CN111264388A - Method for improving tissue culture rooting efficiency of camellia nitidissima - Google Patents
Method for improving tissue culture rooting efficiency of camellia nitidissima Download PDFInfo
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention relates to a method for improving the tissue culture rooting efficiency of golden camellia, and belongs to the technical field of golden camellia tissue culture seedling. The method comprises the following steps: 1) inserting the camellia chrysantha tissue culture subculture bud seedlings into a rooting culture medium for one-stage rooting culture; the selection standard of the tissue culture subculture bud seedling and the components of a rooting culture medium are limited; 2) adding nutrient solution, and performing two-stage rooting culture; the components of the nutrient solution are also limited; 3) hardening and transplanting the seedlings to obtain the camellia chrysantha tissue culture seedlings. The tissue culture rooting method disclosed by the invention is high in efficiency, can greatly improve the rooting rate and the transplanting survival rate of tissue culture bud seedlings, shortens the rooting culture time, improves the seedling culture efficiency, reduces the seedling culture cost, and solves the problem of tissue culture rapid propagation of golden camellia plants with difficult rooting.
Description
Technical Field
The invention relates to the technical field of camellia nitidissima tissue culture seedling culture, and particularly relates to a method for improving the tissue culture rooting efficiency of camellia nitidissima.
Background
Camellia chrysantha (Hu) Tuyama) is the only rare ornamental plant with golden flower in the Camellia of Theaceae, and the golden flower is unique and has luster, so the golden flower is called as panda in plant kingdom and queen in tea family.
However, the long rooting time and low rooting rate of tissue culture are the bottleneck of the industrialization of the camellia nitidissima tissue culture seedling, especially some species which grow slowly and are difficult to root in vitro culture, such as lemon yellow camellia nitidissima, Dongxing camellia nitidissima, hibiscus camellia nitidissima or clusteria camellia nitidissima.
Disclosure of Invention
The invention aims to provide a method for improving the tissue culture rooting efficiency of golden camellia. The tissue culture rooting method is high in efficiency, the rooting rate and the transplanting survival rate of tissue culture bud seedlings can be greatly improved, the rooting culture time is shortened, the seedling culture efficiency is improved, the seedling culture cost is reduced, and the problem of tissue culture rapid propagation of golden camellia plants with difficult rooting is solved.
The invention provides a method for improving the tissue culture rooting efficiency of golden camellia, which comprises the following steps:
1) inserting the camellia chrysantha subculture bud into a rooting culture medium for carrying out first-stage rooting culture for 30-35 d to obtain a bud seedling with a root eye at the base; the subculture bud seedling is a single bud with a terminal bud, a seedling height of more than 2.0cm, a seedling diameter of more than 0.5mm, more than 3 unfolding leaves, a leaf length of more than 0.5cm, a leaf turning from light red to green or light green, and a smooth base cut; the rooting culture medium is a solid matrix soaked in a liquid culture medium, and the solid matrix comprises fine river sand and vermiculite; before the insertion, cutting off the leaves of the part below 1.0cm of the base of the subculture bud seedlings;
2) after obtaining bud seedlings with root eyes at the base parts, adding nutrient solution into the rooting culture medium in the step 1), and performing two-stage rooting culture for 30-35 d to obtain golden camellia tissue culture rooting bottle seedlings; the nutrient solution is prepared from sucrose or white sugar 15.0g/L and K2HPO415.0mg/L aqueous solution;
3) hardening and transplanting the camellia chrysantha tissue culture rooting bottle seedlings obtained in the step 2) to obtain camellia chrysantha tissue culture seedlings.
Preferably, the golden camellia comprises lemon yellow golden camellia, Dongxing golden camellia, hibiscus golden camellia or clusterin golden camellia.
Preferably, in the step 1), the liquid culture medium takes 1/2B5 as a basic culture medium, 0.5mg/L of ABT and 0.5mg/L of IBA are added, and the pH value is 5.5-6.5.
Preferably, the particle size of the fine river sand is 1-2 mm, and the particle size of the vermiculite is 2-3 mm.
Preferably, the preparation method of the rooting medium comprises the following steps:
① mixing the cleaned and drained fine river sand and vermiculite according to the volume ratio of 1:1 to obtain a solid matrix;
② soaking the solid matrix in the liquid culture medium until the liquid culture medium absorbed by the solid matrix is saturated, filtering, and removing the liquid culture medium to obtain rooting culture medium.
Preferably, the depth of the secondary bud seedling in the step 1) inserted into the rooting medium is 1/3-1/2 of the height of the secondary bud seedling.
Preferably, the addition amount of the culture solution in the step 2) is 1/2-2/3 of the volume of the primary root culture medium.
Preferably, the volume ratio of the matrix transplanted in the step 3) is 3: 1.
Preferably, the culture temperature in the first stage rooting culture, the second stage rooting culture and the seedling exercising is 22-26 ℃.
Preferably, the light intensity is 150-300 lx in the first 15 days of the first stage rooting culture, and the light intensity is 1200-1500 lx in the first 16-35 days of the first stage rooting culture; in the process of the two-stage rooting culture, the light intensity is 1800-2300 lx; and in the seedling hardening stage before transplanting, natural scattered light is kept, and direct sunlight is avoided.
