CN101361455A - Cluke cherry high-grade breed 'Juhong' in vitro regeneration system - Google Patents

Abstract

The invention belongs to the biotechnology field, and more particularly relates to a sweet cherry fine variety 'Juhong' in vitro regeneration system; the processes compositing the system is that: (1) a stem section is taken as an explant, and sterilization treatment is carried out; (2) the explant after sterilization is treated with primary culture, and the screening of a culture medium is carried out to determine the optimum primary culture medium; (3) secondary culture is carried out on the basis of the primary culture, and the screening of the culture medium is carried out to determine the optimum secondary culture medium; (4) rootage culture is carried out on the basis of the secondary culture, and the screening of the culture medium is carried out to determine the optimum rootage culture medium; and (5) domestication. The system has the beneficial effects that the sweet cherry fine variety 'Juhong' in vitro regeneration system is firstly established, with stable system, fast reproduction speed, simple and convenient technical system and is easy to be mastered. The cultured seedling has low cost and can be used for a plurality of usages.

Description

The cluke cherry high-grade breed ' Juhong ' in vitro regenerating system

Affiliated technical field

The invention belongs to biological technical field, especially sweet cherry improved seeds " huge red " Regeneration in Vitro system.

Background technology

In recent years, the fruit tree tissue culture technique has obtained using widely on producing, and becomes an important new and high technology in the fruit tree biotechnology gradually, causes the extensive attention and the concern of various circles of society.In general, the application of fruit tree tissue culture and current situation have the following aspects:

1. breeding fast

Owing to adopt the method for tissue culture can make the fruit tree plant test-tube plantlet have higher reproduction speed, therefore this method be called quick breeding.Quick propagating technology has not only solved the supply problem of fruit nursery stock, also provides important means for long preservation and the good fruit tree germ plasm resource of application simultaneously.The nursery stock of breeding has advantages such as growing way unanimity, root system is good, growth is fast fast.

The seedling propagation number can be formulated as: Y=m * x ⁿWherein, Y is a year breeding number, and m is aseptic maternal plant number, and x is the multiple of each cultivation cycle propagation, and n is annual fertile cycle times.As can be seen, seedling propagation speed is with geometric progression propagation from formula, and Here it is adopts the method for tissue culture can realize the fruit nursery stock key of breeding fast.

2. nursery stock detoxification

At present, people have recognized the harmfulness of virus for fruit tree plant, and owing to the reason of virus can cause the fruit deformity, the orchard underproduction may cause the mortality harm of having no harvest even.Obtained general application in the fruit tree virus-free nursery stock agricultural production at home and abroad by plant tissue culture course's production.It is generally acknowledged, do not contain virus in the fruit nursery stock apical meristem or only contain a spot of virus; Be higher than under the normal temperature, a lot of viruses in the fruit tree tissue can be by part or all of passivation.Therefore, if adopt little stem apex technology to combine, just can reach the purpose of fruit tree detoxification with heat treatment technics or with these two technology.At present, set up the detoxification technology of fruit tree kinds such as jujube tree, apple, grape, strawberry, improved output, resistance and the quality of fruit greatly.In recent years, means such as employing immunology correspondingly, molecular biology detect the research work of fruit tree virus also in the middle of actively carrying out.

3. be used for genetic breeding, cultivate distant hybrid

Because fruit tree plant is a perennial plant, reasons such as seed vigor is not strong tended to cause conventional distant hybridization work difficulty bigger after not affinity, hybridization are grown, hybridized to the distant hybrid progeny growth cycle, if plant tissue culture technique is combined with conventional distant hybridization technology, the rataria that distant hybridization is formed carries out the in vitro tissue cultivation, make it under the condition that exsomatizes, to form complete test tube plantlet, just may cultivate good fruit tree of new species.Liaoning Province's fruit tree research utilizes this method to obtain the filial generation of apple and pears, and this offspring has the characteristic of apple and pears concurrently.

Flower pesticide and pollen are cultivated, and can be used for haploid breeding, the dliploid that obtains isozygotying.Protoplast is cultivated, somatocyte hybriding technology makes people carry out genetic recombination in a wider context, creates very rare kind and kind in breeding.Aspects such as the inducing of other breeding at the elite germplasm material, preservation and mutant, screening are compared with conventional breeding, in time or all want much superior on the space.

4. shortening breeding time

The fruit tree plant great majority are cross-pollinatd plants, generally will could obtain the more stable pure line cultivar of genetic character through the selfing of 7～8 generations; Fruit tree through the breeding of growing directly from seeds also approximately needed 10～20 years even the longer time to yielding positive results from seed sprouting, obtain the necessary selfing of stable pure lines, also need considerable time, and self-fruitful rate was also often lower.Method by tissue culture, pollen, flower pesticide, unfertilized ovary and ovule to fruit tree plant are cultivated, obtain after the haplobiont, adopt the method for chromosome artificial doubling to make its recovery become liploid plant again, become new fruit variety thereby cultivate.This method has shortened breeding cycle greatly, has simplified the procedure of breeding, has improved efficiency of selection, and for example, the conventional breeding seed selection phase of lichee needs 7 years, and after adopting tissue culture method, the breeding seed selection phase then only needs 1 year.

