CN101015279B - Tissue culture method for fast propagation of primula poissonii - Google Patents

Tissue culture method for fast propagation of primula poissonii Download PDF

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Publication number
CN101015279B
CN101015279B CN2007100640559A CN200710064055A CN101015279B CN 101015279 B CN101015279 B CN 101015279B CN 2007100640559 A CN2007100640559 A CN 2007100640559A CN 200710064055 A CN200710064055 A CN 200710064055A CN 101015279 B CN101015279 B CN 101015279B
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primula
poisonii
culture
spring
seedling
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CN101015279A (en
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张启翔
李翠娟
潘会堂
梁树乐
程堂仁
孙明
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Beijing Forestry University
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Beijing Forestry University
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Abstract

The invention provides a method for tissue culturing and fast propagating Primula poisonii, comprising explant selection, surface disinfection, adventitious bud induction, subculture, rooting culture and test-tube seedling transplanting. The method is characterized in that one axillary bud can induce about 40-50 sprouts, the multiplication coefficient is about 3- 4 after 30- 40 days subculture, the rooting rate reaches above 95%, and the replanting survival rate is nearly 100%. The invention realizes preservation for single Primula poisonii, meanwhile, greatly increases seedling capacity for the Primula poisonii, reduces seedling time and cost, and provides technique support for Primula poisonii application in large areas.

