CN106171969A - A kind of clone culture technique of Herba Dendrobii embryo microgranule - Google Patents
A kind of clone culture technique of Herba Dendrobii embryo microgranule Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Mushroom Cultivation (AREA)
Abstract
The present invention discloses clone's culture technique of a kind of Herba Dendrobii embryo microgranule, is mainly used in the large-scale production of the high-quality Herba Dendrobii protocorm with medicinal, edibility.The present invention selects the mellow fruit pod of Herba Dendrobii, after Aseptic sterilisation, take out the micro-material of embryo access clone's fluid medium, ventilation low temperature light culture is after one week, proceed to continue ventilation suspension culture at illumination 27 DEG C three weeks, complete to sprout to embryo microgranule, expand, turn green, forming the little protocorm of taper, this can be as the germplasm materials of Herba Dendrobii medical raw bulb large-scale production.The present invention uses Herba Dendrobii protocorm Special ventilating suspension culture device to carry out clone and cultivates, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and feed supplement in real time, promote embryo fast-growth, whole cultivation cycle is only one month, compared with needing 3-4 month with tradition culture technique, substantially reduce the production cycle, and single batch production scale is big, yield is high, low cost, owing to being application clone technology, can be with the hereditary stability of effective guarantee plant and safety.
Description
Technical field
The invention belongs to plant cell engineering field, particularly relate to clone's culture technique of a kind of Herba Dendrobii embryo microgranule.
Background technology
Herba Dendrobii (Dendrobium officinale
Kmuraet Migo) it is China's tradition rare Chinese medicine, sweet in the mouth, cold nature, there is the effects such as nourishing the stomach of promoting the production of body fluid, nourishing YIN and clearing away heat, lung moistening kidney tonifying, the strong waist of improving eyesight.Recording according to state-promulgated pharmacopoeia, its main pharmacodynamics composition is dendrobium polysaccharide and dendrobine.
Herba Dendrobii medical raw bulb is through liquid Clone formation bulb protocorm by the embryo microgranule in Herba Dendrobii fruit pod, this stage, protocorm can be made to be enriched substantial amounts of nascent and secondary metabolite, the stage that wherein content of dendrobine, EN and polysaccharide is the highest in being its whole growth course by nutrition adjustment and the induction of special cultivation bar.Prove that this protocorms has bar fresh with commercially available Herba Dendrobii and the equal medicinal and health value of Herba Dendrobii extract through clinical research.
Embryo microgranule refers to powdery in Herba Dendrobii mellow fruit pod, tiny faint yellow microgranule, it is the most genetic that each micro-material of embryo all carries plant female parent, under lowered in field environment, it need to be combined with certain fungal elicitor and could sprout, therefore nature germination rate is extremely low, in laboratory environments, Herba Dendrobii protocorm and Herba Dendrobii plant can be can be formed directly in by the way of clone induces.
At present, the traditional mode of production mode of protocorm is that the induction utilizing implant leaf stem section outer to Herba Dendrobii produces small cell cluster, again small cell cluster is carried out expanding propagation and continuous successive transfer culture and obtain, for stricti jurise, this protocorms should be referred to as protocorms group, owing to being the cell induction from outer planting, and constantly successive transfer culture and make its inherited character have randomness and relatively macromutation probability, the whole hereditary characters of female parent can not be fully retained, thus cause its express spectra can not be completely the same with female parent, main active ingredient such as polysaccharide, the expressions such as dendrobine are low or expression product changes, protocorms group is made all to can not be guaranteed on medicinal or edibility and safety.
In view of this, the present inventor furthers investigate for drawbacks described above, then has this case to produce.Protocorm in the present invention is single embryo microgranule induced synthesis, and protocorm is formed without multiple irregular cell group during propagation, but growth promoter becomes bigger plant embryoid, has plant feature.Each embryo microgranule only carries out autologous growth growth during induced synthesis medical raw bulb, without being divided into multiple variant cell group, thus intactly remains the inherited character of female parent.
