CN104719165A - Rapid tissue culture method for lycium ruthenicum murr - Google Patents
Rapid tissue culture method for lycium ruthenicum murr Download PDFInfo
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Abstract
The invention discloses a rapid tissue culture method for lycium ruthenicum murr, belonging to the technical field of plant tissue culture. The rapid tissue culture method comprises the following steps: explants sterilization, induction culture, rooting culture, subculture, acclimatization and transplantation. According to the rapid tissue culture method, the rooting culture can be carried out during the subculture, so that the tissue culture is high, the operation is simple and convenient, the period is short, and complete plants can be rapidly acquired; the tissue culture process is not influenced by environmental factors.
Description
Technical field
The invention belongs to field of plant tissue culture technique, relate to a kind of tissue culture method, be specifically related to a kind of quickly tissue culture method of black fruit lyceum.
Background technology
Black fruit lyceum (
lycium ruthenicum Murr.) be Solanaceae Lycium, being the raw medicinal plant of a kind of salt, is many quils shrub.Black fruit lyceum taste is sweet, property is put down, not only be rich in the multiple nutritional components such as protein, lycium barbarum polysaccharide, amino acid, vitamin, mineral matter, trace element, also containing abundant black fruit pigment-natural procyanidins, wherein haw matrimony vine is not containing OPC, the procyanidin content of black fruit lyceum exceedes blueberry, is the wild plant that the procyanidin content that finds so far is the highest.Meanwhile, black fruit lyceum has drought resisting, cold-resistant, Salt And Alkali Tolerance, root turion are strong and the plurality of advantages such as resistance to soil depletion, be Saline-alkali Field Control, bank protection, the good seeds that prevent erosion.Therefore, black fruit lyceum no matter in economic product exploitation, or all has market prospects and potentiality to be exploited widely in ecological environment treatment, can become the Important Economic seeds of medicines and health protection, establishment of economic forest and saline land biological control.
At present, black fruit lyceum is mainly wild is main, but wild resource main area natural conditions are severe, and vegetation degeneration is serious, dead even in flakes, causes serious threat to the normal existence of black fruit lyceum, is substantially in Critical Condition.Now, the research overwhelming majority about black fruit lyceum concentrates on its physiological ecological, analysis of effective component, exploitation and administers in desertification and wild provenance domestication, and little about the research report of the tissue culture technology of black fruit lyceum.Great this grade of benevolence tower (2005) utilizes the stem section of band axillalry bud to carry out breaking up to black fruit lyceum and to take root and callus induction adventitious bud proliferation is studied, and only relates to tissue cultures, does not study its fast culture; Number of patent application 201310094393.2 relates to the method for black fruit lyceum high quality seedling Fast-propagation, needs to select black fruit lyceum high quality seed, uses 75% alcohol disinfecting, aseptic water washing 3 ~ 4 times; Again with 0.1% mercuric chloride sterilization, after aseptic water washing 3 ~ 4 times, seed is inoculated in MS medium and cultivates aseptic seedling, cultivate after 1 month, the cane of aseptic seedling is cut, be cut into the stem section of 0.5cm length, access MS+KH
2pO
4proceed in 13.5mg/L+NAA0.1mg/L medium to cultivate, the tissue cultures of its explant is not described in detail.
Summary of the invention
The present invention utilizes tissue culture technique, carry out explant cultivation and obtain black fruit lyceum whole plant, provide a kind of quickly tissue culture method of black fruit lyceum, group training simple flow, not by seasonal climate variable effect, for black fruit lyceum industrialization nursery provides technical support.
First the present invention utilizes plant tissue culture technique to obtain black fruit lyceum plantlet in vitro, then by directly inducing tender stem segments and stem apex axillary bud growth, axillalry bud culture of rootage, cutting stem section or Shoot Tip Culture breeding.
