CN111492978A - Tissue culture rapid propagation method of Eucalyptus macrocarpa 1212 variety - Google Patents
Tissue culture rapid propagation method of Eucalyptus macrocarpa 1212 variety Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention relates to the technical field of eucalyptus cultivation, in particular to a tissue culture rapid propagation method of a Eucalyptus macrocephala 1212 variety. The tissue culture rapid propagation method of the eucalyptus 1212 big inflorescence variety has the advantages of low operation difficulty, low production cost, stable growth of an aseptic propagation system, high propagation coefficient of subculture seedlings, good repeatability, high rooting rate of rooted seedlings, high survival rate of the produced tissue culture seedlings, stable genetic character, tidy forest phase, and the seedlings can keep the excellent genetic characteristic of superior trees, are suitable for large-area planting and are easy to produce and popularize on a large scale.
Description
Technical Field
The invention relates to the technical field of eucalyptus cultivation, in particular to a tissue culture rapid propagation method of a Eucalyptus macrocarpa 1212 variety.
Background
Eucalyptus grandis (with the scientific name of Eucalyptus cloeziana F. Muell.) is a tree species of a large tree in the genus of Eucalyptus in the family of Myrtaceae, and is naturally distributed in the middle and north of Queensland, Australia, with the height of the tree in the origin being 35-40 m, up to 40-55 m, and the diameter at breast height being 1-2 m. The tree grows fast, and has straight and round trunk, and good natural pruning. The wood is tawny, straight in texture, uniform in structure, heavy, firm, high in hardness and durable, is a good sawn wood, is called as rosewood in eucalyptus, and can be used for high-grade furniture, solid wood floors, mechanical components, buildings, ore pillars, pit trees and the like. The eucalyptus grandis has the advantages of drought resistance, barren property, strong disease and pest resistance, rapid growth, especially obvious later growth advantage, and is a large-diameter tree species with cultivation value.
The introduction of Eucalyptus macrocephala in China began in 1972, and introduction tests were conducted in Guangxi provinces, Guangdong provinces, Hainan provinces, Fujian provinces, Sichuan provinces and other provinces. In 1982-1989, the project of the eastern eucalyptus tree demonstration forest of the technical cooperation of China and Australia is respectively established 2 large-inflorescence eucalyptus provenance tests in 1983 and 1989 in the eastern forest farm of Guangxi, but only the test forest established in 1989 is completely stored, and the test forest refers to 11 provenance tests. The Eucalyptus datura was introduced in the county of Kyowa province in the 1990's, 14 plants grown in 7 years had the average breast height of 20.5cm and the average tree height of 22.4m, and was subjected to the low-temperature weather test at-4 ℃, so that the Eucalyptus datura was not frozen. Guangxi forestry science research institute established large-inflorescence eucalyptus provenance/family trial forests at 2 sites of Yulin, Qinzhou, Guangxi in 2004. The seed source of the 5.5-year-old eucalyptus robusta has extremely obvious growth difference among the 2 test points, the seed source difference is extremely obvious, and the interaction effect of the seed source and the site is also extremely obvious. The introduction of the eucalyptus closterium is also researched by the Guangdong, and the research in the middle area of Guangdong shows that the volume growth rate of the 5-year-old eucalyptus closterium is still continuously increased, is most obvious compared with other eucalyptuses such as eucalyptus urophylla and the like, and is considered to have the cultivation potential of large-diameter-grade materials (sawn timber).
At present, eucalyptus forestation varieties are few, single clone large-area continuous planting is very common, and serious pest and disease disasters in forest stands are high in risk. In addition, the cultivation target is single, most of the cultivation targets are raw materials of pulp materials or rotary-cut boards, and the market risk resistance is weak. The big inflorescence eucalyptus grows rapidly, the later growth advantage is obvious, the felling period is slightly longer than that of the eucalyptus urophylla and the eucalyptus urophylla, the superiority of the big inflorescence eucalyptus is very clear, the eucalyptus afforestation tree species can be enriched by developing the big inflorescence eucalyptus urophylla, the forest stand diversity is increased, the serious occurrence risk of the forest stand pest disasters can be reduced, the sawn timber variety is increased simultaneously, and the market risk resistance of an afforestation owner is improved. The big-inflorescence eucalyptus has a long operating cycle, and the influence of afforestation measures on water, soil and vegetation can be well recovered, so that the artificial forest is more environment-friendly, the prejudice of people on the eucalyptus forest can be gradually changed, various misunderstandings of the eucalyptus can be slowly eliminated, and the development of the eucalyptus artificial forest is facilitated.
