CN102246697B - Method for growing cell embryos of Dendrobium huoshanense - Google Patents
Method for growing cell embryos of Dendrobium huoshanense Download PDFInfo
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- CN102246697B CN102246697B CN201110179435A CN201110179435A CN102246697B CN 102246697 B CN102246697 B CN 102246697B CN 201110179435 A CN201110179435 A CN 201110179435A CN 201110179435 A CN201110179435 A CN 201110179435A CN 102246697 B CN102246697 B CN 102246697B
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Abstract
The invention relates to a method for growing cell embryos of Dendrobium huoshanense, which comprises the following operating steps: (1) inducing sterile in-vitro cuttings; (2) inducing embryonic calli; (3) inducing somatic embryos; and (4) performing synchronous propagation of somatic embryos to obtain a large amount of cell embryos of Dendrobium huoshanense. In the method, morphological lower ends of the jointed stem segments of the sterile in-vitro cuttings are used as explants, the oxidation-reduction potential of the induction culture medium is controlled, exogenous hormone is not required to be added to induce the embryonic calli of Dendrobium huoshanensem, and the exogenous hormone is prevented from being enriched into complete plants through somatic cells and endangering health of human body. By controlling ambient humidity, embryonic calla are grown into somatic embryos, the consistency of spherical cell embryos is over 90 percent, and the survival rate of regenerated plants is over 85 percent. In the invention, the process is simple and not limited by time and seasons, and industrial automatic production of g cell embryos of Dendrobium huoshanense and in-vitro cuttings of Dendrobium huoshanense can be carried out in door for satisfying needs for production and planting.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to the method that a kind of Dendrobidium huoshanness somatic embryo takes place.
Background technology
Dendrobidium huoshanness (
Dendrobium huoshanenseC. Z. Tang et S. J. Cheng); Have another name called suddenly the stem of noble dendrobium, dry measure used in former times, rice dry measure used in former times suddenly, be subordinate to the orchid family Dendrobium, have beneficial smart reinforcing yin essence, promote the production of body fluid to quench thirst, qi-restoratives is won, remove the effect of the fire of deficiency type in the stomach; Praised highly top grade in the stem of noble dendrobium by successive dynasties books on Chinese herbal medicine, receive the favor of people in modern deeply.But the growth that Huoshan stone is separated is extremely strict to environmental requirement, in addition successive dynasties medicinal herb grower's collection and modern age ecotope change, it is endangered that wild Huoshan stone is separated resource, has only minute quantity to be transplanted in Huoshan the county magistrate towards the medicinal material field now.Though Dendrobidium huoshanness is bloomed many, the result is few, and the seed embryo of Dendrobidium huoshanness does not break up, no endosperm, causes germination rate extremely low.Can't satisfy the demand of large-scale artificial cultivation.Somatic embryo generation technique in the plant tissue culture technique can provide the somatic embryo that breeding quantity is big, reproduction speed fast, the descendant inheritting proterties is stable.Somatic embryo has metabolism and the morphological development potential same with plant, can be used to the active substance that replaces crude drug production relevant.Therefore, the somatic embryo generation technique has received extensive concern.The generation technique of Dendrobidium huoshanness somatic embryo is that the sustainable utilization of Dendrobidium huoshanness medical value and effective protection of germ plasm resource provide a kind of important means.
Stem of noble dendrobium somatic embryo be indirect mode; Be that somatic embryo forms from healing tissue development; Current; Not enough below this occurring mode exists: the derived need of (1) embryo callus is added exogenous hormone, and exogenous hormone will be through the somatic embryo enrichment in whole plant, clones exogenous hormone residual in the plant to the health serious harm that lies dormant; (2) the somatic embryo development stage is inconsistent, synchronism is very little (being lower than 50%), difference in size is big, the survival rate very low (less than 10%) of regeneration plant.Therefore, be difficult to satisfy the rule automated production of Dendrobidium huoshanness somatic embryo and the needs of large tracts of land artificial cultivation seedling.This patent through control medium redox potential to induce the generation embryo callus; Embryo callus occurs as the Dendrobidium huoshanness somatic cell in the environment of the certain humidity of control; Obtain a large amount of synchronized Dendrobidium huoshanness somatic embryos through crossing sieve method, starvation method or later stage liquid culture method, the technology of this respect is not appeared in the newspapers at present as yet.
