阅读说明：本技术 一种橡胶草遗传转化方法 (Genetic transformation method for kokstroemia indica ) 是由 彭存智 常丽丽 仝征 王丹 徐兵强 于 2019-10-30 设计创作，主要内容包括：本发明提供一种橡胶草遗传转化方法,包括:(1)取新疆橡胶草种质的幼嫩叶片,切割处理,放于:MS+1.5～1.6mg/L6-BA+0.1mg/LNAA+20g/L蔗糖,pH5.8芽诱导培养基中培养；50天后,挑选出适宜组培的橡胶草优异种质；转至:1/2MS+20g/L蔗糖,pH5.8的生根培养基,成为再生橡胶草组培幼苗；(2)取幼苗叶片切割处理,在芽诱导培养基上预培养2天；(3)加入农杆菌侵染液浸染后,转至芽诱导培养基中,黑暗共培养3天；转至含400mg/L Car的芽诱导培养基培养一周；转至MS+1.5～1.6mg/L 6-BA+0.1mg/L NAA+20g/L蔗糖+400mg/L Car和8～10mg/L Hyg,pH5.8筛选培养基上,诱导抗性芽的再生；转至1/2MS+20g/L蔗糖+400mg/L Car和8～10mg/L Hyg,pH5.8新的生根培养基上,得到橡胶草抗性再生植株；本发明的橡胶草遗传转化方法,抗性再生芽诱导率>30％,转基因再生苗植株畸形率低,对转基因橡胶草阳性检出率>90％。(The invention provides a method for genetic transformation of hevea brasiliensis, which comprises (1) cutting young leaves of the germplasm of the hevea brasiliensis, and culturing in an inducing culture medium containing MS + 1.5-1.6 mg/L6-BA +0.1mg/LNAA +20g/L sucrose and pH5.8 bud; after 50 days, selecting excellent germplasm of the rubber grass suitable for tissue culture; transferring to 1/2MS +20g/L sucrose and a rooting culture medium with pH of 5.8 to obtain regenerated hevea brasiliensis tissue culture seedlings; (2) cutting seedling leaves, and pre-culturing on a bud induction culture medium for 2 days; (3) adding agrobacterium infection solution for dip dyeing, transferring to a bud induction culture medium, and culturing in the dark for 3 days; transferring to a bud induction culture medium containing 400mg/L Car for culturing for one week; transferring to a screening culture medium of MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg at pH5.8 to induce the regeneration of resistant buds; transferring to a new rooting culture medium of 1/2MS +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, and pH5.8 to obtain a hevea brasiliensis resistance regeneration plant; the genetic transformation method of the hevea brasiliensis has the advantages that the inductivity of the resistant regeneration buds is more than 30%, the plant aberration rate of the transgenic regeneration seedlings is low, and the positive detection rate of the transgenic hevea brasiliensis is more than 90%.)

1. A method for genetic transformation of kokstroemia indica, which is characterized in that: the method comprises the following steps:

(1) screening and obtaining of plant material genetically transformed with kokstroemia indica

a. Collecting and selecting Xinjiang rubber grass germplasm, cutting complete young and tender leaves, sterilizing and cutting, and placing in a formula comprising: MS + 1.5-1.6 mg/L6-BA +0.1mg/LNAA +20g/L sucrose, and culturing in a bud induction culture medium with pH of 5.8; transferring the culture medium to a new same culture medium after 28-32 days of culture, and continuing to grow for 20-30 days; selecting excellent germplasm of the Xinjiang rubber grasses suitable for tissue culture according to the regeneration rate and the growth state of the regenerated buds cultured on a bud induction culture medium for 48-52 days;

b. the selected excellent germplasm of the Xinjiang rubber plant is cut off when the plant height reaches 4-6 cm, and the process is transferred to a formula comprising the following steps: 1/2MS +20g/L sucrose, and in the rooting culture medium with pH5.8 to culture roots, becoming complete regeneration rubber grass tissue culture seedlings;

(2) pre-culture of Hedychium plant explants

Cutting the leaves of the tissue culture seedlings of the hevea brasiliensis and pre-culturing the cut leaves on a bud induction culture medium for 2 days;

