CN104263752A - Transgenic method for adult orange stem - Google Patents
Transgenic method for adult orange stem Download PDFInfo
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- CN104263752A CN104263752A CN201410528306.4A CN201410528306A CN104263752A CN 104263752 A CN104263752 A CN 104263752A CN 201410528306 A CN201410528306 A CN 201410528306A CN 104263752 A CN104263752 A CN 104263752A
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Abstract
The invention provides a transgenic method for an adult orange stem. The transgenic method comprises the following steps: performing transfection on agrobacterium and inducing callus. The transgenic method is characterized in that a culture medium induced by the callus comprises 1.8-2.2mg/L of isopentene adenine and 0.4-0.6mg/L of indoleacetic acid. By adopting the transgenic method, the heredity conversion rate of 7.8% can be obtained. Therefore, an obtained transgenic plant can effectively avoid the juvenile phase and can blossom and bear fruits within 2-3 years after being transplanted to the field.
Description
Technical field
The present invention relates to a kind of transgenic method of citrus Adult form stem section.It is mainly used in the genetic transformation of sweet orange class citrus Adult form stem section.
Background technology
The explant material of citrus transgenic research early application mainly contains protoplastis, the epicotyl, cotyledon etc. of aseptic seedling.The Protoplast cuhnre cycle due to kind a lot of in orange tree is long and regeneration is difficult, and cost is also higher, so there be limited evidence currently of has laboratory to adopt in this way; And utilize the epicotyl of aseptic seedling, cotyledon for explant owing to can obtain higher genetic transformation rate and adopt so far always, but transfer-gen plant obtained thus just can will blossom and bear fruit through the very long virgin phase.
In order to accelerate Character Evaluation and the application of citrus transfer-gen plant, the Study on Genetic Transformation of carrying out for explant with Adult form stem section in recent years more and more comes into one's own.But it is generally speaking all lower for explant material carries out the genetic transformation rate that genetic transformation obtains with Adult form stem section.External Cervera etc. (1998) utilize the Adult form stem section of pineapple sweet orange to carry out transgenic research first, obtain 6.1% genetic transformation rate, that after this carries out is explant with citrus Adult form stem section transgenic research does not exceed this genetic transformation rate.Domestic Yang Li etc. (2009) obtain the genetic transformation rate of 7.6% when utilizing sweet orange class citrus variety crystal sugar orange mature internode stems to carry out transgenic research.The 7.6% genetic transformation rate that this institute obtains is undertaken adding up by the number of the number/resistant buds of the positive bud of PCR, and calculate the number of the number/inoculation explant of number that the general method of genetic transformation rate is positive bud or transfer-gen plant.In an experiment, because the regeneration frequency of indefinite bud is only about 48%, thus calculate according to the method for general genetic transformation rate, its genetic transformation rate far below 7.6%, only about 3.6%.Thus the document with regard to having delivered at present, is up to 6.1% with the genetic transformation rate that citrus Adult form stem section obtains for explant material.
The genetic transformation of citrus Adult form stem section one can be divided into 2 stages, i.e. Dual culture phase and the adventitious bud inducing phase.In the adventitious bud inducing phase, callus induction phase (light culture) and indefinite bud can be divided into again to sprout according to the presence or absence of illumination and vegetative period (see light cultivation).In each incubation period above-mentioned, use the microbiotic of the plant hormone of different sorts, concentration and different sorts, concentration, finally genetic transformation rate is all likely impacted.In research in the past, explant before Dual culture all not through preculture.And in the adventitious bud inducing phase, the plant hormone (combination) of use is BA, or BAP, or BAP+NAA, or ZR+GA; The working concentration of kantlex (Km) is 0, or 30 mg/L, or 100mg/L.The plant hormone of these kinds (combination) and kantlex (Km) concentration is used to make the transgenic research of citrus Adult form stem section obtain lower genetic transformation rate.
Summary of the invention
The object of the invention is to overcome the low grade of existing citrus Adult form stem section genetic transformation rate not enough, a kind of transgenic method of efficient citrus Adult form stem section is provided.
