CN112655553A - Rapid sterile short-shoot propagation method for Orthosiphon aristatus - Google Patents
Rapid sterile short-shoot propagation method for Orthosiphon aristatus Download PDFInfo
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Abstract
The invention relates to the technical field of plant tissue culture and rapid propagation, in particular to a method for rapidly propagating aseptic short branches of Chinese alpine rush, which is completed by the steps of pretreatment and cultivation of the Chinese alpine rush, cleaning of explants, disinfection of the explants, primary culture, secondary culture, rooting culture, hardening off and transplanting of tissue culture seedlings and the like. The invention adopts various spectral bactericides to carry out omnibearing disinfection treatment on the substrate, the transplanted seedling and the growth space for cultivating the Chinese alpine rush explant, thereby avoiding the influence of pathogenic bacteria and other interfering substances carried by the explant on tissue culture. The stem section with axillary buds obtained by pre-culturing the Orthosiphon aristatus as the explant material is screened out the explant disinfection method suitable for the rapid propagation of sterile brachycephala and the culture medium formulas in different culture stages, so that the efficiency and the yield of the rapid propagation of the Orthosiphon aristatus are improved. The technology of the invention has the characteristics of short period, quick seedling formation, stable genetic character, simple culture process, consistent seedling growth and the like, and can be used for large-scale industrialized production of the Chinese alpine rush to meet the market demand.
Description
Technical Field
The invention relates to the technical field of plant tissue culture and rapid propagation, in particular to a method for rapidly propagating Chinese alpine rush aseptic short branches.
Background
Clerodendranthus spicatus (Cleriodendanthus spicatus. C.Y.Wu) is a perennial herb of Clerodendranthus of Labiatae, with upright stem, square edge, purple or white corolla, and flowering and fruiting period of 5-11 months. The Orthosiphon aristatus is widely distributed in tropical and subtropical regions, and China is mainly distributed in southern Guangdong, southern Guangxi, southern Yunnan, Taiwan and Fujian regions; the soil is preferably loose and fertile sandy loam which is fond of warm and humid climate and is grown in a wet place under a forest, can be planted on flat ground and gentle slope, has low requirement on illumination, can be cultivated under full illumination and can grow better under certain shading conditions. The Chinese herbal medicine is used for treating acute and chronic nephritis, cystitis and lithangiuria in folk, and has good effect on rheumatic arthritis. Modern researches show that the Orthosiphon aristatus has seven medical health care functions of diuresis, calculus removal, antibiosis, inflammation diminishing, kidney strengthening, chronic renal failure improvement, immunity improvement and the like. As a plant with great development prospect, the market demand is increasingly expanded due to important pharmacological action of the Chinese alpine rush. However, the fruiting rate of the Orthosiphon aristatus is low, the seed life is only 15 days, the germination rate is only 40%, the wild medicinal material resources are gradually exhausted, and the traditional production and propagation mode is difficult to meet the market demand. The problem of providing healthy seedlings required by the large-scale planting of the Chinese alpine rush by adopting a plant tissue culture and rapid propagation technology is urgently needed to be solved.
In recent years, researches on tissue culture of the Orthosiphon aristatus have been made in China, but the research on in-vitro rapid propagation of the Orthosiphon aristatus by adopting an aseptic short-shoot rapid propagation technology is rare by a technical route of regenerating plants through a callus generation or cluster bud generation way. The sterile short branch rapid propagation technology is similar to micro cuttage, and refers to a propagation method that an explant carries a stem section with axillary buds, in-vitro culture is carried out on the stem section with the axillary buds in an artificial culture medium and under proper conditions, so that a new branch grows out, then the new branch is cut into the stem section with the axillary buds, and the stem section is subjected to subculture and then rooted into seedlings. The sterile short-branch rapid propagation technology has the advantages of fast seedling formation, no callus induction stage, stable hereditary character, simple culture process and easy survival after transplantation, thereby being more suitable for large-scale industrial production.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for quickly propagating aseptic short branches of Orthosiphon aristatus.
