CN115644060A - Eucalyptus grandis tissue culture method - Google Patents
Eucalyptus grandis tissue culture method Download PDFInfo
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Abstract
The invention discloses a tissue culture method of eucalyptus grandis, which comprises the following operation steps: (1) selecting an explant; (2) cleaning and disinfecting explants; (3) inoculating and starting culture; (4) propagation culture; (5) rooting culture; (6) Hardening and transplanting the seedlings until new leaves grow out of the tissue culture seedlings, and obtaining complete Eucalyptus grandis seedlings. The method systematically researches the influence of plant nutrient components on the growth of the cultured seedlings, comprehensively improves the components and the content of the culture medium, adopts a transverse inoculation mode during propagation culture, ensures that the propagated bud seedlings have more growth space, and overcomes the problem that the growth of the lateral buds is limited because the space between nodes of the eucalyptus grandis with large inflorescence is shorter in the prior art; the culture method is more suitable for the growth of tissue culture seedlings of the eucalyptus cloeziana, and the tissue culture seedlings of the eucalyptus cloeziana obtained by culture grow strongly and have high proliferation rate and rooting rate.
Description
Technical Field
The invention relates to the technical field of plant cell engineering, in particular to a tissue culture method of eucalyptus grandis.
Background
Eucalyptus grandis (Eucalyptus cloeziana F.Muell.) belongs to family Myrtaceae (Myrtaceae) genus Eucalyptus (Eucalyptus), a tall arbor. The tree has strong top end advantage, the trunk is tall, big and straight, the side branches are few and are easy to fall off, and the height of an adult tree can reach 35-45 meters. The eucalyptus grandis has a common crown size, dense leaves and considerable control capability in a site. The trees can bloom 7-8 years after being planted in the juvenile state, the flowering period is 3 years apart, and mature seeds begin to be produced after 9 years of the trees. The Eucalyptus robusta seeds have strong disease and pest resistance, high preservation rate and certain guarantee on wood quality. The growth speed is high, and particularly, the growth advantage in the later period is very obvious. The eucalyptus grandis is also called as Australia grandis, is straight in dry shape, yellow brown in wood, uniform in structure, clear in texture, high in hardness, durable and firm, and excellent in final water content and uniformity of the dried wood veneer, can be widely applied to furniture, decoration, buildings, pot trees and the like, and is a tree species with planting and cultivating values.
The Eucalyptus dahula is still in the incomplete mature stage in China, the seed sources are few, and the flowering and fruiting rates are low. And the eucalyptus grandis experimental forest is mainly cultured by a plurality of provenances, the tree species belongs to the heteroflorus arbors species, the hybridization is easy to occur among different species, the excellent characters cannot be stably inherited, and the differentiation of filial generation is obvious. Therefore, the Eucalyptus robusta is propagated by a tissue culture method, the randomness of Eucalyptus robusta breeding can be effectively solved, a stable propagation system is established, and the maximum benefit is obtained. Currently, there are research reports on tissue culture studies of eucalyptus closterium, and the tissue culture system of eucalyptus closterium has been studied by people such as tangqinglan (2006), tang regeneration (2006, 2018), dawn (2010), and zhouxiua (2018, 2019), and certain results have been obtained. However, the main problems are that the multiplication coefficient is low, the rooting rate is low, and the tissue culture seedlings are weak, so that the large-scale culture of the eucalyptus grandis still has limitations.
Disclosure of Invention
The invention aims at the problems of weak tissue culture seedling, low proliferation rate, low rooting rate and the like of eucalyptus grandis in tissue culture in the prior art, provides a tissue culture method of eucalyptus grandis, and aims to obtain a tissue culture method of eucalyptus grandis with strong growth, high proliferation rate and high rooting rate of tissue culture seedlings.