The object of the invention is to be realized by following measures:
A kind of transgenic method of citrus Adult form stem section, comprise Agrobacterium infection, callus induction step, it is characterized in that: the substratum of described callus induction comprises 1.8-2.2mg/L 2-ip (isopentenyl gland purine), 0.4-0.6mg/LIAA (indolylacetic acid).Preferably, 40-60mg/LKm (kantlex) is also added in substratum.Described 2-ip and IAA plant hormone is used to combine in the callus induction stage, and kantlex is as selective agent, and each concentration of component is controlled in appropriate scope, explant wound can be made to induce more callus, make callus cell accumulate a certain amount of phytokinin simultaneously, thus the sprouting of indefinite bud and growth after being conducive to citrus Adult form stem Duan Jianguang.Simultaneously also for the germination and growth of positive indefinite bud provides favourable condition, make it have growth vigor.
The transgenic method of above-mentioned citrus Adult form stem section, carries out preculture before Agrobacterium infection, it is characterized in that described preculture is shaking culture 3.5-4.5 hour in containing the liquid nutrient medium of plant hormone.Described plant hormone is 1.8-2.2mg/L 2-ip and 0.4-0.6mg/L IAA.The present invention makes the cell on citrus Adult form stem section wound be in a kind of impression state being easy to accept external information, thus be conducive to transforming, prevent because anoxic causes citrus Adult form stem section brownization and death simultaneously, and the injury of Agrobacterium to Adult form stem section can be alleviated, improve transformation efficiency.
The transgenic method of above-mentioned citrus Adult form stem section, also comprises sprouting and the growth step of indefinite bud after callus induction, it is characterized in that the sprouting of indefinite bud and growth step substratum are WPM solid medium.Preferably, the sprouting of indefinite bud and growth step substratum are made up of fungistat and WPM solid medium.Described fungistat is preferably 250mg/L vancomycin and 250mg/L cephamycin.Plant hormone and selective agent is not added in substratum.From the indefinite bud robust growth that callus is sprouted in the present invention, avoid occurring the phenomenons such as bud is very thin, growing way is weak, improve surviving rate and the genetic transformation rate of indefinite bud.
The transgenic method of above-mentioned citrus Adult form stem section, comprises the following steps:
● the preculture of citrus Adult form stem section: before agroinfection is carried out to citrus Adult form stem section, the Adult form stem section being about 0.8-1.2cm is put into the MS liquid nutrient medium shaking culture 3.5-4.5 hour containing 1.8-2.2mg/L 2-ip and 0.4-0.6mg/L IAA;
● the callus induction stage: it is 1.8-2.2mg/L 2-ip and 0.4-0.6mg/L IAA that culture medium prescription comprises plant hormone, and selective agent is 40-60mg/L Km.
● the sprouting of indefinite bud and growth phase: culture medium prescription is WPM solid medium and fungistat; Described fungistat is Van (vancomycin) and Cef (cephamycin).
The transgenic method of above-mentioned citrus Adult form stem section, comprises the following steps:
(1) preparation of explant
Choose the citrus Adult form branch of robust growth in 3-4 month in the first year or 7-8 month, by bud grafting full on branch on citrange stock.After grafting survival, wait sprout first time the tip or first time tip cutting back after the second time tip sprouted grow to and be about 20 centimetres time cut with secateurs, remove blade and thorn, as the explant material of genetic transformation;
(2) preparation of engineering bacteria liquid
By single colony inoculation of agrobacterium strains in 10ml LB or YEB liquid nutrient medium, under 28 DEG C of dark, 220r/min shaking culture is to OD
600for 0.8-1.0; The above-mentioned identical liquid nutrient medium of Agrobacterium bacterium liquid is diluted to OD
600be 0.1, under above-mentioned identical culture condition, continue shaking culture to OD
600be 0.5;
(3) explant sterilization
By removing blade and thorn after explant with 0.1% mercuric chloride sterilize 10 minutes, sterile water wash 4-5 time;
(4) explant preculture
After sterilization, removed at the two ends of explant, then remove the axillalry bud on branch in the mode of " V-shape ", otch is too not dark, explant is not cut off, only removes axillalry bud, and remaining part is cut into the stem section of 0.8-1.2cm; Stem section is put into and MS liquid nutrient medium is housed and the triangular flask of additional 1.8-2.2mg/L 2-ip and 0.4-0.6mg/LIAA, at 28 DEG C, shaking culture 3.5-4.5 hour under 100r/min;
