CN109735562B

CN109735562B - Construction method of economic plant effective root system transgenic system

Info

Publication number: CN109735562B
Application number: CN201910133371.XA
Authority: CN
Inventors: 付玉杰; 孟冬; 牛丽丽; 杨清; 董碧莹; 宋治华
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2019-02-22
Filing date: 2019-02-22
Publication date: 2020-09-29
Anticipated expiration: 2039-02-22
Also published as: CN109735562A

Abstract

The invention provides a construction method of an economic plant effective root system transgenic system, belonging to the technical field of plant transgenosis. Carrying out solid-liquid separation on the bacterial liquid containing the target recombinant vector agrobacterium rhizogenes to obtain a bacterial body containing the target recombinant vector agrobacterium rhizogenes for heavy suspension, injecting the obtained agrobacterium rhizogenes suspension to the stem of the economic plant seedling, and growing callus at the wound of the stem of the economic plant seedling 10-12 days after injection; and continuously culturing the seedlings for 28-35 days, and cutting off the original hairy root system when the callus is differentiated into a regenerated hairy root. The construction method is suitable for various economic plants, and the regeneration rate of the hairy roots and the regeneration rate of the callus are high. The construction method provides very quick and effective gene system function analysis, secondary metabolite engineering and plant stress response research. The constructed root system transgenic system has only transgenic root and not whole plant, and this is also one excellent system for studying signal transduction between root and stem.

Description

Construction method of economic plant effective root system transgenic system

Technical Field

The invention belongs to the technical field of plant transgenosis, and particularly relates to a construction method of an effective root system transgenic system of an economic plant.

Background

Agrobacterium-mediated plant transformation systems have been the most widespread and successful method in plant genetic engineering in recent decades (Gelvin, 2003; Vain, 2007; Matveeva and Lutova, 2014). Agrobacterium tumefaciens can grow tumors in plants by wounding and transferring a tumor-inducing (Ti) plasmid to the plant nucleus. Based on this theory, the tumor-inducing gene is converted into the desired gene by modifying and removing the Ti plasmid and carries any desired gene into the chromosome of the plant cell. Since the first demonstration in 1983 that Agrobacterium-mediated plant transformation methods were widely used to successfully transfer foreign genes into tobacco, gene function studies and plant genetic modifications of model plants and some crops have been established in more efficient transgenic systems (Zhang et al, 2006; Wenzhler et al, 1989; Stone field et al, 1996).

However, much effort has been expended in the development of Agrobacterium-mediated model plant stable transformation protocols, so far limited to a few plants (Klauso & Bent, 1998; Hoekema et al, 1989; Murzzova Ell, 2014). Due to their uniqueness or long growth cycle, most medicinal and woody plants lack an efficient rapid screening system for gene function analysis and have greatly limited the development of these related research fields.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel, simple, fast and efficient method for constructing an economic plant root system transgenic system, which has the characteristic of high regeneration rate of hairy roots.

The invention provides a construction method of an economic plant effective root system transgenic system, which comprises the following steps:

1) carrying out solid-liquid separation on the bacterial liquid containing the target recombinant vector agrobacterium rhizogenes to obtain heavy suspension of the bacterial body containing the target recombinant vector agrobacterium rhizogenes, so as to obtain agrobacterium rhizogenes suspension;

2) injecting the agrobacterium rhizogenes suspension to the stem of the economic plant seedling, and growing callus at the wound of the stem of the economic plant seedling 10-12 days after injection;

3) culturing the economic plant seedlings with the callus for 28-35 d, and cutting off the original hairy root system when the callus is differentiated into a regeneration hairy root.

Preferably, the Agrobacterium rhizogenes strain of step 1) comprises K599, MSU440, C58C1 or ArA 4.

Preferably, OD of Agrobacterium rhizogenes suspension in step 1)₆₀₀The value is 0.2 to 0.6.

Preferably, the economic plants in step 2) include herbaceous plants, shrubs and arbors.

Preferably, the economic plants in step 2) include pigeon pea, safflower, cassia seed, isatis root, abelmoschus manihot, okra, castor bean, caragana and agilawood.

