CN101575612B - Transgenic method for orange - Google Patents
Transgenic method for orange Download PDFInfo
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- CN101575612B CN101575612B CN2009101041008A CN200910104100A CN101575612B CN 101575612 B CN101575612 B CN 101575612B CN 2009101041008 A CN2009101041008 A CN 2009101041008A CN 200910104100 A CN200910104100 A CN 200910104100A CN 101575612 B CN101575612 B CN 101575612B
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Abstract
The invention relates to a transgenic method for orange, which comprises preparation of test-tube rootstocks and explants, preparation of grafted seedlings, wound infection of the grafted seedlings, grafting of sprouts to be transformed as well as molecular biological identification of plants to be transformed so as to obtain transgenic plants, and the grafting of the transgenic plants; and the characteristics lie in that the preparation of the grafted seedlings in the method selects the grafted seedlings of the second generation and the third generation as initial materials. The invention relates to a transgenic method capable of effectively solving annually implement genetic transformation operation of the oranges.
Description
Technical field
The present invention relates to a kind of transgenic method of citrus.It is mainly used in the breed improvement of orange tree.
Background technology
It is a lot of that citrus variety is improved applied transgenic method, mainly contains direct conversion method of foreign DNA and Agrobacterium tumefaciens mediated conversion method.
The employed explant of the direct conversion method of foreign DNA is a protoplastis, and the long and regeneration difficulty of the protoplastis culture cycle of a lot of kinds in the orange tree, cost is also higher, so there be limited evidence currently of has the laboratory to adopt this method.
The employed explant of agrobacterium tumefaciens-mediated transformation is a lot, and epicotyl segment, cotyledon stripping and slicing, bearing tree stem section and the field bearing tree axillalry bud etc. of aseptic seedling are arranged.Utilize the epicotyl segment, cotyledon stripping and slicing of aseptic seedling genetic transformation frequency height, but the transfer-gen plant that obtains to just can blossom and bear fruit through the very long virgin phase for explant.And be that transfer-gen plant that explant was obtained can be avoided the virgin phase with bearing tree stem section, but its bud ratio and transformation efficiency are extremely low.Li Wei etc. have delivered " citrus gene transformation Study on new method " (1997 on " Botany Gazette ", 39 (8): 782-784), they are to be explant with field bearing tree axillalry bud, adopt attached body axillalry bud to transform-exsomatize the method that expands numerous evaluation and transform citrus, and the transformation efficiency that is obtained is 5.26%.But this method need adding the substratum that sprouts on the cotton balls and preserved moisture 3 days frequently, and the acquisition transfer-gen plant need be walked this approach of in vitro taking root in culturing process altogether.But citrus was only taken out the tip in 1 year three or four times, thereby directly transformed easily in the field and to be subject to seasonal restrictions, troublesome poeration, and orange tree in vitro take root very difficult, the safety issue of also bringing plasmid to discharge simultaneously to environment.
He Yongrui etc. have delivered " Agrobacterium tumefaciens mediated citrus genetic transformation technical study " (2004,25 (1): 11-16) on " tropical crops journal ".This paper to citrus bearing tree axillalry bud handle at different B A, IAA mass concentration, different wound site and whether lure bud and the transformation efficiency aspect these three in paraffin band parcel wound mouth study, set up one general, simply, citrus genetic transformation technical system efficiently.But in the operating process of reality, operation easier with paraffin band parcel wound mouth is big, and the operating time is long, thereby it is very big to the injury of plant, if but without paraffin band parcel wound mouth, add the phytokinin (10mg/l) of high density simultaneously in diluent, then the wound mouth is easy to dehydration and forms callus, is difficult to be differentiated to form bud again; Simultaneously, this method be with first-generation Graft as the parent material that transforms, and in the process of grafting, need the citrus bearing tree branch of lignifying or semi-lignified, thereby draw materials and be subjected to season limit easily, can not the anniversary carry out the operation of genetic transformation.
Summary of the invention
But the object of the present invention is to provide the transgenic method of the citrus that carries out the genetic transformation operation a kind of easy anniversary.
The object of the present invention is achieved like this: a kind of transgenic method of citrus, comprise the preparation of test tube stock, explant, the preparation of Graft, the wound infection of Graft, intend transforming the grafting of bud, intend the molecular biology identification of transformed plant, to obtain transfer-gen plant, the grafting of transfer-gen plant is characterized in that: the preparation of Graft is to select for use second and third generation grafting seedling as parent material in the described method.
