CN104686363B - The asepticization processing method of the outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae) - Google Patents
The asepticization processing method of the outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae) Download PDFInfo
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Abstract
The present invention relates to Chinese herbal medicine cultivation technology field, specifically the asepticization processing method of the outer implant of a kind of Pseudobulbus Bletillae (Rhizoma Bletillae), step includes, and excavates, Sha Pei and aseptic process.The damage of the inventive method artificial mechanical tissue is less, effectively saves plant tissue's integrity；In group training process, brownization of plant, the more traditional cultivation of vitrification significantly reduce, and are effectively saved a large amount of economic losses caused by brownization, vitrification, hence it is evident that improve the work efficiency in this field；Along with the increase of group training subculture number, individual plants stabilization characteristics of genetics, value-added effect is obvious；After processing due to asepticization, outer implant thalline infection rate is effectively controlled within 2.5%, is substantially less than traditional 30～50%；Through the process of this technical step, for artificially effectively choosing healthy and strong, healthy bletilla seed source provides premise guarantee.The seedling in group training later stage: root, stem and leaf is healthy and strong, plant ball expands substantially, field-transplanting survival rate more than 95.67%, the late growing stage impetus is notable.
Description
Technical field
The present invention relates to Chinese herbal medicine cultivation technology field, specifically the asepticization processing method of the outer implant of a kind of Pseudobulbus Bletillae (Rhizoma Bletillae).
Background technology
The Pseudobulbus Bletillae (Rhizoma Bletillae) [Betillastriata (Thunb.) Reichb.f.] is the orchid family (Orchidaceae), belongs to herbaceos perennial.It it is one of China's folk tradition Chinese crude drug.The huge market demand, in its natural state, breeding difficulty.In recent years, artificially excessively excavate and suffer that destruction makes wild Pseudobulbus Bletillae (Rhizoma Bletillae) resource sharply reduce with natural ecological environment, endangered.The Pseudobulbus Bletillae (Rhizoma Bletillae) is by one of China's Wild Medicinal being classified as focused protection.Pseudobulbus Bletillae (Rhizoma Bletillae) has astringing to arrest bleeding, the function of detumescence and promoting granulation, can control the disease such as mucosa injury after hemoptysis haematemesis, pulmonary tuberculosis haematemesis, pertussis, radiotherapy.It is distributed in the ground such as East China, south China and Henan, Shaanxi, Sichuan, Yunnan.In its natural state, few Germination And Seedling.The tradition modes of reproduction such as division propagation, not only breeding coefficient is relatively low, and market consumption quantity is big.Being grown under the hillside thick grass of height above sea level 500～1500m, cheuch, small stream limit and sparse woods, happiness is born on more shady and cool moistening fertile soil and sandy loam, likes warm, shady and cool and moistening climatic environment, can not resist cold.The plantation supply capacity in the whole nation at present, far from meeting the market demand face expanded gradually.Therefore the Developing Extension implantation in large scale Pseudobulbus Bletillae (Rhizoma Bletillae), meets the market demand, imperative.
According to this Project Investigation, the outer implant aseptic process method adopted in current Pseudobulbus Bletillae (Rhizoma Bletillae) Fast-propagation has following several respects research to report: tuber lateral bud is after liquid detergent and tap water are cleaned, insert in 75% ethanol and process 10～30 minutes, then insert sterilization in 0.1%HgCl solution and clean (2012, to China)；Pseudobulbus Bletillae (Rhizoma Bletillae) capsule detergent immersion 10min, with washing 5min from the beginning, then 75% alcohol disinfecting 5min, then inserts the 15min that sterilizes in 0.1%HgCl solution and cleans (2012, Li Weiping)；Pseudobulbus Bletillae (Rhizoma Bletillae) tissue-culturing rapid propagation Study on Seedling Cultivation Technique progress (2010, Guan Changdong), the impact (2011, Zhao Manli) that Pseudobulbus Bletillae (Rhizoma Bletillae) group is trained by additive；Pseudobulbus Bletillae (Rhizoma Bletillae) group culturation rapid propagating technology Review Study (2014, plant refreshing)；Pseudobulbus Bletillae (Rhizoma Bletillae) tissue culture technique (2008, Shi Yunping)；Wild Pseudobulbus Bletillae (Rhizoma Bletillae) tissue-culturing rapid propagation research (2005, Yu Chaoxiu)；Fast propagation techniques of Beltilla ochracea by means of tissue cultures (1999, Zhu Yuqiu)；Pseudobulbus Bletillae (Rhizoma Bletillae) rapid seedling cultivation research (2009, Yuan Ning)；Pseudobulbus Bletillae (Rhizoma Bletillae) tissue culture quick breeding research (2006, Tian Yingcui)；The axenic germination of the Pseudobulbus Bletillae (Rhizoma Bletillae) and tissue culture (2010, Ye Jing)；Pseudobulbus Bletillae (Rhizoma Bletillae) group trains the screening study (2008, Wei Kaya) of outer implant；The group culturation rapid propagating technology (2007, He Junrong) of the Pseudobulbus Bletillae (Rhizoma Bletillae)；Pseudobulbus Bletillae (Rhizoma Bletillae) tissue culture and Fast-propagation research (2013, Zou Na)；Treasure the research such as medical material Pseudobulbus Bletillae (Rhizoma Bletillae) tissue rapid propagation and satellite primers exploitation (2012, Li Jing) and all use asepticization process.
