CN103734016A - Breeding method for in-vitro induction of autotetraploid brassica oleracea l. var. italica plenck - Google Patents

Abstract

The invention belongs to the field of crop breeding and discloses a breeding method for in-vitro induction of autotetraploid brassica oleracea l. var. italica plenck. The breeding method comprises the following steps: reserving a 0.4-0.6-cm seedling of a hypocotyl as an explant in the cotyledon period of the brassica oleracea l. var. italica plenck, inoculating the explant to a bilayer differentiation culture medium containing colchicine and performing induction culture for 45-50h, and directly inducing to grow a tetraploid bud, thus further obtaining the autotetraploid brassica oleracea l. var. italica plenck rapidly and stably. According to the breeding method disclosed by the invention, an asexual polyploidization breeding way is adopted for the first time, the bilayer induction based on the liquid culture medium (with addition of colchicine) and the solid culture medium (without addition of colchicine) is adopted, and the 0.4-0.6-cm seedling of the hypocotyl reserved in the cotyledon period of the brassica oleracea l. var. italica plenck is processed in a combinational way to obtain a new germplasm of the tetraploid brassica oleracea l. var. italica plenck. The breeding method has the advantages of high induction rate, fewer chimera, low death rate and low contamination rate, a large quantity of tetraploid plants can be obtained by performing meristematic proliferation on the obtained tetraploid material, and thus, the induction time is greatly shortened.

Description

A kind of breeding method of in-vitro inducing autotetraploid broccoli

Technical field

The invention belongs to crop breeding field, relate to a kind of breeding method of in-vitro inducing autotetraploid broccoli, be exclusively used in the seed selection of tetraploid broccoli.

Background technology

Broccoli (Brassica oleraceaL var italica Plenck) has another name called broccoli, cauliflower, broccoli, being under the jurisdiction of Cruciferae rape belongs to, be a mutation in wild cabbage in Cruciferae, it mainly take green bouquet as main product organ.Its natural kind and each cultivated species are all dliploid, i.e. 2n=2x=18.Broccoli be a kind of suitable chromosome doubling improvement crop (Ji Dandan, the antitumaous effect of the bright .2007. broccoli of Zhao Xiao and medicinal polyploid breeding Research Prospects. China's Vegetable, (8): 39-41.).At present China uses the breeding methods such as some heterosis breedings, conventional breeding, tissue cultivation to cultivate the dliploid broccoli kind of many high-qualitys, but deficienter for the cultivation of polyploid broccoli kind with larger using value.In prior art, have inventor at home and abroad first induced mutations tetraploid broccoli " spring is green " new varieties (outstanding. the initiative of autotetraploid broccoli and the research of biological property [D] thereof. Nanjing: Agricultural University Of Nanjing, 2006.); Utilize colchicine in-vitro inducing obtain tetraploid broccoli " sea hereby " new varieties (Zhang Shuning, Zhang Lili, Tang Jun, Kong Yane, marquis likes woods .2009. colchicine in-vitro inducing autotetraploid broccoli. gardening journal, 36(11): 1681-1684.).Broccoli nutritive value and abundant, particularly its medical value, it contains a kind of material " sulforaphen " with stronger antitumaous effect.Therefore, for the research of broccoli polyploid breeding, be very significant, and prospect is boundless.

