阅读说明：本技术 一种以酸枣愈伤组织为受体的遗传转化方法 (Genetic transformation method taking wild jujube callus as receptor ) 是由 孙俊 马福利 李亚梅 黄锦秋 周军永 陆丽娟 孙其宝 于 2020-12-29 设计创作，主要内容包括：本发明公开了一种以酸枣愈伤组织为受体的遗传转化方法,包括遗传转化受体的获取,农杆菌培养与浸染液制备,愈伤浸染,共培养,除菌,抑菌培养,筛选培养,抗性愈伤的鉴定。本发明利用酸枣茎段、叶片、子叶、胚轴诱导的愈伤为受体,建立酸枣遗传转化的方法。愈伤来源广泛,转化效率高,可重复性高,操作步骤简单；借助荧光标记方法辅助筛选,方便快捷；该体系为枣基因功能解析提供了便利,同时也为枣的遗传改良提供了可能。(The invention discloses a genetic transformation method taking wild jujube callus as a receptor, which comprises the steps of obtaining a genetic transformation receptor, preparing agrobacterium culture and a staining solution, staining callus, co-culturing, sterilizing, culturing with bacteriostasis, screening and culturing, and identifying resistant callus. The invention relates to a method for establishing wild jujube genetic transformation by using wild jujube stem segment, leaf, cotyledon and hypocotyl induced callus as a receptor. The callus source is wide, the transformation efficiency is high, the repeatability is high, and the operation steps are simple; the screening is assisted by a fluorescent marking method, so that the method is convenient and quick; the system provides convenience for the function analysis of the jujube gene and provides possibility for the genetic improvement of jujubes.)

1. A genetic transformation method taking wild jujube callus as a receptor is characterized by comprising the following steps:

(1) acquisition of genetic transformation receptors

Sowing wild jujube sterile seeds, selecting wild jujube stem segments, leaves, cotyledons and hypocotyls when aseptic seedlings grow true leaves, respectively cutting into explants of 0.5-1 cm multiplied by 0.5-1 cm, placing the explants in a semisolid MS callus induction culture medium, and inducing wild jujube callus to serve as a genetic transformation material;

(2) agrobacterium culture and dip-dyeing liquid preparation

Inoculating agrobacterium tumefaciens liquid GV3101 carrying plant expression vector pCAMBIA2300-GFP to a liquid LB culture medium, and culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm in a dark mode until OD is reached₆₀₀0.6 to 0.8;

subjecting the OD to₆₀₀Resuspending 0.6-0.8 of the agrobacterium liquid by using a liquid MS to obtain a staining solution, and adding 200 mu mol/L acetosyringone into the liquid MS in a super clean bench before;

(3) and (3) callus dip dyeing:

co-culturing the dip dyeing solution obtained in the step (2) and the wild jujube callus in a constant temperature shaking table at 28 ℃ and 100-150 rpm in the dark for 30-35 min to obtain a dip dyed explant;

(4) co-culturing:

draining the soaked wild jujube callus obtained in the step (3), placing the soaked wild jujube callus in a semisolid MS co-culture medium, and co-culturing for 2-3 days under a dark condition;

(5) and (3) degerming:

washing the calluses of the wild jujubes obtained in the step (4) with sterile water containing 250mg/L cefotaxime sodium for 7-8 min, and repeating for 3-4 times;

(6) bacteriostatic culture:

placing the wild jujube callus obtained in the step (5) in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days;

(7) screening and culturing:

placing the callus of wild jujube obtained in the step (6) after bacteriostatic culture in a semisolid MS screening culture medium, and obtaining homozygous transgenic wild jujube callus after 3-5 generations of screening culture for 3-4 weeks each;

(8) identification of resistant calli:

and detecting the callus of the wild jujube after genetic transformation by using an RT-PCR method, and determining the integration condition of the target gene according to whether a report gene strip exists or not.

2. The genetic transformation method of claim 1, wherein the semisolid MS callus induction culture medium comprises 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20-30 g/L sucrose, 6-7 g/L agar and a semisolid MS culture medium with pH of 5.8-6.0.

3. The genetic transformation method taking zizyphus jujube callus as an acceptor according to claim 1, wherein the inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB contained 50mg/L kanamycin and 50mg/L rifampicin.

4. The genetic transformation method of claim 1, wherein in step (2), the agrobacterium is GV3101 and the plasmid vector carried by the agrobacterium is pCAMBIA2300-GFP green fluorescence expression vector.

5. The genetic transformation method of claim 1, wherein the semisolid MS co-culture medium comprises 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20-30 g/L sucrose, 6-7 g/L agar and a semisolid MS culture medium with pH of 5.8-6.0.

6. The genetic transformation method of claim 1, wherein the semisolid MS antibacterial culture medium comprises 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 250mg/L cephamycin, 250mg/L timentin, 20-30 g/L sucrose, 6-7 g/L agar and a semisolid MS culture medium with pH of 5.8-6.0.

7. The genetic transformation method of claim 1, wherein the semisolid MS screening culture medium comprises a semisolid MS culture medium with a pH value of 5.8-6.0 and 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20mg/L kanamycin, 250mg/L cephamycin, 250mg/L timentin, 20-30 g/L sucrose, 6-7 g/L agar.

8. The genetic transformation method using wild jujube callus as receptor according to claim 1, wherein the culture conditions in steps (1), (4), (6) and (7) are 23-25 ℃, 12-14 h/d photoperiod and 2500-2800 lx illumination intensity.

Technical Field

The invention relates to the technical field of plant cell engineering and molecular biology, in particular to a genetic transformation method taking wild jujube callus as a receptor.

Background

The jujube (ZizipHus jujuba Mill.) is perennial woody crop of Rhamnaceae (Rhamnaceae) jujube (Zizyhpus), and is a special fruit tree in China. The jujube has various varieties and rich nutritive value, has the effects of resisting oxidation, inhibiting bacteria, diminishing inflammation, soothing nerves, helping sleep, nourishing yin, enriching blood and the like, and has wide application value in the aspects of food and medicine. Wild jujube (ZizipHus jujuba mill. var. spinosa) is widely distributed in China as a wild species of jujube.