In sum, no matter it is tuber, or seed capsules, the aseptic process taked, all substantially passing through: " clean-alcohol-pickled-0.1% sterilization-clean " such a process, for seed through such process, aseptic result is (but known according to now research at present 60～70%, the training hereditary character of bletilla seed subgroup is unstable, and seedling field production invertion rate of green plants is up to more than 80%)；Process for bletilla seed ball lateral bud, owing to processing procedure uses ethanol, HgCl etc. are with the disinfection drug of strong and stimulating, it is sterilized ectoplast and is brought secondary damage to a certain extent, though the aseptic result of about 30～50% can be reached, but the increase along with disinfecting time, the difference of operator, the difference of reagent concentration, sampling sites are different, the increase of wash number, capital brings destructive injury to the interior tissue cell of kind of ball and lateral bud, even if the lateral bud survived, can be influenced by the lengthening of culture period, and cause serious brownization, finally dead.To scientific research, produce and bring irremediable tremendous economic to lose, seriously hinder development and the research in this field.Train due to the group of kind of ball and lateral bud and there is this feature of stabilization characteristics of genetics, research and production are significant.Therefore, plant ball and optimizing bletilla seed source, maintain gene stable, numerous soon, expand the aspects such as Pseudobulbus Bletillae (Rhizoma Bletillae) community superiority and there is remarkable result.Thus, a large amount of asepticization process kind ball and lateral bud propose new historic requirement.
Summary of the invention
It is an object of the invention to for above-mentioned Problems existing, there is provided one to optimize bletilla seed source, maintain that gene are stable, provide safeguard for Pseudobulbus Bletillae (Rhizoma Bletillae) industrialization seedling quality, there is bud ratio height, the processing method of seedling is healthy and strong, disease resistance the is strong outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae) simultaneously.
For achieving the above object, the technical solution used in the present invention is: the asepticization processing method of the outer implant of a kind of Pseudobulbus Bletillae (Rhizoma Bletillae), and step is as follows:
(1) field plant is excavated: the kind ball that tuber growth is well-balanced is chosen in field, retains complete root, stem, leaf, and clear water is cleaned, label registration, load and be placed in paper bag under dark environment, treats that kind of a napiform root system bleaches；
(2) adopted plant is carried out husky training
1. husky training material shifts to an earlier date preparation in 2～3 days, and clear water is cleaned and is filtered dry, and desired amount of sand is carried out high temperature sterilize, and sterilising temp controls at 121～126 DEG C, sterilizing 35min, after cooling, is laid in cultivation seedbed, and sand thickness controls at 6cm, in order to stand-by；
2. daily management is carried out during cultivating
Regulate sand body pH value 5.6～5.8；Spraying the Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid that mass concentration is 1 ‰ to moisten to sand body, sand body humid control is 60%, and the light regulating culture environment impinges upon 1500～2600lx, and temperature is at 20～25 DEG C；
