CN115644056A - Tissue culture industrialized production method of nepenthes - Google Patents
Tissue culture industrialized production method of nepenthes Download PDFInfo
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Abstract
The invention discloses a nepenthes tissue culture industrialized production method, which is characterized in that seedlings are inversely inserted and inoculated on a rooting culture medium, the rooting speed is greatly improved, the seedlings can root after being cultured for 8-12 days, moreover, the rooting speed is high, the rooting rate is high, the seedlings emerge in 18-22 days, the rooting rate is 100%, the growth cycle of the nepenthes is greatly shortened, and the production efficiency of the seedlings can be quickly improved. The method is simple and easy to operate, and can obviously shorten the culture time and accelerate the propagation speed. The method has the advantages of high rooting rate, strong stability, good seedling uniformity, strong resistance and the like, has wide application prospect, and is suitable for large-area popularization and application. In addition, the liquid rooting culture medium can be reused for 2 times, so that the investment of a large amount of manpower, material resources and financial resources is reduced, and a large amount of time and cost are saved.
Description
Technical Field
The invention belongs to the technical field of tissue culture, and particularly relates to an industrial production method for nepenthes tissue culture.
Background
Nepenthes (lour.) Druce) is a perennial herb semi-woody liana of the genus Nepenthes of the family Piperaceae, also known as Leptolepis petiolus, chenopodium palustris, chenopodium pigeonia and the like. Nepenthes are mainly distributed in central and south peninsula asia, africa (madagasca), northern australia and the pacific islands, and there are about 160 species worldwide. Most varieties of nepenthes are distributed abroad, and China only has 1 nepenthes (mirabilis) which are distributed in the south of the sea, the southwest of the Guangdong, the Guangxi and the like and grow in swamps, roadside, hillsides or forest lands with the altitude of 50-400 m. The nepenthes have unique leaf shape and bright color, are metamorphosis to develop into a bag-shaped insect catching cage, the cage bodies are different in shape and size, some of the cage bodies are like small wine cups, some of the cage bodies are like small pots, the ornamental performance is extremely high, the nepenthes are usually used for microlandschaft or potted plant ornamental, the shape is unique, and the nepenthes are elegant and pleasant. In addition, the pitcher plant can trap insects, is one of the most popular species among the current insect-feeding plants, and has extremely high popular science value. The product has effects in relieving inflammation and cough, clearing away heat, promoting urination, removing food stagnation, and removing calculus, and can be used as refreshing tea in summer to relieve summer heat. In recent years, pitcher plant integrating viewing, medicine and science popularization is more and more concerned by people, and has great economic value and broad market application prospect.
However, wild resources thereof are seriously damaged and the number is sharply reduced. Therefore, a method for the tissue culture and industrial production of nepenthes needs to be researched, and the market demand is met. The common nepenthes belong to male and female heteroplants, are not easy to pollinate, have very low maturing rate, slow seed germination, low germination rate, slow rooting speed of cutting propagation, are difficult to survive and cannot meet the production requirement. In production, a common seedling raising method of pitcher plant is tissue culture, but the existing tissue culture seedling raising method has long rooting time, low rooting rate and high energy consumption, is difficult to save deficient germplasm resources and fills the market vacancy. Therefore, the method takes the new buds or the tender stems of the common nepenthes as test materials, uses a liquid culture medium, and inoculates the seedlings in the rooting culture medium in an in vitro tissue culture method in an inverted way, so that the rooting speed is greatly improved, the production efficiency of the seedlings can be rapidly improved, the increasing market demand of the seedlings is met, and an effective way is provided for large-scale production and seedling culture.
