CN104542302B - A kind of method for quickly breeding of CAULIS MARSDENIAE TENACISSIMAE - Google Patents
A kind of method for quickly breeding of CAULIS MARSDENIAE TENACISSIMAE Download PDFInfo
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- CN104542302B CN104542302B CN201510043975.7A CN201510043975A CN104542302B CN 104542302 B CN104542302 B CN 104542302B CN 201510043975 A CN201510043975 A CN 201510043975A CN 104542302 B CN104542302 B CN 104542302B
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- marsdeniae tenacissimae
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Abstract
The present invention discloses a kind of method for quickly breeding of CAULIS MARSDENIAE TENACISSIMAE, belongs to technical field of plant asexual propagation.The method comprise select CAULIS MARSDENIAE TENACISSIMAE then the newborn tip be explant, with running water, washing powder water soaking, alcohol solution dipping and be that 0.1% mercuric chloride solution soaks after 11 ~ 12min by mass fraction concentration, carry out Fiber differentiation, Multiplying culture, strong seedling culture and culture of rootage.The inventive method inductivity reaches more than 88%, and monthly can breed about 2.5 times, rooting rate reaches more than 95%, and the whole breeding cycle only needs 64 days ~ 72 days, achieves the vegetative propagation of CAULIS MARSDENIAE TENACISSIMAE, suitability for scale production.
Description
Technical field
The invention belongs to technical field of plant asexual propagation, be specifically related to a kind of method for quickly breeding of traditional Chinese medicine CAULIS MARSDENIAE TENACISSIMAE.
Background technology
CAULIS MARSDENIAE TENACISSIMAE (Marsdeniatenocissima (Roxb) WightetArm) is Asclepiadaceae Marsdenia liane, has another name called glaucescent fissistigma root.Begin to be loaded in " the southern regions of the Yunnan Province book on Chinese herbal medicine ", be distributed widely in the subtropical and tropical zones in Asia.Its complete stool all has clearing heat and detoxicating, anti-inflammatory analgetic and relieving cough and asthma effect.Black bone carcinocidin (C contained by CAULIS MARSDENIAE TENACISSIMAE
21-steroidal glucoside and black bone total alkali) have very strong destruction to the root-microtubule arrays of cancer cell division, the mitosis of cancer cell capable of blocking, thus effectively cut off cancer cell diffusion, transfer, cancer therefore atrophy until disappear completely.Can be used for the treatment of the various late malignant tumours such as advanced lung cancer, acute leukemia, the cancer of the esophagus, cancer of the stomach, liver cancer, laryngocarcinoma, nasopharyngeal carcinoma.Take CAULIS MARSDENIAE TENACISSIMAE as the Chinese medicine Xiaoaiping preparation against cancers that primary raw material is prepared from, curative effect is good.
At present, CAULIS MARSDENIAE TENACISSIMAE mainly relies on natural breed and the supply of limited wild resource.Because CAULIS MARSDENIAE TENACISSIMAE wild resource is seriously damaged, and medicinal material demand still improves year by year, the biological nature of CAULIS MARSDENIAE TENACISSIMAE self There are few many fruits of flower, and its plant natural propagation power is low.In addition, the felling of high strength is gathered, and cause the sharply atrophy of current CAULIS MARSDENIAE TENACISSIMAE wild resource amount, artificial breeding is imperative.The quick obtaining of high quality seedling is that CAULIS MARSDENIAE TENACISSIMAE cultivates the most key basic link.And the seedling that seminal propagation obtains, kind is easily degenerated, and growth cycle is long, and ripening rate is low; Although secondly cottage propagation survival rate is high, the later stage is seriously susceptible, is difficult to the demand meeting market.Therefore, explore a kind ofly to have fast, the CAULIS MARSDENIAE TENACISSIMAE high quality seedling group training asexual reproduction method of proper scale, to ensureing that the lasting supply of CAULIS MARSDENIAE TENACISSIMAE raw medicinal material is significant.
Summary of the invention
The object of the invention is to solve that CAULIS MARSDENIAE TENACISSIMAE seminal propagation rate is low, growth cycle is long, variability is comparatively large, the technical problem that cottage propagation seedling is easily susceptible, provides the method for quickly breeding of a kind of suitability for scale production, breeding is fast, survival rate is high CAULIS MARSDENIAE TENACISSIMAE.