With the development of functional genomics of fruit trees, researchers hope to further understand genetic background and gene function information of jujube trees so as to improve jujube tree varieties better. However, the jujube flower is small, the artificial emasculation is difficult, the fruit setting rate is low, the embryo abortion phenomenon is serious, and the like, so that the jujube tree is difficult to crossbreed, and the asexual propagation is carried out for a long time, so that people still know little about the genetic background and gene function information of the jujube tree. Sequencing of jujube whole genome is completed, and genetic transformation receptors with wide sources and efficient genetic transformation systems are urgently needed to analyze jujube genome information and identify gene functions. Wild jujube is used as wild jujube species, has wide sources, and callus of wild jujube is tissue in a dedifferentiation state, is a good genetic transformation receptor, and is a preferred material for analyzing jujube genome information and carrying out functional genomics research.

Regarding the genetic transformation of jujube, although some researches are conducted on different varieties by predecessors, the former people focus on the establishment of a transformation regeneration system and focus on obtaining genetic transformation plants, so that the sources of genetic transformation receptors are limited, the transformation efficiency is low, and the requirements of genetic background and gene function analysis on a large number of genetic transformation receptors and high genetic transformation efficiency of jujube cannot be met. Gu, X.F obtained a transgenic line from winter jujube stem tips as explants by Agrobacterium-mediated transformation (Gu, X.F., Meng, H., Qi, G., & Zhang, J.R. (2008). Hoheqi (Hoheqi. Agrobacterium mediated jujube tree genetic transformation research [ D ]. Hebei agriculture university, 2012.) and Juanqi (Juanqi, Guohui, Yangyaodong, Yangming, Malanqing, Wangcheng. Agrobacterium tumefaciens mediated giant knotweed stilbene synthase gene genetic transformation research [ J ]. forestry science, 2015,51(10): 101) 109.) and the like utilize jujube leaves as explants to respectively obtain insect-resistant and disease-resistant transgenic strains of the jujube tree. Weiwei takes leaves of jujubes as explants to obtain a transgenic line of the wheat phosphorus transporter gene with positive PCR detection (Weiwei, jujube exogenous wheat phosphorus transporter gene (TaPT2) genetic transformation research [ D ]. Hebei agricultural university, 2013.). Pueraria obtains ZjTCP6 and ZjTCP16 transgenic strains positive in PCR detection (Pueraria. jujube germplasm resource collection and preservation and genetic transformation of ZjTCP6 and ZjTCP16 genes [ D ]. Henan agricultural university, 2019.).

The genetic transformation aims at realizing genetic targeted improvement of a certain variety of the jujube tree, so that the genetic transformation efficiency is low, and the genetic transformation is obviously not suitable for the requirement of a large number of genetic transformation receptors and high genetic transformation efficiency for gene function analysis.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to realize the mass production of jujube genetic transformation, provides a genetic transformation and visual rapid identification method taking wild jujube callus as a receptor.

The invention solves the technical problems through the following technical scheme, and the invention comprises the following steps:

(1) acquisition of genetic transformation receptors

Sowing wild jujube sterile seeds, selecting wild jujube stem segments, leaves, cotyledons and hypocotyls when aseptic seedlings grow true leaves, respectively cutting into explants of 0.5-1 cm and 0.5-1 cm multiplied by 0.5-1 cm, placing the explants in a semisolid MS callus induction culture medium, and inducing wild jujube callus to serve as a genetic transformation material;

(2) agrobacterium culture and dip-dyeing liquid preparation

Inoculating agrobacterium liquid GV3101 carrying expression vector pCAMBIA2300 to a liquid LB culture medium, and culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm until OD₆₀₀0.6 to 0.8;

subjecting the OD to₆₀₀Resuspending 0.6-0.8 of the agrobacterium liquid by using a liquid MS to obtain a staining solution, and adding 200 mu mol/L acetosyringone into the liquid MS in a super clean bench before;

(3) and (3) callus dip dyeing:

co-culturing the dip dyeing solution obtained in the step (2) and the callus in a constant temperature shaking table at 28 ℃ and 100-150 rpm in the dark for 30-35 min to obtain a dip dyed explant;

(4) co-culturing:

draining the impregnated explants obtained in the step (3), placing the drained explants in a semisolid MS co-culture medium, and co-culturing for 2-3 days under a dark condition;

(5) and (3) degerming:

washing the calluses of the wild jujubes obtained in the step (4) with sterile water containing 250mg/L cefotaxime sodium for 7-8 min, and repeating for 3-4 times;

(6) bacteriostatic culture:

placing the wild jujube callus obtained in the step (5) in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days;

(7) screening and culturing:

placing the callus of the zizyphus jujube obtained in the step (6) after bacteriostatic culture in a semisolid MS screening culture medium, and obtaining homozygous callus of the zizyphus jujube after 3-5 generations of screening culture for 3-4 weeks each;

(8) identification of resistant calli:

the wild jujube resistant callus is detected by means of fluorescent marker screening and an RT-PCR method, and the integration and expression conditions of target genes are determined according to whether the target gene bands exist or not.

The semi-solid MS callus induction culture medium comprises 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB contained 50mg/L kanamycin and 50mg/L rifampicin.

In the step (2), the agrobacterium is GV3101, and the carried plasmid vector is pCAMBIA2300 green fluorescence expression vector.

The semisolid MS co-culture medium comprises 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semisolid MS antibacterial culture medium comprises 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semisolid MS screening culture medium comprises 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20mg/L of kanamycin, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The culture conditions of the steps (1), (4), (6) and (7) are 23-25 ℃, the photoperiod is 12-14 h/d, and the illumination intensity is 2500-2800 lx.

Compared with the prior art, the invention has the following advantages: the invention relates to a method for establishing wild jujube genetic transformation by using wild jujube stem, leaf, cotyledon and hypocotyl-induced callus as a receptor. The callus source is wide, the transformation frequency is high, the repeatability is high, and the operation steps are simple; meanwhile, the screening is assisted by the fluorescent marker, so that the method is convenient and quick. The system provides convenience for the function analysis of the jujube gene and provides possibility for the genetic improvement of jujubes.

