CN103270951B - Method for obtaining dwarfed early gold sweet orange regeneration plant through agrobacterium rhizogenes - Google Patents
Method for obtaining dwarfed early gold sweet orange regeneration plant through agrobacterium rhizogenes Download PDFInfo
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- CN103270951B CN103270951B CN201310240707.5A CN201310240707A CN103270951B CN 103270951 B CN103270951 B CN 103270951B CN 201310240707 A CN201310240707 A CN 201310240707A CN 103270951 B CN103270951 B CN 103270951B
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Abstract
The invention discloses a method for obtaining dwarfed early gold sweet orange regeneration plant through agrobacterium rhizogenes. According to the characteristic of dwarfed plant type of plant transformed and regenerated through the agrobacterium rhizogenes, polyembryony breed of early gold sweet orange is transformed through agrobacterium rhizogenes NSU440, hairy roots are induced, then complete plant is obtained through a way of germination of hairy roots and radication of regeneration buds, and finally a dwarfed breed suitable for labor-saving cultivation is cultivated, so that the fruit garden labor efficiency is improved, and the labor expenditure is reduced. Transformation receptor material adopts early gold sweet orange aseptic seedling epicotyl, and as sweet orange belongs to a polyembryony breed, regeneration plant can keep parental excellent quality.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of utilizing Agrobacterium rhizogenes to obtain dwarfing golden sweet orange regeneration plant morning.
Background technology
Oranges and tangerines are the important fruit trees in the world, tool Food and Argriculture OrganizationFAO statistics (FAO), and within 2009, world's oranges and tangerines cultivated area reaches 871.9 ten thousand hectares, approximately 1.24 hundred million tons of ultimate productions; The same period, China's oranges and tangerines cultivated area was about 2,000,000 hectares, and output reaches 0.25 hundred million ton, all ranks first in the world.But along with the development of China's Urbanization Construction, rural laborer shifts in a large number, Orange Producing labor cost improves, and affects orchard worker's enthusiasm for production; In addition, orangery laborer aging increasingly, the pattern of only having development to simplify cultivation is just applicable to rural area Aspects In The Development of Citrus Industry.
Cultivate bush type culture kind, can facilitate fruit harvesting and daily cultivation management, reduce labour intensity, simplify cultivation for oranges and tangerines and improve resource.Agrobacterium rhizogenes is Gram-negative bacteria, can T-DNA on Ri plasmid be integrated into host's nuclear gene group by infecting plants wound, and bring out the generation of hairly root; The plant being regenerated by hairly root has the phenotype of internode cripetura and plant dwarfing conventionally, this is confirmed in the regenerative plants of hairy root cultures of apple rootstock Malus baccata (.2008. Agrobacterium rhizogenes mediation Malus baccata genetic transformation and the root of hair regeneration plant such as Wu Jiao. gardening journal, 35(7): 959-966.).Agrobacterium rhizogenes is applied to the research of oranges and tangerines genetic improvement and reports less, utilize Agrobacterium rhizogenes induction oranges and tangerines to produce hairly root, set up hairly root plant regeneration method in oranges and tangerines, cultivate dwarfted varieties, the development that can be rural area Orange Producing simplification cultivation under the new situation and laborsaving cultivation mode provides the support on variety source.
Summary of the invention
The object of the invention is to be to provide a kind of utilizes Agrobacterium rhizogenes to obtain the method for downgrading golden sweet orange regeneration plant morning.The present invention is higher mainly for China's Orange Producing manpower shortage, labor cost, and the present situation that oranges and tangerines price fluctuates, conventionally possess according to the plant of Agrobacterium rhizogenes transformation tissue culture the feature that plant type is downgraded, proposition utilizes Agrobacterium rhizogenes MSU440 to transform early golden sweet orange of polyembryony kind, induce and sprout by hairly root after hairly root and the regeneration bud approach of taking root obtains complete regenerated plant, finally cultivate the dwarfted varieties that is applicable to laborsaving cultivation, thereby improve orchard efficiency, reduced labour consumption.Transformation receptor material of the present invention is golden sweet orange aseptic seedling epicotyl morning, and because sweet orange is polyembryony kind, therefore regeneration plant can retain parent's fine quality.