The Eucalyptus dahliae 1212 is a good Eucalyptus dahliae variety obtained by screening good single plants from a good seed source stand of the Eucalyptus dahliae introduced in Australia, felling down to promote germination and carrying out vegetative propagation. The eucalyptus grandis is a perennial cross-pollinated native plant, the conventional seedling is large in seedling raising and afforestation differentiation, the seedlings are irregular in phase and grow unevenly, and the excellent characteristics of superior trees cannot be guaranteed. The invention can obtain a large number of seedlings with consistent hereditary characters in a short time by utilizing a tissue culture technology, and solves the problems of seedling shortage and large afforestation differentiation of the eucalyptus grandis 1212.
Disclosure of Invention
The invention aims to provide a tissue culture and rapid propagation method of a Eucalyptus robusta 1212 variety, which is characterized in that excellent single plants of excellent Eucalyptus robusta resources are adopted to cut down and promote germination, bud strips are collected to induce aseptic buds, aseptic buds are directly induced in vitro by bud organs for tissue culture without a callus approach, and a large number of regenerated plants are rapidly obtained through subculture, rooting culture and light matrix seedling culture. The regenerated plant obtained by the method has stable result and good repeatability, can be popularized and cultured on a large scale, has high survival rate of tissue culture seedlings and stable hereditary character, and the seedlings can keep the excellent hereditary character of superior trees and are not easy to mutate.
In order to realize the purpose of the invention, the following technical scheme is adopted:
a tissue culture and rapid propagation method of Eucalyptus macrocephala 1212 variety comprises adopting excellent single plants of excellent seed source of Eucalyptus macrocephala, felling and promoting germination, collecting bud strips to induce aseptic buds, performing subculture, rooting culture and light matrix seedling culture on the aseptic buds to obtain regenerated plants; the tissue culture rapid propagation method of the eucalyptus grandis 1212 variety specifically comprises the following steps:
(1) establishing an explant: screening good single plants from good seed forest stand of Eucalyptus robusta introduced in Australia, cutting down to promote germination, shearing bud strips, removing leaves of the collected bud strips, shearing into stem sections of 3-5cm, washing with tap water for 30-35min, washing with saturated washing powder solution for 10-12min, and washing with tap water;
(2) explant disinfection: placing the explant obtained in the step (1) in a sterile bottle subjected to high-pressure sterilization treatment, soaking for 40S by using an alcohol solution with the volume concentration of 75%, pouring off alcohol, washing for 2-3 times by using sterile water, placing in a mercuric chloride solution with the volume concentration of 0.1%, properly stirring, disinfecting for 8min, pouring off mercuric chloride into a special collection tank, washing for 3-4 times by using sterile water, and then soaking for 28-32min in a bactericide S106 with the volume concentration of 0.5%;
(3) sterile bud induction: draining water, placing on an inoculating dish subjected to high-pressure sterilization, cutting off cuts at two ends of a bud stick contacting with the liquid medicine by using forceps and a scalpel subjected to sterilization, cutting the bud stick into stem sections with buds with the length of 1-2cm, inoculating the stem sections on an induction culture medium taking a test tube as a carrier, carrying out dark culture for 7 days, carrying out light culture, and carrying out light culture at the illumination intensity of 1500-;
(4) subculturing: culturing for 25-30 days, starting the explant to sprout, selecting a pollution-free sterile bud when the sterile bud grows to be more than 1cm, extracting the explant from a test tube by using a forceps, transferring the sterile bud into a subculture multiplication medium taking a wide-mouthed bottle as a carrier after cutting, culturing for 7-10 days with the illumination intensity of 1500 plus materials of 2000lux, increasing the illumination intensity to 2000 plus materials of 3000lux to promote the multiplication and growth of the cluster buds, and when the cluster buds grow to be 1-2cm, transferring the culture medium to a new multiplication medium for subculture to obtain a large number of subculture seedlings;
(5) rooting culture: when the subculture seedling reaches a certain amount, starting rooting culture, and cutting 1.1-1.4cm of terminal buds to be inoculated into a rooting culture medium;
(6) hardening seedlings: culturing in a culture room for 7 days, then, making root points or short roots emerge from the cut, picking up bottle seedlings with roots, and transferring the bottle seedlings to a greenhouse for hardening seedlings;
(7) transplanting: hardening the seedlings for 34-36 days, growing the roots to 1-3cm, and the seedlings are higher than 3cm, washing off the culture medium, transplanting the seedlings into a light matrix nutrition cup, and culturing for 85-95 days to obtain regenerated plants;
(8) fertilizer management after transplanting: fertilizing after the nursery stock is moved into a nutrition cup for 14-16 days, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2% on leaf surfaces, immediately cleaning the nursery stock with clear water after spraying, spraying for 1 time every 7 days, and prolonging the time when the nursery stock is in low temperature or rainy days; along with the growth of the seedling, when the seedling height reaches 12-13 cm, the fertilizing concentration can be increased, the using amount of the potassium fertilizer can be properly increased to promote the lignification of the seedling, and when the seedling height reaches 20 cm, the fertilizing is stopped.
