JP7607903B2 - Method for inducing multiple shoots into which a target gene of quinoa has been introduced and multiple shoots obtained by said induction method - Google Patents

Description

The present invention relates to a method for inducing multiple shoots into which a target gene of quinoa has been introduced, and to multiple shoots obtained by said induction method.

Quinoa is a grain (or more accurately, a pseudocereal) of the Chenopodiaceae family that has attracted worldwide attention in recent years due to its exceptional resistance to environmental stress and excellent nutritional properties. For example, quinoa can be grown in salt water that is about half the salt of seawater, and because of its high nutritional value, the National Academy of Sciences selected quinoa as a promising low-resource crop for the future (Non-Patent Document 1), and NASA has also focused on quinoa as a food source for astronauts (Non-Patent Document 2).

Genome editing is a technology that will become an effective breeding method within a few years, and currently, the creation of regulations regarding its use is progressing at a rapid pace. Crops created through genome editing are expected to be approved for cultivation and sale in Japan, and because it is possible to change targeted DNA sequences in the genome, it will be easy to accumulate mutant genes, which was previously difficult. Genome editing uses transformation techniques such as Agrobacterium. For this reason, callus formation and regeneration of plants are often necessary during transformation. Callus formation and regeneration systems have been reported for quinoa to date, but the previously reported callus formation and regeneration systems have poor reproducibility. In addition, the two steps of callus formation and regeneration are time-consuming and costly.

There have been no reports of quinoa transformation to date. In addition, the two steps of callus formation and regeneration require high time and financial costs.

Non-patent literature 3 reports on the callus formation and regeneration system of quinoa.

Non-Patent Document 4 reports on the induction of multiple shoots in quinoa, but does not mention transformation, and it takes as long as four weeks after sowing for multiple shoots to be induced.
However, none of the documents discloses or suggests the construction of the method of the present invention.

National Academy of Sciences (1975) Quinoa.In:Underexploited tropical plants with economic value. Washington DC, pp 20-23 Schlick G, Bubenheim, DL (1993) Quinoa: anemerging“new” crop with potential for celss. NASA Technical Paper 3422:1-7 M. Hesami and M.H. Daneshvar (2016)Development of a regenerationprotocol through indirect organogenesis inChenopodium quinoa willd. Indo-Am.J. Agric. Vet. Sci. 4:25-32. M. Hesami et al. (2018) Optimizing sterilization conditions and growth regulator effects on in vitro shootregeneration through directorganogenesis in Chenopodium quinoa. Biotechnologia99:49-57.

Plant transformation often requires callus formation and regeneration, but in quinoa, shoot regeneration from callus formation is difficult and poorly reproducible. Therefore, the objective of the present invention is to provide a novel method for transforming quinoa that does not require callus formation.

As a result of intensive research to solve the above problems, the inventors came up with the idea of producing multiple shoots from the part of the cotyledon that contains the cotyledonary node, from the tip to the hypocotyl, without going through callus formation, and constructed a method for transforming quinoa that does not require the callus formation/regeneration system, thus completing the present invention.

