CN113598046A - Efficient tissue culture breeding method for ficus microcarpa - Google Patents
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Abstract
The invention discloses a high-efficiency tissue culture breeding method of ficus microcarpa, which comprises the steps of plant pretreatment, explant selection and sterilization, primary generation induction culture, subculture proliferation culture, rooting culture and hardening seedling transplantation; selecting a robust, disease and insect pest-free and excellent-property young and tender stem section of the current year of the ficus auriculata as an explant material source, sterilizing and trimming the explant, inoculating the explant into an initial generation induction culture medium to obtain an initial bud, then inoculating the initial bud into a subculture multiplication culture medium to form a cluster bud, cutting a single bud, vertically inserting the single bud into a rooting culture medium to induce rooting, and finally carrying out hardening seedling and transplanting to obtain a tissue culture of the ficus auriculata into a live seedling; the invention establishes the high-efficiency tissue culture breeding method of the ficus auriculata, has the advantages of simple operation process, short culture period, high proliferation coefficient and rooting rate and good seedling quality, provides technical support for large-scale seedling production and artificial planting and utilization of the ficus auriculata, and has better economic benefit, social benefit and ecological benefit.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to an efficient tissue culture breeding method for ficus microcarpa.
Background
The Ficus hirta Vahl is also called Ficus hirta, Ficus milk, Astragalus membranaceus, milk wood, Caulerpa, etc., is perennial shrub or deciduous small tree of Ficus of Moraceae (Moraceae), has thick hair on the whole plant, has latex on branches, stems and leaves and fragrant root bark, naturally grows in hillsides, wastelands, valleys or shrubs, and is mainly distributed in southeast parts of China, such as Jiangxi, Guangxi, Fujian, Hainan province, etc., and southeast Asia countries of Malaysia, Burma, Vietnam, Thailand, etc. The crude Ficus microcarpa root can be used as a medicine, has sweet and pungent taste and even nature, has the effects of benefiting qi, strengthening spleen, promoting qi circulation, resolving stagnation, relaxing muscles and tendons, eliminating dampness, removing blood stasis and relieving swelling, is a raw material of various Chinese patent medicines such as antiphthisic pills, asthmatic pills, compound fritillaria cirrhosa cough syrup, kidney nourishing and mind tranquilizing pills and the like, and is a common herbal medicine for multiple nationalities in south China; meanwhile, the Roughleaf Ficus microcarpa is also a plant with homology of food and medicine, called as "radix astragali" and "Guangdong ginseng", the root of the Roughleaf Ficus microcarpa is used for cooking pig bones, spareribs and chicken bone soup to be served as soup for health care and food therapy, the health care consciousness of people is gradually enhanced along with the development of economy and improvement of living standard of China, particularly, the rise of the trend that food requires natural green products in recent years leads the market demand of Roughleaf Ficus microcarpa root and deep-processed products to increase day by day, a plurality of health care foods and soup bases such as Roughleaf Ficus microcarpa instant powder and the like are developed, and the annual consumption of the Roughleaf Ficus microcarpa exceeds 1 ten thousand tons.
However, at present, the ficus microcarpa is mainly wild, few manual cultivation is performed, and market supply basically depends on digging wild resources. Under natural conditions, the ficus microcarpa depends on seeds for propagation, the seeds have low ground natural propagation rate and long growth period, the ficus microcarpa can be harvested and dug after 8-10 years generally, the root system is not developed, the yield is very low, a large amount of wild resources are abundantly harvested and dug with increasing demand, the wild resources are gradually fatigue, and the current situation is that the seeds are singly bredThe requirement of domestic and foreign markets can not be met by wild resources, and artificial planting, particularly wild-like planting of excellent clones in forests is a necessary trend for ensuring sustainable utilization of the resources of the coarse-leaf banyans. The ficus microcarpa is shrub, has few branches and low yield of single medicinal material, can obtain higher economic benefit only by high-density planting, and has the seed consumption of 2.7 ten thousand plants/hm2If the price of the seedling is too high, the investment is easily larger than the output, so that the manual planting can not be smoothly carried out. The method can breed a large number of seedlings with excellent properties and consistency in a short time through tissue culture breeding of the seedlings without the limitation of factors such as breeding seasons and the like, so that the research and the application of the ficus auriculata tissue culture breeding method are the key for developing large-scale artificial planting of ficus auriculata.
The ficus microcarpa plants are thick and have hard bristles on the surface, so that callus and adventitious buds are not easy to induce, and tissue culture seedlings are difficult to obtain. At present, reports on the aspects of the tissue culture research of the ficus auriculata are very limited, only in the exploration stage, the ex-vivo regeneration research is carried out on Jianling and the like by taking a ficus auriculata stem node as an explant, the bud proliferation culture research is carried out on Huangsai and the like by taking a terminal bud as the explant, the primary culture, the secondary culture and the rooting culture test are carried out on the stem tip explant of the cycus auriculata, the different hormone comparison research is carried out on the cycus auriculata and the like by taking a leaf, a leaf stalk and a stem section as the explant induced callus, the yoga and the like take fresh ficus auriculata seeds as test materials, the influence of 6-BA, NAA and the like on the induction rate and the root occurrence rate of the callus is compared, the invention patents with the publication numbers of CN 104106468B, CN 104106468A, CN 111213588A and CN 112088775A also disclose the tissue culture breeding method of the ficus auriculata, and the above technologies mostly adopt the indirect ex-vivo regeneration approach for breeding, the problems of low inductivity and proliferation coefficient, long seedling growing period, high production cost and poor seedling quality generally exist, and the method is difficult to be applied to large-scale seedling breeding. Therefore, an efficient tissue culture breeding method of ficus auriculata, which is short in seedling culture period, high in multiplication coefficient and rooting rate and good in seedling quality, is urgently needed, and provides technical support for large-scale seedling production and artificial planting utilization of ficus auriculata.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides the high-efficiency tissue culture breeding method of the ficus microcarpa with short seedling culture period, high multiplication coefficient and rooting rate and good seedling quality.
