitious bud proliferation culture: The adventitious buds induced in the dark were transferred to light culture at a temperature of 25°C, a photoperiod of 16 / 8 hours, and a light intensity of 2800 lux. After about 40 days, clustered buds were obtained. Figure 2 The average proliferation coefficient of clustered shoots is 5.5.

[0050] The proliferation medium is WPM medium as the basic medium, supplemented with 0.5 mg / L 6-benzyladenine, 0.1 mg / L indolebutyric acid (IBA), and 0.1 mg / L gibberellin (GA 3 )0.5mg / L; activated carbon 0.4g / L, sucrose 20g / L, agar 7g / L, the pH value of the culture medium is 5.6-5.8.

[0051] 5) Rooting culture of clustered buds: The induced clustered buds were cut and inoculated into rooting medium. They took root after 30 days of culture. Figure 3 , the rooting rate reached over 85%.

[0052] The rooting medium contains MS, and is supplemented with 1.0 mg / L indolebutyric acid, 0.2 mg / L naphthaleneacetic acid, 0.4 g / L activated carbon, 30 g / L sucrose, and 6-8 g / L agar, and the pH value of the medium is 5.7-5.8. The culture temperature is 25°C, the photoperiod is 16 / 8 hours, and the light intensity is 2800 lux.

[0053] 6) Hardening and transplanting of tissue culture seedlings: Open the cap of the culture medium bottle of the rooted cherry seedlings, harden them at room temperature for 4 days, take out the seedlings, wash off the culture medium on the roots, and transplant them into a plug tray filled with peat soil and perlite (1:1). Maintain the temperature at 30°C and cultivate them in a shade shed with a shading rate of about 50%. Spray water once in the morning and evening to maintain humidity. After 25 days, well-tamed seedlings will be obtained. See Figure 4 .

[0054] In this embodiment, the regeneration cycle of the cherry blossom plant takes about 125 days.

[0055] The formula of the modified MS medium described in the examples is shown in Table 1, where mg / L represents the milligrams of each component contained in each liter of the modified MS medium.

[0056] Table 1

[0057]

[0058] The WPM medium formula in the embodiment is shown in Table 2, and mg / L represents the milligrams of each component contained in each liter of WPM medium.

[0059] Table 2

[0060]

Claims

1. A method for efficiently regenerating adventitious buds of a new cherry variety "Himalaya", comprising the following steps: 1) Explant pretreatment Select the leaves of the tissue culture seedlings of Sakura "Himalaya" as explants, and cut them into small cubes with dissecting scissors; 2) Induce adventitious bud regeneration The cut leaf cubes were inoculated into the induction medium and cultured for 50 to 60 days. Induce adventitious shoot regeneration; The induction medium is based on modified MS as the basic medium, and is supplemented with 2.0-3.0 mg / L of 6-benzyladenine, 0.01-0.05 mg / L of naphthaleneacetic acid, and 0.4-0.5 g / L of hydrolyzed casein; wherein the modified MS is an MS medium in which the contents of NH4NO3, KNO3, CaCl2·2H2O, MgSO4·7H2O, and KH2PO4 are reduced by half; 3) Proliferation culture The adventitious buds are transferred to a proliferation medium and cultured under light for 30 to 45 days to form clustered buds; The proliferation medium is based on WPM as the basic medium, and is supplemented with 0.2-0.5 mg / L of 6-benzyladenine, 0.05-0.1 mg / L of indolebutyric acid, 0.4-0.6 mg / L of gibberellin, and 0.2-0.5 g / L of activated carbon; 4) Rooting culture The clustered buds are cut into individual plants, inoculated into a rooting medium, and rooted after culturing for 25 to 30 days to obtain rooted seedlings; The rooting medium is based on MS as the basic medium, and is supplemented with 0.5-1 mg / L indolebutyric acid, 0.2-0.5 mg / L naphthaleneacetic acid, and 0.2-0.5 g / L activated carbon; 5) Hardening and domestication Open the tissue culture bottle cap of the cherry rooting seedlings, harden the seedlings at room temperature for 4 to 7 days, transplant them into the substrate, maintain the temperature at 25 to 30°C, spray twice a day, maintain the air humidity above 85%, cover with a shade net with a shading rate of 50%, and cultivate them for 25 to 30 days to obtain seedlings.

2. The efficient adventitious bud regeneration method of the new cherry variety "Himalaya" according to claim 1, characterized in that: The culture conditions in step 2) are darkness and 22-25°C.

3. The efficient adventitious bud regeneration method of the new cherry variety "Himalaya" according to claim 1 is characterized in that: The culture conditions in steps 3) and 4) are a temperature of 23-26° C., a light exposure time of 15-16 hours, and a light intensity of 2500-3000 lux.

4. The efficient adventitious bud regeneration method of the new cherry variety "Himalaya" according to claim 1 is characterized in that: 25-30 g / L sucrose and 6.5-7 g / L agar powder are added to the induction medium, proliferation medium and rooting medium in steps 2) to 4) respectively, and the pH value of the medium is 5.75-5.

85.

5. The efficient adventitious bud regeneration method of the new cherry variety "Himalaya" according to claim 1, characterized in that: The matrix in step 5) is: peat soil: perlite = 1:1 (volume ratio).

6. The method for efficient adventitious bud regeneration of the new cherry variety "Himalaya" according to any one of claims 1 to 4, characterized in that: In the improved MS culture medium, the NH4NO3 content is 800-850 mg / L, the KNO3 content is 900-1000 mg / L, the CaCl2·2H2O content is 200-240 mg / L, the MgSO4·7H2O content is 170-200 mg / L, and the KH2PO4 content is 80-90 mg / L.

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