Polyploid refers to have the individuality that 2 above basic chromosomes form.The cumulative effect that a plurality of chromosome sets of polyploid produce increases its biomass, mineral matter and nutrient component, in proterties, show as leaf large, spend in large, the thick color depth of stem, cell the higher and resistance of Cucumber content to strengthen (Lv Xinye, Wang Ji people .1997. China grain supply and demand prediction [J]. agricultural modernization research, 18(1): 12-16.).And at present Polyploid Induction Methods is used maximum be exactly colchicine-induced method.Colchicine-induced can be divided into the induction of in vitro and the induction of non-in vitro.The induction of non-in vitro has: infusion process, injection, agar method, drip method etc.Zhang Hongliang etc. utilize colchicine drop to process cotyledon period dliploid radish shoot tip meristem, thereby acquisition " punching is red " Autotetraploid Radish (Zhang Hongliang. the initiative of " punching is red " Autotetraploid Radish and physio-biochemical characteristics research [D] thereof. Nanjing: Agricultural University Of Nanjing, 2007.); Liu Xue Min etc. by drip method and soak two kinds of methods of root method to dliploid Chinese cabbage " east summer king " carry out colchicine double induction, find that the inductivity of drip method is with respect to soaking root method, better effects if, inductivity higher (Liu Xuemin, Cao Caixia, Wang Yuhai, Mu Jingui, Wang Mingqiu, the research of Liu Xiao east .2004. Chinese cabbage tetraploid induction method. Hebei Agricultural Sciences, 8 (3): 14-16.); Although these methods are producing the effect doubling in varying degrees, there is the problems such as the polyploid strain number obtaining is few, and inductivity is low, and environment is wayward, and workload is large, and chimera is more.With respect to the induction of non-in vitro, the induction of in vitro may be a better approach.In vitro induction cultured cell or tissue staining body double, its approach mainly contains two: the one, and infusion method, first use colchicine solution (Gu Xiaofeng, the sweet persimmon in sieve positive flourish .2003. colchicine processing-Luotian. obtain 12 times of body regeneration plants. gardening journal, 30 (3): 325-327.) or the liquid nutrient medium (Zhang Suzhi that contains colchicine, the tetraploid research of Li Ji Rong .2006. colchicine-induced garlic. nuclear agricultural science report, 20 (4): 303-308.) process culture materials, then proceed to differential medium; The 2nd, mixed training method, at the solid culture medium containing colchicine, cultivate a period of time, induction doubles, proceed to again differential medium (Sun Minhong, Zhang Shuning, the screening of Deng Yun .2005. induction dliploid and autotetraploid Chinese cabbage cytoplasmic male sterile line indefinite bud medium. Plant Physiology Communications, 41 (6): 741-744.).In vitro induction polyploid is all by infusion method mostly, because mixed training method induction time is long, inductivity is low, infusion method can obtain more polyploid strain number, inductivity is higher, can greatly shorten induction required time, and can overcome the difficult problem that conventional mutation breeding is subject to environmental influence.But it can exist easy pollution, and lethality is high, also have long immersion treatment may cause the problems such as anoxic.Model Guoqiang etc. by Double-Medium (on, lower floor is respectively direct liquid (not adding agar powder) and the solid culture medium (spreading 2 layers of aseptic filter paper above) occurring of organ that 10ml is identical with 30ml colchicine concentration) blade of Paulownia elongata is induced and doubled, Dan Yinqi lower floor solid culture medium also contains the colchicine of same concentrations, so this kind of method is similar to infusion method, can not obviously reduce the toxic action of colchicine to a certain extent, and this class methods levels function unreasonable distribution, the sphere of action of the solid culture medium of lower floor in bilayer induction is too narrow.Utilize blade as explant simultaneously, if colchicine excessive concentration, can be in incubation brownization dead, and using blade as explant, be through generation callus, then produce embryoid, process is comparatively loaded down with trivial details.(model Guoqiang, Yang Zhiqing, Cao Yanchun, Liu Fei, Jia Feng .2006. Autotetraploid Induction of Paulownia Elongata With Colchione. nuclear agricultural science report, 20 (6): 473-476.).The induction of in vitro, the selection of explant material mostly is blade, ball stem, from bud etc.; So far also not under in vitro condition with cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm induce the report doubling.The approach that obtains polyploid by vitro tissue culture and inducement is not only relevant with induction mode and explant, goes back and kind, and culture environment, colchicine concentration and induction time etc. are relevant.Colchicine concentration and induction time that different cultivars, different explants and different induction mode are corresponding different.And its suitableeest culture environment of different kinds is also different, in its suitableeest culture environment, it is induced and doubles to increase the chromosomal efficiency that doubles.So for the deficiency in prior art, we propose a kind of new asexual polyploidization breeding technique.In prior art, also do not adopt liquid nutrient medium (interpolation colchicine) and the double-deck new tetraploid asexual polyploidization breeding technique of seedling induction dliploid broccoli acquisition of inducing and retaining plumular axis 0.4 ?0.6cm in conjunction with processing cotyledon period of solid culture medium (not adding colchicine) both at home and abroad at present.

Summary of the invention

The object of the invention is the above-mentioned deficiency for prior art, a kind of breeding method of in-vitro inducing autotetraploid broccoli is provided.