The invention provides a method for improving the tissue culture rooting efficiency of golden camellia. The method can improve the rooting efficiency of the camellia nitidissima species which are difficult to tissue culture and root, low in rooting rate and long in rooting time. Compared with the conventional culture medium, the culture medium uses 'fine river sand and vermiculite' to replace agar as a substrate, does not need boiling, is convenient to operate and has good performanceAir permeability and water retention (good water retention, water drainage and air capacity), is favorable for inducing root to germinate and improving rooting rate. The method carries out sugar-free culture in the early stage of rooting culture, the pollution rate can be controlled to be within 0.5 percent (the pollution rate of conventional tissue culture is 5-10 percent), the pollution rate in the culture process is greatly reduced, and the great loss of tissue culture seedlings is reduced. And thirdly, the early stage of rooting culture is performed without sugar so as to reduce the influence of osmotic pressure of a culture medium on root primordium induction, shorten rooting time, improve the rooting rate of in-vitro culture of plants difficult to root, facilitate promotion of bud seedling induction of root primordium and shorten the rooting time by 15-20 days. The invention has high quality requirement on the bud seedlings, under the condition that carbon sources such as cane sugar or white sugar and the like are not added in the earlier stage of a culture medium, the small bud seedlings survive in an autotrophic mode with weak photosynthetic capacity, more than 3 unfolding blades are utilized, the weak photosynthesis can be carried out to synthesize the carbon sources, the thick and strong bud seedlings enable the cells to contain more protoplasm, callus is easily formed on the base parts, and the differentiation of root primordia is facilitated; when the rooting culture is carried out, the leaves of the part below 1.0cm of the bud seedling base are cut off, so that the bud seedling base can be conveniently inserted into a matrix (fine river sand and vermiculite), and the leaves can be prevented from directly contacting the matrix for a long time in the culture process, so that the bud seedling can be rooted conveniently. And adding 15.0g/L of cane sugar or white sugar in the two-stage rooting culture stage, so that a carbon source required by heterotrophic rooting of the bud seedlings can be provided, and yellowing and falling off of leaves of the bud seedlings, poor growth of the bud seedlings or death of the bud seedlings are avoided. Sixthly, K is added in the two-stage rooting culture stage2HPO415.0mg/L, can ensure the thick green leaves of the seedlings and the stout seedlings, improve the lignification degree and the resistance of the stems of the rooting bottle seedlings and improve the transplanting survival rate. Compared with the conventional culture medium, the invention uses 'fine river sand and vermiculite' to replace agar as a substrate, and 15.0g/L of cane sugar or white sugar and K are added in the two-stage rooting culture stage2HPO415.0mg/L, the bud seedlings are thick and strong, the root systems of the rooting bottle seedlings are developed, and the transplanting survival rate of the rooting bottle seedlings is high. Eighthly, compared with the conventional culture medium, the method uses 'fine river sand and vermiculite' to replace agar as the matrix, does not need to be cleaned before the bottle seedlings with roots are transplanted, takes out the bottle seedlings with roots and directly transplants the bottle seedlings with roots on the transplanting matrix, and reduces the number of the bottle seedlings with rootsThe root agar is cleaned before transplanting, so that root damage or root breakage is caused, and the transplanting survival rate of the rooting bottle seedlings is improved. And ninthly, combining the optimization technology, the transplanting survival rate of the rooting bottle seedlings reaches more than 98%.
The tissue culture rooting method is high in efficiency, the rooting rate and the transplanting survival rate of tissue culture bud seedlings can be greatly improved, the rooting culture time is shortened, the seedling culture efficiency is improved, the seedling culture cost is reduced, and the problem of tissue culture rapid propagation of golden camellia plants with difficult rooting is solved.
Drawings
FIG. 1 is a diagram showing the growth conditions (low rooting rate, thick and brittle leaves) of a rooted bud cultured for 65d by agar (conventional method 1) provided by the invention;
FIG. 2-A is a graph showing the rooting condition of 65d rooted bottle seedlings cultured with agar (conventional method 1) provided by the present invention;
FIG. 2-B is a diagram showing the growth of the root seedling before the seedling is taken out from the rooting bottle and transplanted after the agar (conventional method 1) provided by the present invention is cultured for 65 d;
fig. 3 is a fine river sand provided by the present invention: a growth condition diagram of rooting bottle seedlings cultured for 65d by vermiculite (1:1) and supplementary nutrient solution;
fig. 4 is a fine river sand provided by the present invention: vermiculite (1:1) + supplementary nutrient solution, culturing for 65d, and drawing the root system condition of the tissue culture seedling before transplanting.
Detailed Description
The invention provides a method for improving the tissue culture rooting efficiency of golden camellia, which comprises the following steps:
1) inserting the camellia chrysantha subculture bud into a rooting culture medium for carrying out first-stage rooting culture for 30-35 d to obtain a bud seedling with a root eye at the base; the subculture bud seedling is a single bud with a terminal bud, a seedling height of more than 2.0cm, a seedling diameter of more than 0.5mm, more than 3 unfolding leaves, a leaf length of more than 0.5cm, a leaf turning from light red to green or light green, and a smooth base cut; the rooting culture medium is a solid matrix soaked in a liquid culture medium, and the solid matrix comprises fine river sand and vermiculite; before the insertion, cutting off the leaves of the part below 1.0cm of the base of the subculture bud seedlings;
2) obtaining a bud seedling with a root bud eye at the base part, and then growing the bud seedling in the step 1)Adding nutrient solution into the root culture medium, and performing two-stage rooting culture for 30-35 d to obtain golden camellia tissue culture rooting bottle seedlings; the nutrient solution is prepared from sucrose or white sugar 15.0g/L and K2HPO415.0mg/L aqueous solution;
3) hardening and transplanting the camellia chrysantha tissue culture rooting bottle seedlings obtained in the step 2) to obtain camellia chrysantha tissue culture seedlings.
