CN103081605A - Method for collecting and storing bletilla striata seeds - Google Patents
Method for collecting and storing bletilla striata seeds Download PDFInfo
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- CN103081605A CN103081605A CN2012105916388A CN201210591638A CN103081605A CN 103081605 A CN103081605 A CN 103081605A CN 2012105916388 A CN2012105916388 A CN 2012105916388A CN 201210591638 A CN201210591638 A CN 201210591638A CN 103081605 A CN103081605 A CN 103081605A
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Abstract
The invention provides a method for collecting and storing bletilla striata seeds. The method comprises the following steps of: when the bletilla striata seeds in bletilla striata capsularfruits are sampled and the vitality of the bletilla striata seeds is detected to be up to more than 80%, cutting off the bletilla striata capsularfruits with fruit stems by a pair of shears; cleaning the collected bletilla striata capsularfruits; carrying out disinfection treatment with 75% of alcohol and 0.1% of HgCl2 on the cleaned bletilla striata capsularfruits; and drying till the moisture content is less than 15% at a temperature of 30-35 DEG C, and storing in a sealed container. The method for collecting and storing the bletilla striata seeds has the main beneficial effects that (1) the collecting time of the seeds is definite; if the bletilla striata seeds are prematurely collected, the seeds are premature or incompletely mature, are low in seed vitality and are not high in germination rate, and sprouted seedlings are bad in growth vitality even if the seeds can sprout; if the collecting time of the seeds is too late, the capsularfruits are broken, on one hand, the seeds are easy for contamination, and on the other hand, a part of or all of the seeds are flied away, so that the seeds with high quality are difficultly collected; and (2) the storage method of the treated seeds is simple and practical, and the vitality of the bletilla striata seeds can be effectively maintained, so that the method for collecting and storing the bletilla striata seeds has a wide application prospect.
Description
(1) technical field
The present invention relates to a kind of collection and store method of bletilla seed.
(2) background technology
Bletilla is orchid bletilla Bletilla sfriata(Thunb.) dry tuber of Reiehb.f., has astringing to arrest bleeding, the effects such as detumescence and promoting granulation, be used for the treatment of spitting of blood, spit blood, traumatism and bleeding, sore swollen toxin, the diseases such as chapped skin (Chinese Pharmacopoeia Commission. Pharmacopoeia of People's Republic of China (an one) [S]. Beijing: Chinese Medicine science and technology publishing house, 2010:95).The bletilla pharmacological action is extensive, is a kind of common astringing to arrest bleeding Chinese medicine.The researchs such as Wu Jiujian find that bletilla water soluble part and n-butanol portion are the main active components of its anastalsis, and ethyl acetate extract mainly works the effect of invigorating blood circulation; They further studies show that the Rabbit Blood Platelets MA that can significantly raise bletilla n-butanol extract part and water soluble part adenosine diphosphate induces, and the ethyl acetate extraction position can significantly suppress the platelet aggregation (Wu Jiujian that ADP induces, Yuan Mengliang, Zou Lihua, Deng. the bletilla the different extracted parts shows [J] to mouse styptic activity experimental study. the pharmacy practice magazine, 2011,29 (3): 206-209.).Except anastalsis, bletilla also is widely used in the illnesss such as treatment hemoptysis, haematemesis, sore swollen toxin, chapped skin, the withered film disease in oral cavity clinically, and is evident in efficacy.Except being widely used in Chinese medicine compound prescription, bletilla has been made into various Chinese patent drugs, as bletilla particle, KUAIWEI PIAN, Yunnan Baiyao, compound bletilla cream etc.As natural macromolecular material, functional slow-release, local anelasticity, self degradability, nonirritant, have no side effect, the characteristic of aboundresources, the auxiliary material such as cheap and easy to get, make bletilla glue become present purposes the most natural pharmaceutic adjuvant.
Except have the irreplaceable effect of other drug at field of medicaments, bletilla also is widely used in food industry, tobacco industry, chemical industry (comprising high-grade cosmetics) etc.Bletilla is beauty treatment good medicine from ancient times, is described as " whitening fairy maiden ", adding that it generally has no side effect, is particularly suitable as the function ingredients of natural cosmetics.At present, a lot of cosmetics and commodity take bletilla as raw material have entered into market, as thousand seek all bletilla flower cell face beautifying creams, bletilla South Korea Manual perfumed soap, bletilla is spent clean white stoste etc.With the bletilla toothpaste that Bletilla striata is made, can be used for oral cavity and the dental disorders such as anti-treating dental ulcer, abscess of throat, periodontitis, gingivitis, preventing decayed tooth, oral peculiar smell, meet back to nature, advocate green development trend.In industrial aspect, bletilla as the adhesive of dying cloth, is also senior cigarette plume adhesive, and the disconnected palpus of wild ginseng renovation agent is mounted the Chinese paintings and calligraphic works adhesive, gastroscopic protectant, beauty mask etc.
