CN115873895A - Non-tissue culture method for efficiently transforming trifoliate orange hairy roots - Google Patents
Non-tissue culture method for efficiently transforming trifoliate orange hairy roots Download PDFInfo
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- CN115873895A CN115873895A CN202211686956.2A CN202211686956A CN115873895A CN 115873895 A CN115873895 A CN 115873895A CN 202211686956 A CN202211686956 A CN 202211686956A CN 115873895 A CN115873895 A CN 115873895A
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- 241001522083 Citrus trifoliata Species 0.000 title claims description 21
- 235000000404 Poncirus trifoliata Nutrition 0.000 title claims description 21
- 230000001131 transforming effect Effects 0.000 title claims description 9
- 238000012136 culture method Methods 0.000 title claims description 8
- 241000589156 Agrobacterium rhizogenes Species 0.000 claims abstract description 25
- 230000001580 bacterial effect Effects 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 244000183685 Citrus aurantium Species 0.000 claims abstract description 21
- 235000007716 Citrus aurantium Nutrition 0.000 claims abstract description 21
- 208000015181 infectious disease Diseases 0.000 claims abstract description 17
- 230000009261 transgenic effect Effects 0.000 claims abstract description 15
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 238000005286 illumination Methods 0.000 claims abstract description 7
- 239000011159 matrix material Substances 0.000 claims abstract description 7
- 238000011426 transformation method Methods 0.000 claims abstract description 6
- 230000001939 inductive effect Effects 0.000 claims abstract description 4
- 239000010455 vermiculite Substances 0.000 claims abstract description 4
- 229910052902 vermiculite Inorganic materials 0.000 claims abstract description 4
- 235000019354 vermiculite Nutrition 0.000 claims abstract description 4
- 235000008584 Hovenia dulcis Nutrition 0.000 claims abstract description 3
- 244000010000 Hovenia dulcis Species 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000003242 anti bacterial agent Substances 0.000 claims description 4
- 229940088710 antibiotic agent Drugs 0.000 claims description 4
- 230000012010 growth Effects 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000012880 LB liquid culture medium Substances 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 235000010987 pectin Nutrition 0.000 claims description 3
- 229920001277 pectin Polymers 0.000 claims description 3
- 239000001814 pectin Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000004114 suspension culture Methods 0.000 claims description 3
- 230000006698 induction Effects 0.000 abstract description 7
- 206010020649 Hyperkeratosis Diseases 0.000 abstract description 4
- 230000008595 infiltration Effects 0.000 abstract description 4
- 238000001764 infiltration Methods 0.000 abstract description 4
- 241000196324 Embryophyta Species 0.000 description 7
- 235000020971 citrus fruits Nutrition 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 2
- 241000207199 Citrus Species 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
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Abstract
The invention relates to a non-tissue culture hovenia dulcis high-efficiency hairy root transformation method, which comprises the following steps: 1. culturing seeds of the immature bitter orange to obtain immature bitter orange seedlings for infection; 2. preparing agrobacterium rhizogenes bacterial liquid for infection; 3. and (2) placing the immature bitter orange seedlings obtained in the step (1) into the agrobacterium rhizogenes bacterial liquid obtained in the step (2), then transferring the immature bitter orange seedlings into a breathable and moisture-retaining culture container containing a vermiculite matrix after vacuum infiltration for more than 25min at 30-40psi, culturing for 3-4 days under a dark condition, then transferring into a 16-hour illumination/8-hour dark condition for alternate culture, and inducing rooting to obtain transgenic hairy roots. White callus can be seen after the invention is co-cultured for about 15 days, transgenic positive hairy roots can be obtained after about 25 days, the induction efficiency of the transgenic hairy roots can reach more than 90 percent, and the time consumption is short.
Description
Technical Field
The invention relates to the technical field of plant hairy root transformation methods, in particular to a non-tissue culture trifoliate high-efficiency hairy root transformation method.
Background
The hairy root is a pathological growth formed when plant tissues are infected by agrobacterium rhizogenes at a wound part, and is called as a hairy root because a plurality of branched adventitious roots are formed at the wound. After the infection, the T-DNA fragment on the Ri plasmid of the agrobacterium rhizogenes is inserted into a plant genome, and the T-DNA fragment contains an auxin synthetic gene, so that the plant can be induced to generate a transgenic root system with a target gene. The hairy root has the advantages of high growth speed, no need of exogenous hormone, stable heredity and the like, and has great application prospect.
