CN1276423A

CN1276423A - Tissue culture method for breeding aloe and its culture medium

Info

Publication number: CN1276423A
Application number: CN 99107921
Authority: CN
Inventors: 刘敏; 薛淮; 张纯花; 陈宇红
Original assignee: Institute of Genetics of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 1999-06-02
Filing date: 1999-06-02
Publication date: 2000-12-13
Anticipated expiration: 2019-06-02
Also published as: CN1117857C

Abstract

A tissue culture method for breeding aloe includes inoculating the meristem of aloe to inducing culture medium, culturing for 60-80 days, shearing the young seedling from root, transplanting in root-growing culture medium, culturing for 20-30 days until white root is grown, and transplanting it conventionally.

Description

Aloetic tissue culture propagation and employed substratum

The present invention relates to aloetic tissue culture propagation and employed substratum.

Aloe (Aloe Liliaceae) has another name called oily green onion, Long Jiao, the wolf's fang palm, is the evergreen fleshy-juicy plant of Liliaceae Aloe.It contains Barbaloin the scientific research proof, protein, 20 seed amino acids, multivitamin, Schuttgelb, kind of composition surplus organized enzyme and the trace element 100, can treat intestines, stomach trouble, hypertension, diabetes, heart trouble, hepatitis, ephrosis, flu, asthma, the constipation cancer, external application can be treated scald, burn, knife wound, sprain, frostbite, the ringworm of the foot, neural dermatitis, hemorrhoid, chap, eczema, odontopathy, rhinitis, mosquito bite, prickly heat etc., industrial in daily use chemicals be " natural makeup; " again have sun-proof, skin care, beauty treatment, effects such as removing freckle wrinkle, 80% contains the aloetic composition in the superior cosmetics.In recent years, magical pharmaceutical use and industrial use that aloe had more and more caused people's interest, were called as " plant health beauty treatment doctor ".

Though aloe has great vitality, self is irreproducible, can only lean on plant division and cuttage, breeds aloe if therefore adopt the method for plant tissue culture, carries out industrial seedling rearing, will accelerate widely the aloetic production cycle, produces bigger benefit.

Plant tissue culture technique is the totipotency of utilizing cell (is each cell all potential develop into the ability of new individuality), take a cell mass or a block organization on the plant materials, by artificial preparation Different Nutrition composition and hormone regulating and controlling, make these tissues thousands of plant of formation and preserved whole good characters in the parent, this method can obtain a large amount of test-tube plantlets at short notice, can in the workshop, streamline operration carry out, thereby be called batch production production.Since produce seedling and be in vitro carry out and be placed between the constant-temperature house of illumination condition in, the four seasons all can carry out, and can carry out scale operation in few area, thereby land occupation not, greatly reduce cost.

Yet, bigger with tissue culture method breeding aloe difficulty, because that aloe contains the phenols quinones substance is many, very easy oxidation browning, thus make brownization of substratum influence differentiation rate, so that group is trained effect is very difficult satisfactory.At the deficiencies in the prior art, the invention provides a kind of can be fast, large-scale breeding aloetic tissue culture method.The machine examination rope finds no the report about the aloe tissue culture propagation as calculated.

The invention provides aloe, the tissue culture method of Aloe.arborescens (A.barbadersis) and Aloe Liliaceae (A.arborescens) especially may further comprise the steps:

1, induces and break up: utilize embryoid group training approach to induce.With the aloetic meristematic tissue, be seeded on the inducing culture, this substratum is conventional minimum medium (the Marashige ﹠amp of MS; The Skoog substratum, hereinafter to be referred as MS), wherein added 6-benzyl purine 0.5-5 mg/litre, be preferably 0.5-3 mg/litre, 6-furfuryl group purine (kinetin) 0.5-5 mg/litre, be preferably 0.5-3 milliliter, 2,4 dichlorophenoxyacetic acid 0.1-2 mg/litre, be preferably 0.1-1 mg/litre, naphthylacetic acid 0.1-2 mg/litre, be preferably the 0.1-1 mg/litre, this substratum also contains vitamins C 1-2 milligram and sucrose 30-60 gram in addition, is preferably the 40-50 grams per liter.After cultivating about 60-80 days, seedling grows to routine when taking root the program desired height, enters next step.

2, take root: the neat root of the first step gained seedling is taken off, be transferred in the root media, this substratum with above-mentioned induce and division culture medium identical, just do not add VITAMIN, but added gac 0.1-0.5 gram, and having added indolebutyric acid is the 0.1-0.5 mg/litre, is preferably the 0.1-0.2 mg/litre.After 20-30 days, the seedling base portion begins to occur the short root of white and grows complete root system very soon, can transplant according to a conventional method to grow up to normal aloe plant.

Characteristics of the present invention are: induce by the embryoid approach, effect is fine, and in addition, the substratum sucrose concentration that the normal tissue cultivation is used in atomization is 30 grams, we have improved sucrose concentration greatly, and have added vitamins C at brownization of substratum.Can be the high frequency differentiation than higher sucrose concentration more nutrition is provided, and increase the osmotic pressure of substratum, thereby make the hormone of substratum better act on cell interior culture; The use of Vc can prevent cell brownization in culturing process.In root media, can in above-mentioned scope, improve the content of naphthylacetic acid greatly, so better hestening rooting.

The substratum of this invention has following advantage: it is fast to start early, emerge, and synchronism is good, differentiation frequency height, Miao Qizhuan, well-grown.

Embodiment 1.

1. draw materials: get the Aloe Liliaceae upper end or inhale bud.