Selecting for use second and third generation grafting seedling as parent material in the aforesaid method, is that first-generation Graft level from the scion is downcut, and carries out grafting second time, acquisition s-generation Graft; Or the terminal bud of s-generation Graft carried out grafting once more, obtain third generation Graft; With second or third generation Graft as transforming parent material, between scion and the stock with paraffin band looping.The wound healing of scion and stock is very firm like this, and the phenomenon that just can not occur coming off when carrying out Infection Action; Through repeatedly grafting, can obtain more to transform parent material simultaneously, carry out the purpose of conversion operation with the realization anniversary.
In order to accelerate the cycle of genetic transformation, in the set-up procedure of test tube stock, when planting skin, the seed stripping to fix with disinfectant absorbent cotton.
Seed inoculation in the above-mentioned test tube stock set-up procedure: with scalper fruit is scratched under the aseptic condition, taken out seed, seed is placed on the absorbent cotton, peel off kind of a skin, being seeded in, disinfectant is equipped with in the test tube of MS solid medium; 28 ± 2 ℃ of dark down cultivations, stock is long long with standby to 7-10cm.
In order to obtain higher transformation efficiency, the present invention adopts the 5mg/l phytokinin in the wound infection process of Graft, do not carry out looping; The indefinite bud grafting on the test tube stock, to intending the DNA of transformed plant, is carried out PCR and Southernbloting and analyzed, obtain transfer-gen plant; Practice Miao Yizhou, as intermediate stock, grafting is on the stock of field with the test tube stock, and bagging is preserved moisture 1-2 week.
In the above-mentioned wound infection process, transform parent material and kept two leaves, from top to bottom, the axillalry bud of second leaf will carry out complete wound.After the complete wound of the axillalry bud of second leaf, be easy to sprout indefinite bud on the wound mouth of first axil bud, indefinite bud also can be sprouted in the wound wound of the axillalry bud of second leaf, so just can obtain more the plan and transform bud, helps the raising of transformation frequency.
Beneficial effect of the present invention:
1, in the preparation of test tube stock, when planting skin, the seed stripping fixes with disinfectant absorbent cotton, and to plant skin and peel off easily, the sprouting speed of seed is fast, the cycle that can accelerate genetic transformation;
2, select second and third generation grafting seedling as transforming parent material, the wound healing of scion and stock is very firm, and both are with paraffin band looping, the problem with regard to not occurring coming off when carrying out Infection Action.If adopt first-generation Graft as transforming parent material, the axillalry bud of sprouting in the scion in first-generation Graft is not very firm with combining of scion, if directly carry out wound infection on first-generation Graft, probably has 80% axillalry bud to come off.
3, in conversion process, the stalwartness growth of keeping whole plant is to obtain the high prerequisite that lures the bud rate, and petiole base can come to harm what in the process that infects, if this moment is again with paraffin band looping, be easy to make leaf abscission, be unfavorable for the growth of whole plant; The formation of callus on the wound, the present invention adopts the way that reduces phytokinin in the Agrobacterium diluent, adopts the phytokinin of 5mg/l and does not carry out looping.Why the present invention carries out complete wound to the axillalry bud of second and third generation grafting seedling second leaf from top to bottom of having kept 2 leaves, be because, if not like this, for second and third generation grafting seedling that has excised terminal bud, the axillary bud sprouting speed of second leaf is very fast, growing way, the nutrition priority of supply, then the wound is difficult for sprouting indefinite bud.
4, the present invention does not directly carry out the PCR evaluation to indefinite bud, be because longer indefinite bud is easy to be polluted by plasmid from the wound, simultaneously limited because be used to extract the blade quantity of DNA, cannot carry out Southernbloting and analyze, cause false positive rate to increase; Invisible spectro indefinite bud is very tender, and water content is also high, if with the direct grafting of indefinite bud on the stock of field, then be easy to dehydration after the grafting and here wither, cause graft survival rate to reduce.The present invention adopts the indefinite bud grafting on the test tube stock, along with progressively growing up of plant, blade also progressively increases (if it is not enough to be used to extract the blade of DNA, can get the terminal bud of intending transformed plant and carry out once grafting in vitro again), so just can carry out the Southernbloting analysis to intending transformed plant, to what detect to transfer-gen plant, practice Miao Yizhou, with the test tube stock as intermediate stock, grafting is on the stock of field, bagging is preserved moisture 1-2 week, and such graft survival rate can be up to more than 95%.