Citrus fruits are the largest yields of fruit in the world. Citrus is used as a tree seed for asexual propagation, and the trifoliate orange is a stock which is most widely applied and has excellent characteristics of cold resistance and the like, but the trifoliate orange is not salt-alkali resistant and drought resistant, so that the trifoliate orange is required to be subjected to resistance breeding improvement by combining a molecular technical means. Agrobacterium mediated genetic transformation has been widely applied to citrus, firstly, agrobacterium tumefaciens is used to infect internode stems to obtain transgenic positive regeneration buds which are then rooted to obtain transgenic strains; secondly, agrobacterium rhizogenes is used for infecting the incision of the epicotyl to obtain the transgenic hairy root, the two methods need to maintain strict aseptic environment during operation, subculture is needed every 20 days or so, the transgenic plant can be obtained after 2-3 subcultures, the operation is troublesome, the consumed time is long, and the transformation efficiency can only reach about 50%. At present, a plurality of plants have non-tissue culture hairy root induction culture systems, but the induction culture systems of each plant are different, and the species of the same culture system is difficult to succeed when one species is changed, and a clear non-tissue culture method for successfully obtaining transgenic hairy roots on poncirus trifoliata does not exist. The invention provides a non-tissue culture method for transforming the high-efficiency hairy roots of trifoliate orange, which is characterized in that a target gene is transferred into agrobacterium rhizogenes and infects trifoliate orange, and the induction efficiency of transgenic hairy roots can reach more than 90 percent.
Disclosure of Invention
According to the problems existing in the prior art, the invention discloses a non-tissue culture method for efficiently transforming trifoliate orange hairy roots
A non-tissue culture method for transforming the high-efficiency hairy roots of trifoliate orange comprises the following steps:
step 1, culturing seeds of the immature bitter oranges to obtain immature bitter orange seedlings for infection;
step 2, preparing agrobacterium rhizogenes bacterial liquid for infection;
and 3, placing the immature bitter orange seedlings obtained in the step 1 into the agrobacterium rhizogenes bacterial liquid obtained in the step 2, then transferring the immature bitter orange seedlings into a breathable and moisture-preserving culture container containing a vermiculite matrix after vacuum infiltration for more than 25min at 30-40psi, culturing the immature bitter orange seedlings for 3-4 days under a dark condition, then transferring the immature bitter orange seedlings into a 16-hour illumination/8-hour dark condition for alternative culture, and inducing rooting to obtain transgenic hairy roots.
Further, the step 1 specifically includes the following steps:
step 1.1, taking fresh seeds of trifoliate orange fruits, cleaning, and treating for 15min by using 1mol/L sodium hydroxide to remove pectin;
step 1.2, placing the seeds in a 30% sodium hypochlorite solution for disinfection for 10min, placing the seeds on a wet gauze, and carrying out dark culture for several days at the temperature of 25-30 ℃;
and step 1.3, transferring the seeds into a matrix after the seeds germinate, and placing the seeds in a growth room at the temperature of 26 +/-2 ℃ for 16 hours of illumination/8 hours of darkness until the branches are lignified to obtain immature bitter orange seedlings for infection.
Further, the step 2 specifically includes the following steps:
2.1, transferring the exogenous gene or the plasmid inserted with the exogenous gene into agrobacterium rhizogenes, and mixing the bacterial liquid of the agrobacterium rhizogenes with the ratio of 1:200 are inoculated into 40-50mL LB liquid culture medium containing antibiotics, and cultured for more than 12 hours under the conditions of 28 ℃ and 200r/min shaking table to obtain standby bacterial liquid;
and 2.2, centrifuging the standby bacterial liquid for 5min at the room temperature under the condition of 4000r/min, discarding the supernatant, resuspending the supernatant by using 40mL MT suspension culture medium, adding 50mg/L acetosyringone, and adjusting the OD600 of the agrobacterium rhizogenes bacterial liquid to be 0.6-1.0 to obtain the agrobacterium rhizogenes bacterial liquid for infection.
Further, the culture container in the step 3 is a tissue culture bottle with adjustable air holes.