In the tissue culture and rapid propagation method of the eucalyptus grandis 1212 variety, the superior eucalyptus grandis seed source introduced from australia is subjected to cultivation test, determination analysis, measurement of breast height, tree height, accumulation, successive year growth amount, wood property and dry shape index, and comprehensive comparison, and then the superior tree is selected from the seed sources suitable for local growth.
In the tissue culture rapid propagation method of the Eucalyptus macrocephala 1212 variety, in the step (3) of sterile bud induction, the induction culture medium is modified MS +6-BA1.5 mg/L + KT0.5 mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, and the pH value is 5.8-6.2.
In the tissue culture and rapid propagation method of the Eucalyptus macrocephala 1212 variety, in the step (4) of subculture, the subculture propagation medium is modified MS +6-BA1.0 mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, and the pH value is 5.8-6.2.
The tissue culture and rapid propagation method of the eucalyptus grandis 1212 variety comprises the following steps of (5) rooting culture, wherein the rooting culture medium comprises: 1/3 improved MS + ABT13.0 mg/L + IAA 1.0 mg/L + vitamin C5 mg/L + active carbon 20 mg/L + sucrose 15 g/L + agar 4 g/L, pH 5.8-6.2.
In the tissue culture and rapid propagation method of the Eucalyptus macrocephala 1212 variety, in the step (7) of transplanting, the light matrix used in the light matrix nutrition cup is KMnO4And (3) mixing and uniformly stirring the sterilized coconut chaff, peat soil, carbonized rice husk/carbonized sawdust =6:3:1 in volume ratio to prepare the seedling culture substrate.
The tissue culture rapid propagation method of the Eucalyptus macrocephala 1212 variety further comprises pest control after seedling transplantation, and specifically comprises the following steps: the leaves in the seedling stage are small and have few diseases, the seedlings grow to 10-15 cm, the leaves are gradually enlarged, and the seedling bed is ventilated and has poor air permeability, and stem rot is easy to obtain; after 15 days of seedling transplanting, the leaves gradually fall off after root cutting, and the leaves are adhered to leaf stalks, so that stem rot easily occurs in a large area; for preventing and treating the stem rot, nitrogen fertilizer is applied little at first to prevent the seedlings from growing and tendering and improve the disease resistance of the seedlings; spraying the bactericide for 1 time every 7 days in high-temperature and high-humidity season with disease occurrence, and spraying the root of the nursery stock with 800 times of liquid of fenaminosulf, thiophanate methyl or zineb 600-; for the nursery stock with the disease, the nursery stock is removed in time and is burnt to thoroughly eliminate the germs and prevent the nursery stock from being infected again; the insect pests in the seedling stage of the Eucalyptus dahliae mainly comprise the Fangziworm and the cabbage caterpillar, and the Fangziworm is irrigated by 1400-time liquid of chlorpyrifos missible oil 1200-; the cabbage caterpillar is prevented and controlled by a 1000-time liquid of 800-.
The compound of each component for preparing the culture medium can be searched from a chemical dictionary or a textbook of crop cultivation to obtain the name and the effect of the compound, and the compound is combined to prepare the culture medium for different periods, so that the culture medium is most suitable for tissue culture and rapid propagation of Eucalyptus macrocephala 1212 varieties and is finally determined after multiple experimental screening.