3. cultivating first day, utilizing mass concentration is that 1 ‰ thiophanate methyls spray plant to completely moistening, culture bed overhung yellow plate, and maintains the circulation of air, and after 3～4 days, is 3～8 ‰ tweens by mass concentration successively, and mass concentration is 0.1～1.5 ‰ KMnO4After solution carries out root, stem, foliar spray execute 5min, then soak 2.5min respectively with same Concentration Reagent, each process 1 time after stand-by；
(3) the process plant in (2) is put into 3～5 day time of cultivation in aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid, it is that 1～3% sodium hypochlorite carries out alternate immersion at interval of 20h with the streptomycin that mass concentration is 0.5～2 ‰ and mass concentration afterwards, amount to and carry out 3～6 times, each 10～30min；
(4) step (3) gained plant is cut root, leaf is put into and is carried out shaking table gnotobasis cultivation in 2～3 days in sterile liquid culture fluid；
1. by gained plant in (3) with, after sterile water wash 3～6 times, being soaked in sterilized water, it is put on aseptic operating platform standby；
2. with aseptic nipper, operating scissors, scalpel, inoculation dish, other parts beyond excision bletilla seed ball, often process a Seedling completely, change tweezers, operating scissors, scalpel and inoculation dish；
3. the bletilla seed ball cut is respectively put in ready aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid culture fluid, 4～6 every bottle, is then placed in gnotobasis and carries out shaking table cultivation in 2～3 days；
(5), after repeating step (4) 1～2 times except part sense strain ball, it is 0.1～1.5 ‰ KMnO by the tween that mass concentration is 3～8 ‰, mass concentration successively4Solution, mass concentration are 1～3% sodium hypochlorite solution alkaline, after carrying out the bulb immersion treatment 1～2 time of interval 10h, by a small amount of repeatedly cleaned standby seam of sterilized water；
(6) plant is soaked in 1～10% aseptic ascorbic acid solution after 20min, takes out plant in solid medium, carry out aseptic culture, namely reach asepticization and process requirement.
Further: in step (1), described kind of ball be sized to 10*10mm.
Further: in step (2), it is analytical pure citric acid for regulating the material of described sand body pH.
Further: in step (2), described Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid is by NH3NO3、Ca3(PO4)2、K2SO4, KCI and KH2PO4Be configured to weight nitroxide percentage ratio be 10～15%, phosphorus percentage by weight be 10～15%, potassium percentage by weight be the aqueous solution of 15～25%.
Further: in step (3), described Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid is by NH3NO3、Ca3(PO4)2、K2SO4、KCl、KH2PO4、MgSO4、K2HPO4、Ca(NO3)2、CaCl2、H2BO3、KI、CoCl2And FeC6H5O7·NH4OH、TDZ、BA、NAA、FeSO4·7H2O、CuSO4·5H2O be configured to weight nitroxide percentage ratio be 8～13%, phosphorus percentage by weight be 7～14%, potassium percentage by weight be the aqueous solution of 15～25%.
In the present invention, the effect of each disinfectant solution is as follows:
Tween: effectively protection plant tissue albumen, exempts from the secondary injury of other exogenous chemical materials, strengthens plant resistance；
KMnO4: effectively control the bacterium colony colony number around individual plants；
Sodium hypochlorite, streptomycin: some harmful microorganisms are suppressed or kills；
Ascorbic acid: effectively control plant and suffer O2Unfavorable oxidation Deng oxidant.