The invention relates to a method for rapidly propagating aseptic short branches of Orthosiphon aristatus, which specifically comprises the following steps:
step one, pretreatment and cultivation of the Chinese alpine rush:
s1: selecting healthy and strong cat grass without diseases and insect pests, carefully digging up the cat grass, shaking off soil at roots, washing with clear water, pruning and shearing off old leaves at the positions of branches 3-4 cm away from the ground to keep leafstalks, and soaking and disinfecting the pruned branches for 1-2 hours by using imidazole amide with the concentration of 0.4% or mancozeb liquid medicine with the concentration of 0.2%;
s2: after soaking, transplanting the seedlings into a flowerpot filled with a culture medium sterilized by 350 times of 50% water-soluble amobam solution, and transferring the seedlings into a sealable plastic greenhouse;
s3: after the transplanted seedlings are planted, mixed solution of 0.4% miamide and 0.2% mancozeb is alternately used every 7 days, or the 0.4% miamide is singly used, or the 0.2% mancozeb liquid is singly used for spraying seedling plants, the ground and the periphery of a plastic greenhouse once; when young bud segments are newly drawn out from the top of the Chinese alpine rush, cutting the young bud segments from the base part to be used as explants for the sterile short branch rapid propagation;
step two, cleaning explants: taking the explant obtained by pretreatment and cultivation of the Chinese alpine rush back to a laboratory, cutting off leaves, washing with clear water once, cutting into 2-3 cm stem sections with a pair of axillary buds, soaking for 5 minutes with a detergent solution, washing for 20 minutes with running water, draining water, filling into a sterilized empty bottle, and putting into a super clean bench for later use;
step three, disinfection of explants: transferring the explant to a new sterilized empty bottle in an ultraclean workbench, pouring 75% alcohol to soak the explant for 10-20 s, washing the explant once with sterilized water, soaking the explant with 0.1% mercuric chloride solution for 4-8 min, continuously shaking the explant during the period, washing the explant with sterilized water for 6-8 times, and inoculating the explant after the explant is dried by using sterilized paper;
step four, primary culture: cutting off the outer edge part of the sterilized explant by using an inoculating knife in a super-clean workbench, inoculating the sterile short branch stem section with a pair of axillary buds into an inoculating disc filled with a primary culture medium according to polarity by using tweezers, and culturing for 15-30 days in a culture room; then growing into 2-5 cm tissue culture seedlings; MS minimal medium is selected for primary culture, and 0.5-2.0 mg/L, NAA 0.1.1-1.0 mg/L of 6-BA, 20-30 g/L of cane sugar and 7g/L of agar are added; adjusting the pH value to 5.8-6.0;
step five, subculturing: shearing the primary culture tissue culture seedlings obtained in the fourth step into sterile short branch stem sections with a pair of axillary buds, inoculating the sterile short branch stem sections into an inoculating tray filled with a subculture medium according to polarity, and culturing for 15-30 days in a culture room; then growing into 2-5 cm tissue culture seedlings; the subculture medium is MS minimal medium with 0.01-0.1 mg/L, IBA 0.1.1-1.0 mg/L TDZ, 20-30 g/L sucrose and 7g/L agar; adjusting the pH value to 5.8-6.0;
step six, rooting culture: cutting off the subculture tissue culture seedlings in the fifth step from the base, placing the cut subculture tissue culture seedlings in a rooting culture medium, and culturing for 15-30 days in a culture room; the root length can reach 4-6 cm, and the root hair is white; the rooting culture medium takes 1/2MS as a basic culture medium, and 0.5-2.0 mg/L, IBA 0.5.5-2.0 mg/L of NAA, 2-3 g/L of active carbon, 20-30 g/L of sucrose and 7g/L of agar are added; adjusting the pH value to 5.8-6.0;
seventhly, hardening and transplanting the tissue culture seedlings: when the seedlings in the rooting culture medium grow to 3-5 cm high and the number of roots reaches 6-10, moving the seedlings into a plastic greenhouse, and hardening the seedlings under natural light for 7-10 days; and taking out the culture flask, cleaning a culture medium at the bottom of the culture flask, soaking the culture flask in a carbendazim solution for 5-7 min, transplanting the culture flask into a sterilized culture medium, and growing new leaves of the orthosiphon aristatus to show that the culture flask is transplanted to survive.
Further, before the sealable plastic greenhouse of S2 in the first step is moved in, the ground and the surroundings are sterilized with 0.4% of imamide and 0.2% of mancozeb.
Further, in the step two, when young bud segments newly extracted from the tops of the orthosiphon aristatus are grown to 8-10 cm in S3, the young bud segments are cut off from the base part at two o' clock in the afternoon on a sunny day and are used as explants for the rapid propagation of sterile short branches;
further, 1-2 sterile brachytic stems are inoculated in each bottle, and each sterile brachytic stem has a pair of axillary buds.
Further, the culture conditions in the fourth step, the fifth step and the sixth step are all culture temperature of 23-28 ℃ and illumination intensity of 2000-2500 lx.