The using value that bletilla is good is that the consumption of bletilla constantly increases, and resource is worsening shortages also.The output of China's bletilla is difficult to the demand of satisfying the market at present, and the problem of bletilla shortage of resources has restricted the development of bletilla industry, adds unlimited the digging excessively of natural world, and the bletilla germplasm is degenerated year by year.(the Shi Jing such as Shi Jing, Luo Yibo, Song Xiqiang. China's bletilla striata MarketingResearchandAnalysis [J]. Chinese gardening digest, 2010, (8): 48-51) to China's bletilla cultivating and growing, manage sale enterprise information and the medicinal market price etc. and investigate and analyse, result shows excessively to excavate with habitat destruction and causes bletilla wild resource amount sharply to descend.China's bletilla still relies on wild at present mostly, and wild bletilla resource is limited, digs fewer and fewer.Mad adopting excessively year after year dug, and adds the impact of natural calamity, wild bletilla output sharply descended, imbalance between supply and demand expanding day, resource worsening shortages.Bletilla market demand in 2007 just breaks through 1500 tons, and market aggregate demand in 2010 is more than 2000 tons, all last year China the bletilla gross yield between 800 ~ 900 tons, can not satisfy the demand of industry far away.Further widening of bletilla demand, falling sharply year by year of resource causes the bletilla market price to continue unprecedented soaring always, and high.The eighties in 20th century, it is little that bletilla belongs to the bletilla survival dose of the three little kinds of class medicinal material, and price is many to fluctuate at 4 yuan ~ 6 yuan.Enter the nineties in 20th century, the formula along with bletilla in Chinese patent drug is used, the corresponding increase of its consumption, and price begins to raise up.But the whole nineties in 20th century, the demand of bletilla is relatively stable, and price steadily maintains between 8 yuan ~ 12 yuan.Enter 21 century, scientific and technological develop rapidly, the purposes of bletilla no longer is confined to field of medicaments, and its price also rises rapidly with the continuous increase of usage amount.Since 06 year, the bletilla price progressively went up, 09 year second half year the bletilla price begin to rise suddenly and sharply, the bletilla price had risen to 140 yuan every kilogram in 2010.Recognize on Agriculture of Zhejiang Province leading enterprise exchanging meeting, the market average price of bletilla surpasses 160 yuan of per kilograms at present, and indivedual places are even up to 200 yuan of per kilograms.
For a long time, bletilla be on the spot gather-reserve seed for planting on the spot-cultivate again on the spot such original loop state.Cultivate prolonged and repeatedly, and bletilla itself because of environment, self etc. reason morph constantly, so the bletilla germplasm can't guarantee its growth performance and pharmaceutical characteristic at all.And, bletilla seed seedling quality aspect systematic research report is not yet arranged at present, bletilla resources development and utilization bottleneck is the provenance cultivation at present, wherein bletilla seedling breeding technology is now domestic there is no the large tracts of land test and carries out, the small size cultivation is take vegetative propagation as main, vegetative propagation can't provide enough seedlings in a short time, there is no extensive seedling, and extensive bletilla planting base construction is exactly water without a source.Be the seed resource of research bletilla, realize the Fast-propagation of bletilla, carry out effectively preserving particularly important to the bletilla seed.
(3) summary of the invention
The purpose of this invention is to provide a kind of bletilla seed collection and store method, the method is simple and easy to do, and cost is low, can effectively keep the bletilla seed vitality, further haves laid a good foundation for the artificial propagation of bletilla.
The technical solution used in the present invention is:
A kind of collection of bletilla seed and store method, described method comprises:
(1) gather: the vitality that sampling detects bletilla seed in the bletilla capsule reaches 80% when above, the capsule of bletilla is connected carpopodium one reinstate scissors and cut; In Zhejiang, the acquisition time of bletilla seed was advisable with mid or late August to early October;
(2) preserve pre-treatment: the bletilla capsule that gathers is cleaned, first used 75%(v/v) alcohol disinfecting 2 ~ 3min, then aseptic water washing uses 0.1% (w/w) HgCl
2Sterilization 12 ~ 15min, aseptic water washing, with the capsule surface moisture aseptic paper bag that blots, packs into, 30 ~ 35 ℃ are dried to water content less than 15%(2% ~ 15%) time, drying is completed;
(3) preserve: dried bletilla striata capsule is placed in closed container preserves.