(3) acquisition of a resistant regenerated plant of RUBENCAO

a. Collecting pre-cultured rubber grass explants, putting the rubber grass explants into a sterile culture bottle, adding a pre-prepared agrobacterium infection solution until the rubber grass explants are completely immersed, and carrying out dip dyeing for 30 minutes;

b. after the explant is subjected to bacterium liquid sucking-drying, transferring the explant to a bud induction culture medium, carrying out co-culture for 3 days at the temperature of 22-23 ℃ in the dark, wherein the pH is 5.2-5.3; taking out and washing with sterile water, and then washing with an MS liquid culture medium added with 395-405 mg/L Car; transferring the culture medium to a bud induction culture medium added with 395-405 mg/L Car for culturing for one week;

c. transferring the explant to the formula comprises: MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/LCar and 8-10mg/L Hyg, inducing the regeneration of resistant buds on a screening culture medium with pH of 5.8, and subculturing once every 2 weeks;

d. when the resistant regenerated shoots grew to 3-5cm, the transfer to the formula included: 1/2MS +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, and culturing on new rooting culture medium with pH of 5.8 to root until becoming complete resistant regenerated rubber grass plant.

2. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: the cutting processing method for the blade in the steps (1) and (2) comprises the following steps: cutting the leaf into 2-3cm length, scratching the leaf 1/2-3/4 width across the vein every 0.8-1.2 cm, and cutting the petiole into 1-1.5cm length.

3. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (1), selecting a root system of a tissue culture seedling with the diameter of more than or equal to 1mm from the rubber grass seedlings after 30 days, cutting the root system to the length of 2.8-3.2 cm, and transferring to a formula as follows: 1/2MS, germinating adventitious buds on the root system until rooting to form complete tissue culture seedling, and taking tender leaf for the second time as the plant material for genetic transformation of Hevea brasiliensis.

4. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the preparation method of the agrobacterium infection liquid comprises the following steps:

(1) selecting a single agrobacterium colony of a target gene to be cultured in 10mL of YEP culture medium with corresponding resistance at 28 ℃ and 200rpm overnight;

(2) adding 1mL of overnight culture liquid into 100mL of YEP culture medium with the same resistance, and culturing at 28 ℃ and 200rpm for 10-12 h;

(3) centrifuging with 50mL sterile centrifuge tube at 4 deg.C and 6000rpm for 10min to collect thallus; resuspending the Agrobacterium tumefaciens precipitate with MS liquid culture medium, centrifuging at 4 deg.C and 6000rpm for 10min, and collecting thallus; re-suspending the agrobacterium tumefaciens precipitate by using an MS liquid culture medium, and adjusting OD600 to 0.6 to prepare an agrobacterium tumefaciens infection liquid; the MS liquid culture medium comprises the following components in percentage by weight: MS +20g/L sucrose, pH 5.8.

5. The method of claim 3, wherein the genetic transformation of kokstroemia indica comprises: in the step (3), acetosyringone with the final concentration of 100mg/L is added into the agrobacterium tumefaciens dip dyeing solution before dip dyeing.

6. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (1), the culture conditions of bud induction and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.

7. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the steps (2) and (3), the conditions of pre-culture, induction of resistant buds and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.

8. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (1), the formula of the bud induction medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose, pH 5.8; the formula of the rooting culture medium comprises: 1/2MS +20g/L sucrose, pH 5.8.

9. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the formula of the screening medium comprises: MS +1.5mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 10mg/LHyg, pH 5.8.

10. The method of claim 1, wherein the genetic transformation of hevea brasiliensis is performed by: in the step (3), the formula of the new rooting medium comprises: 1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.

Technical Field

The invention relates to the technical field of plant biology, in particular to a genetic transformation method of hevea brasiliensis.

Background

The Hevea brasiliensis is also known as Taraxacum kok saghyz Rodin, is a plant of Taraxacum of Cichorium of Compositae, and is native to Hassakestan, Europe, Xinjiang, etc. The roots of the rubber grasses contain high-quality rubber with the content of more than 20 percent, and the rubber grasses are gradually used as a second natural rubber resource.

In recent years, scholars at home and abroad have conducted some researches on the tissue culture technology of the kochia scoparia. For example, the leaf of kochia scoparia is used as an explant to induce adventitious buds or callus, but the strain of the kochia scoparia is seriously mixed due to the self-incompatible breeding characteristics of the kochia scoparia. Due to the difference of tissue culture conditions and methods among different strains, the problems of low bud induction rate, low regeneration capacity of regenerated seedlings and the like are easily caused, the genetic transformation of the hevea brasiliensis is difficult to carry out, and in the process of the genetic transformation, the induction rate of the resistant regenerated buds is extremely low, the genetic transformation period is long, the aberration rate is high, and the hevea brasiliensis transgenic strains are difficult to effectively obtain.