The technical scheme of the method for quickly breeding of a kind of CAULIS MARSDENIAE TENACISSIMAE provided by the present invention is as follows:
(1) selection of explant and sterilization treatment
Choose the CAULIS MARSDENIAE TENACISSIMAE newborn tip then without damage by disease and insect, the stem-segment with single bud being cut into a band bud is explant, carries out primary sterilization in the following order: use running water 2.5h, and putting into mass fraction is that 5% washing powder water soaks 5min, rinses 25min under discharge condition; Be 75% alcohol solution dipping 1min by the clearance rattan section volume fraction through primary sterilization process, then be that 0.1% mercuric chloride solution soaks after 11 ~ 12min with mass fraction, again with sterile water washing 3 ~ 5 times, blot stem section surface moisture with aseptic paper for subsequent use;
(2) Fiber differentiation
The explant of step (1) sterilization treatment to be cut into length be 0.5cm ~ 1.0cm is inoculated in inducing culture with the stem section of a bud, condition of culture is: temperature 25 DEG C of scholars 2 DEG C, intensity of illumination 2000-2500Lx, photoperiod is 12h/12h, and relative air humidity remains on 70% ~ 80%; Described inducing culture is: MS+6-BA2.0mg/L+NAA0.5mg/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8;
(3) Multiplying culture
When the bud that Fiber differentiation is formed grows to 2 ~ 3cm, cut the stem section with a bud, be inoculated in proliferated culture medium, condition of culture is identical with step (2); Described proliferated culture medium is: MS+6-BA1.0mg/L+NAA0.3mg/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8;
(4) strong seedling culture
The propagation seedling that Multiplying culture is formed is cut into the stem section of a band bud, be inoculated in strong seedling culture base, condition of culture is identical with step (2); Described strong seedling culture base is: MS+6-BA1.0mg/L+NAA0.3mg/L++NH
4nO
30.66g/L+NaH
2pO
40.56g/L+K
2sO
41.78g/L+ banana 100g/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8;
(5) culture of rootage
When the seedling of step (4) grows to more than 1.5cm, shear and grow into 0.8cm ~ 1.2cm, be at least with the stem section of a bud to be inoculated in root media, condition of culture is identical with step (2); Described root media is: 1/2MS+6-BA0.5mg/L+IAA0.2mg/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8, acclimatization and transplants in time taking root and grow 4 ~ 5 young leaves.
The invention has the beneficial effects as follows:
1, the present invention is that CAULIS MARSDENIAE TENACISSIMAE artificial breeding provides new approach.
The method of CAULIS MARSDENIAE TENACISSIMAE tissue cultures of the present invention, make CAULIS MARSDENIAE TENACISSIMAE can keep the merit of parent, shorten breeding cycle, improve reproduction coefficient, improve seed output and quality, alleviate market medication pressure, solve the ripening rate that CAULIS MARSDENIAE TENACISSIMAE causes due to There are few many fruits of flower low, the seed growing cycle is long, the defect of easily variation.
2, the present invention is by lot of experiments and research, filter out the culture medium prescription of each physiological periods of CAULIS MARSDENIAE TENACISSIMAE growth desired nutritional and each parameter, reach inductivity high (more than 88%), proliferation times high (monthly can breed 2 ~ 2.5 times), rooting rate is high, can reach more than 95%.The coordinated of each group of training formula; the tissue culture propagation cycle is short; reproduction rate is high; the whole breeding cycle only needs 64 days ~ 72 days; achieve CAULIS MARSDENIAE TENACISSIMAE vegetative propagation fast and effectively; reach suitability for scale production, breeding fast, have the object that resistance, survival rate are high, to the popularization of the rattan choiceness that speeds passenger flow with ensure that anticancer bulk drug is supplied continually and steadily and had very important function and significance.
3, the inventive method can also improve the resistance of CAULIS MARSDENIAE TENACISSIMAE nursery stock to damage by disease and insect, solves because CAULIS MARSDENIAE TENACISSIMAE plant rattan is loose porous, includes a large amount of milk, shortcoming easily susceptible after cottage propagation, for CAULIS MARSDENIAE TENACISSIMAE artificial cultivation provides high quality seedling.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.Following embodiment is organized training routinely and is required to carry out sterilizing to various glassware, medium, and carries out Fast-propagation under the group training aseptic condition of routine.Each embodiment is conventional method without specified otherwise.
Embodiment 1
(1) selection of explant and process
Explant derives from the CAULIS MARSDENIAE TENACISSIMAE plant in field, Mengzi County, Yunnan Province, choose the newborn then tip without damage by disease and insect CAULIS MARSDENIAE TENACISSIMAE, the stem-segment with single bud being cut into a band bud is explant, carry out primary sterilization in the following order: use running water 2.5h, putting into mass fraction is that 5% washing powder water soaks 5min, rinses 25min under discharge condition; Be 75% alcohol solution dipping 1min by the clearance rattan section volume fraction through primary sterilization process, then be that 0.1% mercuric chloride solution soaks after 12min with mass fraction, again with sterile water washing 3 ~ 5 times, blot described clearance rattan section surface moisture with aseptic paper for subsequent use.