In order to achieve the purpose, the technical scheme of the invention is as follows:
an efficient tissue culture breeding method for ficus microcarpa is characterized by comprising the following steps: plant pretreatment, explant selection and sterilization, primary generation induction culture, subculture proliferation culture, rooting culture and hardening seedling transplantation; selecting robust, disease and insect pest-free and excellent-property Ficus benjamina plants as explant material sources, pretreating the Ficus benjamina plants, selecting young and tender stem segments of the current year as explants, sterilizing and trimming the explants, inoculating the young and tender stem segments into an initial generation induction culture medium to obtain initial buds, inoculating the initial buds into a subculture multiplication culture medium to form cluster buds, cutting single buds to vertically insert the single buds into a rooting culture medium to induce rooting, and finally hardening and transplanting the seedlings to obtain the Ficus benjamina plants which are cultured into live seedlings; the main operation steps are as follows:
(1) plant pretreatment: selecting robust, disease and insect pest-free and excellent-property ficus microcarpa plants as explant material sources, if transplanted small plants are in a transplanting pot, if the transplanted plants are large and inconvenient to transplant into the pot, bagging the stem sections of the young plants, placing the transplanted plants in a greenhouse or an artificial climate box for maintenance, spraying sterilizing agents on the plants every 5-7 days, and selecting the young plants as explants after new branches germinate;
(2) explant selection and sterilization: collecting young and tender stem segments of the current year in sunny days, cutting off leaves and petioles, and reserving stem segments with buds of 4-6 cm in length as explant materials; washing the surface silt with clear water, scrubbing the explant with a soft hairbrush, placing the explant into detergent with the volume concentration of 1%, shaking and soaking for 5-10 min, washing away the detergent with tap water, placing the explant into new benzalkonium bromide disinfectant with the volume concentration of 2%, soaking for 30-60 min, washing for 1-2 h with running water, shaking and cleaning for 10min in an ultrasonic cleaning machine, placing the explant into a multifunctional fruit and vegetable cleaning machine for disinfection for 10min, placing the explant into an alcohol solution with the volume concentration of 75% on an ultraclean workbench, soaking for 10-30 s, washing for 3-5 times with sterile water, and then placing into HgCl with the volume concentration of 0.1%2Shaking and soaking in the solution for 8min-12min, washing with sterile water for 3-5 times, placing the explant material on sterilized filter paper, and draining;
(3) primary induction culture: cutting off 1mm of each end of the explant obtained in the step (2) by using scissors, cutting the cut explant into stem sections with 1 bud-2 buds, vertically inserting the stem sections into a prepared primary induction culture medium for culture, and culturing 1 plant in each bottle;
(4) subculture multiplication culture: cutting off large leaves when the initial bud grows to 3-8 cm, keeping the top bud and partial petiole, trimming into single buds with the height of 1.0-2.5 cm, transferring into a prepared subculture multiplication medium, culturing 6-8 plants in each bottle for 20-25 days, and multiplying to form cluster buds; when subculture multiplication culture is carried out again, cutting off callus at the bottom of the cluster buds, cutting into single buds or reserving a small cluster with 2-3 buds, inoculating the small cluster into a subculture multiplication culture medium for culture, and culturing 6 strains/cluster-8 strains/cluster in each bottle;
(5) rooting culture: vertically inserting the terminal bud single bud with the height of more than or equal to 1cm obtained in the step (4) into a prepared rooting culture medium for culture, and inserting 12-15 plants into each bottle;
(6) hardening and transplanting seedlings: and (3) when the root length of the single buds cultured in the step (5) is more than or equal to 1.0cm, moving the single buds to an outdoor seedling raising shed for hardening seedlings, opening a culture bottle cap after 5-10 days, opening the cover for hardening the seedlings for 1-2 days, then transplanting the seedlings to a seedling raising container filled with a seedling raising matrix, draining water thoroughly, and then managing the seedlings.
Supplying water to the transplanted plantlets in the step (1) by adopting a pot soaking method, wherein the bags used in the big plant bagging method are breathable non-woven bags; the sterilizing agent is 600 times to 1000 times of chlorothalonil wettable powder with the mass concentration of 75 percent.
The diameter of the stem section with the buds in the step (2) is 0.5cm-1.0 cm; rotating speed of a horizontal rotating oscillator is 160r/min during oscillation soaking; the ultrasonic power during ultrasonic cleaning is 80W, and the water in the ultrasonic cleaning machine and the multifunctional fruit and vegetable cleaning machine is sterilized water.
The stem section length in the step (3) is 1cm-1.5cm, and the formula of the primary induction culture medium is as follows: MS +2-IP 0.3mg/L-0.5mg/L + NAA 0.5mg/L + KT 0.3mg/L-0.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the culture vessel was a 240mL straight glass bottle having a height of 9.5cm, a diameter of 6.6cm and a diameter of 6.3cm, and 50mL of the culture medium was added to each bottle.
The formula of the subculture multiplication medium in the step (4) is as follows: MS +2-IP 0.2mg/L-0.4mg/L + NAA 0.1mg/L-0.2mg/L + IAA 0.5mg/L-1.0mg/L + KT 1.5mg/L-2.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture container is a 650mL small-mouth glass bottle with the height of 14cm, the diameter of 9.5cm and the caliber of 5.3cm, and 100mL of culture medium is added into each bottle.