In order to solve the technical problems, the invention adopts the following technical scheme:
the beneficial effects of the invention are as follows: the invention provides a non-tissue culture hovenia dulcis high-efficiency hairy root transformation method, which has the following advantages: (1) The method can be completed under the non-tissue culture condition without an aseptic environment, and is simple and convenient to operate; (2) The culture container has the characteristics of high moisture retention and ventilation, enough water is poured at the beginning, and watering is not needed after 20 days, so that much labor is not needed; (3) Compared with the traditional tissue culture condition that sterile hairy roots can be obtained only in two months, the induction rate is about 50%, white callus can be seen after co-culture is carried out for about 15 days, transgenic positive hairy roots can be obtained after about 25 days, the induction efficiency of the transgenic hairy roots can reach over 90%, and the time consumption is short.
The present invention will be described in detail below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a co-cultivation diagram of trifoliate orange branches placed in a culture vessel after infection;
FIG. 2 is a white callus image grown after co-cultivation for about 15 d;
FIG. 3 is a comparison of the fluorescent field and the bright field of hairy roots growing in about 60 days of co-culture.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
A non-tissue culture method for transforming the high-efficiency hairy roots of trifoliate orange comprises the following steps:
(1) Obtaining seedlings for infestation:
taking fresh seeds of the trifoliate orange, cleaning, and treating with 1mol/L sodium hydroxide for 15min to remove pectin. Then the seeds are sterilized in 30% sodium hypochlorite solution for 10min, and shaken for several times. Placing the seeds on moist gauze, carrying out dark culture at 28 ℃ for several days, transferring the seeds into a matrix after the seeds germinate as shown in figure 1, wherein the used culture container is a tissue culture bottle purchased from Shanghai Zhi Ji apparatus limited company, and because the moisture retention is better, the seeds can be watered enough at the beginning, the seeds can be kept for about 20 days without watering, and the seeds are placed in a growth chamber at 26 +/-2 ℃ for illumination/dark alternate culture (16 hours of illumination/8 hours of dark) until branches are lignified and then can be used for infection; immature bitter orange seedlings for infection cannot be too tender, and can be used for infection only after branches are lignified, and the hairy root induction efficiency is high at the moment.
(2) Preparing agrobacterium rhizogenes bacterial liquid with target genes:
the Agrobacterium rhizogenes strain employed in the present invention is MSU440, provided by Shanghai Diego Biotechnology Ltd. Transforming the plasmid with the GFP fluorescent label into agrobacterium rhizogenes, coating the agrobacterium rhizogenes on a flat plate containing corresponding antibiotics, carrying out inverted culture in an incubator at 28 ℃ for 2d, picking up plaque and shaking bacteria, and carrying out PCR (polymerase chain reaction) to identify whether the vector is successfully transformed into the agrobacterium rhizogenes. The plasmid was successfully transferred into agrobacterium rhizogenes in a ratio of 1:200 are inoculated into 40-50mL LB liquid culture medium containing antibiotics, and are cultured for about 12 hours at 28 ℃ and 200r/min by shaking for standby. Centrifuging the bacterial liquid at 4000r/min at room temperature for 5min, discarding the supernatant, resuspending with 40mL MT suspension culture medium, adding 50mg/L acetosyringone, measuring the concentration of the bacterial liquid of the agrobacterium with an ultraviolet spectrophotometer, and adjusting OD600 to 0.6-1.0 to be used for infection.
(3) And (3) turning the immature bitter orange:
cutting off the trifoliate orange branches which can be used for infection, pricking 5-6 holes at the roots of the branches after sucking the bacterial liquid by using an injector, directly injecting the bacterial liquid into the trifoliate orange branches in the process of pricking the holes, then immersing the branches into the agrobacterium rhizogenes bacterial liquid, and carrying out vacuum infiltration for about 30min at 30 psi.
(4) Transferring the trifoliate orange branches after vacuum infiltration into a breathable and moisture-retaining culture container containing an inert vermiculite matrix, culturing for 3-4 days under a dark condition, then transferring into alternate light for culturing, and inducing rooting to obtain transgenic hairy roots. As shown in FIG. 2, white callus can be seen to grow at the puncture of the trifoliate orange branch after culturing under alternating light for about 15 days, and roots grow after culturing for about 25 days. As shown in FIG. 3, since the objective vector contains a GFP fluorescent tag, the hairy roots grow, and then the roots are observed by a hand-held fluorescent lamp to emit green fluorescence.
The foregoing is illustrative of the best mode contemplated for carrying out the present invention and the details not specifically mentioned are within the knowledge of one of ordinary skill in the art. The scope of the present invention is defined by the appended claims, and any equivalent modifications based on the technical teaching of the present invention are also within the scope of the present invention.