The method have the benefit that: asepticization carried out according to above technical step processes plant, and chemical damage is minimum, the later stage tissue culture of plant is had better effects, is mainly reflected in the following aspects:
(1) artificial mechanical tissue damage is less, effectively saves plant tissue's integrity；
(2), in group training process, brownization of plant, the more traditional cultivation of vitrification significantly reduce, and are effectively saved a large amount of economic losses caused by brownization, vitrification, hence it is evident that improve the work efficiency of this neighborhood；
(3) along with the increase of group training subculture number, individual plants stabilization characteristics of genetics, value-added effect is obvious；
(4), after processing due to asepticization, outer implant thalline infection rate is effectively controlled within 2.5%, is substantially less than traditional 30～50%；
(5) through the process of this technical step, for artificially effectively choosing healthy and strong, healthy bletilla seed source provides premise guarantee.The seedling in group training later stage: root, stem and leaf is healthy and strong, plant ball expands substantially, field-transplanting survival rate more than 95.67%, the late growing stage impetus is notable.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1
(1) draw materials the Pseudobulbus Bletillae (Rhizoma Bletillae) completely outer implant, sampled point chooses the main place of production of the Pseudobulbus Bletillae (Rhizoma Bletillae): the bletilla seed source on three ground such as township and Zhong Ping town, Lv Quan county, Kunming is sought as culture materials by unity township, Kunming, Yunnan western suburb, Panlong District, Kunming A Zi, each point plant amounts to 2400 strains, each 800 strains of each point, the sampling time is on April 5th, 2012.Clean through clear water, be placed in dark situation stand-by；
(2) adopted plant is carried out husky training
1. husky training material shifts to an earlier date preparation in 2 days, and clear water is cleaned and is filtered dry, and desired amount of sand is carried out high temperature sterilize, and sterilising temp controls at 121 DEG C, sterilizing 35min, after cooling, is laid in cultivation seedbed, and sand thickness controls at 6cm, in order to stand-by；
2. daily management is carried out during cultivating
Analytical pure citric acid is utilized to regulate sand body pH value 5.6；Spraying the Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid that mass concentration is 1 ‰ to moisten to sand body, sand body humid control is 60%, and the light regulating culture environment impinges upon 1500lx, and temperature is at 20 DEG C；
3. cultivating first day, utilizing mass concentration is that 1 ‰ thiophanate methyls spray plant to completely moistening, culture bed overhung yellow plate, and maintains the circulation of air, and after 3 days, is 3 ‰ tweens by mass concentration successively, and mass concentration is 0.1 ‰ KMnO4After solution carries out root, stem, foliar spray execute 5min, then soak 2.5min respectively with same Concentration Reagent, each process 1 time after stand-by；
(3) the process plant in (2) is carried out the 3 day time of cultivation in aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid, and be that 1% sodium hypochlorite carries out alternate immersion at interval of 20h with the streptomycin that mass concentration is 0.5 ‰ and mass concentration, amount to and carry out 3 times, each 10min；
(4) step (3) gained plant is cut root, leaf is put into and is carried out shaking table gnotobasis cultivation in 2 days in sterile liquid culture fluid；
1. by gained plant in (3) with, after sterile water wash 3 times, being soaked in sterilized water, it is put on aseptic operating platform standby；
2. with aseptic nipper, operating scissors, scalpel, inoculation dish, other parts beyond excision bletilla seed ball, often process a Seedling completely, change tweezers, operating scissors, scalpel and inoculation dish；
3. the bletilla seed ball cut is respectively put in ready aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid culture fluid, 4 every bottle, is then placed in gnotobasis and carries out shaking table cultivation in 2 days；
(5), after repeating step (4) 1 times except part sense strain ball, it is 0.1 ‰ KMnO by the tween that mass concentration is 3 ‰, mass concentration successively4Solution, mass concentration are 1% sodium hypochlorite solution alkaline, after carrying out the bulb immersion treatment 1 time of interval 10h, by a small amount of repeatedly cleaned standby seam of sterilized water；
(6) plant is soaked in 1% aseptic ascorbic acid solution after 20min, sterile water wash 1 time, takes out plant in solid medium, carry out aseptic culture, every bottle of 5 strains.
Adding up according to this experimental data, outer planting body infection rate is 4.5%；Appreciation rate is 637.6%；During to the 5th successive propagation, all not occurring that kind of a source substantially breaks up, character pair waits kind of a source property variation than prominent.Become transplantation of seedlings land for growing field crops after 40 days survival rate 95.41%；Plant ball field production after 110 days, enlarged volume 144%；Susceptible gene controls 0.94%.