Summary of the invention
Present invention aim at providing clone's culture technique of a kind of Herba Dendrobii embryo microgranule, be mainly used in the large-scale production of the high-quality Herba Dendrobii protocorm with medicinal, edibility.Patent of the present invention selects the mellow fruit pod of Herba Dendrobii, after Aseptic sterilisation, take out the micro-material of embryo access clone's fluid medium, ventilation low temperature light culture is after one week, proceed to continue ventilation suspension culture at illumination 27 DEG C three weeks, complete to sprout to embryo microgranule, expand, turn green, forming the little protocorm of taper, this can be as the germplasm materials of Herba Dendrobii medical raw bulb large-scale production.The present invention uses Herba Dendrobii protocorm Special ventilating suspension culture device to carry out clone and cultivates, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and feed supplement in real time, promote embryo fast-growth, whole cultivation cycle is only one month, compared with needing 3-4 month with tradition culture technique, substantially reduce the production cycle, and single batch production scale is big, yield is high, low cost, owing to being application clone technology, can be with the hereditary stability of effective guarantee plant and safety.
In order to reach above-mentioned purpose, the solution of the present invention is:
A kind of clone culture technique of Herba Dendrobii embryo microgranule, this technology selects the Herba Dendrobii mellow fruit pod of artificial growth, really pod is full, dark green or green purple, gathers during October to November, first wrap by dried towel, putting into the vial with cover having desiccant, after processing 3 days at low temperature 4 DEG C, the unnecessary part of fruit pod is excised by taking-up knife blade, only stay the fruit stem of 1～2cm length, weigh and record.
Further, the fruit pod handled well is moved in the most sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, processes 10min in 10% sodium hypochlorite, aseptic water washing 3 times, it is then immersed in 40s in 70% ethanol, aseptic water washing 3 times, is placed in inoculation dish bottom incision fruit pod, it is sown into equably in the aerobic culture bottle equipped with clone's fluid medium, finish the fruit pod planted to take out, weigh and record, calculate the grams being sown into embryo microgranule.
Further, first it is inserted in finishing the aerobic culture bottle planted in lucifuge bag, places on culturing rack, connect ventilation pump, ventilation 5L/min, dissolved oxygen coefficient 2%, cultivation temperature 20 DEG C, carries out light culture 1 week, then removes lucifuge bag, enter illumination cultivation, ventilation 10L/min, dissolved oxygen coefficient 2%, cultivation temperature 27 DEG C, intensity of illumination 2500Lux, cultivate 4 weeks, period carries out a fluid infusion week about, totally 3 times.
The formula of the clone's liquid culture in the present invention is: modified MS medium, wherein ammonium nitrate is replaced with ammonium sulfate, and addition halves, and calcium chloride addition halves；Plant growth regulator: 6 benzyladenine 0.02 deals；Osmolyte regulator, carbon source: sucrose 40 deal；Amino acid supplements, nitrogen source: acid hydrolyzed casein 0.2 deal, natural organic additive: potato juice 150 deal, add ultra-pure water and be configured to 1000 deals.
Feed supplement formula is: 1/4 modified MS medium, and single amount infused is the 1/3 of culture medium total amount, altogether fluid infusion 3 times.
Compared with the prior art, the present invention uses Herba Dendrobii protocorm Special ventilating suspension culture device to carry out clone and cultivates, can be that incubation constantly provides fresh dissolved oxygen, effective stirring and feed supplement in real time, promote embryo fast-growth, whole cultivation cycle is only 35 days, compared with needing 3-4 month with tradition culture technique, substantially reduce the production cycle, and single batch production scale is big, yield is high, low cost, owing to being application clone technology, can be with the hereditary stability of effective guarantee plant and safety.The little protocorm of the produced acquisition of the present invention can be as the optimal germplasm materials of Herba Dendrobii medical raw bulb large-scale production, this supports for the factorial praluction offer technology and raw material realizing Herba Dendrobii medical raw bulb, the development process of Herba Dendrobii industrialization can be quickly propelled, product safety and effectiveness is ensured by science and technology, and by effectively reducing production cost and retail cost, allow more people consume can play the Herba mesonae chinensis Herba Dendrobii among the people once exclusively enjoyed only for imperial family.
