CN102550416B - Cultivation method for producing hupenine A through in vitro induction proliferation of thallus of huperzia serrata - Google Patents
Cultivation method for producing hupenine A through in vitro induction proliferation of thallus of huperzia serrata Download PDFInfo
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Abstract
The invention discloses a cultivation method for producing hupenine A through in vitro induction proliferation of thallus of huperzia serrata. The method comprises the following steps: selecting a wilding huperzia serrata plant which is healthy and has no plant disease or insect pests, and taking a new branch which grows in the current year and is 3 cm in length; producing thallus through induction, proliferating into a piece of thalline tissue block with the diameter of 3 cm, and using the two culture mediums as follows: a thalline differentiation launching culture medium and a thalline mulitiplication culture medium; and dividing and subculturing thalline. The cultivation method and a substrate formula provide essential environmental conditions to in vitro induction and proliferation of thallus of huperzia serrata in production of hupenine A. When the method provided by the invention is used for in vitro culture of huperzia serrata, a great number of medicinal plant resources of thalline of huperzia serrata can be obtained. Significant economic values can be produced when the method is used for enlarging the scale of extracting produced medicine hupenine A (Hup-A) through in vitro culture of thalline.
Description
Technical field
The present invention relates to a Plant Biotechnology, namely the cultural method of in-vitro inducing and propagation relates in particular to the cultural method that a kind of stripped Huperzia serrata thallus of producing huperzine is induced and bred.
Background technology
Huperzia serrata Huperzia serrata (Thunb.ex Murray) Trev. has another name called serrate clubmoss herb, serrate clubmoss herb, feet added to a snake by an ignorant artist grass etc., is Huperziaceae stone araucaria pteridophyte, all herbal medicine.Alkaloid-huperzine (hupenine A, Hup-A) in this plant of domestic reported first in 1972 has the striated muscle relaxation effect, and later on further investigation finds that this alkaloid is a kind of potent, reversible and high selectivity acetylcholinesteraseinhibitors inhibitors.Under low dosage Hup-A, acetylcholinesterase (Ach E) there is powerful inhibitory action, acetylcholine (Ach) content in nerve synapse gap in the area is obviously raise, thereby strengthen the neuronal excitation conduction, the effect that improves cognitive function, enhancing memory maintenance and promote memory represents is played in the excitation in intensified learning and memory brain district.It is the present medicine of domestic the most successful treatment Alzheimer disease (senile dementia).Huperzine Hup-A medicine is mainly derived from natural Huperzia serrata herb, because the special efficacy of huperzine, Chinese scholars is complete synthetic to its, derivative and analog synthetic done a large amount of work, also because of the special molecular structure of Hup-A, artificial synthetic wasting time and energy, cost is high, so far almost do not obtain the better derivative of the natural Hup-A of specific activity and analog, therefore artificial synthetic Hup-A and analog thereof are produced from commercialization and are also had longer distance, society arrives along with astogeny, Alzheimer disease (senile dementia) has become the human the fourth-largest serious illness, directly cause the supply breach of Hup-A on the international market increasing, natural serrate clubmoss herb scarcity of resources highlights.
Because the Huperziaceae plant growths such as Huperzia serrata are in remote, thickly forested mountains, habitat conditions is special, is difficult to solve the large-area artificial cultivation of Huperziaceae plant.Take Huperziaceae Vitro Plant tissue to cultivate and to develop Hup-A resource in the plants of Huperzia, but because of explant sterilization and Growth and Differentiation difficulty thereof, not yet break through at present its effective technology both at home and abroad.And the Huperzia serrata cultured in vitro is the important technological platform that its former plant fast propagation of exploitation or biotechnology are produced natural huperzine (Hup-A) resource, must the effective cultured in vitro asexual reproduction of model system.
Summary of the invention
The cultural method that the object of the present invention is to provide a kind of stripped Huperzia serrata thallus of producing huperzine to induce and breed, the method is simple, is convenient to use of large-scale production.
The present invention is achieved in that method step is:
(1.1) material: (Huperzia serrata (Thunb.ex Murray) Trev. selects stalwartness without the wild Huperzia serrata plant in the Mount Lushan of damage by disease and insect to Huperzia serrata, gets the then long shoot of the about 3cm of pumping;
(1.2) to the explant cleaning and sterilizing;
(1.3) explant is seeded in thallus differentiation and starts and be cultured to incision on the medium and green small salient point occurs, continuation starts medium in the thallus differentiation and cultivated 10～15 days, green small salient point develops into the thallus tissue that can distinguish, and the thallus tissue of this moment is propagation rapidly; The thallus differentiation starts medium: 1/2 (6,7-V)+NAA0.1～1.0mg/L, dosage of sucrose 20～30g/L;
(1.4) will induce the thallus tissue segmentation of propagation to become fritter, every bottle of 45ml left and right sides culture medium inoculated 1.0g to 1.5g, be transferred on the thallus shoot proliferation medium and cultivate, thallus shoot proliferation medium: 6,7-V+NAA0.1～1.5mg/L or IAA0.1～1.0mg/L, dosage of sucrose 20～30g/L;
(1.5) thallus is cut apart subculture; The subculture fritter is made in the cutting of sub-fraction thallus, is inoculated into subculture cultivation on the shoot proliferation medium, turns generation once in later per 40～50 days, and per generation thallus biomass fresh weight increases by 8～13 times than inoculum concentration, builds up thallus clone; Another most of thallus results can be extracted huperzine.