That is, the present invention is as follows.
1. A method for producing adventitious shoots, adventitious shoots and/or multiple shoots of quinoa into which a target gene has been introduced, comprising the steps of:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest; and (2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds.
2. The method according to item 1 above, wherein the step (1) includes ultrasonic treatment and/or reduced pressure treatment.
3. The method according to item 1 or 2 above, wherein the step (1) comprises a vacuum treatment subsequent to the ultrasonic treatment.
4. The method according to any one of items 1 to 3 above, wherein the culturing in the step (2) includes a selection step.
5. The method according to any one of claims 1 to 4, wherein the quinoa is US National Plant Germplasm System Accession No. PI 614882 or PI 614883.
6. The method according to any one of the preceding items 1 to 5, which does not include a callus formation step and/or a regeneration step.
7. An adventitious shoot, adventitious shoot and/or multiple shoot into which a gene of interest has been introduced, produced by the method according to any one of the preceding paragraphs 1 to 6.
8. A method for producing an adult quinoa plant into which a gene of interest has been introduced, comprising the steps of:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest;
(2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds; and (3) culturing or growing the adventitious buds, adventitious buds and/or multiple buds to obtain adults. Or,
(1) A step of culturing or growing adventitious buds, adventitious buds and/or multiple buds obtained by the production method according to any one of the preceding paragraphs 1 to 6 to obtain adults;
Or,
(1) A step of culturing or growing the adventitious buds, adventitious buds and/or multiple buds according to the preceding paragraph 7 to obtain adults;
The method includes:
9. An adult animal into which a gene of interest has been introduced, the adult animal being produced by the method described in the above item 8.
10. A method for producing seeds of quinoa into which a target gene has been introduced, comprising the steps of:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest;
(2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds;
(3) culturing or growing the adventitious buds, adventitious buds and/or multiple buds to obtain adults; and
(4) growing the adults until the seeds can be harvested to obtain seeds;
Or,
(1) A step of culturing or growing adventitious buds, adventitious buds and/or multiple buds obtained by the production method according to any one of the preceding paragraphs 1 to 6 to obtain adults; and
(2) growing the adventitious buds, adventitious buds and/or multiple buds until the seeds can be harvested to obtain seeds; or
(1) A step of culturing or growing the adventitious buds, adventitious buds and/or multiple buds described in the preceding paragraph 7 to obtain adults; and
(2) growing the adult plant until the seeds can be harvested to obtain the seeds; or
(1) A step of growing the adult obtained by the production method described in the preceding paragraph 8 or the adult described in the preceding paragraph 9 until the seeds can be harvested to obtain seeds;
The method includes:
11. The method according to any one of items 1 to 5 above, comprising the steps of:
(1) placing the explants cultured on a multiple shoot induction medium in an infection solution, subjecting them to ultrasonic treatment and decompression treatment, and then culturing them on a coculture medium;
(2) culturing the culture in a sterilized medium after the culture in step (1);
(3) culturing the culture in step (2) on a multiple shoot induction medium;
(4) after the culture in step (3), culturing in a selection medium; and (5) after the culture in step (4), culturing in a post-selection shoot elongation medium to obtain adventitious buds, adventitious buds and/or multiple shoots, or culturing in a post-selection shoot elongation medium and further growing in a basal medium or MS medium to obtain adventitious buds, adventitious buds and/or multiple shoots. 12. A method for producing adventitious buds, adventitious buds and/or multiple shoots of quinoa, comprising the steps of:
(1) culturing an explant including a cotyledonary node and hypocotyl of a quinoa seedling on a multiple shoot induction medium;
(2) after the culturing in step (1), culturing in a medium for shoot elongation after selection; and (3) after the culturing in step (2), culturing or growing in a basal medium or MS medium to obtain adventitious buds, adventitious buds and/or multiple buds.

The quinoa multiple shoot induction method and transformation method of the present invention have one or more of the following effects.
(1) It is possible to produce quinoa transformants without going through callus formation.
(2) Multiple shoots can be induced in a shorter period of time than before.
(3) Multiple shoot induction and transformation can be performed simultaneously.
(4) The target gene can be introduced into quinoa in a shorter time than with conventional transformation methods using callus formation and regeneration systems.
(5) It is possible to avoid culture mutations (somatic mutations) that are a problem with conventional callus formation and regeneration.

Shoots (approximately 1 cm long) were subcultured on selection medium containing no BA and cultured for 14 days. GUS staining of hypocotyls two days after infection with Agrobacterium (strain C58). The area indicated by the circle is GUS stained. GUS staining of multiple shoots 20 days after infection with Agrobacterium (strain C58). The area indicated by the circle is GUS stained.

The present invention relates to a method for inducing multiple shoots into which a target gene of quinoa has been introduced, and to multiple shoots obtained by said induction method. The present invention is described in detail below.

