CN108967195B - Culture method for proliferation and rejuvenation of tissue culture subculture bud of pinus massoniana - Google Patents
Culture method for proliferation and rejuvenation of tissue culture subculture bud of pinus massoniana Download PDFInfo
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Abstract
The invention provides a culture method for tissue culture subculture bud proliferation and rejuvenation of pinus massoniana, which comprises the procedures of subculture bud selection, axillary bud induction and rejuvenation culture, wherein pinus massoniana subculture single buds subjected to conventional disinfection, inoculation and subculture are selected and transferred into a subculture bottle filled with a solid culture medium I for axillary bud induction, and then a double-layer double-phase culture mode is adopted, and a liquid culture medium II is added into an original subculture bottle for rejuvenation culture of the subculture buds to obtain pinus massoniana subculture cluster buds with vigorous growth. The invention not only simplifies the transfer step in the traditional masson pine tissue culture subculture, but also obtains the masson pine subculture bud which has high subculture multiplication coefficient, vigorous bud seedling activity and difficult aging, realizes the large-scale in-vitro tissue culture rapid propagation and utilization of the excellent masson pine germplasm, and has better economic, social and ecological benefits.
Description
Technical Field
The invention belongs to the technical field of plant propagation, relates to a tissue culture seedling raising technology, and particularly relates to a culture method for tissue culture subculture bud proliferation and rejuvenation of pinus massoniana.
Background
Masson pine (A) and (B)Pinus massonianan) The native product of China is one of important barren mountain greening, paper making raw material forest, grease collecting forest and natural landscape forest in the south of China, and the distribution range of China is extremely wide. As a tree species with high comprehensive utilization value and large popularization potential, the masson pine can be used for producing not only three plates, papermaking and chemical fiber industrial manufacture, but also industrial raw materials such as rosin, turpentine and the like. In recent years, with the increasing demand of people and the stimulation of the shortage of wood resources and the gradual depletion of non-renewable resources such as petroleum, the sustainable development of ecology, economy and society is receiving more and more social attention. Based on the problems of ecological safety, resource safety, social safety and the like in China, the masson pine is taken as one of main industrial material tree species and biomass energy tree species in China, and the vigorous development of the masson pine artificial forest is necessary.
Since the science and technology of the 'six five' country have been concerned in China, great breakthroughs are made in the aspects of the fine variety breeding of the pinus massoniana, the fast-growing and high-yield cultivation technology and the like. At present, the improved pinus massoniana seeds mainly adopt sexual propagation of a seed garden and a mother forest. However, sexual reproduction has a problem of genetic differentiation, and cannot maximize the gain of genetic improvement, while vegetative reproduction can overcome the above-mentioned disadvantages of sexual reproduction.
Pinus massoniana tissue culture-difficult tree species. Numerous scholars in China carry out a great deal of research on masson pine tissue culture seedling raising technology, wherein the masson pine tissue culture seedling raising technology mainly focuses on the research on the aspects of explant initial bud induction, subculture proliferation and adventitious root induction. However, in the in vitro tissue culture process of the pinus massoniana, the pinus massoniana is easy to be physiologically aged, the physiological activity of the subculture bud is poor, the subculture multiplication is difficult, the effective bud multiplication coefficient is low, the vitrification and browning are obvious, and the development of the excellent germplasm aseless seedling culture industrialization of the pinus massoniana is severely limited.
Disclosure of Invention
The invention aims to provide a culture method for the proliferation and rejuvenation of tissue culture subculture buds of pinus massoniana, which has the advantages of high subculture proliferation coefficient, vigorous bud seedling activity and capability of realizing large-scale in-vitro tissue culture rapid propagation and utilization of excellent germplasm of pinus massoniana, mainly aiming at the current situations of easy physiological aging, low proliferation coefficient, severe vitrification and browning of the tissue culture subculture buds of the pinus massoniana.