The rooting medium formula in the step (5) is as follows: 1/2MS + IBA 0.75mg/L-1.0mg/L + IAA 1.0mg/L-1.5mg/L + activated charcoal 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2, the culture container is a 240mL straight glass bottle with height 9.5cm, diameter 6.6cm and caliber 6.3cm, 50mL culture medium is added into each bottle.
The culture control conditions in the steps (3), (4) and (5) are all as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, and the illumination time is 12h/d-16 h/d.
The seedling raising substrate in the step (6) is rice husk: coconut husk: the volume ratio of the peat soil is 1:3: 5; the specification of the seedling culture container is a non-woven fabric cup with the diameter of 8cm and the height of 10cm, before transplanting, potassium permanganate solution with the volume concentration of 0.5% -0.8% is used for thoroughly leaching and disinfecting the seedling culture substrate, and after 1 day, clear water is used for leaching and washing away liquid medicine.
The nursery stock management method in the step (6) comprises the following steps: the nursery stock is placed in an outdoor seedling raising greenhouse with a shading net, water is sprayed for 1-2 times every day, the transmittance is kept between 40% and 50% within one week after transplanting, the light is gradually increased after one week, the transmittance is increased to 80% to 90% after transplanting for one month, a compound fertilizer aqueous solution with the mass concentration of 0.1% to 0.2% is sprayed on the leaf surface, the leaf surface is immediately washed by clear water after fertilization, and the compound fertilizer is applied for 1 time every month, wherein the N: P: K of the compound fertilizer is 15:15: 15.
The culture medium in each stage of tissue culture and breeding in the steps (3) to (5) is preferably combined as follows: the formula of the initial bud induction culture medium is as follows: MS +2-IP 0.3mg/L + NAA 0.5mg/L + KT 0.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the formula of the subculture multiplication medium is as follows: MS +2-IP 0.2mg/L + NAA 0.1mg/L + IAA 0.5mg/L + KT 2.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the rooting medium comprises the following components: 1/2MS + IBA 0.75mg/L + IAA 1.0mg/L + charcoal 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2.
Compared with the prior art, the invention has the following advantages and positive effects:
1. the method is applied to the high-efficiency tissue culture of the ficus auriculata, the proliferation coefficient, the rooting rate and the seedling quality of ficus auriculata seedlings are obviously improved, a large number of ficus auriculata seedlings with consistent characters and suitable for field cultivation can be cultivated in a short time, large-scale production is realized, the production requirement is met, the problem that wild resources of ficus auriculata are less and even the wild seedlings are prone to exhaustion and seed removal can be solved, farmers in a mountain area can be promoted to expand medicine sources, and the method has good economic benefits and social benefits for developing characteristic economy and developing emerging industries of resources in the mountain area.
2. The method takes the current-year stem segment of robust ficus microcarpa as the explant source, and the explant source material is sufficient; the method comprises the following steps of pretreating the plant before the explant is sterilized, supplying water by a pot soaking method to maintain the plantlets, avoiding the overground part of the plant from being stained with water, spraying sterilizing agent, selecting less explant material with bacteria after a period of maintenance, and remarkably improving the success rate of sterilization; the method that multiple modes such as ultrasonic cleaner and multi-kinetic energy fruit vegetables cleaning machine combined together have been adopted in explant sterilization process to put vibrations on the horizontal rotation oscillator when the germicide soaks, make each position of germicide full contact explant, the difficult abluent position such as stem segment area bud thoroughly washs, greatly reduced the pollution rate.
3. According to the growth conditions of the ficus auriculata at different growth stages, the culture medium formula is screened after a large number of experiments are carried out, the combination mode of 2-IP and KT is adopted as cytokinin for culturing at the successive propagation stage to replace the mode of singly using 6-BA in the prior art, the seedling callus is reduced, meanwhile, the auxin IAA with proper concentration is added, the seedling growth height is not influenced while the bud propagation is carried out, the combination mode of the IAA and IBA is adopted at the rooting culture stage, the rooting rate is improved, meanwhile, the base part callus caused by NAA is avoided, and the number of roots is obviously increased.
4. According to the method, callus is not required to be induced to redifferentiate and sprout in the process of in vitro plant regeneration, the method of bud propagation is directly adopted for propagation, then rooting and seedling formation, operation steps are saved, operation is simpler and more convenient, meanwhile, glass bottles with different specifications are used in a matching mode, a 650mL small-mouth glass bottle is adopted in a successive propagation stage, the culture space is increased, the problem of edema of the ficus microcarpa in the propagation stage due to insufficient space is effectively solved, seedlings in each stage grow robustly, and the effect is ideal.
5. The method disclosed by the invention can be used for culturing under the culture medium formula obtained by screening and a specific light temperature condition, can adapt to the growth characteristics of the ficus auriculata tissue culture seedlings, promotes the growth of the seedlings, has the initial bud induction rate of 92.6-95.3%, can form cluster buds after 20-25 days of subculture proliferation culture, has the proliferation coefficient of 6.9-8.1, roots after 8-12 days after single buds are inoculated into a rooting culture medium, has the rooting rate of 100% in 15 days, the root length of 2.0-2.5 cm, 13-18 root systems, strong root systems and the transplanting survival rate of 95.4-96.7%, greatly shortens the seedling culture period, and can obtain a large number of ficus auriculata tissue culture seedlings in a short period.
Drawings
FIG. 1 shows the induction of bud in the tissue culture stem of Ficus benjamina in example 1.
FIG. 2 shows the example 1 of the subculture bottle seedlings of Ficus benjamina.
FIG. 3 is the bottom photograph of the seedlings of the ficus microcarpa tissue culture rooting culture bottle in example 1.
FIG. 4 shows the tissue culture rooting bottle seedlings of Ficus benjamina after the seedlings are hardened for 10d in example 1.
