CN114586684A - Tissue culture rapid propagation method of triploid eucalyptus new variety' Jinggui eucalyptus I - Google Patents
Tissue culture rapid propagation method of triploid eucalyptus new variety' Jinggui eucalyptus I 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
- A01H4/002—Culture media for tissue culture
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses a tissue culture and rapid propagation method of a novel triploid eucalyptus variety 'Jinggui eucalyptus I', which comprises the following operation steps: (1) cultivating high-quality explant raw materials; (2) sterilizing explants; (3) primary culture; (4) subculturing; (5) rooting culture; (6) hardening seedlings; (7) transplanting; (8) and (5) strong seedling culture. The invention adopts the combination of a plurality of rooting promoters, and determines the tissue culture and rapid propagation method, which can effectively improve the rooting rate and the seedling quality; the compound is most suitable for a new variety of triploid eucalyptus, namely the Jinggui eucalyptus I, is determined after being debugged and screened for many times, and has stable and consistent effects after being used.
Description
Technical Field
The invention relates to the technical field of eucalyptus tissue culture, in particular to a tissue culture rapid propagation method of a triploid eucalyptus new variety 'Jinggui eucalyptus I'.
Background
Polyploids are individuals that contain three or more sets of chromosome sets within the somatic cells of a plant. In the process of plant species formation and evolution, polyploidization plays an important role. In the process of the polyploidization of a plant body, the number of chromosomes in a cell nucleus is increased, the form of the organism is increased, and the gene activity and the diversity of enzymes are continuously enhanced, so that the lower transcription level can be maintained, the stronger photosynthetic efficiency can be ensured, and the stress resistance of the plant can be improved. The duplication of chromosome set in plant nucleus usually brings morphological and physiological changes, and has advantages in improving growth speed, improving quality, enhancing resistance and improving content of target metabolites.
Eucalyptus is a diploid plant (2 n-2X-22) and is an important timber tree species in the world, the planting area in China exceeds 6750 ten thousand acres, more than one quarter of timber yield is provided for China every year, and eucalyptus has an important prominent position in forestry production and ecological construction. However, the difficulty in reproductive isolation of eucalyptus and the limitation of traditional artificial crossbreeding lead to that no significant progress has been made in the aspect of genetic improvement for many years, and the introduction of a polyploid breeding technology with both heterosis and ploidy advantages has great significance for genetic improvement of eucalyptus. A triploid eucalyptus, Jinggui eucalyptus I, is a new species of triploid eucalyptus, which is the cooperation of Tomen forest farm in Guangxi and Beijing forestry university, and is prepared through using Eucalyptus urophylla as mutagenesis female parent, treating megasporocyte in the division stage with colchicine and high temperature to obtain diploid female gamete, combining with haploid male gamete through natural free pollination to obtain triploid seed, sowing to form seedling, and ploidy detection to determine triploid plant. The 'Jinggui eucalyptus I' has the advantages of straight dry shape, small branch angle, no deformity, no plant diseases and insect pests, good stress resistance and great development potential in the growth measurement of three years.
In the process of obtaining the triploid plants, the number of chromosomes is varied, so that the fertility of the triploid plants is reduced, the triploid plants are difficult to continue to be propagated in a sexual reproduction mode, and the excellent characters of the triploid plants are difficult to maintain. Therefore, only asexual propagation can be adopted, and asexual propagation methods such as cuttage, grafting, layering and the like have low propagation speed, are not beneficial to popularizing a new variety in a large amount in a short time, so the tissue culture rapid propagation is the best choice. The Wangbeiqi develops the research of the in vitro rapid propagation of the poplar hybrid triploid, and establishes a stem section regeneration system and a leaf adventitious bud induction regeneration technical system; the Dendrobii dendranthema et al develops the tissue culture technical research of the triploid loquat, establishes a regeneration system and lays a foundation for the genetic improvement of the regeneration system; the Yuanxiuhui develops the tissue culture technical research of triploid Chinese white poplar, and cultivates the tissue culture seedling with good quality and high transplanting survival rate and standard industrialized production. The plant tissue rapid propagation technology belongs to the branch of biotechnology and is an important component of bioengineering, and the technology is very suitable for propagating the 'Jinggui No. one eucalyptus' which is named as a famous, excellent, new, special and rare variety. According to statistics, more than 60 kinds of eucalyptus tissue culture are successful in the world at present, and the eucalyptus pellita No. 1 (1981), the eucalyptus grandis (1989), the eucalyptus globulus rubra (1992), the eucalyptus urophylla (2012), the eucalyptus robusta (2017) and the like are mainly cultivated in China. However, a few varieties such as eucalyptus grandis and eucalyptus urophylla are mainly used for tissue culture and large-scale industrial seedling raising. After the tissue culture of the forest trees is successful, the realization of mass rapid propagation is also limited by various factors. One is the redifferentiation type of culture material: the seedlings obtained by the callus differentiation seedling approach need to be subjected to a dedifferentiation process, in which individual seedlings are easy to change and are easy to differentiate after being planted, so that the forest looks are irregular. Secondly, the tree species has the characteristics that: the rapid propagation difficulty of different varieties is greatly different, and different specific culture medium formulas and management methods need to be developed. Thirdly, the tissue culture rapid propagation technical level and the production conditions are as follows: for example, the subculture multiplication rate is low or the success rate is not high, or the culture material is not easy to be polluted, browned, vitrified and transplanted, so that the seedling raising cost is too high to be popularized.