In order to realize the purpose, the technical scheme provided by the invention is as follows:
a tissue culture method of Eucalyptus grandis comprises the following steps:
(1) Selecting an explant: selecting bud of a big inflorescence eucalyptus cutting pile as an explant, selecting a new strong disease-free semi-lignified branch as the explant, and shearing the bud by using clean scissors and bringing the bud back to a laboratory;
(2) Cleaning and disinfecting explants: removing leaves of the stem segments of the explants obtained in the step (1), cleaning surface dust with clear water, soaking for 10 minutes with a washing powder solution, carefully scrubbing with a soft brush pen, particularly paying attention to gaps at branches, washing for more than 30 minutes with running water after scrubbing, transferring to an ultra-clean bench, rinsing twice with sterile water, soaking in alcohol for 15-30 seconds, washing for 2 times with sterile water, placing the explants in a mercuric chloride solution, adding one drop (about 0.05 mL) of Tween 80 (polysorbate-80), continuously shaking, soaking for 8-10 minutes, and washing for 5-6 times with sterile water, thus finishing cleaning and disinfection;
(3) Inoculation and start-up culture: cutting off a small amount of incisions at two ends of the explant subjected to disinfection in the step (2) to avoid serious wound browning, cutting the explant into a short stem section with at least one axillary bud from a long stem section, vertically inserting the short stem section into a starting culture medium, performing primary starting culture, wherein the starting culture medium is based on an improved B5 culture medium, then adding 0.1-0.2 mg/L6-benzyladenine (6-BA), 0.05-0.1 mg/L naphthylacetic acid (NAA) and 30g/L sucrose, the starting culture time is 30-40 d, culturing by using black cloth for the first 7-10 d, and then opening the black cloth to receive normal illumination;
(4) And (3) proliferation culture: carefully cutting off lateral buds germinated in the starting culture process in the step (3), transferring the lateral buds into a multiplication culture medium for multiplication culture, cutting off 4/5 of the whole leaf of each leaf before inoculation, reserving a small part of leaf and leaf stalk, flatly paving a short stem section on the surface of the multiplication culture medium, slightly pressing the stem section to enable about 1/2 of the short stem section to be immersed into the multiplication culture medium, enabling the newly-increased bud seedlings to grow upwards in an inoculation mode and have enough growth space, reserving 2 buds during inoculation, having higher multiplication success rate than that of single bud inoculation, adding 0.2-0.4 mg/L6-BA, 0.1-0.2mg/L NAA and 30g/L cane sugar into the improved B5 culture medium in the multiplication culture medium, after inoculation, shading culture is carried out for 6-8 days by using black cloth, then opening the black cloth to receive normal light, and obtaining a group seedling for multiplication growth after 30-35 days of multiplication culture;
(5) Rooting culture: cutting off the tissue culture seedlings cultured in the step (4) one by one when the tissue culture seedlings grow to be more than 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.1-0.2mg/L NAA, 0.1-0.3mg/L indoleacetic acid (IAA), 0.8-1.5 mg/L indolebutyric acid (IBA) and 20g/L sucrose into the rooting culture medium for an improved B5 culture medium, shading and culturing the tissue culture seedlings by using black cloth 6-8 days after inoculation, then opening the black cloth to receive normal illumination, and performing rooting culture for 30-35 days to obtain rooted eucalyptus grandis tissue culture seedlings;
(6) Hardening and transplanting seedlings: placing the tissue culture seedling of the eucalyptus grandis rooted in the step (5) in an outdoor shading and hardening seedling for 8-10d and 5-6 d, then removing the tissue culture seedling, wherein the tissue culture seedling cannot be directly pulled out during removal, kneading the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the tissue culture seedling in potassium permanganate with the mass volume concentration of 0.05% for 10min, transplanting the tissue culture seedling into a sterilized medium sprayed with carbendazim with the mass volume concentration of 0.1%, shading and moisturizing for a few days before transplanting, moving the tissue culture seedling out of a shading net once every day until new leaves grow out, and carrying out sprinkler irrigation to obtain the complete eucalyptus grandis seedling.
Preferably, the sprout of the eucalyptus grandis cut stump in the step (1) is selected as an explant, and the explant is collected in clear weather with continuous sunshine for more than 5 days (without rainy days).
