CN100510062C - Inductor of mycorrhizal fungi for cultivating protocorm solid of Tiepi stem of noble dendrobium - Google Patents
Inductor of mycorrhizal fungi for cultivating protocorm solid of Tiepi stem of noble dendrobium Download PDFInfo
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- CN100510062C CN100510062C CNB2005100730490A CN200510073049A CN100510062C CN 100510062 C CN100510062 C CN 100510062C CN B2005100730490 A CNB2005100730490 A CN B2005100730490A CN 200510073049 A CN200510073049 A CN 200510073049A CN 100510062 C CN100510062 C CN 100510062C
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Abstract
This invention discloses a technique for applying MF24 (Mycena sp.) in the culture of protocorm in Dendrobium candidum. The technique comprises: (1) culturing protocorm in Dendrobium candidum and MF24 (Mycena sp.), respectively; (2) manufacturing MF24 (Mycena sp.) into inducer, and adding into protocorm in Dendrobium candidum for co-culture. The cultured protocorm in Dendrobium candidum has higher yield and contents of effective components, and can be used to replace Dendrobium candidum as a pharmaceutical material. The method is simple, and does not need addition of any hormone.
Description
Technical field
The invention belongs to technical field of bioengineering.Relate to the application of a kind of fungal elicitor in Herba Dendrobii protocorm solid culture and the practical technique of raising Herba Dendrobii protocorm active constituent content specifically.
Background technology
The stem of noble dendrobium is a kind of traditional rare Chinese herbal medicine, and the beginning is stated from Shennong's Herbal, classifies as top grade.The stem of noble dendrobium is a orchid family Dendrobium plant, all herbal medicine, and it is sweet to distinguish the flavor of, and cold nature has nourishing the stomach to promote the production of body fluid, the effect of enriching yin heat extraction.Herba Dendrobii (Dendrobium candidum Wall.ex Ldl.) is one of five stem of noble dendrobium kinds recording of China's pharmacopeia, is considered to the treasure of enriching yin help since ancient times.Compendium of Material Medica record Herba Dendrobii has effects such as Yin-nourishing and body fluid promoting, and the yin deficiency of the moon being hindered body fluid deficiency has very high pharmaceutical use.The modern pharmacological research proof stem of noble dendrobium has anti-tumor activity; Has immunoloregulation function; The cataractous effect of control is arranged; Effect to cardiovascular systems shows platelet aggregation-against, suppresses heart, vasodilation; Promote gastrointestinal function; Have effects such as anti-oxidant and antimutagenic activity.
Herba Dendrobii mainly is distributed in South Asian nations such as provinces such as Yunnan on the south China Qinling Mountains, Guizhou, Guangxi, Sichuan and Burma, Vietnam.The Herba Dendrobii plant generally is grown on sylvan life the water other precipitous crag or ancient tree, and growing environment is dark and damp, and is with the humid air nice and cool, drought-resistant and severe cold, and common and lichens, liver moss and pteridophyte mix and give birth to, and form evergreen ground storey.The ecotope that stem of noble dendrobium growth requires is more special, and growth cycle is long, and the natural propagation rate is low.Because people ceaselessly excavate and the continuous forfeiture of natural ecological environment, the natural resources of wild Herba Dendrobii is endangered.1987, Herba Dendrobii was listed in the wild plant of special-protection-by-the-State.Herba Dendrobii is listed in endangered species in " Chinese Plants Red Data Book " (version in 1991).Because Herba Dendrobii natural ecological condition uniqueness, breeding potential is low, and the commercialization of test-tube plantlet is produced and also had certain difficulty, makes the resource problem of Herba Dendrobii can not get all the time solving.
Protocorm (Protocorm) is that proembryo is sprouted and expanded in the Herba Dendrobii seed germination process, the light yellow roundlet taper embryonal connective tissue that the prominent back of breaking in the seed coat forms.Processes such as the explant blade tip of Herba Dendrobii, stem apex, basic point are induced also and can be formed the embryonal connective tissue that is similar to protocorm.Protocorm is the bud into pseudobulb further, sends out roots then and leaf, forms complete plant.But protocorm forms ripe plant limited by several factors, and required time is also long.The protocorm growth of Herba Dendrobii is quick, and the oneself is mitogenetic vigorous, and vigor is lasting, is a kind of substitute of ideal Herba Dendrobii medicinal material., contain less secondary metabolite, also have certain distance from the alternative medicinal material that really becomes Herba Dendrobii because protocorm is the tender plant materials of children.