Further, the culture substrate in the first step and the seventh step is prepared by mixing the components in a volume ratio of 1: 1: 1, the sterilization mode is as follows: 50 percent of water-soluble amobam 350 times solution is uniformly sprayed with 3 kilograms of diluent per square meter of culture medium.
Further, the 75% alcohol and 0.1% mercuric chloride are prepared one day before inoculation; the sterile water, the sterile paper, the inoculation tray, the empty bottle, the forceps and the inoculation knife are sterilized at 121 ℃ for 35min, and the culture medium is sterilized at 121 ℃ for 18 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, the Chinese alpine rush is pretreated and cultivated before the sterile short branches are rapidly propagated, and the matrix, the transplanted seedlings and the growth space for cultivating the Chinese alpine rush explant are subjected to comprehensive disinfection treatment by adopting various spectral bactericides, so that the influence of interference substances such as germs carried by the explant on tissue culture is avoided.
(2) According to the invention, the axillary bud stem section obtained by pre-culturing the Orthosiphon aristatus is taken as the explant material, and the explant disinfection method suitable for the rapid propagation of sterile brachycephala and the culture medium formulas in different culture stages are screened out, so that the efficiency and the yield of the rapid propagation of the Orthosiphon aristatus are improved.
(3) The sterile short-branch rapid propagation technology adopted by the invention has the characteristics of short period, rapid seedling formation, stable hereditary character, simple culture process, consistent seedling growth and the like, and can be used for large-scale industrialized production of the eupatorium odoratum to meet the market demand.
Drawings
FIG. 1 shows the explant material for the sterile short shoot rapid propagation of Orthosiphon aristatus;
FIG. 2 is a schematic diagram showing the explant of sterile brachypodium distichum which is newly extracted from young shoots of rapid propagation;
FIG. 3 shows the cultivation of sterile short branches of Orthosiphon aristatus in primary medium for 5 days;
FIG. 4 shows the cultivation of sterile short branches of Orthosiphon aristatus in primary medium for 20 days;
FIG. 5 shows the cultivation of sterile short Clerodendranthus spicatus in a subculture medium for 15 days;
FIG. 6 shows the white root hairs of the sterilized short branches of Orthosiphon aristatus growing in the rooting medium;
FIG. 7 shows the transplanted sterile short-shoot tissue-cultured plantlets of Orthosiphon aristatus.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The invention relates to a method for rapidly propagating aseptic short branches of Orthosiphon aristatus, which specifically comprises the following steps:
step one, pretreatment and cultivation of the Chinese alpine rush:
s1: selecting healthy and strong cat grass without diseases and insect pests, carefully digging up the cat grass, shaking off soil at roots, washing with clear water, pruning and shearing off old leaves at the positions of branches 3-4 cm away from the ground to keep leafstalks, and soaking and disinfecting the pruned branches for 1.5 hours by using imidazole amide with the concentration of 0.4% or mancozeb liquid medicine with the concentration of 0.2%;
s2: after soaking, transplanting the seedlings into a flowerpot filled with a culture substrate sterilized by 350 times of 50% ambam water solution, uniformly spraying 3 kilograms of diluent per square meter of the culture substrate, and transferring the culture substrate into a sealable plastic greenhouse; before the plastic greenhouse is moved in, the ground and the periphery are disinfected by 0.4 percent of miamide and 0.2 percent of mancozeb;
s3: after the transplanted seedlings are planted, mixed solution of 0.4% miamide and 0.2% mancozeb is alternately used every 7 days, or the 0.4% miamide is singly used, or the 0.2% mancozeb liquid is singly used for spraying seedling plants, the ground and the periphery of a plastic greenhouse once; when young bud segments newly drawn out from the tops of the orthosiphon aristatus are grown to 8-10 cm, the young bud segments are cut off from the base part at 2 o' clock in the afternoon of a sunny day and are taken back to a laboratory to serve as explants for sterile short-shoot rapid propagation;