Inserting the camellia chrysantha subculture bud into a rooting culture medium to perform one-stage rooting culture for 30-35 d to obtain a bud seedling with a root eye at the base; the subculture bud seedling is a single bud with a terminal bud, a seedling height of more than 2.0cm, a seedling diameter of more than 0.5mm, more than 3 unfolding leaves, a leaf length of more than 0.5cm, a leaf turning from light red to green or light green, and a smooth base cut; the rooting culture medium is a solid matrix soaked in a liquid culture medium, and the solid matrix comprises fine river sand and vermiculite; before the insertion, the leaf of the part below 1.0cm of the base of the subculture bud is cut off. The method is specially used for camellia nitidissima species with long rooting time and low rooting rate, and particularly, in the method, the camellia nitidissima preferably comprises lemon yellow camellia nitidissima, Dongxing camellia nitidissima, hibiscus camellia nitidissima or pistil camellia nitidissima. The method for obtaining the subculture sprouts is not particularly limited, and the subculture sprouts can be obtained by adopting a conventional subculture method well known to those skilled in the art. The subculture sprouts of the invention are preferably harvested under aseptic conditions using a scalpel. In the invention, the liquid culture medium preferably takes 1/2B5 as a basic culture medium, 0.5mg/L ABT and 0.5mg/L IBA are added, and the pH value is 5.5-6.5, more preferably 5.8. In the invention, the particle size of the fine river sand is preferably 1-2 mm, and the particle size of the vermiculite is preferably 2-3 mm.
In the invention, the preparation method of the rooting medium comprises the following steps:
① mixing the cleaned and drained fine river sand and vermiculite according to the volume ratio of 1:1 to obtain a solid matrix;
② soaking the solid matrix in the liquid culture medium until the liquid culture medium absorbed by the solid matrix is saturated, filtering, and removing the liquid culture medium to obtain rooting culture medium.
The method mixes the fine river sand and vermiculite which are cleaned and drained in a volume ratio of 1:1 to obtain the solid matrix. The method of washing in the present invention is not particularly limited, and a method of washing with clean water known to those skilled in the art may be used.
Soaking the solid matrix in a liquid culture medium until the liquid culture medium absorbed by the solid matrix is saturated, filtering, and removing the liquid culture medium to obtain the rooting culture medium. In the invention, the dosage of the liquid culture medium is preferably based on that the solid matrix is completely immersed, and the immersion time is preferably 20-50 min, more preferably 30min, based on that the solid matrix is in a water saturation state. The filtering of the invention is preferably to use a filter screen to take out the solid matrix from the liquid culture medium and filter out the redundant liquid, and the filter screen is preferably held by hands to prevent water from dripping. Before the rooting culture medium is used, the rooting culture medium is preferably subpackaged, preferably in culture bottles, and the subpackaged thickness is preferably 1.5-3.0 cm, and more preferably 2.0 cm. After subpackaging, sealing and sterilizing for later use are preferred.
Before the rooting culture medium is used, the rooting culture medium is preferably sterilized, and the method for sterilizing the rooting culture medium is preferably high temperature and high pressure (the temperature is 121 ℃, and the pressure is 1.1 kg/cm)2) Sterilizing for 30 min. The conventional culture medium for the tissue culture and rooting of the golden camellia is as follows: 1/2B5+ ABT0.5mg/L + IBA0.5mg/L + agar 5.5-6.5 g/L + white sugar 5.0-15 g/L + activated carbon 3.0g/L or 1/2B5+ ABT0.5mg/L + IBA0.5mg/L + agar 5.5-6.5 g/L + white sugar 5.0-15 g/L, and the pH of the culture medium is 5.8. Compared with the conventional culture medium, the culture medium uses 'fine river sand and vermiculite' to replace agar as a matrix, does not need to be boiled, and only needs to be elutriated and mixed with the matrix for subpackage (the conventional culture medium needs to be boiled by adding water into the agar until the agar is completely dissolved); in addition, the invention uses 'fine river sand and vermiculite' to replace agar as a matrix, has good air permeability and water retention, is beneficial to the induced germination of roots and improves the rooting rate. Fine river sand and vermiculite belong to porous inorganic substances, fine river sand dust has little good porosity and uniform gap distribution, but the water retention is poor; the vermiculite with moderate particles can increase the water retention of the matrix, and the porosity is moderate. The fine river sand (with the grain diameter of 1.0-2.0 mm) and vermiculite (with the grain diameter of 2.0-3.0 mm) are mixed to form a matrixThe fertilizer has good water retention, water drainage and air capacity, the pH value is about 5.5-6.5, the pH value is easy to regulate, and the water retention (nutrient solution) and air permeability are more suitable for root induction and growth. In addition, the invention uses 'river sand and vermiculite' as a substrate to replace agar, and the substrate is sugar-free culture in the early rooting culture period, the pollution rate can be controlled within 0.5 percent, and the pollution rate in the culture process is greatly reduced (once the conventional tissue culture uses sugar as a carbon source, and uses agar or carrageenan as a substrate, which are organic matters, and the sugar and the agar are the optimal culture medium for bacteria and fungi once infected by the bacteria or the fungi in the culture process, so that the pollution rate of the conventional tissue culture is 5-10 percent, and the great loss of tissue culture seedlings is caused. Finally, the method is characterized in that sugar-free culture is carried out at the early stage of rooting culture, which is favorable for promoting bud seedlings to induce root primordium, and the rooting time is shortened by 15-20 d (aiming at the conventional tissue culture of plants which are easy to root in vitro culture, cane sugar with the concentration of 2-5% is usually added into a culture medium so as to provide a carbon skeleton and energy required by the growth of the plants in an isolated culture state, and simultaneously the osmotic pressure of the culture medium is maintained.