The used medium of the inventive method is as follows:
(1) applicable minimal medium: 6,7-V, and dosage of sucrose scope 20～30g/L,
(2) the thallus differentiation starts medium: 1/2 (6,7-V)+NAA0.1～1.0mg/L;
(3) thallus shoot proliferation medium: 6,7-V+NAA0.1～1.5mg/L or IAA0.1～1.0mg/L; The pH value 5.8～6.0 of above medium, agar powder mass fraction 0.6%, and the 25min that under the HTHP of 118～120 ℃ of temperature and pressure 0.8～1.0 kg/cm, sterilizes.
Condition of culture of the present invention is as follows:
(1) culture parameters: 24 ± 2 ℃ of constant temperature, relative moisture 60%～70%, intensity of illumination are 1000～1500lx, sunshine duration 11～14h/d;
(2) technical parameter: the explant pollution rate is lower than 10%～20%; Thallus inductivity 50%～80%; Per generation thallus biomass fresh weight increases by 8～13 times than inoculum concentration; Subculture cycle 40～50d; The stripped asexual reproduction that the present invention has set up the former plant regeneration thallus of the Huperzia serrata tissue of producing huperzine is this lobate physical efficiency productive accumulation huperzine.
In the inventive method in the explant disinfecting process with behind the dust on the running water flush away branches and leaves, soak 6～10min with detergent liquid again, then scrub branches and leaves with banister bruss, in the running water thread water rinsing clean, filter paper blots the sterile chamber of packing into behind the water droplet; With the ethanol disinfection 0.5min--1min of mass fraction 75%, sterile water is cleaned again, blot water after, again with mass fraction 0.1%～0.5%HgCl
2Add 1 Tween 80 soaking disinfection material 6～10min, behind the liquid that inclines, use aseptic water washing 6--8 time.
Technique effect of the present invention is: the regeneration of Huperzia serrata cultured in vitro thallus does not have successfully report.In view of the Huperzia serrata cultured in vitro is that its former plant fast propagation of exploitation or biotechnology are produced the important technological platform that former botanical is used huperzine (Hup-A) resource, the stripped asexual reproduction that the present invention has set up the former plant regeneration thallus of the Huperzia serrata tissue of producing huperzine is to have broken through a key technology of similar research.Cultural method of the present invention and matrix formulations provide essential condition for the lobate soma of the former plant of Huperzia serrata in-vitro propagate asexual reproduction.Carry out the Huperzia serrata cultured in vitro with method of the present invention, can obtain a large amount of Huperzia serrata thallus resources of medicinal plant.The medicine huperzine (Hup-A) that utilizes this invention to enlarge scale cultured in vitro thallus extraction production will produce earthshaking economic worth.
Description of drawings
The former vegetable material of Fig. 1 Huperzia serrata and cultured in vitro thereof induce thallus to occur and vegetative map;
The thin-layer chromatogram of the thallus extract of Fig. 2 cultured in vitro and huperzine standard items, the contrast of former plant extract.
Embodiment
The present invention is described in detail below in conjunction with embodiment.
Embodiment 1:
＜one〉selects stalwartness without the wild Huperzia serrata plant of damage by disease and insect, get the then long shoot of the about 3cm of pumping;
＜two〉with behind the dust on the running water flush away branches and leaves, soak 6～10min with detergent liquid again, then scrub branches and leaves with banister bruss, in the running water thread water rinsing clean, filter paper blots and carries out surface sterilizing in the sterile chamber of packing into behind the water droplet and process; Go on the superclean bench, again with the ethanol disinfection 0.5min--1min of mass fraction 75%, sterile water is cleaned, blot water after, again with mass fraction 0.1%～0.5%HgCl
2Add 1 Tween 80 soaking disinfection material 6～10min, behind the liquid that inclines, use aseptic water washing 6--8 time.Then under aseptic condition, the Huperzia serrata shoot cut apart to be cut into length be stem segment about 0.5～1cm, be inoculated into ready thallus differentiation and start on the medium and cultivate;
＜three〉explant is cultured to incision and green small salient point occurs, continuation starts medium in the thallus differentiation and cultivated 10～15 days, green small salient point develops into the thallus tissue that can distinguish, the thallus tissue of this moment is propagation rapidly, the thallus of growth has been surrounded explant gradually, grows up to the thallus tissue block of a 3cm diameter;
To induce the thallus tissue segmentation of propagation to become fritter, every bottle of 45ml left and right sides culture medium inoculated 1.0g to 1.5g be transferred on the thallus shoot proliferation medium, cultivates 40～50 days;
＜four〉thallus is cut apart the subculture cultivation, the subculture fritter is made in the cutting of sub-fraction thallus, is inoculated into subculture cultivation on the shoot proliferation medium, turns generation once in later per 40～50 days, the thallus fresh weight that per generation breeds increases by 8～13 times than inoculum concentration, builds up thallus clone; Another most of thallus results can be extracted huperzine.