Herba Dendrobii is the plant with mycosymbiosis.Fungal elicitor (elicitor) is meant the sterile solution that the radicula byssoidea with separation and Culture is prepared into, and it can promote the growth of plant or inducing plant produces and the accumulation secondary metabolite.Studies show that in recent years: be used to elicitor composition from fungi, can inducing plant culturing cell or plant seedlings generation or the interior relevant effective constituent of raising body.
With respect to dendrobium officinale test-tube plantlet, it is fast that the Herba Dendrobii protocorm has propagation, and growth cycle is short, and characteristic of simple is cultivated in breeding.We expect the effect by fungal elicitor and Herba Dendrobii protocorm, improve the output and the quality of Herba Dendrobii protocorm, make it to become the alternate resources of Herba Dendrobii, thereby alleviate the disparities between supply and demand of Herba Dendrobii, protection Herba Dendrobii resource.
Summary of the invention
The purpose of this invention is to provide fungal elicitor preparation method, Herba Dendrobii protocorm solid culture method and fungal elicitor and Herba Dendrobii protocorm co-cultivation method, but this method technology is simple, cost is low, cycle short large-scale industrial production Herba Dendrobii protocorm.
Another object of the present invention is to improve the active constituent content of Herba Dendrobii protocorm,, solve Herba Dendrobii medicine resource problem in short supply so that the Herba Dendrobii protocorm of extensive solid culture can substitute the Herba Dendrobii medicinal material.
For achieving the above object, the technology that the present invention adopts is to cultivate the Herba Dendrobii protocorm earlier, adds fungal elicitor, co-cultivation for some time in its growth later stage.Fungal elicitor preparation method provided by the invention adopts fungi MF24 (Mycena sp.) fermentation culture, refabrication elicitor.The fungi that the present invention adopts obtains by separating in the wild Herba Dendrobii root, through culture identification is fungus Mycenae sp. MF24 (Mycena sp.), this bacterial classification on April 20th, 2005 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, depositary institution address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.1350.Preservation and survival proof are seen Appendix.
Specifically, technological step method of the present invention is as follows:
1. the cultivation of fungi
Above-mentioned fungi behind the slant tube actication of culture of cryopreservation, is transferred in the plate that contains the PDA substratum,, punch into the bacterium sheet at colony edge respectively, and insert liquid nutrient medium with fine grained chippings and cultivate and ferment in 25 ℃ of constant temperature culture 18-26 days.
The substratum that fungi liquid is cultivated: glucose 1.5-4%, KH
2PO
40.1-0.4%, MgSO
40.1-0.3%; Natural goods: wheat bran 1-5% (liquor), above component is percentage composition calculating by weight all, medium pH 5.0-6.5, triangular flask or other container shaking culture.Container loading amount 30-60% inserts above-mentioned fungi behind the autoclaving.Culture condition: 100-130 rev/min of vibration rotating speed; 22-26 ℃ of dark the cultivation gathered in the crops in 18-25 days.
2. the preparation of fungal elicitor
Above-mentioned fungi is after fermentation culture, with 80-120 order nylon net filter, separation of mycelial and fermented liquid; Be prepared into different fungal elicitors by following method;
(1) mycelium elicitor: 20-80 restrains bright mycelium and inserts in the sterilized water of 150-230ml, and 121 ℃ of sterilizations are 20 minutes after the homogenate, the mycelium elicitor;
(2) fermented liquid elicitor: the fermented liquid packing, sterilized 20 minutes, and made fungal elicitor for 121 ℃;
(3) mix elicitor: 20-80 and restrain bright mycelium and insert in 150-230ml self fermented liquid, 121 ℃ of sterilizations are 20 minutes after the homogenate, must mix elicitor;
(4) mix the clear liquid elicitor: 20-80 restrains bright mycelium and inserts in 150-230ml self fermented liquid, sterilizes 20 minutes for 121 ℃ after the homogenate, gets its supernatant liquor, must mix the clear liquid elicitor;
3. the solid culture of Herba Dendrobii protocorm
Get the growth 10 week-18 weeks Herba Dendrobii protocorm subculture in 1/2MS potato solid medium, inoculum size 1-5% (W/V), in 22-26 ℃ of illumination cultivation 10-15 week, intensity of illumination: 1500-5000Lux, light application time: 8-12 hour/day.