In order to achieve the above object, the technical solution of the present invention is as follows:
a culture method for proliferation and rejuvenation of tissue culture subculture buds of pinus massoniana is characterized by comprising the following steps: the method comprises the following steps of selecting subculture buds, inducing axillary buds and performing rejuvenation culture, namely selecting pinus massoniana subculture single buds subjected to conventional disinfection, inoculation and subculture to transfer the pinus massoniana subculture single buds into a subculture bottle filled with a solid culture medium I for axillary bud induction, and then adding a liquid culture medium II into an original subculture bottle to perform rejuvenation culture of the subculture buds by adopting a double-layer double-phase culture mode to obtain pinus massoniana subculture cluster buds with vigorous growth; the main operation steps are as follows:
(1) selecting subculture buds: selecting pinus massoniana subculture buds which are subjected to conventional disinfection, inoculation and subculture for more than 3 times, have spread needle leaves and longer leaf nodes and are 3.5-4.5 cm high;
(2) axillary bud induction: cutting off a single subculture bud which grows strongly and is 2.5-3.5 cm high, transferring the subculture bud into a subculture bottle filled with a solid culture medium I, culturing for 7-10 days under a specific light temperature condition, and inducing formation of an axillary bud of the subculture bud;
(3) rejuvenation culture: and (3) when the macroscopic axillary buds in the subculture bottle in the step (2) are formed, adding a liquid culture medium II into the subculture bottle in the step (2) in a double-layer double-phase culture mode, and carrying out rejuvenation culture for 20-30 d under appropriate conditions to promote the elongation of the axillary buds of the subculture bottle and obtain the masson pine subculture cluster buds with vigorous growth.
The solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.5-0.8 mg.L-1、6-BA 1.0-1.5 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And modified MS medium.
The liquid culture medium II comprises the following raw materials in percentage by weight: IAA 2.0-3.0 mg.L-1、NAA 0.5-1.0 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 100-250 mg. L-1Glutamine 50-100 mg.L-1Phloroglucinol (PG) 50-100 mg.L-130000 mg/L of sucrose-1500-1250 mg-L active carbon-1And modified MS medium.
The improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1。
The light-temperature conditions in the step (2) are as follows: the temperature is 25 +/-1 ℃, the light source is a red-blue LED lamp with 650 nm red light, 450 nm blue light =3:5, the illumination intensity is 3000-.
The suitable conditions in the step (3) are as follows: the temperature is 28 +/-0.5 ℃, the light source is a red, blue and white LED lamp with 650 nm red light, 450 nm blue light and white light =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d.
The subculture bottle is a 500 mL colorless glass triangular bottle; when the culture medium is used, 100 mL of the solid culture medium I is added into a subculture bottle, axillary bud induction is carried out after conventional high-temperature and high-pressure disinfection, when visible axillary buds in the subculture bottle are formed, the solid culture medium I is cut into an X shape by using a pair of tweezers, and then 50 mL of the liquid culture medium II which is conventionally filtered and disinfected is directly added into the subculture bottle for rejuvenation culture.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention improves the MS basic culture medium, reduces K, Ca and ammonium nitrogen content, improves the ratio of nitrate nitrogen to ammonium nitrogen, and also mainly adjusts B, Mn, Mo and other microelements closely related to photosynthetic physiological metabolism in the growth of the masson pine secondary bud, so that the culture medium is more scientific, is more suitable for masson pine secondary bud culture, slows down bud seedling lignification, and has obvious effect.
2. The masson pine subculture bud multiplication method firstly transfers masson pine tissue culture subculture single buds into a solid culture medium I containing low-concentration cytokinin MT, 6-BA and an improved MS culture medium, and performs axillary bud induction culture under LED red and blue light, so that bud seedlings are strong, axillary buds are formed only by inducing for 7-10 days, and the subculture period is greatly shortened.
3. Based on the problems of easy physiological aging, vitrification, browning and the like in the masson pine subculture, the invention adopts a double-layer double-phase culture method, and directly adds a liquid culture medium II rich in acid hydrolyzed casein, glutamine, sodium thiosulfate, phloroglucinol and other seedling strengthening active substances into an original subculture bottle after axillary buds are formed, thereby not only simplifying the transfer step in the traditional masson pine tissue culture subculture, but also obtaining the masson pine subculture buds which have high subculture multiplication coefficient, vigorous bud seedling vitality and difficult aging, realizing the excellent large-scale in-vitro tissue culture and rapid propagation and utilization of the masson pine germplasm, and having better economic, social and ecological benefits.
Drawings
FIG. 1 shows tissue-cultured cluster buds of Pinus massoniana.
FIG. 2 shows tissue-cultured cluster buds of pinus massoniana obtained by conventional single-phase subculture.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1:
performing conventional disinfection, inoculation and subculture according to Guangxi local standard of the masson pine tissue culture rapid propagation, and performing subculture propagation and rejuvenation on the masson pine tissue culture bud by using a subculture bud as an experimental material, wherein the subculture bud has 3 times of subculture/subculture period of 40 d, extended needle leaves, longer leaf nodes and a height of 3.5-4.5 cm.
