CN113142051A - Banana agilawood tissue culture method - Google Patents
Banana agilawood tissue culture method Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a tissue culture method of linaloe, which comprises the following steps: (1) selecting an explant; (2) cleaning and disinfecting explants; (3) inoculating and culturing; (4) carrying out proliferation culture; (5) rooting culture; (6) hardening and transplanting the seedlings until new leaves grow out of the transplanted seedlings, moving the seedlings out of the shading net to receive normal illumination, and obtaining complete Bara aquilaria plants. The invention systematically studies the influence of plant nutrient components on the growth of the linaloe tissue culture seedlings, comprehensively improves the components and the content of the culture medium, and additionally adds riboflavin, calcium pantothenate, ascorbic acid, L-cysteine and folic acid into the culture medium. Wherein the riboflavin and ascorbic acid are not only organic components, but also play a role in reducing browning; the culture method is more suitable for growth of tissue culture test-tube seedlings of linaloe, the tissue culture seedlings of linaloe obtained by culture grow strongly, the proliferation rate and the rooting rate are high, and technical support is provided for large-scale production of linaloe.
Description
Technical Field
The invention belongs to the technical field of plant cell engineering, and particularly relates to a linaloe tissue culture method.
Background
Linaloe (Aquilaria banaensis) is a plant of the genus Aquilaria of the family Thymelaeaceae, a tropical subtropical evergreen tree, naturally distributed in hills and mountains in Vietnam, and is the main tree species for the production of linaloe and linaloe oil in southeast Asia countries. Due to excessive deforestation, the tree species have become endangered in many natural forests. The artificial cultivation of the linaloe is the most effective method for solving the shortage of the linaloe, and in recent years, the problems of insufficient seedlings are still faced when the linaloe is introduced from Vietnam to cultivate in Guangdong, Guangxi and the like. At present, the propagation of linaloe by tissue culture has not been reported.
Disclosure of Invention
Aiming at the technical problems, the invention provides a linaloe tissue culture method, and aims to obtain a linaloe tissue culture seedling method with robust growth, high proliferation rate and high rooting rate.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a method for tissue culture of linaloe comprises the following operation steps:
(1) selecting an explant: selecting the newly grown strong and healthy stem with no plant diseases and insect pests and semi-lignification bud of the adult balsawood in the current year as an explant, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), and shearing by using clean scissors;
(2) cleaning and disinfecting explants: removing leaves of the stem segments of the explants collected 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, rinsing with clear water, washing for more than 30 minutes with running water, transferring to an ultra-clean bench, soaking in alcohol for 15-30 s, washing with sterile water for 2 times, then placing the explants in a 0.1% mercuric chloride solution, adding 1-2 drops of Tween 80 (polysorbate-80), continuously shaking for 6-8 min, and washing with sterile water for 5-6 times, namely completing disinfection;
(3) Inoculation and culture: cutting off a small amount of incisions at two ends of the sterilized explant in the step (2) to avoid serious wound browning, cutting the stem section of the explant into short stem sections with at least one axillary bud, vertically inserting the short stem sections into a starting culture medium, and carrying out primary starting culture, wherein the starting culture medium is an improved B5 culture medium added with 0.1-0.2 mg/L6-BA, 0.05-0.1 mg/L NAA and 30g/L sucrose, and the starting culture time is 30-40 d;
(4) and (3) proliferation culture: carefully cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds to an enrichment culture medium for enrichment culture, wherein the enrichment culture medium is an improved B5 culture medium added with 0.2-0.4 mg/L6-BA, 0.1-0.2mg/L NAA and 30g/L sucrose, and obtaining bud seedlings for enrichment growth after enrichment culture for 30-35 d;
(5) rooting culture: cutting off the tissue culture seedlings generated by the 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, wherein the rooting culture medium is an improved B5 culture medium, 0.8-1.5 mg/L IBA, 0.1-0.2mg/L NAA, 0.1-0.3mg/L IAA and 20g/L cane sugar are added, and the rooting culture is carried out for 30-35 d to obtain rooted linaloe tissue culture seedlings;
(6) hardening and transplanting seedlings: and (3) moving the radication balsawood tissue culture seedlings obtained in the step (5) out of a tissue culture room, placing the tissue culture seedlings outside the room for shading and hardening for 8-10d, then opening a cover, injecting a proper amount of sterile water into the culture medium for moisturizing, continuing shading and hardening the seedlings, moving the balsawood tissue culture seedlings out after 5d, avoiding directly pulling the balsawood tissue culture seedlings out, pinching the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the balsawood tissue culture seedlings in 0.05% of potassium permanganate for 10min, transplanting the balsawood tissue culture seedlings into a sterilized substrate (garden soil: vermiculite: 4:1:1) sprayed with 0.1% of carbendazim, paying attention to shading for several days before transplanting, sprinkling once a day, moving the shading net out until new leaves grow out of the transplanted seedlings, and receiving normal illumination to obtain complete balsawood plants.