Object of the present invention is achieved through the following technical solutions:

A kind of breeding method of in-vitro inducing autotetraploid broccoli, comprise: the broccoli cotyledon period of take retain plumular axis 0.4 ?the seedling of 0.6cm be explant, by explant be seeded to containing induction on the double-deck differential medium of colchicine cultivate 45 ?50h, through regeneration propagation, directly produce tetraploid bud again, and then obtain autotetraploid broccoli through follow-up cultivation; Wherein, the described double-deck differential medium containing colchicine is by upper strata: MS minimal medium+NAA0.05 ?0.1mg/L+6 ?BA2.5-3mg/L+50 ?60ml/L Coconut Juice+300 ?350mg/L colchicine+30g/L sucrose, pH5.8; And lower floor: MS minimal medium+NAA0.05 ?0.1mg/L+6 ?BA2.5-3mg/L+50 ?Coconut Juice+30g/L sucrose+7.8g/L agar of 60ml/L, pH5.8 forms.

The breeding method of described autotetraploid broccoli, described method comprises the steps:

1) Double layer culture induction polyploid: cut broccoli cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm, after preculture 2d, be seeded to containing in the double-deck differential medium of colchicine, be placed on shaking table, process 45 ?50h, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

2) regeneration propagation: the explant after previous step is processed forwards differentiation and proliferation on differentiation and proliferation medium to and obtains tetraploid bud after aseptic water washing 3 times, the differentiation and proliferation medium using for MS minimal medium+NAA0.05 ?0.1mg/L+6 ?BA2.5-3mg/L+ Coconut Juice 50 ?60ml/L+ sucrose 30g/L+ agar 7.8g/L, pH5.8; Cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

3) acquisition of regeneration plant: after 40d, by tetraploid bud cultivate in MS minimal medium+80 without hormone ?in the medium of 100ml/L Coconut Juice+0.5g/L acid hydrolyzed casein+sucrose 30g/L+ agar 7.8g/L 10 ?15d, obtain regeneration plant, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

4) the taking root and tame of regeneration plant: by regeneration plant proceed to 1/2MS minimal medium+NAA0.2mg/L+ Coconut Juice 20 ?in the medium of 30ml/L+ sucrose 30g/L+ agar 7.8g/L, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx cultivation 15～20d and takes root, and then tames, practices seedling and transplant;

5) evaluation of regeneration plant: before transplanting, get the tip of a root of regeneration plant, carry out Chromosome Identification after flow cytometer is identified, transplant after 1 month regeneration plant is carried out to Morphological Identification, Stomatal appraisal and flow cytometer evaluation.

In the breeding method of described autotetraploid broccoli, described is preferably 300mg/L containing colchicine concentration in the double-deck differential medium of colchicine, and explant is being induced the preferred 48h of incubation time containing on the double-deck differential medium of colchicine.

In the breeding method of described autotetraploid broccoli, the described double-deck differential medium containing colchicine at the middle and upper levels the ratio of the volume of liquid nutrient medium and the volume of lower floor's solid culture medium be 2:9 ?2:7, the hypocotyl of explant is vertically inserted in solid culture medium, cotyledon period shoot tip meristem is immersed in supernatant liquid medium, and cotyledon is exposed to above liquid nutrient medium.

The breeding method of described autotetraploid broccoli, described broccoli cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm preferably obtain by the following method: by 75% alcohol processing 1min for broccoli seeds, 0.1% mercuric chloride is processed 15min, aseptic water washing 3 ?4 times, be inoculated into without the MS minimal medium+Coconut Juice 20 of hormone ?cultivate 6d in the medium of 30ml/L+ sucrose 30g/L+ agar 7.8g/L, seedling cotyledon launches completely, take out aseptic seedling, apart from cotyledon petiole end 0.4 ?0.6cm hypocotyl place cut, leave strip part cotyledonous is as explant; Cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx.

The breeding method of described autotetraploid broccoli, described preculture method is preferred: cut cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm be inoculated into MS minimal medium+NAA0.05 ?0.1mg/L+6 ?BA2.5-3mg/L+ Coconut Juice 50 ?preculture 2 days in the medium of 60ml/L+ sucrose 30g/L+ agar 7.8g/L, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx.