The Eucalyptus dahliae 1212 is a good Eucalyptus dahliae variety obtained by screening good single plants from a good seed source stand of the Eucalyptus dahliae introduced in Australia, felling down to promote germination and carrying out vegetative propagation. In a Eucalyptus grandis provenance/family test forest established in Dongmen forest farm in 2004, dominant wood comparison method is adopted to select 3-5 dominant trees, and average height, breast diameter and volume are measured and calculated. The candidate superior tree growth indexes exceed the average standard (the breast diameter is more than 20%, the tree height is more than 5%, and the volume of wood is more than 50%), and then the candidate superior tree can be selected; and in 2018, selecting a superior tree to be fell down and promote germination, and establishing a tissue culture rapid propagation system of the eucalyptus robusta 1212 through a rapid asexual propagation technology. The eucalyptus grandis 1212 variety has the advantages of straight dry shape, rapid growth, obvious later growth advantage and suitability for cultivating large-diameter wood.
The invention has the beneficial effects that:
1. in the tissue culture rapid propagation method, the tissue culture aseptic buds are obtained by in vitro inducing cluster buds from bud organs (stem sections with latent buds) without a callus approach or a dedifferentiation process in the aseptic bud induction process, so that the cultured tissue culture seedlings have stable genetic characters, can completely maintain the genetic characteristics of a mother tree and are not easy to mutate.
2. The tissue culture rapid propagation method of the eucalyptus grandis 1212 variety has the advantages of low operation difficulty, low production cost, stable growth of an aseptic propagation system, high multiplication coefficient of subculture seedlings, good repeatability, high rooting rate of rooted seedlings, about 90% of rooting rate, high survival rate of produced tissue culture seedlings, stable genetic character, regular forest phase, and nursery stocks capable of keeping excellent genetic characteristics of superior trees, is suitable for large-area planting, can be applied to large-scale commercialized seedling culture, and is easy for large-scale production and popularization.
3. By adopting the tissue culture and rapid propagation method of the eucalyptus grandiflorum 1212 variety, a large number of regeneration plants which are tidy and consistent and have stable genetic characters can be obtained in a short time, and the problems of nursery stock shortage and large afforestation differentiation of the eucalyptus grandiflorum 1212 are solved.
Drawings
FIG. 1 shows the subculture propagated seedlings of Eucalyptus robusta 1212 sterile buds obtained by the subculture step in example 2 of the present invention;
FIG. 2 shows the endophytic seedlings of Eucalyptus robusta 1212 bottles obtained by rooting culture in example 2 of the present invention;
FIG. 3 is a bottom view of an endophytic seedling in a bottle 1212 of Eucalyptus robusta obtained by rooting culture in example 2 of the present invention;
fig. 4 shows the cultured eucalyptus robusta 1212 plantlets after the plantlets are transplanted into the light medium nutrition cup in the transplanting step of embodiment 2 of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided to enable those skilled in the art to more readily understand the advantages and features of the invention, and to clearly and unequivocally define the scope of the invention.
Example 1
A tissue culture and rapid propagation method of Eucalyptus macrocephala 1212 variety comprises adopting excellent single plants of excellent seed source of Eucalyptus macrocephala, felling and promoting germination, collecting bud strips to induce aseptic buds, performing subculture, rooting culture and light matrix seedling culture on the aseptic buds to obtain regenerated plants;
the tissue culture rapid propagation method of the eucalyptus grandis 1212 variety specifically comprises the following steps:
(1) establishing an explant: selecting excellent single plants from the excellent seed forest of the Australian-introduced Eucalyptus robusta (namely, selecting excellent trees from the seed forest suitable for local growth after measuring breast diameter, tree height, accumulation, successive year growth amount, wood material property and dry form indexes and comprehensively comparing through cultivation test and determination analysis of the excellent seed sources of the Australian-introduced Eucalyptus robusta), cutting down and promoting germination, shearing bud strips, removing leaves of the collected bud strips, cutting into stem sections of 3-5cm, washing for 35min with tap water, washing for 11min with saturated washing powder solution, and then washing cleanly with tap water;
