CN113122569B

CN113122569B - Construction method of eucommia transgenic plant regeneration system

Info

Publication number: CN113122569B
Application number: CN202110454403.3A
Authority: CN
Inventors: 孟冬; 杨清; 杨琬珑; 付玉杰; 刘腾跃; 杜婷婷; 牛丽丽; 董碧莹; 曹红燕
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2022-07-08
Anticipated expiration: 2041-04-26
Also published as: CN113122569A

Abstract

The invention provides a construction method of a regeneration system of eucommia transgenic plants, belonging to the technical field of plant transgenosis. The construction method of the eucommia transgenic plant regeneration system provided by the invention comprises the following steps: injecting agrobacterium rhizogenes suspension containing a target gene recombinant vector into the stem of the eucommia ulmoides seedling, until callus at the wound of the stem of the eucommia ulmoides seedling is differentiated into a regenerated hairy root, disinfecting by taking the regenerated hairy root as an explant, and carrying out tissue culture until a complete eucommia ulmoides transgenic seedling is obtained. According to the invention, the eucommia Hariy root transgenic plant regeneration system established by using a molecular regulation mechanism can be used for quickly and efficiently culturing the eucommia into transgenic plants, so that the efficient transformation and the quick regeneration of the eucommia are realized. The construction method is simple, the problems of difficult transgenosis, complex operation and low transformation rate of woody plants are solved, the cultured tissue culture seedlings have the same seedling age, excellent properties, consistent and good growth state and higher differentiation rate and rooting rate.

Description

Construction method of eucommia transgenic plant regeneration system

Technical Field

The invention belongs to the technical field of plant transgenosis, and particularly relates to a construction method of a eucommia transgenic plant regeneration system.

Background

Eucommia ulmoides (academic name Eucomia ulmoides Oliver) is a unique economic woody plant resource in China and has important economic value and scientific value. The demand of China for natural rubber is extremely high every year, and the quantity of the natural rubber consumed every year is the first world. The natural rubber in China mainly takes the trefoil rubber in Hainan as a raw material, but because the trefoil rubber belongs to tropical plants and the territorial area in tropical regions in China is small, the trefoil rubber is very narrow in the habitatable area in China, can be cultivated only in Hainan island, Xishuangbanna and the like, and the productivity of the trefoil rubber reaches the limit at present, so that the trefoil rubber resource in China is very deficient at present. To meet rubber requirements, China relies heavily on imported rubber. In recent years, it has been found that fruits, bark, leaves and roots of eucommia ulmoides plants are rich in natural rubber. The structure of the composite is trans-polyisoprene, the composite has the advantages of thermoplasticity, thermal elasticity, rubber elasticity and the like, the environmental conditions of many areas in China are suitable for the growth of eucommia ulmoides, the eucommia ulmoides planting area in China reaches more than 99% of the eucommia ulmoides planting area in the world, and the composite has strong cultivation advantages compared with other countries. Therefore, the gutta-percha can be used as an excellent substitute resource of Hevea hainanensis, and becomes a strategic resource of natural rubber in China. Due to the high economic value of eucommia ulmoides, mass culture also becomes a problem which is worthy of attention in recent years, however, eucommia ulmoides belongs to woody plants, the traditional planting has high requirements on growth environment conditions, the growth period is long, the yield of gutta percha is too low at the present stage, the production cost is relatively too high, and the application and industrial development of the gutta percha are limited to a great extent.

At present, the asexual propagation modes of eucommia ulmoides in China mainly adopt cuttage and grafting, and the modes are long in time and low in efficiency. In recent years, genetic engineering of plants has been developed, so that transformation systems of plants have been increasingly improved. However, the woody plant transgenosis in the field generally has the defects of difficult transgenosis, complex operation, easy bacterial contamination and low success rate. Therefore, it is necessary to find a method for constructing a regeneration system of eucommia transgenic plants, which is simple, efficient and has high success rate.

Disclosure of Invention

In view of the above, the invention aims to provide a method for constructing a regeneration system of a transgenic eucommia ulmoides plant, which is simple to operate, can quickly and efficiently obtain a large number of transgenic eucommia ulmoides plants with the same seedling age of tissue culture seedlings, excellent properties, consistent and good growth states, and high conversion rate, differentiation rate and rooting rate.