Beneficial effect of the present invention:

The present invention adopts the double-deck induction of liquid nutrient medium (interpolation colchicine) and solid culture medium (not adding colchicine) combination to process the asexual polyploidization breeding approach that cotyledon period retains the seedling of plumular axis 0.4 ?0.6cm first, obtains tetraploid broccoli new germ plasm.To compare inductivity higher with the method for Sexual polyploid, a large amount of tetraploid plants of acquisition that can fast and stable.In modern polyploidization technical research, colchicine concentration and induction time are the keys that affects inductivity always.Colchicine concentration is higher, and the processing time is longer, larger to the inhibition degree of plant tissue.The present invention finds by a large amount of experiments, preculture two days, with 300-350mg/L colchicine, organize the double-deck induction of training in vitro, both can strengthen and process material to the adaptability of environment of living in and the resistance to colchicine toxic action thereof, again can successful induce chromosome redoublement.Particularly the present invention organizes the medium of the double-deck induction of training in vitro, by supernatant liquid, cultivate to reach colchicine-induced effect, by lower floor, without the solid culture of colchicine, alleviating or alleviate colchicine poisons, can reduce colchicine and poison accumulation, can recover or improve doubling rear explant regeneration ability, and then induce its differentiation and proliferation to expand numerous tetraploid.

In bilayer induction is cultivated, by broccoli cotyledon period retain plumular axis 0.4 ?the hypocotyl of seedling of 0.6cm be vertically inserted in solid culture medium, object is that fixedly explant reaches that inductive effect is carried out on upper strata and lower floor alleviates or alleviate colchicine poisons, the nutrition supplying plant that can absorb in solid culture medium by hypocotyl grows normally, secondly, cotyledon period shoot tip meristem is immersed in supernatant liquid medium, and cotyledon is exposed to above liquid nutrient medium, the one, cotyledon can provide seedling development required nutritive element by photosynthesis, the 2nd, can prevent the hypoxia death causing due to long-time immersion treatment, reduce lethality.

The present invention adopt direct processing cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm, can directly produce bud without the stage that produces callus, greatly shorten induction time.

The explant lethality that the present invention processed is low, and average survival rate is 98.41%, and value-added coefficient is 2.19, and inductivity is high, and tetraploid induction rate is up to 77.78%, and tetraploid plant is through differentiation and proliferation, can expand rapidly numerous a large amount of tetraploids that go out.

Accompanying drawing explanation

Fig. 1. dliploid pore (* 40)

Fig. 2. tetraploid pore (* 40)

Fig. 3. dliploid chromosome (* 100)

Fig. 4. tetraploid chromosomes (* 100)

Fig. 5. dliploid flow cytometer figure

Fig. 6. tetraploid flow cytometer figure

Fig. 7. the tetraploid plant (LQ-23) that just group training tames out

Fig. 8. be transplanted to farm two, tetraploid plant contrast (LQ-23, a left side is tetraploid, the right side is dliploid)

Embodiment

Embodiment 1

1 materials and methods

1.1 for examination material

Experiment material is dliploid broccoli (2n=2x=18) LQ-2(" green elegant 80 days ", manufacturer is that Xiamen City's Xiang is equipped with power vegetable seedling station), S139-2(" Nan Xiu 366 ", manufacturer U.S. Sheng Nisi) and LQ-23(" wolves' eyes ", manufacturer U.S. Sheng Nisi).Tested year January in September, 2012 to 2012 and carry out at Agricultural University Of Nanjing's crop genetic and germplasm innovation National Key Laboratory.

1.2 abductive approach

1) 75% alcohol for seed is processed to 1min, 0.1% mercuric chloride is processed 15min, and aseptic water washing 3-4 time is inoculated in the medium without MS minimal medium+30ml/L Coconut Juice+sucrose 30g/L+ agar 7.8g/L of hormone and cultivates 6d, and seedling cotyledon launches completely.Cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

2) cut cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm be inoculated into MS minimal medium+NAA0.1mg/L+6 ?preculture 2 days on BA2.5mg/L+ Coconut Juice 50ml/L+ sucrose 30g/L+ agar 7.8g/L medium, in order to doubling, process.Cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

3) Double layer culture induction polyploid: after preculture 2d, by cotyledon period retain plumular axis 0.4 ?the seedling of 0.6cm be seeded on the double-deck differential medium containing colchicine, be placed on shaking table and process, wherein, the described double-deck differential medium containing colchicine is by upper strata: MS minimal medium+NAA0.1mg/L+6 ?Coconut Juice+colchicine+30g/L sucrose of BA2.5mg/L+50ml/L, pH5.8 (10ml); And lower floor: MS minimal medium+NAA0.1mg/L+6 ?Coconut Juice+30g/L sucrose+7.8g/L agar of BA2.5mg/L+50ml/L, pH5.8 (45ml) forms, colchicine concentration is 100,300,450mg/L, is placed in and on shaking table, processes 24h, 48h and 96h, the same preculture of condition; Three explants of each tissue culture bottle inoculation, each explant is in triplicate;