Before the bud seedling is inserted, the bud seedling is preferably punched in a rooting culture medium, more preferably a sterile inoculating needle is used for punching, and because the leaves of the golden camellia are large, the bud seedling is preferably punched by about 10 holes on a culture bottle with the diameter of 5cm and the capacity of 200 mL; about 15 holes were punched in a flask having a diameter of 7.1cm and a capacity of 250 mL. The insertion process of the present invention is preferably performed using forceps. In the invention, the insertion depth is 1/3-1/2 of the height of the subculture sprout. After the insertion operation is complete, the present invention preferably caps the flasks and gently consolidate the substrate on the table so that the sprouts are in intimate contact with the substrate base. In the invention, in the first-stage rooting culture, the culture temperature is preferably 22-26 ℃. After the rooting culture at the first stage, most of the bud seedling base parts have germinated root buds (bud eyes).
The invention has high requirements on the quality of the bud seedlings. In the invention, carbon sources such as cane sugar or white sugar are not added in the early stage of rooting culture (one-stage rooting culture), the plantlet needs to survive in an autotrophic mode with weak photosynthetic capacity, and the transferred plantlet contains more than 3 unfolding leaves and can be used for synthesizing the carbon source under the action of weak photosynthesis. In the isolated culture and rooting stage of woody plants with difficult rooting, tissues are full (bud seedlings are thick and strong), cells contain more protoplasm, and the base parts are easy to form callus, so that the differentiation of root primordia is facilitated. When the rooting culture is carried out, the leaves of the part below 1.0cm of the bud seedling base are cut off, so that the bud seedling base can be conveniently inserted into a matrix (fine river sand and vermiculite), and the leaves are prevented from directly contacting the matrix for a long time in the culture process, so that the bud seedling is not favorable for rooting.
After the bud seedlings with root eyes on the bases are obtained, nutrient solution is added into the rooting culture medium, two-stage rooting culture is carried out for 30-35 d, and golden camellia tissue culture rooting bottle seedlings are obtained; the nutrient solution is prepared from sucrose or white sugar 15.0g/L and K2HPO415.0mg/L of aqueous solution. In the present invention, the sterilization conditions of the nutrient solution are preferably 121 ℃ and 1.1kg/cm of pressure2Sterilizing for 20 min. In the invention, the addition amount of the nutrient solution is preferably 1/2-2/3 of the volume of the original rooting culture medium. The adding mode of the nutrient solution is not specially limited, preferably under the aseptic condition, the culture bottle cap is opened, the sterilized pipette is used for transferring the nutrient solution, the nutrient solution is added into a culture bottle containing a rooting culture medium, and after the bottle cap is covered, two-stage rooting culture is carried out. In the invention, 15.0g/L of sucrose or white sugar is added in the two-stage rooting culture stage, so that a carbon source required by heterotrophic culture of the rooted seedlings can be provided (if 15.0g/L of sucrose or white sugar is not added after the seedlings are rooted, the leaves of the seedlings are yellowed and shed, the seedlings grow badly or die, and the like, and the carbon sources such as sucrose or white sugar are added in a culture medium, so that the requirements of cell nutrition, metabolism and growth under the heterotrophic condition of the seedlings are met). In the invention, K is added in the two-stage rooting culture stage2HPO415.0mg/L, thick green bud leaves and strong (K)2HPO4Rich in K+，K+Can obviously improve the absorption and utilization of nitrogen by plants and promote the synthesis of protein; can also promote photosynthesis; also improves the stress resistance of the plants. The golden camellia is used as a woody plant, and under the condition of in vitro culture, the cultivation of the stout rooting bud is favorable for bud seedling transplantation. Supplement of K at later stage of rooting culture2HPO415.0mg/L, can improve the lignification degree and the resistance of the stems of the rooting bottles). In the invention, in the two-stage rooting culture, the culture temperature is preferably 22-26 ℃. After the first-stage and second-stage rooting culture, the golden camellia rooting bottle seedlings are thick and strong, the leaves are dark green, the root systems are developed, the root lengths can reach 1.0-1.5 cm, the seedlings are about 3.0cm high, and 2-3 main roots are provided (see fig. 3 and fig. 4).
And (4) hardening and transplanting the tissue culture rooting bottle seedlings of the golden camellia to obtain the tissue culture seedlings of the golden camellia. In the invention, the cover of the culture bottle is preferably opened to improve the illumination intensity of the culture chamber or utilize natural scattered light. In the invention, the seedling exercising time is preferably 7-10 days. According to the method, the 'fine river sand and vermiculite' are used as the substrate instead of agar, the substrate does not need to be cleaned before the bottle seedlings with roots are transplanted, the bottle seedlings with roots are taken out and directly transplanted on the transplanting substrate, the root damage or root breakage caused by cleaning of the root agar before the bottle seedlings with roots are transplanted is also reduced, and the transplanting survival rate of the bottle seedlings with roots is improved. After the rooting culture in the first stage and the second stage, the seedling is hardened, and the transplanting survival rate of the bottle seedlings with roots reaches more than 98 percent. In the present invention, the matrix for transplantation preferably comprises loess and coconut coir mixed in a volume ratio of 3: 1. The disinfection and sterilization treatment of the transplanting matrix of the invention is preferably the same as that of other tissue culture seedling transplanting matrices. In the invention, the culture temperature is preferably 22-26 ℃ in the seedling hardening process.