To achieve the above object, the present invention adopts following technical measures:
Utilize Agrobacterium rhizogenes to obtain and downgrade an early method for golden sweet orange regeneration plant, comprise the following steps:
1. early golden sweet orange aseptic seedling is prepared:
Early in golden sweet orange fruit, take out seed, tap water soaks 10min after rinsing well in 1M NaOH solution, and flowing water rinses removes pectin; Adopt 3%(v/v) chlorine bleach liquor's soaking disinfection 15-20min, after peelling off interior exosper, be seeded on MT sowing substratum, 24-26 ℃ of dark cultivation after 4 surroundings, takes out 1500-2000lx illumination 5-7d, selects healthy and strong aseptic seedling for transforming until slightly turning after green.
2. Agrobacterium rhizogenes bacterium liquid is prepared:
Get the Agrobacterium rhizogenes MSU440 bacterial strain of-80 ℃ of preservations rules on LB solid medium, 28 ℃ of dark places are inverted and are cultivated the single bacterium colony of 36-48h acquisition activation, the single bacterium colony of Agrobacterium rhizogenes of picking activation, line again on LB solid medium, 28 ℃ of dark 36-48h that cultivate, with the cultured thalline of knife blade scraping in MT suspension medium, after 180rpm, 28 ℃ of shaking culture 1.5-2h by bacterium liquid OD
600value is adjusted to after 0.6-0.8 stand-by.
3. explant infects with hairly root and induces:
Cut epicotyl segment and infect 20-30min in Agrobacterium bacterium liquid, on sterilizing filter paper, blot, going to MT is total on substratum, 21 ℃ of dark places are cultivated 3d altogether, by explant rinsed with sterile water 3 times, filter paper blots rear switching in inducing hairly root degerming containing 24-26 ℃ of dark place on the MT minimum medium of 400 μ g/L cephamycins.In the present invention, in hairly root inducing culture, do not add hormone.
4. hairly root succeeding transfer culture:
When hairly root is stretched to 2cm left and right, cut 1-1.5cm with tip of a root segment subculture to containing on the MT minimum medium of 400mg/L cephamycin, 20d left and right subculture 1 time, can remove residual Agrobacterium after 24-26 ℃ of continuous succeeding transfer culture in dark place 2-3 time substantially.
5. hairly root is sprouted:
Hairly root is cut into the segment of 1cm left and right, goes to and sprout on substratum, 24-26 ℃, the lower induction of the dark treatment condition of 16h illumination every day (1500-2000lx) 8h is sprouted.First form fine and close embryoid at the hairly root on substratum of sprouting, grow afterwards Multiple Buds.
6. regeneration bud extends:
Regeneration bud bulk-growth during to 0.5-1cm, goes in bud elongation medium, to promote the rapid elongation of sprout.
7. regeneration bud is taken root:
When sprout grows to 1.5cm left and right, cut off separately and go to the root induction of MT root media.Within two weeks, the visible otch in left and right sends adventive root.
Compared with prior art, the present invention has the following advantages:
The invention has the beneficial effects as follows: the aseptic seedling epicotyl of choosing is Agrobacterium rhizogenes good receptor, when hairly root regeneration bud, can produce a large amount of Multiple Buds, thereby can obtain a large amount of clonal offsprings; Regeneration bud is taken root easily, and well developed root system, transplants easily survival after white silk seedling; Because hairly root is generally unicellular origin, there is not mosaic, inheritance stability in regeneration plant therefore; In addition, gold sweet orange plant morning of Agrobacterium rhizogenes transformation tissue culture is downgraded obviously, regenerative plants of hairy root cultures moves to greenhouse plant height (stem apex is to rhizome intersection) average out to 5.1cm after six months, and contrasts (non-transformed regeneration plant of contemporaneously) plant height average out to 10.1cm; Regenerative plants of hairy root cultures leaf morphology is normal, robust growth; ' early gold ' sweet orange is polyembryony kind, and therefore hairly root regeneration dwarfed plant can retain parent's fine quality, can be used as the resource of breeding wheat for semidwarfness.
