阅读说明：本技术 一种带有筛选标记桉树遗传转化方法 (Genetic transformation method for eucalyptus with screening marker ) 是由 欧阳乐军 王泽琛 李莉梅 陈凯钊 刘智超 潘景音 梁楚炎 吴宇朋 于 2020-06-07 设计创作，主要内容包括：本发明公开了一种带有筛选标记桉树遗传转化方法,以尾巨桉无菌苗茎段为外植体诱导愈伤组织,经带有荧光标记基因mCherry的农杆菌转化、不定芽诱导、芽增殖、不定芽伸长、生根及移栽,得到带有可视化荧光标记基因mCherry的再生植株；本发明特点是以带有可视化荧光标记基因mCherry的农杆菌对尾巨桉无性系幼苗下胚轴为外植体经愈伤组织培养的转化,再经不定芽诱导、不定芽伸长、芽增殖以及生根培养、移栽,可以得到可视化荧光标记基因mCherry的再生植株。采用本发明的方法可得到可视化荧光标记基因mCherry的再生植株,结果稳定,转化率良好,能应用于观察目的基因是否成功导入,也为桉树基因育种奠定了基础。(The invention discloses a genetic transformation method for eucalyptus with a screening marker, which comprises the steps of taking stem segments of aseptic seedlings of eucalyptus urophylla as explants to induce callus, and obtaining regeneration plants with a visual fluorescent marker gene mCherry through agrobacterium transformation, adventitious bud induction, bud multiplication, adventitious bud elongation, rooting and transplantation of the fluorescence marker gene mCherry; the invention is characterized in that agrobacterium with a visible fluorescence labeling gene mCherry transforms hypocotyl of clonal seedling of Eucalyptus urophydis by callus culture, and then the regenerated plant of the visible fluorescence labeling gene mCherry can be obtained by adventitious bud induction, adventitious bud elongation, bud multiplication, rooting culture and transplantation. The method can obtain the regeneration plant of the visible fluorescence labeling gene mCherry, has stable result and good transformation rate, can be used for observing whether the target gene is successfully introduced, and lays a foundation for eucalyptus gene breeding.)

1. A genetic transformation method for eucalyptus with a screening marker is characterized by comprising the following steps:

s1, obtaining eucalyptus urophydis aseptic bottle seedlings;

s2, cutting stem segments of the aseptic seedlings into small segments with the length of 0.5-1 cm, inoculating the small segments on a callus induction culture medium, and carrying out dark culture for one week at the culture temperature of 25 +/-2 ℃;

then the illumination is switched to 12 h.d^-1Culturing for 2-3 weeks under the conditions that the illumination intensity is 2000-500 lx and the temperature is 25 +/-2 ℃;

s3, transferring the callus cultured on the callus induction culture medium into a dip-dyeing culture medium, performing ultrasonic wave for 30S, adding agrobacterium containing a visible fluorescence labeling gene mCherry, keeping away from light at 28 ℃, and shaking for 3h at 200 r;

s4, transferring the impregnated callus onto an adventitious bud induction culture medium, culturing in the dark at the temperature of 25 +/-2 ℃ for 1 week, and then culturing for 2-3 weeks under the condition that the illumination intensity is 2000-500 lx;

s5, transferring the callus cultured by the adventitious bud induction culture medium to a bud multiplication culture medium, cutting off buds, observing whether red fluorescence exists under a fluorescence microscope, and culturing for 2-3 weeks at 25 +/-2 ℃ under the illumination intensity of 2000-2500 lx;

s6, transferring the callus cultured by the bud multiplication culture medium to an adventitious bud elongation culture medium, and culturing at 25 +/-2 ℃ under the illumination intensity of 2000-2500 lx to promote bud elongation;

s7, transferring the adventitious bud to a rooting culture medium for culture when the bud extends to 3-4 cm, so as to promote the bud to root;

s8, transferring the well rooted seedlings from the constant-temperature illumination incubator to an outdoor natural environment for hardening for 7d, uncovering a bottle sealing film, hardening for 2d, and transplanting the seedlings to sterilized yellow-heart soil for growing to obtain regenerated plants with the visible fluorescence labeling gene mCherry.

2. The genetic transformation method for eucalyptus with the selection marker as claimed in claim 1, wherein the callus induction medium is: MS culture medium, LC 1.0mL/mL, IAA 0.5mg/mL, Vc 1mg/mL, sucrose 30g/L, agar 7 g/L and pH 5.8-5.9.

3. The genetic transformation method for eucalyptus with the selection marker according to claim 1, wherein the staining culture medium is: MS culture medium + sucrose 30 g/L.

4. The genetic transformation method for eucalyptus with the selection marker according to claim 1, wherein the adventitious bud induction culture medium is: MS culture medium +6-BA 0.5mg/mL + naphthylacetic acid (NAA) 0.5mg/mL + putrescine 500mmol/L + spermidine 100mmol/L + vitamin C (Vc) 1mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9.