5, empirical tests uses citrus transgenic method transformation frequency of the present invention to reach about 10%.Peng Aihong etc. are published in " Molecular Plant Breeding " (2009,7 (1): the paper 51-55) " Agrobacterium tumefaciens mediated hepatitis A virus (HAV) capsid protein fusion gene transform citrus grow up the research of material " though in also mention 10% genetic transformation rate, but that is breadboard statistics, turning in the process in field by the laboratory, having suitable a part of transfer-gen plant to want dead (as table 1).
Table 1: the difference of the genetic transformation rate in breadboard genetic transformation rate and field
| Breadboard transfer-gen plant (strain) | Breadboard transformation frequency | The transfer-gen plant in field (strain) | The transformation frequency in field | |
| The paper method | 25 | 10％ | 10 | 4％ |
| The inventive method | 25 | 10％ | 24 | 9.6％ |
In a word, use the transformation frequency in citrus transgenic method of the present invention field can reach about 10%, the present invention simultaneously carried out indoor before the acquisition transfer-gen plant, thereby can avoid plasmid effectively to the virgin phase that may and can avoid citrus effectively that environment discharges, grafting 2-3 behind the field just can blossom and bear fruit.
Embodiment
The invention will be further described below in conjunction with embodiment, but the present invention is not limited to this.
Embodiment:
1, the preparation of test tube stock.
The preparation of fruit: choose sophisticated, the intact fruit of citrus and clean the surface, on Bechtop, use the alcohol surface sterilization with tap water;
The preparation of aseptic absorbent cotton: with the absorbent cotton about scissors clip one block length wide each 10cm, be immersed in 75% the alcohol about 10 minutes, wring out, with standby;
Seed inoculation: aseptic condition is scratched fruit with disinfectant scalper down, takes out seed, and seed is placed on the absorbent cotton, takes scalper on the other hand, by absorbent cotton, peels off kind of a skin on the other hand, and being seeded in, disinfectant is equipped with in the test tube of MS solid medium; 28 ℃ ± 2 ℃ dark down cultivations, stock is long long with standby to 7-10cm.
The MS solid medium that this is routine is the sucrose of additional 30g/l on the basis of MS substratum and the agar of 8.5g/l, PH5.8.The prescription of MS substratum: (1) macroelement NH
4NO
31650; KNO
31900; CaCl
22H
2O 440; MgSO
47H
2O370; KH
2PO
41700; (2) trace element: KI 0.83; H
3BO
36.2; MnSO
44H
2O 22.3; ZnSO
47H
2O8.6; Na
2MnO
42H
2O 0.25; CuSO
45H
2O 0.025; CoCl
26H
2O 0.025; FeSO47H
2O (27.8)+Na
2-EDTA2H
2O (37.3); (3) organic composition: inositol 100; Nicotinic acid 0.5; Pyridoxine hydrochloride (vitamins B
6) 0.5; Vitamin (vitamins B
1) 0.5; Glycine 2, above unit are mg/l.
2, the preparation of explant.Choose healthy and strong field bearing tree branch, remove blade, branch is cut short, length is 5-10cm, cleans the surface, and rinses well with tap water.On the Bechtop with the mercuric chloride of 0.1% (g/l) sterilization about 10 minutes, clean with aseptic water washing then, be contained in the disinfectant culture dish, with standby.
3, the preparation of test tube Graft.On Bechtop, cut axillalry bud (method that cuts is equivalent to the method for field cut-grafting) on the branch with grafting knife; With tweezers stock is taken out from test tube, be placed in the culture dish; With grafting knife test tube stock upper part level is removed, open a grafting mouth then in the centre, the degree of depth of mouth is decided according to the size of axillalry bud, then axillalry bud is mounted in the grafting mouth of stock, uses the complete looping grafting mouth of disinfectant paraffin band (straight alcohol soaks more than 6 hours), be placed in the test tube that the MS liquid nutrient medium is housed and (include the filter paper bridge), 28 ± 2 ℃ of dark down cultivations, until axillary bud sprouting and to grow to about 1cm long, see light (28 ± 2 ℃ of temperature, illuminance 40 μ mol/m
2S, light application time 12h/d, as follows), obtain first-generation Graft; After seeing light 4-5 days, whole bud level on the first-generation Graft is downcut, on stem with the grafting knife epidermal area of pruning, expose form layers, the bud grafting on the test tube stock, with paraffin band looping grafting mouth (being equivalent to the cut-grafting of field grafting), is placed in the test tube that the MS liquid nutrient medium is housed and (includes the filter paper bridge), under illumination condition, continue to cultivate, obtain s-generation Graft; Or with the excision of the terminal bud of s-generation Graft, grafting obtains third generation Graft (method of grafting is the same, and culture condition is the same) on the test tube stock.