The tissue culture seedling raising of eucalyptus has been realized for more than 30 years, the tissue culture rapid propagation research of eucalyptus is continuously carried out, the technology is continuously improved, and the tissue culture rapid propagation technology of DH32-26 which is currently propagated in large quantity and popularized to be widely cultivated (application with the authorized public number CN 104304028B: the tissue culture rapid propagation method of eucalyptus urophylla DH32-26 variety) is used for in vitro culture of bud organs to directly form seedlings, has no callus seedling formation process, and is an advanced and practical tissue culture rapid propagation method of forest trees. However, the method has many defects, firstly, the explant is not specially cultured and is directly collected from the field and then disinfected, because the explant has a lot of mixed bacteria and is difficult to disinfect and sterilize, higher drug concentration and longer disinfection time are needed, the explant in a tender meristematic state which is easy to sprout is dead, the survival explant is a relatively old explant and is damaged, and the sprouting rate and the sprouting quality are influenced; secondly, an MS culture medium is used as a basic culture medium, a special culture medium for the clone is not prepared, and the rapid propagation capacity of the clone is not optimized to the maximum extent; and thirdly, a transplanting link, namely firstly transplanting the seedlings into a sand bed, hardening the seedlings and then transplanting the seedlings to a seedling cup, wherein a process is added in the process, the seedling loss is increased, and the seedling cost is increased. As mentioned above, a large number of new eucalyptus species are popularized and propagated, the tissue culture rapid propagation technology is quite mature, but the space for continuous research and exploration is still large. The tissue culture and rapid propagation of the novel triploid eucalyptus, namely the Jinggui eucalyptus I, has no precedent at home and abroad at present, realizes tissue culture, obtains regenerated plants and rapidly propagates the regenerated plants in a large scale, and needs to be groped. The method has great significance for meeting the market demand and establishing and tissue culture rapid propagation demonstration of later triploid eucalyptus varieties.
Disclosure of Invention
Aiming at the technical problems, the invention provides a tissue culture and rapid propagation method of a novel triploid eucalyptus variety 'Jinggui No. one eucalyptus', aiming at improving the success rate of tissue culture, rapidly propagating and producing seedlings of the triploid eucalyptus 'Jinggui No. one eucalyptus' in a large scale in an industrialized manner; provides a tissue culture and rapid propagation method of triploid eucalyptus, namely, eucalyptus globulus I, which can be used for seedling formation without a callus approach, is directly cultured in vitro by bud organs, is used for propagating buds by buds, has stable hereditary characters of regenerated plants, high propagation coefficient, easy operation and low cost.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a tissue culture rapid propagation method of a triploid eucalyptus new variety 'Jinggui No. one eucalyptus', comprises the following operation steps:
(1) cultivating high-quality explant raw materials: cutting off a tip section of a triploid eucalyptus 'Jinggui No. one eucalyptus' plant, promoting germination, transferring to a greenhouse for culture, manually and conventionally controlling illumination, temperature and humidity after the germination grows out, taking pest control measures, when the germination grows to 6-8 cm and upper bud eyes are full but lateral buds do not grow, picking lower bud strips in the morning of sunny days, removing leaves, obtaining explant materials, and bringing the explant materials back to a laboratory for later use;
(2) explant disinfection: cleaning the explant material obtained in the step (1) with purified water for 3 times, soaking for 2 minutes with benzalkonium bromide solution, cleaning for 3 times with purified water, pre-treating, transferring to an ultra-clean workbench, shearing to a semi-lignified cut segment with 1-2 latent buds and 1.5-2.0cm in length under aseptic condition, soaking with alcohol, cleaning for 3 times with sterile water, draining off water, placing in sodium hypochlorite solution for disinfection, cleaning for 3 times with sterile water, sucking surface water with sterile filter paper, placing in mercuric chloride solution for disinfection for 3 minutes, allowing the surface of the explant to fully contact with the mercuric chloride solution, pouring out the mercuric chloride solution, and cleaning the explant with sterile water for 5-6 times;
(3) primary culture: sucking water on the surface of the explant sterilized in the step (2) by using sterile filter paper, cutting off stem sections with the length of 1.0-1.5 cm at two ends, inoculating the stem sections onto a primary culture medium JG1, culturing in full darkness for 8-10 days after inoculation, moving to illumination culture after germination, wherein the illumination time is 10-12h/d, and the light source is natural scattered light;
(4) subculturing: after the explant cultured by the primary generation sprouts, lateral buds form cluster buds, the cluster buds are cultured for 15-20 days, healthy and strong aseptic buds are selected and cut and transferred to a subculture medium JG2, the illumination time is 10-12h/d, the light source is natural scattered light, and when the illumination is insufficient in rainy days, the cluster buds are supplemented by an artificial light source; culturing for 18-22 days under the illumination intensity of 2000-;
(5) rooting culture: cutting strong buds from the subculture seedlings expanded and propagated in the step (4), inserting the buds into a rooting culture medium JG3 for culture, wherein the illumination intensity of the cultured buds in 7-9 days is 1000Lux, and when short roots emerge from the cut, the illumination is increased to 3000Lux, and the culture temperature is 26-35 ℃; culturing for 25-35 days, when the root length is 1.5-2.0cm and the seedling height is 2.5-3.0cm, hardening the seedling;
(6) hardening seedlings: moving the pre-transplanted rooting bottle seedlings together with the bottles to a greenhouse, enhancing the illumination to be more than 5000Lux, improving the photosynthesis of the seedlings, enhancing the autotrophic ability and promoting the lignification of the seedlings; 3-5 days before transplanting, uncovering the cap of the rooting bottle, reducing the humidity in the bottle, enhancing the adaptability of the nursery stock to the natural environment, and simultaneously pouring 30-50ml of purified water or cold boiled water into the bottle to prevent the culture medium from drying and hardening;