Disclosure of Invention
The invention aims to provide a nepenthes tissue culture industrialized production method, which uses a liquid culture medium and adopts a mode of inverted grafting and rooting, can effectively improve the rooting rate of seedlings, greatly shortens the growth period of nepenthes, saves a large amount of time, manpower, material resources and financial resources, and is beneficial to industrialized mass production.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a nepenthes tissue culture industrialized production method comprises the following steps:
(1) Explant selection and sterilization
Selecting a halepys nepalensis plant which grows strongly and has no disease or insect pest, soaking the whole plant for 15-25 minutes by adding water into detergent, then washing the plant for 10-20 minutes by tap water, shearing off redundant leaves by using disinfection scissors, and taking a stem section with buds as an explant; sterilizing the treated pitcher plant material;
(2) Induced culture
The explant in the step (1) is dried by using sterile filter paper, and is inoculated to an inoculation culture medium for culture, and the explant starts to grow after being cultured for 7-8 days;
(3) Proliferation culture
Inoculating the bud cluster cultured for 35-40 days into a proliferation culture medium for culturing for 40-50 days;
(4) Rooting culture
Inversely inserting the plantlets grown to 3-5cm in the step (3) into a rooting culture medium with the pH value of 5.8, culturing for 8-12 days to start rooting, completing 18-22 days of rooting to meet the rooting requirement, and ensuring that the rooting rate of seedlings is 100 percent;
(5) Transplanting and managing tissue culture seedlings
And (4) taking out the tissue culture seedlings rooted in the step (4), washing the tissue culture seedlings clean with clear water, wrapping roots with moss soaked in advance, wrapping the roots with moss which must be wrapped tightly, transplanting the seedlings into a flowerpot, placing the flowerpot into a seedling raising plug tray with a moisture preservation cover, and keeping the air humidity around the seedlings to be more than 60% and the temperature to be 25-30 ℃ to obtain the pitcher plant pot seedlings.
Preferably, the specific method of the sterilization treatment is: sterilizing with 75% alcohol for 30 s, washing with sterile water for 3 times, adding into 0.1% mercuric chloride solution for 10 min, and washing with sterile water for 5-6 times.
Preferably, the formula of the inoculation medium is as follows: huabao I1.6-3.2g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar; more preferably, the inoculation medium formulation is: huabao I1.6g-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar.
Preferably, the formula of the proliferation medium is: huabao I number 2.0-3.6g +6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar; more preferably, the formulation of the proliferation medium is: huabao I2.0g +6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar.
Preferably, the formula of the rooting medium is as follows: huabao I1.2-2.8g + NAA0.3mg/L +3% sugar; more preferably, the rooting medium has the following formula: huabao I1.2g BiNAA0.3mg/L +3% sugar.
The sugar used in the above culture medium may be a sugar commonly used in culture media such as sucrose.
Preferably, the culture conditions of the inoculation culture medium, the multiplication culture medium and the rooting culture medium are all as follows: the illumination intensity is 2000lx, the temperature is 23-25 ℃, and the illumination time is 12 hours.
The nepenthes tissue culture large-scale production method provided by the invention has the following advantages and effects:
(1) According to the problems of the nepenthes tissue culture, the culture medium and the culture conditions are optimized, and the optimal culture conditions are adopted, so that the rooting and survival of the nepenthes are facilitated.
(2) The inventor finds that the seedling is reversely inserted and inoculated on the rooting culture medium through long-term experimental results, the rooting speed is greatly improved, the seedling starts to root after about 10 days of culture, the rooting speed is high, the rooting rate is high, the rooting requirement is met after the rooting is finished in 18-22 days, the rooting rate of the seedling is 100%, the time is greatly shortened, and the production efficiency of the seedling can be rapidly improved.
(3) The method is simple and easy to operate, and can obviously shorten the culture time and accelerate the propagation speed. The method has the advantages of high rooting rate, strong stability, good seedling uniformity, strong resistance and the like, has wide application prospect, and is suitable for large-area popularization and application.
(4) The method has the advantages that the seedlings emerge on the workbench through aseptic operation, the liquid culture medium can be reused for 2 times, the investment of a large amount of manpower, material resources and financial resources is reduced, a large amount of time and cost are saved, the method has small pollution to the environment, the production cost is greatly reduced on the premise of ensuring the rooting and the seedling formation, and the method has good popularization value.
Drawings
FIG. 1 is a schematic diagram showing the inverted rooting using a liquid medium;
FIG. 2 shows the survival of tissue-cultured seedlings after batch transplantation;
FIG. 3 is the inverted rooting using solid medium;
FIG. 4 is a normal placement rooting case;
FIG. 5 is a view of the bottom of a normal setting for observing rooting;
FIG. 6 is a comparison of rooting with normal placement (left), liquid medium inversion (middle), solid medium inversion (right).