Preferably, the alcohol in the step (2) is 75% alcohol by volume concentration; the mercuric chloride solution is a calcium chloride solution with the mass volume concentration of 0.1%.
Preferably, the modified B5 medium described in step (3), step (4) and step (5) comprises macro-elements, micro-elements and organic components; what is needed isThe specific components and contents of the improved B5 culture medium are (1) macroelements: KNO 3 2000mg/L，(NH 4 ) 2 SO 4 134mg/L，NaH 2 PO 4 200mg/L，MgSO 4 ·7H 2 O 250mg/L，CaCl 2 ·2H 2 O200 mg/L; (2) trace elements: KI 0.75mg/L, H 3 BO 3 3.0mg/L，MnSO 4 ·4H 2 O 15mg/L，ZnSO 4 ·7H 2 O 5mg/L，Na 2 MoO 4 ·2H 2 O 0.25mg/L；CuSO 4 ·5H 2 O 0.025mg/L，CoCl 2 ·6H 2 O 0.025mg/L，FeSO 4 ·7H 2 O(27.8mg/L)+Na 2 -EDTA·2H 2 O (37.3 mg/L); (3) organic components: 100mg/L of inositol, 1.0mg/L of nicotinic acid, 1.0mg/L of pyridoxine hydrochloride (vitamin B6), 5mg/L of thiamine hydrochloride (vitamin B1), 2mg/L of glycine, 10mg/L of riboflavin (vitamin B2), 1.1mg/L of calcium pantothenate, 10mg/L of ascorbic acid, 5mg/L of L-cysteine and 0.2mg/L of folic acid.
Preferably, the substrate in step (6) is garden soil: vermiculite: perlite = 4.
Compared with the prior art, the invention has the following beneficial effects:
the method systematically studies the influence of plant nutrient components on the growth of the cultured seedlings, comprehensively improves the components and the content of the culture medium, adopts a transverse inoculation mode during the propagation culture, ensures that the bud seedlings generated by the propagation have more growth space, and overcomes the problem that the growth of the lateral buds is limited because the internode of the eucalyptus robusta is shorter in the past; the culture method is more suitable for the growth of tissue culture seedlings of the eucalyptus grandis, and the tissue culture seedlings of the eucalyptus grandis obtained by culture are robust in growth and high in proliferation rate and rooting rate.
Drawings
FIG. 1 shows the new germinated axillary buds of the explant stem of example 1 of the present invention.
FIG. 2 is a photograph showing the proliferation culture of sprouts according to example 1 of the present invention.
FIG. 3 is a photograph showing tissue-cultured seedlings which are proliferation-cultured in example 1 of the present invention taken out and placed on a tray.
FIG. 4 is a picture of a tissue culture seedling rooted in example 1 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 mass-volume concentration refers to the concentration of a solute after a certain mass of the solute is dissolved in a certain volume of the solvent, for example, 1% mass-volume concentration, which is obtained by dissolving 1g of the solute in 100ml of the solvent.
In the examples, explant retention = (1-number of contaminated explants/total number of inoculated explants) × 100%; explant activation rate = (number of explants that sprout new shoots/number of explants that remain after inoculation) × 100%; proliferation factor = total number of shoots at statistical time/number of shoots at inoculation time; rooting rate = (number of rooted tissue culture seedling/number of tissue culture seedling at inoculation) × 100%; transplant survival rate = (number of seedlings with new leaves grown after transplantation/total number of seedlings at transplantation) × 100%.