(7) transplanting: taking out the hardened rooting bottle seedling, washing off the culture medium adhered to the root, transplanting to a yellow core vermiculite matrix seedling culture cup or a light matrix seedling culture bag sterilized by potassium permanganate solution for culture, performing management and protection work such as shading, moisture preservation, pest control and the like, and culturing for 20-25 days;
(8) strong seedling culture: grading according to height and ground diameter of seedling, transferring to seedling raising tray with larger seedling spacing for culturing when seedling grows to 15-20cm and ground diameter is 2.4-2.7mm, wherein seedling density is 625 plants/m2The concentration is reduced to 356 strains/m2(ii) a Keeping the plantlets with the height of 12-15cm and the ground diameter of 2.1-2.3mm in the original seedling raising tray, enlarging the illumination space, timely eliminating a small amount of plantlets with weak growth vigor, and culturing for 40-60 days under the full illumination natural state, and taking out the plantlets for forestation when the plantlets are 25-30cm high;
in the tissue culture and rapid propagation method step (3) of the triploid eucalyptus, namely the first-generation eucalyptus globulus, the primary culture medium JG1 comprises the following components: based on MS culture medium, the improvement is to adjust the mass ratio of the content of macroelements of nitrogen, phosphorus and potassium to be 8:1:13, wherein KH in macroelements2PO4Adjusting the concentration to 270mg/L to form a special basal culture medium suitable for the growth of the 'Jinggui No. one eucalyptus', and adding N61.0mg/L of benzyladenine (6-BA), 0.5mg/L of KT, 0.5mg/L of NAA, 2.0mg/L of vitamin C and 7.0mg/L of riboflavin, wherein 30g/L of commercially available high-quality white sugar is used as a carbon source, 3.75g/L of agar powder is used as a support, and the pH value is adjusted to be 5.8-6.0, so that the primary culture medium JG1 is obtained;
in the tissue culture and rapid propagation method step (4) of the triploid eucalyptus, namely the first Jinggui eucalyptus, the subculture medium JG2 is as follows: on the basis of JG1, the content of nitrogen, phosphorus and potassium elements in the major elements is improved, the content of the nitrogen, phosphorus and potassium elements is respectively improved by 17.3 percent, 10.0 percent and 19.0 percent in percentage by mass, and a special basic culture medium suitable for the subculture multiplication of triploid eucalyptus, namely 'Jinggui No. one eucalyptus'; then adding N6Benzyladenine (6-BA)0.5mg/L + NAA0.2mg/L + vitamin C2.0 mg/L + riboflavin 7.0 mg/L; using 30g/L of commercially available high-quality white sugar as a carbon source, using 3.75g/L of agar powder as a support, and adjusting the pH value to 5.8-6.0 to obtain a subculture medium JG 2;
in the step (5) of the tissue culture and rapid propagation method of the triploid eucalyptus, namely the first Jinggui eucalyptus, the rooting culture medium JG3 is as follows: 1/3MS culture medium is combined with H culture medium, biotin 0.05mg/L and nicotinic acid 0.5mg/L are added into H culture medium, and NH ions for inhibiting rooting are removed4 +To form a special rooting basic culture medium for' Jinggui No. one eucalyptusAdding plant growth regulator ABT11.2mg/L + IBA0.2mg/L + IAA0.1mg/L + 1.2% amino-oligosaccharin, using commercial white sugar 15g/L as carbon source and agar powder 4.0g/L as support, adjusting pH value to 6.0-6.2, and obtaining the rooting culture medium JG 3.
Preferably, the mass concentration of the benzalkonium bromide liquid in the step (2) is 0.1%; the alcohol with the volume concentration of 75% is soaked for 5-8 s; the mass concentration of the sodium hypochlorite solution is 2%, and the sodium hypochlorite solution is disinfected for 20 minutes; the mercuric chloride solution has the mass concentration of 0.1 percent and is disinfected for 3 minutes.
Preferably, the culture in step (3) is performed in full darkness for 8-10 days, and after germination, the culture is performed under the condition that the illumination intensity is 1000Lux, and the culture temperature is 26 +/-2 ℃.
Preferably, the subculture temperature in step (4) is 26. + -. 2 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the hormone KT in the primary culture medium of the invention has weaker cell division promoting effect than 6-BA, is easy to decompose when exposed to light, can promote the internode elongation of the bud seedling and the thickening of the stem, is coordinated with the 6-BA for use, is beneficial to the starting of aseptic bud germination and enhances the seedling quality; the vitamin C and the riboflavin mainly play a role in resisting oxidation, preventing browning and promoting the growth of sprouts; the plant growth regulator ABT in the rooting culture medium1The plant growth regulator can promote the synthesis of biological molecules, induce the morphogenesis of adventitious roots or adventitious buds of plants, regulate the metabolic action strength of the plants and improve the rooting rate of tissue culture seedlings by strengthening and regulating the content of endogenous hormones and the activity of important enzymes of the plants; IBA can promote the growth of main roots of plants and improve the germination rate and survival rate; IAA is auxin and can promote plant rooting and high growth, and is generally denatured and inactivated after being exposed to light or heated, but has better effect in practical application; the amino-oligosaccharin has the functions of improving the stress resistance and bacteriostasis of the bud seedlings, is beneficial to reducing the pollution of mixed bacteria when being used in a rooting culture medium, and can effectively improve the rooting rate and the seedling quality by combining the multiple rooting promoters; the compound is most suitable for a new variety of triploid eucalyptus, namely the Jinggui eucalyptus I, is determined after being debugged and screened for many times, and has stable and consistent effects after being used.
Drawings
FIG. 1 is explant raw material culture of the present invention.
FIG. 2 is the induction of sterile shoots by explants of the invention.
FIG. 3 shows the proliferation of sterile shoots according to the invention.
FIG. 4 shows the subculture of the present invention.
FIG. 5 shows rooting culture according to the present invention.
FIG. 6 shows the transplanting of the plantlets in step (7) of the present invention.
FIG. 7 shows the strong seedling culture in step (8) of the present invention.
Detailed Description
The following detailed description is to be read in connection with the accompanying drawings, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were all commercially available unless otherwise specified. The compounds in other media of the invention may be retrieved from chemical dictionaries or textbooks of crop cultivation to their names and effects.