The vitality of step (1) bletilla seed detects as follows: get TTC and be mixed with 0.5% concentration with 0.1mol/L pH7.2 phosphoric acid buffer, obtain dyeing liquor, the bletilla seed is joined in dyeing liquor, 40 ℃ of oven 24h, then counting dyes and undyed seed under light microscope, calculates as follows the vitality of bletilla seed:
Triphenyltetrazolium chloride (triphenyltetrazolium chloride, TTC) also claims red tetrazolium (Red tetrazoline, RT).Can be used to measure seed vitality, be reduced to red TTF by the reducing substances in seed (dehydrase), thereby make seed embryo alive redness and endosperm is not colored.And blind seed is not colored fully.
Step (3) is preserved three kinds of modes: (1) is freezing preservation in liquid nitrogen; Dried bletilla fruit is put into cryopreservation tube or other suitable utensils, with PVS2, cryopreservation tube is filled up, put into that liquid nitrogen is freezing to be got final product; (2) be no more than under 25 ℃ of conditions in room temperature and preserve; (3) preserve at 4 ℃ of lower low temperature.
Beneficial effect of the present invention is mainly reflected in: the acquisition time of (1) seed is definite; If the bletilla seed collection is too early, seed is not yet ripe or not exclusively ripe, and such seed vitality is low, and germination rate is not high, namely enables to germinate, and the seedling growing way of sending is not good enough yet; If acquisition time is too late, capsule breaks, the easy microbiological contamination of seed on the one hand, and seed partly or entirely flies on the other hand, is difficult to collect the seed of high-quality, and the present invention provides acquisition time very accurately by careful research.(2) store method is simple and practical after treatment for seed; This store method is simple to operate, can effectively keep the vigor of bletilla seed, has broad application prospects.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
Gather the bletilla seed in late August, and measure by the following method the vitality of bletilla seed.
1. operating procedure:
1.1 the configuration of buffer solution
0.1mol/L the configuration of pH7.2 phosphate buffer:
Solution A: get 27.8g NaH
2PO
4, accurately weighed, after the distilled water dissolving, adding distil water is settled to 1000ml and namely gets 0.2mol/L NaH
2PO
4Solution.Solution B: get 53.65Na
2HPO
47H
2O, accurately weighed, after the distilled water dissolving, adding distil water is settled to 1000ml and had both got 0.2mol/L Na
2HPO
4Solution.Get 28ml A solution, 72ml B solution adds the distilled water of 100ml simultaneously, after mixing both 0.1mol/L pH7.2 phosphate buffer.
1.2 quadrature is investigated the dyeing condition of bletilla seed the best
Investigate dyeing time, TTC concentration, dyeing temperature to the impact of coloration result.Get TTC, with 0.1mol/L pH7.2 phosphate buffer be configured to respectively 0.1%, 0.5% and 1.0%(w/w) concentration.Simultaneously, adopt L
93
4Orthogonal table is investigated best dyeing condition.Table specific as follows:
Table 1: the investigation of bletilla seed dyeing condition
After having added reagent by upper table, put into respectively the baking oven of different temperatures and process, then 4*10 doubly counts down dyeing and undyed seed under light microscope, calculates as follows seed vigor.
1.3 the bletilla seed vigor is measured: the vitality of measuring the seed that gathers according to the condition of above-mentioned optimum.
1.4 data statistics
Experimental result with
Form represent.
2. result
Experimental result shows, different dyeing conditions is larger to the determination influences of bletilla seed vigor, and concrete outcome sees the following form:
Table 2: different disposal seed vigor measurement result
Table 3: quadrature is investigated bletilla seed optimum dyeing conditional outcome
Show from above-mentioned result, the dyeing condition of bletilla seed the best is A
3B
2C
3, the 24h that namely dyes, TTC concentration is 0.5%, dyeing temperature is 40 ℃.The factor that bletilla seed coloration result is had the greatest impact is dyeing time, is secondly the temperature of dyeing, is the concentration of coloring agent again.With the above-mentioned condition 24h that namely dyes, TTC concentration is 0.5%, and dyeing temperature is that 40 ℃ of vitalitys that determine are 98.01 ± 0.67%.
Embodiment 2:
Get the bletilla capsule, use detergent immersion 10min, rinse 5min with running water.Then use 75% ethanol disinfection 2min, aseptic water washing, 0.1% mercuric chloride sterilization 15min, sterile water is rinsed residual mercuric chloride well, and aseptic blotting paper blots the water of capsule remained on surface, in the aseptic paper bag of packing into.