Selecting a 500 mL colorless glass triangular flask as a secondary flask, adding 100 mL solid culture medium I into the secondary flask, performing conventional high-temperature and high-pressure sterilization, cutting off a secondary bud which grows robustly and is 2.5-3.5 cm high singly, transferring the secondary bud into the secondary flask filled with the solid culture medium I, and culturing for 7 d under the conditions that the temperature is 25 +/-1 ℃, the light source is a 650 nm red light, a 450 nm blue light =3:5 red-blue LED lamp, the illumination intensity is 3000 and 3500 lx, and the illumination time is 16 h/d, so that secondary bud axillary buds are formed. Wherein, the solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.5 mg. L-1、6-BA 1.0 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And modified MS medium.
When macroscopic axillary buds form in a secondary bottle, a double-layer double-phase culture mode is adopted, a solid culture medium I is cut off in an X shape in an original secondary bottle by using tweezers, then 50 mL of a liquid culture medium II which is subjected to conventional filtering and disinfection is directly added into the secondary bottle, rejuvenation culture is carried out for 20 d under the conditions that the temperature is 28 +/-0.5 ℃, the light source is a red light with the wavelength of 650 nm, the blue light with the wavelength of 450 nm, the white light with the wavelength of =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d, the elongation of the axillary buds of the secondary buds is promoted, and the pinus massoniana secondary cluster buds with vigorous growth are obtained, and the effective buds are subjected to secondary propagationThe coefficient reaches 7.6. Wherein the liquid culture medium II comprises the following raw materials in percentage by weight: IAA 2.0 mg.L-1、NAA 0.5 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 100 mg. L-1Glutamine 50 mg.L-1Phloroglucinol (PG) 100 mg.L-130000 mg/L of sucrose-1500 mg. L of activated carbon-1And modified MS medium.
The improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1。
Example 2:
performing conventional disinfection, inoculation and subculture according to Guangxi local standard of the masson pine tissue culture rapid propagation, and performing subculture propagation and rejuvenation on the masson pine tissue culture bud by using subculture buds as experimental materials, wherein the subculture buds have 5 times of subculture/subculture period of 40 d, extended needle leaves, longer leaf nodes and a height of 3.5-4.5 cm.
Selecting 500 mL colorless glass triangular flask as a secondary flask, adding 100 mL solid culture medium I into the secondary flask, sterilizing at high temperature and high pressure, cutting off secondary buds with strong growth and height of 2.5-3.5 cm, and transferring to a medium containing solid culture medium IIn a secondary transfer bottle, culturing for 8 d under the conditions that the temperature is 25 +/-1 ℃, the light source is a red-blue LED lamp with 650 nm red light and 450 nm blue light =3:5, the illumination intensity is 3000 and 3500 lx, and the illumination time is 16 h/d, and forming secondary bud axillary buds. Wherein, the solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.5 mg. L-1、6-BA 1.5 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And modified MS medium.
When macroscopic axillary buds form in a secondary bottle, a double-layer double-phase culture mode is adopted, a solid culture medium I is cut off in an X shape in an original secondary bottle by using tweezers, then 50 mL of a liquid culture medium II which is subjected to conventional filtering and disinfection is directly added into the secondary bottle, and rejuvenation culture is carried out for 20 d under the conditions that the temperature is 28 +/-0.5 ℃, the light source is a red light with the wavelength of 650 nm, a blue light with the wavelength of 450 nm, a white light with the wavelength of =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d, so that the elongation of the axillary buds of the secondary buds is promoted, the pinus massoniana secondary cluster buds with vigorous growth are obtained, and the effective bud secondary multiplication coefficient reaches 7.8. Wherein the liquid culture medium II comprises the following raw materials in percentage by weight: IAA 2.0 mg.L-1、NAA 1.0 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 200 mg. L-1Glutamine 50 mg.L-1Phloroglucinol (PG) 50 mg.L-130000 mg/L of sucrose-11000 mg. L of active carbon-1And modified MS medium.
The improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1。
Example 3:
performing conventional disinfection, inoculation and subculture according to Guangxi local standard of the masson pine tissue culture rapid propagation, and performing subculture proliferation and rejuvenation on the masson pine tissue culture bud by using subculture buds with the subculture period of 40 d, extended needle leaves, longer leaf nodes and the height of 3.5-4.5 cm as experimental materials.