(2) explant disinfection: placing the explant obtained in the step (1) in a sterile bottle subjected to high-pressure sterilization treatment, soaking for 40S by using an alcohol solution with the volume concentration of 75%, pouring off alcohol, washing for 2 times by using sterile water, placing in a mercuric chloride solution with the volume concentration of 0.1%, properly stirring, disinfecting for 8min, pouring off mercuric chloride to a special collection tank, washing for 3 times by using sterile water, and then soaking for 28min in a bactericide S106 with the volume concentration of 0.5%;
(3) sterile bud induction, namely draining water, placing the bud on an inoculating dish subjected to high-pressure sterilization, cutting off two ends of a bud strip contacted with a liquid medicine by using forceps and a scalpel subjected to sterilization, cutting the bud strip into stem sections with buds with the length of 1-2cm, and inoculating the stem sections with the buds to an induction culture medium taking a test tube as a carrier, wherein the induction culture medium is modified MS + 6-BA1.5mg/L + KT0.5mg/L + NAA0.3mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, the pH value is 5.8-6.2, dark culture is carried out for 7 days, and then light culture is carried out, the light intensity is 1500-2000lux, the temperature is 25 +/-2 ℃, and the culture is carried out for 28 days;
(4) subculture, namely culturing for 28 days, starting the explant to sprout, selecting a non-polluted sterile bud when the sterile bud grows to be more than 1cm, extracting the explant from a test tube by using a forceps, cutting and transferring the sterile bud into a subculture multiplication medium taking a wide-mouthed bottle as a carrier, wherein the subculture multiplication medium is modified by MS +6-BA1.0 mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, has pH of 5.8-6.2, has full light culture light intensity of 1500 plus 2000lux, is cultured for 8 days, increases the light intensity to 2000 plus 3000lux to promote the proliferation and growth of the cluster buds, and is transferred to a new proliferation medium for subculture when the cluster buds grow to be 1-2cm to obtain a large number of subculture seedlings;
(5) rooting culture: when the subculture seedling reaches a certain amount, starting rooting culture, cutting 1.1-1.4cm of terminal buds, and inoculating into a rooting culture medium, wherein the rooting culture medium comprises: 1/3 improved MS + ABT13.0 mg/L + IAA 1.0 mg/L + vitamin C5 mg/L + active carbon 20 mg/L + sucrose 15 g/L + agar 4 g/L, pH 5.8-6.2;
(6) hardening seedlings: culturing in a culture room for 7 days, then, making root points or short roots emerge from the cut, picking up bottle seedlings with roots, and transferring the bottle seedlings to a greenhouse for hardening seedlings;
(7) transplanting: hardening the seedlings for 34-36 days, growing the roots to 1-3cm, and the seedlings are higher than 3cm, washing off the culture medium, transplanting the seedlings into a light matrix nutrition cup, and culturing for 85 days to obtain regenerated plants; the light matrix used in the light matrix nutrition cup is KMnO4The disinfected seedling culture substrate is prepared by uniformly mixing various substrates according to the volume ratio of coconut coir to peat soil to carbonized chaff =6:3: 1;
(8) fertilizer management after transplanting: fertilizing after the nursery stock is moved into a nutrition cup for 15 days, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2% on leaf surfaces, immediately cleaning the nursery stock with clear water after spraying, spraying for 1 time every 7 days, and prolonging the time when the nursery stock is in low temperature or rainy days; along with the growth of the seedling, when the seedling height reaches 12-13 cm, the fertilizing concentration can be increased, the using amount of potassium fertilizer can be properly increased, the lignification of the seedling is promoted, and when the seedling height reaches 20 cm, the fertilizing is stopped;
(9) and (3) pest control: for preventing and treating the stem rot, nitrogen fertilizer is applied little at first to prevent the seedlings from growing and tendering and improve the disease resistance of the seedlings; spraying the bactericide for 1 time every 7 days in high-temperature high-humidity disease epidemic seasons, and spraying the root and stem parts of the nursery stock with 800 times of thiophanate methyl solution; for the nursery stock with the disease, the nursery stock is removed in time and is burnt to thoroughly eliminate the germs and prevent the nursery stock from being infected again; the large inflorescence eucalyptus seedling stage insect pest Fangzi is irrigated with 1200 times of chlorpyrifos missible oil for prevention and treatment; the cabbage caterpillar is prevented and treated by 1000 times of aqueous emulsion of 4.5 percent cypermethrin, and is sprayed for 1 time at intervals of 9 days and is continuously sprayed for 3 times.