Example 1
A tissue culture rapid propagation method of a triploid eucalyptus new variety 'Jinggui No. one eucalyptus', comprises the following specific operation steps:
(1) cultivating high-quality explant raw materials: cutting off a tip section of a triploid eucalyptus 'Jinggui No. one eucalyptus' plant, promoting germination, moving the triploid eucalyptus 'Jinggui No. one eucalyptus' plant to a greenhouse for culture, manually controlling illumination, temperature and humidity after the germination grows out, taking pest control measures, when the germination grows to 6 cm and the upper bud eye is full but no lateral bud is generated, picking a lower bud strip in the morning of sunny days, removing leaves, obtaining an explant material, and returning the explant material to a laboratory for later use;
(2) explant disinfection: cleaning the explant material obtained in the step (1) with purified water for 3 times, soaking the explant material in 0.1% by mass of benzalkonium bromide solution for 2 minutes, cleaning with purified water for 3 times, pre-treating, transferring to an ultra-clean workbench, shearing a semi-lignified cut segment which grows to 1.5cm and is reserved with 1 latent bud under aseptic condition, soaking the cut segment in 75% by volume of alcohol for 5 seconds, cleaning with sterile water for 3 times, draining off sodium hypochlorite, sterilizing in 2% by mass of sodium hypochlorite solution for 20 minutes, cleaning with sterile water for 3 times, sucking surface moisture with sterile filter paper, placing in 0.1% by mass of mercuric chloride solution, sterilizing for 3 minutes and shaking gently to make the surface of the explant fully contact with the mercuric chloride solution, then pouring off the mercuric chloride solution, cleaning the explant with sterile water for 5 times, and each time for 8 minutes;
(3) primary culture: absorbing water on the surface of the explant sterilized in the step (2) by using sterile filter paper, cutting off stem sections with two ends remaining 1.0 cm long, inoculating the stem sections onto a primary culture medium JG1, culturing in full darkness for 8 days after inoculation, moving to the condition that the illumination intensity is 1000Lux after germination, culturing at the temperature of 26 +/-2 ℃, the illumination time is 10-12h/d, and the light source is natural scattered light;
(4) subculturing: after the explant cultured by the primary generation sprouts, lateral buds form cluster buds, the cluster buds are cultured for 15 days, strong and pollution-free sterile buds are selected, the cut sterile buds are transferred to a subculture medium JG2, the culture temperature is 26 +/-2 ℃, the illumination time is 10-12h/d, the light source is natural scattered light, and when the illumination is insufficient in rainy days, the cluster buds are supplemented by an artificial light source; culturing for 18 days under the illumination intensity of 2000Lux, cutting sterile buds growing to 2.0cm into sections with stem nodes, cutting the bud cluster into small bud clusters, transferring the bud clusters into a newly prepared subculture medium to promote the multiplication of the clustered buds and the elongation of single buds, and then repeatedly carrying out subculture in such a way that the number of the sterile buds is continuously increased and the subculture multiplication coefficient is 4.5;
(5) rooting culture: cutting strong buds from the subculture seedlings expanded and propagated in the step (4), inserting the buds into a rooting culture medium JG3 for culture, wherein the illumination intensity of the cultured buds in 7-9 days is 1000Lux, when short roots emerge from the cut, the illumination is increased to 3000Lux, the culture temperature is 26-35 ℃, the buds are cultured for 30 days, the roots are 1.5cm long, and the seedlings are hardened when the seedlings are 2.5cm high;
(6) hardening seedlings: moving the pre-transplanted rooting bottle seedlings together with the bottles to a greenhouse, enhancing the illumination to be more than 5000Lux, improving the photosynthesis of the seedlings, enhancing the autotrophic ability and promoting the lignification of the seedlings; 3 days before transplanting, uncovering the cap of the rooting bottle, reducing the humidity in the bottle, enhancing the adaptability of the nursery stock to the natural environment, and simultaneously pouring 30ml of purified water into the bottle to prevent the culture medium from drying and hardening;
(7) transplanting: taking out the hardened rooting bottle seedlings, washing off the culture medium adhered to the roots, transplanting the seedlings into a yellow core soil vermiculite matrix seedling culture cup sterilized by potassium permanganate solution for culture, building a small arched shed, covering a shading net with the transmittance of 70 percent, shading well, keeping the humidity to be more than 90 percent, using a broad-spectrum bactericide to prevent and control the plant diseases and insect pests, frequently observing the situation of the plant diseases and insect pests, eliminating the plant diseases and insect pests in time when the plant diseases and insect pests occur, performing management and protection work such as shading, moisturizing, plant disease and insect pest prevention and control, culturing for 20 days, and gradually growing the seedlings;
(8) strong seedling culture: grading according to the height and ground diameter of the seedling, transferring to seedling raising tray with larger seedling spacing for culturing when the seedling grows to 15-20cm in height and the ground diameter is 2.4-2.7mm, and the seedling density is 625 plants/m2Reduced to 356 strains/m2(ii) a Keeping the plantlets with the height of 12-15cm and the ground diameter of 2.1-2.3mm in the original seedling raising tray, timely eliminating a small amount of plantlets with weak growth vigor, culturing for 45 days in a full-light natural state, and taking out of the nursery for forestation when the plantlets are 25cm high;
in the step (3) of the tissue culture and rapid propagation method of the triploid eucalyptus 'Jinggui No. I eucalyptus', a primary culture medium JG1 comprises the following components: based on MS culture medium, the improvement is to adjust the mass ratio of the content of macroelements of nitrogen, phosphorus and potassium to be 8:1:13, wherein KH is2PO4Adjusting the concentration to 270mg/L to form a special basal culture medium suitable for the growth of the 'Jinggui No. one eucalyptus', and adding N61.0mg/L of benzyladenine (6-BA), 0.5mg/L of KT, 0.5mg/L of NAA, 2.0mg/L of vitamin C and 7.0mg/L of riboflavin; using commercially available high-quality white sugar 30g/L as a carbon source, using agar powder 3.75g/L as a support, and adjusting the pH value to 5.8-6.0 to obtain a primary culture medium JG 1;
in the tissue culture and rapid propagation method step (4) of the triploid eucalyptus, namely the first Jinggui eucalyptus, the subculture medium JG2 is as follows: on the basis of JG1, the content of nitrogen, phosphorus and potassium elements in the major elements is improved, the content of the nitrogen, phosphorus and potassium elements is respectively improved by 17.3 percent, 10.0 percent and 19.0 percent in percentage by mass, and a special basic culture medium suitable for the subculture multiplication of triploid eucalyptus, namely 'Jinggui No. one eucalyptus'; then adding N6Benzyladenine (6-BA)0.5mg/L + NAA0.2mg/L + vitamin C2.0 mg/L + riboflavin 7.0 mg/L; using commercial high-quality white sugar 30g/L as carbon source and agar powder 3.75g/L as support, adjusting pH value5.8-6.0, namely a subculture medium JG 2;
in the step (5) of the tissue culture and rapid propagation method of the triploid eucalyptus, namely the first Jinggui eucalyptus, the rooting culture medium JG3 comprises the following components: 1/3 the MS culture medium is combined with the H culture medium, biotin 0.05mg/L and nicotinic acid 0.5mg/L are added in the H culture medium, and NH which inhibits rooting is removed4 +Forming a special rooting basal culture medium for 'Jinggui No. one eucalyptus', and adding a plant growth regulator ABT11.2mg/L + IBA0.2mg/L + IAA0.1mg/L + 1.2% amino-oligosaccharin, using commercial white sugar 15g/L as carbon source and agar powder 4.0g/L as support, and adjusting pH value to 6.0-6.2, namely the rooting culture medium JG 3.