(5) explant infects
1. the collection of bacterium liquid: pour in the centrifuge tube of 50mL by engineering bacteria liquid, at 28 DEG C, under the condition of 5000r/min centrifugal 10 minutes; Supernatant liquor is clean, with MS liquid nutrient medium Eddy diffusion Agrobacterium;
2. the collection of explant: outwelled by the liquid nutrient medium in triangular flask, sandwiches in culture dish with tweezers by the explant in bottle;
3. the infection of explant: pour in culture dish by the Agrobacterium bacterium liquid of Eddy diffusion, move with have gentle hands jog, makes all explants all immerse in Agrobacterium bacterium liquid, infects 15 minutes;
4. the removal of unnecessary bacterium liquid: infect after 15 minutes, explant tweezers are sandwiched in the culture dish containing medicated napkin; With medicated napkin wiping explant repeatedly lightly, till moisture is can't see on explant surface;
(6) Dual culture
Metainfective explant is placed on be added with 2mg/L 2-ip, 2mg/L IAA, 2mg/L 2,4-D (2,4 dichlorophenoxyacetic acid) and 100 μMs of AS (acetyl cloves copper) MS solid medium on, light culture 3 days under 26 DEG C of conditions;
(7) induction of indefinite bud
1. the induction of callus: explant, after 3 days, proceeds in screening culture medium by Dual culture; Screening culture medium is MS solid medium and adds 1.8-2.2mg/L 2-ip, 0.4-0.6mg/L IAA, 40-60mg/L Km, 250mg/LVan and 250mg/L Cef; Light culture 15 days under 28 DEG C of conditions;
2. the sprouting of indefinite bud and growth: after light culture terminates, proceeded to by explant in adventitious bud induction culture base, adventitious bud induction culture base is that WPM solid medium adds 250mg/L Van and 250mg/L Cef; Culture temperature is 28 DEG C, the photoperiod is 16 hours;
(8) indefinite bud grafting
When the stem of positive indefinite bud is about 0.5cm, under horizontal cutting, test tube micrografting, on citrange stock, obtains transfer-gen plant;
(9) the field grafting (secondary grafting) of transfer-gen plant
Transfer-gen plant from the base portion horizontal cutting of citrange stock, according to the method for field cut-grafting by its grafting on the citrange stock of field, with 2 week of plastics bag moisturizing; Plastics bag is removed after it survives.
The genetic transformation of citrus Adult form stem section one can be divided into 2 stages, i.e. Agrobacterium Dual culture phase and the adventitious bud inducing phase.In the adventitious bud inducing phase, callus induction phase (light culture) and indefinite bud can be divided into again to sprout according to the presence or absence of illumination and vegetative period (see light cultivation).In each incubation period above-mentioned, use the microbiotic of the plant hormone of different sorts, concentration and different sorts, concentration, finally genetic transformation rate is all impacted.
Beneficial effect
The present invention can obtain the genetic transformation rate of 7.8%.Transfer-gen plant obtained thus effectively can avoid the virgin phase, and after being transplanted to field, 2-3 just can blossom and bear fruit.
Accompanying drawing explanation
Fig. 1: citrus Adult form stem section;
Fig. 2: the preculture of citrus Adult form stem section;
Fig. 3: the Dual culture of citrus Adult form stem section and Agrobacterium;
Fig. 4: the induction of callus;
Fig. 5: the sprouting of indefinite bud and growth;
The control group of Fig. 6: indefinite bud GUS dyeing;
The positive bud of Fig. 7: indefinite bud GUS dyeing;
Fig. 8: the in vitro grafting of indefinite bud;
Fig. 9, Figure 10: the field grafting of transfer-gen plant.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment
1, the preparation of explant
Choose the blood orange Adult form branch of robust growth in 3-4 month in the first year or 7-8 month, by bud grafting full on branch on citrange stock.After grafting survival, wait sprout first time the tip or first time tip cutting back after the second time tip sprouted grow to and be about 20 centimetres time cut with secateurs, remove blade and thorn, as the explant material (see Fig. 1) of genetic transformation.
2, the preparation of engineering bacteria liquid
Select single colony inoculation of agrobacterium strains EHA105 before conversion in 10mlLB liquid nutrient medium, under 28 DEG C of dark, 220r/min shaking culture is to OD
600for 0.8-1.0; The above-mentioned identical LB liquid nutrient medium of Agrobacterium bacterium liquid is diluted to OD
600be 0.1, under above-mentioned identical culture condition, continue shaking culture to OD
600be 0.5.