Detailed description of the invention
In order to technical scheme is explained further, below in conjunction with the embodiment of the present invention and existing solid culture embodiment, the present invention will be described in detail in agitated submerged culture enforcement.
Embodiment one: this case is invented
Select the Herba Dendrobii mellow fruit pod of artificial growth, really pod is full, dark green or green purple, is gathered October 15, first wraps by dried towel, put into the vial with cover having desiccant, after processing 3 days at low temperature 4 DEG C, with knife blade, the unnecessary part of fruit pod is excised, only stay the fruit stem of 1.5cm length, weigh and record, obtain 1.63g.
The fruit pod handled well is moved in the most sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, process 10 ~ 20min, aseptic water washing 3 times in 10% sodium hypochlorite, it is then immersed in 40smin in 70% ethanol, aseptic water washing 3 times, is placed in inoculation dish bottom incision fruit pod, is sown into equably in the aerobic culture bottle equipped with clone's fluid medium, finish the fruit pod planted to take out, weighing and record, obtain 0.93g, it is 0.7g that calculating is sown into the grams of embryo microgranule.
First it is inserted in finishing the aerobic culture bottle planted in lucifuge bag, places on culturing rack, connect ventilation pump, ventilation 5L/min, dissolved oxygen coefficient 2%, cultivation temperature 20 DEG C, carry out light culture 1 week, then remove lucifuge bag, enter illumination cultivation, ventilation 10L/min, dissolved oxygen coefficient 2%, cultivation temperature 25 DEG C, intensity of illumination 2500Lux, cultivating 4 weeks, period carries out a fluid infusion week about, totally 3 times.
Little protocorm is taken out in cultivation after terminating, outward appearance is recorded as: yellow green, and form is full, and taper is spherical, and bulb is bigger；After drain well, weigh to obtain 81.23g, and calculating proliferation times is 115.7.
The formula of clone's liquid culture of the present embodiment is: modified MS medium, wherein ammonium nitrate is replaced with ammonium sulfate, and addition halves, and calcium chloride addition halves；Plant growth regulator: 6 benzyladenine 0.2mL；Osmolyte regulator, carbon source: sucrose 40g；Amino acid supplements, nitrogen source: acid hydrolyzed casein 0.2g, natural organic additive: potato juice 150g, add ultra-pure water and be settled to 1L.
Feed supplement formula is: 1/4 modified MS medium, and single amount infused is the 350mL of culture medium total amount, altogether fluid infusion three times.
Embodiment two: solid culture
Select the Herba Dendrobii mellow fruit pod of artificial growth, really pod is full, dark green or green purple, is gathered October 15, first wraps by dried towel, put into the vial with cover having desiccant, after processing 3 days at low temperature 4 DEG C, with knife blade, the unnecessary part of fruit pod is excised, only stay the fruit stem of 1.5cm length, weigh and record, obtain 1.45g.The fruit pod handled well is moved in the most sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, process 10 ~ 20min, aseptic water washing 3 times in 10% sodium hypochlorite, it is then immersed in 40smin in 70% ethanol, aseptic water washing 3 times, is placed in inoculation dish bottom incision fruit pod, is sown into equipped with in the tissue culture bottle of solid medium equably, finish the fruit pod planted to take out, weighing and record, obtain 0.81g, it is 0.64g that calculating is sown into the grams of embryo microgranule.
The tissue culture bottle planted will be finished, place darkroom and cultivate one week, cultivation temperature 20 DEG C, then enter illumination cultivation, and cultivation temperature 25 DEG C, intensity of illumination 2500Lux, cultivate 8 weeks.
Little protocorm is taken out in cultivation after terminating, outward appearance is recorded as: top layer bulb is yellow green, and bottom bulb is yellow-white, and bulb is less, for elliptic conic shape；Weigh to obtain 25.12g, and calculating proliferation times is 39.2.
