A method for rapid propagation of Xinjiang wild apple tissue culture

Abstract

The present invention belongs to the field of apple tissue culture, and specifically discloses a method for rapid propagation of Xinjiang wild apple tissue culture, comprising the following steps: (1) explant preparation and initiation of culture: (2) rooting culture: first placed in a rooting medium under high temperature dark culture conditions and cultured for 10 to 14 days; then placed under natural scattered light conditions and cultured for 10 to 15 days to obtain rooted tissue culture seedlings; (3) hardening: when the rooted tissue culture seedlings grow to a height of 2 to 4 cm and have 2 to 3 unfolded leaves, they are removed from the culture chamber and placed in an outdoor cool place with natural scattered light conditions, hardened for 5 to 7 days to obtain rooted seedlings; (4) transplanting: taking out the rooted seedlings, washing off the root culture medium, and transplanting the rooted tissue culture seedlings to a transplanting medium. The present invention improves the formula of the rooting medium, adds 5AzaC10-20 g / L and trans-2-dodecenedioic acid 3-6 g / L therein, can greatly accelerate the acquisition of rooted tissue culture seedlings, and accelerate the tissue culture breeding of wild apples.

Description

Technical Field

[0001] The invention belongs to the field of apple tissue culture, and specifically discloses a Xinjiang wild apple tissue culture rapid propagation method. Background Art

[0002] Apple is a perennial woody plant. Xinjiang wild apple (Malussieversii) is a tree of the genus Malus in the Rosaceae family. It is a relict species of the Tertiary period. It is only distributed in Ili, Tacheng and other places in Xinjiang in China. It is listed as a second-level endangered key protected plant in China. Domestic and foreign research on Xinjiang wild apples mainly focuses on morphology, palynology, cytology and molecular biology. Xinjiang wild apples are important wild fruit tree germplasm resources in my country. Its importance is mainly reflected in the fact that Xinjiang wild apples are one of the original ancestors of modern cultivated apples. It provides a "living fossil" specimen for studying the origin and trait evolution of modern cultivated apples, and is also a natural apple germplasm gene bank.

[0003] Apple tissue culture is a method of using aseptic culture technology to culture organs such as stem tips, axillary buds, leaves and their tissue slices from excellent plants in vitro, so as to obtain a large number of genetically consistent individuals in a short period of time. It has the advantages of short cycle, easy artificial control of culture conditions, fast reproduction speed, small space occupation, high economic benefits, etc. At the same time, it also accelerates the promotion and application of new apple varieties. Culture medium is an important material for plant tissue culture and a nutrient for explant growth. Only by preparing a suitable culture medium can tissue culture be successful. The existing rooting culture medium usually takes more than 40 days to culture from buds to rooted seedlings, which greatly reduces the reproduction speed. It is necessary to further improve the culture medium and culture methods to increase the breeding speed of wild apples. Summary of the invention

[0004] In order to solve the above problems, the invention discloses a method for rapid propagation of Xinjiang wild apple tissue culture.

[0005] The technical solution of the present invention is as follows:

[0006] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0007] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple tree branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 0.5 to 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.2 to 0.4 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on an induction medium and cultured in the dark for 10 to 15 days to obtain a cluster of buds.

[0008] (2) Rooting culture: first, culture the seedlings in a rooting medium under high temperature and dark conditions for 7 to 10 days; then culture the seedlings under natural scattered light conditions for 10 to 15 days to obtain rooted tissue culture seedlings;

[0009] (3) Hardening the seedlings: When the rooted tissue culture seedlings grow to a height of 2 to 4 cm and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 5 to 7 days to obtain rooted seedlings;

[0010] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0011] Furthermore, in the above-mentioned Xinjiang wild apple tissue culture rapid propagation method, the induction culture medium is 0.5-0.7 times the concentration of MS culture medium with the addition of 10-20 g / L 5-AzaC, 3-6 g / L trans-2-dodecenedioic acid and 3-5 g / L plant gel.

[0012] Furthermore, in the above-mentioned Xinjiang wild apple tissue culture rapid propagation method, the rooting medium is MS medium with the addition of 10-15 g / L of trans-2-dodecenedioic acid, 20-30 g / L of sucrose and 5-8 g / L of plant gel.