＜five〉condition of culture
1. culture medium prescription
Each constituents such as the macroelement in the medium, trace element, organic principle, plant hormone, carbohydrate are carried out repeatedly obtain on the basis of orthogonal design and gradient test.The minimal medium 6 that the present invention is suitable for, 7-V, the differentiation of (1) thallus starts medium: 1/2 (6,7-V)+NAA0.1mg/L, dosage of sucrose 25g/L (the thallus differentiation of other embodiment starts the medium dosage of sucrose all together); (2) thallus shoot proliferation medium: 6,7-V+NAA0.5mg/L, dosage of sucrose 25g/L.The pH value 5.8～6.0 of above medium, agar powder mass fraction 0.6%, and at 118～120 ℃ of temperature and pressure: 25min sterilizes under the HTHP of 0.8～1.0 kg/cm.
2. culture environment
Culture environment is provided: constant temperature (24 ± 1) ℃, relative moisture 60%～70%, intensity of illumination are about 1000～1500lx, order according to duration 11～14h (hour)/d (my god).
Embodiment 2:
Select healthy and strong without the wild Huperzia serrata plant of damage by disease and insect, get the then long shoot of the about 3cm of pumping, to behind the explant cleaning and sterilizing under aseptic condition, the Huperzia serrata shoot cut apart to be cut into length be stem segment about 0.5～1cm, be inoculated into ready thallus differentiation and start on the medium and cultivate.(1) the thallus differentiation starts medium: 1/2 (6,7-V)+IAA0.5mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+NAA0.5, dosage of sucrose 20g/L, other is identical with embodiment 1.
Embodiment 3:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+IAA1.0mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+NAA1.0, dosage of sucrose 25g/L, other is identical with embodiment 1.
Embodiment 4:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+IAA2.0mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+NAA1.5, dosage of sucrose 30g/L, other is identical with embodiment 1.
Embodiment 5:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+IAA0.1mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+NAA2.0, dosage of sucrose 30g/L, other is identical with embodiment 1.
Embodiment 6:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+NAA0.5mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+IAA0.1, dosage of sucrose 25g/L, other is identical with embodiment 1.
Embodiment 7:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+NAA1.0mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+IAA0.5, dosage of sucrose 25g/L, other is identical with embodiment 1.
Embodiment 8:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+NAA2.0mg/L (same under the unit); (2) thallus shoot proliferation medium: 6,7-V+IAA1.0, dosage of sucrose 25g/L, other is identical with embodiment 1.
Embodiment 9:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+6-BA1.0mg/L (same under the unit)+IAA0.5; (2) thallus shoot proliferation medium: 6,7-V+IAA2.0, dosage of sucrose 25g/L, other is identical with embodiment 1.
Embodiment 10:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+ZT1.0mg/L (same under the unit)+IAA0.5; (2) thallus shoot proliferation medium: 6,7-V+IAA0.5, dosage of sucrose 30g/L, other is identical with embodiment 1.
Embodiment 11:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+ZT1.0mg/L (same under the unit)+NAA0.5; (2) thallus shoot proliferation medium: 6,7-V+IAA0.5, dosage of sucrose 35g/L, other is identical with embodiment 1.
Embodiment 12:
(1) the thallus differentiation starts medium: 1/2 (6,7-V)+ZT2.0mg/L (same under the unit)+NAA0.5; (2) thallus shoot proliferation medium: 6,7-V+IAA0.5, dosage of sucrose 20g/L, other is identical with embodiment 1.
Experimental result such as table 1, table 2:
Table 1: the impact that hormone starts Huperzia serrata thallus
On the basis of table 1 the selection result, continue to have furtherd investigate nutrient media components and hormone combinations subculture and cultivate the thallus effect, found out the medium matrix component of suitable thallus shoot proliferation, the different culture media composition increases the thallus biomass obvious difference.Part test the results are shown in Table 2.