Selecting a 500 mL colorless glass triangular flask as a secondary flask, adding 100 mL solid culture medium I into the secondary flask, performing conventional high-temperature and high-pressure sterilization, cutting off a secondary bud which grows robustly and is 2.5-3.5 cm high singly, transferring the secondary bud into the secondary flask filled with the solid culture medium I, and culturing for 10 d under the conditions that the temperature is 25 +/-1 ℃, the light source is a 650 nm red light, a 450 nm blue light =3:5 red-blue LED lamp, the illumination intensity is 3000 and 3500 lx, and the illumination time is 16 h/d, so that secondary bud axillary buds are formed. Wherein, the solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.8 mg. L-1、6-BA 1.0 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And modified MS medium.
When macroscopic axillary buds form in a secondary bottle, a double-layer double-phase culture mode is adopted, a solid culture medium I is cut into an X shape in an original secondary bottle by using tweezers, then 50 mL of a liquid culture medium II which is subjected to conventional filtering and disinfection is directly added into the secondary bottle, and rejuvenation culture is carried out for 25 d under the conditions that the temperature is 28 +/-0.5 ℃, the light source is a red light with the wavelength of 650 nm, a blue light with the wavelength of 450 nm, a white light with the wavelength of =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d, so that the elongation of the axillary buds of the secondary buds is promoted, the pinus massoniana secondary cluster buds with vigorous growth are obtained, and the effective bud secondary multiplication coefficient reaches 8.4. Wherein the liquid culture medium II comprises the following raw materials in percentage by weight: IAA2.5 mg·L-1、NAA 0.5 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 250 mg. L-1100 mg.L of glutamine-1Phloroglucinol (PG) 50 mg.L-130000 mg/L of sucrose-11000 mg. L of active carbon-1And modified MS medium.
The improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1。
Example 4:
performing conventional disinfection, inoculation and subculture according to Guangxi local standard of the masson pine tissue culture rapid propagation, and performing subculture propagation and rejuvenation on the masson pine tissue culture bud by using a subculture bud as an experimental material, wherein the subculture bud has 18 times of subculture/subculture period of 40 d, extended needle leaves, longer leaf nodes and a height of 3.5-4.5 cm.
Selecting a 500 mL colorless glass triangular flask as a secondary flask, adding 100 mL solid culture medium I into the secondary flask, sterilizing at high temperature and high pressure, cutting off strong secondary buds 2.5-3.5 cm in height, transferring into the secondary flask filled with the solid culture medium I, and culturing at 25 + -1 deg.C with a 650 nm red light source and a 450 nm blue light =3:5And (3) culturing the axillary buds of the subculture buds for 10 days under the conditions of the red and blue LED lamp, the illumination intensity of 3000-. Wherein, the solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.8 mg. L-1、6-BA 1.5 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And modified MS medium.
When macroscopic axillary buds form in a secondary bottle, a double-layer double-phase culture mode is adopted, a solid culture medium I is cut off in an X shape in an original secondary bottle by using tweezers, then 50 mL of a liquid culture medium II which is subjected to conventional filtering and disinfection is directly added into the secondary bottle, and rejuvenation culture is carried out for 30 d under the conditions that the temperature is 28 +/-0.5 ℃, the light source is a red light with the wavelength of 650 nm, a blue light with the wavelength of 450 nm, a white light with the wavelength of =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d, so that the elongation of the axillary buds of the secondary buds is promoted, the pinus massoniana secondary cluster buds with vigorous growth are obtained, and the effective bud secondary multiplication coefficient reaches 8.0. Wherein the liquid culture medium II comprises the following raw materials in percentage by weight: IAA 3.0 mg.L-1、NAA 1.0 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 250 mg. L-1100 mg.L of glutamine-1Phloroglucinol (PG) 100 mg.L-130000 mg/L of sucrose-11250 mg. L of active carbon-1And modified MS medium.
The improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1。
The proliferation and rejuvenation culture conditions of the tissue culture subculture bud of Pinus massoniana in the above examples are shown in Table 1.
TABLE 1 tissue culture subculture bud proliferation and rejuvenation culture conditions of Pinus massoniana
The results in table 1 show that the tissue culture bud subculture multiplication coefficient of the masson pine with different subculture times reaches more than 7.6 and can reach 8.4 at most, the seedling has spread needle leaves, long leaf nodes, no vitrification and water stain phenomena and high effective bud coefficient. The invention better solves the problems of easy physiological aging, difficult proliferation, poor physiological activity and the like of subculture buds in the tissue culture and rapid propagation of the pinus massoniana, and provides a culture method for tissue culture subculture proliferation and rejuvenation for the industrialized production of pinus massoniana tissue culture regeneration plants.