Object of the present invention is achieved through the following technical solutions: a kind of quickly tissue culture method of black fruit lyceum, and it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 3 ~ 5min, 75% alcohol sterilizing 4 ~ 7s, sterile water wash 2 ~ 3 times, 0.1% mercuric chloride solution sterilizing 13 ~ 17min, sterile water wash 4 ~ 6 times;
(2) Fiber differentiation: be inoculated in inducing culture by the tender stem section after sterilizing and stem apex, cultivates 25 ~ 45 days axillalry buds and is stretched to 3 ~ 5cm in culturing room;
(3) culture of rootage: cut by axillalry bud, is inoculated in root media, within 25 ~ 40 days, obtains the black fruit lyceum plantlet in vitro that length is 6 ~ 10cm, and root length is 5 ~ 10cm;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 2 ~ 3 bud points, be inoculated in subculture medium and carry out subculture, carry out culture of rootage simultaneously, cultivate in culturing room and within 29 ~ 32 days, obtain being highly the black fruit lyceum whole plant of 6.8 ~ 8.0cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed under natural daylight bottle and take exercise 5 ~ 8 days, hardening of at room temperature uncapping 3 ~ 5 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions to root sterilization 3 ~ 5min, dry and plant in matrix, be transplanted in soil when the new root system that seedling grows reaches 3 ~ 5cm and grows side root.
Medium described in step (2) is MS+0.5mg/L 6-BA+0.1mg/L NAA.
Cultivation indoor conditions described in step (2) is: intensity of illumination 1500LX, light application time 12 ~ 16h/ days, temperature 24 ~ 26 DEG C.
Step (3) and the medium described in step (4) are 1/2MS+0.1mg/L NAA.
Step (3) and the cultivation indoor conditions described in step (4): intensity of illumination 3000LX, light application time 12-16h/ days, temperature 24 ~ 26 DEG C.
Matrix described in step (5) is perlite and peat mixing, and both weight ratios are 1:1.5 ~ 2.5.
Matrix 0.1% disinfecting solution of potassium permanganate described in step (5).
Described to be transplanted in soil after cultivate and see young leaves after 3 ~ 5 weeks and start growth, show that the black fruit lyceum of transplanting survives.
The present invention has following beneficial effect:
(1) in the present invention while black fruit lyceum plantlet in vitro squamous subculture, can culture of rootage be carried out, decrease operating procedure, improve the efficiency of group training, can obtain robust growth, complete plantlet in vitro that genetic character is consistent fast, be a kind of effective way of black fruit Chinese holly quick Qi group training;
(2) in the present invention, step (3) is identical with the medium that step (4) is inoculated, and decreases the process that medium is changed, and simplify group training flow process, easy and simple to handle, the cycle is short, can obtain black fruit lyceum whole plant fast;
(3) the present invention utilizes tissue culture technique, carries out explant cultivation and obtains black fruit lyceum whole plant, by the impact of seasonal climate change, natural calamity, for black fruit lyceum industrialization nursery provides technical support;
(4) the invention solves that black fruit lyceum fertility is low, the problem of scarcity of resources and residue of pesticide; alleviate wild black fruit lyceum resource nervous; protecting wild black fruit lyceum resource, protecting abundant rational exploitation and utilization on basis, the economic worth of black fruit lyceum can be played.
Accompanying drawing explanation
Fig. 1 is axillalry bud in black fruit lyceum Fiber differentiation of the present invention;
When Fig. 2 is black fruit lyceum culture of rootage of the present invention 10 days, axillalry bud is taken root situation;
When Fig. 3 is black fruit lyceum culture of rootage of the present invention 30 days, axillalry bud is taken root situation;
Fig. 4 is the black fruit lyceum plantlet in vitro obtained in black fruit lyceum culture of rootage of the present invention;
Fig. 5 is culture of rootage situation in black fruit lyceum squamous subculture of the present invention.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated.