Summary of the invention
Dendrobidium huoshanness is a kind of tradition and famous and precious health care medicinal material, and its wild resource is endangered.In order to solve the derived need exogenous hormone of the callus that exists in the existing Dendrobidium huoshanness somatic embryo generation technique; Problems such as the survival rate that the somatic embryo development stage is inconsistent, synchronism is very little, difference in size causes regeneration plant more greatly is very low, the method that a kind of Dendrobidium huoshanness somatic embryo provided by the invention takes place.
The technical solution that realizes above-mentioned purpose is following:
The method that a kind of Dendrobidium huoshanness somatic embryo takes place comprises following operating procedure:
(1) the sterile test tube seedling induces
Get the Dendrobidium huoshanness ripening fruits; Using earlier mass fraction is that 1% aqueous sodium hypochlorite solution carries out surface sterilization 60 min, washes 3～5 times with aseptic DDW again, cuts fruit then; Take out seed; And seed is sprinkling upon on the solid MS medium, under 22 ± 2 ℃ of temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day, cultivated 6～7 months, obtain the long Dendrobidium huoshanness sterile test tube seedling of 4-6 cm;
(2) embryo callus induces
Get the morphology lower end of stem section of the band joint of sterile test tube seedling, insert redox potential and be-0.03～-0.02 volt solid and lead thoroughly in the medium, inducing culture is 20～25 days in 22 ± 2 ℃ of temperature, dark, must embryo callus;
(3) somatic embryo induces
Embryo callus is transferred in the solid MS medium; Be 70～85% in relative moisture, inducing culture after 3 weeks under 22 ± 2 ℃ of the temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; Be transferred in the solid MS medium, be 70～85% in relative moisture, 3 weeks of inducing culture under 22 ± 2 ℃ of the temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; The operating condition of inducing by this step is transferred 10～15 times continuously, and the switching medium is a solid MS medium, gets somatic embryo;
(4) synchronization of somatic embryo propagation
Somatic embryo is transferred in liquid MS medium or the solid MS medium; Under 22 ± 2 ℃ of temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; Adopt sieve method or starvation method synchronization enrichment culture, got the Dendrobidium huoshanness somatic embryo.
It is that the adding mass fraction is the sodium phosphate of 50～100 mg/L on the basis of solid MS medium that said solid is led medium thoroughly.
The said sieve method of crossing is operated as follows: through 10 mesh sieves, the somatic embryo continuation that collection sees through is cultivated and is passed through 20 mesh sieves after 5 days again, collects the somatic embryo of holding back with 25 days somatic embryo of liquid culture elder generation; Said starvation method is operated as follows: somatic embryo is cultivated 25 days in fresh MS medium after; Filter with 10 aseptic mesh sieves; The somatic embryo that sees through filters with 20 mesh sieves again; During the MS that the somatic embryo of holding back is transferred to no molysite cultivates hungry cultivate 2 days after, change over to and continue in the fresh MS medium to cultivate 5 days.
Compared with prior art, method of the present invention has following advantage:
(1) the morphology lower end of the stem section of the band joint of the present invention through getting the sterile test tube seedling is for growing body outward; And the redox potential of control inducing culture; Do not induce acquisition Dendrobidium huoshanness embryo callus and need not add exogenous hormone, avoided exogenous hormone in whole plant, to be detrimental to health through the somatic embryo enrichment;
(2) the present invention makes embryo callus occur as somatic embryo through the humidity that controls environment, and reaches spherical blast uniformity more than 90%, and the regeneration plant survival rate is more than 85%;
(3) operating procedure of the present invention is simple; Do not receive time, season limit; Can produce Dendrobidium huoshanness somatic embryo and test-tube plantlet for the needs of producing cultivation at indoor factory automation, realize the sustainable utilization of Dendrobidium huoshanness medical value and effective protection of germ plasm resource.