In this example, the length that stock keeps is that axillalry bud should not be too little about 3cm; Substratum is the MS liquid nutrient medium, the sucrose of additional 50g/l on the basis of MS substratum, PH5.8 (as follows); The upholder of Graft is the filter paper bridge, folding method is: folding with a foursquare filter paper diagonal form, a mouth is cut at the top, open then, (mouth will be in the centre of test tube bottom to wrap in the test tube bottom, this test tube should be littler than the test tube of putting Graft), vestige along the filter paper diagonal form is entangled in filter paper packet on the test tube then, take off, be placed in vitro (height of filter paper bridge be approximately test tube length about 1/3) that to put Graft, mouthful up, Graft is just put down root by this mouthful, and the part more than the cotyledon is more than mouth.The first-generation Graft that obtains, the stem length of seedling will have more than the 0.5cm.Wherein grafting knife, culture dish, tweezers, MS liquid nutrient medium all need sterilization.
4, the wound of second and third generation grafting seedling, infection.
A) parent material of Gan Raning: treat the wound healing of second and third generation grafting seedling, and when having the complete blade of two complete unfolded, second and third generation grafting seedling just can be used as the parent material of infection;
B) preparation of engineering bacteria liquid: single colony inoculation of selecting an agrobacterium strains before the conversion is in the 5ml liquid nutrient medium, under 28 ℃, dark, 120r/min is cultured to logarithmic phase (OD
600=0.8-1.0), with 5 times of its dilutions, diluent is MS+BA5mg/l+IAA2.5mg/l (PH7.2) during conversion;
C) preparation of cotton pellet: frustrate one by one cotton pellet with absorbent cotton, be placed in the culture dish, it is standby to sterilize;
D) wound, infection: cotton pellet is placed in the engineering bacteria liquid that has diluted; On Bechtop, will transform parent material and from test tube, take out, be placed in the culture dish; To transform parent material with tweezers and pick up, excise along axil place level with the terminal bud of grafting knife with parent material, and with the complete wound of axillalry bud; Cotton pellet looping wound with being soaked with bacterium liquid and diluent is placed in the test tube that the MS liquid nutrient medium is housed and (includes the filter paper bridge); Under the condition of dark, cultivated 28 ± 2 ℃ of temperature altogether 2-3 days; On Bechtop, remove cotton pellet then, under the condition of illumination, continue to cultivate.Wherein tweezers, culture dish, grafting knife, MS liquid nutrient medium all need sterilization.
5, intend transforming the grafting of bud.
When the stem for the treatment of the indefinite bud that grows on the wound has 0.5cm long, on Bechtop plant is taken out from test tube, be placed in the culture dish, with grafting knife the indefinite bud level is downcut, grafting obtains to intend transformed plant on the test tube stock, cultivates under illumination condition.Culture dish wherein, grafting knife, tweezers, MS liquid nutrient medium all need sterilization.
6, intend the molecular biology identification of transformed plant.
On Bechtop, to intend transformed plant from vitro taking out, cut a part of blade (remaining part still is placed in the MS liquid nutrient medium) of intending transformed plant, be used to extract the DNA that intends transformed plant with grafting knife, carry out PCR and Southernbloting and analyze, obtain transfer-gen plant.Culture dish wherein, grafting knife, tweezers, MS liquid nutrient medium all need sterilization.
7, the field grafting of transfer-gen plant:, test-tube plantlet is placed on practices Miao Yizhou on the windowsill to being defined as transfer-gen plant; Downcut transfer-gen plant from the cotyledon level of test tube stock then, according to the method for field cut-grafting with its grafting on the stock of field, preserve moisture 1-2 week with plastics bag.
The present invention is kept at citrus fruit in 4-8 ℃ the freezer or refrigerator, to reach the purpose of year-round supply citrus stock.The present invention is as the parent material that transforms with second and third generation grafting seedling, the axillalry bud of citrus is a compound bud, after the whole bud level of first-generation Graft is downcut, (downcut part and be used for grafting, obtain s-generation Graft), first-generation Graft is placed in the test tube that the MS liquid nutrient medium is housed (includes the filter paper bridge) again, the dark cultivation, temperature is the same, then also can sprout the bud that makes new advances, carry out grafting once more, also can obtain s-generation Graft from axillalry bud; The terminal bud that also can get simultaneously s-generation Graft carries out grafting (remaining s-generation Graft continues to cultivate under the condition of illumination, and temperature is the same) once more, obtains third generation Graft; Thereby can obtain more to transform parent material, also can carry out the purpose of genetic transformation operation simultaneously the realization anniversary.