embodiment 1:a quickly tissue culture method for black fruit lyceum, it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 3min, 75% alcohol sterilizing 7s, sterile water wash 3 times, 0.1% mercuric chloride solution sterilizing 13min, sterile water wash 4 times;
(2) Fiber differentiation: the tender stem section after sterilizing and stem apex are inoculated in MS+0.5mg/L 6-BA+0.1mg/L NAA medium, be 1500LX in intensity of illumination, light application time is 16h/ days, and temperature is cultivate 25 days in the culturing room of 24 DEG C, and axillalry bud average elongation is to 3cm;
(3) culture of rootage: axillalry bud is cut, being inoculated in 1/2MS+0.1mg/L NAA medium, is 3000LX in intensity of illumination, light application time is 16h/ days, temperature is cultivate 25 days in the culturing room of 24 DEG C, obtains the black fruit lyceum plantlet in vitro of height average out to 6cm, the long average out to 5cm of root;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 2 bud points, be inoculated in 1/2MS+0.1mg/L NAA medium, carry out subculture, carry out culture of rootage, be 3000LX in intensity of illumination simultaneously, and light application time is 16h/ days, temperature is cultivate 29 days in the culturing room of 24 DEG C, obtains the black fruit lyceum whole plant of height average out to 6.8cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed bottle under natural daylight and tempers 5 days, hardening of at room temperature uncapping 3 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions, root is sterilized 3min, dry and plant in the matrix after with 0.1% disinfecting solution of potassium permanganate, be transplanted in soil when the new root system that seedling grows on average reaches 3cm and grows side root, its mesostroma is perlite and peat mixing, and both weight ratios are 1:1.5.
Cultivate after being transplanted in soil and see young leaves after 3 weeks and start growth, show that the black fruit lyceum of transplanting survives, investigated the survival rate of black fruit lyceum after 2 weeks, survival rate average out to 75%.
embodiment 2:a quickly tissue culture method for black fruit lyceum, it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 5min, 75% alcohol sterilizing 4s, sterile water wash 2 times, 0.1% mercuric chloride solution sterilizing 17min, sterile water wash 6 times;
(2) Fiber differentiation: the tender stem section after sterilizing and stem apex are inoculated in MS+0.5mg/L 6-BA+0.1mg/L NAA medium, be 1500LX in intensity of illumination, light application time is 12h/ days, and temperature is cultivate 45 days in the culturing room of 26 DEG C, and axillalry bud average elongation is to 5cm;
(3) culture of rootage: axillalry bud is cut, being inoculated in 1/2MS+0.1mg/L NAA medium, is 3000LX in intensity of illumination, light application time is 12h/ days, temperature is cultivate 40 days in the culturing room of 26 DEG C, obtains the black fruit lyceum plantlet in vitro of height average out to 10cm, the long average out to 10cm of root;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 3 bud points, be inoculated in 1/2MS+0.1mg/L NAA medium, carry out subculture, carry out culture of rootage, be 3000LX in intensity of illumination simultaneously, and light application time is 12h/ days, temperature is cultivate 32 days in the culturing room of 26 DEG C, obtains the black fruit lyceum whole plant of height average out to 8cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed bottle under natural daylight and tempers 8 days, hardening of at room temperature uncapping 5 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions, root is sterilized 5min, dry and plant in the matrix after with 0.1% disinfecting solution of potassium permanganate, be transplanted in soil when the new root system that seedling grows on average reaches 5cm and grows side root, its mesostroma is perlite and peat mixing, and both weight ratios are 1:2.5.
Cultivate after being transplanted in soil and see young leaves after 5 weeks and start growth, show that the black fruit lyceum of transplanting survives, investigated the survival rate of black fruit lyceum after 2 weeks, survival rate average out to 75%.
embodiment 3:a quickly tissue culture method for black fruit lyceum, it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 4min, 75% alcohol sterilizing 5s, sterile water wash 2 times, 0.1% mercuric chloride solution sterilizing 14min, sterile water wash 5 times;
(2) Fiber differentiation: the tender stem section after sterilizing and stem apex are inoculated in MS+0.5mg/L 6-BA+0.1mg/L NAA medium, be 1500LX in intensity of illumination, light application time is 14h/ days, and temperature is cultivate 35 days in the culturing room of 25 DEG C, and axillalry bud average elongation is to 4cm;
(3) culture of rootage: axillalry bud is cut, being inoculated in 1/2MS+0.1mg/L NAA medium, is 3000LX in intensity of illumination, light application time is 14h/ days, temperature is cultivate 31 days in the culturing room of 25 DEG C, obtains the black fruit lyceum plantlet in vitro of height average out to 7cm, the long average out to 7cm of root;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 2 bud points, be inoculated in 1/2MS+0.1mg/L NAA medium, carry out subculture, carry out culture of rootage, be 3000LX in intensity of illumination simultaneously, and light application time is 14h/ days, temperature is cultivate 30 days in the culturing room of 25 DEG C, obtains the black fruit lyceum whole plant that average height is 7.2cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed bottle under natural daylight and tempers 6 days, hardening of at room temperature uncapping 4 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions, root is sterilized 4min, dry and plant in the matrix after with 0.1% disinfecting solution of potassium permanganate, be transplanted in soil when the new root system that seedling grows on average reaches 4cm and grows side root, its mesostroma is perlite and peat mixing, and both weight ratios are 1:2.0.