FIG. 5 shows the tissue culture and transplantation of cup seedlings of Ficus benjamina in example 1.
FIG. 6 shows the tissue culture and transplantation of large seedlings of Ficus benjamina in example 1.
FIG. 7 shows the example 3 of the tissue culture subculture proliferation culture bottle seedlings of Ficus benjamina.
FIG. 8 is the bottom photograph of the seedlings of the ficus microcarpa tissue culture rooting culture bottle in example 3.
FIG. 9 shows the tissue culture rooting bottle seedlings of Ficus benjamina after the seedlings are hardened for 10d in example 3.
FIG. 10 shows the bottle seedlings of Ficus benjamina tissue culture subculture propagation in comparative example 1.
FIG. 11 is the bottom photograph of the seedlings of the ficus microcarpa tissue culture rooting culture bottle of comparative example 1.
FIG. 12 shows the Ficus benjamina tissue culture rooting bottle seedlings after the seedlings are hardened for 10 days in comparative example 1.
FIG. 13 shows the bottle seedlings of Ficus benjamina tissue culture subculture propagation in comparative example 2.
FIG. 14 is the bottom photograph of the seedlings of the ficus microcarpa tissue culture rooting culture bottle of comparative example 2.
FIG. 15 shows the Ficus benjamina tissue culture rooting bottle seedlings after hardening for 10d in comparative example 2.
FIG. 16 shows the bottle seedlings of Ficus benjamina tissue culture subculture propagation in comparative example 3.
FIG. 17 is the bottom photograph of the seedlings of the ficus microcarpa tissue culture rooting culture bottle of comparative example 3.
FIG. 18 shows the Ficus benjamina tissue culture rooting bottle seedlings after the seedlings are hardened for 10 days in comparative example 3.
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present invention and are not intended to limit the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Example 1:
(1) plant pretreatment: selecting robust Ficus benjamina plants without diseases and insect pests and with excellent characters as explant material sources, transplanting small plants into a pot, transplanting the small plants into the pot, placing the small plants into a greenhouse or an artificial climate box for maintenance, adopting a pot soaking method for water supply, spraying 600 times of liquid of chlorothalonil wettable powder with the mass concentration of 75% on the plants every 5 days, and selecting the plants as explants after new branches germinate;
(2) explant selection and sterilization: collecting young and tender stem segments of the current year in sunny days, cutting off leaves and petioles, and reserving a stem segment with buds and a length of 4-6 cm and a diameter of 0.5-1.0 cm as an explant material; washing sediment on the surface with clear water, scrubbing explants with a fur brush, then placing the explants into a detergent with a volume concentration of 1% for oscillation and soaking for 10min, adopting a horizontal rotation oscillator with a rotation speed of 160rpm during oscillation and soaking, washing off the detergent with tap water, placing the explants into a new benzalkonium bromide disinfectant with a volume concentration of 2% for soaking for 60min, flushing for 2h with running water, performing oscillation and cleaning for 10min in an ultrasonic cleaner, and performing ultrasonic cleaningThe ultrasonic power is 80W, the explant is put into a multifunctional fruit and vegetable cleaning machine to be disinfected for 10min, the water used in the ultrasonic cleaning machine and the multifunctional fruit and vegetable cleaning machine is sterilized water, the explant is put into 75% alcohol solution by volume concentration on an ultra-clean workbench to be soaked for 30s, then the explant is washed for 5 times by sterile water, and then HgCl with 0.1% volume concentration is put into the explant2Shaking and soaking in the solution for 8min, washing with sterile water for 5 times, and placing the explant material on sterilized filter paper to absorb water;
(3) primary induction culture: cutting off 1mm of two ends of the explant obtained in the step (2) by using scissors, trimming the explant into stem sections with 1 bud-2 buds, keeping the length of the stem sections to be 1cm-1.5cm, vertically inserting the stem sections into a prepared primary induction culture medium for culture, wherein 1 strain is cultured in each bottle, and the formula of the primary induction culture medium is as follows: MS +2-IP 0.3mg/L + NAA 0.5mg/L + KT 0.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then is subpackaged into 240mL straight glass bottles with the height of 9.5cm, the diameter of 6.6cm and the caliber of 6.3cm, 50mL of culture medium is added into each bottle, and the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, and the induction rate is 95.3%;
(4) subculture multiplication culture: when the initial bud grows to 3cm-8cm, cutting off large leaves, reserving terminal buds and partial petioles, trimming into single buds with the height of 1.0cm-2.5cm, transferring the single buds into a prepared subculture multiplication medium for culture, wherein 6 strains are cultured in each bottle, and the formula of the subculture multiplication medium is as follows: MS +2-IP 0.2mg/L + NAA 0.1mg/L + IAA 0.5mg/L + KT 2.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then subpackaged in 650mL small-mouth glass bottles with height of 14cm, diameter of 9.5cm and caliber of 5.3cm, 100mL of culture medium is added into each bottle, and after 20 days of culture, cluster buds are formed by proliferation, and the proliferation coefficient is 8.1; when subculture is carried out again, cutting off callus at the bottom of the cluster buds, cutting into single buds or reserving a small cluster with 2-3 buds, inoculating the small cluster into a subculture multiplication culture medium for culture, wherein 6 strains/cluster are cultured in each bottle under the culture control conditions: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, and the illumination time is 12h/d-16 h/d;
(5) rooting culture: vertically inserting the terminal bud single bud with the height of more than or equal to 1cm obtained in the step (4) into a prepared rooting culture medium for culture, inserting 15 plants into each bottle, wherein the formula of the rooting culture medium is as follows: 1/2MS + IBA 0.75mg/L + IAA 1.0mg/L + activated carbon 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2, the culture container is a 240mL straight glass bottle with height 9.5cm, diameter 6.6cm and caliber 6.3cm, 50mL of culture medium is poured into each bottle, and the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, the roots grow after 8 days, the rooting rate is 100% in 15 days, the roots grow by 2.0cm-2.5cm, and the number of root systems is 14-18;
(6) hardening and transplanting seedlings: preparing a seedling culture medium of rice husks, coconut chaff and peat soil in a volume ratio of 1:3:5, filling the seedling culture medium into a non-woven fabric cup with the diameter of 8cm and the height of 10cm, before transplanting, thoroughly spraying a potassium permanganate solution with the volume concentration of 0.5% to disinfect the seedling culture medium, after 1 day, spraying clear water to remove liquid medicine, when the length of a root system of a single bud cultured in the step (5) is more than or equal to 1.0cm, transferring the seedling to an outdoor seedling culture shed for hardening, after 5 days, opening a culture bottle cap, after 1 day of uncovering and hardening, transplanting the seedling to the seedling culture medium, thoroughly spraying water, then placing the seedling in the outdoor seedling culture shed with a shading net, spraying water 2 times every day, after transplanting, within one week, gradually increasing the light after one week, after transplanting, increasing the light transmittance to 80%, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2%, immediately washing the leaf surfaces with clear water after fertilizing, and then applying the compound fertilizer for 1 time per month, the N, P and K of the compound fertilizer are 15:15:15, and the transplanting survival rate is 96.7%.