Preferably, the culturing method before and after injection in the step 2) is to perform tissue culture on economic plant seeds for 2-5 weeks, directly inject the obtained tissue culture seedlings, and then plant the seedlings in a soil medium.

Preferably, the culturing method before and after injection in the step 2) is to inject the economic plant seeds when the economic plant seeds grow in the soil medium for 3-8 weeks, and then continue to grow in the soil medium.

Preferably, the injection position in the step 2) is located at a stem part of 0-1.5 cm above the original hairy root system of the seedling.

Preferably, the position of the injection in the step 2) is a stem part which is 0.1-1.0 cm above the original hairy root of the seedling.

Preferably, the vector of the target recombinant vector in step 1) includes pROK 2; the target gene in the target recombinant vector comprises GFP.

The invention provides a construction method of an economic plant effective root system transgenic system, which comprises the following steps: 1) carrying out solid-liquid separation on the bacterial liquid containing the target recombinant vector agrobacterium rhizogenes to obtain heavy suspension of the bacterial body containing the target recombinant vector agrobacterium rhizogenes, so as to obtain agrobacterium rhizogenes suspension; 2) injecting the agrobacterium rhizogenes suspension to the stem of the plant seedling, and growing callus at the wound of the stem of the plant seedling 10-12 days after injection; 3) culturing the plant seedlings with the callus for 28-35 d, and cutting off the original hairy root system when the callus is differentiated into a regeneration hairy root. Experiments show that the regeneration rate of the hairy roots is 15-45% by adopting the construction method provided by the invention. Meanwhile, the method constructed by the invention has higher callus regeneration rate, specifically 16-85%.

Meanwhile, the construction method provided by the invention has the characteristic of wide application range, and particularly nine kinds of selected economic plants in medicine. The phenotypes of four of the nine plants showed that they had higher root regeneration rates. The reverse transcription polymerase chain reaction result and GFP signal prove that the target Gene (GFP) is expressed in the transgenic hairy root, and the target gene is transferred into the hairy root. All results indicate that the hairy root method has wide application, including herbaceous and woody plants.

Further, the present invention further defines the species of Agrobacterium rhizogenes strain, and Agrobacterium rhizogenes strain K599, MSU440, C58C1 and Ar4 were selected to determine the transformation efficiency of seedlings, and the results showed that Agrobacterium rhizogenes strain K599 showed high efficiency in both callus induction (80%) and hairy root induction (30%). The transformation efficiencies of the other three strains were relatively low compared to K599. For calli, the induction efficiency of strain C58C1 was 15%, followed by strain MSU440 (10%) and ArA4 (8%). For hairy roots, the induction efficiency of the three strains is 5-8%. This suggests that K599 induction should be more efficient in hairy root transgenic systems and may be more appropriate for pigeon pea.

Furthermore, the invention further defines the concentration of the agrobacterium tumefaciens suspension, and researches the influence of the injection concentration on the callus induction rate, the hairy root induction rate and the transgenic rate. Injection concentrations were 0.2,0.3,0.4,0.5 and 0.6OD₆₀₀Values were selected for testing. The results showed that OD2(OD value 0.3) and OD3(OD value 0.4) were the best values to obtain high callus induction rate (OD2: 58% + -2%; OD3: 60% + -4%). OD2 was also the best value (33% + -4%) to obtain the best regeneration rate of hairy roots, followed by OD3 (26% + -4%). In addition, transgenic roots obtained by injecting agrobacterium solution with different concentrations were not different. In combination, the optimal callus and hairy root induction concentration is 0.3 OD.

Furthermore, the injection position is further limited, the stem part is injected at the position of 0-1.0 cm above the original hairy root system of the seedling, the callus and the hairy root are about 60%, and relatively 1.0-1.5 cm has higher efficiency.

Further, the invention further defines the injection age of the tissue culture seedlings. After 15 days, 30 days, the seedlings cultured in the tissue culture flasks were used for injection and then transferred to the soil environment. The results showed that the incidence of infestation was higher for the seedlings (15 and 30 days) than for the old seedlings (45 days).