What the present invention created is that the agrobacterium tumefaciens-mediated transformation of explant is 28 ± 2 ℃ of temperature, the illuminance 40 μ mol/m that are provided with artificial with the bearing tree axillalry bud
2Carry out under the condition of s, light application time 12h/d, can break the natural dormancy period of citrus, thereby can carry out the genetic transformation operation anniversary; Moreover, in the process of cultivating altogether, only need the cotton pellet that is soaked with bacterium liquid and diluent (MS+BA5mg/l+IAA2.5mg/l) is placed on the wound, thereby simplified schedule of operation; The three, the indefinite bud grafting that grows from wound after testing, is defined as remarrying of transfer-gen plant and receives on the stock of field, thereby avoided this approach of in vitro taking root to the test tube stock.
Claims (3)
1. the transgenic method of a citrus, comprise the preparation of test tube stock, explant, the preparation of Graft, the wound infection of Graft, intend transforming the grafting of bud, intend the molecular biology identification of transformed plant, to obtain transfer-gen plant, the grafting of transfer-gen plant is characterized in that: in the described method preparation of Graft be select for use second or/and the three generations Graft as parent material; Describedly select second for use, carry out grafting second time, acquisition s-generation Graft or/and the three generations Graft as parent material, is that first-generation Graft level from the scion is downcut; Or the terminal bud of s-generation Graft carried out grafting once more, obtain third generation Graft; With second or third generation Graft as transforming parent material, between scion and the stock with paraffin band looping; Adopt the 5mg/l phytokinin in the wound infection process of described Graft, do not carry out looping; The indefinite bud grafting on the test tube stock, to intending the DNA of transformed plant, is carried out PCR and Southern blotting and analyzed, obtain transfer-gen plant; Practice Miao Yizhou, as intermediate stock, grafting is on the stock of field with the test tube stock, and bagging is preserved moisture 1-2 week; In the wound infection process of described Graft, transform parent material and kept two leaves, from top to bottom, the axillalry bud of second leaf will carry out complete wound.
2. the transgenic method of citrus as claimed in claim 1 is characterized in that: the preparation of test tube stock in the described method: fix with disinfectant absorbent cotton when skin is planted in the seed stripping.
3. the transgenic method of citrus as claimed in claim 2, it is characterized in that: the seed inoculation in the described test tube stock set-up procedure: with scalper fruit is scratched under the aseptic condition, take out seed, seed is placed on the absorbent cotton, peel off kind of a skin, be seeded in the test tube that own disinfectant is equipped with the MS solid medium; 28 ± 2 ℃ of dark down cultivations, stock length to 7 10cm is long with standby.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101041008A CN101575612B (en) | 2009-06-16 | 2009-06-16 | Transgenic method for orange |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101041008A CN101575612B (en) | 2009-06-16 | 2009-06-16 | Transgenic method for orange |
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| Publication Number | Publication Date |
|---|---|
| CN101575612A CN101575612A (en) | 2009-11-11 |
| CN101575612B true CN101575612B (en) | 2011-01-26 |
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ID=41270685
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101041008A Expired - Fee Related CN101575612B (en) | 2009-06-16 | 2009-06-16 | Transgenic method for orange |
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| CN (1) | CN101575612B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293829B (en) * | 2014-10-20 | 2017-03-29 | 广东省农业科学院果树研究所 | It is a kind of that quick transgenic method of the seedling of growing directly from seeds as material is broadcast with Citrus soil |
| CN105112444B (en) * | 2015-09-22 | 2019-01-29 | 中国农业科学院柑桔研究所 | It is a kind of based on double bacterial strains/double-mass model genetic transformation citrus co-transformation method of particle |
| CN105613062B (en) * | 2016-03-22 | 2018-09-11 | 中国热带农业科学院橡胶研究所 | A kind of rubber select tree tender tip grafting childrenization method for culturing seedlings |
| CN105917976B (en) * | 2016-05-17 | 2018-09-28 | 木崇凤 | A kind of engrafting method of pear tree |
| CN110972871B (en) * | 2019-12-04 | 2021-09-21 | 新疆农业科学院植物保护研究所 | Novel method for evaluating survival competitive capacity of transgenic cotton wasteland in extreme arid region |
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2009
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