Bletilla capsule in aseptic paper bag is put into 35 ℃ of baking ovens dry by the fire respectively 3d, 4d, 5d, 6d, 7d, 8d.The bletilla capsule of dry different number of days takes out respectively, and after 0.5%TTC dyeing 16h, the microscopically counting is pressed the vitality that embodiment 1 formula calculates the bletilla seed.In the bletilla seed, the mensuration of water content is carried out according to GB/T 3543.6 aquametries under People's Republic of China's crop seeds inspection procedure item.
Result shows, after the dry different number of days of bletilla, vitality is all higher, specifically sees the following form.
Table 4: different disposal bletilla seed vitality result
| Dry number of days | Water content | Vitality (%) |
| 3d | 26.3% | 97.56±1.74 |
| 4d | 19.8% | 98.18±0.96 |
| 5d | 17.9% | 96.50±4.72 |
| 6d | 13.4% | 97.24±1.03 |
| 7d | 4.28% | 97.71±0.31 |
| 8d | 2.97% | 97.59±0.42 |
| Blank | 44.51% | 98.01±0.67 |
Embodiment 3:
Above-mentioned dried seed room temperature preservation 6 months, is measured its vitality, specifically sees the following form:
Table 5: room temperature preservation bletilla different time vitality test result
| Dry number of days | Water content | 1 month | 3 months | 6 months |
| 3d | 26.3% | 96.26±1.24% | 77.56±0.93% | 52.15±3.44% |
| 4d | 19.8% | 97.11±0.56% | 78.18±1.22% | 59.87±2.92% |
| 5d | 17.9% | 97.50±1.71% | 82.99±1.12% | 70.47±1.65% |
| 6d | 13.4% | 97.14±0.93% | 92.09±1.98% | 91.24±1.21% |
| 7d | 4.28% | 98.21±1.37% | 95.71±1.21% | 92.33±1.31% |
| 8d | 2.97% | 97.29±1.42% | 96.59±0.64% | 95.59±0.42% |
| Blank | 44.51% | 98.21±1.01% | 75.03±1.27% | 50.98±3.39% |
Found that moisture below 15%, can preserve in room temperature and can keep vitality preferably in 6 months.And the too high seed of moisture, namely moisture is shorter in the holding time more than 15%, only has one month, so moisture is factor comparatively crucial during the bletilla seed is preserved.
Embodiment 4:
Above-mentioned dried seed was preserved 12 months at 4 ℃, is measured vitality wherein, specifically see the following form:
Table 6: low temperature is preserved bletilla different time vitality test result
Found that moisture below 15%, can preserve in room temperature and can keep vigor preferably in 12 months, it is shorter that moisture surpasses holding time of 15%.
Embodiment 5:
Above-mentioned dried seed is put into cryopreservation tube, then formed by the 0.4mol/L sucrose of 30% glycerine, 15% ethylene glycol, 15%DMSO and 40% with PVS2(, v/v) fill up, put into liquid nitrogen freezing, hatch after freezing completing, measure vitality.
Table 7: liquid nitrogen is preserved bletilla seed vitality result
| Dry number of days | Water content | Deposit survival rate in liquid nitrogen |
| 3d | 26.3% | 43.21±1.11% |
| 4d | 19.8% | 47.22±0.72% |
| 5d | 17.9% | 56.84±6.16% |
| 6d | 13.4% | 97.60±1.42% |
| 7d | 4.28% | 97.47±0.17% |
| 8d | 2.97% | 97.03±1.07% |
| Blank | 44.51% | 15.43±1.57% |
Found that moisture below 15%, melt after freezing, the bletilla seed can also keep high viability (more than 97%), so under this preservation condition, the bletilla seed can long preservation; And moisture surpass 15% freezing after melt, vigor is lower, illustrates to be not suitable for freezing preservation under this condition.
Claims (5)
1. the collection of a bletilla seed and store method, described method comprises:
(1) gather: the vitality that sampling detects bletilla seed in the bletilla capsule reaches 80% when above, the capsule of bletilla is connected carpopodium one reinstate scissors and cut;
(2) preserve pre-treatment: the bletilla capsule that gathers is cleaned, first used 75% alcohol disinfecting 2 ~ 3min, then aseptic water washing uses 0.1% HgCl
2Sterilization 12 ~ 15min, aseptic water washing, with the capsule surface moisture aseptic paper bag that blots, packs into, 30 ~ 35 ℃ are dried to water content less than 15% the time, and drying is completed;
(3) preserve: dried bletilla striata capsule is placed in closed container preserves.
