CN1216148C

CN1216148C - Culturing method for 'cell-tissue'

Info

Publication number: CN1216148C
Application number: CN 03135614
Authority: CN
Inventors: 陈锐平
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-08-16
Filing date: 2003-08-16
Publication date: 2005-08-24
Anticipated expiration: 2023-08-16
Also published as: CN1490402A

Abstract

The present invention belongs to the field of biological technique, which has the purpose of providing a cell-tissue culture method. The present invention is characterized in that plant tissues are added with a few liquid culture mediums, ground in a mortar, inoculated and put into a rotary rocking bed, the culture temperature is 25(+/-)1 DEGC, the plant tissues are carried out cell suspension culture under the condition of dark, the proliferative plant cell suspension is poured in a buchner filter with filter paper to pump off the culture solution, and washed several times by distilled water, the cultured cells are carried out cell growth measurement, the cells whose growth is static, are inoculated with solid culture mediums to carry out propagation culture of clumpy buds, and the well-grown buds are inoculated to carry out radication culture, and finally emerged. The method solves the problem of culturing certain plants by a traditional tissue culture method, particularly the problems of culture difficulty and low growth rate of some medicinal plants and ligneous plants caused by containing special metabolite. Simultaneously clone descendants keep high conformance of good characteristics with maternal plants, the method has the advantages of substantially raising the growth rate, reducing the culture cost, and achieving 'cell-tissue' culture industrialized production.

Description

A kind of preparation method of seedlings of Dendrobium officinale

Technical field

The present invention relates to biological technical field, specifically a kind of preparation method of seedlings of Dendrobium officinale.

Background technology

The a large amount of culture techniques of vegetable cell are to grow up on the basis that plant tissue culture is bred fast.Concrete way is exactly that vegetable cell is transferred in the large fermentation tank of microbial fermentation in test tube or triangular flask, giving suitable condition cultivates, make vegetable cell as microorganism breeding in a large number in fermentor tank, in the vegetable cell of a large amount of propagation, directly extract useful material then.

At present, the scientist of states such as the countries in the world such as the U.S., Japan, Germany, Russia, Britain, business circles all stepping up research, are being paid close attention to a large amount of trainings of vegetable cell.

China has also obtained good achievement in a large amount of culture technique researchs of vegetable cell, obtained many scientific payoffss aspect the cultivation in a large number at medicinal plant cells such as genseng, the coptis, Radix Panacis Quinquefolii, pseudo-ginseng.For example, with the culture that a large amount of culture techniques of cell are extracted from ginseng-cell, main content of effective reaches about 70%, and is more much higher than the effective constituent of tame genseng.

Plant tissue culture is exactly the part tissue of separating plant body, as root, stem section, leaf, flower, rataria etc., in sterile test tube, cooperates conditions such as certain nutrition, hormone, temperature, illumination, makes it produce whole plant.Because its condition can strict be controlled, growth is one-period about 1 month rapidly, therefore in the production of plant significant application value is arranged.

Biotechnology is China, also is the technical field that the world gives priority to 21 century.Though a large amount of culture techniques of vegetable cell have obtained many achievements since 1970, especially cell proliferation speed is fast, and pollution rate is low, and is noticeable.But no matter on the cultivation cost of cell, or in the improvement of technology, all exist a lot of problems.Though and the group training is called as quick breeding, its reproduction speed is too slow with regard to what show from our theoretical value and expected value, and the pollution rate height, so array training chamber is that quantity or cost all have very big gap with our social desirability mostly.Be to solve cell cultures cost height, whether the slow-footed problem of tissue culture can find the advantage of a kind of existing cell cultures and tissue culture, can overcome the method for cell cultures and tissue culture shortcoming again, invents problem to be solved with regard to becoming this.

Summary of the invention

The preparation method who the purpose of this invention is to provide a kind of a kind of seedlings of Dendrobium officinale that can increase reproduction speed, reduce cost.

Technical scheme of the present invention is: the preparation method of seedlings of Dendrobium officinale carries out according to the following steps: a). and the plant Herba Dendrobii is organized in to add in the mortar body inoculates after a little liquid nutrient medium grinds, enter rotary shaking table, 25 ± 1 ℃ of culture temperature are carried out the Herba Dendrobii cell suspension culture under the dark condition; B). pour the plant Herba Dendrobii cell suspending liquid of propagation the B of filter paper into, take out nutrient solution, use the distilled water wash several times again, the Herba Dendrobii cell of cultivating is carried out the cell growth measurement; C). choose cell that the Herba Dendrobii cell grows into stationary phase and insert the Herba Dendrobii solid medium again and carry out clump bud multiplication culture; D). select well-grown bud grafting to go into solid medium and carry out root culture, produce Herba Dendrobii section seedling at last: wherein said plant Herba Dendrobii cell suspension liquid substratum is formed by MS+BA 0.3 ~ 0.6mg/L, NAA 1 ~ 5mg/L, LH 0.5 ~ 1.5mg/L, sucrose 19 ~ 22g/L mixed preparing; Described Herba Dendrobii solid medium adds the 6B A of 0.2-2mg/L for the MS basic medium and the NAA of 0.1-0.9mg/L makes.