4) regeneration propagation: after processing, explant forwards differentiation and proliferation medium to after aseptic water washing 3 times: the upper differentiation and proliferation of MS minimal medium+NAA0.1mg/L+6-BA2.5mg/L+50ml/L Coconut Juice+sucrose 30g/L+ agar 7.8g/L, obtain tetraploid bud, the same preculture of condition of culture;

5) acquisition of regeneration plant: after 40d, tetraploid bud is cultivated to 15d in the Coconut Juice+0.5g/L of the MS minimal medium+100ml/L without hormone acid hydrolyzed casein medium, obtain regeneration plant, the same preculture of condition of culture.

6) the taking root and tame of regeneration plant: regeneration plant is proceeded to and cultivate 15d in the medium of 1/2MS minimal medium+NAA0.2mg/L+ Coconut Juice 30ml/L+ sucrose 30g/L+ agar 7.8g/L and take root, the same preculture of condition of culture; Then according to conventional method domestication, white silk seedling transplanting;

The Ploidy Identification of 1.3 regeneration plants

1.3.1 tetraploid morphology screening

When blade reaches 6-7 sheet leaf, the plant that the plant discovery leaf-shrinkage that observation was processed and color burn, blade are widened the leaf change circle of thickening can just be decided to be variant.

1.3.2 Stomatal appraisal

Pore opening is measured: tear and get blade one fritter lower epidermis, put on slide, drip 1 0.5% concentration I-KI solution, cover slide, dispel bubble, Olympus light microscope (object lens 40 *, eyepiece 10 *) under, size and the density of mensuration pore, get 10 samples and add up.Not add colchicine, process in contrast.

1.3.3 flow cytometer is identified

Use the flow cytometer of U.S. Beckman company to carry out DNA content mensuration.Weigh 0.2g blade, put into culture dish, add 2ml dissociation solution, use that sharp single-edge blade is disposable to be shredded leaf tissue fast, through double wall funnel 400 mesh filter screens, be filled in 2ml centrifuge tube, extract the liquid to be measured that 500 μ l filter, be put in new 2ml centrifuge tube, adding 1.5ml PI (iodate the third ingot) dyeing liquor, fully concussion, mix, dyeing 60min, carries out upper machine mensuration subsequently.Above all under 4 ℃ of conditions, carry out in steps.

1.3.4 chromosome counting

The chromosome counting tip of a root is from the regeneration induction plant that is grown in greenhouse.Get 0.002M8-oxyquinoline pretreatment 3-4h for the 1-2cm tip of a root, with Kano fixer (ethanol: acetic acid=3:1) fixedly after 24h, at 4 ℃, 70% alcohol is preserved for a long time.Get the tip of a root fixing, distilled water cleans 3 times, hypotonic 25min before 0.075M KCL, putting into the 1M HCl60 ℃ 8min that dissociates, after distilled water, dripping to ooze and process 7min, cutting most advanced and sophisticated 2mm and be placed on slide, with the precious dye liquor dyeing of improvement card 5-8min, covered, strikes sheet, at Olympus optical microphotograph Microscopic observation, counts.

2, results and analysis

2.1 colchicines on Double layer culture cotyledon period retain plumular axis 0.4 ?the seedling survival of 0.6cm and the impact of propagation

Utilize three kinds herein, 243 explants (not very contrast) are inoculated on the Double-Medium of the colchicine that contains variable concentrations and process 24h altogether, 48h and 96h, process the death condition of observing explant after 3 weeks, 239 explant survivals (not very contrast) altogether, only LQ-2 has the dead and S139-2 of two explants to have an explant death, and survival rate is minimum 77.78%, is up to 100% (table 1).After colchicine is processed, each explant bottom can become black gradually, and dead explant is all finally to cause cotyledon to turn to be yellow gradually because terminal bud suppresses growth, death.With contrast growth coefficient and compare, the explant differentiation of processing and growth be slowing down in various degree to some extent all, and its growth coefficient is decreasing in various degree also, and along with colchicine concentration increases gradually, time extends gradually, and the growth coefficient between each processing of each kind also reduces accordingly.When the processing time, be 24h, when colchicine concentration is 100,300,450mg/L, the growth coefficient of LQ-2 and S139-2 is respectively 3.00,2.22,1.56 and 3.22,2.56,2 (table 1).When the processing time, be 48h, when colchicine concentration is 450mg/L, the growth coefficient of LQ-2 and S139-2 is respectively 1.11 and 1.71 (table 1).LQ-2, the growth coefficient scope of S139-2 and LQ-23 is respectively 1.11-3.38,1-3.22 and 1-3.33 (table 1).Therefore colchicine concentration and processing time are the key factors that affects broccoli bud point propagation.