Example 2
A tissue culture and rapid propagation method of Eucalyptus macrocephala 1212 variety comprises adopting excellent single plants of excellent seed source of Eucalyptus macrocephala, felling and promoting germination, collecting bud strips to induce aseptic buds, performing subculture, rooting culture and light matrix seedling culture on the aseptic buds to obtain regenerated plants;
the tissue culture rapid propagation method of the eucalyptus grandis 1212 variety specifically comprises the following steps:
(1) establishing an explant: selecting excellent single plants from the excellent seed forest of the Australian-introduced Eucalyptus robusta (namely, selecting excellent trees from the seed forest suitable for local growth after measuring breast diameter, tree height, accumulation, successive year growth amount, wood material property and dry form indexes and comprehensively comparing through cultivation test and determination analysis of the excellent seed sources of the Australian-introduced Eucalyptus robusta), cutting down and promoting germination, shearing buds, removing leaves of the collected buds, cutting into stem sections of 3-5cm, washing for 30min with tap water, washing for 10min with saturated washing powder solution, and then washing cleanly with tap water;
(2) explant disinfection: placing the explant obtained in the step (1) in a sterile bottle subjected to high-pressure sterilization treatment, soaking for 40S by using an alcohol solution with the volume concentration of 75%, pouring off alcohol, washing for 3 times by using sterile water, placing in a mercuric chloride solution with the volume concentration of 0.1%, properly stirring, disinfecting for 8min, pouring off mercuric chloride to a special collection tank, washing for 4 times by using sterile water, and then soaking for 30min in a bactericide S106 with the volume concentration of 0.5%;
(3) sterile bud induction, namely draining water, placing the bud on an inoculating dish subjected to high-pressure sterilization, cutting off two ends of a bud strip contacted with a liquid medicine by using forceps and a scalpel subjected to sterilization, cutting the bud strip into stem sections with buds with the length of 1-2cm, and inoculating the stem sections with the buds to an induction culture medium taking a test tube as a carrier, wherein the induction culture medium is modified MS + 6-BA1.5mg/L + KT0.5mg/L + NAA0.3mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, the pH value is 5.8-6.2, dark culture is carried out for 7 days, and then light culture is carried out, the light intensity is 1500-2000lux, the temperature is 25 +/-2 ℃, and the culture is carried out for 30 days;
(4) subculture, namely culturing for 30 days, starting the explant to sprout, selecting a pollution-free sterile bud when the sterile bud grows to be more than 1cm, extracting the explant from a test tube by using a forceps, cutting and transferring the sterile bud into a subculture multiplication medium taking a wide-mouthed bottle as a carrier, wherein the subculture multiplication medium is modified by MS +6-BA1.0 mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, has pH of 5.8-6.2, has full light culture light intensity of 1500 minus 2000lux, is cultured for 7 days, increases the light intensity to 2000 minus 3000lux to promote the proliferation and growth of the cluster buds, and is transferred to a new multiplication medium for subculture when the cluster buds grow to be 1-2cm to obtain a large number of subculture seedlings;
(5) rooting culture: when the subculture seedling reaches a certain valueAfter the amount is measured, starting rooting culture, cutting 1.1-1.4cm of terminal buds, and inoculating into a rooting culture medium, wherein the rooting culture medium comprises: 1/3 improved MS + ABT13.0 mg/L + IAA 1.0 mg/L + vitamin C5 mg/L + active carbon 20 mg/L + sucrose 15 g/L + agar 4 g/L, pH 5.8-6.2;
(6) hardening seedlings: culturing in a culture room for 7 days, then, making root points or short roots emerge from the cut, picking up bottle seedlings with roots, and transferring the bottle seedlings to a greenhouse for hardening seedlings;
(7) transplanting: hardening the seedlings for 35 days, growing the roots to 1-3cm, and keeping the height of the seedlings to be more than 3cm, washing off the culture medium, transplanting the seedlings into a light matrix nutrition cup, and culturing for 90 days to obtain regenerated plants; the light matrix used in the light matrix nutrition cup is KMnO4The disinfected seedling culture substrate is prepared by uniformly mixing various substrates according to the volume ratio of coconut coir to peat soil to carbonized sawdust =6:3: 1;