Description of drawings
The Dendrobidium huoshanness sterile test tube seedling figure that Fig. 1 obtains for step 1.
The embryo callus figure that Fig. 2 obtains for step 2.
The somatic embryo figure that Fig. 3 obtains for step 3.
The Dendrobidium huoshanness somatic embryo figure that Fig. 4 cultivates for the present invention.
Embodiment
Through embodiment, the present invention is done explanation further below.
Embodiment 1:
The method that a kind of Dendrobidium huoshanness somatic embryo takes place comprises following operating procedure:
Inducing of step 1, sterile test tube seedling
Get the wild Dendrobidium huoshanness ripening fruits that originates in peaceful field township, Huoshan County, Anhui Province, using mass fraction earlier is that 1% aqueous sodium hypochlorite solution carries out surface sterilization 60 min, washes 3 times with aseptic DDW again; Cut fruit then; Take out seed, and seed is sprinkling upon on the MS solid culture medium, under 22 ± 2 ℃ of temperature, intensity of illumination 2000 luxs, 12 hours condition of illumination every day, cultivated 7 months; Obtain the long Dendrobidium huoshanness sterile test tube seedling of 4 cm, see Fig. 1.
Inducing of step 2, embryo callus
Get the morphology lower end of stem section of the band joint of sterile test tube seedling, insert the control redox potential and lead thoroughly in the medium for-0.02 volt solid, inducing culture is 20 days in 22 ± 2 ℃ of temperature, dark, gets embryo callus, sees Fig. 2.It is that the adding mass fraction is the sodium phosphate of 100 mg/L on the basis of solid MS medium that solid is led medium thoroughly.
Inducing of step 3, somatic embryo
In the fresh solid MS medium that embryo callus is transferred to; Be 70% in relative moisture, under 22 ± 2 ℃ of the temperature, intensity of illumination 3000 luxs, 12 hours condition of illumination every day behind the inducing culture; Be transferred in the fresh solid MS medium, be 70% in relative moisture, 3 weeks of inducing culture under 22 ± 2 ℃ of the temperature, intensity of illumination 3000 luxs, 12 hours condition of illumination every day; The operating condition of inducing by this step is transferred 10 times continuously, and the switching medium is a solid MS medium, gets somatic embryo, sees Fig. 3.
The synchronization propagation of step 4, somatic embryo
Somatic embryo is transferred in the fresh solid MS medium; Under 22 ± 2 ℃ of temperature, intensity of illumination 3000 luxs, 12 hours condition of illumination every day; Adopt starvation method synchronization enrichment culture, concrete operations are following: somatic embryo is cultivated 25 days in fresh solid MS medium after, filter with 10 aseptic mesh sieves; The somatic embryo that sees through filters with 20 mesh sieves again, and hungry cultivation was after 2 days during the solid MS that the somatic embryo of holding back is transferred to no molysite cultivated.Change over to and continue in the fresh solid MS medium to cultivate 5 days, get the Dendrobidium huoshanness somatic embryo, see Fig. 4.
Embodiment 2:
The method that a kind of Dendrobidium huoshanness somatic embryo takes place comprises following operating procedure:
Inducing of step 1, sterile test tube seedling: get the wild Dendrobidium huoshanness ripening fruits that originates in peaceful field township, Huoshan County, Anhui Province; Using mass fraction earlier is that 1% aqueous sodium hypochlorite solution carries out surface sterilization 60 min, with aseptic DDW flushing 5 times, cuts fruit then again; Take out seed; And seed is sprinkling upon on the MS solid culture medium, under 22 ± 2 ℃ of temperature, intensity of illumination 2500 luxs, 12 hours condition of illumination every day, cultivated 7 months, obtain the long Dendrobidium huoshanness sterile test tube seedling of 5 cm.