The formula of the fixing cultivation of the present embodiment is: modified MS medium, and wherein ammonium nitrate is replaced with ammonium sulfate, and addition halves, and calcium chloride addition halves；Plant growth regulator: 6 benzyladenine 0.2mL；Osmolyte regulator, carbon source: sucrose 40g；Amino acid supplements, nitrogen source: acid hydrolyzed casein 0.2g, natural organic additive: potato juice 150g, 8 grams of agar, add ultra-pure water and be settled to 1L.
Embodiment three: agitated submerged culture
Select the Herba Dendrobii mellow fruit pod of artificial growth, really pod is full, dark green or green purple, is gathered October 15, first wraps by dried towel, put into the vial with cover having desiccant, after processing 3 days at low temperature 4 DEG C, with knife blade, the unnecessary part of fruit pod is excised, only stay the fruit stem of 1.5cm length, weigh and record, obtain 1.65g.The fruit pod handled well is moved in the most sterilized superclean bench, first with cotton ball soaked in alcohol wiping fruit pod surface, process 10 ~ 20min, aseptic water washing 3 times in 10% sodium hypochlorite, it is then immersed in 40smin in 70% ethanol, aseptic water washing 3 times, is placed in inoculation dish bottom incision fruit pod, is sown into equipped with in the tissue culture bottle of fluid medium equably, finish the fruit pod planted to take out, weighing and record, obtain 0.86g, it is 0.79g that calculating is sown into the grams of embryo microgranule.
The tissue culture bottle planted will be finished, light culture one week, 100 turns/min of rotating speed, cultivation temperature 20 DEG C in placement shaken cultivation case, then enter illumination cultivation, and cultivation temperature 25 DEG C, 150 turns/min of rotating speed, intensity of illumination 2500Lux, cultivate 4 weeks.
Little protocorm is taken out in cultivation after terminating, outward appearance is recorded as: bulb is yellow green, and bulb is less, has hyalinization；Weigh to obtain 48.31g, and calculating proliferation times is 54.8.
The formula of the fixing cultivation of the present embodiment is: modified MS medium, and wherein ammonium nitrate is replaced with ammonium sulfate, and addition halves, and calcium chloride addition halves；Plant growth regulator: 6 benzyladenine 0.2mL；Osmolyte regulator, carbon source: sucrose 40g；Amino acid supplements, nitrogen source: acid hydrolyzed casein 0.2g, natural organic additive: potato juice 150g, add ultra-pure water and be settled to 1L.
To sum up, as shown in table 1, the present invention is compared with existing solid tissue culture technology and agitated submerged culture technology, and its growth coefficient improves notable, protocorm quality better.
The parameter comparison of 1 three kinds of Protocorm technology of table
| Technical scheme | Inoculum concentration | Cultivation cycle | Outward appearance describes | Propagation weight | Proliferation times |
| This case | 0.70g | 35 days | Green, form is full, and taper is spherical, and bulb is bigger | 81.23g | 115.7 |
| Solid culture | 0.64g | 35 days | Top layer bulb is yellow green, and bottom bulb is yellow-white, and bulb is less, for elliptic conic shape | 25.12g | 39.2 |
| Agitated submerged culture | 0.79g | 35 days | Bulb is yellow green, and bulb is less, has hyalinization | 48.31g | 54.8 |
Claims (6)
1. clone's culture technique of a Herba Dendrobii embryo microgranule, it is characterised in that include herein below: 1. select the embryo microgranule in Herba Dendrobii mellow fruit pod as inoculation material；2. Herba Dendrobii embryo microgranule is accessed in clone's fluid medium suspension culture of ventilating；3. light culture of first ventilating after completing inoculation proceeds to after one week continue ventilation suspension culture at illumination 27 DEG C three weeks；4. start to carry out a feed operation week about to cultivation terminal procedure from incubation time；5. cultivate and sprout completely to embryo microgranule, expand, turn green, form taper bottom set.