Example 2
A tissue culture rapid propagation method of a triploid eucalyptus new variety 'Jinggui No. one eucalyptus', comprises the following specific operation steps:
(1) cultivating high-quality explant raw materials: cutting off a tip section of a triploid eucalyptus 'Jinggui No. one eucalyptus' plant, promoting germination, moving the triploid eucalyptus 'Jinggui No. one eucalyptus' plant to a greenhouse for culture, manually controlling illumination, temperature and humidity after the germination grows out, taking pest control measures, when the germination grows to 8cm and the upper bud eye is full but the lateral bud is not germinated, picking a lower bud strip in the morning on sunny days, removing leaves, obtaining an explant material, and taking the explant material back to a laboratory for later use;
(2) explant disinfection: cleaning the explant material obtained in the step (1) with purified water for 3 times, soaking for 2 minutes with 0.1% of benzalkonium bromide solution, cleaning for 3 times with purified water, moving to an ultra-clean workbench after pretreatment, shearing a semi-lignified cut segment which grows to 2.0cm and is reserved with 2 latent buds under aseptic condition, soaking for 8 seconds with 75% of alcohol by volume concentration, cleaning for 3 times with sterile water, draining off sodium hypochlorite, placing in 2% of solution by mass concentration for disinfection for 20 minutes, cleaning for 3 times with sterile water, sucking surface moisture with sterile filter paper, placing in 0.1% of mercuric chloride solution by mass concentration, disinfecting for 3 minutes and shaking gently to allow the surface of the explant to be in full contact with the mercuric chloride solution, then pouring off the mercuric chloride solution, cleaning the explant with sterile water for 6 times, and 5 minutes each time;
(3) primary culture: absorbing water on the surface of the explant sterilized in the step (2) by using sterile filter paper, cutting off stem sections with two ends remaining 1.5cm long, inoculating the stem sections onto a primary culture medium JG1, culturing in full darkness for 10 days after inoculation, transferring to the condition that the illumination intensity is 1000Lux after germination, culturing at the temperature of 26 +/-2 ℃, the illumination time is 10-12h/d, and the light source is natural scattered light;
(4) subculturing: after the explant cultured by the primary generation sprouts, lateral buds form cluster buds, the cluster buds are cultured for 20 days, healthy and strong aseptic buds are selected and cut and then transferred to a subculture medium JG2, the culture temperature is 26 +/-2 ℃, the illumination time is 10-12h/d, the light source is natural scattered light, and when the illumination is insufficient in rainy days, the cluster buds are supplemented by an artificial light source; culturing for 22 days under the illumination intensity of 2000Lux, cutting sterile buds growing to 2.5cm into sections with stem nodes, cutting the bud cluster into small bud clusters, transferring the bud clusters into a newly prepared subculture medium to promote the proliferation of the clustered buds and the elongation of single buds, and then repeatedly subculturing in such a way, wherein the number of the sterile buds is continuously increased, and the subculture multiplication coefficient is 4.5;
(5) rooting culture: cutting strong buds of the subculture seedlings expanded and propagated in the step (4), inserting the strong buds into a rooting culture medium JG3 for culture, wherein the illumination intensity of the cultured buds in 7-9 days is 1000Lux, when short roots emerge from a cut, the illumination is increased to 3000Lux, the culture temperature is 35 ℃, the seedlings are cultured for 25 days, the roots are 2.0cm long, and the seedlings are hardened when the seedlings are 3.0cm high;
(6) hardening seedlings: moving the pre-transplanted rooting bottle seedlings together with the bottles to a greenhouse, enhancing the illumination to be more than 5000Lux, improving the photosynthesis of the seedlings, enhancing the autotrophic ability and promoting the lignification of the seedlings; 3 days before transplanting, uncovering the cap of the rooting bottle, reducing the humidity in the bottle, enhancing the adaptability of the nursery stock to the natural environment, and simultaneously pouring 50ml of cold boiled water into the bottle to prevent the culture medium from drying and hardening;
(7) transplanting: taking out the acclimatized rooting bottle seedlings, washing off the culture medium adhered to the roots, transplanting the seedlings to a yellow-core vermiculite matrix seedling culture cup or a light matrix seedling culture bag disinfected by potassium permanganate solution for culture, building a small arched shed, covering a shading net with the transmittance of 70 percent, shading, keeping the humidity over 90 percent, using a broad-spectrum bactericide for preventing and treating plant diseases and insect pests, frequently observing the condition of the plant diseases and insect pests, eliminating the plant diseases and insect pests in time when the plant diseases and insect pests appear, well shading, preserving the moisture, performing management and protection work for preventing and treating the plant diseases and insect pests and the like, culturing for 25 days, and gradually growing the seedlings;
(8) strong seedling culture: grading according to the height and ground diameter of the seedling, transferring to seedling raising tray with larger seedling spacing for culturing when the seedling grows to 15-20cm in height and the ground diameter is 2.4-2.7mm, and the seedling density is 625 plants/m2Reduced to 356 strains/m2The illumination space is enlarged; keeping the seedlings with the height of 12-15cm and the ground diameter of 2.1-2.3mm in the original seedling raising tray; timely eliminating a small amount of weak seedlings, culturing for 45 days in a full-light natural state, and taking out of nursery for forestation when the seedlings are 25cm high;
in the step (3) of the tissue culture and rapid propagation method of the triploid eucalyptus 'Jinggui No. I eucalyptus', a primary culture medium JG1 comprises the following components: based on MS culture medium, the improvement is to adjust the mass ratio of the content of macroelements of nitrogen, phosphorus and potassium to be 8:1:13, wherein KH in macroelements2PO4Adjusting the concentration to 270mg/L to form a special basal culture medium suitable for the growth of the 'Jinggui No. one eucalyptus', and adding N61.0mg/L of benzyladenine (6-BA), 0.5mg/L of KT, 0.5mg/L of NAA, 2.0mg/L of vitamin C and 7.0mg/L of riboflavin; using commercially available high-quality white sugar 30g/L as a carbon source, using agar powder 3.75g/L as a support, and adjusting the pH value to 5.8-6.0 to obtain a primary culture medium JG 1;