Drawings

FIG. 1 is a diagram of a pigeon pea hairy root transgene system; wherein FIG. 1(a) Agrobacterium rhizogenes (Agrobacterium rhizogenes) solution containing recombinant vector is injected into the stem of one month old passaged pigeon pea seed; FIG. 1(b) to FIG. 1(e) process for callus and hairy root regeneration; on the left is a schematic diagram of each stage and on the right is a picture of this stage; FIG. 1(f) shows that primitive roots can be cut after transgenic hairy roots grow well;

FIG. 2 shows the OD value of the selected bacterial liquid and the identification of the transgenic line; OD1 to OD5 represent OD values of 0.2,0.3,0.4,0.5 and 0.6, respectively; wherein FIG. 2(a) is the regeneration rate of callus after injection of Agrobacterium K599 of different OD values; FIG. 2(b) shows the regeneration rate of hairy roots in Agrobacterium solutions of different OD values; FIG. 2(c) is the transgene rate for regenerating hairy roots at different OD values; FIGS. 2(d) -2 (e) are photographs of the primitive root of a transgenic hairy root; FIG. 2(g) is a photograph showing the stem regenerated callus after pigeon pea injection; FIGS. 2(f) to 2(h) show transgenic hairy roots after cutting of the original root; the scale bar of FIGS. 2(d) to 2(h) is 1 cm; FIG. 2(i) is a RT-PCR analysis of the transgenic rate of hairy roots; FIG. 2(j) shows GFP signals in transgenic hairy roots, Control shows Agrobacterium injection Control, and the scale bar in FIG. 2(j) is 50 μm;

FIG. 3 is an optimum injection condition test for the seedling stage and the injection site, wherein FIG. 3(a) is a picture of pigeon pea sown for fifteen days; from the bottom of the stem, three sites were selected for injection, designated as sites C to a. each site was about 0.5cm in width; FIG. 3(b) is the regeneration rate of callus and hairy root injected at different sites; FIG. 3(c) phenotype of different seedling ages of sown seeds; FIG. 3(d) regeneration rates of callus and hairy roots after using seeds of different seedling ages; wherein the scale bar in fig. 3(a) and 3(c) is 0.5 cm;

FIG. 4 is a hairy root transgenic approach used in four typical economic plants; 4(a) -4 (d) four selected economic plants with higher regeneration rates from 9 economic plants showing hairy root phenotype of Abelmoschus manihot, Abelmoschus esculentus, Isatis root, Cajanus cajan, respectively; RT-PCR and Western blot analysis of each transgenic hairy root line are listed at the right hand site (T1 and T2 represent two transgenic lines) on a scale of 1 cm; FIG. 4(e) regeneration rates of calli and hairy roots in four plants;

FIG. 5 is a flow chart of a method for transgenic herbaceous plants, shrubs and tree hairy roots; type I, seedlings were first cultured in MS medium and then removed from the flask for injection. And II, taking out the culture flask before injection, and planting subculture seedlings in a soil culture medium for about 3-5 days. Type III, plant seeds are planted directly in the soil before injection; the text box shows the range of possible applications for the hairy root transgene system.

Detailed Description

The method comprises the steps of carrying out solid-liquid separation on a bacterial liquid containing the target recombinant vector agrobacterium rhizogenes to obtain a thallus containing the target recombinant vector agrobacterium rhizogenes for heavy suspension, and obtaining an agrobacterium rhizogenes suspension.

The strain of Agrobacterium rhizogenes of the present invention is not particularly limited, and any Agrobacterium rhizogenes strain known in the art may be used. The strain of agrobacterium rhizogenes preferably comprises K599, MSU440, C58C1 or ArA4, more preferably K599. Agrobacterium rhizogenes strains K599, MSU440, C58C1 and ArA4 were all purchased from Shanghai Diego Biotechnology Ltd.