Example 2:
(1) plant pretreatment: selecting robust, disease and insect pest-free and excellent-property ficus microcarpa plants as explant material sources, transplanting larger plants into a pot inconveniently, bagging stems of the large plants by adopting a non-woven bag, spraying 1000 times of liquid of chlorothalonil wettable powder with the mass concentration of 75% on the plants every 7 days, and selecting the large plants as explants after new branches germinate;
(2) explant selection and sterilization: collecting young and tender stem segments of the current year in sunny days, cutting off leaves and petioles, and reserving a stem segment with buds and a length of 4-6 cm and a diameter of 0.5-1.0 cm as an explant material; washing the surface with clear water, scrubbing explant with a brush, adding into 1% detergent, and shaking for 10min at rotation speed of horizontal rotation shaking instrument160r/min, washing detergent with tap water, soaking in 2% volume concentration benzalkonium bromide disinfectant for 30min, washing with running water for 1h, washing with ultrasonic cleaner for 10min, washing with ultrasonic cleaner at ultrasonic power of 80W, disinfecting in multifunctional fruit and vegetable cleaner for 10min, washing with sterilized water for 3 times, soaking explant in 75% volume concentration alcohol solution for 30s, and washing with 0.1% volume concentration HgCl2Shaking and soaking in the solution for 12min, washing with sterile water for 3 times, and placing the explant material on sterilized filter paper to absorb water;
(3) primary induction culture: cutting off 1mm of two ends of the explant obtained in the step (2) by using scissors, trimming the explant into stem sections with 1 bud-2 buds, keeping the length of the stem sections to be 1cm-1.5cm, vertically inserting the stem sections into a prepared primary induction culture medium for culture, wherein 1 strain is cultured in each bottle, and the formula of the primary induction culture medium is as follows: MS +2-IP 0.5mg/L + NAA 0.5mg/L + KT 0.3mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then is subpackaged into 240mL straight glass bottles with the height of 9.5cm, the diameter of 6.6cm and the caliber of 6.3cm, 50mL of culture medium is added into each bottle, and the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, and the induction rate is 92.6 percent;
(4) subculture multiplication culture: when the initial bud grows to 3cm-8cm, cutting off large leaves, reserving terminal buds and partial petioles, trimming into single buds with the height of 1.0cm-2.5cm, transferring the single buds into a prepared subculture multiplication medium for culture, wherein 8 strains are cultured in each bottle, and the formula of the subculture multiplication medium is as follows: MS +2-IP 0.4mg/L + NAA 0.2mg/L + IAA 1.0mg/L + KT 1.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then subpackaged in 650mL small-mouth glass bottles with height of 14cm, diameter of 9.5cm and caliber of 5.3cm, 100mL of culture medium is added into each bottle, and after 25 days of culture, cluster buds are formed by proliferation, and the proliferation coefficient is 6.9; when subculture is carried out again, cutting off callus at the bottom of the cluster buds, cutting into single buds or reserving a small cluster with 2-3 buds, inoculating the small cluster into a subculture multiplication culture medium for culture, wherein 8 strains/cluster are cultured in each bottle under the culture control conditions: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, and the illumination time is 12h/d-16 h/d;
(5) rooting culture: vertically inserting the terminal bud single bud with the height of more than or equal to 1cm obtained in the step (4) into a prepared rooting culture medium for culture, inserting 12 plants into each bottle, wherein the formula of the rooting culture medium is as follows: 1/2MS + IBA 1.0mg/L + IAA 1.5mg/L + activated carbon 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2, the culture container is a 240mL straight glass bottle with height 9.5cm, diameter 6.6cm and caliber 6.3cm, each bottle is added with 50mL culture medium, the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, the roots grow after 12 days, the rooting rate is 100% in 15 days, the roots grow by 2.0cm-2.5cm, and the number of the roots is 13-16;
(6) hardening and transplanting seedlings: preparing a seedling culture medium of rice husks, coconut chaff and peat soil in a volume ratio of 1:3:5, filling the seedling culture medium into a non-woven fabric cup with the diameter of 8cm and the height of 10cm, before transplanting, thoroughly spraying a potassium permanganate solution with the volume concentration of 0.8% to disinfect the seedling culture medium, after 1 day, spraying clear water to remove liquid medicine, when the length of a root system of a single bud cultured in the step (5) is more than or equal to 1.0cm, transplanting the seedling into an outdoor seedling culture shed for hardening, after 10 days, opening a culture bottle cap, after uncovering and hardening the seedling for 2 days, transplanting the seedling into the seedling culture medium, thoroughly spraying water, then placing the seedling into the outdoor seedling culture shed with a shading net, spraying 1 time of water every day, after transplanting, gradually increasing the light transmittance after one week, after transplanting, increasing the light transmittance to 80% after one month, spraying a compound fertilizer aqueous solution with the mass concentration of 0.2%, immediately washing the leaf surfaces with clear water after fertilizing, and then applying the compound fertilizer for 1 time per month, the N, P and K of the compound fertilizer are 15:15:15, and the transplanting survival rate is 95.4%.