The components and contents of the modified B5 culture medium in the examples are as follows:
(1) macroelements: KNO 3 2000mg/L，(NH 4 ) 2 SO 4 134mg/L，NaH 2 PO 4 200mg/L，MgSO 4 ·7H 2 O 250mg/L，CaCl 2 ·2H 2 O 200mg/L；
(2) Trace elements: KI 0.75mg/L, H 3 BO 3 3.0mg/L，MnSO 4 ·4H 2 O 15mg/L，ZnSO 4 ·7H 2 O 5mg/L，Na 2 MoO 4 ·2H 2 O 0.25mg/L；CuSO 4 ·5H 2 O 0.025mg/L，CoCl 2 ·6H 2 O 0.025mg/L，FeSO 4 ·7H 2 O(27.8mg/L)+Na 2 -EDTA·2H 2 O(37.3mg/L)；
(3) Organic components: 100mg/L of inositol, 1.0mg/L of nicotinic acid, 1.0mg/L of pyridoxine hydrochloride (vitamin B6), 5mg/L of thiamine hydrochloride (vitamin B1), 2mg/L of glycine, 10mg/L of riboflavin (vitamin B2), 1.1mg/L of calcium pantothenate, 10mg/L of ascorbic acid, 5mg/L of L-cysteine and 0.2mg/L of folic acid.
Example 1
A tissue culture method of Eucalyptus robusta (Roxb.) Roxb comprises the following specific steps:
(1) Selecting an explant: selecting bud strips of eucalyptus grandis cutting piles as explants, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), selecting new strong disease-free semi-lignified branches as the explants, and shearing the explants by using clean scissors to bring the explants back to a laboratory;
(2) Cleaning and disinfecting explants: removing leaves of the stem segments of the explants obtained in the step (1), cleaning surface dust with clear water, soaking for 10 minutes with a washing powder solution, carefully scrubbing with a soft brush pen, particularly paying attention to gaps at branches, washing for more than 30 minutes with running water after being cleaned, rinsing twice with sterile water after being transferred to an ultra-clean bench, then soaking for 15-30 s in 75% alcohol, washing for 2 times with sterile water, then placing the explants into 0.1% mercuric chloride solution, adding one drop (about 0.05 mL) of Tween 80 (polysorbate-80), continuously shaking for 8min, and washing for 5 times with sterile water, thus completing washing and disinfection;
(3) Inoculation and initiation culture: cutting off a small amount of incisions at two ends of the explant subjected to disinfection in the step (2) to avoid serious wound browning, cutting the explant into a short stem section with at least one axillary bud from a long stem section, vertically inserting the short stem section into a starting culture medium, performing primary generation starting culture, wherein the starting culture medium is obtained by adding 0.2 mg/L6-benzyladenine (6-BA), 0.1mg/L naphthylacetic acid (NAA) and 30g/L sucrose on the basis of an improved B5 culture medium, the starting culture time is 35d, the first 7d is subjected to shading culture by using black cloth, and then the black cloth is opened to receive normal illumination; the illumination intensity is 2000LX, the illumination period is 16h/d, the temperature is 26 +/-1 ℃, the humidity is 60%, and the explant is statistically obtained after 35d, wherein the explant storage rate is 73%, and the starting rate is 82%;
(4) And (3) proliferation culture: carefully cutting off lateral buds germinated in the starting culture process in the step (3), transferring the lateral buds into a multiplication culture medium for multiplication culture, cutting off 4/5 of the whole leaf area of each leaf before inoculation, reserving a small part of leaves and leaf stalks, flatly paving short stem sections on the surface of the multiplication culture medium, slightly pressing stem sections to enable 1/2 of the short stem sections to be immersed into the multiplication culture medium, enabling newly-increased bud seedlings to grow upwards in an inoculation mode and have enough growth space, reserving 2 buds during inoculation, having higher multiplication success rate than single bud inoculation, adding 0.2 mg/L6-BA, 0.5mg/L NAA and 30g/L cane sugar into an improved B5 culture medium in the multiplication culture medium, performing shading culture on the seedlings for the first 6d after inoculation by using a black cloth, then opening the black cloth to receive normal light, and obtaining tissue culture seedlings for multiplication growth after the multiplication culture for 35d, wherein the multiplication coefficient is 4.0 by statistics;