Cultivate after being transplanted in soil and see young leaves after 4 weeks and start growth, show that the black fruit lyceum of transplanting survives, investigated the survival rate of black fruit lyceum after 2 weeks, survival rate average out to 75%.
embodiment 4:a quickly tissue culture method for black fruit lyceum, it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 4min, 75% alcohol sterilizing 6s, sterile water wash 3 times, 0.1% mercuric chloride solution sterilizing 16min, sterile water wash 5 times;
(2) Fiber differentiation: the tender stem section after sterilizing and stem apex are inoculated in MS+0.5mg/L 6-BA+0.1mg/L NAA medium, be 1500LX in intensity of illumination, light application time is 15h/ days, and temperature is cultivate 40 days in the culturing room of 24 DEG C, and axillalry bud average elongation is to 4.5cm;
(3) culture of rootage: axillalry bud is cut, being inoculated in 1/2MS+0.1mg/L NAA medium, is 3000LX in intensity of illumination, light application time is 15h/ days, temperature is cultivate 35 days in the culturing room of 24 DEG C, obtains the black fruit lyceum plantlet in vitro of height average out to 8cm, the long average out to 8.5cm of root;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 3 bud points, be inoculated in 1/2MS+0.1mg/L NAA medium, carry out subculture, carry out culture of rootage, be 3000LX in intensity of illumination simultaneously, and light application time is 15h/ days, temperature is cultivate 30 days in the culturing room of 24 DEG C, obtains that on average arrive be highly the black fruit lyceum whole plant of 7.5cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed bottle under natural daylight and tempers 8 days, hardening of at room temperature uncapping 5 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions, root is sterilized 5min, dry and plant in the matrix after with 0.1% disinfecting solution of potassium permanganate, be transplanted in soil when the new root system that seedling grows on average reaches 5cm and grows side root, its mesostroma is perlite and peat mixing, and both weight ratios are 1:2.2.
Cultivate after being transplanted in soil and see young leaves after 5 weeks and start growth, show that the black fruit lyceum of transplanting survives, investigated the survival rate of black fruit lyceum after 2 weeks, survival rate average out to 75%.
Draw from embodiment 1 ~ 4: the present invention utilizes tissue culture technique to obtain black fruit lyceum plantlet in vitro, again by directly inducing tender stem segments and stem apex axillary bud growth, axillalry bud culture of rootage, cut into stem section or Shoot Tip Culture breeding obtains the whole plant of black fruit lyceum, the black fruit lyceum survival rate after acclimatization and transplants is to 75%.
Claims (7)
1. a quickly tissue culture method for black fruit lyceum, it is characterized in that, it comprises the following steps:
(1) explant sterilization: get the raw then tender stem segments on adult black fruit lyceum branch and stem apex running water 3 ~ 5min, 75% alcohol sterilizing 4 ~ 7s, sterile water wash 2 ~ 3 times, 0.1% mercuric chloride solution sterilizing 13 ~ 17min, sterile water wash 4 ~ 6 times;
(2) Fiber differentiation: be inoculated in inducing culture by the tender stem section after sterilizing and stem apex, cultivates 25 ~ 45 days axillalry buds and is stretched to 3 ~ 5cm in culturing room;
(3) culture of rootage: cut by axillalry bud, is inoculated in root media, within 25 ~ 40 days, obtains the black fruit lyceum plantlet in vitro that length is 6 ~ 10cm, and root length is 5 ~ 10cm;
(4) squamous subculture: with black fruit lyceum plantlet in vitro for parent, be cut to stem section or the stem apex of band 2 ~ 3 bud points, be inoculated in subculture medium and carry out subculture, carry out culture of rootage simultaneously, cultivate in culturing room and within 29 ~ 32 days, obtain being highly the black fruit lyceum whole plant of 6.8 ~ 8.0cm;
(5) acclimatization and transplants: the black fruit lyceum whole plant obtained is closed under natural daylight bottle and take exercise 5 ~ 8 days, hardening of at room temperature uncapping 3 ~ 5 days, take out the medium that single seedling cleans root, with 1000 times of carbendazim solutions to root sterilization 3 ~ 5min, dry and plant in matrix, be transplanted in soil when the new root system that seedling grows reaches 3 ~ 5cm and grows side root.
2. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, the medium described in step (2) is MS+0.5mg/L 6-BA+0.1mg/L NAA.
3. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, the cultivation indoor conditions described in step (2) is: intensity of illumination 1500LX, light application time 12 ~ 16h/ days, temperature 24 ~ 26 DEG C.
4. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, step (3) and the medium described in step (4) are 1/2MS+0.1mg/L NAA.
5. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, step (3) and the cultivation indoor conditions described in step (4): intensity of illumination 3000LX, light application time 12 ~ 16h/ days, temperature 24 ~ 26 DEG C.
6. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, the matrix described in step (5) is perlite and peat mixing, and both weight ratios are 1:1.5 ~ 2.5.
7. the quickly tissue culture method of a kind of black fruit lyceum according to claim 1, is characterized in that, matrix 0.1% disinfecting solution of potassium permanganate described in step (5).
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| CN105766646A (en) * | 2016-03-31 | 2016-07-20 | 内蒙古自治区农牧业科学院 | Tissue culture method for physochlaina |
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| CN105850733A (en) * | 2016-04-07 | 2016-08-17 | 甘肃省治沙研究所 | Lycium ruthenicum regenerated seedling cultivation method |
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| CN106538331A (en) * | 2016-11-02 | 2017-03-29 | 甘肃省治沙研究所 | A kind of hardening off method of black fruit lyceum plantlet in vitro |
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| CN107484665A (en) * | 2017-09-29 | 2017-12-19 | 黑龙江省林业科学研究所 | A kind of method using black fruit fructus lycii resting shoot seedling |
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| CN105684901B (en) * | 2016-01-22 | 2018-07-13 | 甘肃省治沙研究所 | A kind of rapid propagation method of Desert Regions medicinal plant black fruit fructus lycii |
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| CN105766646A (en) * | 2016-03-31 | 2016-07-20 | 内蒙古自治区农牧业科学院 | Tissue culture method for physochlaina |
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| CN105850733A (en) * | 2016-04-07 | 2016-08-17 | 甘肃省治沙研究所 | Lycium ruthenicum regenerated seedling cultivation method |
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| CN106538331A (en) * | 2016-11-02 | 2017-03-29 | 甘肃省治沙研究所 | A kind of hardening off method of black fruit lyceum plantlet in vitro |
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| CN106718912A (en) * | 2016-12-23 | 2017-05-31 | 庆阳敦博科技发展有限公司 | A kind of tissue culture and rapid propagation method of lycium ruthenicum |
| CN106718912B (en) * | 2016-12-23 | 2019-01-11 | 潘智渊 | A kind of tissue culture and rapid propagation method of lycium ruthenicum |
| CN107484665A (en) * | 2017-09-29 | 2017-12-19 | 黑龙江省林业科学研究所 | A kind of method using black fruit fructus lycii resting shoot seedling |
| CN107926711B (en) * | 2017-12-22 | 2020-12-29 | 沈阳农业大学 | In-vitro rapid propagation method for lycium ruthenicum |
| CN107926711A (en) * | 2017-12-22 | 2018-04-20 | 沈阳农业大学 | A kind of black fruit fructus lycii method for in-vitro rapid propagation |
| CN108012930A (en) * | 2017-12-28 | 2018-05-11 | 甘肃省治沙研究所 | A kind of black fruit fructus lycii tissue culture adventitious bud outside sprout-cultivating-bottle method |
| CN108012930B (en) * | 2017-12-28 | 2019-10-15 | 甘肃省治沙研究所 | A kind of black fruit fructus lycii tissue culture adventitious bud outside sprout-cultivating-bottle method |
| CN116606880A (en) * | 2023-05-25 | 2023-08-18 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
| CN116606880B (en) * | 2023-05-25 | 2023-12-08 | 中国科学院华南植物园 | Method for producing anthocyanin by using lycium ruthenicum callus |
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