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

the invention provides a construction method of a eucommia transgenic plant regeneration system, which comprises the following steps: injecting agrobacterium rhizogenes suspension containing a target gene recombinant vector into the stem of the eucommia ulmoides seedling, until callus at the wound of the stem of the eucommia ulmoides seedling is differentiated into a regenerated hairy root, and performing tissue culture by taking the regenerated hairy root as an explant until a complete eucommia ulmoides transgenic seedling is obtained.

Preferably, the strain of agrobacterium rhizogenes comprises K599, MSU440, C58C1 or ArA 4.

Preferably, the eucommia ulmoides seedlings are seedlings growing for 10-20 days.

Preferably, the injection site is located at the stem 0.5-1.5cm above the original hairy root line of the seedling.

Preferably, the OD of said Agrobacterium rhizogenes suspension₆₀₀The value is 0.3-0.7.

Preferably, the temperature of the tissue culture is 23-27 ℃, the illumination period is 16h, and the illumination intensity is 1800 Lx.

Preferably, the callus induction medium for tissue culture is: basic culture medium +2-2.5 mg/L6-BA +1-1.5mg/LNAA +700 +800mg/L proline +500 +600mg/L LH (hydrolyzed milk protein) +30g/L sucrose +8g/L agar, pH 5.8.

Preferably, the callus differentiation medium for tissue culture is: basic culture medium +1.0-1.5 mg/L6-BA +1.0-1.5mg/LIAA +30g/L sucrose +8g/L agar, pH 5.8.

Preferably, the rooting medium for tissue culture is: basic culture medium +30-35g sucrose, pH 5.8-6.

The invention also provides a eucommia transgenic plant constructed according to any one of the construction methods.

The invention has the beneficial effects that:

according to the invention, the root system of the eucommia ulmoides is quickly and efficiently cultured into a transgenic plant through the root system transgenic gene of the root system mediated by the agrobacterium rhizogenes and a regeneration system of the root system, so that the efficient transformation and the quick regeneration of the eucommia ulmoides are realized. The method is simple and convenient to operate and high in conversion rate, and solves the problems of difficult transgenosis, complex operation and low conversion rate of woody plants. By adopting the construction method of the eucommia transgenic plant regeneration system, the cultured tissue culture seedlings have the same seedling age, excellent shape, consistent and good growth state, no bacterial contamination, difficult browning and higher differentiation rate and rooting rate.

Description of the drawings:

FIG. 1 is a schematic diagram of eucommia ulmoides obtaining transgenic roots through Hairyroot and regenerating transgenic plants through the transgenic roots;

FIG. 2 shows the growth states of transgenic eucommia roots at different stages during regeneration;

FIG. 3 shows RT-PCR and Western blot analysis of transgenic hairy root lines of eucommia ulmoides (T1 and T2 represent two transgenic lines).

Detailed Description

The schematic diagram of the construction method of the eucommia ulmoides transgenic plant regeneration system is shown in figure 1. The growth states of the eucommia transgenic root in different stages in the regeneration process are shown in figure 2. The invention has no special limitation to the types of target genes and vectors, and can select proper target genes and corresponding vectors according to the transgenic purposes. In the embodiment of the invention, in order to facilitate subsequent screening of transgenic roots, the target gene is set as Green Fluorescent Protein (GFP), and pROK2 is selected as the corresponding vector. In the present invention, the Agrobacterium rhizogenes strain is preferablyIncluding K599, MSU440, C58C1 or ArA4, more preferably K599. The specific mode for introducing the recombinant vector containing the target gene into the agrobacterium rhizogenes is not particularly limited, and any conventional introduction mode in the field can be adopted. After successful introduction, the bacterial liquid of the agrobacterium rhizogenes containing the recombinant vector is centrifuged, and the deposited thallus is resuspended to obtain the agrobacterium rhizogenes suspension containing the target gene recombinant vector₆₀₀The value is preferably 0.3 to 0.7, more preferably 0.4 to 0.6, most preferably 0.5.