In the invention, the light intensity is 150-300 lx in the first 15 days of the first stage rooting culture, and the light intensity is 1200-1500 lx in the first 16-35 days of the first stage rooting culture; in the process of the two-stage rooting culture, the light intensity is 1800-2300 lx; in the seedling exercising process, a fluorescent lamp is preferably used for assisting illumination to improve illumination intensity, or natural scattered light is used for avoiding direct sunlight. According to the method, 15d before a rooting culture stage, the golden camellia is cultured by adopting the light intensity of 150-300 lx, the browning rate of the bud seedlings can be greatly reduced, and the golden camellia is a strong shade plant and is extremely sensitive to illumination intensity, so that the bud seedlings can die due to strong light.
The tissue culture method has high efficiency, can greatly improve the rooting rate of tissue culture bud seedlings, shortens the rooting culture time, improves the seedling culture efficiency and reduces the seedling culture cost.
The method for improving the tissue culture rooting efficiency of the camellia nitidissima is further described in detail with reference to specific embodiments, and the technical scheme of the invention includes but is not limited to the following embodiments.
Example 1
Species of the golden camellia:
selecting and culturing 3 kinds of Camellia Chysantha with long rooting time and low rooting rate, such as lemon yellow Camellia Chysantha, Dongxing Camellia Chysantha, and hibiscus Camellia Chysantha.
Material (subculture sprout specification)
Single bud with top bud, seedling height above 2.0cm, seedling diameter above 0.5mm, more than 3 extended leaves with leaf length above 0.5cm, green or light green leaves, and smooth base cut.
Media preparation
A conventional culture medium for tissue culture and rooting of golden camellia: 1/2B5+ ABT0.5mg/L + IBA0.5mg/L + agar 5.5-6.5 g/L + white sugar 15 g/L; a conventional culture medium II, wherein the conventional culture medium I + 3.0g/L of active carbon; the pH of the culture medium is 5.8, and the culture medium is subjected to high temperature and high pressure (temperature is 121 deg.C, pressure is 1.1 kg/cm)2) Sterilizing for 30 min.
The one-stage rooting culture medium comprises the following components: fine river sand, vermiculite, 1/2B5, ABT, 0.5mg/L and IBA, 0.5mg/L and matrix pH of 5.8, and subjecting to high temperature and high pressure (temperature 121 deg.C, pressure 1.1 kg/cm)2) Sterilizing for 30 min. The two-stage nutrient solution of the invention contains 15.0g/L of cane sugar or white sugar and K2HPO415.0mg/L of an aqueous solution, and subjecting the aqueous solution to high temperature and high pressure (temperature 121 ℃ C., pressure 1.1 kg/cm)2) Sterilizing for 20 min.
The preparation method comprises the specific preparation operation processes of ① fine river sand (with the grain diameter of 1.0-2.0 mm) being washed by clear water and dried by filtration, ② dried fine river sand and vermiculite (with the grain diameter of 2.0-3.0 mm) being mixed according to the volume ratio of 1:1, ③ being prepared into 1/2B5+ ABT0.5mg/L + IBA0.5mg/L solution, ④ being prepared into the mixed fine river sand and vermiculite, soaking for 30min by taking the liquid amount as the basis for immersion, taking ⑤ the solid matrix from the liquid by using a filter screen in the state of water saturation, filtering out excessive water, taking hands as the basis for no water dripping, ⑥ being prepared into culture bottles, subpackaging the solid matrix with the thickness of about 2.0cm, sealing the culture bottles by ⑦ for later use after sterilization.
Inoculation operation flow
① under aseptic condition, cutting the camellia chrysantha subculture bud seedling into single buds with terminal buds and seedlings with height of more than 2.0cm by using an operating knife, ② cutting off leaves of the part below 1.0cm of the bud seedling base part for later use, ③ opening a culture bottle, punching holes on a culture substrate (fine river sand and vermiculite) by using a sterilized inoculating needle, wherein 10 holes are punched in the culture bottle with larger camellia chrysantha leaf blade, the diameter of which is usually 5cm and the capacity of which is 200mL, about 15 holes are punched in the culture bottle with the diameter of 7.1cm and the capacity of which is 250mL, ④ directly inserting the bud seedling into the culture substrate hole by using a forceps, the inserting depth is 1/3-1/2 of the bud seedling height, slightly pressing the substrate on a table top after ⑤ is covered with a bottle cover so that the bud seedling is in close contact with the substrate base part, ⑥ is placed in a culture rack for culture for 30-35 d, and most of the bud seedling base part is germinated (bud eye.
Carbohydrate supplementation protocol
Culturing for 30-35 days, after most bud seedling base roots, ① preparing cane sugar or white sugar 15.0g/L + K2HPO415.0mg/L solution, high-temperature and high-pressure sterilizing for later use, opening a culture bottle cap under ② aseptic conditions, transferring the solution into a culture bottle by using a sterilized pipette, adding the solution into the culture bottle, adding the nutrient solution into the culture bottle in an amount which is 1/2-2/3 of the volume of the solid matrix, putting back a culture shelf after covering the bottle cap with ③, continuously culturing the solution for 30-35 days by ④, keeping the leaves dark green and the root length of the solution to be 1.0-1.5 cm, keeping the ⑤ seedlings at 3.0cm high and having 2-3 main roots, and opening the bottle cap to harden the seedlings for 7-10 days.