(Quinoa)
Quinoa (Chenopodium quinoa) is a plant of the genus Chenopodium, subfamily Chenopodiaceae, family Amaranthaceae. The quinoa used in the present invention is not particularly limited, but is preferably US National Plant Germplasm System accession number PI 614882 (line #79 in the following example) and PI 614883 (line #80 in the following example).

(Agrobacterium)
Agrobacterium has the ability to integrate an expression vector into a plant cell. Agrobacterium has the ability to integrate an expression vector preferably into the nucleus, and more preferably into a chromosome.
The Agrobacterium used in the transformation of the present invention is not particularly limited, but may be Agrobacterium tumefaciens or Agrobacterium rhizogenes, which are bacteria of the genus Agrobacterium. The Agrobacterium tumefaciens is not particularly limited, but may be, for example, the LBA4404 strain or the C58 strain. The Agrobacterium rhizogenes is not particularly limited, but may be, for example, the A13 strain.

(Binary Vector)
A preferred transformation method using Agrobacterium is the binary vector method, which is a method in which a plasmid having borders (LB and RB) of the T-DNA region of the plasmid is introduced into Agrobacterium, which is then used to infect a plant, thereby inserting the target gene into the plant genome.
An expression cassette using the binary vector method may contain, in the T-DNA region, a foreign gene to be transformed (e.g., a drought-resistance gene, a cold-resistance gene, etc.), as well as a promoter, a terminator, a marker gene, and a reporter gene for expressing the gene.
A binary vector can be prepared by ligating a desired recombinant gene to a desired vector for recombination, for example, a vector for plant transformation, in a conventional manner.
The vector used in the present invention is not particularly limited as long as it can introduce a target nucleic acid into a plant via Agrobacterium, and for example, a pBI-based vector can be used. Examples of pBI-based vectors include pBI121, pBI101, pBI101.2, pBI101.3, and pBI221, and preferably pBI101 and pIG121Hm. As pBI101, for example, pIG121Hm incorporating a CaMV35S promoter (CaMV35S), a hygromycin resistance gene (HPT), and a nopaline synthase terminator (NosT) cassette can be used, but is not particularly limited. pIG121Hm has an NPTII gene and is basically a vector for gene recombination (transformation) of plants, derived from Escherichia coli RK2 strain and Agrobacterium tumefaciens C58 strain.
The binary vector is transformed into Agrobacterium by the desired transformation method, for example, electroporation.

(Binary Vector Expression Cassette)
An expression cassette using the binary vector method can contain, in the T-DNA region, a foreign gene to be transformed, as well as a promoter, terminator, selection marker gene, and reporter gene for expressing the gene.
The foreign gene or endogenous gene whose expression is improved (sometimes referred to as a gene of interest) for transformation is not particularly limited as long as it is a gene that can be expressed in a plant cell, and may be a gene that expresses a new trait or a gene that controls the expression of an endogenous gene, for example, environmental stress resistance genes such as salt resistance genes, drought resistance genes, cold resistance genes, high temperature resistance genes, and insect resistance genes, lipid synthesis genes, etc. The gene of interest can be isolated and used by performing genome editing, overexpression and/or suppression of expression of the gene of interest, etc. The introduced gene of interest is incorporated into the genome of the plant and further exists in seeds obtained from the plant.
The promoter is not particularly limited as long as it is a promoter that can induce the expression of a target gene in a plant body, and any promoter known per se can be applied. Examples of such promoters include the 35S cauliflower mosaic virus (CaMV35S) promoter, the nopaline synthase (NOS) promoter derived from Agrobacterium, drug-inducible promoters (e.g., the alcohol dehydrogenase (alcA) promoter, the UAS promoter, etc.), and plant gene promoters (e.g., the ubiquitin promoter, etc.), and the CaMV35S promoter is preferred.
The terminator may be any sequence capable of terminating transcription of a gene transcribed by a promoter, and examples thereof include the nopaline synthase (NOS) gene terminator (NosT), octovin synthase (OCS), and CaMV35SRNA gene terminator, with NosT being preferred.
The selection marker gene (selective drug resistance gene) is not particularly limited as long as it enables the selection of transformants and non-transformants, and any selection marker gene known per se can be applied. Examples of such a selection marker gene include an NPTII gene, a geneticin resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a paromomycin B resistance gene, or resistance genes to herbicides such as glufosinate and glyphosate, and preferably the geneticin resistance gene or the hygromycin resistance gene.
The reporter gene that can visually identify the transformant is not particularly limited, and any reporter gene known per se can be applied. For example, a gene that expresses a colored or fluorescent protein such as luciferase or green fluorescent protein (GFP), or a gene that expresses β-glucuronidase or GUS, for which various color-producing substrates are known, can also be used.