Description

The tissue culture and rapid propagation method that Hai Xian is heralded spring
Technical field
The present invention relates to Plant Tissue Breeding, specifically, relate to the herald spring quick breeding method for tissue culture of (Primulapoisonii) of Hai Xian.
Background technology
Hai Xian is heralded spring (Primula poisonii) for the Primulaceae primula, is a kind of very beautiful ornamental plantation flowers.Hai Xian herald spring (Primula poisonii) be the perennial herb flowers, whole plant is rosette-stape, the root-like stock prosperity, upwards give birth to 5 to a plurality of leafages, based on seed propagation, generally in spring after planting, through nourishing and growing in beginning May next year flower, it is slower to grow, and reproduction coefficient is low.Except that seed propagation, the primula vegetative propagation can with method have: plant division (leafage) breeding, tissue culture propagating, the perennial kind of minority can adopt cottage propagation, however the method for division propagation far can not satisfy the production demand.
Tissue culture is an important channel of breeding the good plant kind fast, the existing many relevant reports of having carried out tissue culture of planting of primula, the tissue culture of (Primulapoisonii) does not but have correlative study and report but Hai Xian is heralded spring, therefore need the research Hai Xian tissue culture of (Primula poisonii) of heralding spring, and form the special fast breeding technique system of a cover.
Summary of the invention
The purpose of this invention is to provide the herald spring tissue culture and rapid propagation method of (Primula poisonii) of a kind of Hai Xian, improve Hai Xian herald spring reproduction coefficient, rooting rate and the transplanting survival rate of (Primula poisonii), and, make the herald spring seedling production, quality saving etc. of (Primulapoisonii) of Hai Xian can access better guarantees in conjunction with other relevant technical measures.
In order to realize the object of the invention, the herald spring tissue culture and rapid propagation method of (Primula poisonii) of a kind of Hai Xian of the present invention, explant selection, surface sterilization, adventitious bud inducing, successive transfer culture, culture of rootage and test-tube seedling transplanting.
Described explant is the herald spring axillalry bud of (Primula poisonii) of Hai Xian.
The medium of described adventitious bud inducing and subculture is MS+2.0mg/l 6-BA+0.1mg/lNAA, and pH is 5.8~5.9.Its illumination condition is that natural scattering light 2500~3000Lux adds artificial fill-in light 1500~2000Lux.Lux (lux) is the unit of illumination.
Described root media is MS or MS+0.2mg/l NAA, is preferably MS+0.2mg/lNAA, and pH is 5.8~5.9.
Its illumination condition is that natural scattering light 2500~3000Lux adds artificial fill-in light 2500~3000Lux.
Through cultivated for 2~3 weeks in root media after, hardening was transplanted and is cultivated to the thin turfy soil in greenhouse in 3~4 days.
Explant adopts 0.1% mercuric chloride solution sterilization, 3~4min.
Tissue cultivating seedling suitable culture temperature is between 20~23 ℃, the tissue cultivating seedling flavescence above 25 ℃ even dead gradually.
The transplanting preference temperature of tissue cultivating seedling is between 18~25 ℃, too high or too lowly all is unfavorable for surviving of test-tube plantlet.
Tissue culture and rapid propagation method is particularly: selecting the herald spring axillalry bud of (Primula poisonii) of Hai Xian is explant, rinse well through water, adopt 0.1% mercuric chloride solution sterilization, 3~4min, under aseptic condition, clean repeatedly through distilled water, be seeded among the inducing culture MS+2.0mg/l6-BA+0.1mg/l NAA, pH is 5.8, and to be natural scattering light 2500~3000Lux add artificial fill-in light 1500~2000Lux with interior to illumination condition; Move to same medium propagation afterwards, subculture 2~3 times, root media are MS+0.2mg/l NAA, and pH is 5.8, and illumination condition is that natural scattering light 2500~3000Lux adds artificial fill-in light 2500~3000Lux; Temperature remains between 20~23 ℃; In 2~3 weeks of culture of rootage, hardening moved in the thin turfy soil and cultivates after 3~4 days, kept humidity to be higher than 80%, sprayed water every day 2~3 times.Progressively throwing off coverlay after 2 weeks normally cultivates.
In general with the axillalry bud be explant, bigger to the destructiveness of plant, but relatively precious for some, and be badly in need of expanding numerous material, it is numerous with the axillalry bud to be that explant expands, and can obtain a large amount of regeneration plants at short notice; By the Hai Xian of axillalry bud regeneration (Primulapoisonii) test-tube plantlet growth stalwartness of heralding spring, growth of later stage observed find, can keep its good strains of seeds constant substantially.The test-tube plantlet of tissue-culturing rapid propagation in process of growth, is not vulnerable to infecting of damage by disease and insect, but in order further to prevent the generation of damage by disease and insect, it is for 3~4 times good should spraying carbendazim, flolimat etc. in growth season; Temperature during test-tube seedling transplanting should be controlled between 18~25 ℃, too high or too lowly all is unfavorable for surviving of test-tube plantlet, and should not carry out the transplanting of test-tube plantlet summer.
Tissue culture and rapid propagation method of the present invention is bred Hai Xian herald spring (Primula poisonii), the internal breeding coefficient reached 3~4 in 2 months, rooting rate reaches more than 95%, transplanting survival rate is almost 100%, greatly improved the herald spring reproduction coefficient of (Primula poisonii) of Hai Xian, shorten into the seedling cycle, can reduce cost again, for large tracts of land Landscape Application and factorial seedling growth provide very effective method from now on; Simultaneously, be that explant is bred and numerous soon with the axillalry bud, can keep the herald spring kind of (Primula poisonii) of Hai Xian constant, for the preservation of the comparatively precious rare mutation that occurs in the rearing new variety provides guarantee.
Compared with prior art, the herald spring advantage of tissue culture and rapid propagation method of (Primula poisonii) of Hai Xian of the present invention is:
1. set up a kind of Hai Xian and heralded spring the tissue culture and rapid propagation method of (Primula poisonii), compare traditional primula propagation method of growing seedlings, as seed propagation and division propagation, greatly improved the herald spring reproduction coefficient of (Primula poisonii) of Hai Xian, for scientific research research with produce cultivation technical support is provided;
2. the seedling that obtains by tissue-culturing rapid propagation, it is strong to grow, and the leaf look dark green, and uniformity is strong, becomes seedling easy, and adaptability is stronger, and damage by disease and insect is few, is easy to management;
3. utilize tissue culture and rapid propagation method to breed Hai Xian and herald spring (Primula poisonii), removed the restriction of season and weather, thereby shortened growing-seedling period, increased breeding amount;
4. utilize tissue culture and rapid propagation method to breed Hai Xian and herald spring (Primula poisonii), can keep the characteristic of good species or kind constant, thereby provide effective method for making a variation to preserve in the breeding of new variety research.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
With Hai Xian (Primula poisonii) blade of heralding spring is that explant is probed into the adventitious bud inducing optimum medium: select for use the seedling leaves that grows fine to probe into adventitious bud inducing optimal medium prescription as explant.Get the herald spring young leaflet tablet running water flushing 2~3h of (Primula poisonii) of Hai Xian, drip cleaning agent concussion 15min removing surface impurity in water, running water is rinsed well repeatedly; Surface sterilization: 70% alcohol disinfecting, 10~15s, aseptic water washing 4~5 times, mercuric chloride sterilization 3~4min of 0.1% uses aseptic water washing 4~5 times again.The blade that to sterilize is cut into 1cm then 2Fritter (band portion vein) be seeded on the inducing culture of the bud of growing thickly, 5~6 of every bottle graft kinds, every kind of medium connects 10 bottles, the dark cultivation.Wherein, the concentration gradient of 6-BA: 0.5mg/l, 1.0mg/l and 2.0mg/l; The concentration gradient of NAA: 0.05mg/l, 0.1mg/l and 0.5mg/l, medium pH is 5.8, adopts the design of two factor completely randomized experiments.
The herald spring differentiation of (Primula poisonii) blade of different 6-BA and NAA concentration affects Hai Xian.Inoculate after 4~5 days, blade begins the swelling shrinkage, and 15 days left and right sides incision thickening of growing fat after 30 days, has medium kind blade to begin the generation that produces callus and a small amount of indefinite bud is arranged.The optium concentration of differentiation adventitious buds is 6-BA 2.0mg/l and NAA 0.1mg/l, and this medium Shanghai celestial being (Primula poisonii) inductivity of heralding spring reaches 22.5%, and the part differentiation and bud formation.
Embodiment 2
With Hai Xian (Primula poisonii) axillalry bud of heralding spring is that explant carries out just cultivating and successive transfer culture: choose the axillalry bud on the plant of robust growth, running water flushing 2~3h, the same blade of sterilization process, be seeded on the MS+6-BA 2.0mg/l+NAA 0.1mg/l inducing clumping bud medium 2~3 of every bottle graft kinds; Illumination condition is that natural scattering light 3000 Lux add artificial secondary light source 1800 ± 200Lux, light application time 14 h.
When axillalry bud is seeded on the differential medium, constantly extract young leaves out, beginning in 10 days, the petiole of extracting young leaves out is sturdy, and base portion reddens, and forms the light green color callus gradually, begins to have the differentiation of indefinite bud after 2 weeks; Inoculate after 40 days, on the new petiole of extracting out and blade of axillalry bud, differentiate a large amount of indefinite buds.
After 2 months, on average to differentiate the number of indefinite bud be 40~50 to each axillalry bud to statistical result showed in inoculation on the medium of MS+6-BA 2mg/l+NAA 0.1mg/l, significantly the quantity of the indefinite bud of inducing more than blade.Simultaneously, temperature plays very crucial effect for inducing with the growth of tissue cultivating seedling of indefinite bud, and between 20~23 ℃, tissue cultivating seedling leaf look dark green, robust growth; Surpass 25 ℃, the tissue cultivating seedling flavescence is also dead gradually.Under such condition of culture, 2 months value-added coefficient of test-tube plantlet reaches 3~4, has greatly improved the herald spring reproduction coefficient of (Primula poisonii) of Hai Xian.
Embodiment 3
The herald spring culture of rootage of (Primula poisonii) tissue cultivating seedling of Hai Xian: when indefinite bud grows 2~3 leaves on differential medium, it is excised from callus, be transferred to root media, prescription: among MS, the MS+NAA (0.1mg/l, 0.2mg/l, 0.5mg/l), pH is 5.8; Illumination condition is that natural scattering light 3000Lux adds artificial secondary light source 3000Lux, light application time 14h.
From taking root the needed time, the required time of taking root in medium MS and MS+0.2mg/l NAA is the shortest, it is 8~10 days, but in MS+0.2mg/l NAA, tissue cultivating seedling amount of taking root and growing way are significantly better than the MS minimal medium, the tissue cultivating seedling average height can reach 4.8cm, and rooting rate is more than 95%.
Embodiment 4
The herald spring transplanting of (Primula poisonii) tissue cultivating seedling of Hai Xian: culture of rootage is after two weeks, treats that the average every strain of tissue cultivating seedling takes root 4~5, when root reaches 1cm, can begin to prepare for transplanting.Hardening is the transition before the test-tube seedling transplanting, can help test-tube plantlet progressively to adapt to external environment, makes test-tube plantlet more healthy and stronger, and the leaf look more dark green, thereby improves survival rate.
Hai Xian was heralded spring (Primula poisonii) test-tube plantlet hardening after 3~4 days, transplanted to the thin turfy soil in greenhouse, and turfy soil is paved with the cave dish in advance, and clear water soaks into; Keep humidity more than 80% behind the test-tube seedling transplanting, cover and spray water film and every day 2~3 times, 2 weeks can progressively be carried out normal management later, and transplanting survival rate is almost 100%.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (1)