Transplanting of rooted bottle seedlings
① the bottle seedlings of the birth roots are taken from the culture bottle and directly transplanted to the prepared transplanting substrate, ② the transplanting substrate is yellow core soil and coconut chaff (3: 1), ③ the transplanting method, the substrate disinfection and sterilization method and the management measures after the bottle seedlings of the birth roots are transplanted are similar to other tissue culture seedling transplanting management.
The requirements of illumination and temperature for rooting culture
The temperature of the culture room is 24 +/-2 ℃, a light source does not need to be specially supplemented above the culture shelf before culturing for 15 days, and the light intensity is 150-300 lx; culturing for 16-35 days, and supplementing a light source with the light intensity of 1200-1500 lx; in the two-stage rooting culture stage, the light intensity can be improved to 1800-2300 lx; before transplanting, hardening seedlings, natural scattered light can be fully utilized, and direct sunlight is avoided.
Compared with 2 conventional culture media (or culture methods), the rooting culture effect of the invention is as follows:
① the invention has high requirement for sprout quality, and can ensure high rooting efficiency, the specific sprout specification and shearing requirement include single sprout with top bud and height of more than 2.0cm, extended leaves with more than 3, leaf length of more than 0.5cm, green or light green leaf, smooth cut at base, and seedling diameter of more than 0.5mm, and leaf cutting at root of less than 1.0 cm.
Description of the drawings: in the invention, carbon sources such as sucrose or white sugar are not added in the earlier stage of the culture medium, and the plantlets need to survive in an autotrophic mode with weak photosynthetic capacity, and the transferred plantlets contain more than 3 leaves which are stretched and can be used for synthesizing the carbon source under the action of weak photosynthesis; in the isolated culture and rooting stage of the woody plant with difficult rooting, the tissue is fully recovered (the bud seedling is thick and strong), the cell contains more protoplasm, the base part is easy to form callus, and the differentiation of the root primordium is facilitated; when the rooting culture is carried out, the leaves of the part below 1.0cm of the bud seedling base are cut off, so that the bud seedling base can be conveniently inserted into a matrix (fine river sand and vermiculite), and the leaves are prevented from directly contacting the matrix for a long time in the culture process, so that the bud seedling is not favorable for rooting.
② the invention uses 'fine river sand and vermiculite' instead of agar as substrate, without boiling culture medium, only needs to elutriate, mix and split charging the substrate, the conventional culture medium needs to be boiled with water until the agar is completely dissolved.
③ the agar is replaced by the fine river sand and vermiculite as the substrate, which has good air permeability and water retention property, is beneficial to inducing the root to germinate and improves the rooting rate.
Description of the drawings: fine river sand and vermiculite belong to porous inorganic substances, fine river sand dust has little good porosity and uniform gap distribution, but the water retention is poor; the vermiculite with moderate particles can increase the water retention of the matrix, and the porosity is moderate. The mixed matrix of the fine river sand (with the particle size of 1.0-2.0 mm) and the vermiculite (with the particle size of 2.0-3.0 mm) has good water retention, water drainage and air capacity, the pH value is about 5.5-6.5, the pH value is easy to regulate, and the water retention (nutrient solution) and the air permeability are more suitable for root induction and growth. The results are shown in table 1, which is a comparison result of rooting rates and rooting effects of 3 golden camellia such as lemon yellow golden camellia, Dongxing golden camellia, and Fuseius golden camellia in different culture mediums (culture modes), wherein the average rooting rate of the 3 golden camellia subculture seedlings cultured in agar (conventional culture medium I) for 65d is 33%, the average rooting rate of the seedlings cultured in conventional culture medium II for 65d is 25.3%, and the rooting rate of conventional culture medium II is slightly lower than that of conventional culture medium I, and the rooting bottle seedlings cultured in 2 conventional culture mediums (culture modes) have few main roots, short roots and no fibrous roots; the fine river sand of the invention: the vermiculite (1:1) and the supplementary nutrient solution are cultured, the average rooting rate of 3 golden camellia subculture seedlings is 88%, the root system is developed, the number of main roots is more than 2.8 on average, and the number of fibrous roots is large.
FIGS. 1 to 4 show the growth and rooting of 65d seedlings cultured in different culture media (culture modes). FIG. 1 is a diagram showing the growth conditions of the rooted seedlings cultured for 65d by agar (conventional medium I), the rooting rate is low, and the seedlings have thick and crisp leaves; the growth vigor and root system condition of 65d seedlings cultured by agar (conventional medium I) are shown as follows: FIG. 2-A is a diagram showing the condition of rooting and root system of a bottle seedling for rooting, and FIG. 2-B is a diagram showing the condition of growth before the bottle seedling for rooting is taken out and transplanted, wherein the bottle seedling for rooting has few main roots, short and thin roots and no fibrous roots; fig. 3 is fine river sand: the growth condition of the bud seedlings (rooting bottle seedlings) cultured for 65d by vermiculite (1:1) and supplementary nutrient solution is shown, and the bud seedlings are thick and strong; fig. 4 is fine river sand: taking out the growth vigor and rooting condition diagram before transplanting of the rooting bottle seedlings cultured for 65 days by vermiculite (1:1) and supplementary nutrient solution, wherein the seedlings are thick and strong, and the root system is developed. According to the results, the roots of the sprouts on the agar culture medium are thin and short, the leaves of the sprouts are light green and small, and the fine river sand of the invention: the vermiculite (1:1) and the supplementary nutrient solution are cultured, the rooting bottle seedlings are developed in root systems, the number of main roots is more than 2.8 on average, and the number of fibrous roots is large.