In the present invention, the vector in the objective recombinant vector preferably includes pROK 2. The pROK2 vector was purchased from Biovector plasmid vector strain cell Gene Collection. The invention does not specially limit the types of target genes in the target recombinant vector, and any target gene can be adopted. In order to illustrate the successful establishment of an effective root system transgenic system, in the embodiment of the invention, the target gene comprises GFP, the GFP gene is transferred to express the target protein in the system, and the successful transformation of the target gene is detected by detecting a luminescent signal of a fluorescent protein in the system. The preparation method of the agrobacterium rhizogenes containing the target recombinant vector is preferably carried out by adopting an electric shock transformation method.

In the invention, the bacterial liquid containing the target recombinant vector agrobacterium rhizogenes is obtained by carrying out shake culture on the target recombinant vector agrobacterium rhizogenes in a liquid culture medium at 180rpm and 28 ℃ for 12-14 h. The liquid culture medium is YEP liquid culture medium containing 20mg/L rifampicin and 50mg/L kanamycin. Culturing to OD of bacterial liquid₆₀₀Ending the culture when the value is 0.2-0.6. The solid-liquid separation is preferably centrifugation. The rotation speed of the centrifugation is preferably 8,000 rpm. The time for the centrifugation is preferably 10 min. The supernatant was collected and the cells were resuspended in the same volume of buffer as the supernatant. The buffer is preferably MES buffer; the MES buffer comprises the following components in percentage by weight: 10mmol/L MES-KOH, 10mmol/L MgCl₂And 100. mu. mol/L acetosyringone aqueous solution with pH 5.2. OD of Agrobacterium rhizogenes suspension₆₀₀The value is preferably 0.2 to 0.6, more preferably 0.3 to 0.5.

After the agrobacterium rhizogenes suspension is obtained, the agrobacterium rhizogenes suspension is injected to the stem of the economic plant seedling, and callus grows at the wound of the stem of the economic plant seedling 10-12 days after injection.

In the present invention, the economic plants preferably include herbaceous plants, shrub plants and arbor plants. In the invention, the economic plants comprise nine selected economic plants, more preferably pigeon pea, safflower, cassia seed, isatis root, abelmoschus manihot, okra, castor-oil plant, caragana and agilawood. All nine economic plants were successfully induced to produce callus. In addition to cassia seed, castor bean and aquilaria sinensis, all six of them can successfully induce hairy roots by the method with only slight modification.

In the invention, in order to optimize the influence of seedling culture conditions on callus induction and hairy root regeneration rate, two seedling culture methods are provided: the first method is as follows: the method comprises the steps of carrying out tissue culture on economic plant seeds for 2-5 weeks, directly injecting obtained tissue culture seedlings, and then planting the seedlings in a soil medium. The culture medium for tissue culture is preferably MS culture medium. The economic plant seeds are preferably sterilized prior to tissue culture. The method of sterilization in the present invention is not particularly limited, and a sterilization method known in the art may be used. The temperature of the tissue culture is preferably 24-26 ℃, and more preferably 25 ℃. The test period of the seedling raising method for the abelmoschus manihot and the pigeon pea is shorter, and the induction rate of the hairy roots is higher than that of the two other methods.

The second method is that plant seeds are injected when the plant seeds grow in a soil medium for 3-8 weeks, and then the plant seeds continue to grow in the soil medium, wherein the soil medium is a mixture of soil and sand, the volume ratio of the soil to the sand in the mixture of the soil and the sand is preferably 3:1, the particle size of the sand is preferably 0.35-0.5 mm, the soil medium is preferably filled in a pot with the diameter of 10cm × and the height of 9cm, the temperature of the growth is preferably 25 ℃, the humidity of the growth environment is preferably 85-90%, and the illumination intensity during the growth period is preferably 50 mu mol photon m^-2s^-1. The photoperiod during the growth was 16 h. The second method results in lower hairy root induction than the first method, but involves high survival rates.

In the invention, the injection position is preferably located at the stem position of 0-1.5 cm above the original hairy root system of the seedling, and is particularly divided into three regions, wherein the height of each region is 0.5cm, for example, the stem position of (0-0.5) cm above the original hairy root system of the seedling is a C region, and the stem position of [ 0.5-1.0) cm above the original hairy root system of the seedling is a B region; the 1.0-1.5 cm stem position on the original hairy root system of the seedling is an A region, and the B region and the C region are more preferable.