(Explant)
In the present invention, an explant containing a cotyledonary node and hypocotyl of a young plant is used as a material for multiple shoot induction culture. The explant containing a cotyledonary node and hypocotyl is not particularly limited as long as it contains a cotyledonary node and hypocotyl, but is preferably a portion from the tip of the cotyledon including the cotyledonary node to the hypocotyl, or a portion from the cotyledon including the cotyledonary node but not including the cotyledons to the hypocotyl.
In conventional multiple shoot induction, 4-week-old seedlings were used, but by using seedlings that are 1 to 14 days old after sowing, for example, 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days old, 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, or any range selected therefrom, for example, 3 to 8 days old, preferably 4 to 6 days old, more preferably 5 days old, the period for multiple shoot induction can be shortened.
For example, 0.1 to 20 mm or 0.5 to 15 mm, preferably 1 to 10 mm, more preferably 2 to 5 mm, from the tip of the cotyledonary part including the cotyledonary node of a 4- to 6-day-old seedling is used as an explant.

(Adventitious buds, adventitious buds and multiple buds)
"Apical bud" refers to the first bud that emerges from the apical meristem found in the cotyledonary node and its surroundings, and "lateral bud" refers to a bud that originates from the same apical meristem and forms in the leaf axil. The apical bud and lateral bud are collectively called "adventitious bud". "Adventitious bud" refers to a bud that forms in a part other than the apical meristem where buds are not usually formed, such as in leaves, roots, callus, etc. "Multiple bud body" refers to a collective formation of multiple adventitious buds and adventitious buds. In the present invention, an explant with a total number of adventitious buds and/or adventitious buds of 2 or more is defined as a multiple bud body.

(Shoot)
A shoot is a unit of a plant body (above ground) having a shoot apex, a leaf and a stem. In the present invention, adventitious buds and adventitious buds elongated to 1 cm or more were counted as shoots.

(Preparation of Axenic Explants)
The method for producing the axenic explants of the present invention is described below.
The aseptically sown quinoa is grown for 1 to 10 days, preferably 3 to 7 days, more preferably 5 days to obtain seedlings. For example, quinoa seeds are sterilized with an aqueous hypochlorous acid solution, and then sown and grown on a sterilized basic medium (MS medium) in a clean bench.
An explant is obtained by cutting out a portion of the seedling from the tip of the cotyledonary part including the cotyledonary node to the hypocotyl, the length being 0.1 to 20 mm, for example 0.5 to 15 mm, preferably 1 to 10 mm, more preferably 2 to 5 mm.
The explants are cultured in a medium containing a multiple shoot inducer (multiple shoot induction medium) for 1 hour to 7 days, preferably 12 hours to 5 days, more preferably 1 to 2 days.
The basal medium for culturing explants is not particularly limited as long as it is a medium that can be used for plant tissue culture, and is preferably MS (Murashige and Skoog) medium or a modified medium thereof, etc. Various media used for culturing explants in the present invention are prepared by adding necessary components such as a gelling agent to the basal medium and then adjusting the pH to pH 4.0 to pH 8.0, preferably pH 5.0 to pH 7.0, more preferably pH 5.5 to pH 6.1, and even more preferably pH 5.8.
An example of the gelling agent is gellan gum, etc. The concentration of gellan gum in the medium is 0.1 to 20 g/L, preferably 0.5 to 10 g/L, and more preferably 3.0 g/L.
Examples of multiple shoot inducers include 6-benzyladenine (BA), forchlorfenuron (CPPU), thidiazuron (TDZ), etc. The concentration of the multiple shoot inducer in the medium is not particularly limited as long as it can induce multiple shoots, but in the case of BA, it is 0.1 to 10 mg/L, preferably 1 to 5 mg/L, and more preferably 2 mg/L.
Before infection with Agrobacterium, the cells are stored at 10° C. to 1° C., preferably 4° C., for 1 hour to 2 days, preferably 2 hours to 1 day.