The substratum of Herba Dendrobii protocorm succeeding transfer culture: potato 150-250g/L (boiled 20 minutes, and got juice), 1/2MS substratum, sucrose 15-35g/L, inositol 80-120mg/L, pH 5.0-6.5, agar powder 6-9g/L, culture vessel loading amount 10%-50%.
4. Herba Dendrobii protocorm and fungal elicitor co-cultivation
In the Herba Dendrobii protocorm substratum in growth 10-16 week, add the fungal elicitor of any one sterilization, adding dosage is the 180-300mL/L solid medium.
5. the results of Herba Dendrobii protocorm
The Herba Dendrobii protocorm continues illumination cultivation 5-8 week after adding elicitor, results.Dry in the shade or be lower than 60 ℃ of oven dry.
Description of drawings
Accompanying drawing is the HPLC-MS base peak ionic current color atlas of Herba Dendrobii protocorm and Herba Dendrobii medicinal material.
001: add the Herba Dendrobii protocorm that MF24 (Mycena sp.) fungal elicitor is handled;
T01: Herba Dendrobii medicinal material;
002: blank Herba Dendrobii protocorm.
Embodiment
Embodiment:
1. the cultivation of fungi
(1) with fungus Mycenae sp. (Mycena sp.) behind the slant tube actication of culture of cryopreservation, transfer in ф 90mmPDA plate, 25 ℃ of constant temperature culture are after 24 days, with the punch tool of ф 9mm respectively the colony edge in plate punch into the bacterium sheet, and insert liquid nutrient medium with fine grained chippings and cultivate and ferment.
(2) substratum of fungi liquid cultivation: glucose 2%, KH
2PO
40.3%, MgSO
40.15%; Natural goods: wheat bran 3% (liquor), pH5.6.
(3) culture condition: above-mentioned substratum 500mL shaking table culturing bottle loading amount 250mL, sterilized 30 minutes for 121 ℃, insert fungus Mycenae sp. (Mycena sp.).Culture condition: 120 rev/mins of shaking speed, temperature: 25 ℃, secretly cultivate 21 days results.
2. the preparation of fungal elicitor
After Mycena (Mycena sp.) fungi fermentation is cultivated and finished,, sterilized 20 minutes, and made the fermented liquid elicitor for 121 ℃ with 100 order nylon net filters, separation of mycelial and fermented liquid, fermented liquid packing.
3. the solid culture of Herba Dendrobii protocorm
(1) substratum of Herba Dendrobii protocorm solid culture: 1/2MS, sucrose 3%, inositol 0.01%; Natural goods: potato 20% (peeling was boiled 20 minutes, got juice), pH5.8, agar powder 0.75%.
(2) the bottled amount 50mL of above-mentioned substratum 300mL plastic culture sterilized 20 minutes for 121 ℃, inserted the Herba Dendrobii protocorm in 15 weeks of growth, and inoculum size 2% (W/V) was in 24-26 ℃ of 14 weeks of illumination cultivation, intensity of illumination: 2000Lux, light application time: 10h/ days.
4. Herba Dendrobii protocorm and fungal elicitor co-cultivation
Add the fermented liquid elicitor of sterilization in the Herba Dendrobii protocorm substratum in 14 weeks of growth, adding dosage is 25% (V/V).The Herba Dendrobii protocorm continues with the old terms illumination cultivation after adding fungal elicitor.
5. the results of Herba Dendrobii protocorm
The Herba Dendrobii protocorm continues to cultivate for 6 weeks after adding fungal elicitor, isolation medium and Herba Dendrobii protocorm, and protocorm is in 55 ℃ of oven dry.
