CN103098714A - Method for improving regeneration in vitro and conversion rate of cucumber cotyledonary node - Google Patents
Method for improving regeneration in vitro and conversion rate of cucumber cotyledonary node Download PDFInfo
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Abstract
The invention provides a method for improving regeneration in vitro and conversion rate of a cucumber cotyledonary node. The method comprises the following steps of: (1) selecting two cucumber cotyledonary nodes with two cotyledons which are rightly released as explants; (2) slightly scratching wounds on roots of the cucumber cotyledonary nodes; (3) inoculating the cucumber cotyledonary nodes into a bud induction culture medium to be cultured; and (4) carrying out bud induction, elongation and rooting to finally obtain a complete plant. According to the invention, an optimal seedling state and bud induction culture medium for improving the regeneration in vitro and conversion rate of the cucumber cotyledonary node are provided to optimize a cucumber S52 cotyledonary node regeneration system; the method is simple and easy to operate; and meanwhile, the quantity of cultivated adventitious buds is large, the quality is good, seedlings are strong, the vitrification is low, the regeneration rate is high and the regeneration period is short. The method disclosed by the invention provides firm technical supports for cucumber genetic transformation and molecular modification and has very important practical significance for innovating cucumber germplasm and new species by using a modern genetic engineering technology.
Description
Technical field
The present invention relates to plant genetic engineering, particularly a kind ofly do explant by the optimum seedling attitude of screening cucumber, improve the method for cucumber cotyledons joint Regeneration in Vitro and conversion ratio.
Background technology
Cucumber (Cucumis sativus L.) is Curcurbitaceae (Cucurbitaceae) Cucumis, annually overgrows or climbs up by holding on to herbaceous plant.Its fruit instant, tender and crisp succulence, unique flavor, the fruit and vegetable dual-purpose, nutritive value is high, even can be used for beauty treatment, liked by the modern.In recent years, the breed cucumber target tightening is obtained larger economic benefit to increase the yield per unit area aspect quality, degeneration-resistant and Resistant.Because the cucumber hereditary basis is narrow, there is the distant hybridization obstacle, therefore utilizing technique for gene engineering will be the effective means of cucumber variety improvement from now on.In addition, various because of the differentiation of cucumber sexual type, cucumber becomes the type material of research plant sexual type differentiation gradually.Current, increasing researcher joins cucumber molecular breeding and the correlation molecule biological study field take cucumber as research material.Especially completing of Cucumber germplasm order-checking, accelerated the process of cucumber molecular breeding and correlation molecule biological study, and plant genetic engineering is the core means of carrying out above-mentioned research.At present, generally take on plant to carry out transgeneic procedure with agriculture bacillus mediated genetic transformation system, therefore, ripe and stable plant tissue isolated regeneration culture and genetic conversion system become and utilize plant genetic engineering to carry out the bottleneck of genetic improvement and molecular breeding.
The isolated regeneration culture of cucumber and Study on Genetic Transformation start from 1979, the factor that affects cucumber regeneration and genetic transformation is many, comprises the use, preculture time, time of infection of genotype, explant type, seedling attitude, plant growth regulator, agrobacterium strains and activity, external force (as hyperacoustic use), aldehydes matter, the selection of inducing of incubation time, bud is pressed, taken root and select to press etc. altogether.At present, the researcher has carried out the research of regeneration plant to cucumber cotyledons, cotyledonary node, true leaf, hypocotyl, radicle etc., but still have the inferior series of problems of bud ratio, regeneration rate and low conversion rate, seriously hindered the application of plant genetic engineering in the improvement of cucumber molecular genetic.
