Method for inducing hairy roots of sesbania cannabina

Abstract

The invention provides an induction method of sesbania hairy roots, which comprises the following steps: beveling hypocotyl of a sesbania seedling, dipping recombinant agrobacterium rhizogenes in the oblique incision of the seedling on the ground, inserting the seedling into vermiculite, and culturing until a new root system is generated, namely the new root system is a sesbania transgenic root system. The method is simple to operate, low in cost and short in time consumption, avoids tedious sterile operation, provides important technical support for molecular biology research of sesbania, and has a relatively great application prospect.

Description

Technical Field

[0001] The invention relates to a plant biological induction method, in particular to a sesbania hairy root induction method. Background Art

[0002] Sesbania tian Sesbania cannabina ) is a plant of the genus Sesbania, Fabaceae, Fabaceae subfamily. It has excellent characteristics such as waterlogging resistance, salt-alkali resistance and barrenness resistance. It has high yield and is rich in protein. Sesbania can be used as high-quality protein forage to feed livestock, and can also be used as a green manure crop. It is an excellent pioneer plant for improving saline-alkali land.

[0003] Agrobacterium rhizogenes Agrobacterium rhizogenes ) is a Gram-negative bacterium widely present in the soil. It can induce the production of hairy roots by introducing the rol gene in the Ri plasmid into plant cells and stably integrating it into the plant genome, and integrate the exogenous genes carried in T-DNA into the plant genome, thereby achieving transgenic.

[0004] At present, the lack of perfect transgenic technology for Sesbania sesbania restricts the research on its gene function. Therefore, the establishment of a method for inducing hairy roots in Sesbania sesbania is of great significance for the research on Sesbania sesbania, but there is no report on the induction of hairy roots in Sesbania sesbania. Summary of the invention

[0005] The present invention aims to provide a method for inducing hairy roots of sesbania serrata, which is simple to operate, short in time and high in efficiency. The present invention utilizes recombinant Agrobacterium rhizogenes carrying three key genes CYP76AD1 (nucleotide sequence as shown in SEQ ID NO: 1), DODA (nucleotide sequence as shown in SEQ ID NO: 2) and glucosyltransferase (nucleotide sequence as shown in SEQ ID NO: 3) for betalain biosynthesis and a novel reporter system (RUBY), and successfully establishes a method for inducing hairy roots of Sesbania sesbania by infecting the hypocotyls of Sesbania sesbania seedlings and then culturing them.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A method for inducing hairy roots of Sesbania sesbania, the method comprising the following steps: S1 transfers the binary vector carrying RUBY into Agrobacterium rhizogenes and cultures the Agrobacterium rhizogenes plate for later use; S2 Take the Sesbania sesbania seedlings, cut them obliquely at the hypocotyl of the Sesbania sesbania seedlings, and keep the aerial part for later use; S3 takes the aerial part of the Sesbania sesbania seedlings retained in S2, and uses the oblique cut edge to dip the recombinant Agrobacterium rhizogenes carrying the target plasmid cultured in S1; S4 inserts the sesbania seedlings into vermiculite to complete the process of recombinant Agrobacterium rhizogenes infecting the sesbania seedlings; S5 cultivates the Sesbania sesbania seedlings that were successfully infected in S4 until they grow new roots, which are the transgenic roots.

[0007] Furthermore, the Agrobacterium rhizogenes described in step S1 is Agrobacterium rhizogenes K599.

[0008] Furthermore, the Sesbania sesbania seedlings described in step S2 are seedlings that are 12 to 15 days old, 4 to 5 cm in height, and have one compound leaf.

[0009] Furthermore, in step S2, when the hypocotyls of the sesbania seedlings are cut obliquely, the hypocotyls are cut obliquely at 0.5 cm above the aerial part.

[0010] Further, in step S4, the process is performed in a moisturizing environment.

[0011] Furthermore, the successfully infected Sesbania seedlings in step S5 are covered with a tray lid and then placed in a culture greenhouse for culture at a temperature of 28° C. and 16 h light / 8 h dark.

[0012] The present invention has the following beneficial effects: The present invention provides a method for inducing hairy roots of Sesbania sesbania for the first time. The method of the present invention is simple to operate, low in cost, short in time and avoids tedious aseptic operation, thus providing important technical support for the molecular biology research of Sesbania sesbania. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The present invention is a flowchart of the operation process of the method for inducing hairy roots of Sesbania sesbania; wherein, Figure A is a Sesbania sesbania seedling; Figure BC is an oblique cut of the hypocotyl of the Sesbania sesbania seedling; Figure D is a plate of recombinant Agrobacterium rhizogenes; Figure EF is an oblique cut of the hypocotyl of the Sesbania sesbania seedling dipped in Agrobacterium rhizogenes; Figure G is a Sesbania sesbania seedling that has been infected and cultured in a moisturizing environment; and Figure H is a Sesbania seedling with induced hairy roots.