Embodiment 2
(1) draw materials the Pseudobulbus Bletillae (Rhizoma Bletillae) completely outer implant, sampled point chooses the main place of production of the Pseudobulbus Bletillae (Rhizoma Bletillae): the bletilla seed source on three ground such as township and Zhong Ping town, Lv Quan county, Kunming is sought as culture materials by unity township, Kunming, Yunnan western suburb, Panlong District, Kunming A Zi, each point plant amounts to 2400 strains, each 800 strains of each point, the sampling time is on April 6th, 2012.Clean through clear water, be placed in dark situation stand-by；
(2) adopted plant is carried out husky training
1. husky training material shifts to an earlier date preparation in 3 days, and clear water is cleaned and is filtered dry, and desired amount of sand is carried out high temperature sterilize, and sterilising temp controls at 123 DEG C, sterilizing 35min, after cooling, is laid in cultivation seedbed, and sand thickness controls at 6cm, in order to stand-by；
2. daily management is carried out during cultivating
Sand body pH value is regulated 5.7 with analytical pure citric acid；Spraying the Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid that mass concentration is 1 ‰ to moisten to sand body, sand body humid control is 60%, and the light regulating culture environment impinges upon 2000lx, and temperature is at 23 DEG C；
3. cultivating first day, utilizing mass concentration is that 1 ‰ thiophanate methyls spray plant to completely moistening, culture bed overhung yellow plate, and maintains the circulation of air, and after 4 days, is 5 ‰ tweens by mass concentration successively, and mass concentration is 0.7 ‰ KMnO4After solution carries out root, stem, foliar spray execute 5min, then soak 2.5min respectively with same Concentration Reagent, each process 1 time after stand-by；
(3) the process plant in (2) is carried out the 4 day time of cultivation in aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid, and be that 2% sodium hypochlorite carries out alternate immersion at interval of 20h with the streptomycin that mass concentration is 1 ‰ and mass concentration, amount to and carry out 4 times, each 20min；
(4) step (3) gained plant is cut root, leaf is put into and is carried out shaking table gnotobasis cultivation in 2 days in sterile liquid culture fluid；
1. by gained plant in (3) with, after sterile water wash 4 times, being soaked in sterilized water, it is put on aseptic operating platform standby；
2. with aseptic nipper, operating scissors, scalpel, inoculation dish, other parts beyond excision bletilla seed ball, often process a Seedling completely, change tweezers, operating scissors, scalpel and inoculation dish；
3. the bletilla seed ball cut is respectively put in ready aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid culture fluid, 5 every bottle, is then placed in gnotobasis and carries out shaking table cultivation in 3 days；
(5), after repeating step (4) 2 times except part sense strain ball, it is 1.0 ‰ KMnO by the tween that mass concentration is 6 ‰, mass concentration successively4Solution, mass concentration are 2% sodium hypochlorite solution alkaline, after carrying out the bulb immersion treatment 2 times of interval 10h, by a small amount of repeatedly cleaned standby seam of sterilized water；
(6) plant is soaked in 5% aseptic ascorbic acid solution after 20min, sterile water wash 3 times, takes out plant in solid medium, carry out aseptic culture, every bottle of 5 strains.Adding up according to this experimental data, outer planting body infection rate is 4.5%；Appreciation rate is 637.6%；During to the 5th successive propagation, all not occurring that kind of a source substantially breaks up, character pair waits kind of a source property variation than prominent.Become transplantation of seedlings land for growing field crops after 40 days survival rate 95.41%；Plant ball field production after 110 days, enlarged volume 150%；Susceptible gene controls 0.99%.
Embodiment 3
(1) draw materials the Pseudobulbus Bletillae (Rhizoma Bletillae) completely outer implant, sampled point chooses the main place of production of the Pseudobulbus Bletillae (Rhizoma Bletillae): the bletilla seed source on three ground such as township and Zhong Ping town, Lv Quan county, Kunming is sought as culture materials by unity township, Kunming, Yunnan western suburb, Panlong District, Kunming A Zi, each point plant amounts to 2400 strains, each 800 strains of each point, the sampling time is on April 7th, 2012.Clean through clear water, be placed in dark situation stand-by；
(2) adopted plant is carried out husky training
1. husky training material shifts to an earlier date preparation in 3 days, and clear water is cleaned and is filtered dry, and desired amount of sand is carried out high temperature sterilize, and sterilising temp controls at 126 DEG C, sterilizing 35min, after cooling, is laid in cultivation seedbed, and sand thickness controls at 6cm, in order to stand-by；