The preparation method of seedlings of Dendrobium officinale of the present invention (cultural method), can the past tissue culture method propagation plant speed improve more than 20 times, cost descends 50%.According to statistics, 80% of Chinese medicine derives from plant, and most medicinal plant is that harvesting is wild, and this just means one day can be wild medicinal plant daylighting.Now existing hundreds of medicinal plant is in short supply or be in Critical Condition, promptly uses the tame method of seed to produce medicinal plant, and breeding quantity also is limited.Therefore, use this culture technique, the plant seedling production rate that can make cultivation is than fast 20 times of traditional group training speed.Method of the present invention also can be used for the cultivation of Herba Erigerontis.

Combine cell cultures and tissue culture are organic, give full play to the advantage of cell cultures and tissue culture, be combined into " cell-tissue " cultivation and just show its great vitality, maximum innovative point just of the present invention, and can develop into an emerging technical field.

Embodiment

Embodiment 1: the cultivation of Herba Erigerontis (Erigeron breviscapus)

1, the selection of Herba Erigerontis explant and cultivation:

By the culture that the stock plant of high yield is set up, its offspring's output is higher than the culture of being set up by the plant of low yield.

A, selection: by wild plant 20 strains of picked at random Herba Erigerontis, measure scutellarin content, shown in following table (table 1):

The content of scutellarin in the table 1 20 strain Herba Erigerontiss

Tabl The scutellarin content in 20 individual Erigreron breviscapus (mg/g)

D ₁	D ₂	D ₃	D ₄	D ₅	D ₆	D ₇	D ₈	D ₉	D ₁₀
D ₁	D ₂	D ₃	D ₄	D ₅	D ₆	D ₇	D ₈	D ₉	D ₁₀	0.315	0.438	0.512	0.491	0.387	0.514	0.547	0.425	0.641	0.448
D ₁₁	D ₁₂	D ₁₃	D ₁₄	D ₁₅	D ₁₆	D ₁₇	D ₁₈	D ₁₉	D ₂₀	0.315	0.438	0.512	0.491	0.387	0.514	0.547	0.425	0.641	0.448
D ₁₁	D ₁₂	D ₁₃	D ₁₄	D ₁₅	D ₁₆	D ₁₇	D ₁₈	D ₁₉	D ₂₀	0.487	0.574	0.653	0.396	0.469	0.391	0.479	0.592	0.473	0.427
On average	4.79			Maximum	6.53			Minimum	3.15	0.487	0.574	0.653	0.396	0.469	0.391	0.479	0.592	0.473	0.427

Select the highest strain (D of scutellarin content ₆=6.53mg/g) and minimum strain (D ₁₅=3.15mg/g) carry out callus induction, set up clone, be labeled as D respectively _MaxAnd D _Min

B, cultivation evoked callus and scutellarin assay

Get the Herba Erigerontis blade, after cleaning up with detergent water, wash 10min under flowing water, forward to then on the clean bench, suck dry moisture with 70% alcohol sterilization 30s, changes 0.1% HgCl over to earlier again ₂The 6min that sterilizes in the solution, sterile distilled water flushing 5 times, petiole is cut into the long segment of 0.4cm, and blade is cut into 0.5 * 0.5cm ²Fritter, inoculate sub-callus inducing medium MS+6-BA0.2mg/L+NAA2mg/L+ sucrose 30g/L, solidify with 0.7% agar, pH is adjusted to 5.8,25 ± 1 ℃ of culture temperature before the high-temperature sterilization.Promptly as seen obviously expand about explant inoculation one week of back, form the callus about the about 1cm of diameter about 20d.Carry out the shoot proliferation cultivation on the same substratum with forwarding to after the callus cutting.F ₁For the time, every bottle graft kind 1.0g (DW) callus (dry weight 0.12g, the rate of giving money as a gift is 88%), every kind of callus inoculated 10 bottles, altogether 1.2g (DW).Later on the callus of propagation is all inoculated the parent material of breeding as next time, be 18d proliferating cycle.Subculture is respectively got 50g (fresh weight) after 3 generations, measures the content of scutellarin.

Scutellarin content in the different clone callus of table 2

Callus harvest yield (FW) (g/ bottle) callus harvest yield (DW) (g/ bottle) callus growth rate (dry weight) (%)	D _max	D _min
	D _max	D _min	F1 F2 F3 29.54 63.34 207.06 3.230 7.724 20.364 169.28 138.82 163.65	F1 F2 F3 24.38 61.12 190.66 2.942 7.113 20.816 145.36 141.77 192.64
	Scutellarin content (mg/g)	0.040			0.015

The result shows that the callus growth that derives from two strain Herba Erigerontiss there is no significant difference, and the content of scutellarin is starkly lower than former plant in the callus, and derives from the callus of different plants, the content difference of its scutellarin.The content of scutellarin in the callus that derives from the high-content plant (Dmax) is apparently higher than the callus that derives from the low levels plant (Dmin).