2. materials disinfection method: the material that takes off washes in flowing water after 5 minutes and soaked 30 seconds, puts into 0.1% mercuric chloride solution sterilization 15 minutes (on Bechtop with aseptic water washing 3-4 time afterwards filter paper suck dry moisture) then.

3. inoculation: on Bechtop, to sterilize afterwards with conventional aseptic technique method, material centre portions (vegetative point and near tissue) is cut into the little finger size, insert in the substratum along the former direction of growth, this substratum is MS+6-BA 2mg/L+KT 2mg/L+NAA 0.1mg/L+2.4-D 0.1mg/L+Vc2mg/L+ sucrose 45 gram/L, 25 ℃ ± 2 ℃ of culture temperature, intensity of illumination is 1500-2000Lux.

4. differentiation: visible budlet sprouts after 20 days, grows the seedling subculture three times, and about 60 days, seedling can grow to 3 centimeters high.

5. take root, seedling is gone to MS+IBA 0.2mg/L+NAA 0.5mg/L+ sucrose 30 grams, 2 gram gacs, after about 20 days, the lower end grows white rootlet.

6. transplant:, during seedling, it is transplanted in the matrix that contains 1/2 turfy soil and 1/2 vermiculite when test-tube plantlet grows to the complete root of tool.

Method for transplanting: the test tube mouth is opened, opened wide bottleneck 24 hours at normal temperatures, with long tweezers are carefully taken out seedling from test tube, stick in nutrient agar on the root with the tap water flush away, transplanting medium is put into flowerpot, carefully plant seedling in the engagement, water permeable back covered with plastic film and open plastics film after 24 hours, took a breath 2 hours, prolong the time of opening plastic film later every day, till not covering fully, according to local air themperature and humidity, proper extension or reduce the cover time, wait seedling to survive fully really after, amount is executed some potash fertilizer and nitrogenous fertilizer routinely, the assurance seedling is strong, Miao Qi., we in this laboratory and agricultural demonstration base, Shunyi, the suburb of Beijing respectively transplanted 100 strain Aloe Liliaceae seedlings, survive strain number 81 strains and 83 strains respectively, surviving rate reaches more than 80%.Embodiment two:

Get Aloe.arborescens, cultivate and transplant by embodiment one same procedure, its result has also obtained healthy and strong aloe seedling.We in this laboratory and agricultural demonstration base, Shunyi, capital respectively transplanted 100 strain Aloe.arborescens seedlings, survive the strain number and be respectively 87 strains and 84 strains, surviving rate all reaches more than 80%.

Claims

1. an aloetic tissue culture propagation comprises the steps:

A. the aloetic meristematic tissue is seeded on the inducing culture, this inducing culture is the conventional minimum medium of MS, 6-benzyl purine 0.5-5 mg/litre, 6-furfuryl group purine (kinetin) 0.5-5 mg/litre, 2 have wherein been added, 4-dichlorphenoxyacetic acid 0.1-2 mg/litre, naphthylacetic acid 0.1-2 mg/litre, this substratum also contains vitamins C 1-2 milligram and sucrose 30-60 gram in addition; Meristematic tissue was cultivated 60-80 days on this substratum;

B. the neat root of gained seedling in the above-mentioned steps is wiped out, be transferred in the root media, this substratum is identical with above-mentioned inducing culture, does not just add VITAMIN, but has added gac 0.1-0.5 gram, and to have added indolebutyric acid be the 0.1-0.5 mg/litre; After 20-30 days, the seedling base portion begins to occur the short root of white and grows complete root system, transplants according to a conventional method to grow up to normal aloe plant.

2. in accordance with the method for claim 1, wherein, described aloe is Aloe.arborescens (A.barbadersis) or Aloe Liliaceae (A.arborescens).

3. in accordance with the method for claim 1, wherein, the 6-benzyl purine is that 0.5-3 mg/litre, 6-furfuryl group purine (kinetin) are that 0.1-1 mg/litre, naphthylacetic acid are the 0.1-1 mg/litre for 0.5-3 milliliter, 2,4 dichlorophenoxyacetic acid in the described inducing culture, and sucrose is the 40-50 grams per liter; Indolebutyric acid is the 0.1-0.2 mg/litre in the described root media.

4. inducing culture that is used for the aloetic tissue culture propagation, this substratum is the conventional minimum medium of MS, 6-benzyl purine 0.5-5 mg/litre, 6-furfuryl group purine (kinetin) 0.5-5 mg/litre, 2 have wherein been added, 4-dichlorphenoxyacetic acid 0.1-2 mg/litre, naphthylacetic acid 0.1-2 mg/litre, this substratum also contains vitamins C 1-2 milligram and sucrose 30-60 gram in addition.

5. according to the described substratum of claim 4, wherein, the 6-benzyl purine is that 0.5-3 mg/litre, 6-furfuryl group purine (kinetin) are that 0.1-1 mg/litre, naphthylacetic acid are the 0.1-1 mg/litre for 0.5-3 milliliter, 2,4 dichlorophenoxyacetic acid, and sucrose is the 40-50 grams per liter.

6. according to the described substratum of claim 4, wherein, described aloe is Aloe.arborescens (A.barbadersis) or Aloe Liliaceae (A.arborescens).

7. root media that is used for the aloetic tissue culture propagation, its composition is identical with the described inducing culture of claim 1, just do not add VITAMIN, but added gac 0.1-0.5 gram, and to have added indolebutyric acid be the 0.1-0.5 mg/litre.

8. according to the described root media of claim 7, wherein, indolebutyric acid is the 0.1-0.2 mg/litre.

9. according to the described root media of claim 7, wherein, described aloe is Aloe.arborescens (A.barbadersis) or Aloe Liliaceae (A.arborescens).