2. daily management is carried out during cultivating
Analytical pure citric acid is utilized to regulate sand body pH value 5.8；Spraying the Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid that mass concentration is 1 ‰ to moisten to sand body, sand body humid control is 60%, and the light regulating culture environment impinges upon 2600lx, and temperature is at 25 DEG C；
3. cultivating first day, utilizing mass concentration is that 1 ‰ thiophanate methyls spray plant to completely moistening, culture bed overhung yellow plate, and maintains the circulation of air, and after 4 days, is 8 ‰ tweens by mass concentration successively, and mass concentration is 1.5 ‰ KMnO4After solution carries out root, stem, foliar spray execute 5min, then soak 2.5min respectively with same Concentration Reagent, each process 1 time after stand-by；
(3) the process plant in (2) is carried out the 5 day time of cultivation in aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid, and be that 3% sodium hypochlorite carries out alternate immersion at interval of 20h with the streptomycin that mass concentration is 2 ‰ and mass concentration, amount to and carry out 6 times, each 30min；
(4) step (3) gained plant is cut root, leaf is put into and is carried out shaking table gnotobasis cultivation in 3 days in sterile liquid culture fluid；
1. by gained plant in (3) with, after sterile water wash 6 times, being soaked in sterilized water, it is put on aseptic operating platform standby；
2. with aseptic nipper, operating scissors, scalpel, inoculation dish, other parts beyond excision bletilla seed ball, often process a Seedling completely, change tweezers, operating scissors, scalpel and inoculation dish；
3. the bletilla seed ball cut is respectively put in ready aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid culture fluid, 6 every bottle, is then placed in gnotobasis and carries out shaking table cultivation in 3 days；
(5), after repeating step (4) 2 times except part sense strain ball, it is 1.5 ‰ KMnO by the tween that mass concentration is 8 ‰, mass concentration successively4Solution, mass concentration are 3% sodium hypochlorite solution alkaline, after carrying out the bulb immersion treatment 2 times of interval 10h, by a small amount of repeatedly cleaned standby seam of sterilized water；
(6) plant is soaked in 10% aseptic ascorbic acid solution after 20min, sterile water wash 4 times, takes out plant in solid medium, carry out aseptic culture, every bottle of 5 strains.Adding up according to this experimental data, outer planting body infection rate is 4.5%；Appreciation rate is 637.6%；During to the 5th successive propagation, all not occurring that kind of a source substantially breaks up, character pair waits kind of a source property variation than prominent.Become transplantation of seedlings land for growing field crops after 40 days survival rate 95.41%；Plant ball field production after 110 days, enlarged volume 156%；Susceptible gene controls 1.1%.
In the present invention, also outer for the Pseudobulbus Bletillae (Rhizoma Bletillae) of 2400 strains on the three ground implant traditionally pattern of sterilizing is carried out control treatment.1. clean through liquid detergent and tap water, put after rinsing 10～30min under flowing water, be placed in 75% ethanol and process 10～30s, then then through using aseptic water washing after the HgCl disinfectant solution 10min of 0.1%, standby after cutting the part of outer implant variable color.2. outer implant is put in identical solid medium and cultivate, every bottle of 5 strains.Cultivate the 4th day, start the infection rate occurring 15%；By the 15th day, starting universality brownization occur, infection rate is up to 36.61%；To when cultivating the 35th day, infection rate is up to 54.32%, during to the 5th successive propagation, has begun to occur wide leaf, spire, leafy, character variation such as " leaf line skill ".Overall appreciation rate is 597.4%；Become transplantation of seedlings land for growing field crops after 40 days survival rate 75.41%；Planting ball field production after 110 days, enlarged void volume is 104～126%；Susceptible gene controls 6.94～11.1%.
In above three embodiment, Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid is by NH3NO3、Ca3(PO4)2、K2SO4, KCI and KH2PO4Be configured to weight nitroxide percentage ratio be 10%, phosphorus percentage by weight be 10%, potassium percentage by weight be the aqueous solution of 20%.
Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid is by NH3NO3、Ca3(PO4)2、K2SO4、KCl、KH2PO4、MgSO4、K2HPO4、Ca(NO3)2、CaCl2、H2BO3、KI、CoCl2And FeC6H5O7·NH4OH、TDZ、BA、NAA、FeSO4·7H2O、CuSO4·5H2O be configured to weight nitroxide percentage ratio be 10%, phosphorus percentage by weight be 10%, potassium percentage by weight be the aqueous solution of 20%.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.