[0013] Furthermore, in the above-mentioned Xinjiang wild apple tissue culture rapid propagation method, the transplanting matrix includes 100-200 parts of Northeast black charcoal soil and peat soil, 100-200 parts of vermiculite with a pore size of 3-6 mm, and 50-100 parts of animal manure.

[0014] Furthermore, in the above-mentioned Xinjiang wild apple tissue culture rapid propagation method, the temperature in the induction culture in step 1) is 20-25°C.

[0015] Furthermore, in the above-mentioned Xinjiang wild apple tissue culture rapid propagation method, the temperature of the high-temperature dark culture in step 2) is 35-37°C.

[0016] The invention also discloses the use of the rapid propagation method in preparing and cultivating Xinjiang wild apple seedlings.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention discloses a method for rapid propagation of Xinjiang wild apple tissue culture. The invention discloses a technical scheme for combining 5-AzaC (5-azacytidine nucleoside) and trans-2-dodecenedioic acid. The use of the scheme in combination with a suitable rapid propagation method can greatly accelerate the growth of Xinjiang wild apple rooted tissue culture seedlings, improve the rooting rate and root length of the wild apple tissue culture seedlings, improve the survival rate of the wild apple tissue culture seedlings, and accelerate the tissue culture breeding of wild apples, which has significant progress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the comparison of rooting rate (%) in the test cases;

[0020] Figure 2 is the comparison of the mean root length (cm) in the test case;

[0021] Figure 3 Comparison of the mean root circumference (mm) in the test case. DETAILED DESCRIPTION

[0022] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0023] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple tree branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 0.5 to 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.2 to 0.4 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on an induction medium and cultured in the dark for 10 to 15 days to obtain a cluster of buds.

[0024] (2) Rooting culture: first, culture the seedlings in a rooting medium under high temperature and dark conditions for 7 to 10 days; then culture the seedlings under natural scattered light conditions for 10 to 15 days to obtain rooted tissue culture seedlings;

[0025] (3) Hardening the seedlings: When the rooted tissue culture seedlings grow to a height of 2 to 4 cm and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 5 to 7 days to obtain rooted seedlings;

[0026] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0027] The induction medium is 0.5-0.7 times the concentration of MS medium with 10-20 g / L 5-AzaC, 3-6 g / L trans-2-dodecenedioic acid and 3-5 g / L plant gel added;

[0028] The rooting medium is prepared by adding 10-15 g / L of trans-2-dodecenedioic acid, 20-30 g / L of sucrose and 5-8 g / L of plant gel to the MS medium;

[0029] The transplanting matrix comprises 100-200 parts of Northeastern black charcoal soil, 100-200 parts of vermiculite with a pore size of 3-6 mm, and 50-100 parts of livestock manure;

[0030] The temperature in the induction culture in step 1) is 20-25°C.

[0031] In the high temperature dark culture in step 2), the temperature is 35-37°C.

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] The reagents or instruments used in the examples of the present invention without indicating the manufacturer are all conventional reagent products that can be obtained through commercial purchase.

[0034] Example 1

[0035] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0036] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 0.5 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.2 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on the induction medium and cultured in the dark for 10 days to obtain a cluster of buds.

[0037] (2) Rooting culture: First, culture the seedlings in a rooting medium under high temperature and dark conditions for 7 days; then culture them under natural scattered light conditions for 10 days to obtain rooted tissue culture seedlings;

[0038] (3) Hardening: When the rooted tissue culture seedlings grow to 2 to 4 cm in height and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 5 days to obtain rooted seedlings.

[0039] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0040] The induction medium is 0.5 times the concentration of MS medium with 10 g / L 5-AzaC, 3 g / L trans-2-dodecenedioic acid and 3 g / L plant gel added;

[0041] The rooting medium is MS medium with 10 g / L trans-2-dodecenedioic acid, 20 g / L sucrose and 5 g / L plant gel added;

[0042] The transplanting matrix includes 100 parts of Northeastern black charcoal soil, 100 parts of vermiculite with a pore size of 3-6 mm, and 50 parts of livestock manure;

[0043] The temperature in the induction culture in step 1) is 20°C.