Zhao Junliang [1996, the Shanxi Agricultural science, the cucumber cotyledons Regenerated Plantlets] cotyledon and the hypocotyl (medium is the MS medium of BA and NAA) of six cucumber varieties is studied, found that the cotyledon base portion can directly differentiate indefinite bud, the upper part of cotyledon can not be regenerated, hypocotyl can not break up, and thinks that optimum cucumber explant is cotyledonary node.Hou Aiju [2003, gardening journal, the research of the direct organ generation of inducing cucumber major influence factors] and Du Shengli [2000, Tianjin agricultural science, seedling age, genotype and explant type are on the organogenetic impact of Cucumber In Vitro] etc. also obtain similar results.In cucumber cotyledons joint Regeneration System, ABA matches with kinetin can promote Plantlet Differentiation.Sekioka etc. studies show that in the tissue of cucumber is cultivated, low concentration ABA can promote the normal development [1981 of embryoid, Hort Science Differentiation in callus culture of cucumber (Cucumis sativus L.)], control embryoid and be in globular embryo or globular embryo later stage [Chen Zheng etc., 2001, the Tianjin agricultural science, the research that improves cucumber Agrobacterium genetic conversion system regeneration frequency].In plant tissue culture course, Growth of Cells can produce and accumulate ethene, and then affects the differentiation of cell, Ag in silver nitrate
+Be the ethene activity inhibitor, can be incorporated into competitively the ethylene action position, alleviated the effect of ethene, thereby promote plant organ to occur and somatic embryo generation [Mohiuddin A K M, 1997, Plant Cell, Tissue and Organ Culture, Influences of silver nitrate oncucumber in vitro shoot regeneration; Gao Wujun, 2002, Chinese agronomy circular, the influence of silver nitrate in the rape genetic transformation].Cao Lixian etc. studies show that, AgNO
3Can improve significantly the cucumber shoot regeneration frequency, with 2.0mg/L AgNO
3Effect is [2001, Gansu Agriculture University,'s journal, the promotion effect of silver nitrate to Cucumber In Vitro Cotyledon culture shoot regeneration] the most obviously.The size of explant is one of important internal factor that affects the cotyledon node regeneration frequency.Explant size research aspect is mainly take seedling age as foundation, think best seedling age be 1-7d report all arranged, explant due to same seedling age, its physiological status is not identical, and generally the germination rate of cucumber seeds and germination vigor are also variant, so adopt different seedling attitude cucumber cotyledons joints more rational.
Summary of the invention
Purpose of the present invention, for a kind of method that improves cucumber cotyledons joint Regeneration in Vitro and transformation efficiency is provided, study different seedling attitudes to the situation that affects of cucumber genetic transformation efficiency, filter out the best seedling attitude of cucumber genetic transformation, and the high best medium of regeneration rate, enrich the Cucumber In Vitro regenerating system, for the foundation of cucumber genetic conversion system from now on provides theory and technology basic.
The technical solution used in the present invention is as follows:
A kind of method that improves cucumber cotyledons joint Regeneration in Vitro and genetic transformation rate comprises the following steps:
(1) the cucumber cotyledons joint of selecting two cotyledons just to break away from kind of shell is external body;
(2) draw gently upper wound at step (1) gained cucumber cotyledons joint base portion;
(3) step (2) gained cucumber cotyledons joint is received in the bud inducing culture cultivated, this bud inducing culture is by MS medium and 6-BA, ABA, AgNO
3Formulated, wherein the concentration of 6-BA is 1.5mg/L, and the concentration of ABA is 0.5mg/L, AgNO
3Concentration be 2.0mg/L;
(4) cucumber cotyledons joint is received the bud inducing culture after, induce, extend, take root through bud, obtain at last whole plant.
In said method, described MS medium is the medium that contains 5.8g/L agar, 30g/L sucrose, and pH is 5.8.
The present invention saves as explant with cucumber cotyledons, study different seedling attitudes to the impact of Cucumber In Vitro regeneration and transformation efficiency, comprised screening, the gus transient expression under different seedling attitude of seedling attitude, the optimization of plant growth regulator (being in the method the bud inducing culture) proportioning.Specifically follow these steps to carry out:
1, the screening of seedling attitude
4 seedling attitudes are defined as: (1), cotyledon shelling 1/2-2/3; (2), two cotyledons have just broken away from kind of a shell; (3), hypocotyl is upright, but cotyledon does not flatten; (4), two cotyledons just flatten (referring to Fig. 1).Cut the cotyledon with the 1-2mm petiole, every cotyledon removal growing point of cutting sth. askew, and the 1/3-1/2 of excision cotyledon the first half, with the cotyledonary node adaxial and its surface up 75 degree angle oblique cuttings enter (MS+1.5mg/L 6-BA+1.5mg/L ABA+2.0mg/LAgNO in the bud inducing culture
3), every 2 all subcultures 1 time count bud explant number, bud number after sprouting, and result filters out optimum seedling attitude accordingly.