The injection amount is not particularly limited in the invention, and the conventional injection amount in the field can be adopted. In the present invention, the eucommia ulmoides seedling is preferably a seedling grown for 10 to 20 days, and more preferably an eucommia ulmoides seed is injected for 13 to 17 days in a wet soil. The injection site is preferably a stem located 0.5-1.5cm above the original hairy root system of the seedling, more preferably a stem located 1-1.5cm above the original hairy root system of the seedling. After injection, the plant can continue to grow in the original soil, the culture temperature is preferably 25 ℃, the illumination period is preferably 16h, and the illumination intensity is preferably 1800 Lx. And (5) growing callus at the wound of the stem of the eucommia ulmoides seedling 12-15 days after injection, continuously culturing for 7-10 days, and differentiating the callus into a regenerated hairy root.

The regenerated hairy root is cut off to be used as an explant, and the regenerated hairy root is used as the explant to carry out tissue culture. The regenerated hairy roots are disinfected in a sterile environment, preferably, the regenerated hairy roots are soaked in 75% alcohol for 30s in an ultra-clean bench, soaked in sodium hypochlorite for 5 minutes and washed 5 times by sterile water. After the disinfection is finished, the hairy roots are cut into 0.9-1.1cm root segments for standby.

And performing tissue culture on the prepared eucommia transgenic root material, wherein the tissue culture temperature is preferably 23-27 ℃, more preferably 25-26 ℃, the illumination period is preferably 16h, and the illumination intensity is preferably 1800 Lx. Inoculating the prepared eucommia transgenic root material into a callus induction culture medium in a sterile environment, and inducing callus for 7-10 days, wherein the callus induction culture medium is preferably selected from the following components: basal medium +2-2.5 mg/L6-BA +1-1.5mg/LNAA +700 +800mg/L proline +500 +600mg/L LH (hydrolyzed milk protein) +30g/L sucrose +8g/L agar, pH 5.8, more preferably: basic culture medium +2.0mg/L6-BA +1.0mg/L NAA +700mg/L proline +500mg/L LH +30g/L sucrose +8g/L agar PH 5.8.

Cutting and transferring the strongly growing callus onto a callus differentiation culture medium, increasing the callus after 10-15 days, generating punctiform bulges on the surface, then growing buds about 1cm, and waiting for the growth of buds; transferring the differentiating callus to the same differentiation culture medium every 10-15 days for continuous differentiation, and finally differentiating the transgenic eucommia seedlings. The callus differentiation medium is preferably: basal medium +1.0-1.5 mg/L6-BA +1.0-1.5mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, more preferably: basic medium +1.0 mg/L6-BA +1.0mg/L IAA +30g/L sucrose +8g/L agar PH 5.8.

When transgenic seedlings grow to 5-7cm in the differentiation culture medium, selecting strong seedlings, and transferring the strong seedlings to a rooting culture medium for rooting. The rooting medium is preferably: basal medium +30-35g sucrose, pH 5.8-6, more preferably: basal medium +30g sucrose, PH 5.8.

The present invention is not particularly limited to the basic culture medium of the callus induction culture medium, the callus differentiation culture medium, and the rooting culture medium, and in a specific embodiment of the present invention, the basic culture medium comprises the following components: potassium nitrate, ammonium nitrate, monopotassium phosphate, magnesium sulfate, calcium chloride, potassium iodide, boric acid, manganese sulfate, zinc sulfate, sodium molybdate, copper sulfate, cobalt chloride, disodium ethylenediamine tetraacetic acid, ferrous sulfate, inositol, glycine, thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, sucrose and agar.

