CN106613938A - Effective huperzia serrata callus culture method - Google Patents
Effective huperzia serrata callus culture method Download PDFInfo
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- CN106613938A CN106613938A CN201610814179.3A CN201610814179A CN106613938A CN 106613938 A CN106613938 A CN 106613938A CN 201610814179 A CN201610814179 A CN 201610814179A CN 106613938 A CN106613938 A CN 106613938A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses an effective huperzia serrata callus culture method. The method includes: adopting a detergent to wash a huperzia serrata stem tip twice, adopting sterile water to wash the same for 4 times, adopting 0.15% mercuric chloride liquid to sterilize for 5min, and adopting sterile water to wash for 4-6 times; taking down a micro stem tip of 0.1-0.2mm in length from the huperzia serrata stem tip under an anatomical lens, adopting an MS culture medium, adding 0.1uM of IBA, 1.5uM of KT and 3mg/L glutamine, and performing photoperiod culture at constant temperature of 25 DEG C for 14h. A method of repeated sterilization combined with AAS to inhibit endophyte is adopted, so that the problem of endophytic fungi pollution in the process of huperzia serrata callus culture is solved, and callus induction rate is higher than 25% generally.
Description
Technical field
The invention belongs to agricultural technology field, specifically, is related to a kind of effective Huperzia serrata callus tissue culture side
Method.
Background technology
Huperzia serrata (Huperzia serrata (Thunb) Trev.) is pteridophyte, belongs to stone China fir (Huperiaceae),
Stone araucaria (Huperzia Bernh), also known as it is help king, serrate clubmoss herb, invaluable.It is among the people for treating carbuncle furuncle poison, traumatic injury
Deng.All parts of the country are distributed in, the ground such as Cuba, the Mexico in Asia other area, Oceania and Central America are also widely distributed in[1].It is many
Be born under the thick forest of mountain region or cheuch it is dark and damp soil in, height above sea level 400m～1600m. groups upper layer trees seeds mainly have mao bamboon
(Phyllostachys heterocyc la), cepehalotaxus fortunei (Cephalotaxus fortunei), shrub species has the disconnected wood of wood
(Loropetalum chinense), honeysuckle (Lonicera japonica), azalea (Rhododendron
Mariesii), the Japanese ardisia (Maesa japonica), Eurya muricata (Eurya muricata), herbaceous plant has fern, David's-harp
(Polygonatum cyrtonema), aster trinervius (Aster geratoides), Herba Lycopi (Eupatorium japonicum)
And the thick cylinder lettuce tongue in Zhejiang Anhui (Briggsia chienii), Huperzia serrata accompanying plant has pigeon-wheat (Polytrichum
) and the mosses such as warm ground great Ye moss (Rhodobryum giganteum) commune.Plant is the herbaceous plant for growing thickly, high 10cm
～24cm, stem top is red or green. and adventitious root is born in stem base portion, stretches in soil or matrix. and stem is upright, or bottom is oblique
Rise, cylinder, single or one to several times dichotomies, spring its top Chang Sheng has brood cell, outer by 1～3 green and brown color ovates
Lanceolar perula piece, brood cell lands into new talent. and leaf helical form raw, ellipticity lanceolar, there is an obvious short handle, the long 1cm of leaf～
3cm, wide 2mm～4mm, the sharp point in top, base portion gradually narrow wedgewise, there is irregular sharp sawtooth at edge；Middle arteries are obvious；Leaf thin paper
Matter, two sides is smooth. sporophyll and trophophyll similar shape or abnormity, green；Spore cystic kidney shape, is singly born between axil, not integrated fringe, many
The top of plant is born in, faint yellow, smooth, transverse fissure when ripe；The type of spore one, spherical shape tetrahedron[2]。
Huperzia serrata is a kind of precious medicinal plant, and the alkaloid from the domestic reported first plant in 1972 is in animal
After having striated muscle relaxation effect in test, 13 alkaloid monomers are therefrom got. pharmacological evaluation proves that huperzine and second have
The activity for having very strong suppression cholinesterase, the effect for improving learning and memory and improving memory of elderly person function, and can be used to control
Treat myasthenia gravis. in view of its important medical value, is extensively paid attention to its research[2]。
In recent years, because people recognize：The huperzine that Huperzia serrata contains has high anticholinesterase activity, for
Improve memory, treatment person in middle and old age are dull-witted and myasthenia gravis has good curative effect and toxic and side effect is little, before market potential
Scape is wide, therefore, the plant is increasingly subject to people's attention.But because Huperzia serrata resource is disperseed, and many relative collection
In the place of production be respectively positioned in the range of nature reserve area and scenic spot, meanwhile, Huperzia serrata is a kind of perennial, poky little
Draft, year the rate of rise of net biomass be extremely limited, grow up to the seedling of a high 12cm, 6 are taken around under field conditions (factors)
The time of～7 years, the resource updates cycle is longer, can not large scale mining, a kind of effective Huperzia serrata is badly in need of in prior art
Callus culture method.