5. The genetic transformation method for eucalyptus with the selection marker according to claim 1, wherein the bud multiplication medium is: 1/2 MS culture medium, 6-BA 0.5mg/mL, naphthylacetic acid (NAA) 0.5mg/mL, sucrose 30g/L, agar 7 g/L, pH 5.8-5.9.

6. The genetic transformation method for eucalyptus with the selection marker according to claim 1, wherein the rooting medium is: 1/2 MS culture medium, 1mg/mL indolebutyric acid (IBA), 30g/L sucrose and 7 g/L agar, and the pH value is 5.8-6.0.

7. The genetic transformation method for eucalyptus with the selection marker as claimed in claim 5 or 6, wherein the 1/2 MS culture medium is: macroelements are half of MS culture medium, the rest are unchanged, sucrose is 30g/L + agar is 7 g/L, and pH is 5.8-5.9.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a genetic transformation method for eucalyptus with a screening marker.

Background

Agrobacterium is a gram-negative bacterium ubiquitous in soil and capable of chemotactic infection of wounded parts of most dicotyledonous plants under natural conditions and induction of crown gall or hairy roots. The cells in the agrobacterium tumefaciens and the agrobacterium rhizogenes respectively contain Ti plasmids and Ri plasmids, a section of T-DNA is arranged on the Ti plasmids and the Ri plasmids, and the T-DNA can be inserted into plant genomes after the agrobacterium tumefaciens enters the cells by infecting plant wounds. Thus, Agrobacterium is a natural plant genetic transformation system. People insert the target gene into the modified T-DNA region, transfer and integration of exogenous genes to plant cells are realized by virtue of agrobacterium infection, and then transgenic plants are regenerated by cell and tissue culture technology.

The scheme transforms plants through agrobacterium transformation with mCherry gene so as to realize the screening of morphological markers of positive transformed plants after plant transformation, thereby becoming visual screening markers.

Disclosure of Invention

The invention aims to provide a genetic transformation method for eucalyptus with a screening marker, which aims to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a genetic transformation method for eucalyptus with a screening marker specifically comprises the following steps:

s1, obtaining eucalyptus urophydis aseptic bottle seedlings;

s2 aseptic seedling stem sectionCutting into 0.5-1 cm long segments, inoculating on callus induction culture medium, dark culturing for one week at 25 + -2 deg.C. Then the illumination is switched to 12 h.d^-1Culturing for 2-3 weeks under the conditions that the illumination intensity is 2000-500 lx and the temperature is 25 +/-2 ℃;

s3, transferring the callus cultured on the callus induction culture medium into a dip-dyeing culture medium, performing ultrasonic wave for 30S, adding agrobacterium containing a visible fluorescence labeling gene mCherry, keeping away from light at 28 ℃, and shaking for 3h at 200 r;

s4, transferring the impregnated callus onto an adventitious bud induction culture medium, culturing in the dark at the temperature of 25 +/-2 ℃ for 1 week, and then culturing for 2-3 weeks under the condition that the illumination intensity is 2000-500 lx;

s5, transferring the callus cultured by the adventitious bud induction culture medium to a bud multiplication culture medium, cutting off buds, observing whether red fluorescence exists under a fluorescence microscope, and culturing for 2-3 weeks at 25 +/-2 ℃ under the illumination intensity of 2000-2500 lx;

s6, transferring the callus cultured by the bud multiplication culture medium to an adventitious bud elongation culture medium, and culturing at 25 +/-2 ℃ under the illumination intensity of 2000-2500 lx to promote bud elongation;

s7, transferring the adventitious bud to a rooting culture medium for culture when the bud extends to 3-4 cm, so as to promote the bud to root;

s8, transferring the well rooted seedlings from the constant-temperature illumination incubator to an outdoor natural environment for hardening for 7d, uncovering a bottle sealing film, hardening for 2d, and transplanting the seedlings to sterilized yellow-heart soil for growing to obtain regenerated plants with the visible fluorescence labeling gene mCherry.

Further, the callus induction medium is: MS, LC 1.0mL/mL, IAA 0.5mg/mL, Vc 1mg/mL, sucrose 30g/L, agar 7 g/L and pH 5.8-5.9.

Further, the staining medium is as follows: MS + sucrose 30 g/L.

Further, the adventitious bud induction medium is as follows: MS +6-BA 0.5mg/mL + naphthylacetic acid (NAA) 0.5mg/mL + putrescine 500mmol/L + spermidine 100mmol/L + vitamin C (Vc) 1mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9.

Further, the bud multiplication medium is: 1/2 MS +6-BA 0.5mg/mL + naphthylacetic acid (NAA) 0.5mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9.

Further, the rooting medium is as follows: 1/2 MS, 1mg/mL indolebutyric acid (IBA), 30g/L sucrose and 7 g/L agar, and the pH value is 5.8-6.0.