Preferably, the process of starting the culture in the step (3) comprises dark culture and light culture, wherein the dark culture time is 7-10 d, the light culture time is 23-33 d, and the total time is 30-40 d; the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%.
Preferably, the proliferation culture in the step (4) and the rooting culture in the step (5) both comprise dark culture and light culture, the dark culture time is 6-8 d, the light culture time is 24-32 d, and the total time is 30-35 d; the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%.
Preferably, the minimal medium of the starting medium, the propagation medium and the rooting medium is a modified B5 medium, and the modified B5 medium comprises the following components in percentage by weight:
macroelements: KNO3 2000mg/L，(NH4)2SO4 134mg/L，NaH2PO4 200mg/L，MgSO4·7H2O 250mg/L，CaCl2·2H2O 200mg/L；
Trace elements: KI 0.75mg/L, H3BO3 3.0mg/L，MnSO4·4H2O 15mg/L，ZnSO4·7H2O 5mg/L，Na2MoO4·2H2O 0.25mg/L；CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L，FeSO4·7H2O(27.8mg/L)+Na2-EDTA·2H2O(37.3mg/L)；
Organic components: 100mg/L inositol, 1.0mg/L nicotinic acid, 1.0mg/L pyridoxine hydrochloride (vitamin B6), 5mg/L thiamine hydrochloride (vitamin B1), 2mg/L glycine, 10mg/L riboflavin (vitamin B2), 1.1mg/L calcium pantothenate, 10mg/L ascorbic acid, 5 mg/L-cysteine, and 0.2 mg/L folic acid.
Preferably, the pH values of the starting culture medium, the multiplication culture medium and the rooting culture medium are all 5.8-6.0; the culture medium is a solid culture medium taking agar as a support, and the addition amount of the agar is 5.8-6.3 g/L.
Compared with the prior art, the invention has the following beneficial effects:
the invention systematically studies the influence of plant nutrient components on the growth of the linaloe tissue culture seedlings, comprehensively improves the components and the content of the culture medium, and additionally adds riboflavin, calcium pantothenate, ascorbic acid, L-cysteine and folic acid in the culture medium. Wherein, the riboflavin and the ascorbic acid are not only organic components, but also play a role in reducing browning; the culture method is more suitable for growth of tissue culture test-tube seedlings of linaloe, the tissue culture seedlings of linaloe obtained by culture grow strongly, the proliferation rate and the rooting rate are high, and technical support is provided for large-scale production of linaloe.
Detailed Description
The following detailed description is to be read in connection with specific embodiments, but it should 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 components and amounts of the modified B5 medium used in the following examples are as follows:
Macroelements: KNO3 2000mg/L，(NH4)2SO4 134mg/L，NaH2PO4 200mg/L，MgSO4·7H2O 250mg/L，CaCl2·2H2O 200mg/L；
Trace elements: KI 0.75mg/L, H3BO3 3.0mg/L，MnSO4·4H2O 15mg/L，ZnSO4·7H2O 5mg/L，Na2MoO4·2H2O 0.25mg/L；CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L，FeSO4·7H2O(27.8mg/L)+Na2-EDTA·2H2O(37.3mg/L)；
Organic components: 100mg/L inositol, 1.0mg/L nicotinic acid, 1.0mg/L pyridoxine hydrochloride (vitamin B6), 5mg/L thiamine hydrochloride (vitamin B1), 2mg/L glycine, 10mg/L riboflavin (vitamin B2), 1.1mg/L calcium pantothenate, 10mg/L ascorbic acid, 5 mg/L-cysteine, 0.2 mg/L folic acid; wherein, the riboflavin, the calcium pantothenate, the ascorbic acid, the L-cysteine and the folic acid are organic components additionally added in the culture method, wherein the riboflavin and the ascorbic acid are not only organic components but also play a role in reducing browning, and the roles in the culture method are very critical; the pH value of the culture medium is 5.8-6.0, the agar is used as a solid culture medium of a support, and the addition amount of the agar is 5.8-6.3 g/L.