step two, cleaning explants: taking the explant obtained by pretreatment and cultivation of the Orthosiphon aristatus back to a laboratory, cutting off leaves by using scissors, washing once by using clear water, cutting into 2-3 cm stem sections (shown in attached figures 1 and 2) with a pair of axillary buds, soaking for 5 minutes by using a detergent solution, washing for 20 minutes by using running water, controlling water content, filling into a sterilized empty bottle, and putting into an ultra-clean workbench for later use;
step three, disinfection of explants: transferring the explant to a new sterilized empty bottle by using a pair of tweezers in a clean bench, pouring 75% alcohol to soak the explant for 20s, washing the explant with sterilized water for one time, soaking the explant with 0.1% mercuric chloride solution for 8min, shaking the explant continuously during the period, washing the explant with sterilized water for 7 times, sucking the explant with sterilized paper, and then inoculating the explant;
step four, primary culture: cutting the outer brown stain part of the sterilized explant by using an inoculating knife in a superclean workbench, inoculating the sterile short-branch stem segments with a pair of axillary buds into an inoculating tray filled with a primary culture medium according to polarity by using tweezers, and inoculating 1-2 sterile short-branch stem segments into each bottle (as shown in attached figure 3); MS basic culture medium is selected for primary culture, and added with 1.0mg/L of 6-BA, 1.0mg/L of NAA, 30g/L of cane sugar and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 20 days under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 20 days (as shown in figure 4);
step five, subculturing: shearing the primary culture tissue culture seedling obtained in the fourth step into sterile short branch stem sections with a pair of axillary buds, inoculating the sterile short branch stem sections into an inoculation tray filled with a subculture medium according to polarity, and inoculating 1-2 sterile short branch stem sections into each bottle; the subculture medium is MS minimal medium with TDZ 0.05mg/L, IBA0.2mg/L, sucrose 20g/L and agar 7 g/L; adjusting the pH value to 5.8-6.0; after inoculation, culturing for 15 days in a culture room under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 15 days (as shown in figure 5);
step six, rooting culture: cutting off the subculture tissue culture seedlings in the fifth step from the base, placing the subculture tissue culture seedlings in a rooting culture medium, and inoculating one sterile short-shoot tissue culture seedling into each bottle; the rooting culture medium takes 1/2MS as a basic culture medium, and is added with 1.0mg/L, IBA 1.0.0 mg/L of NAA, 2.5g/L of active carbon, 20-30 g/L of cane sugar and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 25 days under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, wherein the root length is 4-6 cm, the root hair is white, and the rooting rate is more than 98% (as shown in figure 6);
seventhly, hardening and transplanting the tissue culture seedlings: when the seedlings in the rooting culture medium grow to 3-5 cm in height and the number of the roots of the tissue culture seedlings reaches 6-10, transferring the seedlings into a plastic greenhouse, and training the seedlings for 7 days under natural light; then taking out the culture medium from a culture bottle, cleaning the culture medium at the bottom of the culture medium, soaking the culture medium in a carbendazim solution for 5min, transplanting the culture medium into a sterilized culture medium which is 350 times of 50% ambam water, uniformly spraying 3 kilograms of diluent on each square meter of the culture medium, and growing new leaves of the Chinese alpine rush to show that the Chinese alpine rush is transplanted to survive (see the attached drawing 7 for details), wherein the volume ratio of the culture medium is 1: 1: 1 perlite, fine river sand and peat soil.
Wherein 75% alcohol and 0.1% mercuric chloride are prepared one day before inoculation; sterilizing sterile water, sterile paper, a seed inoculating tray, an empty bottle, tweezers and an inoculating knife for 35min at 121 ℃, and sterilizing the culture medium for 18min at 121 ℃.