After callus induction, the economic plant seedlings with the callus are cultured for 28-35 d, and the original hairy root system is cut off when the callus is differentiated into regenerated hairy roots.

In the present invention, callus gradually increases and becomes dark over time. After about three weeks, small hairy roots, similar to adventitious roots, grew out of the calli. The hairy roots are increased after 1-2 months, which is enough to support the nutrition of the whole plant and is beneficial to the development of the plant.

In the invention, the RT-PCR method and the fluorescence microscope observation are adopted to test the transgenic rate and detect whether the target gene in the regenerated hairy root is expressed in the hairy root. After confirmation of transgenic hairy roots, the original hairy root line can be cut and the transgenic regenerated hairy roots can function to drive the root system (FIG. 1 (f)). The root system transgenic system constructed by the invention is very suitable for quick and effective gene system function analysis, secondary metabolite engineering and plant stress response research. In this system, only the roots are transgenic, not the entire plant. This is also a good system to study signal transduction between roots and stems.

The following examples are provided to illustrate the construction of an economic plant root system transgene system provided by the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

Method for constructing effective root system transgenic system by taking economic plant green soy bean as example

The pigeon pea seeds come from the key laboratory of forest plant ecology education department of northeast forestry university, the surfaces of the pigeon pea seeds are sterilized by 0.1% mercuric chloride for 5-10 min, and then washed five times by sterile water, the sterilized pigeon pea seeds are inoculated in MS culture medium for about 1-2 months, for pigeon pea, the bacteria liquid can be injected into subculture plants after about 30 days of growth, for other plants, the pigeon pea seeds are moved to soil (10 cm (diameter) × 9cm (height) pot, soil and sand in a volume ratio of 3: 1) and grow in a high humidity environment at 25 ℃, and before injection experiments are carried out, the pigeon pea seeds are in a 16-hour light period and grow in a 50 mu mol photon m photon m environment^-2·s^-1Emits light. To select an agrobacterium rhizogenes (a. rhizogenes) strain with higher transformation efficiency in cajan, agrobacterium rhizogenes strains K599, MSU440, C58C1 and Ar4 were selected to determine the transformation efficiency of seedlings. Agrobacterium solution (OD) of 4 recombinant plasmids (pROK2-GFP) containing GFP gene₆₀₀0.3) into 15-day-old young pigeon pea, with hairy roots growing. 4 kinds of Agrobacterium solution containing pROK2 empty vectorThe regeneration rates of the four agrobacterium rhizogenes injection pigeon pea root transgenic systems are counted, wherein the callus induction rate is × 100% of the number of induced callus seedlings/the number of total seedlings, the root regeneration rate is × 100% of the number of rooted seedlings/the number of total seedlings, and the results are shown in table 1.

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

As shown in table 1, the rooting strain K599 showed high efficiency in both callus (80%) and root induction (30%). The transformation efficiencies of the other three strains were relatively low compared to K599. For calli, the induction efficiency of strain C58C1 was only 15%, followed by strain MSU440 (10%) and Ar a4 (8%). For hairy roots, the induction efficiency of the three strains is only 5-8%. The results indicate that K599 induction should be more efficient in hairy root transgenic systems and may be more suitable for pigeon pea.

Induction aspects of transgenic hairy roots of pigeon pea

The invention establishes an effective pigeon pea seedling agrobacterium injection system. As shown in FIG. 1, the constructed vector was transformed into Agrobacterium rhizogenes and then injected into 15-day-old Cajanus seedlings (FIG. 1 (a)). After approximately 7-14 days of growth, callus appeared around the injection site (FIGS. 1(b) and 1 (c)). Over time, the callus gradually increased and became darker. After about three weeks, small hairy roots, similar to adventitious roots, grew out of the calli (FIG. 1 (d)). The hairy roots increased after one to two months enough to support the nutrition of the whole plant and contribute to the development of the plant (fig. 1 (e)).