Drawings

FIG. 1 is a comparison of callus induction conditions at different parts of wild jujube;

FIG. 2 is a map of pCAMBIA2300 plasmid;

FIG. 3 is a diagram of genetic transformation steps of wild jujube stem callus;

FIG. 4 is a diagram of the genetic transformation steps of wild jujube leaf callus;

FIG. 5 is a diagram of the genetic transformation steps of wild jujube cotyledon callus;

FIG. 6 is a diagram of the genetic transformation steps of wild jujube hypocotyl callus;

FIG. 7 is a comparison of genetic transformation rates of transgenic calli of different tissues and organs of wild jujube;

FIG. 8 is RT-PCR detection of resistant calli from different tissue sites of Zizyphus jujuba.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1

This example is the genetic transformation of callus from wild jujube stem segments.

Induction of genetic transformation receptors:

in the embodiment, commercially available wild jujube seeds are selected, soaked in 75% ethanol for 30s and treated with sterile water for 2 min; soaking in 5% NaClO for 7min, treating with sterile water for 2min, repeating for three times, and placing in semisolid MS culture medium. After 30 days, true leaves grow out from the aseptic seedlings, wild jujube stem segments with good growth vigor are cut, the wild jujube stem segments are cut into explants with the size of 0.5-1 cm respectively, and the explants are placed in a semisolid MS callus induction culture medium (shown as C in figure 1). Culturing the explant in dark environment at 23-25 deg.C for 30 days to induce wild jujube stem callus (shown as G in figure 1); after 1-2 generations of subculture for 3-4 weeks each, obtaining a favorable-growth wild jujube stem callus, namely the genetic transformation material (shown as D in figure 3).

The semisolid MS callus induction culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0.

Hot-pressing sterilization of the culture medium and the plant growth regulating substances; filtering and sterilizing active ingredients such as antibiotic, and adding after sterilizing culture medium. The semi-solid MS medium formulation is as follows:

(1) macroelement 20 ×, including mother liquor I (KNO)₃38g/L，NH₄NO₃33g/L，MgSO₄·7H₂O7.4 g/L); mother liquor II (KH)₂PO₄3.4 g/L); mother liquor III (CaCl)₂ 6.64g/L)。

(2) Micro-scale1000×(MnSO₄·H₂O16.9g/L，ZnSO₄·7H₂O 8.6g/L，H₃BO₃6.2g/L，KI 0.83g/L，Na₂MoO₄·2H₂O 0.25g/L，CuSO₄·5H₂O 0.025g/L，CoCl₂·6H₂O 0.025g/L)。

(3) Iron salt 200 × (disodium ethylenediaminetetraacetate 7.46g/L, FeSO)₄·7H₂O 5.56g/L)。

(4) Organic element 200 × (inositol 20g/L, glycine 0.4g/L, nicotinic acid VB)₃0.1g/L, pyridoxine hydrochloride VB₆0.1g/L, thiamine hydrochloride VB₁0.02g/L)。

(5) The semisolid MS culture medium further comprises 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semisolid MS callus induction and semisolid MS co-culture medium refers to: adding 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20-30 g/L sucrose, 6-7 g/L agar and a semisolid MS culture medium with the pH value of 5.8-6.0.

The semisolid MS antibacterial culture medium refers to: adding 2, 4-dichlorophenoxyacetic acid of 0.5-2mg/L, thidiazuron of 0.4mg/L, cefotaxime sodium of 250mg/L, timentin of 250mg/L, cane sugar of 20-30 g/L, agar of 6-7 g/L and semisolid MS culture medium of pH5.8-6.0.

The semisolid MS screening culture medium refers to: adding 2, 4-dichlorophenoxyacetic acid of 0.5-2mg/L, thidiazuron of 0.4mg/L, kanamycin of 20mg/L, cephamycin of 250mg/L, timentin of 250mg/L, cane sugar of 20-30 g/L, agar of 6-7 g/L and semisolid MS culture medium of pH 5.8-6.0.

The agrobacterium culturing and the dip dyeing liquid preparation:

inoculating agrobacterium liquid GV3101 carrying expression vector pCAMBIA2300 to liquid LB culture medium, culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm in a dark mode to enable OD₆₀₀The value is 0.6 to 0.8. Centrifuging the bacterial solution at 28 deg.C and 5000rpm for 5min in a centrifuge to obtain precipitated thallus, re-suspending thallus with liquid MS of the same volume to obtain staining solution, and adding 200 μ M acetosyringone into the liquid MS in a super clean bench. The inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB (yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.0) contained 50mg/L kanamycin and 50mg/L rifampicin.

In this example, the Agrobacterium is Agrobacterium tumefaciens GV3101, available from Shanghai Diego Biotechnology technology Ltd.

The expression vector carried by the agrobacterium GV3101 is pCAMBIA2300, and the plasmid map of the expression vector is shown in FIG. 2. The expression vector takes kanamycin resistance kanamyin resistance gene as a screening marker gene and carries a GFP green fluorescence reporter gene.

Performing wound healing dip dyeing, co-culture and degerming:

and (3) co-culturing the dip dyeing solution and the callus of the wild jujube stem segment in a constant temperature shaking table at 28 ℃ and 120rpm in the dark for 30min to obtain the soaked callus of the wild jujube stem segment.

Draining the soaked wild jujube stem segments, placing the soaked wild jujube stem segments into a semisolid MS co-culture medium, and co-culturing for 2-3 days under a dark condition, wherein the semisolid MS co-culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH.

And (3) washing the callus of the wild jujube stem section obtained after the co-culture for 7-8 min by using sterile water containing 250mg/L cefotaxime sodium, and repeating for 3-4 times.

Bacteriostasis and screening culture:

and placing the obtained wild jujube stem callus in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days. And (5) inhibiting bacteria for 30 days, and counting the genetic transformation rate of the transgenic wild jujube stem callus after the fluorescence signal is stable. The statistical method of the genetic transformation rate comprises the following steps: under LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp, Green Fluorescent Protein (GFP) can be detected by yellow filter, and transgenic wild jujube stem callus can emit green fluorescence (shown in FIG. 3, F). Under the observation of a double-fluorescent protein observation lamp, the ratio of the number of the callus blocks of the wild jujube stem segments emitting green fluorescence is counted, namely the genetic transformation rate.