Example 3:
(1) plant pretreatment: selecting robust Ficus benjamina plants without diseases and insect pests and with excellent characters as explant material sources, transplanting small plants into a pot, placing the small plants in a greenhouse or an artificial climate box for maintenance, supplying water by using a pot soaking method, spraying 600 times of liquid of chlorothalonil wettable powder with the mass concentration of 75% on the plants every 7 days, and selecting the plants as explants after new branches germinate;
(2) explant selection and sterilization: collecting young and tender stem segments of the current year in sunny days, cutting off leaves and petioles, and reserving stem segments with buds of 4-6 cm length and 0.5-1.0 cm diameterAs explant material; washing sediment on the surface with clear water, scrubbing explants with a fur brush, then placing the explants into detergent with the volume concentration of 1%, oscillating and soaking for 10min, when oscillating and soaking, adopting a horizontal rotation oscillator with the rotating speed of 160r/min, washing away detergent with tap water, placing the explants into fresh benzalkonium bromide disinfectant with the volume concentration of 2%, soaking for 30min, flushing with running water for 2h, oscillating and cleaning for 10min in an ultrasonic cleaner, when ultrasonic cleaning, the ultrasonic power is 80W, placing the explants into a multifunctional fruit and vegetable cleaner for disinfection for 10min, using water in the ultrasonic cleaner and the multifunctional fruit and vegetable cleaner as sterilizing water, placing the explants into alcohol solution with the volume concentration of 75% on an ultraclean workbench, soaking for 20s, washing for 5 times with sterile water, and placing HgCl with the volume concentration of 0.1% on an ultraclean workbench2Shaking and soaking in the solution for 12min, washing with sterile water for 5 times, and placing the explant material on sterilized filter paper to absorb water;
(3) primary induction culture: cutting off 1mm of two ends of the explant obtained in the step (2) by using scissors, trimming the explant into stem sections with 1 bud-2 buds, keeping the length of the stem sections to be 1cm-1.5cm, vertically inserting the stem sections into a prepared primary induction culture medium for culture, wherein 1 strain is cultured in each bottle, and the formula of the primary induction culture medium is as follows: MS +2-IP 0.2mg/L + NAA 0.5mg/L + KT 1.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then is subpackaged into 240mL straight glass bottles with the height of 9.5cm, the diameter of 6.6cm and the caliber of 6.3cm, 50mL of culture medium is added into each bottle, and the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, and the induction rate is 84.4 percent;
(4) subculture multiplication culture: when the initial bud grows to 3cm-8cm, cutting off large leaves, reserving terminal buds and partial petioles, trimming into single buds with the height of 1.0cm-2.5cm, transferring the single buds into a prepared subculture multiplication medium for culture, wherein 8 strains are cultured in each bottle, and the formula of the subculture multiplication medium is as follows: MS +2-IP 0.5mg/L + NAA 0.1mg/L + IAA 0.3mg/L + KT 2.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture medium is prepared and then subpackaged in 650mL small-mouth glass bottles with height of 14cm, diameter of 9.5cm and caliber of 5.3cm, 100mL of culture medium is poured into each bottle, and after 30 days of culture, cluster buds are formed by proliferation, and the proliferation coefficient is 5.5; when subculture is carried out again, cutting off callus at the bottom of the cluster buds, cutting into single buds or reserving a small cluster with 2-3 buds, inoculating the small cluster into a subculture multiplication culture medium for culture, wherein 8 strains/cluster are cultured in each bottle under the culture control conditions: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, and the illumination time is 12h/d-16 h/d;
(5) rooting culture: vertically inserting the terminal bud single bud with the height of more than or equal to 1cm obtained in the step (4) into a prepared rooting culture medium for culture, inserting 15 plants into each bottle, wherein the formula of the rooting culture medium is as follows: 1/2MS + IBA1.5 mg/L + IAA 2.0mg/L + activated carbon 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2, the culture container is a 240mL straight glass bottle with height 9.5cm, diameter 6.6cm and caliber 6.3cm, each bottle is added with 50mL culture medium, the culture control conditions are as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, the illumination time is 12h/d-16h/d, the roots grow for 13 days, the rooting rate is 99.4% in 15 days, the root length is 1.5-2.0cm, and the number of root systems is 2-7;
(6) hardening and transplanting seedlings: preparing the rice husks in a volume ratio of 1:3: 5: coconut husk: placing a seedling raising substrate of peat soil into a non-woven fabric cup with the diameter of 8cm and the height of 10cm, before transplanting, using a potassium permanganate solution with the volume concentration of 0.5% to thoroughly disinfect the seedling raising substrate, after 1 day, using clear water to spray and wash away a liquid medicine, when the length of a single-bud root system after rooting culture in the step (5) reaches more than 1.0cm, moving the seedling into an outdoor seedling raising shed for hardening seedlings, after 8 days, opening a culture bottle cap, after opening the cover and hardening the seedlings for 2 days, transplanting the seedlings into the seedling raising substrate, spraying thoroughly, then placing the seedlings into the outdoor seedling raising shed with a shading net, spraying water for 2 times every day, keeping the light transmittance within one week after transplanting, gradually increasing the light after one week, after transplanting for one month, increasing the light transmittance to 80%, using a compound fertilizer aqueous solution with the mass concentration of 0.1% to spray leaf surfaces, immediately washing the leaf surfaces with clear water after fertilizer application, and applying the compound fertilizer in the manner for 1 time every month, wherein N: P: K is 15:15: 15:15, the transplanting survival rate is 89.4%.