Comparison of rooting rate and rooting effect of the Table 13 golden camellia in different culture media (culture modes)
④ the agar is replaced by the substrate of 'fine river sand and vermiculite', and the early stage of rooting culture is sugar-free culture, the pollution rate can be controlled within 0.5%, and the pollution rate in the culture process is greatly reduced.
Description of the drawings: the conventional tissue culture takes sugar as a carbon source and agar or carrageenan as a matrix, which are organic matters; once infected by bacteria or fungi in the culture process, sugar and agar are the optimal culture medium for the bacteria and the fungi, so that the pollution rate of conventional tissue culture is 5-10%, and the great loss of tissue culture seedlings is caused.
In the invention, the fine river sand and the vermiculite are inorganic mineral substances, and no carbon sources such as white sugar and the like are added in the early stage of rooting culture, so that the propagation conditions of bacteria and fungi are avoided in the culture process, and the tissue culture seedling is not easily polluted in a large amount.
⑤ the early stage of rooting culture is sugar-free culture, which is beneficial to promoting the bud seedling to induce root primordium, and the rooting time is shortened by 15-20 days.
Description of the drawings: according to the conventional tissue culture of the plant easy to root in the isolated culture, the sucrose with the concentration of 2-5% is usually added into a culture medium so as to provide a carbon skeleton and energy required by the growth of the plant in the isolated culture state, and meanwhile, the osmotic pressure of the culture medium is maintained. And (4) conventionally culturing (in an agar and sugar culture mode), wherein the rooting culture time of the golden camellia such as lemon yellow golden camellia, Dongxing golden camellia, hibiscus golden camellia or clusterin golden camellia is 75-90 days.
In the early stage of rooting culture, namely before the root primordium is formed, carbon sources such as sucrose or white sugar are not added, so that the influence of the osmotic pressure of the culture medium on the induction of the root primordium is reduced, the rooting time is shortened, and the rooting rate of in-vitro culture of the plant with difficulty in rooting is improved. Taking the tissue culture sprout of the lemon yellow golden camellia as a material, wherein the comparison result of the rooting conditions of the sprout added with sugar and the sprout not added with sugar at the early stage of rooting culture is shown in table 2, the culture time is 25d, 100 sprouts are randomly sampled and checked, the sprout of the sprout without sugar at the early stage of rooting culture reaches 55, the sprout with sugar at the early stage of rooting culture only has 23, and the sprout without sugar at the early stage of rooting culture has 32 more sprouts than the sprout with sugar at the bud of the sprout; the culture time is 35d, 100 seedlings are randomly sampled and checked, the number of the seedlings which are not added with sugar in the early stage of rooting culture and germinate root eyes reaches 84, the number of the seedlings which are added with sugar in the early stage of rooting culture and germinate root eyes reaches 48, and the number of the seedlings which are not added with sugar in the early stage of rooting culture is 36 more than that of the seedlings which are added with sugar and germinate root eyes; in conclusion, the method is favorable for inducing the bud seedlings to root without adding sugar in the early stage of rooting culture.
TABLE 2 comparison of the rooting of seedlings with and without sugar at the early stage of rooting culture
⑥ at the later stage of rooting culture, adding sucrose or white sugar 15.0g/L to provide carbon source for heterotrophic growth of rooted bud.
Description of the drawings: if 15.0g/L of cane sugar or white sugar is not added after the bud seedlings take root, the leaves of the bud seedlings are yellowed and fall off, and the bud seedlings grow badly or die. Carbon sources such as sucrose or white sugar are added into the culture medium, so that the requirements of cell nutrition, metabolism and growth under the heterotrophic condition of the bud seedlings are met. The method comprises the following steps of taking a tissue culture seedling of lemon yellow golden camellia as a material, and randomly sampling and screening 100 seedlings when the culture time is 50d (15 d after adding sugar), wherein the result of comparison of growth conditions of the seedlings with sugar and the seedlings without sugar is shown in Table 3, the seedlings with sugar are 0 in the later stage of rooting culture, and the seedlings with sugar and the yellow leaves are 11 in the later stage of rooting culture; when the culture time is 65d (30 d after adding sugar), randomly sampling and spot-checking 100 seedlings, wherein the seedlings with the yellow leaves added with sugar at the later stage of rooting culture are 0 seedlings, and the seedlings with the yellow leaves without sugar at the later stage of rooting culture are 29 seedlings; in summary, the requirements of cell nutrition, metabolism and growth under the heterotrophic condition of the bud seedling can be met only by adding sugar at the later stage of rooting culture, and the normal growth of the bud seedling is ensured.
TABLE 3 comparison of growth of seedlings with and without sugar at the late stage of rooting culture
⑦ supplement K at the later stage of rooting culture2HPO415.0mg/L, the leaves of the bud seedlings are dark green, the stems of the seedlings are thick, and the transplanting survival rate reaches more than 98 percent.