The semisolid MS antibacterial culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value of 5.8-6.0; the LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp was purchased from Shanghai Luoyang instruments, Inc.

In order to study the difference of the transgenic wild jujube callus genetic transformation rates of different tissues and organs, the transgenic wild jujube callus genetic transformation rates of different tissues and organs are counted in fig. 7; wherein, 42 callus blocks of the wild jujube stem segments are co-dip-dyed, 10 callus blocks of the green fluorescent transgenic wild jujube stem segments account for 23.8 percent, namely, the genetic transformation rate of the transgenic wild jujube stem segment callus is 23.8 percent (as shown in figure 7).

Placing the callus of the wild jujube stem section obtained after bacteriostatic culture in a semisolid MS screening culture medium, transferring each callus of the wild jujube stem section to a new culture medium by the smallest volume when subculturing the wild jujube stem section, and thus, the independent callus of the wild jujube stem section after the first screening culture is called a strain. The semisolid MS screening culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20mg/L of kanamycin, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH; and screening and culturing the wild jujube stem callus for 3-5 generations and 3-4 weeks per generation to obtain homozygous wild jujube stem resistance callus.

And (3) carrying out RT-PCR detection on the wild jujube stem segment resistant callus:

detecting the expression condition of the wild jujube stem segment resistance callus reporter gene by using an RT-PCR method; firstly, continuously screening 3-5 generations to homozygous wild jujube stem segment resistant callus, grinding by liquid nitrogen, extracting RNA, and carrying out reverse transcription to obtain cDNA of wild jujube stem segment resistant callus; with 2300-GFP-F: CCGGGGTGGTGCCCATC, 2300-GFP-R: CTCCAGCTTGTGCCCCAGG as primer, cDNA of wild jujube stem segment resistance callus as template, PCR amplification reaction is carried out. The 2 Xfast Taq PCR system is 25 mu L, and specifically comprises the following components: ddH₂O10. mu.L, 2 Xfast Taq 12. mu.L, Primer R1. mu.L, Primer L1. mu.L, template 1. mu.L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min and denaturation at 95 ℃ for 30 s; annealing at 58 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles(ii) a Final extension at 72 deg.c for 10min, reaction termination at 8 deg.c and preservation at 4 deg.c; the amplification products were detected on a 1% agarose/Gel Red Gel. Plasmid DNA is used as a positive control, and untransformed wild jujube callus is used as a negative control; according to the existence of a reporter gene GFP band (400bp), the integration condition of the target gene is determined.

The RNAprep Pure polysaccharide polyphenol Plant total RNA extraction Kit RNAprep Pure Plant Plus Kit (polysaccharides & Polyphenonics-rich) is purchased from Tiangen Biochemical technology, Inc. (Beijing). DNA Maker (DL2000), 6 × loading buffer, reverse transcription Kit Hifair II 1st Strand cDNA Synthesis Kit (gDNA digaster) was purchased from san assist in Shanghai, Inc.

FIG. 8 is an electrophoresis chart of 2 different lines of wild jujube stem segment resistant callus detected by RT-PCR, all of which detect target bands consistent with the positive control (as shown in FIG. 8, lanes 1 and 3), indicating that the reporter gene has been integrated into the genome of the transgenic wild jujube stem segment callus.

Example 2

This example is the genetic transformation of wild jujube leaf callus

Acquisition of genetic transformation receptors:

soaking wild jujube seed in 75% ethanol for 30s, and treating with sterile water for 2 min; soaking in 5% NaClO for 7min, treating with sterile water for 2min, repeating for 3 times, and placing in semisolid MS culture medium. After 30 days, true leaves grow out from the aseptic seedlings, good-growing wild jujube leaves are cut, the wild jujube leaves are respectively cut into explants of 0.5-1 cm multiplied by 0.5-1 cm, and the explants are placed in a semisolid MS callus induction culture medium (shown in figure 1 and D). Culturing the wild jujube leaves in dark at 23-25 deg.C for 30 days to induce wild jujube leaf callus (shown in figure 1, H); and (3) carrying out subculture on the wild jujube leaf callus for 1-2 generations for 3-4 weeks every generation to obtain the wild jujube leaf callus with good growth vigor, namely the wild jujube leaf callus is shown as a genetic transformation material (shown as a figure 4, D).

The semisolid MS callus induction culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0.

Hot-pressing sterilization of the culture medium and the plant growth regulating substances; filtering and sterilizing active ingredients such as antibiotic, and adding after sterilizing culture medium. The semi-solid MS medium formulation is as follows:

(1) macroelement 20 ×, including mother liquor I (KNO)₃38g/L，NH₄NO₃33g/L，MgSO₄·7H₂O7.4 g/L); mother liquor II (KH)₂PO₄3.4 g/L); mother liquor III (CaCl)₂6.64g/L)。

(2) Trace 1000X (MnSO)₄·H₂O16.9g/L，ZnSO₄·7H₂O 8.6g/L，H₃BO₃6.2g/L，KI 0.83g/L，Na₂MoO₄·2H₂O 0.25g/L，CuSO₄·5H₂O 0.025g/L，CoCl₂·6H₂O 0.025g/L)。

(3) Iron salt 200 × (disodium ethylenediaminetetraacetate 7.46g/L, FeSO)₄·7H₂O 5.56g/L)。

(4) Organic element 200 × (inositol 20g/L, glycine 0.4g/L, nicotinic acid VB)₃0.1g/L, pyridoxine hydrochloride VB₆0.1g/L, thiamine hydrochloride VB₁0.02g/L)。

(5) The semisolid MS culture medium further comprises 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semi-solid MS callus induction culture medium and the semi-solid MS co-culture medium refer to the following components: the semisolid MS culture medium which is added with 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 250mg/L cefamamycin, 250mg/L timentin, 20-30 g/L cane sugar, 6-7 g/L agar and pH5.8-6.0.