Further, the 1/2 MS (Murashige and Skoog) culture medium is: macroelements are half of MS, the rest are unchanged, sucrose is 30g/L + agar is 7 g/L, and the pH value is 5.8-5.9.

Compared with the prior art, the invention has the beneficial effects that:

the invention transforms the plant by agrobacterium transformation with mCherry gene, can realize the screening of the morphological marker of the positive transformed plant after the plant transformation, thereby becoming the transformation method of the visual screening marker.

The invention is characterized in that agrobacterium with a visible fluorescence labeling gene mCherry transforms hypocotyl of clonal seedling of Eucalyptus urophydis by callus culture, and then the regenerated plant of the visible fluorescence labeling gene mCherry can be obtained by adventitious bud induction, adventitious bud elongation, bud multiplication, rooting culture and transplantation. The method can obtain the regeneration plant of the visible fluorescence labeling gene mCherry, has stable result and good transformation rate, can be used for observing whether the target gene is successfully introduced, and lays a foundation for eucalyptus gene breeding.

Detailed Description

The technical solution of the present patent will be described in further detail with reference to the following embodiments.

A genetic transformation method for eucalyptus with a screening marker comprises the steps of taking stem segments of aseptic seedlings of Eucalyptus urophydis as explants to induce callus, and obtaining regenerated plants with a visualized fluorescence marker gene mCherry through agrobacterium transformation with the fluorescence marker gene mCherry, adventitious bud induction, bud multiplication, adventitious bud elongation, rooting and transplantation.

The method specifically comprises the following steps:

s1, obtaining the aseptic seedlings of the eucalyptus urophydis, wherein the aseptic seedlings of the eucalyptus urophydis in the embodiment are aseptic bottle seedlings of the eucalyptus urophydis provided by the research and development center of the national forestry bureau;

s2, removing terminal buds and roots of the aseptic seedlings of the eucalyptus urophydis in the step S1, cutting hypocotyls into small sections with the length of 0.5-1 cm, inoculating the small sections to a callus induction culture medium (MS + LC 1mg/mL + IAA 0.5mg/mL + Vc 1mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9), and culturing in the dark for one week at the culture temperature of 25 +/-2 ℃. Then the illumination is switched to 12 h.d^-1Culturing for 2-3 weeks under the conditions that the illumination intensity is 2000-500 lx and the temperature is 25 +/-2 ℃;

s3, after callus culture, putting the callus obtained in the step S2 in a dip-dyeing culture medium (MS + sucrose 30 g/L), adding agrobacterium with a fluorescence labeling gene mCherry after 30S of ultrasonic wave, and shaking for 3 hours in a shaking table at 28 ℃ and 200r in a dark place to obtain an explant;

s4, after dip dyeing, transferring the explant to an adventitious bud induction culture medium (MS +6-BA 0.5mL/mL + NAA 0.5mg/mL + putrescine 500mmol/L + spermidine 100mmol/L + Vc 1 mg/L + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9), performing dark culture at 25 +/-2 ℃ for 1 week, and then performing dark culture for 2-3 weeks under the condition of illumination intensity of 2000-2500 lx;

s5, after adventitious bud induction culture for 2-3 weeks, observing whether bacteria grow on the periphery of the callus, transferring the callus onto a bud proliferation culture medium (MS +6-BA 0.5mg/mL + NAA 0.5mg/mL + cef 200mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9), and illuminating for 12 h d at 25 +/-2 DEG C^-1Culturing for 2-3 weeks under the condition of illumination intensity of 2000-2500 lx;

and S6, cutting the bud cluster obtained by bud multiplication culture into single plants, and observing whether the bud cluster has red fluorescence or not under a fluorescence microscope. Then transferring the bud cluster to a bud elongation culture medium (1/2 MS + NAA 0.5mg/mL + LC 1mg/mL + Vc 1mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9), and illuminating at 25 +/-2 ℃ for 12 h d^-1Culturing for 2-3 weeks under the condition of illumination intensity of 2000-2500 lx;

s7, transferring aseptic seedlings with the height of 3-5 cm to a rooting medium (1/2 MS + IBA 1mg/mL + sucrose 30g/L + agar 7 g/L, pH 5.8-5.9), and irradiating for 12 h d at 25 +/-2 DEG C^-1Light, lightCulturing for 2-3 weeks under the condition of the intensity of 2000-2500 lx;

s8, transferring the well rooted seedlings from the constant-temperature illumination incubator to an outdoor natural environment for hardening for 7d, uncovering a bottle sealing film, hardening for 2d, and transplanting the seedlings to sterilized yellow-heart soil for growing to obtain regenerated plants with the visible fluorescence labeling gene mCherry.

In this example, the MS medium is the following, 1/2 MS medium: macroelements are half of MS, the rest are unchanged, sucrose is 30g/L + agar is 7 g/L, and the pH value is 5.8-5.9.

Although the preferred embodiments of the present patent have been described in detail, the present patent is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present patent within the knowledge of those skilled in the art.