The invention also provides a eucommia transgenic plant constructed according to any one of the construction methods. The type of the transgenic gene of the present invention is not particularly limited, and any type of transgenic gene known or unknown in the art may be used.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The seed coat of the current-year eucommia is removed, and the eucommia is sowed in the moist soil (length, width, height, 9, 10 cm; nutrient soil: vermiculite: perlite: 3: 1: 1), the illumination period is 16h, and the illumination intensity is 1800 Lx. Using PROK2 as a vector, constructing a gene expression vector of a target gene GFP, transferring the gene expression vector into K599 agrobacterium, centrifuging a bacterium solution of the agrobacterium rhizogenes containing the recombinant vector, and re-suspending deposited bacteria to obtain an agrobacterium rhizogenes suspension (OD) containing the target gene recombinant vector₆₀₀0.5). And (3) carrying out agrobacterium tumefaciens suspension injection on the eucommia ulmoides seedling growing for 15 days, wherein the injection part is positioned at the stem part 1cm above the original root of the eucommia ulmoides, callus grows for 12-15 days, and hairy roots grow for 7-10 days. Whether the target gene was successfully introduced into the hairy roots was examined by RT-PCR and Western blotting, and the results are shown in FIG. 3.

And (3) counting the callus induction rate and the rooting rate of the transgenic roots, wherein the callus induction rate is the number of seedlings of the induced callus/the total seedlings multiplied by 100%, and the regeneration rate of the transgenic roots is the number of rooted seedlings/the total seedlings multiplied by 100%.

Example 2

The difference from example 1 is that the Agrobacterium strain type is MSU440, and the rest is the same as example 1.

Example 3

The difference from example 1 is that the Agrobacterium strain type is C58C1, and the rest is the same as example 1.

Example 4

The difference from example 1 is that the Agrobacterium strain type is ArA4, and the rest is the same as example 1.

The results of callus induction rate and transgenic root rooting rate obtained in examples 1 to 4 are shown in Table 1.

TABLE 1 regeneration rates of four Agrobacterium rhizogenes-injected eucommia root transgene systems

	OD₆₀₀	Type of Strain	Distance from injection site to original root (cm)	Callus induction rate (%)	Rooting percentage (%)
						Example 1	0.5	K599	1	75	36
Example 2	0.5	MSU440	1	13	5
						Example 3	0.5	C58C1	1	20	6
Example 4	0.5	ArA4	1	5	8

Example 5

The difference from example 1 is in OD of Agrobacterium rhizogenes suspension containing recombinant vector of target gene₆₀₀0.3, and the same procedure as in example 1 was repeated.

Example 6

The difference from example 1 is in OD of Agrobacterium rhizogenes suspension containing recombinant vector of target gene₆₀₀This was 0.7, and the same procedure as in example 1 was repeated. The results of callus induction rate and transgenic root rooting rate obtained in examples 1, 5 and 6 are shown in Table 2.

TABLE 2 regeneration rates of eucommia ulmoides root transgene systems injected with different concentrations of Agrobacterium rhizogenes suspensions

	OD₆₀₀	Type of Strain	Distance from injection site to original root (cm)	Callus induction rate (%)	Rooting percentage (%)
						Example 1	0.5	K599	1	75	36
Example 5	0.3	K599	1	30	14
						Example 6	0.7	K599	1	45	25

Example 7

The difference from example 1 was that the injection site was located at the stem portion 0.5cm above the original root of eucommia ulmoides, and the same as example 1 was applied.

Example 8

The difference from example 1 was that the injection site was located at the stem 1.5cm above the original root of eucommia ulmoides, and the same as example 1 was applied. The results of callus induction rate and transgenic root rooting rate obtained in examples 1, 7 and 8 are shown in Table 3.

TABLE 3 regeneration Rate of transgenic eucommia root System at three different injection sites

	OD₆₀₀	Type of Strain	Distance from injection site to original root (cm)	Callus induction rate (%)	Rooting percentage (%)
						Example 1	0.5	K599	1	75	36
Example 7	0.5	K599	0.5	30	10
						Example 8	0.5	K599	1.5	68	30

Example 9

The eucommia transgenic roots grown in example 1 were cut, soaked in 75% alcohol for 30 seconds in a sterile environment, soaked in sodium hypochlorite for 5 minutes, washed 5 times with sterile water, then the transgenic roots were cut into 1cm pieces, inoculated into a root callus induction medium and cultured at a culture temperature of 25 ℃, a light intensity of 1800lx and a light time of 16h per day, and the callus state was counted after 10 days, and the callus induction rate (%) — the number of root pieces from which callus was induced/the number of all root pieces × 100%.