1. tissue culture and rapid propagation method that extra large celestial being is heralded spring, comprise explant selection, surface sterilization, adventitious bud inducing, successive transfer culture, culture of rootage and test-tube seedling transplanting, it is characterized in that, the axillalry bud that selects Hai Xian to herald spring is an explant, rinse well through water, adopt 0.1% mercuric chloride solution sterilization, 3~4min, under aseptic condition, clean repeatedly through distilled water, be seeded among the inducing culture MS+2.0mg/l 6-BA+0.1mg/l NAA, pH is 5.8, and illumination condition is that natural scattering light 2500~3000Lux adds artificial fill-in light 1500~2000Lux; Move to same medium propagation afterwards, subculture 2~3 times, root media are MS+0.2mg/l NAA, and pH is 5.8, and illumination condition is that natural scattering light 2500~3000Lux adds artificial fill-in light 2500~3000Lux; Temperature remains between 20~23 ℃; In 2~3 weeks of culture of rootage, hardening moved in the thin turfy soil and cultivates after 3~4 days, and transplanting temperature is 18~25 ℃, kept humidity to be higher than 80%, sprayed water every day 2~3 times.
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Publication number Priority date Publication date Assignee Title
CN101965796B (en) * 2010-10-14 2012-07-25 北京林业大学 Method for performing tissue culture and rapid propagation on primula saxatilis
CN103070064B (en) * 2012-12-17 2013-12-18 中国科学院昆明植物研究所 Artificial hybrid breeding method of Primula L.
CN107041308A (en) * 2017-04-26 2017-08-15 江苏农林职业技术学院 A kind of primrose root tissue culture propagation method
CN112293251A (en) * 2020-10-19 2021-02-02 云南中医药大学 Artificial efficient primula forbesii breeding method

Non-Patent Citations (4)

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Title
李燕等.高穗花报春的组织培养和快速繁殖.植物生理学通讯41 6.2005,41(6),796.
李燕等.高穗花报春的组织培养和快速繁殖.植物生理学通讯41 6.2005,41(6),796. *
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