2, the gus transient expression under different seedling attitudes
With the cotyledon cutting of sprouting to different seedling attitudes, nearly pommel cotyledonary node carries out respectively three kinds of processing: (1) does not scratch mouth; (2) scratch lightly mouth; (3) scratch mouth than the important place.Receive afterwards preculture 1d on the bud inducing culture, afterwards with the bacterium liquid (OD that contains the pCAMBIA2301-gus expression vector
600=0.3-0.6) infect 20min, go to (bud inducing culture+50 μ mol/L acetosyringones) cultivation 3d on common culture medium after end, after end, explant is gone in the bud inducing culture (adding the 300mg/L Ticarcillin/Clavulanate Acid to suppress the growth of Agrobacterium) that contains kanamycin (Kan) and select to cultivate, after 5d, its transient expression rate of gus staining examine is determined the optimum preculture time.
Gus colouring method: first explant is placed in dyeing liquor (100mmol/L sodium phosphate buffer, 2mmol/LX-glue, the 2mmol/L potassium ferricyanide solution, the 2mmol/L potassium ferrocyanide solution, 0.5% (V/V) TritonX-100) in, dyeing is spent the night, and removes dyeing liquor, adds acetone: ethanol (1: 2, V/V) in mixed liquor, decolouring is spent the night, and dissects Microscopic observation dyeing situation and takes pictures.
3, the optimization of plant growth regulator proportioning
Cotyledonary node take the best seedling attitude of screening cuts as explant, adaxial and its surface 75 degree angle oblique cuttings up enters to add in the MS medium of variable concentrations 6-BA (6-benzyl aminoadenine) and ABA (abscisic acid) and (contains 5.8g/L agar, 30g/L sucrose, pH 5.8, lower same), add appropriate AgNO
3(2.0mg/L AgNO
3Can significantly improve shoot regeneration frequency) carry out bud and induce.6-BA establishes 3 concentration gradients (1.0,1.5,2.0mg/L), and ABA establishes 4 concentration gradients (0.5,2.0,1.5,2.0mg/L), and dual factors are done mutually, totally 12 treatment combinations.Explant is received on the inducing culture that contains 6-BA and ABA, every 2 all subcultures 1 time count bud explant number, bud number after sprouting, select the Optimal Medium formula according to result.When bud grows to 1-2cm, the 1/2MS medium is received in its cutting-out flourishing to root, calculate from being seeded to and obtain the whole plant required time.
4, inductivity and several calculating of on average sprouting
Inductivity=going out bud explant number/explant falls apart * 100%;
Number=total bud number/go out bud explant number on average sprouts.
Total data adopts SPSS 14.0 for Windows softwares to carry out the LSD statistical analysis.
Method of the present invention provides best seedling attitude and the bud inducing culture that improves the regeneration of cucumber cotyledons joint and transformation efficiency, has optimized the cotyledon node regeneration system of cucumber S52, and method is simple, easy to operate; Cultivation indefinite bud number out is many simultaneously, quality good, healthy and strong, vitrifying is low, and regeneration rate is high, and the regeneration period is short.For cucumber genetic transformation and molecular improvement provide solid technical support, will have important practical significance to utilizing modern genetic engineering technological innovation Cucumber Germplasm and new varieties.
Description of drawings
Fig. 1 is four seedling attitudes, and wherein, (1) number seedling attitude is cotyledon shelling 1/2-2/3; (2) number seedling attitude is that two cotyledons have just broken away from kind of a shell; (3) number seedling attitude is that hypocotyl is upright, but cotyledon does not flatten; (4) number seedling attitude is that two cotyledons just flatten.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.