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. However, the specific experimental procedures referred to in the following examples were carried out in a conventional manner or under the conditions recommended by the manufacturer's instructions unless otherwise specified.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials used are commercially available, unless otherwise specified.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Moreover, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Embodiment 1 a nepenthes tissue culture large-scale production method, including the following steps:
(1) Explant selection and sterilization
Selecting a halenia nepalensis plant which grows strongly and has no plant diseases and insect pests, soaking the whole plant for 20 minutes by adding water into a detergent, then washing the plant for 15 minutes by using tap water, and shearing redundant leaves by using disinfection scissors to replace bud stems as explants;
sterilizing the surface of the treated pitcher plant material with 75% alcohol for 30 seconds, washing with sterile water for 3 times, placing in 0.1% mercuric chloride solution for 10 minutes, and washing with sterile water for 5-6 times;
(2) Induced culture
The explant in the step (1) is dried by using sterile filter paper, and is inoculated into an induction culture medium with the pH of 5.8, the illumination intensity of 2000lx, the temperature of 23-25 ℃ and the illumination time of 12 hours, wherein the explant is cultured for 7-8 days to start to grow, and the Huabao I1.6 g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar;
(3) Multiplication culture
Inoculating the bud cluster cultured for about 40 days to Huabao I
2.0g of +6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g of agar in a proliferation medium with the pH value of 5.8, and culturing for 40-50 days under the same culture conditions;
(4) Rooting culture
Inoculating the plantlets grown to 3-5cm in the step (3) to a rooting culture medium with Huabao I No. 1.2g + NAA0.3mg/L +3% sugar and pH of 5.8, pouring the plantlets on the culture medium under the same culture conditions, culturing for about 10 days to root (figure 1), wherein the rooting rate of emergence is up to 100% about 20 days;
(5) Transplanting and managing tissue culture seedlings
And (3) taking out the tissue culture seedlings rooted in the step (4), washing the tissue culture seedlings with clear water, wrapping roots with moss soaked in advance to be as compact as possible, transplanting the seedlings into flowerpots with the width of 8cm and the height of 7cm, placing the flowerpots into seedling culture hole trays with moisture-preserving covers, keeping the air humidity around the seedlings to be more than 60%, keeping the temperature to be 25-30 ℃, and obtaining pot seedlings of the pitcher plant (shown in figure 2), wherein the survival rate can reach 98%.
Embodiment 2 a method for producing pitcher plant in a large scale by tissue culture comprises the following steps:
(1) Explant selection and sterilization
Selecting a halepys nepalensis plant which grows strongly and has no disease or insect pest, soaking the whole plant for 20 minutes by adding water into washing agent, then washing the plant for 15 minutes by tap water, and shearing redundant leaves by using disinfection scissors to replace bud stem segments as explants;
sterilizing the surface of the treated pitcher plant material with 75% alcohol for 30 seconds, washing with sterile water for 3 times, placing in 0.1% mercuric chloride solution for 10 minutes, and washing with sterile water for 5-6 times;
(2) Induced culture
The explant in the step (1) is dried by using sterile filter paper, and is inoculated into an induction culture medium with the pH of 5.8, wherein the culture medium comprises 2.5g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar, the illumination intensity is 2000lx, the temperature is 23-25 ℃, and the illumination time is 12 hours, and the explant starts to grow after being cultured for 7-8 days;
(3) Multiplication culture
Inoculating the bud cluster cultured for about 40 days to Huabao I
2.8g of 6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g of agar, in a proliferation medium with pH of 5.8, and culturing for 40-50 days under the same culture conditions as above;
(4) Rooting culture
Inoculating the plantlet which grows to 3-5cm in the step (3) into a rooting culture medium with Huabao I No. 2.0g + NAA0.3mg/L +3% sugar and pH of 5.8, and pouring the plantlet on the culture medium under the same culture conditions, and culturing for about 10 days to start rooting;
(5) Transplanting and managing tissue culture seedlings
And (5) taking out the tissue culture seedlings rooted in the step (4), washing the tissue culture seedlings with clear water, wrapping roots with moss soaked in advance to be as tight as possible, transplanting the seedlings into flowerpots with the width of 8cm and the height of 7cm, placing the flowerpots into seedling raising hole trays with moisture-preserving covers, and keeping the air humidity around the seedlings to be more than 60% and the temperature to be 25-30 ℃ to obtain the pot seedlings of the nepenthes.
