CN104145819B - A kind of ten number camphor tree tissue cultivation rapid breeding methods - Google Patents

Abstract

The invention discloses a kind of ten number camphor tree tissue cultivation rapid breeding methods, relate to plant tissue culture technique in molecular biology experiment.This method is: 1. explant sterilization; 2. callus of induce; 4. the elongation of seedling; 5. the induction of root; 6. hardening and transplanting.Introduce from Africa ten number camphor trees can be obtained a large amount of seedling by tissue-culturing quick-propagation by the present invention, thus provide sufficient vegetable material for downstream extraction ten number camphor tree active pharmaceutical ingredient, accelerate the development process of oral health-care product.

Description

A kind of ten number camphor tree tissue cultivation rapid breeding methods

Technical field

The present invention relates to plant tissue culture technique in molecular biology experiment, particularly relate to a kind of ten number camphor tree tissue cultivation rapid breeding methods.

Background technology

Ten numbers camphor tree (Warburgia ugandensis) are African peculiar medicinal plants, and the branch of this plant is used as in Africa " tooth rod ", has broad spectrum antibiotic activity.From the hot plant antibacterial agent in sky that ten number camphor trees are extracted, will contribute to developing a series of health products, especially health-care toothpaste, can be used for the health care in oral cavity in the future.The present invention establishes ten number camphor tree Tissue Culture Regeneration Systems, can be downstream extraction and analyzes the vegetable material that medicinal active ingredient provides abundance.Through retrieval, up to the present, the special report for ten number camphor tree tissue cultures is not also had in prior art.

Summary of the invention

Object of the present invention is just to provide a kind of ten number camphor tree tissue cultivation rapid breeding methods, namely by setting up the rapid propagation system of ten number camphor tree tissue cultures, obtain a large amount of ten number camphor tree seedlings, for next step extraction and analysis medicinal active ingredient from ten number camphor trees provides sufficient vegetable material.

The object of the present invention is achieved like this:

1, this method comprises the following steps:

1. explant sterilization

Explant for callus of induce is tender leaf base portion or the tender shoots base portion at close petiole place;

Outwell waste liquid after explant sterilization employing 0.1% mercuric chloride sterilization 6min, outwell waste liquid with after 0.1% new mercuric chloride sterilization 6min again, re-use aseptic water washing 6 ~ 8 times;

2. callus of induce

Callus of induce medium used is: MS+1.6mg/L 6BA+1mg/L IBA+0.1mg/L TDZ;

3. Bud polarization

Bud polarization used medium is: MS+1.6mg/L 6BA+0.2mg/L IBA;

4. the elongation of seedling

Seedling extends used medium: MS+0.4 ~ 0.8mg/L 6BA+0.1mg/L IBA, by the Multiple Buds that Bud polarization medium obtains, is divided into fritter to be inoculated on this medium;

5. the induction of root

The first step, root restriction inducing culture is: 1/2MS+0.4mg/L NAA+0.5mg/L IBA;

Second step, root elongation medium is 1/2MS+0.3% active carbon;

6. hardening and transplanting

Hardening and transplanting are according to a conventional method.

Operation principle:

Plant cell has totipotency, has and breaks up by dedifferentiation the ability generating whole plant again.By applying the exogeneous growth conditioning agent of different ratio in different phase, the such as basic element of cell division, makes plant cell progressively be divided into whole plant.

2, the purposes of this method

Our early-stage Study finds, African traditional medicinal plant---the extract of ten number camphor trees has good antibacterium and antifungic action; And as a kind of natural plant kind antibacterial agent, can not drug resistance be produced.

This method can obtain a large amount of ten number camphor tree seedlings, analyzes medicinal active ingredient and afforestation etc. for downstream extraction.

The present invention has following advantages and good effect:

Introduce from Africa ten number camphor trees can be obtained a large amount of seedling by tissue-culturing quick-propagation, thus provide sufficient vegetable material for downstream extraction ten number camphor tree active pharmaceutical ingredient, accelerate the development process of oral health-care product.

Accompanying drawing explanation

Fig. 1 is the photo of callus;

Fig. 2 .1 is the photo of Multiple Buds---spherical latter two hemispherical photography of Multiple Buds crosscut, and a is the sphere by air, and b is the sphere being positioned at medium inside;

Fig. 2 .2 is the photo of Multiple Buds---the photo of position, spherical Multiple Buds cross section;

Fig. 3 is the photo of tufted seedling;

Fig. 4 .1 is photo---the photo of the white root point that root restriction inducing culture occurs of induction root;

The photo of the root that Fig. 4 .2 induces---root elongation medium is cultivated the photo of about 5 days roots.