Example 2
The invention relates to a method for rapidly propagating aseptic short branches of Orthosiphon aristatus, which specifically comprises the following steps:
step one, pretreatment and cultivation of the Chinese alpine rush:
s1: selecting healthy and strong cat grass without diseases and insect pests, carefully digging up the cat grass, shaking off soil at roots, washing with clear water, pruning and shearing off old leaves at the positions of branches 3-4 cm away from the ground to keep leafstalks, and soaking and disinfecting the pruned branches for 1 hour by using imidazole amide with the concentration of 0.4% or mancozeb liquid medicine with the concentration of 0.2%;
s2: after soaking, transplanting the seedlings into a flowerpot filled with a culture substrate sterilized by 350 times of 50% ambam water solution, uniformly spraying 3 kilograms of diluent per square meter of the culture substrate, and transferring the culture substrate into a sealable plastic greenhouse; before the plastic greenhouse is moved in, the ground and the periphery are disinfected by 0.4 percent of miamide and 0.2 percent of mancozeb;
s3: after the transplanted seedlings are planted, mixed solution of 0.4% miamide and 0.2% mancozeb is alternately used every 7 days, or the 0.4% miamide is singly used, or the 0.2% mancozeb liquid is singly used for spraying seedling plants, the ground and the periphery of a plastic greenhouse once; when young bud segments newly drawn out from the tops of the orthosiphon aristatus are grown to 8-10 cm, the young bud segments are cut off from the base part at 2 o' clock in the afternoon of a sunny day and are taken back to a laboratory to serve as explants for sterile short-shoot rapid propagation;
step two, cleaning explants: taking the explant obtained by pretreatment and cultivation of the Orthosiphon aristatus back to a laboratory, cutting off leaves by using scissors, washing once by using clear water, cutting into 2-3 cm stem sections (shown in attached figures 1 and 2) with a pair of axillary buds, soaking for 5 minutes by using a detergent solution, washing for 20 minutes by using running water, controlling water content, filling into a sterilized empty bottle, and putting into an ultra-clean workbench for later use;
step three, disinfection of explants: transferring the explant to a new sterilized empty bottle by using a pair of tweezers in a clean bench, pouring 75% alcohol to soak the explant for 10s, washing the explant with sterilized water for one time, soaking the explant with 0.1% mercuric chloride solution for 5min, shaking the explant continuously during the period, washing the explant with sterilized water for 7 times, sucking the explant with sterilized paper, and then inoculating the explant;
step four, primary culture: cutting the outer brown stain part of the sterilized explant by using an inoculating knife in a superclean workbench, inoculating the sterile short-branch stem segments with a pair of axillary buds into an inoculating tray filled with a primary culture medium according to polarity by using tweezers, and inoculating 1-2 sterile short-branch stem segments into each bottle (as shown in attached figure 3); MS minimal medium is selected for primary culture with the addition of 0.5mg/L of 6-BA, 0.1mg/L of NAA0.1mg/L, 20g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 20 days under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 20 days (as shown in figure 4);
step five, subculturing: shearing the primary culture tissue culture seedling obtained in the fourth step into sterile short branch stem sections with a pair of axillary buds, inoculating the sterile short branch stem sections into an inoculation tray filled with a subculture medium according to polarity, and inoculating 1-2 sterile short branch stem sections into each bottle; the subculture medium is MS minimal medium with 0.01mg/L of TDZ, 0.1mg/L of IBA0.1mg/L, 20-30 g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing for 15 days in a culture room under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 15 days (as shown in figure 5);
step six, rooting culture: cutting off the subculture tissue culture seedlings in the fifth step from the base, placing the subculture tissue culture seedlings in a rooting culture medium, and inoculating one sterile short-shoot tissue culture seedling into each bottle; the rooting culture medium takes 1/2MS as a basic culture medium, and is added with 2.0mg/L, IBA 2.0.0 mg/L of NAA, 3g/L of active carbon, 25g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 25 days under the conditions that the temperature is 23-25 ℃ and the illumination intensity is 2000-2500 lx, wherein the root length is 4-6 cm, the root hair is white, and the rooting rate is more than 98% (as shown in figure 6);
seventhly, hardening and transplanting the tissue culture seedlings: when the seedlings in the rooting culture medium grow to 3-5 cm in height and the number of the roots of the tissue culture seedlings reaches 6-10, transferring the seedlings into a plastic greenhouse, and training the seedlings for 7 days under natural light; then taking out the culture medium from a culture bottle, cleaning the culture medium at the bottom of the culture medium, soaking the culture medium in a carbendazim solution for 5min, transplanting the culture medium into a sterilized culture medium which is 350 times of 50% ambam water, uniformly spraying 3 kilograms of diluent on each square meter of the culture medium, and growing new leaves of the Chinese alpine rush to show that the Chinese alpine rush is transplanted to survive (see the attached drawing 7 for details), wherein the volume ratio of the culture medium is 1: 1: 1 perlite, fine river sand and peat soil.
Wherein 75% alcohol and 0.1% mercuric chloride are prepared one day before inoculation; sterilizing sterile water, sterile paper, a seed inoculating tray, an empty bottle, tweezers and an inoculating knife for 35min at 121 ℃, and sterilizing the culture medium for 18min at 121 ℃.