Because CAULIS MARSDENIAE TENACISSIMAE spray is close by faint yellow fine hair, the dust thing adhering many above, adopt the sterilization effect that conventional method can not reach, even cause whole group of failure of training, the present invention, through lot of experiments (see table 1), show that the pollution rate that must occur with sterilizing methods of the present invention is minimum, survival rate is the highest situation, again with sterile water washing 3 ~ 5 times, blot clearance rattan section moisture with aseptic paper, be placed on sterilized culture dish for subsequent use.
Table 10.1% mercury chloride is in the impact of different disinfecting time on explant survival rate and medium pollution rate
| Time/project | 8min | 9min | 10min | 11min | 12min | 13min |
| Pollution rate | 27％ | 22％ | 16％ | 9％ | 5％ | 2％ |
| Survival rate | 62％ | 53％ | 68％ | 84％ | 88％ | 47％ |
Test shows: being 0.1% mercury chloride with mass fraction soaks explant 8 ~ 13min in different disinfecting time, along with the increase of disinfecting time, pollution rate reduces gradually, and its reason is the increase along with disinfecting time, kills more thorough to germ entrained on explant.And survival rate increases gradually from sterilization 8min to 12min, reduce suddenly to 13min timesharing, its reason is disinfecting time when increasing to 13min, and thimerosal is the grievous injury histocyte of explant, so survival rate reduces suddenly.Therefore ideal disinfecting time is 11min ~ 12min.
(2) Fiber differentiation
By step (1), through the explant of sterilization treatment, to be cut into length be 0.5cm ~ 1.0cm is inoculated in inducing culture with the stem section of a bud, condition of culture is: temperature 25 DEG C of scholars 2 DEG C, intensity of illumination 2000-2500Lx, photoperiod is the 12h photophase, the 12h dark phase (namely the photoperiod is 12h/12h), and relative air humidity remains on 70% ~ 80%; Described inducing culture is: MS+6-BA2.0mg/L+NAA0.5mg/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8.
(3) Multiplying culture
When the bud of step (2) Fiber differentiation grows to 2 ~ 3cm, be cut into the stem section with a bud, be inoculated in proliferated culture medium, condition of culture is identical with step (2); Described proliferated culture medium is: MS+6-BA1.0mg/L+NAA0.3mg/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8.
(4) strong seedling culture
The propagation seedling that step (3) Multiplying culture is formed is cut into the stem section of a band bud, be inoculated in strong seedling culture base, condition of culture is identical with step (2); Described strong seedling culture base is: MS+6-BA1.0mg/L+NAA0.3mg/L+NH
4nO
30.66g/L+NaH
2pO
40.56g/L+K
2sO
41.78g/L+ banana 100g/L+ sucrose 25g/L+ agar 7.1g/L, pH value 5.8.
(5) culture of rootage
When the seedling of step (4) grows to more than 1.5cm, shear and grow into 0.8cm ~ 1.2cm, be at least with the stem section of a bud to be inoculated in root media, condition of culture is identical with step (2); Described root media is: 1/2MS+6-BA0.5mg/L+IAA0.2mg/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8.Acclimatization and transplants in time taking root and grow 4 ~ 5 young leaves.
Before transplanting, in greenhouse, remove bottle cap hardening 3 ~ 5 days, make seedling contact nature environment, strengthen the resistance of undesirable element to external world, can transplanting survival rate be improved.With tweezers, seedling is taken out in blake bottle during transplanting, and in water, wash away the medium be attached on root, plant the mixed-matrix interior (perlite: the volume ratio of leaf mould is 1:5) into being added with a small amount of perlite and leaf mould again, water permeable after transplanting, note heat and moisture preserving, humidity is about 60%, temperature is at 20 ~ 25 DEG C, transplant the sunshade net sunshade that early stage (within transplanting latter 25 days) with shading rate is 75%, later stage (after transplanting growth 40 days) suitably increases illumination, shading rate is 35%, and survival rate is to more than 95%.
Embodiment 2
Embodiment 2 is except following steps operation difference, and all the other each step operations are identical with embodiment 1, repeat no more.
(1) selection of explant and sterilization treatment
Explant, from the CAULIS MARSDENIAE TENACISSIMAE plant in field, Mengzi County, Yunnan Province, is transplanted to the young sprout grown then after Yunnan Prov Agriculture University greenhouse is tamed.It is consistent with embodiment 1 explant processing method that other disinfects step, being 11min, just can reaching minimum pollution rate unlike being 0.1% mercury chloride disinfecting time with mass fraction.