The semisolid MS screening culture medium refers to: the semisolid MS culture medium with the pH value of 5.8-6.0 is added with 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20mg/L kanamycin, 250mg/L cephamycin, 250mg/L timentin, 20-30 g/L cane sugar, 6-7 g/L agar.

The agrobacterium culturing and the dip dyeing liquid preparation:

inoculating agrobacterium liquid GV3101 carrying expression vector pCAMBIA2300 to liquid LB culture medium, culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm in a dark mode to enable OD₆₀₀0.6 to 0.8; after thatCentrifuging the bacterial liquid in a centrifuge at 28 deg.C and 5000rpm for 5min to obtain precipitated thallus, re-suspending thallus with liquid MS of the same volume to obtain a staining solution, and adding 200 μ M acetosyringone into the liquid MS in a super clean bench. The inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB (yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.0) contained 50mg/L kanamycin and 50mg/L rifampicin.

In this example, the Agrobacterium is Agrobacterium tumefaciens GV3101, available from Shanghai Diego Biotechnology technology Ltd.

The expression vector carried by the agrobacterium GV3101 is pCAMBIA2300, and the plasmid map of the expression vector is shown in FIG. 2. The expression vector takes kanamycin resistance kanamyin resistance gene as a screening marker gene and carries a GFP green fluorescence reporter gene.

Wound healing dip dyeing, co-culture and degerming

And (3) carrying out dark co-culture on the dip dyeing solution and wild jujube leaf callus for 30min in a constant temperature shaking table at 28 ℃ and 120rpm to obtain the dip dyed wild jujube leaf callus.

Draining the soaked and dyed wild jujube leaves, placing the drained wild jujube leaves in a semisolid MS co-culture medium, and co-culturing for 2-3 days under a dark condition, wherein the semisolid MS co-culture medium is a semisolid MS culture medium containing 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20-30 g/L sucrose, 6-7 g/L agar and 5.8-6.0 of pH.

And (3) washing the callus of the co-cultured wild jujube leaves with sterile water containing 250mg/L cefotaxime sodium for 7-8 min, and repeating for 3-4 times.

Bacteriostasis and screening culture

Placing the obtained wild jujube leaf callus in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days. And after the dip dyeing is carried out for 30d and the fluorescence signal is stable, counting the genetic transformation rate of the transgenic wild jujube leaf callus. The statistical method of the genetic transformation rate comprises the following steps: under LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp, Green Fluorescent Protein (GFP) can be detected by observing with yellow filter, and transgenic Ziziphus jujube leaf callus emitting green fluorescence (shown in FIG. 4, F) can be detected. Under the observation of a double-fluorescent protein observation lamp, the ratio of the number of the callus pieces of the green fluorescent wild jujube leaves is counted, namely the genetic transformation rate.

The semisolid MS antibacterial culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value of 5.8-6.0; the LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp was purchased from Shanghai Luoyang instruments, Inc.

In order to study the difference of the transgenic wild jujube callus genetic transformation rates of different tissues and organs, the transgenic wild jujube callus genetic transformation rates of different tissues and organs are counted in fig. 7; wherein 66 callus pieces of wild jujube leaf are co-dip-dyed, 21 callus pieces of green fluorescent transgenic wild jujube leaf are used, accounting for 31.8% (as shown in figure 7), that is, the genetic transformation rate of the transgenic wild jujube leaf callus is 31.8%.

Placing the obtained wild jujube leaf callus subjected to bacteriostatic culture in a semisolid MS screening culture medium, transferring each wild jujube leaf callus to a new culture medium by the smallest volume when subculturing wild jujube leaf callus, and calling the independent wild jujube leaf callus block subjected to primary screening culture as a strain. The semisolid MS screening culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20mg/L of kanamycin, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH; the homozygous wild jujube leaf resistant callus is obtained after 3-5 generations of wild jujube leaf callus is screened and cultured for 3-4 weeks per generation.

RT-PCR detection of wild jujube leaf resistant callus

Detecting the expression condition of the wild jujube leaf resistance callus reporter gene by using an RT-PCR method; firstly, collecting the wild jujube leaf resistance callus from 3-5 generations through resistance screening to homozygous, grinding by liquid nitrogen, extracting RNA, and obtaining cDNA of the wild jujube leaf resistance callus through reverse transcription; with 2300-GFP-F: CCGGGGTGGTGCCCATC, 2300-GFP-R: CTCCAGCTTGTGCCCCAGG as primer, and cDNA of wild jujube leaf resistant callus as template, to proceed PCR amplification reaction. 2 xFast Taq PCR System 25. mu.L, specifically comprising: ddH₂O10. mu.L, 2 Xfast Taq 12. mu.L, Primer R1. mu.L, Primer L1. mu.L, template 1. mu.L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min and denaturation at 95 ℃ for 30 s; annealing at 58 ℃ for 30 s; extension at 72 ℃ for 30 s; 30 cycles; final extension at 72 deg.c for 10min, reaction termination at 8 deg.c and preservation at 4 deg.c; the amplification products were detected on a 1% agarose/Gel Red Gel. Plasmid DNA is used as a positive control, untransformed wild jujube callus is used as a negative control, and the integration condition of the reporter gene is determined according to whether a band (400bp) of the reporter gene GFP exists.

The RNAprep Pure polysaccharide polyphenol Plant total RNA extraction Kit RNAprep Pure Plant Plus Kit (polysaccharides & Polyphenonics-rich) is purchased from Tiangen Biochemical technology, Inc. (Beijing). DNA Maker (DL2000), 6 × loading buffer, reverse transcription Kit Hifair II 1st Strand cDNA Synthesis Kit (gDNA digaster) was purchased from san assist in Shanghai, Inc.

FIG. 8 is an electrophoretogram of 3 different lines of wild jujube leaf resistant callus detected by RT-PCR, all detecting target bands consistent with the positive control (shown as lanes 4, 5 and 6 in FIG. 8), which proves that the reporter gene is integrated into the genome of the transgenic wild jujube leaf callus.