Embodiment 3 a nepenthes tissue culture large-scale production method, including the following steps:
(1) Explant selection and disinfection
Selecting a halenia nepalensis plant which grows strongly and has no plant diseases and insect pests, soaking the whole plant for 20 minutes by adding water into a detergent, then washing the plant for 15 minutes by using tap water, and shearing redundant leaves by using disinfection scissors to replace bud stems as explants;
sterilizing the surface of the treated pitcher plant material by using 75% alcohol for 30 seconds, then washing the plant material by using sterile water for 3 times, then placing the plant material in 0.1% mercuric chloride solution for 10 minutes, and finally washing the plant material by using the sterile water for 6 times;
(2) Induced culture
The explant in the step (1) is dried by using sterile filter paper, and is inoculated into an induction culture medium with the pH value of 5.8, wherein the induction culture medium is 3.2g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar, the illumination intensity is 2000lx, the temperature is 23-25 ℃, and the illumination time is 12 hours, and the explant starts to grow after being cultured for 7-8 days;
(3) Proliferation culture
Inoculating the bud cluster cultured for about 40 days to Huabao I
3.6g of 6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar, in a proliferation medium with pH of 5.8, and cultured for 40-50 days under the same culture conditions as above;
(4) Rooting culture
Inoculating the plantlet which grows to 3-5cm in the step (3) into a rooting culture medium with Huabao I No. 2.8g + NAA0.3mg/L +3% sugar and pH of 5.8, pouring the plantlet on the culture medium under the same culture condition, and culturing for about 10 days to start rooting;
(5) Transplanting and managing tissue culture seedlings
And (5) taking out the tissue culture seedlings rooted in the step (4), washing the tissue culture seedlings with clear water, wrapping roots with moss soaked in advance to be as tight as possible, transplanting the seedlings into flowerpots with the width of 8cm and the height of 7cm, placing the flowerpots into seedling raising hole trays with moisture-preserving covers, and keeping the air humidity around the seedlings to be more than 60% and the temperature to be 25-30 ℃ to obtain the pot seedlings of the nepenthes.
Comparative example 1
A nepenthes tissue culture large-scale production method comprises the following steps:
(1) Selection and treatment of explants
Selecting a halepys nepalensis plant which grows strongly and has no disease or insect pest, soaking the whole plant for 20 minutes by adding water into washing agent, then washing the plant for 15 minutes by tap water, and shearing redundant leaves by using disinfection scissors to replace bud stem segments as explants;
sterilizing the surface of the treated pitcher plant material with 75% alcohol for 30 seconds, washing with sterile water for 3 times, placing in 0.1% mercuric chloride solution for 10 minutes, and washing with sterile water for 5-6 times;
(2) Induced culture
The explants in the step (1) are dried by using sterile filter paper, and inoculated to Huabao I1.6-3.2g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar; culturing the explant to grow for 7 days in a culture medium with the pH of 5.8 under the culture conditions that the illumination intensity is 2000lx, the temperature is 23-25 ℃ and the illumination time is 12 hours;
(3) Proliferation culture
Inoculating the bud cluster cultured for about 40 days to Huabao I
2.0-3.6g of +6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar, in a proliferation medium with pH of 5.8, for 40-50 days under the same culture conditions as above;
(4) Rooting culture
In the rooting stage, the liquid culture medium is changed into a solid culture medium, namely, a plantlet which grows to 3-5cm in the step (3) in the example 1 is inoculated to the Huabao I1.2-2.8g + NAA0.3mg/L +3% sugar to be replaced by the Huabao I1.2-2.8g + NAA0.3mg/L +3% sugar +7g agar, and in this case, the rootage is generated after about 15 days of culture (figure 3), and the rooting rate can be 95% after about 25 days.
Comparative example 2
A nepenthes tissue culture large-scale production method comprises the following steps:
(1) Selection and treatment of explants
Selecting a halepys nepalensis plant which grows strongly and has no disease or insect pest, soaking the whole plant for 20 minutes by adding water into washing agent, then washing the plant for 15 minutes by tap water, and shearing redundant leaves by using disinfection scissors to replace bud stem segments as explants;
sterilizing the surface of the treated pitcher plant material with 75% alcohol for 30 seconds, washing with sterile water for 3 times, placing in 0.1% mercuric chloride solution for 10 minutes, and washing with sterile water for 5-6 times;
(2) Induced culture
The explants in the step (1) are dried by using sterile filter paper, and inoculated to Huabao I No. 1.6-3.2g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar; culturing the explant to grow for 7 days in a culture medium with the pH of 5.8 under the culture conditions that the illumination intensity is 2000lx, the temperature is 23-25 ℃ and the illumination time is 12 hours;
(3) Proliferation culture
Inoculating the bud cluster cultured for about 40 days to Huabao I
2.0-3.6g of 6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar, in a proliferation culture medium with pH of 5.8, culturing for 40-50 days under the same culture conditions;
(4) Rooting culture
In the rooting culture stage, the plantlets which grow to 3-5cm in the step (3) are inoculated into a rooting culture medium with the pH value of 5.8 of Huabao I No. 1.2-2.8g + NAA0.3mg/L +3% sugar +7g agar, and the plantlets under the same culture conditions are normally placed on the culture medium, so that the plantlets do not root in the first two months (figure 4 and figure 5), and root is continuously generated in 2-3 months, and the root generation rate is 82% after 3 months.