Example 1
A method for tissue culture of linaloe comprises the following specific operation steps:
(1) selecting an explant: selecting the newly grown strong and healthy stem with no plant diseases and insect pests and semi-lignification bud of the adult balsawood in the current year as an explant, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), and shearing by using clean scissors;
(2) cleaning and disinfecting explants: removing leaves of the stem segments of the explants collected 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, rinsing with clear water, then washing for more than 30 minutes with running water, transferring to an ultra-clean bench, soaking in 75% alcohol by volume concentration for 15 seconds, cleaning with sterile water for 2 times, then placing the explants in 0.1% mercuric chloride solution, adding 1-2 drops of Tween 80 (polysorbate-80) into a standard burette, continuously shaking during the period, soaking for 8 minutes, and cleaning with sterile water for 5 times, namely completing disinfection;
(3) Inoculation and culture: cutting off a small amount of incisions at two ends of the explant subjected to sterilization in the step (2) to avoid serious wound browning, cutting the stem section of the explant into short stem sections with at least one axillary bud, vertically inserting the short stem sections into a starting culture medium, carrying out primary starting culture, wherein the starting culture medium is an improved B5 culture medium, 0.2 mg/L6-BA, 0.1mg/L NAA and 30g/L cane sugar are added, the black cloth is subjected to shading culture in the first 10d, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is 2000LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%; the starting culture time is 35 d; counting at 35d, wherein the explant storage rate is 73%, and the starting rate is 82%;
(4) and (3) proliferation culture: carefully cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is an improved B5 culture medium, 0.2 mg/L6-BA, 0.15mg/L NAA and 30g/L sucrose are added into the culture medium, after inoculation, the black cloth is used for shading culture for the first 6 days, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, after proliferation culture for-35 days, a bud seedling for proliferation growth is obtained, and the proliferation coefficient is 2.8 after statistics;
(5) Rooting culture: cutting off the tissue culture seedlings generated by the propagation culture in the step (4) one by one when the tissue culture seedlings grow to 1.5cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, adding 0.8mg/L IBA, 0.2mg/L NAA, 0.2mg/L IAA and 20g/L cane sugar into an improved B5 culture medium, performing shading culture on the first 8 days after inoculation by using black cloth, then opening the black cloth to receive normal illumination, wherein the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, and performing rooting culture for 30 days to obtain rooted balsalazide tissue culture seedlings, wherein the rooting rate is 73% through statistics;
(6) hardening and transplanting seedlings: and (3) moving the rooting Bara aquilaria wood tissue culture seedling obtained in the step (5) out of a tissue culture room, placing the rooting Bara aquilaria wood tissue culture seedling outside a room for shading and hardening the seedling for 8d, then opening a cover, injecting a proper amount of sterile water into the culture medium for moisturizing, continuing shading and hardening the seedling, moving the Bara aquilaria wood tissue culture seedling after 5d, avoiding directly pulling the seedling when the seedling is moved out, kneading the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the Bara aquilaria wood tissue culture seedling in 0.05% potassium permanganate for 10min, transplanting the Bara wood tissue culture seedling into a sterilized matrix (fruit garden soil: vermiculite: perlite: 4:1:1(v: v: v)) sprayed with 0.1% carbendazim, shading and hardening moisture for a few days before transplanting, performing sprinkling once a day until a new leaf of the transplanted seedling grows out, moving a shading net and receiving normal light to obtain a complete Bara complete Bara seedling, wherein the transplanting survival rate is 85%.