Claims (1)
1. A culture method for proliferation and rejuvenation of tissue culture subculture buds of pinus massoniana is characterized by comprising the following steps: the method comprises the following steps of selecting subculture buds, inducing axillary buds and performing rejuvenation culture, namely selecting pinus massoniana subculture single buds subjected to conventional disinfection, inoculation and subculture to transfer the pinus massoniana subculture single buds into a subculture bottle filled with a solid culture medium I for axillary bud induction, and then adding a liquid culture medium II into an original subculture bottle to perform rejuvenation culture of the subculture buds by adopting a double-layer double-phase culture mode to obtain pinus massoniana subculture cluster buds with vigorous growth; the main operation steps are as follows:
(1) selecting subculture buds: selecting pinus massoniana subculture buds which are subjected to conventional disinfection, inoculation and subculture for more than 3 times, have spread needle leaves and longer leaf nodes and are 3.5-4.5 cm high;
(2) axillary bud induction: cutting off a single subculture bud which grows strongly and is 2.5-3.5 cm high, transferring the subculture bud into a subculture bottle filled with a solid culture medium I, culturing for 7-10 days under a specific light temperature condition, and inducing formation of an axillary bud of the subculture bud;
(3) rejuvenation culture: when the macroscopic axillary buds in the subculture bottle in the step (2) are formed, adding a liquid culture medium II into the subculture bottle in the step (2) in a double-layer double-phase culture mode, and performing rejuvenation culture for 20-30 d under appropriate conditions to promote the elongation of the axillary buds of the subculture bottle and obtain the pinus massoniana subculture cluster buds with vigorous growth;
the solid culture medium I comprises the following raw materials in percentage by weight: Meta-Topolin (MT) 0.5-0.8 mg.L-1、6-BA 1.0-1.5 mg·L-125000 mg. L of sucrose-110000 mg.L of glucose-17000 mg. L of Qiongfen-1And improving the MS culture medium;
the liquid culture medium II comprises the following raw materials in percentage by weight: IAA 2.0-3.0 mg.L-1、NAA 0.5-1.0 mg·L-1Sodium thiosulfate 100 mg.L-1Acid hydrolyzed casein 100-250 mg. L-1Glutamine 50-100 mg.L-1Phloroglucinol (PG) 50-100 mg.L-130000 mg/L of sucrose-1500-1250 mg-L active carbon-1And improving the MS culture medium;
the improved MS culture medium comprises the following raw materials in percentage by weight: KNO3 1000 mg·L-1；NH4NO3 200 mg·L-1；CaCl2·2H2O 260 mg·L-1；MgSO4·7H2O 250 mg·L-1；Mg(NO3)2·6H2O 470 mg·L-1；Ca(NO3)2·4H2O 220 mg·L-1；KH2PO4 210 mg·L-1；MnSO4·H2O 12 mg·L-1；ZnSO4·7H2O 10 mg·L-1；CuSO4·5H2O 0.1 mg·L-1；H3BO3 5.0 mg·L-1；Na2MoO4·2H2O 0.25 mg·L-1；CoCl2·6H2O 0.025 mg·L-1(ii) a Vitamin B1 1.0 mg·L-1(ii) a Vitamin B6 0.5 mg·L-1(ii) a Nicotinic acid 0.5 mg.L-1(ii) a Glycine 2.0 mg. L-1(ii) a Inositol 300 mg.L-1；
The light-temperature conditions in the step (2) are as follows: the temperature is 25 +/-1 ℃, the light source is 650 nm red light, 450 nm blue light =3:5 red-blue LED lamp, the illumination intensity is 3000-;
the suitable conditions in the step (3) are as follows: the temperature is 28 +/-0.5 ℃, the light source is a red, blue and white LED lamp with 650 nm red light, 450 nm blue light and white light =3:1:7, the illumination intensity is 2000 and 2500 lx, and the illumination time is 16 h/d;
the subculture bottle is a 500 mL colorless glass triangular bottle; when the culture medium is used, 100 mL of the solid culture medium I is added into a subculture bottle, axillary bud induction is carried out after conventional high-temperature and high-pressure disinfection, when visible axillary buds in the subculture bottle are formed, the solid culture medium I is cut into an X shape by using a pair of tweezers, and then 50 mL of the liquid culture medium II which is conventionally filtered and disinfected is directly added into the subculture bottle for rejuvenation culture.
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