Description of the drawings: k2HPO4Rich in K+，K+Can obviously improve the absorption and utilization of nitrogen by plants and promote the synthesis of protein; can also promote photosynthesis; also improves the stress resistance of the plants. The golden camellia is used as a woody plant, and under the condition of in vitro culture, the cultivation of the stout and strong rooted seedlings is undoubtedly favorable for the transplantation of the seedlings. So that K is supplemented in the later stage of rooting culture2HPO415.0mg/L, and improves the lignification degree and the resistance of the stems of the rooted bottle seedlings. Adding or not adding K at the later stage of rooting culture2HPO4The comparison result of the growth potential of the 15.0mg/L bud seedlings is shown in Table 4, the culture time is 65d, 100 bud seedlings are randomly sampled and checked, and K is supplemented at the later stage of rooting culture2HPO4The average stem thickness of 15.0mg/L bud seedlings reaches 2.16mm (the thickest is 2.8mm and the thinnest is 1.8mm), the bud seedlings are transplanted for 50 days, and the transplanting survival rate reaches 98 percent; no addition of K in the later stage of rooting culture2HPO4The average stem thickness of 15.0mg/L bud seedlings is 1.63mm (the thickest is 2.1mm and the thinnest is 1.3mm), the bud seedlings are transplanted for 50 days, and the transplanting survival rate is 87%; to sum up, K is supplemented at the later stage of rooting culture2HPO4No K is added in 15.0mg/L bud seedlings2HPO415.0mg/L of bud seedlings are thick and strong, and the transplanting survival rate is improved by 11 percent.
TABLE 4 addition and non-addition of K at the late stage of rooting culture2HPO4Comparison of growth potential of 15.0mg/L seedlings
⑧ the temperature of the culture room is 24 +/-2 ℃, the light intensity is 150-300 lx without special light source above the culture shelf for 15 days before culture, the light intensity can be supplemented for 16-35 days before culture, the light intensity is 1200-1500 lx, the light intensity can be improved to 1800-2300 lx during the two-stage rooting culture process, and the natural scattered light can be fully utilized to avoid direct sunlight during seedling hardening before transplanting.
Description of the drawings: in the early stage of rooting culture (the first 15 days), the culture is carried out by adopting light intensity of 150-300 lx, the browning rate of the bud seedlings can be greatly reduced, and the golden camellia is a strong shadow plant and is extremely sensitive to illumination intensity, and the bud seedlings can die due to strong light.
The method can effectively improve the tissue culture seedling raising efficiency and the transplanting survival rate, reduce the seedling raising cost and solve the problem of tissue culture and rapid propagation of the golden camellia plants with difficult rooting.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for improving the tissue culture rooting efficiency of golden camellia comprises the following steps:
1) inserting the camellia chrysantha subculture bud into a rooting culture medium for carrying out first-stage rooting culture for 30-35 d to obtain a bud seedling with a root eye at the base; the subculture bud seedling is a single bud with a terminal bud, a seedling height of more than 2.0cm, a seedling diameter of more than 0.5mm, more than 3 unfolding leaves, a leaf length of more than 0.5cm, a leaf turning from light red to green or light green, and a smooth base cut; the rooting culture medium is a solid matrix soaked in a liquid culture medium, and the solid matrix comprises fine river sand and vermiculite; before the insertion, cutting off the leaves of the part below 1.0cm of the base of the subculture bud seedlings;
2) after obtaining bud seedlings with root eyes at the base parts, adding nutrient solution into the rooting culture medium in the step 1), and performing two-stage rooting culture for 30-35 d to obtain golden camellia tissue culture rooting bottle seedlings; the nutrient solution is prepared from sucrose or white sugar 15.0g/L and K2HPO415.0mg/L aqueous solution;
3) hardening and transplanting the camellia chrysantha tissue culture rooting bottle seedlings obtained in the step 2) to obtain camellia chrysantha tissue culture seedlings.
2. The method of claim 1, wherein the Camellia nitidissima comprises lemon yellow Camellia nitidissima, Dongxing Camellia nitidissima, Euseiulus Camellia nitidissima, or Coleus forskohlii Camellia nitidissima.
3. The method as claimed in claim 1, wherein in step 1), the liquid culture medium is 1/2B5 medium as a minimal medium, 0.5mg/L ABT and 0.5mg/L IBA are added, and the pH value is 5.5-6.5.
4. The method according to claim 1, wherein the particle size of the fine river sand is 1 to 2mm, and the particle size of the vermiculite is 2 to 3 mm.
5. The method of claim 1, 3 or 4, wherein the rooting medium is prepared by a method comprising the steps of:
① mixing the cleaned and drained fine river sand and vermiculite according to the volume ratio of 1:1 to obtain a solid matrix;
② soaking the solid matrix in the liquid culture medium until the liquid culture medium absorbed by the solid matrix is saturated, filtering, and removing the liquid culture medium to obtain rooting culture medium.
6. The method according to claim 1, wherein the secondary sprouts of step 1) are inserted into the rooting medium to a depth of 1/3-1/2 of the height of the secondary sprouts.
7. The method according to claim 1, wherein the culture solution of step 2) is added in an amount of 1/2-2/3% of the volume of the primary root medium.
8. The method as claimed in claim 1, wherein the matrix for transplanting in step 3) comprises a mixture of loess and coconut coir in a volume ratio of 3: 1.
9. The method according to claim 1, wherein the temperature for the first stage rooting culture, the second stage rooting culture and the seedling exercising is 22-26 ℃.
10. The method according to claim 1, wherein the light intensity of the first 15d of the first stage rooting culture is 150-300 lx, the light intensity of the first 16-35 d of the first stage rooting culture is 1200-1500 lx; in the process of the two-stage rooting culture, the light intensity is 1800-2300 lx; and in the seedling hardening stage before transplanting, natural scattered light is kept, and direct sunlight is avoided.
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