in the tissue culture and rapid propagation method step (4) of the triploid eucalyptus, namely the first Jinggui eucalyptus, the subculture medium JG2 is as follows: on the basis of JG1, the content of nitrogen, phosphorus and potassium elements in the major elements is improved, the content of the nitrogen, phosphorus and potassium elements is respectively improved by 17.3 percent, 10.0 percent and 19.0 percent in percentage by mass, and a special basic culture medium suitable for the subculture multiplication of triploid eucalyptus, namely 'Jinggui No. one eucalyptus'; then adding N6Benzyladenine (6-BA)0.5mg/L + NAA0.2mg/L + vitamin C2.0 mg/L + riboflavin 7.0 mg/L; using 30g/L of commercially available high-quality white sugar as a carbon source, using 3.75g/L of agar powder as a support, and adjusting the pH value to 5.8-6.0 to obtain a subculture medium JG 2;
in the step (5) of the tissue culture and rapid propagation method of the triploid eucalyptus, namely the first Jinggui eucalyptus, the rooting culture medium JG3 comprises the following components: 1/3MS culture medium is combined with H culture medium, biotin 0.05mg/L and nicotinic acid 0.5mg/L in H culture medium are added, and the process is omittedIonic NH for inhibiting rooting4 +Forming a special rooting basal culture medium for 'Jinggui No. one eucalyptus', and adding a plant growth regulator ABT11.2mg/L + IBA0.2mg/L + IAA0.1mg/L + 1.2% amino-oligosaccharin, using commercial white sugar 15g/L as carbon source and agar powder 4.0g/L as support, and adjusting pH value to 6.0-6.2, namely the rooting culture medium JG 3.
Example 3
A tissue culture rapid propagation method of a novel triploid eucalyptus variety 'Jinggui eucalyptus I', comprises the following specific operation steps:
(1) cultivating high-quality explant raw materials: cutting off a tip section of a triploid eucalyptus 'Jinggui No. one eucalyptus' plant, promoting germination, moving the triploid eucalyptus 'Jinggui No. one eucalyptus' plant to a greenhouse for culture, manually controlling illumination, temperature and humidity after the germination grows out, taking pest control measures, when the germination grows to 7 cm, the upper bud eye is full but the lateral bud is not germinated, picking a lower bud strip in the morning of sunny days, removing leaves, obtaining an explant material, and taking the explant material back to a laboratory for later use;
(2) explant disinfection: cleaning the explant material obtained in the step (1) with purified water for 3 times, soaking for 2 minutes with 0.1% of benzalkonium bromide solution, cleaning for 3 times with purified water, moving to an ultra-clean workbench after pretreatment, shearing a semi-lignified cut segment which grows to 1.8cm and is reserved with 2 latent buds under an aseptic condition, soaking for 5-8 seconds with 75% of alcohol by volume concentration, cleaning for 3 times with sterile water, draining off water, placing in 2% of sodium hypochlorite solution by mass concentration for disinfection for 20 minutes, cleaning for 3 times with sterile water, sucking surface moisture with sterile filter paper, placing in 0.1% of mercuric chloride solution by mass concentration, disinfecting for 3 minutes and shaking slightly to allow the surface of the explant to be in full contact with the mercuric chloride solution, then pouring off the mercuric chloride solution, cleaning the explant with sterile water for 6 times, and 7 minutes each time;
(3) primary culture: the explants sterilized in the step (2) are dried by using sterile filter paper, stem segments with the length of 1.0-1.5 cm left at two ends are cut off and inoculated on a primary culture medium JG1, the inoculated explants are cultured in full darkness for 9 days, the explants are cultured after germination under the condition that the illumination intensity is 1000Lux, the culture temperature is 26 +/-2 ℃, the illumination time is 10-12h/d, and the light source is natural scattered light;
(4) subculturing: after the explant cultured by the primary generation sprouts, lateral buds form cluster buds, the cluster buds are cultured for 15-20 days, healthy and strong aseptic buds are selected and cut and then transferred onto a subculture medium JG2, the culture temperature is 26 +/-2 ℃, the illumination time is 10-12h/d, the light source is natural scattered light, and when the illumination is insufficient in rainy days, the cluster buds are supplemented by an artificial light source; culturing for 20 days under the illumination intensity of 2500Lux, cutting into 2.3cm sterile buds, cutting into sections with stem nodes, cutting the bud cluster into small bud clusters, transferring into a newly prepared subculture medium to promote the proliferation of the clustered buds and the elongation of single buds, and then repeatedly subculturing in such a way, wherein the number of the sterile buds is continuously increased, and the subculture multiplication coefficient is 4.5;
(5) rooting culture: cutting strong buds from the subculture seedlings expanded and propagated in the step (4), inserting the buds into a rooting culture medium JG3 for culture, wherein the illumination intensity of the cultured buds in 7-9 days is 1000Lux, when short roots emerge from the cut, the illumination is increased to 3000Lux, the culture temperature is 30-32 ℃, the seedlings are cultured for 32 days, the roots are 1.8cm long, and the seedlings are hardened when the seedlings are 3.0cm high;
(6) hardening seedlings: moving the pre-transplanted rooting bottle seedlings together with the bottles to a greenhouse, enhancing the illumination to 5000Lux, improving the photosynthesis of the seedlings, enhancing the autotrophic capacity and promoting the lignification of the seedlings; 3-5 days before transplanting, uncovering the cap of the rooting bottle, reducing the humidity in the bottle, enhancing the adaptability of the nursery stock to the natural environment, and simultaneously pouring 40ml of purified water into the bottle to prevent the culture medium from drying and hardening;