Claims (3)
1. the asepticization processing method of the outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae), it is characterised in that step is as follows:
(1) field plant is excavated: the kind ball that tuber growth is well-balanced is chosen in field, retains complete root, stem, leaf, and clear water is cleaned, label registration, load and be placed in paper bag under dark environment, treats that kind of a napiform root system bleaches；
(2) adopted plant is carried out husky training
1. husky training material shifts to an earlier date preparation in 2～3 days, and clear water is cleaned and is filtered dry, and desired amount of sand is carried out high temperature sterilize, and sterilising temp controls at 121～126 DEG C, sterilizing 35min, after cooling, is laid in cultivation seedbed, and sand thickness controls at 6cm, in order to stand-by；
2. daily management is carried out during cultivating
Regulate sand body pH value 5.6～5.8；Spraying the Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid that mass concentration is 1 ‰ to moisten to sand body, sand body humid control is 60%, and the light regulating culture environment impinges upon 1500～2600lx, and temperature is at 20～25 DEG C；
3. cultivating first day, utilizing mass concentration is that 1 ‰ thiophanate methyls spray plant to completely moistening, culture bed overhung yellow plate, and maintains the circulation of air, and after 3～4 days, is 3～8 ‰ tweens by mass concentration successively, and mass concentration is 0.1～1.5 ‰ KMnO4After solution carries out root, stem, foliar spray execute 5min, then soak 2.5min respectively with same Concentration Reagent, each process 1 time after stand-by；
Described Pseudobulbus Bletillae (Rhizoma Bletillae) special nutrient fluid is by NH3NO3、Ca3(PO4)2、K2SO4, KCl and KH2PO4Be configured to weight nitroxide percentage ratio be 10～15%, phosphorus percentage by weight be 10～15%, potassium percentage by weight be the aqueous solution of 15～25%；
(3) the process plant in (2) is put into 3～5 day time of cultivation in aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid, it is that 1～3% sodium hypochlorite carries out alternate immersion at interval of 20h with the streptomycin that mass concentration is 0.5～2 ‰ and mass concentration afterwards, amount to and carry out 3～6 times, each 10～30min；
Described Pseudobulbus Bletillae (Rhizoma Bletillae) liquid nutritional liquid is by NH3NO3、Ca3(PO4)2、K2SO4、KCl、KH2PO4、MgSO4、K2HPO4、Ca(NO3)2、CaCl2、H2BO3、KI、CoCl2、FeC6H5O7·NH4OH、TDZ、BA、NAA、FeSO4·7H2O and CuSO4·5H2O is configured to weight nitroxide percentage ratio to be 8-13%, phosphorus percentage by weight is 7-14%, potassium percentage by weight is the aqueous solution of 15-25%；
(4) step (3) gained plant is cut root, leaf is put into and is carried out shaking table gnotobasis cultivation in 2～3 days in sterile liquid culture fluid；
1. by gained plant in (3) with, after sterile water wash 3～6 times, being soaked in sterilized water, it is put on aseptic operating platform standby；
2. with aseptic nipper, operating scissors, scalpel, inoculation dish, other parts beyond excision bletilla seed ball, often process a Seedling completely, change tweezers, operating scissors, scalpel and inoculation dish；
3. the bletilla seed ball cut is respectively put in ready aseptic Pseudobulbus Bletillae (Rhizoma Bletillae) liquid culture fluid, 4～6 every bottle, is then placed in gnotobasis and carries out shaking table cultivation in 2～3 days；
(5), after repeating step (4) 1～2 times except part sense strain ball, it is 0.1～1.5 ‰ KMnO by the tween that mass concentration is 3～8 ‰, mass concentration successively4Solution, mass concentration are 1～3% sodium hypochlorite solution alkaline, after carrying out the bulb immersion treatment 1～2 time of interval 10h, by a small amount of repeatedly cleaned standby seam of sterilized water；
(6) plant is soaked in 1～10% aseptic ascorbic acid solution after 20min, takes out plant in solid medium, carry out aseptic culture, namely reach asepticization and process requirement.
2. the asepticization processing method of the outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae) according to claim 1, it is characterised in that: in step (1), described kind of ball be sized to 10*10mm.
3. the asepticization processing method of the outer implant of the Pseudobulbus Bletillae (Rhizoma Bletillae) according to claim 1, it is characterised in that: in step (2), it is analytical pure citric acid for regulating the material of described sand body pH.
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| CN103314856A (en) * | 2013-07-08 | 2013-09-25 | 重庆市秀山红星中药材开发有限公司 | Bletilla lateral bud tissue culture propagation method |
| CN103828717A (en) * | 2013-08-15 | 2014-06-04 | 张家界本草科技有限公司 | Rapid propagating and seedling culturing method by virtue of Bletilla tubers |
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| CN103828717A (en) * | 2013-08-15 | 2014-06-04 | 张家界本草科技有限公司 | Rapid propagating and seedling culturing method by virtue of Bletilla tubers |
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