5. cultivate burdo

Adopt cell-fusion techniques can overcome fruit tree plant distant hybridization disaffinity, obtain burdo, and the protoplasm cultivation is the prerequisite of cytomixis, fruit tree plant protoplast culture studies work starting is than later, since nineteen ninety, researcher is cultivated successfully the protoplast of delicious kiwi fruit, has obtained the fruit tree protoplast first and has cultivated regeneration plant; After this, set up the comparatively stable cytomixis system of oranges and tangerines again.At present, the protoplast training of other fruit tree kind also has some reports.

6. germ plasm resource is preserved

In recent years, worked out the method for preserving germ plasm resource with tissue culture and cell culture method low temperature.Fruit tree plant research is in this respect also being carried out and is being obtained some successful examples.Because most fruit tree plant plant is tall and big, the gene pool of therefore foundation routine takes up an area of many, and management is also relatively more difficult, and the method for employing tissue culture is preserved test-tube plantlet can save the space, a 0.28m widely ³Common refrigerator can deposit 2000 test tubes, the apple plant of holding similar number then needs 85 mu of soils.Under cryogenic conditions, fruit tree plant often stop growing (for example, the plantlet that becomes of grape stem segment length need be placed under 9 ℃ the condition and preserve), annual only the needs transfers 1 time, at least can preserve 20 years, and when a large amount of nursery stock of needs, can realize the purpose of breeding fast again at any time.

7. fruit tree plant gene engineering

Traditional fruit tree plant breeding method (for example hybridization) has the shortcoming that wastes time and energy, and therefore, a lot of people have turned to transgenic breeding to sight, tries hard to artificial transfer by foreign gene, thereby obtains having the new varieties of objective trait.Since nineteen eighty-three, the first genetically modified plants were come out, transgenic technology had obtained development rapidly, and genetically modified plants have reached kind more than 120 at present.Compare with other plant, the research of fruit tree plant gene engineering is started late, and people try hard to obtain resistance, output height, colory fruit tree of new species by engineered method.The genes of interest of having separated in the world at present has more or less a hundred, comprise disease-resistant gene, anti insect gene, degeneration-resistant border gene, anti-herbicide gene, raising protein quality gene, chromogene, the ripe gene of delayed fruit, and the promotor that efficiently expresses.Conventional transgenic method comprises agrobacterium mediation converted method, particle bombardment, electric shocking method, PEG mediated transformation method and pollen tube passage method etc.Because fruit tree plant directly offers the edible fruit of people, so draw attention for the bio-safety sexual needs of transgenosis fruit tree plant.Adopt uncontested bio-safety marker gene can get rid of the worry of people for the marker gene safety, the bio-safety marker gene of finding mainly contains green fluorescence protein gene, betaine aldehyde dehydrogenase gene, xylose isomerase gene or the like at present.Different with conventional marker gene, these marker gene do not have antibiotic or Herbicid resistant, are safe to biology comparatively speaking.Along with the progress of functional genomics and proteomics, the fruit tree plant transgenic technology will constantly be rationalized and precision.

8. help pathology, physiology and other theoretical research

Utilize the fruit tree tissue culture technique, all can provide eugonic nursery stock or tissue, cell throughout the year, for the research of subjects such as pathology, physiology, it is the good method of screening bactericide, disease-resistant variety and plant hormone.The tissue culture space is little, condition is easy to control, is not subject to seasonal restrictions, and is all meaningful to accelerating genetics, cytology, biochemical research.

Sweet cherry improved seeds " huge red " Regeneration in Vitro research work at present, same domain scientific and technical personnel do not carry out as yet.

Summary of the invention

The purpose of this invention is to provide a kind of based on the modern plants tissue culture technique, setting up one, to have system stable, reproduction speed is fast, sweet cherry improved seeds " huge red " the Regeneration in Vitro system of low cost and other advantages, for quick breeding, the nursery stock detoxification of improved seeds " huge red " and even cherry, cultivate distant hybrid, shorten breeding time, cultivate burdo, germ plasm resource preservation, gene engineering and pathology, physiology and other theoretical research and lay the foundation.Principle of the present invention is based on the basic principle of totipotency of plant cell, under the suitable culture condition, and the regeneration whole plant

The technical solution adopted for the present invention to solve the technical problems is: the cluke cherry high-grade breed ' Juhong ' in vitro regenerating system, and the process of forming this system is:

(1) gets the stem section as explant.Carry out sterilization treatment;

(2) explant after will sterilizing carries out initial culture, and carries out the screening of medium, determines best initial culture base;

(3) on the basis of initial culture, carry out successive transfer culture, carry out the screening of medium, determine best subculture medium;

(4) on the basis of successive transfer culture, carry out culture of rootage, carry out the screening of medium, determine best root media;

(5) domestication;

Concrete steps are:

(1) explant is handled and initial culture: cut off the full branch of axillalry bud autumn, at indoor aquatic 7 days, the crust band girdle of branch is removed, and remove the outer scale of axillalry bud base portion with single-edge blade, branch is cut into the stem section of 1.5-2cm, and each stem section contains 1 axillalry bud, the stem section is placed on the bromogeramine with 2% soaks 15min in the beaker of 500mL, wash 5min with flowing water, under aseptic technique, carry out explant sterilization; 75% Ethanol Treatment 30s uses aseptic water washing one time then, handles 15min with 2% clorox again, with aseptic water washing 3-5 time, use the aseptic filter paper suck dry moisture, downcut axillalry bud and be inoculated on the initial culture base, cultivating indoor temperature is 22 ℃, and intensity of illumination is 2500LX, and the photoperiod is 12h/d;

(2) successive transfer culture: explant is cut into stem-segment with single bud and is inoculated on the subculture medium, with MS+6-BA through initial culture _1.0+ IBA _0.2, additional agar 7g/L, sucrose 30g/L, stem section growth coefficient maximum is 5;

(3) culture of rootage: with MS+6-BA _0.2+ IBA _0.2, additional agar 7g/L, sucrose 30g/L.It is best to take root, and rooting rate reaches 100%, and the bar of taking root is counted 11 of average out to;

(4) domestication: the survival rate on perlite and the sandy soil all reaches 100%.Transplanting in neutral loam, survival rate 90%.

The invention has the beneficial effects as follows that it is stable to set up sweet cherry improved seeds " huge red " Regeneration in Vitro system, system first, reproduction speed is fast, and technical system is easy, is easy to grasp.The seedling cost of being cultivated is low, can be used for multiple use.

Embodiment

The invention will be further described below in conjunction with embodiment.

The invention process step is:

(1) explant is handled and initial culture: cut off the full branch of axillalry bud autumn, at indoor aquatic 7 days, the crust band girdle of branch is removed, and remove the outer scale of axillalry bud base portion with single-edge blade, branch is cut into the stem section of 1.5-2cm, and each stem section contains 1 axillalry bud, the stem section is placed on the bromogeramine with 2% soaks 15min in the beaker of 500mL, wash 5min with flowing water, under aseptic technique, carry out explant sterilization; 75% Ethanol Treatment 30s uses aseptic water washing one time then, handles 15min with 2% clorox again, with aseptic water washing 3-5 time, use the aseptic filter paper suck dry moisture, downcut axillalry bud and be inoculated on the initial culture base, cultivating indoor temperature is 22 ℃, and intensity of illumination is 2500LX, and the photoperiod is 12h/d;

(2) successive transfer culture: explant is cut into stem-segment with single bud and is inoculated on the subculture medium, with MS+6-BA through initial culture _1.0+ IBA _0.2, additional agar 7g/L, sucrose 30g/L, stem section growth coefficient maximum is 5;

(3) culture of rootage: with MS+6-BA _0.2+ IBA _0.2, additional agar 7g/L, sucrose 30g/L.It is best to take root, and rooting rate reaches 100%, and the bar of taking root is counted 11 of average out to;

(4) domestication: the survival rate on perlite and the sandy soil all reaches 100%.Transplanting in neutral loam, survival rate 90%.

Claims (1)

1, the cluke cherry high-grade breed ' Juhong ' in vitro regenerating system is characterized in that, the process of forming this system is:

(1) gets the stem section as explant, carry out sterilization treatment;

(2) explant after will sterilizing carries out initial culture, and carries out the screening of medium, determines best initial culture base;

(3) on the basis of initial culture, carry out successive transfer culture, carry out the screening of medium, determine best subculture medium;

(4) on the basis of successive transfer culture, carry out culture of rootage, carry out the screening of medium, determine best root media;

(5) domestication;

Concrete steps are:

(1) explant is handled and initial culture: cut off the full branch of axillalry bud autumn, at indoor aquatic 7 days, the crust band girdle of branch is removed, and remove the outer scale of axillalry bud base portion with single-edge blade, branch is cut into the stem section of 1.5-2cm, and each stem section contains 1 axillalry bud, the stem section is placed on the bromogeramine with 2% soaks 15min in the beaker of 500mL, wash 5min with flowing water, under aseptic technique, carry out explant sterilization; 75% Ethanol Treatment 30s uses aseptic water washing one time then, handles 15min with 2% clorox again, with aseptic water washing 3-5 time, use the aseptic filter paper suck dry moisture, downcut axillalry bud and be inoculated on the initial culture base, cultivating indoor temperature is 22 ℃, and intensity of illumination is 2500LX, and the photoperiod is 12h/d;

(2) successive transfer culture: explant is cut into stem-segment with single bud and is inoculated on the subculture medium, with MS+6-BA through initial culture _1.0+ IBA _0.2, additional agar 7g/L, sucrose 30g/L, stem section growth coefficient maximum is 5;

(3) culture of rootage: with MS+6-BA _0.2+ IBA _0.2, additional agar 7g/L, sucrose 30g/L;

(4) domestication.