[0014] Figure 2 The induction efficiency of different rhizogenes Agrobacterium on Sesbania sesbania hairy roots. DETAILED DESCRIPTION

[0015] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0016] Example 1: A method for inducing hairy roots of Sesbania sesbania 1. Cultivation of Sesbania tianxia After the sesbania seeds were peeled, they were sown into the nutrient soil matrix and placed under normal light conditions for cultivation. The cultivation conditions were: the temperature was 28°C during the day and night, and the light was 16 hours per day.

[0017] 2. Transformation and cultivation of Agrobacterium rhizogenes Add the binary vector plasmid with RUBY reporter to K599 rhizogenes competent cells, mix well, stand on ice for 5 min, freeze in liquid nitrogen for 5 min, place in a 37℃ water bath for 5 min, place in an ice bath for 5 min, add 800 µL LB antibiotic-free liquid medium, culture at 220 rpm in a 28℃ constant temperature shaker for 60 min, take 50 µL and spread it on LB solid medium containing antibiotics, culture at 28℃ inverted for 2 d, pick a single colony and add it to 5 mL LB liquid medium containing antibiotics, culture at 220 rpm in a 28℃ constant temperature shaker for 12 h. Use PCR technology to detect RUBY gene to screen positive clones. Pipette 200 µL positive clone bacterial solution and add it to 10 mL LB liquid medium containing antibiotics, culture rhizogenes in a constant temperature shaker until OD600 is 0.8~1, take 0.2 m bacterial solution and evenly spread it on LB solid medium containing antibiotics, culture at 28℃ inverted for use.

[0018] 3. Induction of Sesbania hairy roots Normal growth of 12-15 days, 4-5 cm in height, and a single compound leaf of Sesbania sesbania seedlings ( Figure 1 A) Use a surgical blade to cut the hypocotyl obliquely at 0.5 cm from the soil. Figure 1 B, C). Take the aboveground part, and dip the hypocotyl section of the aboveground part into the prepared Agrobacterium rhizogenes (i.e., take 0.2 mL of bacterial solution with OD600 of 0.8-1 and evenly spread it on the Agrobacterium rhizogenes plate obtained by LB solid medium containing antibiotics) ( Figure 1 E, F), then insert the sesbania seedlings into the sterilized vermiculite and cover the tray with a tray cover to maintain humidity ( Figure 1 G). Transfer to the cultivation greenhouse for cultivation ( Figure 1 H), the culture conditions were: 16 h light / 8 h dark, 28°C. The newly grown roots after 15 days of culture were the transgenic roots.

[0019] Example 2: Effects of different Agrobacterium rhizogenes strains on the induction efficiency of Sesbania sesbania hairy roots The 15-day-old Sesbania seedlings were used as materials, and the infection site was the hypocotyl 0.5 cm above the ground. Four strains of Agrobacterium rhizogenes (Ar1193, C58C1, K599, and MSU440) were used to induce hairy roots in Sesbania. The effects of different strains of Agrobacterium rhizogenes on the induction efficiency of Sesbania hairy roots were compared. Figure 2As shown, the induction efficiency was over 75% when K599 was used, the induction efficiency of MSU440 was 43.33%, while the induction efficiencies of Ar1193 and C58C1 were less than 40%.

[0020] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for inducing hairy roots of Sesbania sesbania, characterized in that: The method comprises the following steps: S1 transfers the binary vector carrying RUBY into Agrobacterium rhizogenes and cultures the Agrobacterium rhizogenes plate for later use; S2 Take the Sesbania sesbania seedlings, cut them obliquely at the hypocotyl of the Sesbania sesbania seedlings, and keep the aerial part for later use; S3 takes the aerial part of the Sesbania sesbania seedlings retained in S2, and uses the oblique cut edge to dip the recombinant Agrobacterium rhizogenes carrying the target plasmid cultured in S1; S4 inserts the sesbania seedlings into vermiculite to complete the process of recombinant Agrobacterium rhizogenes infecting the sesbania seedlings; S5 cultivates the Sesbania sesbania seedlings that were successfully infected in S4 until they grow new roots, which are the transgenic roots.

2. The method according to claim 1, characterized in that: The Agrobacterium rhizogenes described in step S1 is Agrobacterium rhizogenes K599.

3. The method according to claim 1, characterized in that: The sesbania seedlings described in step S2 are seedlings that are 12 to 15 days old, 4 to 5 cm in height, and have one compound leaf.

4. The method according to claim 1, characterized in that: In step S2, when the hypocotyl of the sesbania seedling is cut obliquely, the hypocotyl is cut obliquely at 0.5 cm above the aerial part.

5. The method according to claim 1, characterized in that: In step S4, the process is performed in a moisturizing environment.

6. The method according to claim 1, characterized in that: The successfully infected Sesbania seedlings in step S5 are covered with a tray lid and then placed in a culture greenhouse for culture at a temperature of 28° C. and 16 h light / 8 h dark.

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