1, the acquisition of aseptic seedling
Cucumber S52 is the south China kind, synoecy is got full seed and uniform cucumber seeds, soaking at room temperature 30min to 1h in clear water, then in superclean bench with 70% alcohol immersion 30s, use 1%NaClO solution disinfection 10min, aseptic water washing 3-5 time (each 3min) soaks 4-5h afterwards in sterile water again, be tiled on the 1/2MS medium, 15 seeds of every bottle graft, 25 ± 1 ℃ of cultivation temperature are cultured under dark condition to go to after seed shows money or valuables one carries unintentionally under light and cultivate, sprout.25 ± 1 ℃ of cultivation temperature, light application time 16h, intensity of illumination 2000lx.
2, the impact of different seedling attitudes on the regeneration of cucumber cotyledons joint
4 seedling attitudes are defined as: (1), cotyledon shelling 1/2-2/3; (2), two cotyledons have just broken away from kind of a shell; (3), hypocotyl is upright, but cotyledon does not flatten; (4), two cotyledons just flatten.20 explants of each treatment combination repeat for 3 times totally.
Cut the cotyledon with the 1-2mm petiole, every cotyledon removal growing point of cutting sth. askew, and the 1/3-1/2 of excision cotyledon the first half, with the cotyledonary node adaxial and its surface up 75 degree angle oblique cuttings enter (MS+1.5mg/L6-BA+1.5mg/L ABA+2.0mg/L AgNO in the bud inducing culture
3), every 2 all subcultures 1 time count bud explant number, bud number after sprouting, and as shown in table 1, result filters out optimum seedling attitude accordingly.
As can be seen from Table 1, the cucumber explant inducing clumping bud rate of different seedling attitudes is different, and the too small or regeneration frequency when excessive of cucumber explant and the average every explant number average that sprouts is lower.(1) all can go out indefinite bud by high-frequency induction to the cotyledonary node of (3) seedling attitude, wherein regeneration frequency and the every explant regeneration bud number during seedling attitude (2) is the highest, and regeneration frequency is up to 100%, and the number that on average sprouts reaches 4.13, is significantly higher than other seedling attitudes.Inductivity (48.33%) and every explant regeneration bud number (0.48) during seedling attitude (4) are minimum, significantly lower than other seedling attitudes.In sum, selecting the cotyledonary node of seedling attitude (2) is best explant seedling attitude.
The impact of the different seedling attitude of table 1 explant on the regeneration of cucumber cotyledons joint
Annotate: different letter representation significant differences (P＜0.05) in same column data.
3, the gus transient expression situation of different seedling attitude explants
With the cotyledon cutting of sprouting to different seedling attitudes, nearly pommel cotyledonary node carries out respectively three kinds of processing: (1) does not scratch mouth; (2) draw lightly a little wounds; (3) scratch mouth than the important place.Receive afterwards preculture 1d on the bud inducing culture, afterwards with the bacterium liquid (OD that contains the pCAMBIA2301-gus expression vector
600=0.3-0.6) infect 20min, go to (bud inducing culture+50 μ mol/L acetosyringones) cultivation 3d on common culture medium after end, after end, explant is gone in the bud inducing culture (adding the 300mg/L Ticarcillin/Clavulanate Acid to suppress the growth of Agrobacterium) that contains kanamycin (Kan) and select to cultivate, after 5d, its transient expression rate of gus staining examine is determined the optimum preculture time.
Gus colouring method: first explant is placed in dyeing liquor (100mmol/L sodium phosphate buffer, 2mmol/LX-glue, the 2mmol/L potassium ferricyanide solution, the 2mmol/L potassium ferrocyanide solution, 0.5% (V/V) TritonX-100) in, dyeing is spent the night, and removes dyeing liquor, adds acetone: ethanol (1: 2, V/V) in mixed liquor, decolouring is spent the night, and dissects Microscopic observation dyeing situation and takes pictures.
Coloration result shows, under same cutting method, the explant of seedling attitude (1) and (2) occurs that blue area is larger, and gus instantaneity expression rate is higher, and the dyeing area of seedling attitude (4) is minimum, illustrates that seedling age is larger, and transformation efficiency is lower; And the coloration result of seedling attitude (2) is ideal, and the dyeing area is maximum.Under same seedling attitude, when the slight wound mouth is arranged, gus instantaneity expression rate is the highest, when wound is overweight, almost there is no colors blue, and yellowish-brown even appears in explant, and gus instantaneity expression rate is extremely low, illustrates that when the explant wound is heavy, transformation efficiency is extremely low.Therefore, when drawing the slight wound mouth during cutting explant, gus instantaneity expression rate is the highest, and transformation efficiency is the highest.So the seedling attitude is that two cotyledons break away from kind of shell and when its cotyledonary node base portion was drawn wound gently, its dyeing area was maximum, color is the darkest, and gus transient expression amount is the highest, and Agrobacterium-mediated Transformation is most effective.