In this example, agrobacterium strains EHA105 contains pL35SAATCB carrier.This carrier using antibacterial peptide gene AATCB as goal gene, using NPT II (neomycin phosphotransferase gene) as riddled basins; Contain Reporter gene GUS (beta-glucosiduronatase gene) in this carrier simultaneously.
3, explant sterilization
By removing blade and thorn after explant with 0.1% mercuric chloride sterilize 10 minutes, sterile water wash 4-5 time.
4, explant preculture
After sterilization, removed at the two ends of explant, then remove the axillalry bud (otch is too not dark, explant is not cut off, only removes axillalry bud) on branch in the mode of " V-shape ", remaining part is cut into the stem section of about 1cm; Stem section is put into and MS liquid nutrient medium is housed and the triangular flask of additional 2mg/L 2-ip and 0.5mg/L IAA, 28 DEG C, shaking culture 4 hours (see Fig. 2) under 100r/min.
In this example, MS liquid nutrient medium is the sucrose that MS minimum medium adds 30g/L, PH5.8.MS minimum medium is filled a prescription: (1) macroelement: NH
4nO
31650; KNO
31900; CaCl
22H
2o 440; MgSO
47H
2o370; KH
2pO
41700; (2) trace element: KI0.83; H
3bO
36.2; MnSO
44H
2o22.3; ZnSO
47H
2o 8.6; Na
2mnO
42H
2o 0.25; CuSO
45H
2o 0.025; CoCl
26H
2o0.025; 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 is mg/L.
5, explant infects
(1) collection of bacterium liquid: pour in the centrifuge tube of 50mL by engineering bacteria liquid, at 28 DEG C, under the condition of 5000r/min centrifugal 10 minutes; Supernatant liquor is clean, with MS liquid nutrient medium Eddy diffusion Agrobacterium.
Only PH is different for MS liquid nutrient medium in this example and above-mentioned MS liquid nutrient medium, and remaining composition is identical; PH value in this example is 5.4.
(2) collection of explant: outwelled by the liquid nutrient medium in triangular flask, sandwiches the explant in bottle in culture dish with tweezers.
(3) infection of explant: pour in culture dish by the Agrobacterium bacterium liquid of Eddy diffusion, move with have gentle hands jog, makes all explants all immerse in Agrobacterium bacterium liquid, infects 15 minutes.
(4) removal of unnecessary bacterium liquid: infect after 15 minutes, explant tweezers are sandwiched in the culture dish containing medicated napkin; With medicated napkin wiping explant repeatedly lightly, till moisture is can't see on explant surface.
6, Dual culture
Metainfective explant is placed on the MS solid medium of additional 2mg/L 2-ip, 2mg/L IAA, 2mg/L 2,4-D and 100 μMs of AS.Light culture 3 days (see Fig. 3) under 26 DEG C of conditions.
In this example, MS solid medium is the sugar of the agar that MS minimum medium adds 8g/L, 30g/L, PH5.8, lower same.
7, the induction of indefinite bud
(1) induction of callus: explant, after 3 days, proceeds in screening culture medium by Dual culture; Screening culture medium is MS solid medium and additional 2mg/L 2-ip, 0.5mg/L IAA, 50mg/L Km, 250mg/L Van and 250mg/L Cef; Light culture 15 days (see Fig. 4) under 28 DEG C of conditions.
(2) sprouting of indefinite bud and growth: after light culture terminates, proceeded to by explant in adventitious bud induction culture base, adventitious bud induction culture base is that WPM solid medium adds 250mg/L Van and 250mg/L Cef; Culture temperature is 28 DEG C, the photoperiod is 16 hours (see Fig. 5).
WPM solid medium is the agar of the sugar that WPM minimum medium adds 30g/L, 8g/L.The formula of WPM minimum medium is: 1) macroelement: NH
4nO
3400; K
2sO
4990; Ca (NO
3)
24H
2o 556; MgSO
47H
2o 370; KH
2pO
4170; 2) trace element: H
3bO
36.2; MnSO
44H
2o 22.5; ZnSO
47H
2o 8.6; Na
2mnO
42H
2o 0.25; CuSO
45H
2o 0.25; 3) organic composition: inositol 100; Nicotinic acid 0.5; Pyridoxine hydrochloride (vitamins B
6) 0.5; Vitamin (vitamins B
1) 1.0; Glycine 2.0; 4) molysite: FeSO47H
2o (27.8)+Na
2-EDTA2H
2o (37.3); Above unit is mg/L.