Comparative example 1:
the method is basically the same as the example 1, except that the subculture multiplication and rooting culture medium and the transplanting substrate adopt the optimal formula disclosed in 'CN 104106468B tissue culture rapid propagation method of Ficus Simplicissima', and the transplanting survival rate is 93.8%.
Comparative example 2:
the method is basically the same as the embodiment 1, except that the subculture multiplication and rooting culture medium and the transplanting matrix adopt the optimal formula disclosed in 'CN 111213588A simple and efficient tissue culture propagation method of hispid fig', and the transplanting survival rate is 95.3%.
Comparative example 3:
the method is basically the same as the example 1, except that the subculture multiplication and rooting culture medium and the transplanting matrix adopt the optimal formula disclosed in the Chinese medicinal material 2004, volume 27, stage 8 of the research on the tissue culture of the five-finger wild peach, and the transplanting survival rate is 85.3 percent.
The treatment methods of examples 1 to 3 and comparative examples 1 to 3 of the present invention were respectively adopted to perform treatment, and the experimental results were counted, where the initial bud induction rate/% (number of explants inducing initial buds/total number of inoculated explants) × 100%; the multiplication coefficient is the number of buds which can be cut into the height of 0.5cm-1cm when the buds grow/the total number of single buds are inoculated; rooting rate/% (rooted bud number/total inoculated bud number) × 100%; the shortest seedling period/day of the tissue culture seedlings is the shortest propagation period plus the shortest rooting time, and the processing results are compared and shown in table 1.
Table 1: contrast of seedling raising effect in each treatment
As can be seen from table 1, example 1 is the most preferred example, and the effects are shown in fig. 1 to 6;
compared with the embodiment 1 and the embodiment 2, the culture medium formula of the embodiment 3 adjusts the hormone content, the leaves of the culture medium are slightly browned (the effect is shown in figure 7), the proliferation coefficients are respectively reduced by 2.6 and 1.4, the rooting time is respectively increased by 5 days and 1 day, the rooting rate is reduced by 0.6%, the seedling period of the shortest tissue culture seedling is respectively increased by 10 days and 6 days, the transplanting survival rate is respectively reduced by 7.3% and 6.2%, the number of root systems is less (the effect is shown in figure 8), the seedling quality is poorer after seedling hardening (the effect is shown in figure 9), which shows that the hormone content in an appropriate range has important significance for the growth of seedlings, the proliferation coefficient of the unsuitable hormone content is reduced, the rooting rate is reduced, the root system quality is poor, and the like, and is not beneficial to the growth of the seedlings, and the culture medium formula obtained by optimization of a large number of tests is suitable in hormone content and strong in the seedlings;
compared with the example 1, the method has the advantages that the disclosed preferable proliferation and rooting culture medium and transplanting substrate are respectively adopted in the comparative example 1 (the effect is shown in fig. 10, fig. 11 and fig. 12), the comparative example 2 (the effect is shown in fig. 13, fig. 14 and fig. 15) and the comparative example 3 (the effect is shown in fig. 16, fig. 17 and fig. 18), so that the proliferation coefficient, the rooting time, the rooting rate, the seedling state and the transplanting survival rate of the example 1 are all optimal, and the seedling culture period is shortened.
Claims (10)
1. An efficient tissue culture breeding method for ficus microcarpa is characterized in that: comprises the steps of plant pretreatment, explant selection and sterilization, primary generation induction culture, subculture proliferation, rooting culture and hardening-seedling transplantation; selecting robust, disease and insect pest-free and excellent-property Ficus benjamina plants as explant material sources, pretreating the Ficus benjamina plants, selecting young and tender stem segments of the current year as explants, sterilizing and trimming the explants, inoculating the young and tender stem segments into an initial generation induction culture medium to obtain initial buds, inoculating the initial buds into a subculture multiplication culture medium to form cluster buds, cutting single buds to vertically insert the single buds into a rooting culture medium to induce rooting, and finally hardening and transplanting the seedlings to obtain the Ficus benjamina plants which are cultured into live seedlings; the main operation steps are as follows:
(1) plant pretreatment: selecting robust, disease and insect pest-free and excellent-property ficus microcarpa plants as explant material sources, if transplanted small plants are in a transplanting pot, if the transplanted plants are large and inconvenient to transplant into the pot, bagging the stem sections of the young plants, placing the transplanted plants in a greenhouse or an artificial climate box for maintenance, spraying sterilizing agents on the plants every 5-7 days, and selecting the young plants as explants after new branches germinate;
(2) explant selection and sterilization: collecting young and tender stem segments of the current year in sunny days, cutting off leaves and petioles, and reserving stem segments with buds of 4-6 cm in length as explant materialFeeding; washing the surface silt with clear water, scrubbing the explant with a soft hairbrush, placing the explant into detergent with the volume concentration of 1%, shaking and soaking for 5-10 min, washing away the detergent with tap water, placing the explant into new benzalkonium bromide disinfectant with the volume concentration of 2%, soaking for 30-60 min, washing for 1-2 h with running water, shaking and cleaning for 10min in an ultrasonic cleaning machine, placing the explant into a multifunctional fruit and vegetable cleaning machine for disinfection for 10min, placing the explant into an alcohol solution with the volume concentration of 75% on an ultraclean workbench, soaking for 10-30 s, washing for 3-5 times with sterile water, and then placing into HgCl with the volume concentration of 0.1%2Shaking and soaking in the solution for 8-12 min, washing with sterile water for 3-5 times, and placing the explant material on sterilized filter paper to absorb water;