By comparing the method of the present invention with comparative examples 1 and 2 (FIG. 6), it was found that, in the rooting stage, the liquid medium without agar addition was used, which resulted in a shorter rooting time and a higher rooting rate than the solid medium. By comparing the inverted culture with the normal placement culture, the normal placement of the pitcher plant can be seen to root very slowly, which greatly restricts the industrial production, while the inverted method only needs about 10 days to root, which greatly shortens the time and is suitable for the industrial production compared with the normal placement.
The above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and it is obvious to those skilled in the art that other embodiments can be easily made by replacing or changing the technical contents disclosed in the present specification, and therefore, all changes and modifications made on the principle of the present invention should be included in the claims of the present invention.
Claims (7)
1. The tissue culture industrialized production method of pitcher plant is characterized by comprising the following steps:
(1) Explant selection and sterilization
Selecting a halepys nepalensis plant which grows strongly and has no disease or insect pest, soaking the whole plant for 15-25 minutes by adding water into detergent, then washing the plant for 10-20 minutes by tap water, shearing off redundant leaves by using disinfection scissors, and taking a stem section with buds as an explant; sterilizing the treated pitcher plant material;
(2) Induced culture
The explant in the step (1) is dried by using sterile filter paper, and is inoculated to an inoculation culture medium for culture, and the explant starts to grow after being cultured for 7-8 days;
(3) Proliferation culture
Inoculating the bud cluster cultured for 35-40 days into a proliferation culture medium for culturing for 40-50 days;
(4) Rooting culture
Inversely inserting the plantlets which grow to 3-5cm in the step (3) into a rooting culture medium with the pH value of 5.8, culturing for 8-12 days to start rooting, completing 18-22 days to meet the rooting requirement, and ensuring that the rooting rate of seedlings is 100 percent;
(5) Transplanting and managing tissue culture seedlings
And (5) taking out the tissue culture seedlings rooted in the step (4), washing the tissue culture seedlings clean by clear water, wrapping roots with moss soaked in advance, transplanting the seedlings to a flowerpot, placing the seedlings in a seedling raising plug tray with a moisture-preserving cover, and keeping the air humidity around the seedlings to be more than 60% and the temperature to be 25-30 ℃ to obtain the pitcher seedling of pitcher plant.
2. The nepenthes tissue culture industrialized production method of claim 1, which is characterized in that the specific method of disinfection treatment is as follows: sterilizing with 75% alcohol for 30 s, washing with sterile water for 3 times, adding into 0.1% mercuric chloride solution for 10 min, and washing with sterile water for 5-6 times.
3. The method for the tissue culture and industrial production of pitcher plant according to claim 1, wherein the formula of the inoculation medium is: huabao I1.6-3.2g +6-BA2.0mg/L + NAA0.3mg/L +3% sugar +7g agar.
4. The tissue culture and industrial production method of pitcher plant according to claim 1, wherein the formula of the proliferation medium is: huabao I number 2.0-3.6g +6-BA1.5mg/L + NAA0.1mg/L +3% sugar +7g agar.
5. The nepenthes tissue culture industrialized production method of claim 1, wherein the rooting medium comprises the following formula: huabao I1.2-2.8g + NAA0.3mg/L +3% sugar.
6. The method for the tissue culture and industrial production of pitcher plant according to claim 1, wherein the culture conditions of the inoculation culture medium, the multiplication culture medium and the rooting culture medium are all as follows: the illumination intensity is 2000lx, the temperature is 23-25 ℃, and the illumination time is 12 hours.
7. The method for the tissue culture and industrial production of nepenthes according to any one of claims 3-5, wherein the sugar is sucrose.
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