2. the method for claim 1, the vitality that it is characterized in that step (1) bletilla seed detects as follows: get TTC and be mixed with 0.5% concentration with 0.1mol/L pH7.2 phosphoric acid buffer, obtain dyeing liquor, the bletilla seed is taken out from capsule, join in dyeing liquor, 40 ℃ of oven 24h, then counting dyes and undyed seed under light microscope, calculates as follows the vitality of bletilla seed:
3. the method for claim 1, is characterized in that step (3) freezing preservation in liquid nitrogen.
4. the method for claim 1 is characterized in that step (3) is no more than under 25 ℃ of conditions in room temperature to preserve.
5. the method for claim 1, is characterized in that step (3) is 4 ℃ of lower low temperature preservations.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103797984A (en) * | 2014-03-14 | 2014-05-21 | 湖南农业大学 | Method for harvesting triarrhena lutarioriparia seeds |
| CN104186121A (en) * | 2014-08-08 | 2014-12-10 | 岭南园林股份有限公司 | One-time collecting and sowing method for seeds different in maturation periods |
| CN104718841A (en) * | 2015-02-26 | 2015-06-24 | 李泽诚 | Treatment method of rhizoma bletillae seeds |
| CN105474831A (en) * | 2015-12-16 | 2016-04-13 | 湖南省西瓜甜瓜研究所 | Bletilla striata bare seed disinfection method |
| CN105941392A (en) * | 2016-05-03 | 2016-09-21 | 湖南农业大学 | Bletilla striata capsule preserving method suitable for tissue culture and rapid propagation |
| CN106105586A (en) * | 2016-06-23 | 2016-11-16 | 四川省中医药科学院 | A kind of method of Cryopreservation Radix Angelicae Dahuricae seed |
| CN106941890A (en) * | 2017-03-23 | 2017-07-14 | 杨军 | A kind of storage method of willow seed |
| CN107087457A (en) * | 2017-04-05 | 2017-08-25 | 周兴虎 | A kind of cinnamomum camphora seed poison-removing method |
| CN112514751A (en) * | 2020-11-30 | 2021-03-19 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Long-term storage and recovery method of cassava crossbreeding capsule |
| CN112616657A (en) * | 2020-12-02 | 2021-04-09 | 云南省农业科学院农业环境资源研究所 | Sterile preservation method of bletilla striata seeds |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103797984A (en) * | 2014-03-14 | 2014-05-21 | 湖南农业大学 | Method for harvesting triarrhena lutarioriparia seeds |
| CN103797984B (en) * | 2014-03-14 | 2015-12-02 | 湖南农业大学 | A kind of method gathering in the crops southern reed seed |
| CN104186121A (en) * | 2014-08-08 | 2014-12-10 | 岭南园林股份有限公司 | One-time collecting and sowing method for seeds different in maturation periods |
| CN104718841A (en) * | 2015-02-26 | 2015-06-24 | 李泽诚 | Treatment method of rhizoma bletillae seeds |
| CN105474831B (en) * | 2015-12-16 | 2017-11-17 | 湖南省西瓜甜瓜研究所 | A kind of bletilla striata naked seed sterilization method |
| CN105474831A (en) * | 2015-12-16 | 2016-04-13 | 湖南省西瓜甜瓜研究所 | Bletilla striata bare seed disinfection method |
| CN105941392A (en) * | 2016-05-03 | 2016-09-21 | 湖南农业大学 | Bletilla striata capsule preserving method suitable for tissue culture and rapid propagation |
| CN105941392B (en) * | 2016-05-03 | 2018-10-09 | 湖南农业大学 | A kind of bletilla striata capsule store method suitable for tissue-culturing rapid propagation |
| CN106105586A (en) * | 2016-06-23 | 2016-11-16 | 四川省中医药科学院 | A kind of method of Cryopreservation Radix Angelicae Dahuricae seed |
| CN106941890A (en) * | 2017-03-23 | 2017-07-14 | 杨军 | A kind of storage method of willow seed |
| CN107087457A (en) * | 2017-04-05 | 2017-08-25 | 周兴虎 | A kind of cinnamomum camphora seed poison-removing method |
| CN112514751A (en) * | 2020-11-30 | 2021-03-19 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Long-term storage and recovery method of cassava crossbreeding capsule |
| CN112514751B (en) * | 2020-11-30 | 2022-06-17 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Long-term storage and recovery method of cassava crossbreeding capsule |
| CN112616657A (en) * | 2020-12-02 | 2021-04-09 | 云南省农业科学院农业环境资源研究所 | Sterile preservation method of bletilla striata seeds |
| CN112616657B (en) * | 2020-12-02 | 2022-01-07 | 云南省农业科学院农业环境资源研究所 | Sterile preservation method of bletilla striata seeds |
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