CN113142051B - Tissue culture method for linaloe - Google Patents
Tissue culture method for linaloe Download PDFInfo
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- CN113142051B CN113142051B CN202110339675.9A CN202110339675A CN113142051B CN 113142051 B CN113142051 B CN 113142051B CN 202110339675 A CN202110339675 A CN 202110339675A CN 113142051 B CN113142051 B CN 113142051B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- 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
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) propagation 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 linaloe, the Guangxi and the like are introduced from Vietnam to cultivate, but still face the problem of insufficient seedlings. 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 strong 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 a super 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 during the period, soaking 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 explant subjected to sterilization in the step (2) to avoid serious wound browning, 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, performing primary starting culture, adding 0.1-0.2 mg/L6-BA, 0.05-0.1 mg/L NAA and 30g/L cane sugar into an improved B5 culture medium as the starting culture medium, and starting culture for 30-40 d;
(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, wherein the multiplication 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 multiplication growth after 30-35 d of multiplication culture;
(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, and 0.8-1.5 mg/L IBA, 0.1-0.2mg/L NAA, 0.1-0.3mg/L IAA and 20g/L sucrose are added into the improved B5 culture medium for rooting culture for 30-35 d to obtain rooted balsawood 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 after 5d, and moving the balsawood tissue culture seedlings out when moving out, wherein the balsawood tissue culture seedlings cannot be directly pulled out, but after the culture medium around the root system is firstly pinched and crushed, the residual culture medium is carefully rinsed by clear water, the balsawood tissue culture seedlings are soaked for 10min by 0.05% potassium permanganate and are transplanted into a 0.1% carbendazim sprayed sterilized substrate (garden soil: vermiculite: perlite = 4).
Preferably, the process of starting the culture in the step (3) comprises dark culture and light culture, wherein the time of the dark culture is 7-10 d, the time of the light culture 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 days, the light culture time is 24-32 days, and the total time is 30-35 days; 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 start medium, the multiplication medium and the rooting medium is an improved B5 medium, and the improved B5 medium comprises the following components in percentage by weight:
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；
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)；
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, 5mg/L cysteine, 0.2mg/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; all the solid culture mediums take 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: 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；
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)；
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, 0.2mg/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 a stem section with buds, which is a new strong and healthy stem section without diseases and insect pests and semi-lignification of the adult aquilaria sinensis in the current year, as an explant, collecting in clear weather with continuous sunshine for more than 5 days (in a cloudy and rainy day), 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 a super 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 sucrose are added, black cloth is used for shading culture in the first 10 days, 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 35d; 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 germinated in the starting culture process in the step (3), transferring the lateral buds into 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 improved B5 culture medium, after inoculation, the front 6d of the culture medium is subjected to shading culture by using black cloth, 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 is carried out for-35 d, obtaining bud seedlings for proliferation growth, and the proliferation coefficient is 2.8 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 1.5cm, 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 the rooting culture is performed for 30 days to obtain rooted balsalazide tissue culture seedlings, and the rooting rate is 73% through statistics;
(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 radication balsawood tissue culture seedlings in an outdoor shading and hardening 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 balsawood tissue culture seedlings out after 5d, avoiding directly pulling the balsawood tissue culture seedlings out, carefully rinsing the residual culture medium by using clear water after pinching the culture medium around the root system, soaking the balsawood tissue culture seedlings in 0.05% potassium permanganate for 10min, transplanting the balsawood tissue culture seedlings into a 0.1% carbendazim sprayed sterilized matrix (fruit garden: vermiculite: perlite = 4).