(3) primary induction culture: cutting off 1mm of each end of the explant obtained in the step (2) by using scissors, cutting the cut explant into stem sections with 1 bud-2 buds, vertically inserting the stem sections into a prepared primary induction culture medium for culture, and culturing 1 plant in each bottle;
(4) subculture multiplication culture: cutting off large leaves when the initial bud grows to 3-8 cm, keeping the top bud and partial petiole, trimming into single buds with the height of 1.0-2.5 cm, transferring into a prepared subculture multiplication medium for culturing 6-8 plants in each bottle, and multiplying to form cluster buds after 20-25 days; when subculture multiplication culture is carried out again, cutting off callus at the bottom of the cluster buds, cutting into single buds or reserving a small cluster with 2-3 buds, inoculating the small cluster into a subculture multiplication culture medium for culture, and culturing 6 strains/cluster-8 strains/cluster in each bottle;
(5) rooting culture: vertically inserting the terminal bud single bud with the height of more than or equal to 1cm obtained in the step (4) into a prepared rooting culture medium for culture, and inserting 12-15 plants into each bottle;
(6) hardening and transplanting seedlings: and (3) when the root length of the single buds cultured in the step (5) is more than or equal to 1.0cm, moving the single buds to an outdoor seedling raising shed for hardening seedlings, opening a culture bottle cap after 5-10 days, opening the cover for hardening the seedlings for 1-2 days, then transplanting the seedlings to a seedling raising container filled with a seedling raising matrix, draining water thoroughly, and then managing the seedlings.
2. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: supplying water to the transplanted plantlets in the step (1) by adopting a pot soaking method, wherein the bags used in the big plant bagging method are breathable non-woven bags; the sterilizing agent is 1000 times of 600 times of chlorothalonil wettable powder with the mass concentration of 75 percent.
3. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the diameter of the stem section with the buds in the step (2) is 0.5cm-1.0 cm; rotating speed of a horizontal rotating oscillator is 160rpm r/min during oscillation soaking; the ultrasonic power during ultrasonic cleaning is 80W, and the water in the ultrasonic cleaning machine and the multifunctional fruit and vegetable cleaning machine is sterilized water.
4. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the stem section length in the step (3) is 1cm-1.5cm, and the formula of the primary induction culture medium is as follows: MS +2-IP 0.3mg/L-0.5mg/L + NAA 0.5mg/L + KT 0.3mg/L-0.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the culture vessel was a 240mL straight glass bottle having a height of 9.5cm, a diameter of 6.6cm and a diameter of 6.3cm, and 50mL of the culture medium was added to each bottle.
5. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the formula of the subculture multiplication medium in the step (4) is as follows: MS +2-IP 0.2mg/L-0.4mg/L + NAA 0.1mg/L-0.2mg/L + IAA 0.5mg/L-1.0mg/L + KT 1.5mg/L-2.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2, the culture container is a 650mL small-mouth glass bottle with the height of 14cm, the diameter of 9.5cm and the caliber of 5.3cm, and 100mL of culture medium is added into each bottle.
6. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the rooting medium formula in the step (5) is as follows: 1/2MS + IBA 0.75mg/L-1.0mg/L + IAA 1.0mg/L-1.5mg/L + activated charcoal 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2, the culture container is a 240mL straight glass bottle with height 9.5cm, diameter 6.6cm and caliber 6.3cm, 50mL culture medium is added into each bottle.
7. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the culture control conditions of the step (3), the step (4) and the step (5) are all as follows: the temperature is 25 +/-3 ℃, the illumination intensity is 3000Lx-9000Lx, and the illumination time is 12h/d-16 h/d.
8. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the seedling raising substrate in the step (6) is prepared by mixing rice husk, coconut husk and peat soil in a volume ratio of 1:3: 5; the specification of the seedling culture container is a non-woven fabric cup with the diameter of 8cm and the height of 10cm, before transplanting, potassium permanganate solution with the volume concentration of 0.5% -0.8% is used for thoroughly leaching and disinfecting the seedling culture substrate, and after 1 day, clear water is used for leaching and washing away liquid medicine.
9. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the nursery stock management method in the step (6) comprises the following steps: the nursery stock is placed in an outdoor seedling raising greenhouse with a shading net, water is sprayed for 1-2 times every day, the transmittance is kept between 40% and 50% within one week after transplanting, the light is gradually increased after one week, the transmittance is increased to 80% to 90% after transplanting for one month, a compound fertilizer aqueous solution with the mass concentration of 0.1% to 0.2% is sprayed on the leaf surface, the leaf surface is immediately washed by clear water after fertilization, and the compound fertilizer is applied for 1 time every month, wherein the N: P: K of the compound fertilizer is 15:15: 15.
10. The efficient tissue culture breeding method of ficus microcarpa according to claim 1, wherein the efficient tissue culture breeding method comprises the following steps: the culture medium of each stage of tissue culture breeding is preferably combined as follows: the formula of the initial bud induction culture medium is as follows: MS +2-IP 0.3mg/L + NAA 0.5mg/L + KT 0.5mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the formula of the subculture multiplication medium is as follows: MS +2-IP 0.2mg/L + NAA 0.1mg/L + IAA 0.5mg/L + KT 2.0mg/L + agar 3.7g/L + sugar 30g/L, pH 5.8-6.2; the rooting medium comprises the following components: 1/2MS + IBA 0.75mg/L + IAA 1.0mg/L + charcoal 0.05g/L + agar 3.7g/L + sugar 15g/L, pH 5.8-6.2.
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