Example 3
The invention relates to a method for rapidly propagating aseptic short branches of Orthosiphon aristatus, which specifically comprises the following steps:
step one, pretreatment and cultivation of the Chinese alpine rush:
s1: selecting healthy and strong cat grass without diseases and insect pests, carefully digging up the cat grass, shaking off soil at roots, washing with clear water, pruning and shearing off old leaves at the positions of branches 3-4 cm away from the ground to keep leafstalks, and soaking and disinfecting the pruned branches for 2 hours by using imidazole amide with the concentration of 0.4% or mancozeb liquid medicine with the concentration of 0.2%;
s2: after soaking, transplanting the seedlings into a flowerpot filled with a culture substrate sterilized by 350 times of 50% ambam water solution, uniformly spraying 3 kilograms of diluent per square meter of the culture substrate, and transferring the culture substrate into a sealable plastic greenhouse; before the plastic greenhouse is moved in, the ground and the periphery are disinfected by 0.4 percent of miamide and 0.2 percent of mancozeb;
s3: after the transplanted seedlings are planted, mixed solution of 0.4% miamide and 0.2% mancozeb is alternately used every 7 days, or the 0.4% miamide is singly used, or the 0.2% mancozeb liquid is singly used for spraying seedling plants, the ground and the periphery of a plastic greenhouse once; when young bud segments newly drawn out from the tops of the orthosiphon aristatus are grown to 8-10 cm, the young bud segments are cut off from the base part at 2 o' clock in the afternoon of a sunny day and are taken back to a laboratory to serve as explants for sterile short-shoot rapid propagation;
step two, cleaning explants: taking the explant obtained by pretreatment and cultivation of the Orthosiphon aristatus back to a laboratory, cutting off leaves by using scissors, washing once by using clear water, cutting into 2-3 cm stem sections (shown in attached figures 1 and 2) with a pair of axillary buds, soaking for 5 minutes by using a detergent solution, washing for 20 minutes by using running water, controlling water content, filling into a sterilized empty bottle, and putting into an ultra-clean workbench for later use;
step three, disinfection of explants: transferring the explant to a new sterilized empty bottle by using a pair of tweezers in a clean bench, pouring 75% alcohol to soak the explant for 20s, washing the explant with sterilized water for one time, soaking the explant with 0.1% mercuric chloride solution for 8min, shaking the explant continuously during the period, washing the explant with sterilized water for 8 times, sucking the explant with sterilized paper, and then inoculating the explant;
step four, primary culture: cutting the outer brown stain part of the sterilized explant by using an inoculating knife in a superclean workbench, inoculating the sterile short-branch stem segments with a pair of axillary buds into an inoculating tray filled with a primary culture medium according to polarity by using tweezers, and inoculating 1-2 sterile short-branch stem segments into each bottle (as shown in attached figure 3); MS basic culture medium is selected for primary culture, and 6-BA2.0mg/L, NAA0.5mg/L, cane sugar 25g/L and agar 7g/L are added; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 20 days under the conditions that the temperature is 25-28 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 20 days (as shown in figure 4); step five, subculturing: shearing the primary culture tissue culture seedling obtained in the fourth step into sterile short branch stem sections with a pair of axillary buds, inoculating the sterile short branch stem sections into an inoculation tray filled with a subculture medium according to polarity, and inoculating 1-2 sterile short branch stem sections into each bottle; the subculture medium is MS basic culture medium with 0.1mg/L of TDZ, 1.0mg/L of IBA1, 25g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing for 15 days in a culture room under the conditions that the temperature is 25-28 ℃ and the illumination intensity is 2000-2500 lx, and growing into 2-5 cm tissue culture seedlings after 15 days (as shown in figure 5);
step six, rooting culture: cutting off the subculture tissue culture seedlings in the fifth step from the base, placing the subculture tissue culture seedlings in a rooting culture medium, and inoculating one sterile short-shoot tissue culture seedling into each bottle; the rooting culture medium takes 1/2MS as a basic culture medium, and is added with 0.5mg/L, IBA 0.5.5 mg/L of NAA, 2g/L of active carbon, 30g/L of cane sugar and 7g/L of agar; adjusting the pH value to 5.8-6.0; after inoculation, culturing the seedlings in a culture room for 25 days at the temperature of 25-28 ℃ and the illumination intensity of 2000-2500 lx, wherein the root length reaches 4-6 cm, the root hair is white, and the rooting rate reaches more than 95% (as shown in figure 6);
seventhly, hardening and transplanting the tissue culture seedlings: when the seedlings in the rooting culture medium grow to 3-5 cm in height and the number of the roots of the tissue culture seedlings reaches 6-10, transferring the seedlings into a plastic greenhouse, and training the seedlings for 10 days under natural light; then taking out the culture medium from a culture bottle, cleaning the culture medium at the bottom of the culture medium, soaking the culture medium in a carbendazim solution for 7min, transplanting the culture medium into a sterilized culture medium which is 350 times of 50% ambam water, uniformly spraying 3 kilograms of diluent on each square meter of the culture medium, and growing new leaves of the Chinese alpine rush to show that the Chinese alpine rush is transplanted to survive (see the attached drawing 7 for details), wherein the volume ratio of the culture medium is 1: 1: 1 perlite, fine river sand and peat soil.