2, carry out cell suspension culture:

With 100ml triangular flask (other containers also can, size is not limit), interior Sheng 25ml liquid nutrient medium MS+BA 0.4mg/L+NAA 3mg/L+LH 1.0g/L+ sucrose 20g/L carries out cell suspension culture.To bigger callus lines, in mortar, add earlier a little liquid nutrient medium and grind the back inoculation.Rotary shaking speed 125r/min, 25 ± 1 ℃ of culture temperature, dark condition is cultivated down fully.Pour the cell suspending liquid that to gather in the crops the B of filter paper into, take out nutrient solution, use the distilled water wash several times again, drain standby again.

3, clump bud multiplication culture:

Choose the above-mentioned standby cell that grows into stationary phase and insert solid medium again and carry out clump bud multiplication culture, the optimal concentration of 6BA and NAA is respectively 0.5mg/L, 0.1mg/L.

4, inducing of root:

During root culture, select well-grown bud grafting to go into root media and cultivate, take root and use the IBA0.01mg/L+MS substratum, took root through 14 days.

5, bottle outlet, acclimatization and transplants:

As the long 0.5cm of root, when leaf grows into the 3-5 sheet, need not in culturing bottle, practice seedling, directly the oil lamp flower seedling that takes root is taken out from culturing bottle, move in the pearlite interstitial substance, keep certain atmospheric moisture, temperature, after 2 weeks, move to big Tanaka's cultivation.

Embodiment 2: the cultivation of Herba Dendrobii (Dendrobi candidum)

1, the selection of Herba Dendrobii explant and cultivation:

Select Herba Dendrobii stem section, after cleaning up with detergent, flushing is 2 hours under flowing water, forwards to then on the clean bench, and suck dry moisture with 70% alcohol sterilization 30s, changes 0.1% HgCl over to earlier again ₂The 6min that sterilizes in the solution, sterile distilled water flushing 5 times, the stem section is cut into the long segment of 1cm, inoculate sub-callus inducing medium MS+6-BA3mg/L+NAA2mg/L+ sucrose 30g/L, agar with 0.7% solidifies, and pH is adjusted to 5.8,25 ℃ of culture temperature before the high-temperature sterilization.Visible obviously bud after explant is inoculated back 20 days.

2, Herba Dendrobii cell suspension culture:

Get the bud of above-mentioned cultivation, in mortar, add earlier a little liquid nutrient medium and grind the back inoculation.With 100ml triangular flask (other containers also can, size is not limit), interior Sheng 25ml liquid nutrient medium MS+BA0.5mg/L+NAA 1mg/L+LH 1.0g/L+ sucrose 20g/L.Carry out cell suspension culture.Rotary shaking speed 125r/min, 25 ℃ of culture temperature, dark condition is cultivated down fully.Pour the cell suspending liquid that can gather in the crops the B of filter paper into, take out nutrient solution, use the distilled water wash several times again, drain again, standby.

3, inducing of bud multiplication culture and root:

Choose Herba Dendrobii cell that cell grows into stationary phase and insert solid medium again and carry out clump bud multiplication culture, the optimal concentration of 6BA and NAA is respectively 1mg/L, 0.5mg/L.In the blastogenesis growth process, plant strain growth can then be taken root in this substratum to about 2cm.

4, bottle outlet, acclimatization and transplants:

As the long 1cm of root, need not in culturing bottle, practice seedling, directly the Herba Dendrobii seedling of taking root is taken out from culturing bottle, move in the pearlite interstitial substance, keep certain atmospheric moisture, temperature, after 2 weeks, move to cultivation matrix (first-class) cultivation as trunk.

Claims

1. the preparation method of a seedlings of Dendrobium officinale, it is characterized in that carrying out according to the following steps: a). the plant Herba Dendrobii is organized in to add in the mortar body inoculates after a little liquid nutrient medium grinds, enter rotary shaking table, 25 ± 1 ℃ of culture temperature are carried out the Herba Dendrobii cell suspension culture under the dark condition; B). pour the plant Herba Dendrobii cell suspending liquid of propagation the B of filter paper into, take out nutrient solution, use the distilled water wash several times again, the Herba Dendrobii cell of cultivating is carried out the cell growth measurement; C). choose cell that the Herba Dendrobii cell grows into stationary phase and insert the Herba Dendrobii solid medium again and carry out clump bud multiplication culture; D). select well-grown bud grafting to go into solid medium and carry out root culture, produce seedlings of Dendrobium officinale at last; Wherein said plant Herba Dendrobii cell suspension liquid substratum is formed by MS+BA 0.3～0.6mg/L, NAA1-5mg/L, LH 0.5～1.5mg/L, sucrose 19～22g/L mixed preparing; Described Herba Dendrobii solid medium adds the 6BA of 0.2-2mg/L for the MS basic medium and the NAA of 0.1-0.9mg/L makes.