8, the GUS dyeing of indefinite bud
When the stem of indefinite bud is about 0.5cm, cut the sub-fraction blade near blade tip, immerse in GUS dye liquor, 37 DEG C of overnight incubation; Within second day, to be proceeded to by the blade cut in 70% ethanol and decolour 2 times, when negative control material is white, observe (Fig. 6), incision or the whole part cut, in blue (Fig. 7), are defined as positive bud.
In this example, GUS dye liquor is containing, for example lower composition: NaH
2pO
4100, Na
2hPO
4100, K
4[Fe (CN)
6] 0.5, K
3[Fe (CN)
6] 0.5, EDTA-Na
210, X-Gluc 1, above unit is mM; Sodium azide0.1%, Triton-1000.1%.
9, indefinite bud grafting
By under the indefinite bud horizontal cutting of GUS stained positive, test tube micrografting, on citrange stock, obtains GUS positive plant (see Fig. 8).
10, the molecular biology identification of transfer-gen plant
Extract the DNA of GUS positive plant, carry out pcr analysis, obtain transgenosis blood orange.The method of calculation of genetic transformation rate are the explant sum of the sum/inoculation of PCR positive plant, experiment repetition 3 times.This experiment obtains 36 strain PCR positive plants altogether, and the explant of inoculation is 459, and genetic transformation rate=36/459, is about 7.8%.
11, the field grafting (secondary grafting) of transgenosis blood orange
Transgenosis blood orange from the base portion horizontal cutting of citrange stock, according to the method for field cut-grafting by its grafting on the citrange stock of field (Fig. 9), with 2 week of plastics bag moisturizing (Figure 10).Plastics bag is removed after it survives.
Claims (10)
1. a transgenic method for citrus Adult form stem section, comprises Agrobacterium infection, callus induction step, it is characterized in that: the substratum of described callus induction comprises 1.8-2.2 mg/L isopentenyl gland purine, 0.4-0.6 mg/L indolylacetic acid.
2. the transgenic method of citrus Adult form stem section as claimed in claim 1, also comprises 40-60 mg/L kantlex in the substratum of described callus induction.
3. the transgenic method of citrus Adult form stem section as claimed in claim 1 or 2, carries out preculture before Agrobacterium infection, it is characterized in that: described preculture is shaking culture 3.5-4.5 hour in liquid medium within.
4. the transgenic method of citrus Adult form stem section as claimed in claim 3, carries out preculture before Agrobacterium infection, it is characterized in that: be added with plant hormone in liquid nutrient medium.
5. the transgenic method of citrus Adult form stem section as claimed in claim 4, described plant hormone is 1.8-2.2 mg/L 2-ip and 0.4-0.6 mg/L IAA.
6. the transgenic method of the citrus Adult form stem section as described in as arbitrary in claim 1-5, also comprises sprouting and the growth step of indefinite bud, it is characterized in that: the sprouting of indefinite bud and growth step substratum are WPM solid medium after callus induction.
7. the transgenic method of citrus Adult form stem section as claimed in claim 6, described WPM solid medium adds fungistat.
8. the transgenic method of citrus Adult form stem section as claimed in claim 7, the sprouting of indefinite bud and growth step substratum are WPM solid medium, 250mg/L vancomycin and 250mg/L cephamycin.
9. the transgenic method of citrus Adult form stem section as claimed in claim 1, comprises the following steps:
●the preculture of citrus Adult form stem section: before agroinfection is carried out to citrus Adult form stem section, the Adult form stem section of long 0.8-1.2cm is put into the MS liquid nutrient medium shaking culture 3.5-4.5 hour containing 1.8-2.2mg/L 2-ip and 0.4-0.6mg/L IAA;
●in the callus induction stage: it is 1.8 ~ 2.2mg/L 2-ip and 0.4 ~ 0.6mg/L IAA that culture medium prescription comprises plant hormone, selective agent is 40 ~ 60 mg/L Km;
●the sprouting of indefinite bud and growth phase: culture medium prescription is WPM solid medium and fungistat; Described fungistat is Van and Cef.