4, the impact of plant growth regulator on the regeneration of cucumber cotyledons joint
Cotyledonary node take the best seedling attitude of screening cuts as explant, adaxial and its surface 75 degree angle oblique cuttings up enters to add in the MS medium of variable concentrations 6-BA (6-benzyl aminoadenine) and ABA (abscisic acid) and (contains 5.8g/L agar, 30g/L sucrose, pH 5.8, lower same), add appropriate AgNO
3(2.0mg/L AgNO
3Can significantly improve shoot regeneration frequency) carry out bud and induce.6-BA establishes 3 concentration gradients (1.0,1.5,2.0mg/L), and ABA establishes 4 concentration gradients (0.5,2.0,1.5,2.0mg/L), and dual factors are done mutually, totally 12 treatment combinations.Explant is received on above-mentioned inducing culture, and every 2 all subcultures 1 time count bud explant number, bud number after sprouting, select the Optimal Medium formula according to result.When bud grows to 1-2cm, the 1/2MS medium is received in its cutting-out flourishing to root, calculate from being seeded to and obtain the whole plant required time.
After being connected to explant on medium, inducing, extend, take root through bud, obtain at last whole plant, in table 2, data show, 6-BA and ABA induce S52 strain cucumber cotyledons joint and have a significant impact, various combination, and it is larger that result differs.Inductivity is higher when 6-BA concentration is 2.0mg/L, but in practical operation, when 6-BA was 2.0mg/L, explant vitrifying degree was higher, and its leaf and the tender tip are the transparent or semitransparent water stain shape of crystal, and swelling, chlorosis, frangible are downgraded in whole strain.On average sprout simultaneously several less, and be unfavorable for the foundation of cucumber high-efficiency regeneration system.And be in four groups of processing of 1.5mg/L when 6-BA concentration, when two kinds of hormone concentration and media were 1.5,0.5, inductivity reached 83.33%, and the number that on average sprouts can reach 4.47, be significantly higher than other processing, so select MS+1.5mg/L 6-BA+0.5mg/L ABA+2.0mg/L AgNO
3For utilizing cotyledonary node, S52 strain cucumber carries out the bud inducing culture of adventitious bud inducing regeneration plant.
After the cotyledonary node of 4d seedling age is received the bud inducing culture that the present invention selectes, approximately Multiple Buds appears in 15d, bud grows to 1-2cm (approximately 10d), bud is downcut carry out root induction, approximately 7d can send out roots, the 15d root system is comparatively flourishing, calculates with this, and the shortest 45d of cucumber cotyledons joint isolated regeneration culture can obtain whole plant.
The impact that table 2 6-BA and ABA induce S52 strain cucumber cotyledons nodal bud
Annotate: different letter representation significant differences (P＜0.05) in same column data.
Claims (2)
1. one kind is improved the method that cucumber cotyledons saves Regeneration in Vitro and genetic transformation rate, comprises the following steps:
(1) the cucumber cotyledons joint of selecting two cotyledons just to break away from kind of shell is external body;
(2) draw gently upper wound at step (1) gained cucumber cotyledons joint base portion;
(3) step (2) gained cucumber cotyledons joint is received in the bud inducing culture cultivated, this bud inducing culture is by MS medium and 6-BA, ABA, AgNO
3Formulated, wherein the concentration of 6-BA is 1.5mg/L, and the concentration of ABA is 0.5mg/L, AgNO
3Concentration be 2.0mg/L;
(4) cucumber cotyledons joint is received the bud inducing culture after, induce, extend, take root through bud, obtain at last whole plant.
2. method according to claim 1, is characterized in that, described MS medium is the medium that contains 5.8g/L agar, 30g/L sucrose, and pH is 5.8.
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Application publication date: 20130515 |