Accompanying drawing explanation
Fig. 1 is a kind of hairly root induction schematic diagram.
A is not for infecting contrast; B Agrobacterium rhizogenes is infected the hairly root of rear generation.
Fig. 2 is a kind of hairly root regeneration Multiple Buds schematic diagram.
Fig. 3 is that a kind of Multiple Buds extends schematic diagram.
Fig. 4 is a kind of regeneration bud schematic diagram of taking root.
Fig. 5 is that a kind of regeneration plant root system comparison CK is unconverted contrast schematic diagram.
T is for transforming regenerative plants of hairy root cultures
Fig. 6 is that a kind of regeneration plant strain shape comparison a is side, and b is front schematic view.
CK is non-transformed contrast; T is for transforming regenerative plants of hairy root cultures
Embodiment
Now the each step of the inventive method is described in further detail below:
Embodiment 1: a kind of method of utilizing Agrobacterium rhizogenes to obtain dwarfing golden sweet orange regeneration plant morning, comprises the following steps:
1. explant is prepared:
Early golden sweet orange fruit picks up from Hua Zhong Agriculture University's oranges and tangerines breeding center.In citrus fruit, take out seed, tap water soaks 10min after rinsing well in 1M NaOH solution, and after removal pectin, distilled water flushing is clean; 3%(v/v on super clean bench) chlorine bleach liquor soak 20min, sterile purified water cleans 3 times, each 5min; Seed after sterilization is seeded on MT sowing substratum peel off interior exosper with aseptic operation cutter and tweezers on Bechtop after, 24-26 ℃ of dark cultivation after 4W, take out illumination 5-7d, select healthy and strong aseptic seedling after green epicotyl is cut into 1.5cm left and right until slightly turning, stand-by;
2. Agrobacterium rhizogenes bacterium liquid is prepared:
Get the Agrobacterium rhizogenes MSU440(Limpens E of-80 ℃ of preservations etc., RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.J Exp Bot.2004May; 55 (399): 983-92) bacterial strain is rule on LB solid medium, 28 ℃ of dark places are inverted cultivation 36-48h and are obtained single bacterium colony, picking list bacterium colony is line again on new LB solid medium, under similarity condition, cultivate 36-48h, with all thalline of knife blade scraping in MT suspension medium, 180rpm, after 28 ℃ of shaking culture 1.5-2h by bacterium liquid OD
600value is adjusted to after 0.6-0.8 stand-by.
3. explant infects with hairly root and induces:
Epicotyl segment is infected 20-30min in Agrobacterium bacterium liquid, shakes up 2-3 time during this time; After infecting end, explant is blotted on sterilizing filter paper, going to MT is total in culture medium, cultivate after 3d altogether by explant rinsed with sterile water 3 times 21 ℃ of dark places, after blotting, filter paper goes to MT minimum medium (the Murashige T containing 400 μ g/L cephamycins, Tucher DPH.Growth factor requirement of citrus tissue culture.Proc1st Int Citrus Symp1993,3:1155-1169) upper, hairly root degerming are induced in 24-26 ℃ of dark place:.Control material infects with the MT suspension medium that does not contain Agrobacterium, and all the other processing are identical.Because hairly root itself can synthetic hormone, can independently growth on the MT minimum medium that does not add hormone, therefore in the present invention, hairly root inducing culture does not add hormone.
Agrobacterium rhizogenes MSU440 infects after golden epicotyl segment 8-10d morning, produce white callus and send successively white hairly root from incision, hairly root majority sends from the base portion (morphology lower end) of epicotyl segment, does not infect contrast and sends (Fig. 1) without hairly root.