(5) Rooting culture: cutting off the tissue culture seedlings subjected to propagation culture in the step (4) one by one when the tissue culture seedlings grow to be more than 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.1mg/L NAA, 0.3mg/L indoleacetic acid (IAA), 0.8mg/L indolebutyric acid (IBA) and 20g/L cane sugar into an improved B5 culture medium, shading and culturing by using black cloth in the first 8 days after inoculation, opening the black cloth to receive normal illumination, and performing rooting culture for 35 days to obtain rooted eucalyptus grandis tissue culture seedlings, wherein the rooting rate is 83% by statistics;
(6) Hardening and transplanting seedlings: placing the tissue culture seedlings of the eucalyptus grandis rooted in the step (5) in an outdoor shading and hardening seedling for 8d, and moving out the tissue culture seedlings after 5d, wherein the tissue culture seedlings are forbidden to be directly pulled out during moving out, carefully rinsing the residual culture medium by using clear water after the culture medium around the root system is firstly kneaded and crushed, soaking the tissue culture seedlings in potassium permanganate with the mass volume concentration of 0.05% for 10min, transplanting the tissue culture seedlings into a sterilized substrate (the substrate is obtained by mixing fruit garden soil: vermiculite: perlite =4:1 by volume ratio for several days) with carbendazim with the mass volume concentration of 0.1%, shading and moisturizing the tissue culture seedlings before transplanting, and carrying out spray irrigation once every day until the tissue culture seedlings grow new leaves, and then moving out a shading net to receive normal illumination, so that complete eucalyptus grandis seedlings are obtained, 86 seedlings are transplanted, and survive 82 plants, and the transplanting survival rate is 95.3%.
Example 2
A tissue culture method of Eucalyptus robusta (Roxb.) Roxb comprises the following specific steps:
(1) Selecting an explant: selecting bud strips of eucalyptus grandis cutting piles as explants, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), selecting new strong disease-free semi-lignified branches as the explants, and shearing the explants by using clean scissors to bring the explants back to a laboratory;
(2) Cleaning and disinfecting explants: removing leaves of the stem segments of the explants obtained in the step (1), cleaning surface dust with clear water, soaking for 10 minutes with a washing powder solution, carefully scrubbing with a soft brush pen, particularly paying attention to gaps at branches, washing for more than 30 minutes with running water after being cleaned, rinsing twice with sterile water after being transferred to an ultra-clean bench, then soaking for 15-30 s in 75% alcohol, washing for 2 times with sterile water, then placing the explants into 0.1% mercuric chloride solution, adding one drop (about 0.05 mL) of Tween 80 (polysorbate-80), continuously shaking for 10 minutes, and washing for 6 times with sterile water, thus completing washing and disinfection;
(3) Inoculation and start-up culture: cutting off a small amount of incisions at two ends of the explant subjected to disinfection in the step (2) to avoid serious wound browning, cutting the explant into a short stem section with at least one axillary bud from a long stem section, vertically inserting the short stem section into a starting culture medium, performing primary generation starting culture, wherein the starting culture medium is obtained by adding 0.5 mg/L6-benzyladenine (6-BA), 0.1mg/L naphthylacetic acid (NAA) and 30g/L sucrose on the basis of an improved B5 culture medium, the starting culture time is 35d, the first 7d is subjected to shading culture by using black cloth, and then the black cloth is opened to receive normal illumination; the illumination intensity is 2500LX, the illumination period is 16h/d, the temperature is 26 +/-1 ℃, the humidity is 60%, the counting is carried out at 35d, the explant preservation rate is 76%, and the starting rate is 86%;
(4) And (3) proliferation culture: carefully cutting the lateral buds emerging in the initial culture process in the step (3), transferring the lateral buds into a multiplication culture medium for multiplication culture, cutting off 4/5 of the whole leaf area of each leaf blade before inoculation, reserving a small part of leaf blades and leaf stalks, flatly paving the short stem segments on the surface of the multiplication culture medium, slightly pressing the stem segments to ensure that 1/2 of the short stem segments are immersed into the multiplication culture medium, enabling newly-increased bud seedlings to grow upwards in an inoculation mode and have enough growth space, reserving 2 buds during inoculation, having higher multiplication success rate than single bud inoculation, adding 0.2 mg/L6-BA, 0.2mg/L NAA and 30g/L cane sugar into the improved B5 culture medium in the multiplication culture medium, shading and culturing by using black cloth after inoculation for the first 7d, then opening the black cloth to receive normal light, obtaining the group seedlings for multiplication growth after the multiplication culture 35d, and counting the multiplication coefficient to be 4.2;