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, washing for more than 30 minutes with running water, transferring to a super clean bench, soaking in 75% alcohol by volume concentration for 30s, cleaning for 2 times with sterile water, 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 for 6 times with sterile water, 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, black cloth is used for shading culture in the first 8d, then the black cloth is opened to receive normal illumination, the illumination intensity LX 2500 of the light culture is realized, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%; the start culture time is 35d; counting at 35d, wherein the explant preservation rate is 76%, and the starting rate is 86%;
(4) And (3) proliferation culture: carefully cutting lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds into a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is an improved B5 culture medium, 0.3 mg/L6-BA, 0.2mg/L NAA and 30g/L sucrose are added, after inoculation, shading culture is carried out on the first 8d with black cloth, 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 proliferation culture is carried out for 33d, bud seedlings for proliferation growth are obtained, and the proliferation coefficient is 2.7 through 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 2500LX, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, the humidity is 60%, and the rooting culture time is 34d, so that rooted balsalazide tissue culture seedlings are obtained, and the rooting rate is 80% through statistics;
(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 the seedlings for 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 6d, avoiding directly pulling the seedlings when moving the seedlings out, kneading the culture medium around the root system, carefully rinsing the residual culture medium with clear water, soaking the balsawood tissue culture seedlings for 10min with 0.05% of potassium permanganate, transplanting the seedlings to a medium (orchard soil: 1 (v: v: v)) in a vermiculite-perlite =4 mode, shading and moisturizing are noticed for a few days before transplanting, sprinkling irrigation is carried out once every day until new leaves grow out of transplanted seedlings, the shading net can be moved out to receive normal illumination, complete linaloe seedlings are obtained, and the transplanting survival rate is 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, washing for more than 30 minutes with running water, transferring to a super clean bench, soaking in 75% alcohol by volume concentration for 20s, washing for 2 times with sterile water, 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 washing for 6 times with sterile water, 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 a short stem section with at least one axillary bud, vertically inserting the short stem section into a starting culture medium, performing primary starting culture, adding 0.1 mg/L6-BA, 0.05mg/L NAA and 30g/L cane sugar into the improved B5 culture medium as the starting culture medium, performing shading culture on the first 8d black cloth, then opening the black cloth to receive normal illumination, wherein the illumination intensity LX of the light culture is 2500, the illumination period is 16h/d, the temperature is 25 +/-1 ℃, and the humidity is 60%; the start culture time is 35d; counting at 35d, wherein the explant storage rate is 77% and the starting rate is 87%;
(4) And (3) proliferation culture: carefully cutting lateral buds sprouting in the starting culture process in the step (3), transferring the lateral buds into a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is an improved B5 culture medium, 0.4 mg/L6-BA, 0.2mg/L NAA and 30g/L sucrose are added, after inoculation, black cloth is used for shading culture in 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 proliferation culture is carried out for 30d, bud seedlings for proliferation growth are obtained, and the proliferation coefficient is 2.5 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 1.5-2.0cm, 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 sucrose 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 is carried out for 35d to obtain rooted balsalazide tissue culture seedlings, and the rooting rate is 83% through statistics;
(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 radication balsawood tissue culture seedlings in an outdoor 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 seedlings, moving the balsawood tissue culture seedlings out after 7d, avoiding directly pulling the balsawood tissue culture seedlings out, carefully rinsing the residual culture medium by using clear water after pinching the culture medium around the root system, soaking the balsawood tissue culture seedlings in 0.05% potassium permanganate for 10min, transplanting the balsawood tissue culture seedlings into a 0.1% carbendazim sprayed sterilized matrix (fruit garden: vermiculite: perlite = 4).