Wherein 75% alcohol and 0.1% mercuric chloride are prepared one day before inoculation; sterilizing sterile water, sterile paper, a seed inoculating tray, an empty bottle, tweezers and an inoculating knife for 35min at 121 ℃, and sterilizing the culture medium for 18min at 121 ℃.
Comparative example 1
The difference between the comparative example 1 and the example 1 is that the explant sterilization method is different, and the following steps are specifically adopted: transferring the explant to a new sterilized empty bottle in a clean bench, pouring 75% alcohol to soak the explant for 10s, washing with sterilized water once, soaking with 0.1% mercuric chloride solution for 10min, shaking continuously during the period, washing with sterilized water for 7 times, blotting with sterilized paper, and inoculating.
TABLE 1 germination and contamination rates under different disinfection methods
As can be seen from Table 1, the mercuric chloride disinfection time is proper at 4-6 min, and when the mercuric chloride disinfection time reaches 8min, the germination rate of the explants is obviously reduced.
Comparative example 2
The difference between the comparative example 2 and the example 1 lies in that the formula of the primary culture medium is different, specifically, the MS basic culture medium is added with 1.0mg/L of 6-BA, 0.2mg/L of NAA0, 30g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0.
Comparative example 3
The difference between the comparative example 3 and the examples 1 and 2 is that the formula of the primary culture medium is different, specifically, the MS minimal medium is added with 2.0mg/L, NAA 1.0.0 mg/L of 6-BA, 30g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0 (see Table 2 for details).
TABLE 2 growth of tissue culture seedlings in different primary culture media
As can be seen from Table 2, when 1.0mg/L of 6-BA, 0.5mg/L of NAA0, or 1.0mg/L, NAA 0.5.5 mg/L of 6-BA is added into the primary culture medium, 2-4 tissue culture seedlings can be formed on each sterile short shoot after 20 days of culture, and the stem length of each seedling can reach 2-5 cm.
Comparative example 4
The difference between the comparative example 4 and the example 1 is that the rooting culture medium has different formulas, specifically 1/2MS minimal medium is added with NAA1.5mg/L, IBA 1.5.5 mg/L, 3g/L of active carbon, 20g/L of sucrose and 7g/L of agar; adjusting the pH value to 5.8-6.0.
TABLE 3 rooting conditions of tissue culture seedlings in different rooting media
As can be seen from Table 3, when IBA1.0mg/L, NAA 1.0.0 mg/L or IBA1.0mg/L, NAA 1.0.0 mg/L was added to the rooting medium, the rooting rate was more than 95%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for the sterile short branch rapid propagation of Orthosiphon aristatus is characterized by comprising the following steps:
step one, pretreatment and cultivation of the Chinese alpine rush:
s1: selecting healthy and strong cat grass without diseases and insect pests, carefully digging up the cat grass, shaking off soil at roots, washing with clear water, pruning and shearing off old leaves at the positions of branches 3-4 cm away from the ground to keep leafstalks, and soaking and disinfecting the pruned branches for 1-2 hours by using imidazole amide with the concentration of 0.4% or mancozeb liquid medicine with the concentration of 0.2%;
s2: after soaking, transplanting the seedlings into a flowerpot filled with a culture medium sterilized by 350 times of 50% water-soluble amobam solution, and transferring the seedlings into a sealable plastic greenhouse;
s3: after the transplanted seedlings are planted, mixed solution of 0.4% miamide and 0.2% mancozeb is alternately used every 7 days, or the 0.4% miamide is singly used, or the 0.2% mancozeb liquid is singly used for spraying seedling plants, the ground and the periphery of a plastic greenhouse once; when young bud segments are newly drawn out from the top of the Chinese alpine rush, cutting the young bud segments from the base part to be used as explants for the sterile short branch rapid propagation;
step two, cleaning explants: taking the explant obtained by pretreatment and cultivation of the Chinese alpine rush back to a laboratory, cutting off leaves, washing with clear water once, cutting into 2-3 cm stem sections with a pair of axillary buds, soaking for 5 minutes with a detergent solution, washing for 20 minutes with running water, draining water, filling into a sterilized empty bottle, and putting into a super clean bench for later use;