Inducing of step 2, embryo callus: the morphology lower end of stem section of getting the band joint of sterile test tube seedling; Inserting the control redox potential leads in the medium for-0.03 volt solid thoroughly; Inducing culture is 25 days in 22 ± 2 ℃ of temperature, dark, gets embryo callus; It is that the adding mass fraction is the sodium phosphate of 50 mg/L on the basis of solid MS medium that solid is led medium thoroughly.
Inducing of step 3, somatic embryo: in the fresh solid MS medium that embryo callus is transferred to; Be 80% in relative moisture, under 22 ± 2 ℃ of the temperature, intensity of illumination 2000 luxs, 12 hours condition of illumination every day behind the inducing culture; Be transferred in the fresh solid MS medium, be 80% in relative moisture, 3 weeks of inducing culture under 22 ± 2 ℃ of the temperature, intensity of illumination 2000 luxs, 12 hours condition of illumination every day; The operating condition of inducing by this step is transferred 15 times continuously, and the switching medium is a solid MS medium, gets somatic embryo.
The synchronization propagation of step 4, somatic embryo: somatic embryo is transferred in the fresh liquid MS medium; Under 22 ± 2 ℃ of temperature, intensity of illumination 2000 luxs, 12 hours condition of illumination every day; Adopt starvation method synchronization enrichment culture, concrete operations are following: somatic embryo is cultivated 25 days in fresh liquid MS medium after, filter with 10 aseptic mesh sieves; The somatic embryo that sees through filters with 20 mesh sieves again; During the liquid MS that the somatic embryo of holding back is transferred to no molysite cultivates hungry cultivate 2 days after, change over to and continue in the fresh liquid MS medium to cultivate 5 days, the Dendrobidium huoshanness somatic embryo.
Embodiment 3:
The method that a kind of Dendrobidium huoshanness somatic embryo takes place comprises following operating procedure:
Inducing of step 1, sterile test tube seedling: get the wild Dendrobidium huoshanness ripening fruits that originates in peaceful field township, Huoshan County, Anhui Province; Using mass fraction earlier is that 1% aqueous sodium hypochlorite solution carries out surface sterilization 60 min, with aseptic DDW flushing 4 times, cuts fruit then again; Take out seed; And seed is sprinkling upon on the MS solid culture medium, under 22 ± 2 ℃ of temperature, intensity of illumination 3000 luxs, 12 hours condition of illumination every day, cultivated 6 months, obtain the long Dendrobidium huoshanness sterile test tube seedling of 6 cm.
Inducing of step 2, embryo callus: the morphology lower end of stem section of getting the band joint of sterile test tube seedling; Inserting the control redox potential leads in the medium for-0.02 volt solid thoroughly; Inducing culture is 22 days in 22 ± 2 ℃ of temperature, dark, gets embryo callus.It is that the adding mass fraction is the sodium phosphate of 100 mg/L on the basis of solid MS medium that solid is led medium thoroughly.
Inducing of step 3, somatic embryo: in the fresh solid MS medium that embryo callus is transferred to; Be 85% in relative moisture, under 22 ± 2 ℃ of the temperature, intensity of illumination 2500 luxs, 12 hours condition of illumination every day behind the inducing culture; Be transferred in the fresh solid MS medium, be 85% in relative moisture, 3 weeks of inducing culture under 22 ± 2 ℃ of the temperature, intensity of illumination 2500 luxs, 12 hours condition of illumination every day; The operating condition of inducing by this step is transferred 13 times continuously, and the switching medium is a solid MS medium, gets somatic embryo.
The synchronization propagation of step 4, somatic embryo: somatic embryo is transferred in the fresh liquid MS medium; Under 22 ± 2 ℃ of temperature, intensity of illumination 2500 luxs, 12 hours condition of illumination every day; Adopted sieve method synchronization enrichment culture; Concrete operations are following: somatic embryo was cultivated in fresh liquid MS medium after 25 days, and somatic embryo through 10 aseptic mesh sieves, is collected the somatic embryo that sees through and after fresh liquid MS medium continues to cultivate 5 days, passed through 20 aseptic mesh sieves again; The somatic embryo that collection is held back promptly gets the Dendrobidium huoshanness somatic embryo.