The callus induction culture medium is as follows: basic medium +2.0mg/L6-BA +1mg/L NAA +700mg/L proline +500mg/LLH +30g/L sucrose +8g/L agar, pH 5.8. The basic culture medium is as follows: 1900mg/L potassium nitrate, 1650mg/L ammonium nitrate, 170mg/L monopotassium phosphate, 370mg/L magnesium sulfate, 440mg/L calcium chloride, 0.83mg/L potassium iodide, 6.2mg/L boric acid, 22.3mg/L manganese sulfate, 8.6mg/L zinc sulfate, 0.25mg/L sodium molybdate, 0.025mg/L copper sulfate, 0.025mg/L cobalt chloride, 37.25mg/L disodium edetate, 27.85mg/L ferrous sulfate, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride, 0.5mg/L nicotinic acid, 30g/L sucrose and 7g/L agar.

Example 10

The difference from example 9 is that the callus induction medium is: basal medium +2.5 mg/L6-BA +1.5mg/L NAA +800mg/L proline +600mg/L LH +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 9.

Comparative example 1

The difference from example 9 is that the callus induction medium is: basic medium +1mg/L6-BA +1mg/L NAA +500mg/L proline +300mg/L LH +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 9.

Comparative example 2

The difference from example 9 is that the callus induction medium is: basal medium +1.5 mg/L6-BA +1.3mg/L NAA +600mg/L proline +400mg/L LH +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 9.

Comparative example 3

The difference from example 9 is that the callus induction medium is: basal medium +3mg/L6-BA +2.5mg/L LNAA +900mg/L proline +700mg/L LH +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 9.

The callus induction rates obtained in examples 9 and 10 and comparative examples 1, 2 and 3 are shown in Table 4.

TABLE 4 influence of different callus induction media on callus induction of eucommia ulmoides root

Example 11

The strongly growing callus obtained in example 9 was excised and transferred to a callus differentiation medium to culture at 25 ℃ under an illumination intensity of 1800lx for 16 hours per day with 10-15 days of callus growth, with punctate projections on the surface, with shoots growing about 1cm later, and after 30 days, the callus differentiation state was counted, and the callus differentiation rate (%) was calculated as the number of callus differentiated/total number of callus. Wherein the callus differentiation culture medium is as follows: basic medium +1mg/L6-BA +1mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, the basic medium was the same as that of example 9.

Example 12

The difference from example 11 is that the callus differentiation medium is: basal medium +1.5 mg/L6-BA +1.5mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, the rest being as in example 11.

Comparative example 4

The difference from example 11 is that the callus differentiation medium is: basal medium +0.3 mg/L6-BA +0.3mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 11.

Comparative example 5

The difference from example 11 is that the callus differentiation medium is: basal medium +0.5 mg/L6-BA +0.5mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 11.

Comparative example 6

The difference from example 11 is that the callus differentiation medium is: basal medium +2 mg/L6-BA +2mg/L IAA +30g/L sucrose +8g/L agar, pH 5.8, the rest was the same as in example 11.

The differentiation rates of calli obtained in examples 11 and 12 and comparative examples 4, 5 and 6 are shown in Table 5.

TABLE 5 Effect of different callus differentiation media on callus induction of eucommia ulmoides

Example 13

When the seedling in the differentiation medium of example 11 grows to 5cm, selecting a bud which grows strongly, cutting off and transferring the bud into a rooting medium for rooting culture, wherein the culture temperature is 25 ℃, the illumination intensity is 1800lx, the illumination time is 16h every day, and after 10d, counting the rooting state of the regenerated plant, and the rooting rate (%) (seedling rooting number/seedling total number).

The rooting culture medium comprises: basic medium +30g/L sucrose +8g/L agar, pH 5.8, the same basic medium as in example 9.

Example 14

The difference from example 13 is that the rooting medium is: basal medium +35g/L sucrose +10g/L agar, pH 6, and the same as in example 13.

Comparative example 7

The difference from example 13 is that the rooting medium is: basic medium +25g/L sucrose, +5g/L agar, pH 4.5, and the rest were the same as in example 13.