Example 2
A method for tissue culture of linaloe seedlings comprises the following specific operation steps:
(1) selecting an explant: selecting the newly grown strong and healthy stem with no plant diseases and insect pests and semi-lignification bud of the adult balsawood in the current year as an explant, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), and shearing by using clean scissors;
(2) cleaning and disinfecting explants: removing leaves of the stem segments of the explants collected 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, rinsing with clear water, then washing for more than 30 minutes with running water, transferring to an ultra-clean bench, soaking in 75% alcohol by volume concentration for 30s, cleaning with sterile water for 2 times, then placing the explants in 0.1% mercuric chloride solution, adding 1-2 drops of Tween 80 (polysorbate-80) into a standard burette, continuously shaking during the period, soaking for 8min, and cleaning with sterile water for 6 times, namely completing disinfection;
(3) inoculation and culture: cutting off a small amount of incisions at two ends of the explant subjected to sterilization in the step (2) to avoid serious wound browning, cutting the stem section of the explant into short stem sections with at least one axillary bud, vertically inserting the short stem sections into a starting culture medium, carrying out primary starting culture, wherein the starting culture medium is an improved B5 culture medium, 0.1 mg/L6-BA, 0.05mg/L NAA and 30g/L sucrose are added, the black cloth is cultured in a shading mode in the first 8d, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%; the starting culture time is 35 d; counting at 35d, wherein the explant preservation rate is 76%, and the starting rate is 86%;
(4) And (3) proliferation culture: carefully cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds to an enrichment culture medium for enrichment culture, wherein the enrichment culture medium is an improved B5 culture medium, 0.3 mg/L6-BA, 0.2mg/L NAA and 30g/L sucrose are added into the culture medium, after inoculation, the black cloth is used for shading culture in the first 8d, then the black cloth is opened to receive normal illumination, the illumination intensity of the illumination culture is 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, after the enrichment culture is carried out for 33d, sprouts which are proliferated and grown are obtained, and the proliferation coefficient is 2.7 by statistics;
(5) rooting culture: cutting off the tissue culture seedlings generated by the propagation culture in the step (4) one by one when the tissue culture seedlings grow to 1.5-2.0cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, wherein the rooting culture medium is obtained by adding 1.0mg/L IBA, 0.1mg/L NAA, 0.1mg/L IAA and 20g/L sucrose into an improved B5 culture medium, after inoculation, shading culture is carried out on the first 8d by using black cloth, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is LX 2500, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, and the rooting culture time is 34d to obtain rooted balsalazide tissue culture seedlings, and the rooting rate is 80% by statistics;
(6) hardening and transplanting seedlings: and (3) moving the rooting Bara aquilaria wood tissue culture seedling obtained in the step (5) out of a tissue culture room, placing the rooting Bara aquilaria wood tissue culture seedling outside a room for shading and hardening the seedling for 10d, then opening a cover, injecting a proper amount of sterile water into the culture medium for moisturizing, continuing shading and hardening the seedling, moving the Bara aquilaria wood tissue culture seedling out after 6d, avoiding directly pulling the seedling when moving the seedling out, kneading the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the Bara aquilaria wood tissue culture seedling in 0.05% potassium permanganate for 10min, transplanting the Bara wood tissue culture seedling into a sterilized matrix (fruit garden soil: vermiculite: perlite: 4:1:1(v: v: v)) sprayed with 0.1% carbendazim, shading and hardening moisture for a few days before transplanting, performing sprinkling once a day until a new leaf of the transplanted seedling grows out, moving a shading net and receiving normal light to obtain a complete Bara complete Bara seedling with transplanting survival rate of 86%.
Example 3
A method for tissue culture of linaloe seedlings comprises the following specific operation steps:
(1) selecting an explant: selecting the newly grown strong and healthy stem with no plant diseases and insect pests and semi-lignification bud of the adult balsawood in the current year as an explant, collecting in sunny days with continuous sunshine for more than 5 days (in cloudy and rainy days), and shearing by using clean scissors;
(2) cleaning and disinfecting explants: removing leaves of the stem segments of the explants collected 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, rinsing with clear water, then washing for more than 30 minutes with running water, transferring to an ultra-clean bench, soaking in 75% alcohol by volume concentration for 20s, cleaning with sterile water for 2 times, then placing the explants in 0.1% mercuric chloride solution, adding 1-2 drops of Tween 80 (polysorbate-80) into a standard burette, continuously shaking during the period, soaking for 6 minutes, and cleaning with sterile water for 6 times, namely completing disinfection;