step three, disinfection of explants: transferring the explant to a new sterilized empty bottle in an ultraclean workbench, pouring 75% alcohol to soak the explant for 10-20 s, washing the explant once with sterilized water, soaking the explant with 0.1% mercuric chloride solution for 4-8 min, continuously shaking the explant during the period, washing the explant with sterilized water for 6-8 times, and inoculating the explant after the explant is dried by using sterilized paper;
step four, primary culture: cutting off the outer edge part of the sterilized explant by using an inoculating knife in a super-clean workbench, inoculating the sterile short branch stem section with a pair of axillary buds into an inoculating disc filled with a primary culture medium according to polarity by using tweezers, and culturing for 15-30 days in a culture room; then growing into 2-5 cm tissue culture seedlings; MS minimal medium is selected for primary culture, and 0.5-2.0 mg/L, NAA 0.1.1-1.0 mg/L of 6-BA, 20-30 g/L of cane sugar and 7g/L of agar are added; adjusting the pH value to 5.8-6.0;
step five, subculturing: shearing the primary culture tissue culture seedlings obtained in the fourth step into sterile short branch stem sections with a pair of axillary buds, inoculating the sterile short branch stem sections into an inoculating tray filled with a subculture medium according to polarity, and culturing for 15-30 days in a culture room; then growing into 2-5 cm tissue culture seedlings; the subculture medium is MS minimal medium with 0.01-0.1 mg/L, IBA 0.1.1-1.0 mg/L TDZ, 20-30 g/L sucrose and 7g/L agar; adjusting the pH value to 5.8-6.0;
step six, rooting culture: cutting off the subculture tissue culture seedlings in the fifth step from the base, placing the cut subculture tissue culture seedlings in a rooting culture medium, and culturing for 15-30 days in a culture room; the root length can reach 4-6 cm, and the root hair is white; the rooting culture medium takes 1/2MS as a basic culture medium, and 0.5-2.0 mg/L, IBA 0.5.5-2.0 mg/L of NAA, 2-3 g/L of active carbon, 20-30 g/L of sucrose and 7g/L of agar are added; adjusting the pH value to 5.8-6.0;
seventhly, hardening and transplanting the tissue culture seedlings: when the seedlings in the rooting culture medium grow to 3-5 cm high and the number of roots reaches 6-10, moving the seedlings into a plastic greenhouse, and hardening the seedlings under natural light for 7-10 days; and taking out the culture flask, cleaning a culture medium at the bottom of the culture flask, soaking the culture flask in a carbendazim solution for 5-7 min, transplanting the culture flask into a sterilized culture medium, and growing new leaves of the orthosiphon aristatus to show that the culture flask is transplanted to survive.
2. The method for rapid propagation of sterile Clerodendranthus spicatus of claim 1, wherein the sealable plastic greenhouse of step S2 is sterilized with imazamide 0.4% and mancozeb 0.2% before moving into the greenhouse.
3. The method for rapid propagation of aseptic short branches of Orthosiphon aristatus as claimed in claim 1, wherein in step two, when young shoots newly extracted from top of Orthosiphon aristatus grow to 8-10 cm, in S3, they are cut from the base at two o' clock in the afternoon of sunny day, and used as explants for rapid propagation of aseptic short branches.
4. The method for the rapid propagation of the aseptic short branches of the Orthosiphon aristatus as claimed in claim 1, wherein 1-2 sterile short branch stem segments are inoculated in each bottle, and each sterile short branch stem segment has a pair of axillary buds.
5. The method for rapid propagation of aseptic short branches of Orthosiphon aristatus as claimed in claim 1, wherein the culture conditions in the fourth, fifth and sixth steps are 23-28 ℃ and the illumination intensity is 2000-2500 lx.
6. The method for the rapid propagation of the aseptic brachypodium distichum benth of claim 1, wherein the culture medium in the first step and the seventh step is prepared from the following components in a volume ratio of 1: 1: 1, the sterilization mode is as follows: 50 percent of water-soluble amobam 350 times solution is uniformly sprayed with 3 kilograms of diluent per square meter of culture medium.
7. The method for rapid propagation of sterile short branches of Orthosiphon aristatus as claimed in claim 1, wherein the 75% alcohol and 0.1% mercuric chloride are formulated one day before inoculation; the sterile water, the sterile paper, the inoculation tray, the empty bottle, the forceps and the inoculation knife are sterilized at 121 ℃ for 35min, and the culture medium is sterilized at 121 ℃ for 18 min.
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