English to Chinese:

1,6BA:6-benzylaminopurine;

2, IBA: heteroauxin;

3, TDZ: thiadiazole phenylurea;

4, NAA: methyl α-naphthyl acetate.

Embodiment

Describe in detail below in conjunction with drawings and Examples:

One, method

1. explant sterilization

Explant for callus of induce is tender leaf base portion or the tender shoots base portion at close petiole place;

Many experiments proves that this position healing rate is high, and callus can occur in wound at other position of blade (comprising the tender leaf do not launched), but healing rate is lower.

Outwell waste liquid after explant sterilization employing 0.1% mercuric chloride sterilization 6min, outwell waste liquid with after 0.1% new mercuric chloride sterilization 6min again, re-use aseptic water washing 6 ~ 8 times;

2. callus of induce

Callus of induce medium used is: MS+1.6mg/L 6BA+1mg/L IBA+0.1mg/L TDZ;

Inoculate 1 week and can see that explant starts to expand, progressively form yellowish green callus (there is pitchy local) and see Fig. 1;

3. Bud polarization

Bud polarization used medium is: MS+1.6mg/L 6BA+0.2mg/L IBA;

This medium induced bundle is sprouted ultrahigh in efficiency, and the inoculation fritter profile after bud inducement is spherical, and bud dense distribution, in spherical surface, be difficult to add up number, and lopsided bud is less, sees Fig. 2 .1, Fig. 2 .2;

4. the elongation of seedling

Seedling extends used medium: MS+0.4mg/L 6BA+0.1mg/L IBA, by the Multiple Buds that Bud polarization medium obtains, is divided into fritter to be inoculated on this medium;

Within 2 weeks, can see that seedling is obviously grown up, and sees Fig. 3;

5. the induction of root

Hormone because of high concentration can promote that root restriction is formed, but suppresses the elongation of root.What therefore this step adopted is two step rooting.

The first step, root restriction inducing culture is: 1/2MS+0.4mg/L NAA+0.5mg/L IBA;

Second step, root elongation medium is 1/2MS+0.3% active carbon;

Can take root after ten number camphor tree seedlings produce root restriction in the first step, but root extends very slow after taking root, and many experiments finds that different batches seedling occurs that naked eyes root point time fluctuation used scope is larger, scope is 7 ~ 21 days, therefore when seedling is gone to second step by bad judgement, therefore after being observed visually root point, then seedling is transferred to second step (see Fig. 4 .1, Fig. 4 .2); Every bottle graft kind seedling number 4 ~ 6, each little seedling rooting number 1 ~ 12, root is all sturdy; Different batches rooting rate fluctuation range is 21.1% ~ 37.7%.

6. hardening and transplanting

Hardening and transplanting are according to a conventional method.

Seedling of taking root grows to about 6cm, and stem section normal growth, carries out hardening and transplanting; First remove rubber band during hardening, loosen sealed membrane, after 7 days, progressively remove sealed membrane, keep having a small amount of water to make medium be unlikely to stiff in container, within 2 weeks, remove sealed membrane completely, transplant after 3 weeks; After cleaning seedling root medium, transplant to little basin; Matrix used is humus and the vermiculite of 1:1, uses after high-temperature sterilization; After transplanting, matrix is watered permeable; For preventing excessive water from evaporating, with transparent plastic film cover mouth after transplanting, and staying ventilation finedraw, preventing mould from growing; Progressively expand ventilating opening after 7 days, after 2 weeks, remove plastic sack completely.

Claims (1)

1. ten number camphor tree tissue cultivation rapid breeding methods, is characterized in that comprising the following steps:

1. explant sterilization

Explant for callus of induce is tender leaf base portion or the tender shoots base portion at close petiole place;

Outwell waste liquid after explant sterilization employing 0.1% mercuric chloride sterilization 6min, outwell waste liquid with after 0.1% new mercuric chloride sterilization 6min again, re-use aseptic water washing 6 ~ 8 times;

2. callus of induce

Callus of induce medium used is: MS+1.6mg/L 6BA+1mg/L IBA+0.1mg/L TDZ;

3. Bud polarization

Bud polarization used medium is: MS+1.6mg/L 6BA+0.2mg/L IBA;

4. the elongation of seedling

Seedling extends used medium: MS+0.4 ~ 0.8mg/L 6BA+0.1mg/L IBA, by the Multiple Buds that Bud polarization medium obtains, is divided into fritter to be inoculated on this medium;

5. the induction of root

The first step, root restriction inducing culture is: 1/2MS+0.4mg/L NAA+0.5mg/L IBA;

Second step, root elongation medium is 1/2MS+0.3% active carbon;

6. hardening and transplanting

Hardening and transplanting are according to a conventional method.