The present invention tests whether the target gene was successfully introduced into these hairy roots by RT-PCR or using Western blotting. The transgenic root RNA, DNA extraction and PCR verification analysis method specifically comprises the following steps:

RNA inducing re-rooting was extracted using the CTAB method (Meng et al, 2018). Quantification of RNA was performed by a NanoDrop spectrophotometer (NanoDrop Technologies, Inc). After treatment with RNase-free DNase I, 1. mu.g of total RNA was reverse transcribed into cDNA using Invitrogen TM SuperScript TM (Invitrogen, USA). The reaction system for RT-PCR is shown in Table 2 and the amplification procedure is shown in Table 3. The primer sequences for RT-PCR are listed in Table 4.

TABLE 2 reaction System for RT-PCR

TABLE 3 amplification conditions for RT-PCR

TABLE 4 primer sequences for RT-PCR

After confirming the transgenic hairy root, the original root was cut, and the transgenic hairy root can function to drive the root system (fig. 1 (f)). It is a very fast and effective gene system function analysis, secondary metabolite engineering and plant stress response research. In this system, only the roots are transgenic, not the entire plant. The construction method can provide a good system for researching signal transduction between roots and stems.

Example 2

The influence of the injection concentration of the agrobacterium solution on the callus induction rate, the hairy root induction rate and the transgenic rate is tested. The K599 strain was used as the infecting strain, and concentrations of 0.2,0.3,0.4,0.5 and 0.6OD were set for selection of the optimum concentration of the suspension of the K599 strain₆₀₀Value, using pigeon pea as the target, the hairy root regeneration was performed and calculated according to the method of example 1And (4) rate.

RT-PCR (same operation method as example 1) and an experiment for detecting GFP signals are adopted to verify the transfer condition of the target gene. The experiments for detecting GFP signals are as follows: taking the pigeonpea regenerated hairy root protein, transferring a protein sample separated by PAGE (polyacrylamide gel electrophoresis) to a solid phase carrier (NC membrane), adding rabbit anti-GFP serum to perform immunoreaction, then reacting with horseradish peroxidase, detecting the expression condition of GFP protein, and setting a blank control group.

OD1 to OD5 represent OD values of 0.2,0.3,0.4,0.5 and 0.6, respectively. The results showed that OD2(OD value 0.3) and OD3(OD value 0.4) were the best values to obtain high callus induction rate (OD2: 58% + -2%; OD3: 60% + -4%) (FIG. 2 (a)). OD2 was also the best value to obtain the best regeneration rate of hairy roots (33% ± 4%), followed by OD3 (26% ± 4%) (fig. 2 (b)). In addition, transgenic roots obtained by injection of different concentrations of agrobacterium solution were not different (fig. 2 (c)). In conclusion, the optimal callus and hairy root induction concentration is OD 0.3. The phenotypes of the transgenic hairy roots and the pigeon pea primitive roots before and after the cutting are shown in FIGS. 2(d) to 2 (h). The results of RT-PCR (same procedure as in example 1) and GFP signal confirmed that the target gene had been transferred to the hairy roots (FIGS. 2(i) and 2 (j)).

Example 3

The optimal injection site of the pigeon pea seedlings plants was tested separately.

Firstly, dividing a stem part which is 0.1-1.0 cm above an original hairy root of a seedling into three parts with the height of 0.5 cm; are named positions A, B and C, respectively (fig. 3 (a)). The K599 strain was used as an infecting strain and the method of example 1 was followed.

The results show that: the induction rate of the callus and the hairy root at the B and C positions was about 60%, and the efficiency was higher than that at the A position (FIG. 3 (B)).

Example 4

The optimum age of the young plants of Cajanus cajan was tested separately

Three different ages of pigeon pea seedlings were used to select the optimal injection age of the seedlings, 15 days, 30 days and 45 days after tissue culture, respectively, and the inoculation site was zone B, and the other operations were performed according to the method example in example 3. As shown in FIG. 3(c), after 15 days, 30 days and 45 days, respectively, seedlings cultured in tissue culture flasks were used for injection and then transferred to the soil environment. The results showed that the infection rate of the seedlings (15 days and 30 days) was higher than that of the old seedlings (45 days) (see fig. 3 (d)).