Comparative example 8

The difference from example 13 is that the rooting medium is: basal medium +40g/L sucrose +15g/L agar, pH 7.5, and the rest as in example 13.

The rooting rates obtained in examples 13 and 14 and comparative examples 7 and 8 are shown in Table 6.

TABLE 6 Effect of different rooting media on rooting of eucommia ulmoides

Example 15

The seed coat of the current-year eucommia is removed, and the eucommia is sowed in the moist soil (length, width, height, 9, 10 cm; nutrient soil: vermiculite: perlite: 3; 1; 1), the illumination period is 16h, and the illumination intensity is 1800 Lx. PROK2 is used as a vector to construct a gene expression vector of a target gene GFP, and the gene expression vector is transferred into K599 agrobacterium tumefaciens (OD)₆₀₀0.5), the eucommia seedlings growing for 15 days are injected with agrobacterium, the injection part is positioned 1cm above the original root of the eucommia, callus grows for 12-15 days, and hairy roots grow for about 7-10 days. Whether the target gene was successfully introduced into these hairy roots was tested by RT-PCR and Western blotting.

Cutting transgenic eucommia bark roots, soaking in 75% alcohol for 30s in a sterile environment, soaking in sodium hypochlorite for 5 minutes, washing with sterile water for 5 times, cutting the transgenic eucommia bark roots into sections of about 1cm, and inoculating the sections to a root callus induction culture medium: the method comprises the following steps of (1) adding 6-BA in a basic culture medium +2.0mg/L +1mg/LNAA +700mg/L proline +500mg/LLH +30g/L sucrose +8g/L agar, wherein the pH value is 5.8, the culture temperature is 25 ℃, the illumination intensity is 1800lx, the illumination time is 16h every day, the callus state is counted after 10d, and the callus induction rate (%) < the number of the roots of the induced callus/the number of all the roots.

Cutting the strong callus and transferring the cut callus to a callus differentiation culture medium: the method comprises the steps of adding a basic culture medium, 1mg/L6-BA, 1mg/L IAA, 30g/L sucrose and 8g/L agar, adjusting the pH to 5.8, increasing callus tissue in 10-15 days, enabling the surface of the callus tissue to have punctiform protrusions, growing buds about 1cm, waiting for the growth of buds, culturing at the temperature of 25 ℃, the illumination intensity of 1800lx, the illumination time of 16h every day, counting the differentiation state of the callus tissue after 30 days, and adjusting the differentiation rate (%) of the callus tissue to the number of the differentiated buds/the total number of the callus tissue.

When the plantlets grow to about 5cm in a differentiation culture medium, selecting strong buds, cutting off the strong buds, transferring the strong buds to a rooting culture medium for rooting, wherein the rooting culture medium is a basic culture medium, 30g/L sucrose and 8g/L agar, the pH value is 5.8, the culture temperature is 25 ℃, the illumination intensity is 1800lx, the illumination time is 16h every day, the rooting state of the regenerated plants is counted after 10 days, and the rooting rate (%) -the rooting number of the plantlets/the total number of the plantlets.

Comparative example 9

The traditional transgenic regeneration plant construction method in the field is adopted: the 35s-eGFP-pROK2 vector was constructed. Transforming the constructed vector into GV3101 competent cells by an electrotransformation method; selecting a single colony of the positive clone, shaking the colony in 5ml YEP +20mg/L Rif +50mg/L Kana liquid culture medium at 180rpm in a shaking table at 28 ℃ overnight, transferring the colony into 50ml YEP +20mg/L Rif +50mg/L Kana liquid culture medium on the next day, and amplifying the colony until the concentration OD of the colony is₆₀₀And (3) centrifuging the bacterial liquid at the room temperature at 10000rpm for 10min after the bacterial liquid is 0.5, discarding the supernatant, re-suspending the bacterial liquid by using a basic culture medium, and standing for 2-3h to prepare the transgenic infected bacterial liquid.