10. the transgenic method of citrus Adult form stem section as claimed in claim 1, comprises the following steps:
(1) preparation of explant
Choose the citrus Adult form branch of robust growth in 3-4 month in the first year or 7-8 month, by bud grafting full on branch on citrange stock;
After grafting survival, wait sprout first time the tip or first time tip cutting back after the second time tip sprouted grow to and be about 20 centimetres time cut with secateurs, remove blade and thorn, as the explant material of genetic transformation;
(2) preparation of engineering bacteria liquid
By single colony inoculation of agrobacterium strains in 10ml LB or YEB liquid nutrient medium, under 28 DEG C of dark, 220r/min shaking culture is to OD
600for 0.8-1.0; The above-mentioned identical liquid nutrient medium of Agrobacterium bacterium liquid is diluted to OD
600be 0.1, under above-mentioned identical culture condition, continue shaking culture to OD
600be 0.5;
(3) explant sterilization
By removing blade and thorn after explant with 0.1% mercuric chloride sterilize 10 minutes, sterile water wash 4-5 time;
(4) explant preculture
After sterilization, removed at the two ends of explant, then remove the axillalry bud on branch in the mode of " V-shape ", otch is too not dark, explant is not cut off, only removes axillalry bud, and remaining part is cut into the stem section of 0.8-1.2cm; Stem section is put into and MS liquid nutrient medium is housed and the triangular flask of additional 1.8-2.2mg/L 2-ip and 0.4-0.6mg/L IAA, at 28 DEG C, shaking culture 3.5-4.5 hour under 100r/min;
(5) explant infects
1. the collection of bacterium liquid: pour in the centrifuge tube of 50mL by engineering bacteria liquid, at 28 DEG C, under the condition of 5000r/min centrifugal 10 minutes; Supernatant liquor is clean, with MS liquid nutrient medium Eddy diffusion Agrobacterium;
2. the collection of explant: outwelled by the liquid nutrient medium in triangular flask, sandwiches in culture dish with tweezers by the explant in bottle;
3. the infection of explant: pour in culture dish by the Agrobacterium bacterium liquid of Eddy diffusion, move with have gentle hands jog, makes all explants all immerse in Agrobacterium bacterium liquid, infects 15 minutes;
4. the removal of unnecessary bacterium liquid: infect after 15 minutes, explant tweezers are sandwiched in the culture dish containing medicated napkin; With medicated napkin wiping explant repeatedly lightly, till moisture is can't see on explant surface;
(6) Dual culture
Metainfective explant is placed on be added with 2mg/L 2-ip, 2mg/L IAA, 2mg/L 2,4 dichlorophenoxyacetic acid and 100 μMs of acetyl cloves copper MS solid medium on, light culture 3 days under 26 DEG C of conditions;
(7) induction of indefinite bud
1. the induction of callus: explant, after 3 days, proceeds in screening culture medium by Dual culture; Screening culture medium is MS solid medium and adds 1.8-2.2mg/L 2-ip, 0.4-0.6mg/L IAA, 40-60mg/L Km, 250mg/L Van and 250mg/L Cef; Light culture 15 days under 28 DEG C of conditions;
2. the sprouting of indefinite bud and growth: after light culture terminates, proceeded to by explant in adventitious bud induction culture base, adventitious bud induction culture base is that WPM solid medium adds 250mg/L Van and 250mg/L Cef; Culture temperature is 28 DEG C, the photoperiod is 16 hours;
(8) indefinite bud grafting
When the stem of positive indefinite bud is about 0.5cm, under horizontal cutting, test tube micrografting, on citrange stock, obtains transfer-gen plant;
(9) the field grafting of transfer-gen plant
Transfer-gen plant from the base portion horizontal cutting of citrange stock, according to the method for field cut-grafting by its grafting on the citrange stock of field, with 2 week of plastics bag moisturizing; Plastics bag is removed after it survives.
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| CN106172001A (en) * | 2016-07-26 | 2016-12-07 | 象山宏森源农产品开发有限公司 | A kind of red beauty's Fructus Citri tangerinae Seedling tissue culture propagation technology |
| CN106613993B (en) * | 2017-01-13 | 2018-08-03 | 四川农业大学 | A kind of cultural method of the tissue cultures regrowth of trifoliate orange |
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