(3) inoculation and culture: cutting off a small amount of incisions at two ends of the explant subjected to sterilization in the step (2) to avoid serious wound browning, cutting the stem section of the explant into short stem sections with at least one axillary bud, vertically inserting the short stem sections into a starting culture medium, carrying out primary starting culture, wherein the starting culture medium is an improved B5 culture medium, 0.1 mg/L6-BA, 0.05mg/L NAA and 30g/L sucrose are added, the black cloth is cultured in a shading mode in the first 8d, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%; the starting culture time is 35 d; counting at 35d, wherein the explant storage rate is 77% and the starting rate is 87%;
(4) And (3) proliferation culture: carefully cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds to an enrichment culture medium for enrichment culture, wherein the enrichment culture medium is an improved B5 culture medium, 0.4 mg/L6-BA, 0.2mg/L NAA and 30g/L sucrose are added into the culture medium, after inoculation, black cloth is used for shading culture for the first 7d, then the black cloth is opened to receive normal illumination, the illumination intensity of the illumination culture is 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, after the enrichment culture is carried out for 30d, sprouts which are proliferated and grown are obtained, and the proliferation coefficient is 2.5 by statistics;
(5) rooting culture: cutting off the tissue culture seedlings generated by the propagation culture in the step (4) one by one when the tissue culture seedlings grow to 1.5-2.0cm, vertically transferring the tissue culture seedlings to a rooting culture medium for rooting culture, wherein the rooting culture medium is an improved B5 culture medium, 1.0mg/L IBA, 0.1mg/L NAA, 0.1mg/L IAA and 20g/L cane sugar are added, after inoculation, black cloth is used for shading culture in the first 8 days, then the black cloth is opened to receive normal illumination, the illumination intensity of the light culture is 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, and the rooting culture time is 35d to obtain rooted Balinaya tissue culture seedlings, and the rooting rate is 83% through statistics;
(6) hardening and transplanting seedlings: and (3) moving the rooting Bara aquilaria wood tissue culture seedling obtained in the step (5) out of a tissue culture room, placing the rooting Bara aquilaria wood tissue culture seedling outside a shading and hardening room for 9d, then opening a cover, injecting a proper amount of sterile water into the culture medium for moisturizing, continuing shading and hardening the seedling, moving the Bara aquilaria wood tissue culture seedling out after 7d, avoiding directly pulling the seedling when moving the seedling out, kneading the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the Bara aquilaria wood tissue culture seedling in 0.05% potassium permanganate for 10min, transplanting the seedling into a sterilized matrix (fruit garden soil: vermiculite: perlite: 4:1:1(v: v: v)) sprayed with 0.1% carbendazim, shading and hardening moisture for several days before transplanting, performing sprinkling once a day until the transplanted seedling grows out new leaves, moving the shading net to receive normal light irradiation, obtaining a complete Bara seedling with transplanting survival rate of 85%.
Comparative example 1
In this comparative example 1, the minimal medium was B5 medium, and these components were not added, riboflavin, calcium pantothenate, ascorbic acid, L-cysteine, and folic acid, and the remaining culture methods and culture conditions were completely the same as those in example 1.
The components and contents of the B5 medium used in comparative example 1 were as follows:
macroelements：KNO3 2500mg/L，(NH4)2SO4 134mg/L，NaH2PO4 150mg/L，MgSO4·7H2O 250mg/L，CaCl2·2H2O 150mg/L；
Trace elements: KI 0.75mg/L, H3BO3 3.0mg/L，MnSO4·4H2O 10mg/L，ZnSO4·7H2O 2.0mg/L，Na2MoO4·2H2O 0.25mg/L；CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L，FeSO4·7H2O(27.8mg/L)+Na2-EDTA·2H2O(37.3mg/L)；
Organic components: 100mg/L inositol, 1.0mg/L nicotinic acid, 1.0mg/L pyridoxine hydrochloride (vitamin B6), 10mg/L thiamine hydrochloride (vitamin B1), 2mg/L glycine.
The pH value of the culture medium is 5.8-6.0, and the addition amount of the agar is 5.8-6.3 g/L.
The statistics of the starting culture for 35d shows that the explant storage rate is 53%, the starting rate is 62%, and browning is obvious; after the proliferation culture is carried out for 35d, the bud seedlings which are proliferated and grown are obtained, and the proliferation coefficient is 2.1; and (5) performing rooting culture for 35 days to obtain rooted linaloe tissue culture seedlings, wherein the rooting rate is 65%.
By comprehensively comparing the culture medium formula of the invention, the comparative examples have poor effects from the starting culture stage to the proliferation culture and rooting culture stages, and the browning phenomenon is more common.