The content of the invention
It is an object of the invention to overcome the defect that above-mentioned technology is present, there is provided a kind of effective Huperzia serrata callus
Cultural method.
Its concrete technical scheme is：
A kind of effective Huperzia serrata callus culture method, comprises the following steps：
Step 1, Huperzia serrata stem apex are cleaned 2 times using liquid detergent, and sterile water wash 4 times takes 0.15% liter of mercury solution to go out
Bacterium 5min, sterile water wash 4-6 time；
Step 2, the Micro-stem tip that Huperzia serrata stem apex cleaned in step 1 is taken 0.1-0.2mm length under anatomical lens；
Step 3, Micro-stem tip is carried out into Huperzia serrata callus tissue culture, using MS culture mediums, add 0.1 μM of IBA and
1.5 μM of KT and 3mg/L glutamine, condition of culture is 25 degree of constant temperature, and the 14h photoperiods cultivate.
Further, the scheme of addition suppression endophyte is in incubation in step 3：Penicillin 50000IU/L, streptomysin
50mg/L, amphotericin B 750 μ g/L, malachite green 1.5-1.8mg/L.
Compared with prior art, beneficial effects of the present invention：
The invention provides effective ways and technology of Huperzia serrata artificial organ culture so far, healing rate is reachable
More than 25%, using repeatedly sterilizing and the method for adding endophyte inhibitor AAS, solve Huperzia serrata callus tissue culture mistake
The endogenetic fungus pollution problem produced in journey, is expected to realize the breakthrough on Huperzia serrata callus culture method.
Specific embodiment
Technical scheme is described in more detail with reference to specific embodiment.
1.1 material
Experiment material is picked up from Lushan, Jiangxi, is introduced a fine variety in Jiangxi agricultural university greenhouse in December, 2014 by teacher Hu Songping.
1.2 culture medium
1.2.1 type of culture medium
Four kinds of culture mediums：(1) MS+0.1 μM of IBA+1.5 μM of KT+3mg/L glutamine；(2)1/2MS+0.1μMIBA+
1.5 μM of KT+3mg/L glutamine；(3) 1/2MS+3mg/L glutamine/without hormone；(4) MS+3mg/L glutamine/nothing swashs
Element
2.2.2 medium sterilization
Autoclaving, sterilization time is 15min, and sterilising temp is 121 DEG C
1.3 method
1.3.1 the selection and sterilization of explant
Two kinds of sterilization methods：
(1). liquid detergent cleans 2 times → sterile water wash, 4 times → 0.15% liter mercury solution sterilizing 5 ' → aseptic water washing 4-6
It is secondary.(2) liquid detergent cleaning 2 times → sterile water wash, 4 times → 0.01M HCl immersions 1min → 5%NaOCl immersion 1min →
The alcohol-pickled 30 seconds → 5%NaOCl of 2.4mM lemon acid soak 1min → 75% soaks 2min → 8%H2O2Immersion 8min；
1.3.2 cultural method
The Huperzia serrata stem apex for disinfecting is taken into the Micro-stem tip of 0.1-0.2mm length under anatomical lens, then two kinds of trainings are set
Foster condition：(1) 25 degree of constant temperature, 14h photoperiods.(2) 25 degree of constant temperature, light culture.
2 interpretations of result
Impact of the 2.1 different sterilization methods to callus induction
The present invention adopts (1). and liquid detergent cleans 2 times → sterile water wash, 4 times → 0.15% liter mercury solution sterilizing 5min → nothing
Bacterium water is rinsed 4-6 time.(2) liquid detergent cleaning 2 times → sterile water wash, 4 times → 0.01M HCl immersions 1min → 5%NaOCl leachings
The alcohol-pickled 30 seconds → 5%NaOCl of bubble 1min → 2.4mM lemon acid soak 1min → 75% soaks 2min → 8%H2O2Immersion
8min → aseptic water washing 4-6 time；As a result it is the explant for adopting first method to sterilize ratio second in fungal infection degree
Plant light, therefore, the first sterilization method is more preferable.
Impact of 2.2 different culture medias to callus induction
The present invention is shown in Table 1 using four kinds of culture mediums, and due to the infection for having endogenetic fungus, 16 bottles of No. 1 culture mediums have 4 bottles of successes
Callus is induced, and normal growth shows light green in 6 months to callus；The callus induction rate of the culture scheme
For 25%.
(1) MS+0.1 μM of IBA+1.5 μM of KT+3mg/L glutamine；(2)1/2MS+0.1μM IBA+1.5μM KT+
3mg/L glutamine；(3) 1/2MS+3mg/L glutamine/without hormone；(4) MS+3mg/L glutamine/without hormone
Callus incidence of the Huperzia serrata Micro-stem tip of table 1 on different culture media
Impact of 2.3 condition of culture to callus induction
The topmost environmental condition of callus induction is affected to be temperature and illumination.The temperature of evoked callus typically with
25 degree or so are advisable.Available complete dark, the periodicity illumination of illumination, Full daylight shine.In the present invention, the callus group to Huperzia serrata is drawn
Induction is knitted, periodicity illumination cultivation is better than light culture, this is consistent with general Plant Tissue Breeding performance[3]。
The inhibition of 2.4AAS and malachite green to endogenetic fungus
AAS is made up of antibiotic such as penicillin, streptomysin and both sexes Nysfungin B by different proportion mixing, and antibiotic exists
Just can suppress to kill pathogenic microorganism during very low concentrations.Malachite green is soluble in water and ethanol, with drug effects such as antibacterial, desinsections,
Can be used as pest repellant and bactericide, to kill the external parasite of aquatic livestock, protozoan and fish-egg in mould etc., be anti-
The stronger class of bacterium effect.