Sterilizing root of eucommia ulmoides with alcohol for 30s in sterile environment, sterilizing with sodium hypochlorite for 5min, washing with sterile water for 5 times, cutting sterilized root of eucommia ulmoides into segments of about 1cm, placing in bacterial liquid, and converting at 80rpm of shaking table for 1 h. After transformation, the embryos are eluted with sterile water 2 times and transferred to a basic medium +2mg/L TDZ +0.5mg/L NAA +1mg/L folic acid +30g/L sucrose +10g/L glucose +8g/L agar for 3 days at 25 ℃ in the dark. Washing the embryo with sterile water for 4-6 times, drying the excess liquid on the surface with sterile filter paper, and transferring: and (3) carrying out degerming on a culture medium of basic culture medium, 250mg/L of cefamycin, 10mg/L of hygromycin, 2mg/LTDZ, 0.5mg/LNAA, 1mg/L of folic acid, 30g/L of cane sugar, 10g/L of glucose and 8g/L of agar for 20-30 d.

Transferring the sterile root segments into: the root callus is obtained after the culture of basic culture medium, 5 mg/L2.4-D, 0.5mg/L zeatin, 300mg/L lactoprotein hydrolysate, 500mg/L glutamine, 30g/L sucrose and 8g/L agar for about 30-60 days in a culture medium with the pH value of 5.8, and the callus grows well. Transferring the callus to a culture medium of a basic culture medium, 1mg/L6-BA, 1mg/L IAA, 30g/L sucrose and 8g/L agar for differentiation culture, and completing bud differentiation of the callus within about 40-80 days. And (3) inducing 3-5cm buds to root for 10-20 days on a culture medium with the pH value of 5.8, wherein the culture medium comprises 0.1mg IBA, 0.005mg NAA, 30g cane sugar and 8g agar, so as to obtain transgenic regenerated seedlings.

The different methods for constructing transgenic plants regenerated in example 15 and comparative example 9 show the effect of the methods on transgenic plants regenerated from eucommia ulmoides as shown in Table 7.

TABLE 7 Effect of different regeneration transgenic plant construction methods on transgenic efficiency

As can be seen from Table 7, the callus induction rate, differentiation rate and rooting rate of the regenerated plants of the traditional construction method of the regenerated transgenic plants in the field are relatively low, the time consumption of the transgenic process is long, and the survival rate and the state of the obtained regenerated transgenic plants are relatively low and general. In comparison, in the construction method of the Hariy root regeneration transgenic plant, the callus induction rate, the differentiation rate and the rooting rate of the regeneration plant are relatively high, the time consumption of the transgenic process is short, and the obtained regeneration transgenic plant has high survival rate and excellent growth state.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A construction method of a eucommia transgenic plant regeneration system is characterized by comprising the following steps: injecting agrobacterium rhizogenes suspension containing a target gene recombinant vector into the stem of a eucommia ulmoides seedling, until callus at a wound of the stem of the eucommia ulmoides seedling is differentiated into a regenerated hairy root, and performing tissue culture by taking the regenerated hairy root as an explant until a complete eucommia ulmoides transgenic seedling is obtained;

the injection position is positioned at the stem part 1-1.5cm above the original hairy root system of the seedling;

the OD600 value of the agrobacterium rhizogenes suspension is 0.5-0.7;

the callus induction culture medium for tissue culture comprises: basic culture medium +2-2.5 mg/L6-BA +1-1.5mg/L NAA +700 +800mg/L proline +500 +600mg/L LH +30g/L sucrose +8g/L agar, pH 5.8;

the callus differentiation culture medium for tissue culture comprises: basic culture medium +1.0-1.5 mg/L6-BA +1.0-1.5mg/L IAA +30g/L sucrose +8g/L agar, pH is 5.8;

the rooting culture medium for tissue culture comprises: a basal culture medium plus 30-35g of cane sugar, wherein the pH value is 5.8-6;

the strain of the agrobacterium rhizogenes is K599.

2. The method of claim 1, wherein the eucommia ulmoides oliv seedling is a seedling that grows for 10 to 20 days.

3. The method according to claim 1, wherein the temperature of the tissue culture is 23-27 ℃, the light cycle is 16h, and the light intensity is 1800 Lx.