Claims (4)
1. A non-tissue culture method for transforming the high-efficiency hairy roots of trifoliate orange is characterized by comprising the following steps:
step 1, culturing seeds of the immature bitter oranges to obtain immature bitter orange seedlings for infection;
step 2, preparing agrobacterium rhizogenes bacterial liquid for infection;
and 3, placing the immature bitter orange seedlings obtained in the step 1 in the agrobacterium rhizogenes bacterial liquid obtained in the step 2, then transferring the immature bitter orange seedlings into a breathable and moisture-preserving culture container containing a vermiculite matrix after vacuum permeation for more than 25min at 30-40psi, culturing the immature bitter orange seedlings for 3-4 days under a dark condition, then transferring the immature bitter orange seedlings into a 16-hour illumination/8-hour dark condition for alternative culture, and inducing the immature bitter orange seedlings to grow roots to obtain transgenic hairy roots.
2. The non-tissue culture trifoliate high-efficiency hairy root transformation method according to claim 1, wherein the step 1 specifically comprises the following steps:
step 1.1, taking fresh seeds of trifoliate orange fruits, cleaning, and treating for 15min by using 1mol/L sodium hydroxide to remove pectin;
step 1.2, placing the seeds in a 30% sodium hypochlorite solution for 10min to sterilize, placing the seeds on a wet gauze, and performing dark culture for several days at the temperature of 25-30 ℃;
and step 1.3, transferring the seeds into a matrix after the seeds germinate, and placing the seeds in a growth room at the temperature of 26 +/-2 ℃ for 16 hours of illumination/8 hours of darkness until the branches are lignified to obtain immature bitter orange seedlings for infection.
3. The non-tissue culture hovenia dulcis high-efficiency hairy root transformation method according to claim 1, wherein the step 2 specifically comprises the following steps:
2.1, transferring the exogenous gene or the plasmid inserted with the exogenous gene into agrobacterium rhizogenes, and mixing the bacterial liquid of the agrobacterium rhizogenes with the ratio of 1:200 percent of the strain is inoculated into 40-50mL LB liquid culture medium containing antibiotics, and cultured for more than 12 hours under the conditions of a shaking table at 28 ℃ and 200r/min to obtain standby bacterial liquid;
and 2.2, centrifuging the standby bacterial liquid for 5min at the room temperature under the condition of 4000r/min, discarding the supernatant, resuspending the supernatant by using 40mL MT suspension culture medium, adding 50mg/L acetosyringone, and adjusting the OD600 of the agrobacterium rhizogenes bacterial liquid to be 0.6-1.0 to obtain the agrobacterium rhizogenes bacterial liquid for infection.
4. The method for transforming non-tissue culture trifoliate orange high-efficiency hairy roots according to claim 1, wherein the culture container in the step 3 is a tissue culture bottle with adjustable air holes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146748A (en) * | 2012-04-13 | 2013-06-12 | 天津大学 | Agrobacterium mediated rose bud transgenosis infecting method |
| CN103320464A (en) * | 2013-06-18 | 2013-09-25 | 华中农业大学 | Method for generating hairy roots by efficiently inducing trifoliate orange by agrobacterium rhizogenes and application of method |
| WO2017063060A2 (en) * | 2015-10-14 | 2017-04-20 | Instituto Agronõmico Do Paraná - Iapar | Method for production of transgenic plants and bacteria-resistant plants having the "quorum sensing" system |
| CN111454986A (en) * | 2020-04-13 | 2020-07-28 | 南京农业大学 | Genetic transformation method for pears |
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- 2022-12-26 CN CN202211686956.2A patent/CN115873895B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146748A (en) * | 2012-04-13 | 2013-06-12 | 天津大学 | Agrobacterium mediated rose bud transgenosis infecting method |
| CN103320464A (en) * | 2013-06-18 | 2013-09-25 | 华中农业大学 | Method for generating hairy roots by efficiently inducing trifoliate orange by agrobacterium rhizogenes and application of method |
| WO2017063060A2 (en) * | 2015-10-14 | 2017-04-20 | Instituto Agronõmico Do Paraná - Iapar | Method for production of transgenic plants and bacteria-resistant plants having the "quorum sensing" system |
| CN111454986A (en) * | 2020-04-13 | 2020-07-28 | 南京农业大学 | Genetic transformation method for pears |
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