Example 3

This example is the genetic transformation of wild jujube cotyledon callus

Acquisition of genetic transformation receptors:

soaking wild jujube seed in 75% ethanol for 30s, treating with sterile water for 2min, soaking in 5% NaClO for 7min, treating with sterile water for 2min, repeating for three times, and placing in semisolid MS culture medium. After 30 days, the aseptic seedlings grow true leaves, the wild jujube cotyledons with good growth vigor are cut into explants of 0.5-1 cm multiplied by 0.5-1 cm respectively, and the explants are placed in a semisolid MS callus induction culture medium (shown in figure 1 and E). Culturing the explant in dark environment at 23-25 deg.C for 30 days to induce callus of Ziziphus jujube cotyledon (as shown in figure 1, I); after 1-2 generations of subculture for 3-4 weeks each, obtaining a callus of wild jujube cotyledon with good growth vigor, namely the genetic transformation material (as shown in figure 5, D).

The semisolid MS callus induction culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0.

Hot-pressing sterilization of the culture medium and the plant growth regulating substances; filtering and sterilizing active ingredients such as antibiotic, and adding after sterilizing culture medium. The MS minimal medium formulation is as follows:

(1) macroelement 20 ×, including mother liquor I (KNO)₃38g/L，NH₄NO₃33g/L，MgSO₄·7H₂O7.4g/L); mother liquor II (KH)₂PO₄3.4 g/L); mother liquor III (CaCl)₂6.64g/L)。

(2) Trace 1000X (MnSO)₄·H₂O16.9g/L，ZnSO₄·7H₂O 8.6g/L，H₃BO₃ 6.2g/L，KI 0.83g/L，Na₂MoO₄·2H₂O 0.25g/L，CuSO₄·5H₂O 0.025g/L，CoCl₂·6H₂O 0.025g/L)。

(3) Iron salt 200 × (disodium ethylenediaminetetraacetate 7.46g/L, FeSO)₄·7H₂O 5.56g/L)。

(4) Organic element 200 × (inositol 20g/L, glycine 0.4g/L, nicotinic acid VB)₃0.1g/L, pyridoxine hydrochloride VB₆0.1g/L, thiamine hydrochloride VB₁0.02g/L)。

(5) The semisolid MS culture medium further comprises 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semisolid MS callus induction and semisolid MS co-culture medium refers to: the semisolid MS culture medium with 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0 is added.

The semisolid MS antibacterial culture medium refers to: the semisolid MS culture medium which is added with 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 250mg/L cefamamycin, 250mg/L timentin, 20-30 g/L cane sugar, 6-7 g/L agar and pH5.8-6.0.

The semisolid MS screening culture medium refers to: the semisolid MS culture medium with the pH value of 5.8-6.0 is added with 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20mg/L kanamycin, 250mg/L cephamycin, 250mg/L timentin, 20-30 g/L cane sugar, 6-7 g/L agar.

The agrobacterium culturing and the dip dyeing liquid preparation:

inoculating agrobacterium liquid GV3101 carrying expression vector pCAMBIA2300 to liquid LB culture medium, culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm in a dark mode to enable OD₆₀₀And (3) 0.6-0.8, centrifuging the bacterial liquid in a centrifuge at the rotating speed of 28 ℃ and 5000rpm for 5min to obtain precipitated bacteria, then re-suspending the bacteria by using liquid MS with the same volume to obtain a staining solution, and adding 200 mu M acetosyringone into the liquid MS. The inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB (yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.0) contained 50mg/L kanamycin and 50mg/L rifampicin.

In this example, the Agrobacterium is Agrobacterium tumefaciens GV3101, available from Shanghai Diego Biotechnology technology Ltd.

The expression vector carried by the agrobacterium GV3101 is pCAMBIA2300, and the plasmid map of the expression vector is shown in FIG. 2. The expression vector takes kanamycin resistance kanamyin resistance gene as a screening marker gene and carries a GFP green fluorescence reporter gene.

Wound healing dip dyeing, co-culture and degerming

And (3) co-culturing the staining solution and the callus of the wild jujube cotyledon in a constant temperature shaking table at 28 ℃ and 120rpm in the dark for 30min to obtain the stained wild jujube cotyledon callus.

Draining the soaked spina date seed leaf calluses, putting the calluses into a semisolid MS co-culture medium, and co-culturing for 2-3 days under a dark condition, wherein the semisolid MS co-culture medium is the semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH.

And (3) washing the callus of the wild jujube cotyledon with sterile water containing 250mg/L cefotaxime sodium for 7-8 min, and repeating for 3-4 times.

Bacteriostasis and screening culture

And placing the obtained wild jujube cotyledon callus in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days. And after the dip dyeing is carried out for 30d and the fluorescence signal is stable, counting the genetic transformation rate of the transgenic wild jujube cotyledon callus. The statistical method of the genetic transformation rate comprises the following steps: under LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp, Green Fluorescent Protein (GFP) can be detected by yellow filter, and the callus emitting green fluorescence is wild jujube cotyledon positive callus (as shown in FIG. 5, F). Under the observation of a double fluorescent protein observation lamp, the ratio of the number of the cotyledon calluses emitting the green fluorescent acid is counted, namely the genetic transformation rate.

The semi-solid MS bacteriostatic culture medium is a semi-solid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value of 5.8-6.0; the LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp was purchased from Shanghai Luoyang instruments, Inc.

In order to study the difference of the transgenic wild jujube callus genetic transformation rates of different tissues and organs, the transgenic wild jujube callus genetic transformation rates of different tissues and organs are counted in fig. 7; wherein 129 callus pieces of zizyphus jujube cotyledon are co-dip-dyed, 33 callus pieces of green fluorescent transgenic zizyphus jujube cotyledon account for 25.6%, that is, the genetic transformation rate of the transgenic zizyphus jujube cotyledon callus is 25.6% (as shown in fig. 7).

Placing the obtained wild jujube cotyledon callus after bacteriostatic culture in a semisolid MS screening culture medium, transferring each wild jujube cotyledon callus to a new culture medium by the smallest volume when subculturing the wild jujube cotyledon callus, and then, taking the independent wild jujube cotyledon callus after primary screening culture as a strain. The semisolid MS screening culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20mg/L of kanamycin, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH; and screening and culturing the wild jujube cotyledon callus for 3-5 generations and 3-4 weeks per generation to obtain homozygous wild jujube cotyledon resistance callus.