Comparative example:
1. fungal elicitor is to the influence of Herba Dendrobii protocorm growth:
In the grow Herba Dendrobii protocorm in 14 weeks of 1/2MS potato inoculation of medium, inoculum size 2% (W/V).The inoculation protocorm in 24-26 ℃, the 2000Lux illumination cultivation.In the 16th week of cultivating,, add the back respectively at the 2nd week of continuing to cultivate, the 4th week, the 6th all results in ratio adding elicitor, the distilled water of 250mL/L substratum.Not add the protocorm of handling cultivation is blank.The Herba Dendrobii protocorm of results claims fresh weight, and 55 ℃ of dry backs of sample claim dry weight.Statistic analysis result shows: add fungal elicitor in the Herba Dendrobii protocorm growth later stage, continue can make the results fresh weight of protocorm improve 30%-40% after cultivation for some time, make the results dry weight of protocorm improve 15%-20%.
(1) to the influence (table 1) of fresh weight
According to The results of analysis of variance: compare with blank, adding elicitor and distilled water can improve fresh weight (p＜0.01) by the utmost point significantly, and average mark you can well imagine high 44.9% and 37.6%; Add processing back incubation time fresh weight is not had remarkable influence.
Table 1. fungal elicitor is to the influence of Herba Dendrobii protocorm fresh weight (gram/bottle)
(2) to the influence (table 2) of dry weight
According to The results of analysis of variance: with blank with add distilled water and compare, adding elicitor can improve dry weight (p＜0.01) by the utmost point significantly, has improved 19.8% and 22.4% respectively; Adding processing back incubation time has no significant effect dry weight.
Table 2. fungal elicitor is to the influence of Herba Dendrobii protocorm dry weight (gram/bottle)
2. fungal elicitor is to the influence of Herba Dendrobii protocorm total alkaloid content:
In the grow Herba Dendrobii protocorm in 14 weeks of 1/2MS potato inoculation of medium, inoculum size 2% (W/V).The inoculation protocorm in 24-26 ℃, the 2000Lux illumination cultivation.In the 14th week of cultivating,, add the back in the 3rd all results that continue to cultivate in ratio adding elicitor, the distilled water of high dosage 250mL/L substratum, middle dosage 150mL/L substratum, low dosage 50mL/L substratum.Not add the protocorm of handling cultivation is blank.The Herba Dendrobii protocorm of results claims fresh weight, and 55 ℃ of dry backs of sample claim dry weight.With the dendrobine is standard substance, measures total alkaloid content in the Herba Dendrobii protocorm with acid-dye colorimetry, the results are shown in Table 3.Experimental result shows: when fungal elicitor adds the Herba Dendrobii protocorm with the dosage of 250mL/L substratum, total alkaloid contents is the highest in the Herba Dendrobii protocorm, though the fresh weight that the adding that improves 35.7% distilled water can improve protocorm is shone in comparison, and is little to the influence of total alkaloid content.
Table 3. fungal elicitor is to the influence of Herba Dendrobii protocorm total alkaloid content (%)
3. fungal elicitor joining day and adding back are continued incubation time influences Herba Dendrobii protocorm total alkaloid content:
In the grow Herba Dendrobii protocorm in 15 weeks of 1/2MS potato inoculation of medium, inoculum size 2% (W/V).The inoculation protocorm in 24-26 ℃, the 2000Lux illumination cultivation.Respectively at the 10th week, the 12nd week, the 14th week, the 16th week of cultivating,, gather in the crops after continuing respectively behind the adding elicitor to cultivate 2 weeks, 4 weeks, 6 weeks by the dosage adding fungal elicitor of 25% (V/V).55 ℃ of dryings of Herba Dendrobii protocorm of results.With the dendrobine is standard, measures the total alkaloid content of each sample with acid-dye colorimetry, the results are shown in Table 4.Experimental result shows: add fungal elicitor when 14 weeks of Herba Dendrobii protocorm succeeding transfer culture, 16 weeks, add the back and continue to cultivate for 6 weeks, total alkaloid contents is the highest in the Herba Dendrobii protocorm.Consider the problem that shortens the production cycle, the fungal elicitor joining day was decided to be in the 14th week of succeeding transfer culture adds, continue again after the adding to cultivate for 6 weeks.