Disclosure of Invention

In view of the above, the invention provides a high-transformation-efficiency genetic transformation method for the rubber grass, the inductivity of the resistant regeneration buds is more than 30%, the plant aberration rate of the transgenic regeneration seedlings is low, the growth speed is high, and the positive detection rate of the transgenic rubber grass is more than 90%.

The technical scheme of the invention is realized as follows:

the invention provides a genetic transformation method of kokstroemia indica, which comprises the following steps:

(1) screening and obtaining of plant material genetically transformed with kokstroemia indica

a. Collecting and selecting Xinjiang rubber grass germplasm, cutting complete young and tender leaves, sterilizing and cutting, and placing in a formula comprising: MS + 1.5-1.6 mg/L6-BA +0.1mg/LNAA +20g/L sucrose, and culturing in a bud induction culture medium with pH of 5.8; transferring the culture medium to a new same culture medium after 28-32 days of culture, and continuing to grow for 20-30 days; selecting excellent germplasm of the Xinjiang rubber grasses suitable for tissue culture according to the regeneration rate and the growth state of the regenerated buds cultured on a bud induction culture medium for 48-52 days;

b. the selected excellent germplasm of the Xinjiang rubber plant is cut off when the plant height reaches 4-6 cm, and the process is transferred to a formula comprising the following steps: 1/2MS +20g/L sucrose, and in the rooting culture medium with pH5.8 to culture roots, becoming complete regeneration rubber grass tissue culture seedlings;

(2) pre-culture of explants of Hedychium plant

Cutting the leaves of the tissue culture seedlings of the hevea brasiliensis and pre-culturing the cut leaves on a bud induction culture medium for 3 days;

(3) acquisition of resistant regenerated rubber grass plants

a. Collecting pre-cultured rubber grass explants, putting the rubber grass explants into a sterile culture bottle, adding a pre-prepared agrobacterium infection solution until the rubber grass explants are completely immersed, and performing dip dyeing;

b. after the explant is subjected to bacterium liquid sucking-drying, transferring the explant to a bud induction culture medium, carrying out co-culture for 3 days at the temperature of 22-23 ℃ in the dark, wherein the pH is 5.2-5.3; taking out and washing with sterile water, and then washing with an MS liquid culture medium added with 395-405 mg/L Car; transferring the culture medium to a bud induction culture medium added with 395-405 mg/L Car for culturing for one week;

c. transferring the explant to the formula comprises: MS + 1.5-1.6 mg/L6-BA +0.1mg/L NAA +20g/L sucrose +400mg/L Car and 8-10mg/L Hyg, inducing the regeneration of resistant buds on a screening culture medium with pH of 5.8, and subculturing once every 2 weeks;

d. when the resistant regenerated shoots grew to 3-5cm, the transfer to the formula included: 1/2MS, 20g/L sucrose, 400mg/LCar and 8-10mg/L Hyg, and culturing on a new rooting culture medium with pH of 5.8 to root until becoming a complete resistant regenerated rubber grass plant.

Further, the cutting treatment method for the blade in the step (1) and the step (2) comprises the following steps: cutting the leaf into 2-3cm length, scratching the leaf 1/2-3/4 width across the vein every 0.8-1.2 cm, and cutting the petiole into 1-1.5cm length.

Further explaining, in the step (1), the root system of the tissue culture seedling with the diameter of more than or equal to 1mm is selected from the rubberella seedlings after 30 days, the length of 2.8-3.2 cm is cut, and the formula is transferred as follows: 1/2MS, germinating adventitious buds on the root system until rooting to form complete tissue culture seedling, and taking tender leaf for the second time as the plant material for genetic transformation of Hevea brasiliensis. Is beneficial to keeping the young state of the rubber grass for a long time, keeping the regeneration capacity of the rubber grass and reducing the distortion rate.