The variable concentrations AAS of table 2 and malachite green suppress the effect of endogenetic fungus
Variable concentrations AAS as shown in table 2 is present invention employs, has been carried out to Huperzia serrata endogenetic epiphyte inhibition
Research, as a result shows, both sexes Nysfungin B etc. has no obvious inhibition to endogenetic fungus, and works as peacock green concentration up to 1.5 millis
G/l when most of fungi be suppressed, but the mould of black still has a small amount of growth, so can not be such as Wojciech using AAS[4]It is described to solve the problems, such as that endogenetic fungus pollute；The scheme of AAS optimal inhibition endophytes is in suggestion incubation：Mould
50000 (IU/L) of element, streptomysin 50 (mg/L), amphotericin B 750 (μ g/L), malachite green 1.5-1.8 (mg/L).
The above, preferably specific embodiment only of the invention, protection scope of the present invention not limited to this are any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter of the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Claims (2)
1. a kind of effective Huperzia serrata callus culture method, it is characterised in that comprise the following steps：
Step 1, Huperzia serrata stem apex are cleaned 2 times using liquid detergent, sterile water wash 4 times, take 0.15% liter of mercury solution sterilizing
5min, sterile water wash 4-6 time；
Step 2, the Micro-stem tip that Huperzia serrata stem apex cleaned in step 1 is taken 0.1-0.2mm length under anatomical lens；
Step 3, Micro-stem tip is carried out into Huperzia serrata callus tissue culture, using MS culture mediums, add 0.1 μM IBA and 1.5 μM
KT and 3mg/L glutamine, condition of culture is 25 degree of constant temperature, and the 14h photoperiods cultivate.
2. effective Huperzia serrata callus culture method according to claim 1, it is characterised in that train in step 3
The scheme of addition suppression endophyte is during supporting：Penicillin 50000IU/L, streptomysin 50mg/L, the μ g/ of amphotericin B 750
L, malachite green 1.5-1.8mg/L.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108925427A (en) * | 2018-05-31 | 2018-12-04 | 合肥本盛生物科技有限公司 | A method of improving serrate clubmoss herb tufted seedling breeding coefficient |
| CN110393152A (en) * | 2019-07-26 | 2019-11-01 | 西北大学 | A kind of improvement MS fluid nutrient medium and the sterile Shoot Tip Culture method of Huperzia serrata |
| CN110663547A (en) * | 2018-07-03 | 2020-01-10 | 中国科学院上海生命科学研究院 | Culture method of cryptomeria draconis and picea mucronata explants |
| CN111543325A (en) * | 2020-06-05 | 2020-08-18 | 江西农业大学 | Induction culture method and induction culture medium for slash pine embryonic callus |
| CN115606503A (en) * | 2022-12-19 | 2023-01-17 | 北京花乡花木集团有限公司 | Tissue culture method of aster |
Citations (2)
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| CN101658124A (en) * | 2009-09-01 | 2010-03-03 | 王忠华 | Rapid propagation method of huperzia serrata |
| CN101889551A (en) * | 2010-08-06 | 2010-11-24 | 合肥工业大学 | Tissue culture method of huperizia serrata |
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2016
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| CN101658124A (en) * | 2009-09-01 | 2010-03-03 | 王忠华 | Rapid propagation method of huperzia serrata |
| CN101889551A (en) * | 2010-08-06 | 2010-11-24 | 合肥工业大学 | Tissue culture method of huperizia serrata |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108925427A (en) * | 2018-05-31 | 2018-12-04 | 合肥本盛生物科技有限公司 | A method of improving serrate clubmoss herb tufted seedling breeding coefficient |
| CN110663547A (en) * | 2018-07-03 | 2020-01-10 | 中国科学院上海生命科学研究院 | Culture method of cryptomeria draconis and picea mucronata explants |
| CN110393152A (en) * | 2019-07-26 | 2019-11-01 | 西北大学 | A kind of improvement MS fluid nutrient medium and the sterile Shoot Tip Culture method of Huperzia serrata |
| CN111543325A (en) * | 2020-06-05 | 2020-08-18 | 江西农业大学 | Induction culture method and induction culture medium for slash pine embryonic callus |
| CN115606503A (en) * | 2022-12-19 | 2023-01-17 | 北京花乡花木集团有限公司 | Tissue culture method of aster |
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Application publication date: 20170510 |