The table 4. fungal elicitor joining day reaches and is total to the influence of incubation time to Herba Dendrobii protocorm total alkaloid content (%)
4. the mass ratio of Herba Dendrobii protocorm and Herba Dendrobii medicinal material is:
The Herba Dendrobii protocorm in 15 weeks of growth on 1/2MS potato substratum, the dosage adding fungal elicitor by 25% (V/V) continues to cultivate for 6 weeks results behind the adding elicitor.With with a collection of subculture, the Herba Dendrobii protocorm of cultivating for 21 weeks is a blank.With the Herba Dendrobii medicinal material is contrast.55 ℃ of dryings of sample are pulverized, and cross 40 mesh sieves.Precision takes by weighing 100mg, and is wetting with proper ammonia, after the adding chloroform refluxing extraction, with methanol constant volume.Sample solution detects through HPLC-MS, and Herba Dendrobii protocorm and Herba Dendrobii medicinal material almost do not have difference on composition, on each component content, and difference to some extent.
From above comparative example as can be seen, the present invention has following advantage: fungus Mycenae sp. MF24 (Mycena sp.) has obvious facilitation to the growth of Herba Dendrobii protocorm; Adopt this technology can improve the output of Herba Dendrobii protocorm, improve content of effective; The Herba Dendrobii protocorm that adopts the present invention to produce does not have very big difference with the Herba Dendrobii plant on chemical ingredients, the Herba Dendrobii protocorm has the potentiality as the Herba Dendrobii substitute.Herba Dendrobii protocorm production technology in this invention, cultural method is simple, and less investment need not added any plant hormone, and the Herba Dendrobii protocorm of being produced is a green product.The present invention provides scientific basis for Herba Dendrobii protocorm large-scale industrialized production from now on.
Claims (6)
1. method for preparing the Herba Dendrobii protocorm comprises step:
(1) cultivation of fungi
With deposit number is that the fungus Mycenae sp. MF24 (Mycena sp.) of CGMCC No.1350 is behind the slant tube actication of culture of cryopreservation, transfer in the plate that contains the PDA substratum, in 25 ℃ of constant temperature culture 18-26 days, punch into the bacterium sheet at colony edge respectively, and insert liquid nutrient medium with fine grained chippings and cultivate and ferment;
The substratum that fungi liquid is cultivated consists of glucose, KH
2PO
4, MgSO
4, wheat bran; Each component is glucose 1.5-4% by weight, KH
2PO
40.1-0.4%, MgSO
40.1-0.3%, wheat bran 1-5%; Wherein said wheat bran is got the juice use after must adding poach; Medium pH 5.0-6.5;
The fungi liquid culture condition is: triangular flask or other container shaking culture fungies, container loading amount 30-60%; Above-mentioned fungi is inserted in the sterilization back, 100-130 rev/min of vibration rotating speed; 22-26 ℃ of dark the cultivation gathered in the crops in 18-25 days;
(2) preparation of fungal elicitor
Above-mentioned fungi is after fermentation culture, with 80-120 order nylon net filter, separation of mycelial and fermented liquid; Be prepared into different fungal elicitors by following method;
1. mycelium elicitor: bright mycelium is inserted in the sterilized water, sterilize after the homogenate the mycelium elicitor;
2. fermented liquid elicitor: the fermented liquid packing sterilize fungal elicitor;
3. mix elicitor: bright mycelium is inserted in self fermented liquid, sterilizes to such an extent that mix elicitor after the homogenate;
4. mix the clear liquid elicitor: bright mycelium is inserted in self fermented liquid, sterilizes after the homogenate, gets its supernatant liquor and must mix the clear liquid elicitor;
(3) solid culture of Herba Dendrobii protocorm
The Herba Dendrobii protocorm on 1/2MS potato solid medium, illumination cultivation; Solid medium consists of: potato 150-250g/L, 1/2MS substratum, sucrose 15-35g/L, inositol 80-120mg/L, pH5.0-6.5, agar powder 6-9g/L; Culture vessel loading amount 10%-50%; Wherein said potato must add poach and get the juice use after 20 minutes;
(4) Herba Dendrobii protocorm and fungal elicitor co-cultivation
In the Herba Dendrobii protocorm solid medium of growing, add a kind of fungal elicitor of sterilization, with its co-cultivation;
(5) results of Herba Dendrobii protocorm
The Herba Dendrobii protocorm continues illumination cultivation 5-8 week after adding elicitor, and results are dried in the shade or are lower than 60 ℃ of oven dry.