Table 1 colchicine on Double layer culture cotyledon period retain plumular axis 0.4 ?the seedling survival of 0.6cm and the impact of propagation

The induction result of 2.2 colchicines

In colchicine concentration, be 100 herein, 300, the seedling that on the Double-Medium of 450mg/L, three kinds of cotyledon periods is retained to plumular axis 0.4 ?0.6cm carries out tissue culture and inducement, processing time is 24h, 48h and 96h, by double-deck tissue culture and inducement, 511 group training seedlings have altogether been obtained, through identifying that discovery wherein has 178 for tetraploid material (table 2).Each processing of two kinds of LQ-2 and S139-2 all can obtain tetraploid material, and LQ-23 is 100mg/L in colchicine concentration, and the processing time is without tetraploid, to produce (table 2) in 24h processing.Wherein take colchicine concentration as 300mg/L, and the processing time is that this treatment combination of 48h is best (table 2).In this processing, the explant survival rate of three kinds reaches 100%, growth coefficient be respectively 2,2.33 and 2.67(table 1), obtain tetraploid plant and be respectively 12,15 and 15, tetraploid induction rate be respectively 66.67%, 71.42% and 62.5%(table 2).Table 1 and table 2 demonstrate, and tetraploid induction effect is relevant with colchicine concentration for the treatment of, time and induction mode.Colchicine concentration is higher, induction time is longer, can obtain higher tetraploid inductivity, in table 2, show that tetraploid induction rate is up to 77.78%, but this has a negative impact the propagation to explant, if LQ-23 is 450mg/L in colchicine concentration, the processing time, while being 96h, growth coefficient dropped to 1(table 1 from 3.67).The present invention, by great many of experiments, has found best colchicine concentration and induction time, has both improved inductivity, has alleviated again the adverse effect of colchicine to explant propagation.In this research, Double layer culture induction broccoli survival rate high (average out to 98.41%), the explant of all survivals, through differentiation and proliferation, can obtain a large amount of tetraploid plants fast, has so not only improved tetraploid induction efficiency, has also reduced chimeric generation simultaneously.

The Ploidy Identification of 2.3 regeneration plants

2.3.1 Morphological Identification and Stomatal appraisal

Regenerate variation plant compared with its liploid plant, growth retardation, plant strain growth is healthy and strong; Stem is sturdy; Blade becomes greatly, becomes circle, thickening, and leaf color is deepened, and shrinkage out-of-flatness, and leaf margin is smooth (Fig. 7-8) gradually.The pore of regeneration variation plant has increase in various degree on length and width compared with dliploid, seem obvious increase, and stomatal frequency diminish (Fig. 1-2).

2.3.2 root tip chromosomes is identified

The tetraploid regeneration plant of identifying by flow cytometer is carried out to root tip chromosomes counting.Normal dliploid broccoli body tip of a root mitotic chromosome number is 2n=2x=18, and the chromosome number that is accredited as tetraploid plant is 2n=2x=36(Fig. 3-4).

2.3.3 the mensuration of flow cytometer DNA content

By flow cytometer, doubtful variation regeneration plant is carried out to DNA content determination and analysis.With the regeneration plant without colchicine processing in contrast.Flow cytometer figure (5-6) ordinate Count is by the relative value of cell number, and what abscissa represented is fluorescence channel value, can be by go out the ploidy of the different of peak position and then mensuration plant at abscissa.Research discovery, contrasts diplontic DNA relative amount near 200, and tetraploid relative amount, near 400, is about 2 times of dliploid DNA content.