(8) fertilizer management after transplanting: fertilizing after the nursery stock is moved into a nutrition cup for 15 days, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2% on leaf surfaces, immediately cleaning the nursery stock with clear water after spraying, spraying for 1 time every 7 days, and prolonging the time when the nursery stock is in low temperature or rainy days; along with the growth of the seedling, when the seedling height reaches 12-13 cm, the fertilizing concentration can be increased, the using amount of potassium fertilizer can be properly increased, the lignification of the seedling is promoted, and when the seedling height reaches 20 cm, the fertilizing is stopped;
(9) and (3) pest control: the leaves in the seedling stage are small and have few diseases, the seedlings grow to 10-15 cm, the leaves are gradually enlarged, and the seedling bed is ventilated and has poor air permeability, and stem rot is easy to obtain; after 15 days of seedling transplanting, the leaves gradually fall off after root cutting, and the leaves are adhered to leaf stalks, so that stem rot easily occurs in a large area; for preventing and treating the stem rot, nitrogen fertilizer is applied little at first to prevent the seedlings from growing and tendering and improve the disease resistance of the seedlings; spraying the bactericide for 1 time every 7 days in high-temperature high-humidity disease epidemic seasons, and spraying 700 times of dixon solution on the root and stem parts of the nursery stock; for the nursery stock with the disease, the nursery stock is removed in time and is burnt to thoroughly eliminate the germs and prevent the nursery stock from being infected again; the large inflorescence eucalyptus seedling stage insect pest Fangzi is irrigated with 1200 times of chlorpyrifos missible oil for prevention and treatment; the cabbage caterpillar is prevented and controlled by 900 times of aqueous emulsion of 4.5 percent cypermethrin, and is sprayed for 1 time at intervals of 8 days and continuously sprayed for 2 to 3 times.
The following table 1 shows the breeding results of the eucalyptus grandis 1212 cultivars cultured by the methods of example 1 and example 2 of the present invention:
as can be seen from the above table 1, the tissue culture rapid propagation method of the Eucalyptus macrocephala 1212 variety has the advantages of high propagation coefficient, rooting rate of more than 89%, transplanting survival rate of more than 85%, rapid adaptation to the open air environment after the tissue culture seedling is transplanted, rapid growth and strong capability of resisting the external adverse environment. At present, the tissue culture and rapid propagation method of the eucalyptus grandis 1212 varieties is adopted to produce seedlings of the eucalyptus grandis 1212 varieties in large batches, and great economic, ecological and social benefits are created.
Claims (7)
1. A tissue culture rapid propagation method of Eucalyptus macrocarpa 1212 variety is characterized in that: adopting excellent single plants of excellent seed sources of the eucalyptus grandis, felling the excellent single plants to promote germination, collecting bud strips to induce aseptic buds, and performing subculture, rooting culture and light matrix seedling culture on the aseptic buds to obtain regenerated plants;
the tissue culture rapid propagation method of the eucalyptus grandis 1212 variety specifically comprises the following steps:
(1) establishing an explant: screening good single plants from good seed forest stand of Eucalyptus robusta introduced in Australia, cutting down to promote germination, shearing bud strips, removing leaves of the collected bud strips, shearing into stem sections of 3-5cm, washing with tap water for 30-35min, washing with saturated washing powder solution for 10-12min, and washing with tap water;
(2) explant disinfection: placing the explant obtained in the step (1) in a sterile bottle subjected to high-pressure sterilization treatment, soaking for 40S by using an alcohol solution with the volume concentration of 75%, pouring off alcohol, washing for 2-3 times by using sterile water, placing in a mercuric chloride solution with the volume concentration of 0.1%, properly stirring, disinfecting for 8min, pouring off mercuric chloride into a special collection tank, washing for 3-4 times by using sterile water, and then soaking for 28-32min in a bactericide S106 with the volume concentration of 0.5%;
(3) sterile bud induction: draining water, placing on an inoculating dish subjected to high-pressure sterilization, cutting off cuts at two ends of a bud stick contacting with the liquid medicine by using forceps and a scalpel subjected to sterilization, cutting the bud stick into stem sections with buds with the length of 1-2cm, inoculating the stem sections on an induction culture medium taking a test tube as a carrier, carrying out dark culture for 7 days, carrying out light culture, and carrying out light culture at the illumination intensity of 1500-;