[0044] In the high temperature dark culture in step 2), the temperature is 35°C.

[0045] Example 2

[0046] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0047] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.3 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on the induction medium and cultured in the dark for 12 days to obtain a cluster of buds.

[0048] (2) Rooting culture: First, culture the seedlings in a rooting medium under high temperature and dark conditions for 8 days; then culture the seedlings under natural scattered light conditions for 12 days to obtain rooted tissue culture seedlings;

[0049] (3) Hardening: When the rooted tissue culture seedlings grow to 2 to 4 cm in height and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 6 days to obtain rooted seedlings.

[0050] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0051] The induction medium is 0.6 times the concentration of MS medium with 15 g / L 5-AzaC, 4.5 g / L trans-2-dodecenedioic acid and 4 g / L plant gel added;

[0052] The rooting medium is MS medium with 12 g / L trans-2-dodecenedioic acid, 20-30 g / L sucrose and 6 g / L plant gel added;

[0053] The transplanting matrix includes 150 parts of Northeastern black charcoal soil, 150 parts of vermiculite with a pore size of 3-6 mm, and 75 parts of livestock manure;

[0054] The temperature in the induction culture in step 1) is 22°C.

[0055] In the high temperature dark culture in step 2), the temperature is 36°C.

[0056] Example 3

[0057] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0058] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.4 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on the induction medium and cultured in the dark for 15 days to obtain a cluster of buds.

[0059] (2) Rooting culture: First, culture the seedlings in a rooting medium under high temperature and dark conditions for 10 days; then culture the seedlings under natural scattered light conditions for 15 days to obtain rooted tissue culture seedlings;

[0060] (3) Hardening: When the rooted tissue culture seedlings grow to a height of 2 to 4 cm and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 7 days to obtain rooted seedlings.

[0061] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0062] The induction medium is 0.7 times the concentration of MS medium with 20 g / L 5-AzaC, 6 g / L trans-2-dodecenedioic acid and 5 g / L plant gel added;

[0063] The rooting medium is MS medium with 15 g / L trans-2-dodecenedioic acid, 30 g / L sucrose and 8 g / L plant gel added;

[0064] The transplanting matrix includes 200 parts of Northeast black charcoal soil, 200 parts of vermiculite with a pore size of 3-6 mm, and 100 parts of livestock manure;

[0065] The temperature in the induction culture in step 1) is 25°C.

[0066] In the high temperature dark culture in step 2), the temperature is 37°C.

[0067] Comparative Example 1

[0068] The difference between Comparative Example 1 and Example 2 is that no trans-2-dodecenedioic acid is added to the induction medium and the rooting medium, and the other contents are the same.

[0069] A method for rapid propagation of Xinjiang wild apple tissue culture comprises the following steps:

[0070] (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.3 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on the induction medium and cultured in the dark for 12 days to obtain a cluster of buds.

[0071] (2) Rooting culture: First, culture the seedlings in a rooting medium under high temperature and dark conditions for 8 days; then culture the seedlings under natural scattered light conditions for 12 days to obtain rooted tissue culture seedlings;

[0072] (3) Hardening: When the rooted tissue culture seedlings grow to 2 to 4 cm in height and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 6 days to obtain rooted seedlings.

[0073] (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium.

[0074] The induction medium is 0.6 times the concentration of MS medium with 15 g / L 5-AzaC and 4 g / L plant gel added;

[0075] The rooting medium is prepared by adding 20-30 g / L sucrose and 6 g / L plant gel to the MS medium;

[0076] The transplanting matrix includes 150 parts of Northeastern black charcoal soil, 150 parts of vermiculite with a pore size of 3-6 mm, and 75 parts of livestock manure;

[0077] The temperature in the induction culture in step 1) is 22°C.

[0078] In the high temperature dark culture in step 2), the temperature is 36°C.

[0079] Comparative Example 2

[0080] The difference between Comparative Example 2 and Example 2 is that trans-2-dodecenedioic acid is not added to the induction medium, and the other contents are the same.

[0081] Comparative Example 3

[0082] The difference between Comparative Example 3 and Example 2 is that trans-2-dodecenedioic acid is not added to the rooting medium, and the other contents are the same.