Example 5

Applicability of hairy root transgenic method to other economic plants

To extend this approach, other economic plants, particularly the nine selected economic plants of medicine, including cassia, okra, abelmoschus manihot, and pigeon pea, were selected to test and optimize this root transgene system, and the specific construction of each economic plant is shown in table 5. The hairy root regeneration rate was calculated using the method in example 1.

And verifying whether the target Gene (GFP) is expressed in the transgenic hairy roots by adopting a western blot detection experiment. The specific method comprises the following steps:

extracting proteins of regenerated hairy roots of each economic plant, transferring a protein sample separated by PAGE (polyacrylamide gel electrophoresis) to a solid phase carrier (NC membrane), taking the economic plant proteins as antigens, adding rabbit anti-GFP serum to perform immunoreaction, reacting with horseradish peroxidase secondary antibody, and detecting the expression condition of GFP proteins.

TABLE 59 Uniform look-up table for constructing transgenic root system by economic plants

Note: none indicates that hairy roots are not induced.

As shown in Table 5, all nine plants successfully induced callus. In addition to cassia seed, castor bean and aquilaria sinensis, all six of them can successfully induce hairy roots by the method with only slight modification. Meanwhile, the regeneration rate of the long hair roots is between 15 and 45 percent, and the regeneration rate of the long hair roots relative to the callus is between 16 and 85 percent. The phenotypes of four of the nine plants showed that they had higher root regeneration rates, as shown in fig. 4. Reverse transcription polymerase chain reaction and western blot results confirmed that the gene of interest (GFP) was expressed in transgenic hairy roots. All results indicate that the hairy root method has wide application, including herbaceous and woody plants.

Example 6

In order to obtain an accurate method (herbaceous plants, shrubs or trees) for different kinds of plants, the effect of the conditions of seedling raising was tested. Three culture conditions were evaluated as follows: type I, tissue culture seedlings are directly injected in 2-5 weeks and then planted in a soil medium. And II, planting the seedlings in a soil culture medium after 2-5 weeks of tissue culture. After about 3 to 5 days, growth is restored, and then injection is performed. Type III, seeds are directly planted in a soil culture medium and then injected after 3-8 weeks.

As shown in fig. 5, a flowchart and possible applications of hairy root induction are summarized. The above method was verified for two typical plants, Abelmoschus manihot, Cajanus cajan.

Table 6 shows that three seedling raising methods are used for verifying the conditions of Abelmoschus manihot and pigeon pea

The result shows that the test period of the Abelmoschus manihot and the pigeon pea by the type I method is shorter, and the induction rate of the hairy root is higher than that of the other two methods. However, the survival rate of type I is low. When the type III method is used, hairy root induction rate is lower than that of type I, but has high survival rate. The type III method is best if a simpler and faster method is desired in pigeon pea and Abelmoschus manihot.

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.

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Claims

1. A construction method of an economic plant effective root system transgenic system is characterized by comprising the following steps:

the injection position is located at a stem part of 0.1-1.5 cm above the original hairy root system of the seedling;

the culture method before and after injection comprises the steps of carrying out tissue culture on economic plant seeds for 15-30 d, directly injecting the obtained tissue culture seedlings, and then planting the seedlings in a soil medium;

3) culturing economic plant seedlings with callus for 28-35 days, and cutting off an original hairy root system when the callus is differentiated into a regenerated hairy root;

the economic plants are cajan, safflower, isatis root, abelmoschus manihot, okra and caragana.

2. The method of claim 1, wherein the Agrobacterium rhizogenes strain of step 1) comprises K599, MSU440, C58C1 or ArA 4.

3. The method for constructing a microorganism according to claim 1, wherein the OD of the Agrobacterium rhizogenes suspension in the step 1) is₆₀₀The value is 0.2 to 0.6.

4. The method of claim 1, wherein the injection in step 2) is at a stem 1.0cm above the original hairy root of the seedling.

5. The method for constructing according to claim 1, wherein the vector of the objective recombinant vector in step 1) includes pROK 2; the target gene in the target recombinant vector comprises GFP.