RT-PCR detection of wild jujube cotyledon resistance callus

By RT-PCRDetecting the expression condition of the wild jujube cotyledon resistance callus reporter gene; firstly, collecting and screening 3-5 generations of subcultures to homozygous wild jujube cotyledon resistance callus, grinding by liquid nitrogen, extracting RNA, and carrying out reverse transcription to obtain cDNA of the wild jujube cotyledon resistance callus; with 2300-GFP-F: CCGGGGTGGTGCCCATC, 2300-GFP-R: CTCCAGCTTGTGCCCCAGG as primer, cDNA of wild jujube cotyledon resistance callus as template, PCR amplification reaction. The 2 Xfast Taq PCR system is 25 mu L, and specifically comprises the following components: ddH₂O10. mu.L, 2 Xfast Taq 12. mu.L, Primer R1. mu.L, Primer L1. mu.L, template 1. mu.L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min and denaturation at 95 ℃ for 30 s; annealing at 58 ℃ for 30s, extending at 72 ℃ for 30s, and performing 35 cycles; final extension at 72 deg.c for 10min, reaction termination at 8 deg.c and preservation at 4 deg.c; the amplification products were detected on a 1% agarose/Gel Red Gel. Plasmid DNA is used as a positive control, and untransformed wild jujube callus is used as a negative control; the integration status of the reporter gene was determined based on the presence or absence of the reporter gene GFP band (400 bp).

The RNAprep Pure polysaccharide polyphenol Plant total RNA extraction Kit RNAprep Pure Plant Plus Kit (polysaccharides & Polyphenonics-rich) is purchased from Tiangen Biochemical technology, Inc. (Beijing). DNA Maker (DL2000), 6 × loading buffer, reverse transcription Kit Hifair II 1st Strand cDNA Synthesis Kit (gDNA digaster) was purchased from san assist in Shanghai, Inc.

FIG. 8 is an electrophoresis chart of 3 different lines of wild jujube cotyledon resistant callus detected by RT-PCR, all of which detect target bands consistent with the positive control (as shown in FIG. 8, lanes 7, 8 and 9), and prove that the reporter gene is integrated into the genome of the transgenic wild jujube cotyledon resistant callus.

Example 4

This example is the genetic transformation of calli of hypocotyl of Zizyphus jujuba

Acquisition of genetic transformation receptors:

soaking wild jujube seed in 75% ethanol for 30s, and treating with sterile water for 2 min; soaking in 5% NaClO for 7min, sterilizing with sterile water for 2min, repeating for three times, and placing in semisolid MS culture medium. And after 30 days, growing true leaves from the aseptic seedlings, shearing the wild jujube hypocotyls with good growth vigor, cutting the wild jujube hypocotyls into explants with the size of 0.5-1 cm respectively, placing the explants in a semisolid MS callus induction culture medium (shown as figure 1 and F), placing the explants in a dark environment, culturing at the temperature of 23-25 ℃, inducing the wild jujube hypocotyls to callus after 30 days (shown as figure 1 and J), and carrying out 1-2 generations of subculture for 3-4 weeks each time to obtain the wild jujube hypocotyls with good growth vigor, namely the genetic transformation material (shown as figure 6 and D).

The semisolid MS callus induction culture medium is a semisolid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0.

Hot-pressing sterilization of the culture medium and the plant growth regulating substances; filtering and sterilizing active ingredients such as antibiotic, and adding after sterilizing culture medium. The semi-solid MS medium formulation is as follows:

(1) macroelement 20 ×, including mother liquor I (KNO)₃38g/L，NH₄NO₃33g/L，MgSO₄·7H₂O7.4 g/L); mother liquor II (KH)₂PO₄3.4 g/L); mother liquor III (CaCl)₂ 6.64g/L)。

(2) Trace 1000X (MnSO)₄·H₂O16.9g/L，ZnSO₄·7H₂O 8.6g/L，H₃BO₃6.2g/L，KI 0.83g/L，Na₂MoO₄·2H₂O 0.25g/L，CuSO₄·5H₂O 0.025g/L，CoCl₂·6H₂O 0.025g/L)。

(3) Iron salt 200 × (disodium ethylenediaminetetraacetate 7.46g/L, FeSO)₄·7H₂O 5.56g/L)。

(4) Organic element 200 × (inositol 20g/L, glycine 0.4g/L, nicotinic acid VB)₃0.1g/L, pyridoxine hydrochloride VB₆0.1g/L, thiamine hydrochloride VB₁0.02 g/L)。

(5) The semisolid MS culture medium further comprises 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value is 5.8-6.0.

The semi-solid MS callus induction culture medium and the semi-solid MS co-culture medium refer to the following components: the semisolid MS culture medium with 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH of 5.8-6.0 is added.

The semisolid MS antibacterial culture medium refers to: the semisolid MS culture medium is added with 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and pH value of 5.8-6.0.

The semisolid MS screening culture medium refers to: the semisolid MS culture medium with the pH value of 5.8-6.0 is added with 0.5-2 mg/L2, 4-dichlorophenoxyacetic acid, 0.4mg/L thidiazuron, 20mg/L kanamycin, 250mg/L cephamycin, 250mg/L timentin, 20-30 g/L cane sugar, 6-7 g/L agar.

The agrobacterium culturing and the dip dyeing liquid preparation:

inoculating agrobacterium liquid GV3101 carrying expression vector pCAMBIA2300 to liquid LB culture medium, culturing the liquid in a constant temperature shaking table at 28 ℃ and 200-250 rpm in a dark mode to enable OD₆₀₀The bacterial suspension is 0.6-0.8, then the bacterial suspension is centrifuged in a centrifuge at the rotating speed of 5000rpm at 28 ℃ for 5min to obtain precipitated bacteria, and the bacteria are resuspended by using liquid MS with the same volume to obtain a staining solution; 200 μ M acetosyringone has been added to liquid MS before. The inoculation amount of the agrobacterium liquid GV3101 is 0.25-0.5%; the liquid LB (yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.0) contained 50mg/L kanamycin and 50mg/L rifampicin.