4. hairly root subculture:
Hairly root cuts 1-1.5cm and extremely contains on the MT minimum medium of 400mg/L cephamycin with tip of a root segment subculture while being stretched to 2cm left and right, part hairly root can continue to extend; Choose well-grown hairly root, 20d left and right subculture 1 time, can remove residual Agrobacterium after 24-26 ℃ of continuous succeeding transfer culture in dark place 2-3 time substantially.
5. hairly root is sprouted:
Hairly root is cut into the segment of 1cm left and right, goes to and sprout on substratum, 24-26 ℃, the lower induction of the dark treatment condition of 16h illumination every day (1500-2000lx) 8h is sprouted.Hairly root segment is exposed to next week left and right of illumination condition can turn green, and green bud point appears in about 20d left and right successively, and forms Multiple Buds (Fig. 2).The mode of sprouting by hairly root can produce a large amount of Clonic breeding clones, for follow-up study provides the material that a large amount of genetic background is consistent.
6. regeneration bud extends:
In the time that Multiple Buds grows to 0.5-1cm, cut and separate Multiple Buds and go in bud elongation medium, to promote rapid elongation (Fig. 3 a, b) of sprout.
7. regeneration bud is taken root:
When regeneration bud is stretched to 1.5cm left and right, sprout is cut off separately and go to the root induction of MT root media, the visible otch in about 20d left and right goes out to send adventive root.
8. regeneration plant is cultivated:
When the comparatively prosperity of regeneration plant root system, when plant strain growth is healthy and strong (Fig. 4), take out plant, clean the substratum adhering in order to avoid mould contamination (Fig. 5) appears in follow-up experienced seedling, 24-26 ℃, under the dark treatment condition of 16h illumination every day (1500-2000lx) 8h, in distilled water, practice seedling 7d left and right, go to matrix after sterilizing, indoor matrix is practiced seedling and is moved to greenhouse about 3 days.Regenerative plants of hairy root cultures shows as that root system is more flourishing, lateral root increases the feature such as (Fig. 5), internode cripetura, the dwarfing of strain shape with comparing, but normally (Fig. 6 a, b) of leaf morphology.
Described matrix is bud culture medium (production of Zhenjiang, Jiangsu bud fertilizer company limited)
The substratum of using in experiment is as follows, and wherein MT Medium's PH Value is 5.85; Adjust pH not after LB dissolves:
MT sows substratum: MT+25g/L sucrose+8g/L agar
MT suspension medium: MT+0.5g/L malt extract+1.5g/L glutamine+40g/L sucrose+20mg/L Syringylethanone
MT is culture medium altogether: MT+40g/L sucrose+8g/L agar+20mg/L Syringylethanone
Hairly root inducing culture: MT+40g/L sucrose+8g/L agar+400mg/L cephamycin
The hairly root substratum of sprouting: MT+40g/L sucrose+8g/L agar+1mg/L BA
Bud elongation medium: MT+30g/L sucrose+8g/L agar+0.1mg/L BA+0.1mg/L IAA+0.25mg/L GA
3
MT root media: 1/2MT+25g/L sucrose+8g/L agar+0.5mg/L NAA+0.1mg/L IBA+0.5g/L gac
LB solid medium: yeast extract 5g/L, tryptone 10g/L, sodium-chlor 10g/L, agar 15g/L.