(5) Rooting culture: cutting off the tissue culture seedlings cultured in the step (4) one by one when the tissue culture seedlings grow to be more than 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.1mg/L NAA, 0.1mg/L indoleacetic acid (IAA), 1.0mg/L indolebutyric acid (IBA) and 20g/L cane sugar into an improved B5 culture medium, after inoculation, performing shading culture by using black cloth for the first 8 days, then opening the black cloth to receive normal illumination, performing rooting culture for 35 days to obtain rooted eucalyptus grandis tissue culture seedlings, and counting to obtain the rooting rate of 90%;
(6) Hardening and transplanting seedlings: placing the tissue culture seedlings of the eucalyptus grandis rooted in the step (5) in outdoor shading and hardening seedlings for 10d and 5d, then removing the tissue culture seedlings, wherein the tissue culture seedlings cannot be directly pulled out during removing, kneading the culture medium around the root system, then carefully rinsing the residual culture medium with clear water, soaking the tissue culture seedlings in potassium permanganate with the mass-volume concentration of 0.05% for 10min, transplanting the tissue culture seedlings into a sterilized substrate (the substrate is obtained by mixing the fruit garden soil, vermiculite, perlite =4, and the volume ratio of 1 in a ratio of several days) sprayed with carbendazim with the mass-volume concentration of 0.1%, keeping the shade and moisturizing before transplanting, performing spray irrigation once every day until new leaves grow out of the tissue culture seedlings, then moving the tissue culture seedlings out of a shade net to receive normal illumination, and thus obtaining complete seedlings of the eucalyptus grandis, transplanting 82 plants, survival rate of the seedlings is 79, and the transplanting survival rate is 96.3%.
Example 3
A tissue culture method of Eucalyptus grandis, the specific operation steps are as follows:
(1) Selecting an explant: selecting bud strips of eucalyptus grandis cutting piles as explants, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), selecting new strong disease-free semi-lignified branches as the explants, and shearing the explants by using clean scissors to bring the explants back to a laboratory;
(2) Cleaning and disinfecting explants: removing leaves of the stem segments of the explants obtained in the step (1), cleaning surface dust with clear water, soaking for 10 minutes with a washing powder solution, carefully scrubbing with a soft brush pen, particularly paying attention to gaps at branches, washing for more than 30 minutes with running water after being cleaned, rinsing twice with sterile water after being transferred to an ultra-clean bench, then soaking for 15-30 s in 75% alcohol, washing for 2 times with sterile water, then placing the explants into 0.1% mercuric chloride solution, adding one drop (about 0.05 mL) of Tween 80 (polysorbate-80), continuously shaking for 10 minutes, and washing for 6 times with sterile water, thus completing washing and disinfection;
(3) Inoculation and initiation culture: cutting off a small amount of incisions at two ends of the explant subjected to disinfection in the step (2) to avoid serious wound browning, cutting the explant into a short stem section with at least one axillary bud from a long stem section, vertically inserting the short stem section into a starting culture medium, performing primary generation starting culture, wherein the starting culture medium is obtained by adding 0.2 mg/L6-benzyladenine (6-BA), 0.05mg/L naphthylacetic acid (NAA) and 30g/L sucrose on the basis of an improved B5 culture medium, the starting culture time is 35d, the first 7d is subjected to shading culture by using black cloth, and then the black cloth is opened to receive normal illumination; the illumination intensity is 2200LX, the illumination period is 16h/d, the temperature is 26 +/-1 ℃, the humidity is 60%, the explant is statistically obtained after 35d, the explant storage rate is 77%, and the starting rate is 87%;