In this example, Agrobacterium used was Agrobacterium tumefaciens GV3101, available from Shanghai Diego Biotechnology technology Ltd.

The expression vector carried by the agrobacterium GV3101 is pCAMBIA2300, and the plasmid map of the expression vector is shown in FIG. 2. The expression vector takes kanamycin resistance kanamyin resistance gene as a screening marker gene and carries a GFP green fluorescence reporter gene.

Wound healing dip dyeing, co-culture and degerming

And (3) carrying out dark co-culture on the dip dyeing solution and the wild jujube hypocotyl callus for 30min in a constant temperature shaking table at the temperature of 28 ℃ and the rpm of 120 to obtain the dip dyed wild jujube hypocotyl callus.

Draining the soaked and dyed spina date hypocotyls, putting the soaked and dyed spina date hypocotyls into a semi-solid MS co-culture medium, and co-culturing for 2-3 days under a dark condition, wherein the semi-solid MS co-culture medium is a semi-solid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20-30 g/L of cane sugar, 6-7 g/L of agar and 5.8-6.0 of pH.

And (3) washing the obtained wild jujube hypocotyl callus with sterile water containing 250mg/L cefotaxime sodium for 7-8 min, and repeating for 3-4 times.

Bacteriostasis and screening culture

And placing the obtained wild jujube hypocotyl callus in a semisolid MS bacteriostatic culture medium, and carrying out bacteriostatic culture for 30-45 days. And after the dip dyeing is carried out for 30d and the fluorescence signal is stable, counting the genetic transformation rate of the transgenic wild jujube hypocotyl callus. Under LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp, Green Fluorescent Protein (GFP) can be detected by observing with yellow filter, and transgenic zizyphus jujube hypocotyl callus emitting green fluorescence (shown in FIG. 6, F) can be obtained. The statistical method of the genetic transformation rate comprises the following steps: under the observation of a double-fluorescent protein observation lamp, the ratio of the number of the calluses of the hypocotyls of the wild jujube which emits green fluorescent light is counted.

The semi-solid MS bacteriostatic culture medium is a semi-solid MS culture medium containing 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar, 6-7 g/L of agar and the pH value of 5.8-6.0; the LUYOR-3415RG Hand-Held Lamp double fluorescent protein observation Lamp was purchased from Shanghai Luoyang instruments, Inc.

In order to study the difference of the transgenic wild jujube callus genetic transformation rates of different tissues and organs, the transgenic wild jujube callus genetic transformation rates of different tissues and organs are counted in fig. 7; wherein, 49 pieces of wild jujube hypocotyl callus are dip-dyed, and the green fluorescent wild jujube hypocotyl callus accounts for 24.5% of 12 pieces, i.e. the genetic transformation rate of the transgenic wild jujube hypocotyl callus is 24.5% (as shown in fig. 7).

Placing the obtained wild jujube hypocotyl callus subjected to bacteriostatic culture in a semisolid MS screening culture medium, transferring each wild jujube hypocotyl callus to a new culture medium by the smallest number of blocks during subculture of the wild jujube hypocotyl callus, and calling an independent wild jujube hypocotyl callus block subjected to primary screening culture as a strain. The semisolid MS screening culture medium is a semisolid MS culture medium which contains 0.5-2mg/L of 2, 4-dichlorophenoxyacetic acid, 0.4mg/L of thidiazuron, 20mg/L of kanamycin, 250mg/L of cefamycin, 250mg/L of timentin, 20-30 g/L of cane sugar and 6-7 g/L of agar, and the pH value is 5.8-6.0; and screening and culturing the wild jujube hypocotyl callus for 3-5 generations and 3-4 weeks per generation to obtain homozygous wild jujube hypocotyl resistance callus.

RT-PCR detection of zizyphus jujube hypocotyl resistance callus

Detecting the expression condition of the zizyphus jujube hypocotyl resistance callus reporter gene by using an RT-PCR method; firstly, continuously screening 3-5 generations to homozygous wild jujube hypocotyl resistance callus, grinding by liquid nitrogen, extracting RNA, and carrying out reverse transcription to obtain cDNA of the wild jujube hypocotyl resistance callus; with 2300-GFP-F: CCGGGGTGGTGCCCATC, 2300-GFP-R: CTCCAGCTTGTGCCCCAGG as primer, and cDNA of the wild jujube hypocotyl resistant callus as template, to perform PCR amplification reaction. The 2 Xfast Taq PCR system is 25 mu L, and specifically comprises the following components: ddH₂O10. mu.L, 2 Xfast Taq 12. mu.L, Primer R1. mu.L, Primer L1. mu.L, template 1. mu.L; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min and denaturation at 95 ℃ for 30 s; annealing at 58 ℃ for 30 s; extension at 72 ℃ for 30 s; 35 cycles; final extension at 72 deg.c for 10min, reaction termination at 8 deg.c and preservation at 4 deg.c; the amplification products were detected on a 1% agarose/Gel Red Gel. Plasmid DNA is used as a positive control, and untransformed wild jujube callus is used as a negative control; the integration status of the reporter gene was determined based on the presence or absence of the reporter gene GFP band (400 bp).

The RNAprep Pure polysaccharide polyphenol Plant total RNA extraction Kit RNAprep Pure Plant Plus Kit (polysaccharides & Polyphenonics-rich) is purchased from Tiangen Biochemical technology, Inc. (Beijing). DNA Maker (DL2000), 6 × loading buffer, reverse transcription Kit Hifair II 1st Strand cDNA Synthesis Kit (gDNA digaster) was purchased from san assist in Shanghai, Inc.

FIG. 8 is an electrophoresis chart of 3 different lines of wild jujube hypocotyl resistant callus detected by RT-PCR, all detected target bands consistent with the positive control (as shown in FIG. 8, lanes 10, 11, 12), demonstrating that the reporter gene has integrated into the genome of the transgenic wild jujube hypocotyl callus.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.