(7) transplanting: taking out the acclimatized rooting bottle seedlings, washing off the culture medium adhered to the roots, transplanting the seedlings to a yellow-core vermiculite matrix seedling culture cup or a light matrix seedling culture bag disinfected by potassium permanganate solution for culture, building a small arched shed, covering a shading net with the transmittance of 70 percent, shading, keeping the humidity over 90 percent, using a broad-spectrum bactericide for preventing and treating plant diseases and insect pests, frequently observing the condition of the plant diseases and insect pests, eliminating the plant diseases and insect pests in time when the plant diseases and insect pests appear, well shading, preserving the moisture, performing management and protection work on the plant diseases and insect pests and the like, culturing for 20-25 days, and gradually growing the seedlings;
(8) strong seedling culture: grading according to the height and ground diameter of the seedling, transferring to seedling raising tray with larger seedling spacing when the seedling grows to 15-20cm in height and the ground diameter is 2.4-2.7mmRaising the seedlings with the density of 625 plants/m2Reduced to 356 strains/m2The illumination space is enlarged; keeping the seedlings with the height of 12-15cm and the ground diameter of 2.1-2.3mm in the original seedling raising tray; timely eliminating a small amount of weak seedlings, culturing for 50 days in a full-light natural state, and taking out of nursery for forestation when the height of the seedlings is 25-30 cm;
in the step (3) of the tissue culture and rapid propagation method of the triploid eucalyptus 'Jinggui No. I eucalyptus', a primary culture medium JG1 comprises the following components: based on MS culture medium, the improvement is to adjust the mass ratio of the content of macroelements of nitrogen, phosphorus and potassium to be 8:1:13, wherein KH in macroelements2PO4Adjusting the concentration to 270mg/L to form a special basal culture medium suitable for the growth of' Jinggui No. one eucalyptus61.0mg/L of benzyladenine (6-BA), 0.5mg/L of KT, 0.5mg/L of NAA, 2.0mg/L of vitamin C and 7.0mg/L of riboflavin; taking commercially available high-quality white sugar 30g/L as a carbon source, taking agar powder 3.75g/L as a support, and adjusting the pH value to 5.8-6.0 to obtain a primary culture medium JG 1;
in the tissue culture and rapid propagation method step (4) of the triploid eucalyptus, namely the first Jinggui eucalyptus, the subculture medium JG2 is as follows: on the basis of JG1, the content of nitrogen, phosphorus and potassium elements in the macroelements is increased, the content of nitrogen, phosphorus and potassium elements is respectively increased by 17.3 percent, 10.0 percent and 19.0 percent in mass percentage, and a special basic culture medium suitable for the subculture multiplication of triploid eucalyptus 'Jinggui No. one eucalyptus' is formed; then adding N6Benzyladenine (6-BA)0.5mg/L + NAA0.2mg/L + vitamin C2.0 mg/L + riboflavin 7.0 mg/L; using 30g/L of commercially available high-quality white sugar as a carbon source, using 3.75g/L of agar powder as a support, and adjusting the pH value to 5.8-6.0 to obtain a subculture medium JG 2;
in the step (5) of the tissue culture and rapid propagation method of the triploid eucalyptus, namely the first Jinggui eucalyptus, the rooting culture medium JG3 comprises the following components: 1/3MS culture medium is combined with H culture medium, biotin 0.05mg/L and nicotinic acid 0.5mg/L are added into H culture medium, and NH ions for inhibiting rooting are removed4 +Forming a special rooting basal culture medium for 'Jinggui No. one eucalyptus', and adding a plant growth regulator ABT11.2mg/L + IBA0.2mg/L + IAA0.1mg/L + 1.2% amino-oligosaccharin, wherein commercial white sugar 15g/L is used as a carbon source, agar powder 4.0g/L is used as a support, and the pH value is adjusted to 6.0 to 6.2, namely a rooting culture medium JG 3.
The following table shows the tissue culture and breeding results of the novel triploid eucalyptus variety' Jinggui eucalyptus I:
the above table shows that the tissue culture rapid propagation method of the new triploid eucalyptus variety 'Jinggui eucalyptus I' can effectively induce the explant to sprout, has high successive multiplication coefficient and good seedling quality, can produce the seedlings in large scale and successfully realize the tissue culture rapid propagation of the triploid eucalyptus, and the rooting rate after the inoculation and rooting exceeds 95 percent and the transplanting survival rate is more than 90 percent.
The medicament used in the disinfection process of the culture material is a disinfectant commonly used in medical treatment, and the benzalkonium bromide solution is used at low concentration in the pretreatment process of the culture material to inactivate part of fungi and prevent the fungi from growing on explants. The alcohol with the volume concentration of 75% has the sterilization effect, but can not be sterilized completely, and the air on the material is eliminated by mainly utilizing the wetting effect of the alcohol, so that other disinfectants can be sterilized in a full-covering manner. The alcohol has strong penetrating power and is easy to kill the material, so the disinfection time is required to be short. The sodium hypochlorite is sterilized by chlorine generated by decomposition, so that the sodium hypochlorite is easy to clean and remove, has no toxic or harmful effect on materials, and has good effect, but is still difficult to thoroughly sterilize in practical application. Mercuric chloride is extremely toxic and is thoroughly sterilized, and the problems are that the residual mercury is difficult to remove after sterilization, the mercuric chloride has toxic action on materials and the performance is late. Therefore, it is necessary to shorten the sterilization time and clean it thoroughly when using mercuric chloride. The disinfectants are used in combination for multiple times of disinfection and layer-by-layer sterilization, can kill germs on the material, and do not damage or only slightly damage the material.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (4)
1. A tissue culture rapid propagation method of a triploid eucalyptus new variety 'Jinggui No. one eucalyptus', is characterized by comprising the following operation steps:
(1) cultivating high-quality explant raw materials: cutting off a tip section of a triploid eucalyptus 'Jinggui No. one eucalyptus' plant, promoting germination, transferring to a greenhouse for culture, manually and conventionally controlling illumination, temperature and humidity after the germination grows out, taking pest control measures, collecting the bud when the bud grows to 6-8 cm, the upper bud eye is plump but the lateral bud is not grown, and removing leaves to obtain an explant material for later use;