(4) subculturing: culturing for 25-30 days, starting the explant to sprout, selecting a pollution-free sterile bud when the sterile bud grows to be more than 1cm, extracting the explant from a test tube by using a forceps, transferring the sterile bud into a subculture multiplication medium taking a wide-mouthed bottle as a carrier after cutting, culturing for 7-10 days with the illumination intensity of 1500 plus materials of 2000lux, increasing the illumination intensity to 2000 plus materials of 3000lux to promote the multiplication and growth of the cluster buds, and when the cluster buds grow to be 1-2cm, transferring the culture medium to a new multiplication medium for subculture to obtain a large number of subculture seedlings;
(5) rooting culture: when the subculture seedling reaches a certain amount, starting rooting culture, and cutting 1.1-1.4cm of terminal buds to be inoculated into a rooting culture medium;
(6) hardening seedlings: culturing in a culture room for 7 days, then, making root points or short roots emerge from the cut, picking up bottle seedlings with roots, and transferring the bottle seedlings to a greenhouse for hardening seedlings;
(7) transplanting: hardening the seedlings for 34-36 days, growing the roots to 1-3cm, and the seedlings are higher than 3cm, washing off the culture medium, transplanting the seedlings into a light matrix nutrition cup, and culturing for 85-95 days to obtain regenerated plants;
(8) fertilizer management after transplanting: fertilizing after the nursery stock is moved into a nutrition cup for 14-16 days, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2% on leaf surfaces, immediately cleaning the nursery stock with clear water after spraying, spraying for 1 time every 7 days, and prolonging the time when the nursery stock is in low temperature or rainy days; along with the growth of the seedling, when the seedling height reaches 12-13 cm, the fertilizing concentration can be increased, the using amount of the potassium fertilizer can be properly increased to promote the lignification of the seedling, and when the seedling height reaches 20 cm, the fertilizing is stopped.
2. The tissue culture rapid propagation method of the eucalyptus grandis 1212 variety according to claim 1, characterized in that: a superior seed source of Eucalyptus grandis introduced from Australia is cultivated, tested and analyzed, and after measuring the indexes of diameter at breast height, tree height, accumulation, successive year growth amount, wood quality and dry shape, comprehensively compared, a superior tree is selected from seed sources suitable for local growth.
3. The tissue culture rapid propagation method of Eucalyptus macrocarpa 1212 variety according to claim 1, characterized in that in the step (3) of aseptic bud induction, the induction medium is modified MS + 6-BA1.5mg/L + KT0.5mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, pH 5.8-6.2.
4. The tissue culture rapid propagation method of the Eucalyptus macrocarpa 1212 variety according to claim 1, wherein in the step (4) of subculture, the subculture proliferation medium comprises modified MS + 6-BA1.0mg/L + NAA0.3 mg/L + riboflavin 15 mg/L + sucrose 30 g/L + agar 4 g/L, and has a pH of 5.8-6.2.
5. The tissue culture rapid propagation method of the eucalyptus grandis 1212 variety according to claim 1, characterized in that: in the rooting culture of the step (5), the rooting culture medium is as follows: 1/3 improved MS + ABT13.0 mg/L + IAA 1.0 mg/L + vitamin C5 mg/L + active carbon 20 mg/L + sucrose 15 g/L + agar 4 g/L, pH 5.8-6.2.
6. The tissue culture rapid propagation method of the eucalyptus grandis 1212 variety according to claim 1, characterized in that: in the transplanting step (7), the light matrix used in the light matrix nutrition cup is KMnO4And (3) mixing and uniformly stirring the sterilized coconut chaff, peat soil, carbonized rice husk/carbonized sawdust =6:3:1 in volume ratio to prepare the seedling culture substrate.
7. The tissue culture rapid propagation method of the eucalyptus grandis 1212 variety according to claim 1, characterized in that: still include the pest control after the nursery stock is transplanted, specifically do: for preventing and treating the stem rot, nitrogen fertilizer is applied little at first to prevent the seedlings from growing and tendering and improve the disease resistance of the seedlings; spraying the bactericide for 1 time every 7 days in high-temperature and high-humidity season with disease occurrence, and spraying the root of the nursery stock with 800 times of liquid of fenaminosulf, thiophanate methyl or zineb 600-; for the nursery stock with the disease, the nursery stock is removed in time and is burnt to thoroughly eliminate the germs and prevent the nursery stock from being infected again; the large inflorescence eucalyptus seedling stage insect pest Fangzi is irrigated and controlled by using 1200-fold liquid and 1400-fold liquid of chlorpyrifos emulsifiable concentrate; the cabbage caterpillar is prevented and controlled by a 1000-time liquid of 800-.
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