Further, in the step (3), the preparation method of the agrobacterium infection solution comprises the following steps:

(1) selecting a single agrobacterium colony of a target gene to be cultured in 10mL of YEP culture medium with corresponding resistance at 28 ℃ and 200rpm overnight;

(2) adding 1mL of overnight culture liquid into 100mL of YEP culture medium with the same resistance, and culturing at 28 ℃ and 200rpm for 10-12 h;

(3) centrifuging with 50mL sterile centrifuge tube at 4 deg.C and 6000rpm for 10min to collect thallus; resuspending the Agrobacterium tumefaciens precipitate with MS liquid culture medium, centrifuging at 4 deg.C and 6000rpm for 10min, and collecting thallus; re-suspending the agrobacterium tumefaciens precipitate by using an MS liquid culture medium, and adjusting OD600 to 0.6 to prepare an agrobacterium tumefaciens infection liquid; the MS liquid culture medium comprises the following components in percentage by weight: MS +20g/L sucrose, pH 5.8.

Further explaining, in the step (3), acetosyringone with the final concentration of 100mg/L is added into the agrobacterium tumefaciens dip dyeing solution before dip dyeing.

Further, in the step (1), the culture conditions for bud induction and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.

Further, in the steps (2) and (3), the conditions for preculture, induction of resistant bud and rooting culture are as follows: 22 ℃, the illumination period is 16 hours/day, and the illumination intensity is 1800 and 2000 lux.

Further, in step (1), the formulation of the shoot induction medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose, pH 5.8; the formula of the rooting culture medium comprises: 1/2MS +20g/L sucrose, pH 5.8.

Further, in step (3), the formulation of the screening medium comprises: MS +1.5mg/L6-BA +0.1mg/LNAA +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.

Further, in step (3), the formulation of the new rooting medium comprises: 1/2MS +20g/L sucrose +400mg/L Car and 10mg/L Hyg, pH 5.8.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, through screening of excellent germplasms of the Xinjiang rubber grass, the rubber grass regenerated seedling with strong regeneration capacity is obtained for genetic transformation, and hygromycin resistance screening of different concentrations of the rubber grass is carried out, so that the induction rate of the resistant regenerated bud is improved, and the rubber grass genetic transformation with high transformation rate and low deformity rate is realized. Has the following characteristics:

1. the screened excellent germplasm of the Xinjiang rubber plant has high seed germination rate, and the germination rate in the same batch of seeds reaches 100 percent; the growth speed is high, the continuous growth activity is strong, and the leaves are not easy to age; high bud induction rate, strong regeneration capability and high regeneration seedling forming efficiency, and is favorable for genetic transformation.

2. In the process of genetic transformation, the transformation efficiency is high, and the induction rate of the resistance regeneration bud is more than 30 percent; the genetic transformation period is short, and only about 3 months are needed from the infection of agrobacterium to the acquisition of the complete transgenic regeneration seedlings; the transgenic regenerated seedling has complete plant shape and low aberration rate; the transgenic regeneration seedlings have vigorous activity and high growth speed; the transgenic regenerated seedling has high propagation efficiency and is easy to obtain transgenic strains.

3. In the shoot induction medium for selecting resistant regenerated shoots, callus and shoots induced to be regenerated by explants which have not been successfully genetically transformed have a plurality of manifestation symptoms: (1) growth is severely inhibited; (2) easy vitrification; (3) the tip of the seedling leaf is easy to brown and necrose. According to the characteristics, the regeneration buds which are not successfully genetically transformed can be directly removed, and the positive detection rate of the transgenic rubber grass is over 90 percent finally.

Drawings

FIG. 1 shows the excellent germplasm of Xinjiang rubber grass suitable for tissue culture screened by the embodiment of the invention;

FIG. 2 is an explant of genetically transformed Columba glabra according to an embodiment of the present invention;

FIG. 3 is a graph of a first week of growth of a RUBENCH PLANT on a selection medium in accordance with an embodiment of the present invention;

FIG. 4 is a third week of growth of a RUBENCH PLANT on screening media in accordance with an embodiment of the present invention;

FIG. 5 shows the growth of a Hevea brasiliensis explant on a selection medium for 50 days according to an embodiment of the present invention;

FIG. 6 is a 75 day growth of a Hevea brasiliensis explant on selection medium according to an embodiment of the present invention;

FIG. 7 shows the expanding propagation of transgenic Hevea brasiliensis line according to an embodiment of the present invention;

FIG. 8 is the electrophoresis diagram of the PCR detection of the exogenous DNA of the transgenic hevea brasiliensis according to the embodiment of the present invention;

FIG. 9 shows the regeneration of multiple shoots after 30 days of treatment of RUBENCAO with hygromycin at different concentrations in accordance with the present invention.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.