Claims (1)
1. the method that takes place of a Dendrobidium huoshanness somatic embryo is characterized in that comprising following operating procedure:
(1) the sterile test tube seedling induces
Get the Dendrobidium huoshanness ripening fruits; Using earlier mass fraction is that 1% aqueous sodium hypochlorite solution carries out surface sterilization 60min, washes 3～5 times with aseptic DDW again, cuts fruit then; Take out seed; And seed is sprinkling upon on the solid MS medium, under 22 ± 2 ℃ of temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day, cultivated 6～7 months, obtain the long Dendrobidium huoshanness sterile test tube seedling of 4-6cm;
(2) embryo callus induces
Get the morphology lower end of stem section of the band joint of sterile test tube seedling, insert redox potential and be in-0.03～-0.02 volt the solid inducing culture, inducing culture is 20～25 days in 22 ± 2 ℃ of temperature, dark, must embryo callus; Said solid inducing culture is that the adding mass fraction is the sodium phosphate of 50～100mg/L on the basis of solid MS medium;
(3) somatic embryo induces
Embryo callus is transferred in the solid MS medium; Be 70～85% in relative moisture, inducing culture after 3 weeks under 22 ± 2 ℃ of the temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; Be transferred in the solid MS medium, be 70～85% in relative moisture, 3 weeks of inducing culture under 22 ± 2 ℃ of the temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; The operating condition of inducing by this step is transferred 10～15 times continuously, and the switching medium is a solid MS medium, gets somatic embryo;
(4) synchronization of somatic embryo propagation
Somatic embryo is transferred in liquid MS medium or the solid MS medium; Under 22 ± 2 ℃ of temperature, intensity of illumination 2000～3000 luxs, 12 hours condition of illumination every day; Adopt sieve method or starvation method synchronization enrichment culture, got the Dendrobidium huoshanness somatic embryo;
The said sieve method of crossing is operated as follows: through 10 mesh sieves, the somatic embryo continuation that collection sees through is cultivated and is passed through 20 mesh sieves after 5 days again, collects the somatic embryo of holding back with 25 days somatic embryo of liquid culture elder generation;
Said starvation method is operated as follows: somatic embryo is cultivated 25 days in fresh MS medium after; Filter with 10 aseptic mesh sieves; The somatic embryo that sees through filters with 20 mesh sieves again; During the MS that the somatic embryo of holding back is transferred to no molysite cultivates hungry cultivate 2 days after, change over to and continue in the fresh MS medium to cultivate 5 days.
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| CN103109742B (en) * | 2013-01-31 | 2014-12-24 | 安徽霍山县药王谷林农科技有限公司 | Tissue culture method of Dendrobium huoshanense |
| CN103468633B (en) * | 2013-09-24 | 2015-02-25 | 薛刚 | Inducing method for improving cell synchronization of tendril-leaved fritillary bulbs |
| CN103493737B (en) * | 2013-10-14 | 2015-06-17 | 合肥工业大学 | Method for promoting germination of shoots of protocorms of dendrobium huoshanense |
| CN103688857B (en) * | 2013-12-13 | 2015-05-20 | 奉化市九味圣草堂铁皮石斛专业合作社 | Method for quickly breeding dendrobium officinale |
| CN104082134A (en) * | 2013-12-30 | 2014-10-08 | 石明莉 | Dendrobium embryoid suspension culture process |
| CN107094622B (en) * | 2017-04-18 | 2019-04-26 | 天津农学院 | A method of induction raspberry callus forms globular embryo |
| CN112021180B (en) * | 2020-09-17 | 2022-03-08 | 北京农学院 | Synchronization method for chestnut somatic embryo development and tissue culture seedling rooting method |
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