(4) And (3) proliferation culture: carefully cutting off lateral buds germinated in the starting culture process in the step (3), transferring the lateral buds into a multiplication culture medium for multiplication culture, cutting off 4/5 of the whole leaf area of each leaf before inoculation, reserving a small part of leaves and leaf stalks, flatly paving short stem sections on the surface of the multiplication culture medium, slightly pressing stem sections to enable 1/2 of the short stem sections to be immersed into the multiplication culture medium, enabling newly-increased bud seedlings to grow upwards in an inoculation mode and have enough growth space, reserving 2 buds during inoculation, having higher multiplication success rate than single bud inoculation, adding 0.3 mg/L6-BA, 0.2mg/L NAA and 30g/L cane sugar into the improved B5 culture medium in the multiplication culture medium, performing shading culture on the seedlings for 6d before inoculation, opening a black cloth to receive normal light, and performing multiplication culture for 35d to obtain tissue culture seedlings for multiplication growth, wherein the multiplication coefficient is 4.6 by statistics;
(5) Rooting culture: cutting off the tissue culture seedlings cultured in the step (4) one by one when the tissue culture seedlings grow to be more than 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.1mg/L NAA, 0.2mg/L indoleacetic acid (IAA), 1.2mg/L indolebutyric acid (IBA) and 20g/L cane sugar into an improved B5 culture medium, after inoculation, performing shading culture by using black cloth for the first 7 days, then opening the black cloth to receive normal illumination, performing rooting culture for 35 days to obtain rooted eucalyptus grandis tissue culture seedlings, and counting to obtain the rooting rate of 94%;
(6) Hardening and transplanting seedlings: placing the tissue culture seedlings of the eucalyptus grandis rooted in the step (5) in an outdoor shading hardening seedling 10d and 5d, then moving the tissue culture seedlings out, wherein the tissue culture seedlings are forbidden to be directly pulled out when moving out, after the culture medium around the root system is firstly kneaded and crushed, carefully rinsing the residual culture medium with clear water, soaking the tissue culture seedlings in potassium permanganate with the mass volume concentration of 0.05% for 10min, transplanting the tissue culture seedlings into a sterilized substrate (the substrate is obtained by mixing fruit garden soil: vermiculite: perlite =4:1 in a volume ratio of several days) with carbendazim with the mass volume concentration of 0.1%, shading and moisturizing before transplanting, carrying out spray irrigation once every day until the tissue culture seedlings grow new leaves, then moving out a shading net to receive normal illumination, thus obtaining complete eucalyptus grandis seedlings, transplanting 86 plants, survival 82 plants and transplanting survival rate of 95.3%.
The foregoing description of specific exemplary embodiments of the invention has been presented for the 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. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (5)
1. A tissue culture method of Eucalyptus grandis is characterized by comprising the following steps:
(1) Selecting an explant: selecting bud of a big inflorescence eucalyptus cutting pile as an explant, and selecting a semi-lignified branch as the explant;
(2) Cleaning and disinfecting explants: removing leaves of the stem segments of the explants obtained in the step (1), cleaning, soaking and scrubbing, washing the stem segments clean, washing the stem segments with running water, transferring the stem segments to a super clean bench, rinsing the stem segments with sterile water, soaking the stem segments in alcohol for 15-30 s, cleaning the stem segments with sterile water, then placing the stem segments in a mercuric chloride solution, adding Tween 80, continuously shaking the stem segments, soaking the stem segments for 8-10 min, and cleaning the stem segments with sterile water to finish cleaning and disinfection;