Embodiment 3
Embodiment 3 is except following steps operation difference, and all the other each step operations are identical with embodiment 1, repeat no more.
(1) selection of explant and sterilization treatment
Explant derives from the CAULIS MARSDENIAE TENACISSIMAE plant in field, Maguan County, Yunnan Province, choose the newborn then tip without damage by disease and insect CAULIS MARSDENIAE TENACISSIMAE, the stem-segment with single bud being cut into a band bud is explant, and when be 0.1% mercury chloride disinfecting time being 12min with mass fraction, pollution rate reaches minimum.Its disinfecting time comes from the consistent of Mengzi County, Yunnan Province with embodiment 1 explant, this be all come from field, the dust with a lot of on plant is relevant with germ.
The result of the test of embodiment 1 to embodiment 3 refers to table 2.
The test effect of table 2 embodiment 1-embodiment 3
The present invention is by lot of experiments and research, filter out the culture medium prescription of each physiological periods of CAULIS MARSDENIAE TENACISSIMAE growth desired nutritional and each parameter, make its CAULIS MARSDENIAE TENACISSIMAE explant coming from different regions can reach excellent sterilization effect and the effect of high-servival rate, healthy and strong seedling can be bred, inductivity high (more than 88%), proliferation times is high (monthly can breed 2 ~ 2.5 times, breeding cycle is short, 64 days ~ 72 days whole breeding cycle, rooting rate is high, can more than 95% be reached, create special technique effect.
Claims (1)
1. a method for quickly breeding for CAULIS MARSDENIAE TENACISSIMAE, is characterized in that:
(1) selection of explant and sterilization treatment
Choose the CAULIS MARSDENIAE TENACISSIMAE newborn tip then without damage by disease and insect, the stem-segment with single bud being cut into a band bud is explant, carries out primary sterilization in the following order: use running water 2.5h, and putting into mass fraction is that 5% washing powder water soaks 5min, rinses 25min under discharge condition; Be 75% alcohol solution dipping 1min by the clearance rattan section volume fraction through primary sterilization process, then be that 0.1% mercuric chloride solution soaks after 11 ~ 12min with mass fraction, again with sterile water washing 3 ~ 5 times, blot stem section surface moisture with aseptic paper for subsequent use;
(2) Fiber differentiation
By step (1), through the explant of sterilization treatment, to be cut into length be 0.5cm ~ 1.0cm is inoculated in inducing culture with the stem section of a bud, condition of culture is: temperature 25 DEG C of scholars 2 DEG C, intensity of illumination 2000-2500Lx, photoperiod is 12h/12h, and relative air humidity remains on 70% ~ 80%; Described inducing culture is: MS+6-BA2.0mg/L+NAA0.5mg/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8;
(3) Multiplying culture
When the bud that Fiber differentiation is formed grows to 2 ~ 3cm, cut the stem section with a bud, be inoculated in proliferated culture medium, condition of culture is identical with step (2); Described proliferated culture medium is: MS+6-BA1.0mg/L+NAA0.3mg/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8;
(4) strong seedling culture
The propagation seedling that Multiplying culture is formed is cut into the stem section of a band bud, be inoculated in strong seedling culture base, condition of culture is identical with step (2); Described strong seedling culture base is: MS+6-BA1.0mg/L+NAA0.3mg/L+NH
4nO
30.66g/L+NaH
2pO
40.56g/L+K
2sO
41.78g/L+ banana 100g/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8;
(5) culture of rootage
When the seedling of step (4) grows to more than 1.5cm, shear and grow into 0.8cm ~ 1.2cm, be at least with the stem section of a bud to be inoculated in root media, condition of culture is identical with step (2); Described root media is: 1/2MS+6-BA0.5mg/L+IAA0.2mg/L+ sucrose 25g/L+ agar 7.1g/L, pH5.8, acclimatization and transplants in time taking root and grow 4 ~ 5 young leaves.
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| CN107333550B (en) * | 2017-08-03 | 2020-09-04 | 云南信通植物药业有限公司 | Marsdenia tenacissima seed seedling growing method |
| CN107667795B (en) * | 2017-11-15 | 2020-04-14 | 云南农业大学 | Method for shortening flowering age of marsdenia tenacissima and improving flowering rate |
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| WO2009095059A1 (en) * | 2008-01-31 | 2009-08-06 | Westfälische Wilhelms Universität Münster | Rubber biosynthesis promoters from taraxacum koksaghyz and their use |
| CN103548553A (en) * | 2013-11-25 | 2014-02-05 | 云南圣和植物药业有限公司 | Cutting propagation method of Marsdenia tenocissima (Roxb) Wight et Arm |
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