Clone's culture technique of a kind of Herba Dendrobii embryo microgranule the most as claimed in claim 1, it is characterized in that, described content 1. in first with cotton ball soaked in alcohol wiping fruit pod surface, 10 ~ 20min is processed in 5 ~ 10% sodium hypochlorite, aseptic water washing 3 ~ 5 times, is then immersed in 30s ~ 1min in 70 ~ 75% ethanol, aseptic water washing 3 ~ 5 times, it is placed in inoculation dish bottom incision fruit pod, is sown into equably in the aerobic culture bottle equipped with clone's fluid medium.
Clone's culture technique of a kind of Herba Dendrobii embryo microgranule the most as claimed in claim 1, it is characterized in that, described content 2. in clone liquid culture formula be: modified MS medium, wherein ammonium nitrate is replaced with ammonium sulfate, addition halves, and calcium chloride addition halves；Plant growth regulator: 6 benzyladenine 0.02 deals；Osmolyte regulator, carbon source: sucrose 40 deal；Amino acid supplements, nitrogen source: acid hydrolyzed casein 0.2 deal, natural organic additive: potato juice 150 deal, add ultra-pure water and be configured to 1000 deals.
Clone's culture technique of a kind of Herba Dendrobii embryo microgranule the most as claimed in claim 1, it is characterized in that, the parameter of described content 3. middle ventilation light culture is: ventilation 5L/min, dissolved oxygen coefficient 2%, cultivation temperature 20 DEG C, be inserted in lucifuge bag be placed in group training frame start cultivate；The parameter of illumination cultivation is: ventilation 10L/min, dissolved oxygen coefficient 2%, cultivation temperature 27 DEG C, intensity of illumination 2500Lux, puts and cultivates under illumination cultivation frame.
5. clone's culture technique of a kind of Herba Dendrobii embryo microgranule as described in claim 1,3, it is characterised in that described content 4. in feed supplement formula be: 1/4 modified MS medium, single amount infused is the 1/3 of culture medium total amount, altogether fluid infusion three times.
Clone's culture technique of a kind of Herba Dendrobii embryo microgranule the most as claimed in claim 1, it is characterized in that, described content 5. middle embryo microgranule is after the cultivation of four time-of-weeks, can completely sprout, expand, turn green, form the size little protocorm of taper at 0.2-0.5mm, and using this little protocorm as the germplasm materials of Herba Dendrobii medical raw bulb.
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| CN110771443A (en) * | 2019-11-22 | 2020-02-11 | 阳山县三连阳生态农林开发有限公司 | Yangshan bletilla striata single culture medium formula sterile seeding seedling production process |
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| CN101268759A (en) * | 2008-04-16 | 2008-09-24 | 中国林业科学研究院林业研究所 | Cultivation method for Dendrobium initial species embryo |
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| CN103563741A (en) * | 2012-07-27 | 2014-02-12 | 宁波德健生物科技有限公司 | Aseptic detoxicant culturing method of cracked Dendrobium officinale capsule embryos |
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2015
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Patent Citations (3)
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| CN101268759A (en) * | 2008-04-16 | 2008-09-24 | 中国林业科学研究院林业研究所 | Cultivation method for Dendrobium initial species embryo |
| CN102301953A (en) * | 2011-07-20 | 2012-01-04 | 江苏农林职业技术学院 | Method for efficiently and rapidly propagating Dendrobium candidum test-tube seedlings in large scale |
| CN103563741A (en) * | 2012-07-27 | 2014-02-12 | 宁波德健生物科技有限公司 | Aseptic detoxicant culturing method of cracked Dendrobium officinale capsule embryos |
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| YUAN LIN ET.AL.: "Effects of light quality on growth and development of protocorm-like bodies of Dendrobium officinale in vitro", 《PLANT CELL TISSUE AND ORGAN CULTURE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110771443A (en) * | 2019-11-22 | 2020-02-11 | 阳山县三连阳生态农林开发有限公司 | Yangshan bletilla striata single culture medium formula sterile seeding seedling production process |
| CN110771443B (en) * | 2019-11-22 | 2021-12-21 | 阳山县三连阳生态农林开发有限公司 | Yangshan bletilla striata single culture medium formula sterile seeding seedling production process |
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