(2) explant disinfection: cleaning the explant material obtained in the step (1), soaking and cleaning the explant material by using benzalkonium bromide solution, shearing the explant material to grow into 1.5-2.0cm under the aseptic condition, reserving semi-lignified cuttings with 1-2 latent buds, soaking the semi-lignified cuttings in alcohol, cleaning, draining water liquid, placing the semi-lignified cuttings in a sodium hypochlorite solution for disinfection, cleaning, sucking surface water, placing the semi-lignified cuttings in a mercuric chloride solution, fully contacting the surface of the explant with the mercuric chloride solution, pouring off the mercuric chloride solution, and cleaning the explant for 5-6 times;
(3) primary culture: sucking water on the surface of the explant sterilized in the step (2), cutting off stem sections with the length of 1.0-1.5 cm at two ends, inoculating the stem sections onto a primary culture medium JG1, culturing in full darkness for 8-10 days after inoculation, and moving to illumination culture after germination for 10-12 h/d;
(4) subculturing: after the explant cultured by the primary generation sprouts, lateral buds form cluster buds, the cluster buds are cultured for 15 to 20 days, strong and pollution-free sterile buds are selected, cut and transferred to a subculture medium JG2, and the illumination time is 10 to 12 h/d; culturing for 18-22 days under the illumination intensity of 2000-;
(5) rooting culture: cutting strong buds from the subculture seedlings expanded and propagated in the step (4), inserting the buds into a rooting culture medium JG3 for culture, wherein the illumination intensity of the cultured buds in 7-9 days is 1000Lux, and when short roots emerge from the cut, the illumination is increased to 3000Lux, and the culture temperature is 26-35 ℃; culturing for 25-35 days, when the root length is 1.5-2.0cm and the seedling height is 2.5-3.0cm, hardening the seedling;
(6) hardening seedlings: the pre-transplanted seedlings are illuminated to above 5000Lux, the photosynthesis of the seedlings is improved, the autotrophic capacity is enhanced, and the lignification of the seedlings is promoted; 3-5 days before transplanting, reducing the humidity in the bottle, enhancing the adaptability of the nursery stock to the natural environment, and simultaneously filling water into the bottle;
(7) transplanting: taking out the hardened rooting bottle seedling, transplanting to sterilized yellow-heart vermiculite soil stone matrix or light matrix, and culturing for 20-25 days;
(8) strong seedling culture: grading according to the height and ground diameter of the seedlings, and transferring the seedlings to a seedling raising tray with larger seedling spacing for culture when the seedlings grow to 15-20cm and the ground diameter is 2.4-2.7 mm; the density of the nursery stock is 625 plants/m2Reduced to 356 strains/m2Enlarging the illumination space, keeping the seedlings with the height of 12-15cm and the ground diameter of 2.1-2.3mm in the original seedling raising tray; timely eliminating a small amount of weak seedlings, culturing for 40-60 days in a full-light natural state, and taking out of nursery for forestation when the seedlings are 25-30cm high;
in the tissue culture and rapid propagation method step (3) of the triploid eucalyptus, namely the first-generation eucalyptus globulus, the primary culture medium JG1 comprises the following components: based on MS culture medium, the improvement is to adjust the mass ratio of the content of macroelements of nitrogen, phosphorus and potassium to be 8:1:13, wherein KH in macroelements2PO4Adjusting the concentration to 270mg/L to form a special basal culture medium suitable for the growth of the 'Jinggui No. one eucalyptus', and adding N61.0mg/L of benzyladenine (6-BA), 0.5mg/L of KT, 0.5mg/L of NAA0.5mg/L of vitamin C, 2.0mg/L of vitamin C and 7.0mg/L of riboflavin, wherein 30g/L of white sugar is used as a carbon source, 3.75g/L of agar powder is used as a support, and the pH value is adjusted to be 5.8-6.0, so that the primary culture medium JG1 is obtained;
the tissue culture and rapid propagation method of the triploid eucalyptus' Jinggui eucalyptus I4) In (3), the subculture medium JG2 is: on the basis of JG1, the content of nitrogen, phosphorus and potassium elements in the major elements is improved, the content of the nitrogen, phosphorus and potassium elements is respectively improved by 17.3 percent, 10.0 percent and 19.0 percent in percentage by mass, and a special basic culture medium suitable for the subculture multiplication of triploid eucalyptus, namely 'Jinggui No. one eucalyptus'; then adding N6Benzyl adenine (6-BA)0.5mg/L + NAA0.2mg/L + vitamin C2.0 mg/L + riboflavin 7.0 mg/L; taking 30g/L of white sugar as a carbon source and 3.75g/L of agar powder as a support, and adjusting the pH value to 5.8-6.0 to obtain a subculture medium JG 2;
in the step (5) of the tissue culture and rapid propagation method of the triploid eucalyptus, namely the first Jinggui eucalyptus, the rooting culture medium JG3 is as follows: 1/3 the MS culture medium is combined with the H culture medium, biotin 0.05mg/L and nicotinic acid 0.5mg/L are added in the H culture medium, and NH which inhibits rooting is removed4 +Forming a special rooting basal culture medium for 'Jinggui No. one eucalyptus', and adding a plant growth regulator ABT11.2mg/L + IBA0.2mg/L + IAA0.1mg/L + 1.2% amino-oligosaccharin, taking 15g/L white sugar as a carbon source and 4.0g/L agar powder as a support, and adjusting the pH value to 6.0-6.2 to obtain the rooting culture medium JG 3.
2. The tissue culture rapid propagation method of the new triploid eucalyptus variety 'Jinggui eucalyptus I', according to claim 1, is characterized in that: the mass concentration of the new benzalkonium bromide liquid in the step (2) is 0.1%; the alcohol with the volume concentration of 75% is soaked for 5-8 s; the mass concentration of the sodium hypochlorite solution is 2%, and the sodium hypochlorite solution is disinfected for 20 minutes; the mercuric chloride solution has the mass concentration of 0.1 percent and is disinfected for 3 minutes.
3. The tissue culture rapid propagation method of the new triploid eucalyptus variety 'Jinggui eucalyptus I', according to claim 1, is characterized in that: culturing in full dark for 8-10 days in the step (3), and culturing under the condition that the illumination intensity is 1000Lux after germination, wherein the culture temperature is 26 +/-2 ℃.
4. The tissue culture rapid propagation method of the novel triploid eucalyptus variety 'Jinggui eucalyptus I', according to claim 1, is characterized in that: in the step (4), the subculture temperature is 26 +/-2 ℃.
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