The effect of table 2 colchicine-induced autotetraploid broccoli

Claims (6)

1. the breeding method of an in-vitro inducing autotetraploid broccoli, it is characterized in that comprising: the seedling that the broccoli cotyledon period of take retains plumular axis 0.4-0.6cm is explant, explant is seeded to containing induction on the double-deck differential medium of colchicine and cultivates 45-50h, through regeneration propagation, directly produce tetraploid bud again, and then obtain autotetraploid broccoli through follow-up cultivation; Wherein, the described double-deck differential medium containing colchicine is by upper strata: the Coconut Juice of MS minimal medium+NAA0.05-0.1mg/L+6-BA2.5-3mg/L+50-60ml/L+300-350mg/L colchicine+30g/L sucrose, pH5.8; And lower floor: the Coconut Juice of MS minimal medium+NAA0.05-0.1mg/L+6-BA2.5-3mg/L+50-60ml/L+30g/L sucrose+7.8g/L agar, pH5.8 forms.

2. the breeding method of autotetraploid broccoli according to claim 1, is characterized in that described method comprises the steps:

1) Double layer culture induction polyploid: cut the seedling that broccoli cotyledon period retains plumular axis 0.4-0.6cm, after preculture 2d, be seeded to containing in the double-deck differential medium of colchicine, be placed in and on shaking table, process 45-50h, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

2) regeneration propagation: the explant after previous step is processed forwards differentiation and proliferation on differentiation and proliferation medium to and obtains tetraploid bud after aseptic water washing 3 times, the differentiation and proliferation medium using is MS minimal medium+NAA0.05-0.1mg/L+6-BA2.5-3mg/L+ Coconut Juice 50-60ml/L+ sucrose 30g/L+ agar 7.8g/L, pH5.8; Cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

3) acquisition of regeneration plant: after 40d, tetraploid bud is cultivated to 10-15d in the medium of the MS minimal medium+80-100ml/L Coconut Juice+0.5g/L acid hydrolyzed casein+sucrose 30g/L+ agar 7.8g/L without hormone, obtain regeneration plant, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx;

4) the taking root and tame of regeneration plant: regeneration plant is proceeded in the medium of 1/2MS minimal medium+NAA0.2mg/L+ Coconut Juice 20-30ml/L+ sucrose 30g/L+ agar 7.8g/L, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx cultivation 15-20d and takes root, and then tames, practices seedling and transplant;

5) evaluation of regeneration plant: before transplanting, get the tip of a root of regeneration plant, carry out Chromosome Identification after flow cytometer is identified, transplant after 1 month regeneration plant is carried out to Morphological Identification, Stomatal appraisal and flow cytometer evaluation.

3. the breeding method of autotetraploid broccoli according to claim 2, described in it is characterized in that is 300mg/L containing colchicine concentration in the double-deck differential medium of colchicine, and explant is being induced the preferred 48h of incubation time containing on the double-deck differential medium of colchicine.

4. the breeding method of autotetraploid broccoli according to claim 2, it is characterized in that the described double-deck differential medium containing colchicine at the middle and upper levels the ratio of the volume of liquid nutrient medium and the volume of lower floor's solid culture medium be 2:9-2:7; During inoculation, the hypocotyl of explant is vertically inserted in lower floor's solid culture medium, cotyledon period shoot tip meristem is immersed in supernatant liquid medium, and cotyledon is exposed to above supernatant liquid medium.

5. according to the breeding method of the autotetraploid broccoli described in any one in claim 1-4, the seedling that broccoli cotyledon period described in it is characterized in that retains plumular axis 0.4-0.6cm obtains by the following method: by 75% alcohol processing 1min for broccoli seeds, 0.1% mercuric chloride is processed 15min, aseptic water washing 3-4 time, be inoculated in the medium without MS minimal medium+Coconut Juice 20-30ml/L+ sucrose 30g/L+ agar 7.8g/L of hormone and cultivate 6d, seedling cotyledon launches completely, take out aseptic seedling, apart from cotyledon petiole end 0.4-0.6cm hypocotyl place, cutting, leave strip part cotyledonous is as explant, cultivation temperature maintains 23 ± 2 ℃, the photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx.

6. according to the breeding method of the autotetraploid broccoli described in any one in claim 1-4, it is characterized in that described preculture is to cut in the medium that seedling that cotyledon period retains plumular axis 0.4-0.6cm is inoculated into MS minimal medium+NAA0.05-0.1mg/L+6-BA2.5-3mg/L+ Coconut Juice 50-60ml/L+ sucrose 30g/L+ agar 7.8g/L preculture 2 days, cultivation temperature maintains 23 ± 2 ℃, photoperiod maintain 16/8h white/the black cycle, luminous intensity maintains 2000lx.

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