[0083] Comparative Example 4

[0084] The difference between Comparative Example 4 and Example 2 is that 5-AzaC is not added to the induction medium.

[0085] Test Case

[0086] The test results of wild apple tissue culture seedling rooting in Examples 1-3 and Comparative Examples 1-4 were compared and tested. The results are shown in Tables 1 and Figure 1-3 .

[0087] Table 1 Rooting situation

[0088] Rooting rate (%) Mean root length (cm) Mean root circumference (mm) Example 1 96.2 1.87 2.31 Example 2 98.5 1.92 2.45 Example 3 99.3 1.97 2.51 Comparative Example 1 89.2 1.56 1.91 Comparative Example 2 91.3 1.58 1.94 Comparative Example 3 92.1 1.61 1.83 Comparative Example 4 91.8 1.63 1.87

[0089] From the results in Table 1 above, it can be seen that adding trans-2-dodecenedioic acid to both the induction medium and the rooting medium in combination with 5-AzaC can greatly improve the rooting growth of tissue cultured apple seedlings.

[0090] It can be seen from the above embodiments that the present invention discloses a method for rapid propagation of Xinjiang wild apple tissue culture. The present invention discloses a technical scheme for combining 5-AzaC and trans-2-dodecenedioic acid. The use of the above scheme in combination with a suitable rapid propagation method can greatly accelerate the growth of Xinjiang wild apple rooted tissue culture seedlings, improve the rooting rate and root length of the wild apple tissue culture seedlings, improve the survival rate of the wild apple tissue culture seedlings, and accelerate the tissue culture breeding of wild apples, which has significant progress.

[0091] The description of the limited preferred embodiments of the invention is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the invention, which all belong to the protection scope of the invention.

Claims

1. A method for rapid propagation of Xinjiang wild apple tissue culture, characterized in that: The following steps are involved: (1) Explant preparation and initiation of culture: After buds sprout in spring, Xinjiang wild apple tree branches are taken from the field. The sprouted new buds are rinsed with tap water and then 75% alcohol for 0.5 to 1 min. The leaf sheaths are peeled off on a clean bench and cut into a cylindrical stem with a diameter of 0.5 cm and a height of 1 cm centered on the growth point. A small square with a side length of 0.2 to 0.4 cm is then peeled off from the center of the cylindrical stem, which is the small explant bud block. The small square is inoculated on an induction medium and cultured in the dark for 10 to 15 days to obtain a cluster of buds. (2) Rooting culture: first, culture the seedlings in a rooting medium under high temperature and dark conditions for 7 to 10 days; then culture the seedlings under natural scattered light conditions for 10 to 15 days to obtain rooted tissue culture seedlings; (3) Hardening the seedlings: When the rooted tissue culture seedlings grow to a height of 2 to 4 cm and have 2 to 3 expanded leaves, they are removed from the culture room and placed in a cool outdoor place with natural diffuse light for 5 to 7 days to obtain rooted seedlings; (4) Transplantation: Take out the rooted seedlings, wash off the root culture medium, and transplant the rooted tissue culture seedlings to the transplanting medium; The induction medium is 0.5-0.7 times the concentration of MS medium with 5-AzaC 10-20g / L, trans-2-dodecenedioic acid 3-6g / L and 3-5g / L plant gel; The rooting medium is prepared by adding 10-15 g / L of trans-2-dodecenedioic acid, 20-30 g / L of sucrose and 5-8 g / L of plant gel to the MS medium.

2. The method for rapid propagation of Xinjiang wild apple tissue culture according to claim 1, characterized in that: The transplanting matrix comprises 100-200 parts of Northeastern black charcoal soil and peat soil, 100-200 parts of vermiculite with a pore size of 3-6 mm, and 50-100 parts of livestock manure.

3. The method for rapid propagation of Xinjiang wild apple tissue culture according to claim 1, characterized in that: The temperature in the induction culture in step 1) is 20-25°C.

4. The method for rapid propagation of Xinjiang wild apple tissue culture according to claim 1, characterized in that: In the high temperature dark culture in step 2), the temperature is 35-37°C.

5. Use of the method according to claims 1-4 in cultivating Xinjiang wild apple seedlings.

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