2. the method for claim 1 is characterized in that: the mycelium elicitor is that 20-80 restrains bright mycelium and inserts in the sterilized water of 150-230ml, and sterilization gets final product after the homogenate; The fermented liquid elicitor is that the fermented liquid sterilization gets final product; Mix elicitor and be 20-80 and restrain bright mycelium and insert in 150-230ml self fermented liquid, sterilization gets final product after the homogenate; Mix the clear liquid elicitor and be 20-80 and restrain bright mycelium and insert in 150-230ml self fermented liquid, sterilize after the homogenate, get its supernatant liquor and get final product.
3. cultural method as claimed in claim 1, it is characterized in that: the Herba Dendrobii protocorm of getting 10 week-18 weeks of growth is inoculated in the 1/2MS potato solid medium, inoculum size is counted 1-5% with bulking value, in 22-26 ℃ of illumination cultivation 10-15 week, intensity of illumination: 1500-5000Lux, light application time: 8-12 hour/day.
4. cultural method as claimed in claim 1, it is characterized in that: under aseptic condition, in the Herba Dendrobii protocorm solid medium in growth 10-16 week, can add a kind of fungal elicitor and its co-cultivation of sterilization arbitrarily, adding dosage is the 180-300mL/L solid medium; In 22-26 ℃ of illumination cultivation, intensity of illumination: 1500-5000Lux, light application time: 8-12 hour/day.
5. cultural method as claimed in claim 1 is characterized in that: the Herba Dendrobii protocorm continues illumination cultivation 5-8 week results after adding elicitor, removes impurity such as substratum during results, and the Herba Dendrobii protocorm dries in the shade or is lower than 60 ℃ of oven dry hyoscines.
6. a fungus Mycenae sp. MF24 (Mycena sp.), its deposit number is CGMCC No.1350.
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| CN100510061C (en) * | 2005-05-31 | 2009-07-08 | 中国医学科学院药用植物研究所 | Method for raising quality of protocorm of Tiepi stem of noble dendrobium cultivated in liquid through inductor of mycorrhizal fungi |
| CN103881928A (en) * | 2014-03-13 | 2014-06-25 | 浙江农林大学 | Application of mortierella mycorrhiza fungi to culture of tissue culture seedling of dendrobium officinale |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1433669A (en) * | 2002-01-25 | 2003-08-06 | 中国医学科学院药用植物研究所 | Application of mycorrhizal fungi in dendrobe cultivation |
| CN1872995A (en) * | 2005-05-31 | 2006-12-06 | 中国医学科学院药用植物研究所 | Method for raising quality of protocorm of Tiepi stem of noble dendrobium cultivated in liquid through inductor of mycorrhizal fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1433669A (en) * | 2002-01-25 | 2003-08-06 | 中国医学科学院药用植物研究所 | Application of mycorrhizal fungi in dendrobe cultivation |
| CN1872995A (en) * | 2005-05-31 | 2006-12-06 | 中国医学科学院药用植物研究所 | Method for raising quality of protocorm of Tiepi stem of noble dendrobium cultivated in liquid through inductor of mycorrhizal fungi |
Non-Patent Citations (2)
| Title |
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| Mycena dendrobii, a new mycorrhizal fungus. Guo SX, Fan L, Cao WQ, et al.Mycosystema,Vol.18 No.2. 1999 * |
| 真菌诱导子对铁皮石斛组培物生长的影响. 潘超美等.中医药学刊，第22卷第1期. 2004 * |
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