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: KNO 3 2500mg/L，(NH 4 ) 2 SO 4 134mg/L，NaH 2 PO 4 150mg/L，MgSO 4 ·7H 2 O 250mg/L，CaCl 2 ·2H 2 O 150mg/L；
Trace elements: KI 0.75mg/L, H 3 BO 3 3.0mg/L，MnSO 4 ·4H 2 O 10mg/L，ZnSO 4 ·7H 2 O 2.0mg/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)；
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 preservation rate is 53%, the starting rate is 62%, and the 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 (4)
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 stem section with buds of the linaloe as an explant, and collecting in clear weather with continuous sunshine for more than 5 days;
(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 and 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 to 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 percent, adding 1 to 2 drops of Tween 80, continuously shaking the stem segments, soaking the stem segments for 6 to 8 minutes, and washing the stem segments with the sterile water for 5 to 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, and 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 LNAA 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 into a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is an improved B5 culture medium, and adding 0.2-0.4 mg/L6-BA, 0.1-0.2mg/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, and 0.8-1.5 mg/LIBA, 0.1-0.2mg/LNAA, 0.1-0.3mg/LIAA and 20g/L of cane sugar are added, and performing rooting culture for 30-35 d to obtain rooted linaloe tissue culture seedlings;
(6) Hardening and transplanting seedlings: placing the radication balsamifera tissue culture seedling obtained in the step (5) in outdoor shading and hardening for 8-10d, keeping moisture in a culture medium, continuously shading and hardening the seedling, moving out the balsamifera tissue culture seedling after 5d, first pinching off the culture medium around a root system when moving out, rinsing the residual culture medium with water, soaking the balsamifera tissue culture seedling with 0.05 percent potassium permanganate for 10min, transplanting the balsamifera tissue culture seedling into a sterilized matrix sprayed with 0.1 percent carbendazim, and sprinkling once a day until the transplanted seedling grows out new leaves, then moving out a shading net to receive normal illumination, and obtaining a complete balsamifera plant;
wherein, the basic culture medium of the start culture medium, the multiplication culture medium and the rooting culture medium is an improved B5 culture medium, and the improved B5 culture medium comprises the following components in percentage by weight:
macroelements: KNO 3 2000 mg/L，(NH 4 ) 2 SO 4 134 mg/L，NaH 2 PO 4 200 mg/L，MgSO 4 ·7H 2 O 250mg/L，CaCl 2 ·2H 2 O 200mg/L；
Trace elements: KI 0.75mg/L, H 3 BO 3 3.0 mg/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+Na 2 -EDTA·2H 2 O; wherein, feSO 4 ·7H 2 The concentration of O is 27.8mg/L, na 2 -EDTA·2H 2 The concentration of O is 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 0.2mg/L folic acid.
2. The method of claim 1, wherein: the process of starting culture in the step (3) comprises dark culture and light culture, wherein the dark culture time is 8d, the light culture time is 27d, and the total time is 35d; 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 in the step (4) and the rooting culture in the step (5) comprise dark culture and light culture; the dark culture time of the proliferation culture is 8d, the light culture time is 25d, and the total time is 33d; the dark culture time of the rooting culture is 8d, the light culture time is 26d, and the total time is 34d; 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 pH values of the starting culture medium, the multiplication culture medium and the rooting culture medium are all 5.8-6.0; all the solid culture mediums take agar as a support, and the addition amount of the agar is 5.8-6.3 g/L.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102870683A (en) * | 2012-10-30 | 2013-01-16 | 广东省林业科学研究院 | Microbody propagation expanding method of aquilaria malaccensis |
| CN103348920A (en) * | 2013-07-25 | 2013-10-16 | 中国科学院华南植物园 | Rapid propagation method for high quality seedlings of Kyara |
| CN105010142A (en) * | 2015-07-14 | 2015-11-04 | 中国林业科学研究院热带林业研究所 | Vietnamese Aquilaria agallocha Roxb tissue culture method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102870683A (en) * | 2012-10-30 | 2013-01-16 | 广东省林业科学研究院 | Microbody propagation expanding method of aquilaria malaccensis |
| CN103348920A (en) * | 2013-07-25 | 2013-10-16 | 中国科学院华南植物园 | Rapid propagation method for high quality seedlings of Kyara |
| CN105010142A (en) * | 2015-07-14 | 2015-11-04 | 中国林业科学研究院热带林业研究所 | Vietnamese Aquilaria agallocha Roxb tissue culture method |
Non-Patent Citations (4)
| Title |
|---|
| A New Species Of Aquilaria (Thymelaeaceae) From Vietnam;Le Cong Kiet等;《BLUMEA》;20050422;第50卷;135-141 * |
| 土沉香成熟胚的组织培养及植株再生;兰芹英等;《广西农业生物科学》;20010930;第20卷(第3期);231-232 * |
| 茉莉酸甲酯诱导白木香结香技术研究;江浩;《中国优秀硕士学位论文全文数据库（农业科技辑）》;20200815(第8期);正文第3页表1-1 * |
| 马来沉香组织培养技术研究;张卫华等;《广西植物》;20131105;第34卷(第3期);381-386 * |
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