Claims (1)
1. utilize Agrobacterium rhizogenes to obtain and downgrade an early method for golden sweet orange regeneration plant, comprise the following steps:
1) early golden sweet orange aseptic seedling is prepared:
Early in golden sweet orange fruit, take out seed, tap water soaks 10min after rinsing in 1M NaOH solution, and flowing water rinses removes pectin; Adopt chlorine bleach liquor's soaking disinfection 15-20min of 3%v/v, be seeded on MT sowing substratum after peelling off interior exosper, 24-26 ℃ of dark cultivation after 4 surroundings, takes out 1500-2000lx illumination 5-7 days, selects healthy and strong aseptic seedling for transforming until slightly turning after green;
2). Agrobacterium rhizogenes bacterium liquid is prepared:
Get the Agrobacterium rhizogenes MSU440 bacterial strain of-80 ℃ of preservations rules on LB solid medium, 28 ℃ of dark places are inverted and are cultivated the single bacterium colony of 36-48h acquisition activation, the single bacterium colony of Agrobacterium rhizogenes of picking activation, line again on LB solid medium, 28 ℃ of dark 36-48h that cultivate, the cultured thalline of scraping in MT suspension medium, after 180rpm, 28 ℃ of shaking culture 1.5-2h by bacterium liquid OD
600value is adjusted to after 0.6-0.8 stand-by;
3) explant infects with hairly root and induces:
Cut epicotyl segment and infect 20-30min in Agrobacterium bacterium liquid, on sterilizing filter paper, blot, going to MT is total in culture medium, 21 ℃ of dark places are cultivated 3d altogether, by explant rinsed with sterile water 3 times, filter paper blots rear switching in inducing hairly root degerming containing 24-26 ℃ of dark place on the MT minimum medium of 400 μ g/L cephamycins;
4) hairly root succeeding transfer culture:
When hairly root is stretched to 2cm, cut 1-1.5cm with tip of a root segment subculture to containing on the MT minimum medium of 400mg/L cephamycin, 20d subculture 1 time, 24-26 ℃ of continuous succeeding transfer culture in dark place 2-3 time;
5) hairly root is sprouted:
Hairly root is cut into 1cm segment, goes to and sprout on substratum, 24-26 ℃, 16h illumination every day, 1500-2000lx; Under the dark treatment condition of 8h, induce and sprout;
6) regeneration bud extends:
Regeneration bud bulk-growth during to 0.5-1cm, goes to bud elongation medium;
7) regeneration bud is taken root:
When sprout grows to 1.5cm, cut off separately and go to the root induction of MT root media, within two weeks, visible otch sends adventive root;
Described substratum is as follows:
MT sows substratum: MT+25g/L sucrose+8g/L agar;
MT suspension medium: MT+0.5g/L malt extract+1.5g/L glutamine+40g/L sucrose+20mg/L Syringylethanone;
MT is culture medium altogether: MT+40g/L sucrose+8g/L agar+20mg/L Syringylethanone;
The hairly root substratum of sprouting: MT+40g/L sucrose+8g/L agar+1mg/L BA;
Bud elongation medium: MT+30g/L sucrose+8g/L agar+0.1mg/L BA+0.1mg/L IAA+0.25mg/L GA
3;
MT root media: 1/2MT+25g/L sucrose+8g/L agar+0.5mg/L NAA+0.1mg/L IBA+0.5g/L gac;
LB solid medium: yeast extract 5g/L, tryptone 10g/L, sodium-chlor 10g/L, agar 15g/L.
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| CN1833488A (en) * | 2005-11-28 | 2006-09-20 | 邓子牛 | Method for regeration of citrus internode stem and genetic transformation |
| CN101250549A (en) * | 2008-04-01 | 2008-08-27 | 邓子牛 | Citrus mature internode stems genetic transformation method |
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| CN1833488A (en) * | 2005-11-28 | 2006-09-20 | 邓子牛 | Method for regeration of citrus internode stem and genetic transformation |
| CN100415890C (en) * | 2005-11-28 | 2008-09-03 | 邓子牛 | Method for regeration of citrus internode stem and genetic transformation |
| CN101250549A (en) * | 2008-04-01 | 2008-08-27 | 邓子牛 | Citrus mature internode stems genetic transformation method |
| CN102586317A (en) * | 2011-01-10 | 2012-07-18 | 华中农业大学 | Method for transforming and cultivating citrus transgenic plant by agrobacterium-mediated blade |
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