(3) Inoculation and initiation culture: cutting off cuts at two ends of the explant sterilized in the step (2), cutting the explant into a short stem section with at least one axillary bud from a long stem section, vertically inserting into a starting culture medium, carrying out primary generation starting culture, wherein the starting culture medium is based on an improved B5 culture medium, then adding 0.1-0.2 mg/L6-benzyladenine (6-BA), 0.05-0.1 mg/L naphthylacetic acid (NAA) and 30g/L sucrose, the starting culture time is 30-40 d, the first 7-10 d is carried out shading culture by using black cloth, and then the black cloth is opened to receive normal illumination;
(4) And (3) proliferation culture: cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds into a multiplication culture medium for multiplication culture, cutting off 4/5 of the whole leaf of each leaf blade before inoculation, paving the short stem segments on the surface of the multiplication culture medium and immersing 1/2 of the short stem segments into the multiplication culture medium, reserving 2 buds during inoculation, adding 0.2-0.4 mg/L6-BA, 0.1-0.2mg/L NAA and 30g/L cane sugar into the improved B5 culture medium, shading and culturing the buds for the first 6-8 days after inoculation by using black cloth, then opening the black cloth to receive normal light, and obtaining a tissue culture seedling for multiplication growth after multiplication culture is carried out for 30-35 days;
(5) Rooting culture: cutting off the tissue culture seedlings cultured in the step (4) one by one when the tissue culture seedlings grow to be more than 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.1-0.2mg/L NAA, 0.1-0.3mg/L indoleacetic acid (IAA), 0.8-1.5 mg/L indolebutyric acid (IBA) and 20g/L sucrose into the rooting culture medium for an improved B5 culture medium, shading and culturing the tissue culture seedlings by using black cloth 6-8 days after inoculation, then opening the black cloth to receive normal illumination, and performing rooting culture for 30-35 days to obtain rooted eucalyptus grandis tissue culture seedlings;
(6) Hardening and transplanting seedlings: placing the tissue culture seedling of the eucalyptus grandis rooted in the step (5) in a shady and hardened seedling for 8-10d and 5-6 d, then removing the tissue culture seedling, rinsing the residual culture medium with water, soaking the tissue culture seedling in potassium permanganate with the mass volume concentration of 0.05%, transplanting the tissue culture seedling into a sterilized matrix sprayed with carbendazim with the mass volume concentration of 0.1%, and performing sprinkler irrigation once a day until the tissue culture seedling grows new leaves, thus obtaining the complete eucalyptus grandis seedling.
2. The Eucalyptus dahliae tissue culture method according to claim 1, characterized in that: and (2) selecting sprout strips of eucalyptus grandis cut piles as explants in the step (1), and collecting the sprout strips in clear weather with continuous sunshine for more than 5 days.
3. The Eucalyptus dahliae tissue culture method according to claim 1, characterized in that: the alcohol in the step (2) is 75% alcohol by volume concentration; the mercuric chloride solution is a calcium chloride solution with the mass volume concentration of 0.1%.
4. The tissue culture method of Eucalyptus macrocephala according to claim 1, which comprises the following steps: the improved B5 culture medium in the step (3), the step (4) and the step (5) contains macroelements, microelements and organic components; the improved B5 culture medium comprises the following specific components in percentage by weight (1) macroelements: KNO 3 2000mg/L，(NH 4 ) 2 SO 4 134mg/L，NaH 2 PO 4 200mg/L，MgSO 4 ·7H 2 O 250mg/L，CaCl 2 ·2H 2 O200 mg/L; (2) trace elements: KI 0.75mg/L, H 3 BO 3 3.0mg/L，MnSO 4 ·4H 2 O 15mg/L，ZnSO 4 ·7H 2 O 5mg/L，Na 2 MoO 4 ·2H 2 O 0.25mg/L；CuSO 4 ·5H 2 O 0.025mg/L，CoCl 2 ·6H 2 O 0.025mg/L，FeSO 4 ·7H 2 O(27.8mg/L)+Na 2 -EDTA·2H 2 O (37.3 mg/L); (3) organic components: 100mg/L of inositol, 1.0mg/L of nicotinic acid, 1.0mg/L of pyridoxine hydrochloride (vitamin B6), 5mg/L of thiamine hydrochloride (vitamin B1), 2mg/L of glycine, 10mg/L of riboflavin (vitamin B2), 1.1mg/L of calcium pantothenate, 10mg/L of ascorbic acid, 5mg/L of L-cysteine and 0.2mg/L of folic acid.
5. The Eucalyptus dahliae tissue culture method according to claim 1, characterized in that: the substrate in the step (6) is garden soil: vermiculite: perlite = 4.
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