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 (5)
1. The method for tissue culture of seedlings of linaloe is characterized by comprising the following operation steps:
(1) selecting an explant: selecting a semi-lignified shoot segment of linaloe as an explant, and collecting in a sunny day with more than 5 days of continuous sunshine;
(2) cleaning and disinfecting explants: removing leaves of the stem segments of the explants collected in the step (1), soaking the stem segments in a washing powder solution for 10 minutes, scrubbing the stem segments, rinsing the stem segments clean, washing the stem segments for more than 30 minutes, transferring the stem segments to a clean bench, soaking the stem segments in alcohol for 15-30 seconds, washing the stem segments with sterile water for 2 times, then putting the explant in a mercuric chloride solution with the concentration of 0.1%, adding 1-2 drops of Tween 80, continuously shaking the stem segments, soaking the stem segments for 6-8 minutes, and washing the stem segments with sterile water for 5-6 times, thus completing disinfection;
(3) inoculation and culture: cutting off cuts at two ends of the sterilized explant in the step (2), cutting a stem section of the explant into a short stem section with at least one axillary bud, vertically inserting the short stem section into a starting culture medium, carrying out primary generation starting culture, wherein the starting culture medium is an improved B5 culture medium added with 0.1-0.2 mg/L6-BA, 0.05-0.1 mg/L NAA and 30g/L sucrose, and the starting culture time is 30-40 d;
(4) and (3) proliferation culture: cutting off lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is an improved B5 culture medium added with 0.2-0.4 mg/L6-BA, 0.1-0.2 mg/L NAA and 30g/L sucrose, and obtaining bud seedlings for proliferation growth after proliferation culture for 30-35 d;
(5) Rooting culture: cutting off the tissue culture seedlings generated by the 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, wherein the rooting culture medium is an improved B5 culture medium, 0.8-1.5 mg/L IBA, 0.1-0.2mg/L NAA, 0.1-0.3mg/L IAA and 20g/L cane sugar are added, and the rooting culture is carried out for 30-35 d to obtain rooted linaloe tissue culture seedlings;
(6) hardening and transplanting seedlings: placing the radication balsawood tissue culture seedling obtained in the step (5) in outdoor shade to harden the seedling for 8-10d, keeping the culture medium moist, continuing shade to harden the seedling, moving out the balsawood tissue culture seedling after 5d, first pinching off the culture medium around the root system when moving out, rinsing the residual culture medium with water, soaking the balsawood tissue culture seedling with 0.05% potassium permanganate for 10min, transplanting the balsawood tissue culture seedling into a sterilized matrix sprayed with 0.1% carbendazim, and sprinkling once a day until the transplanted seedling grows out new leaves, then moving out a shade screen to receive normal illumination, and obtaining a complete balsawood plant.
2. The method of claim 1, wherein: the starting culture process in the step (3) comprises dark culture and light culture, the dark culture time is 7-10 d, the light culture time is 23-33 d, and the total time is 30-40 d; the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%.
3. The method of claim 1, wherein: the proliferation culture process in the step (4) and the rooting culture process in the step (5) both comprise dark culture and light culture, the dark culture time is 6-8 d, the light culture time is 24-32 d, and the total time is 30-35 d; the illumination intensity of the light culture is 2000-2500 LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%.
4. The method of claim 1, wherein the minimal medium of the initiation medium, the proliferation medium and the rooting medium is modified B5 medium, and the modified B5 medium comprises the following components in percentage by weight:
macroelements: KNO3 2000mg/L，(NH4)2SO4 134mg/L，NaH2PO4 200mg/L，MgSO4·7H2O 250mg/L，CaCl2·2H2O 200mg/L；
Trace elements: KI 0.75mg/L, H3BO3 3.0mg/L，MnSO4·4H2O 15mg/L，ZnSO4·7H2O 5mg/L，Na2MoO4·2H2O 0.25mg/L；CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L，FeSO4·7H2O(27.8mg/L)+Na2-EDTA·2H2O(37.3mg/L)；
Organic components: 100mg/L inositol, 1.0mg/L nicotinic acid, 1.0mg/L pyridoxine hydrochloride (vitamin B6), 5mg/L thiamine hydrochloride (vitamin B1), 2mg/L glycine, 10mg/L riboflavin (vitamin B2), 1.1mg/L calcium pantothenate, 10mg/L ascorbic acid, 5 mg/L-cysteine, and 0.2 mg/L folic acid.
5. The method of claim 1, wherein: the pH values of the starting culture medium, the multiplication culture medium and the rooting culture medium are all 5.8-6.0; the culture medium is a solid culture medium taking agar as a support, and the addition amount of the agar is 5.8-6.3 g/L.
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