Table 2 different culture media matrix is on the impact of Huperzia serrata thallus shoot proliferation
As shown in Figure 1, A: the former plant of wild Huperzia serrata of taking from Mount Lushan; B: Huperzia serrata cultured in vitro inoculated and cultured; C1 Huperzia serrata cultured in vitro thallus is bred, C2, C3 thallus subculture cultivate system and harvested material thereof.
As shown in Figure 2,1: huperzine standard items (arrow shows); 2: former plant extract; The thallus extract of cultivating in the 3:f medium; The thallus extract of cultivating in the 4:m medium.
Claims (3)
1. cultural method that the stripped Huperzia serrata thallus of producing huperzine is induced and bred is characterized in that: may further comprise the steps:
1) material: select healthy and strong Huperzia serrata plant without damage by disease and insect, get the then long shoot of about 3 ㎝ of pumping;
2) to the explant cleaning and sterilizing;
3) explant is seeded in thallus differentiation and starts and be cultured to incision on the medium and green small salient point occurs, continuation starts medium in the thallus differentiation and cultivated 10～15 days, green small salient point develops into the thallus tissue that can distinguish, and the thallus tissue of this moment is propagation rapidly; The thallus differentiation starts medium: 1/2 (6,7 – V)+NAA 0.1～1.0 mg/L, dosage of sucrose 20～30g/L;
4) will induce the thallus tissue segmentation of propagation to become fritter, every bottle of 45ml left and right sides culture medium inoculated 1.0g to 1.5g, be transferred on the thallus shoot proliferation medium and cultivate, thallus shoot proliferation medium: 6,7 – V+NAA, 0.1～1.5 mg/L or IAA 0.1～1.0 mg/L, dosage of sucrose 20～30g/L;
5) thallus is cut apart subculture; The subculture fritter is made in sub-fraction thallus cutting, is inoculated on the shoot proliferation medium subculture and cultivates, and turns generation once in later per 40～50 days, per generation thallus biomass fresh weight increase by 8～13 times than inoculum concentration, build up thallus clone; Another most of thallus results.
2. the stripped Huperzia serrata thallus of the product huperzine according to claim 1 cultural method of inducing and breeding, it is characterized in that: condition of culture is as follows:
Culture parameters: 24 ± 2 ℃ of constant temperature, relative moisture 60%～70%, intensity of illumination are 1000～1500lx, sunshine duration 11～14 h/d;
Technical parameter: the explant pollution rate is lower than 10%～20%; Thallus inductivity 50%～80%; Subculture cycle 40～50d, the stripped asexual reproduction of setting up the former plant regeneration thallus of Huperzia serrata tissue is; In per generation,, thallus biomass fresh weight increased by 8～13 times than inoculum concentration, the lobate physical efficiency productive accumulation of the Huperzia serrata of cultured in vitro huperzine.
3. the stripped Huperzia serrata thallus of the product huperzine according to claim 1 cultural method of inducing and breeding, it is characterized in that: after using the dust on the running water flush away branches and leaves in the explant disinfecting process, soak 6～10 min with detergent liquid again, then scrub branches and leaves with banister bruss, rinsing is clean in the running water thread water, and filter paper blots the sterile chamber of packing into behind the water droplet; Again with ethanol and the mass fraction 0.1%～0.5%HgCl of mass fraction 75%
2Add successively gradation of 1 Tween 80 sterilization.
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| CN103518599A (en) * | 2013-10-08 | 2014-01-22 | 长沙慧日生物技术有限公司 | Method for hydroponic breeding of huperzine serrate and induced improvement to huperzine a of huperzine serrate |
| CN103975857A (en) * | 2014-05-27 | 2014-08-13 | 江西师范大学 | Hormone-autotrophic huperzia serrata in-vitro thallus cloning line for producing huperzine-a |
| CN105191657A (en) * | 2015-11-06 | 2015-12-30 | 长沙慧日生物技术有限公司 | Rapid breeding method of aseptic huperzia serrata seedling |
| CN105638476B (en) * | 2016-01-27 | 2018-02-13 | 江西师范大学 | A kind of method for inducing the in vitro thallus high yield huperzine of Huperzia serrata |
| CN105532476B (en) * | 2016-02-02 | 2018-03-27 | 江西师范大学 | A kind of precursor aspartic acid of adding improves the Huperzia serrata of huperzine content thallophytic cultural method in vitro |
| CN111893136B (en) * | 2020-08-12 | 2021-08-13 | 江西师范大学 | Method for establishing agrobacterium-mediated huperzia serrata thallus genetic transformation system |
| CN113005079B (en) * | 2021-05-08 | 2022-12-06 | 威海见生生物技术有限公司 | Additive for human bone marrow mesenchymal stem cell in vitro amplification and amplification method |
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