(Preparation of infection solution)
An infection solution is prepared by adjusting the _OD600 of logarithmic growth phase Agrobacterium cells into which a binary vector containing a gene of interest has been introduced to 0.01 to 0.5, preferably 0.05 to 0.2, and more preferably 0.1, with a solution containing a reagent that improves the infection rate of Agrobacterium (solution for preparing an infection solution).
An example of a reagent that improves the infection rate of Agrobacterium is silwet, etc. The concentration of silwet in the infection solution is 0.001 to 1%, preferably 0.01 to 0.1%, and more preferably 0.01%.
Acetosyringone may be added to the infection solution, preferably at a final concentration of 10 to 1000 μM, more preferably at a final concentration of 50 to 150 μM, and even more preferably at a final concentration of 80 to 120 μM.

(Agrobacterium infection)
The explants are immersed in or contacted with an infection fluid. The step of immersing or contacting in an infection fluid preferably includes ultrasonic treatment followed by vacuum treatment.
In the ultrasonic treatment, the explants in the infection solution are treated with ultrasonic waves at 1 to 50 AMP, preferably 10 to 40 AMP, more preferably 20 to 30 AMP for 1 to 30 seconds, preferably 5 to 20 seconds, more preferably 10 to 15 seconds.
In the reduced pressure treatment, the explants in the infection solution are subjected to a reduced pressure of -1200 to -400 psi, preferably -1000 to -600 psi, more preferably -800 psi for 30 seconds to 30 minutes, preferably 2 to 10 minutes, more preferably 5 minutes.
The explants are cultured in a medium containing a nitrogen source (coculture medium) for 12 hours to 7 days, preferably 1 to 3 days, more preferably 2 days (15 to 35°C, preferably 20 to 30°C, more preferably 25°C, in the dark).
Examples of the nitrogen source include casamino acids, etc. The concentration of casamino acids in the medium is 0.1 to 100 g/L, preferably 1 to 50 g/L, and more preferably 10 g/L.
The explants are washed with the infection preparation solution 1 to 5 times, preferably 3 times.

(Induction of multiple shoots and selection of transformants)
The explant is transplanted into a sterilization medium and cultured for 3 to 14 days, preferably 5 to 10 days, more preferably 7 days. The explant can become multiple shoots (the total number of adventitious shoots and/or adventitious shoots can be confirmed to be 2 or more) during culture in a medium containing an agent for sterilizing Agrobacterium (sterilization medium).
Examples of drugs for eradicating Agrobacterium include cefotaxime sodium salt, carbenicillin, etc. The concentration of cefotaxime sodium salt in the medium is 1 to 2000 mg/L, preferably 50 to 500 mg/L, and more preferably 250 mg/L.
The explant (which may be a multiple shoot) is transferred to a medium containing a multiple shoot inducer, a selection agent, and an agent for eliminating Agrobacterium (multiple shoot induction medium further containing a selection agent and an agent for eliminating Agrobacterium) and cultured for 2 weeks under a 12-16 hour day length, preferably a 14 hour day length (light period 20-28°C, preferably 24°C, dark period 14-22°C, preferably 18°C). Explants that have not become multiple shoots can become multiple shoots during culture in the multiple shoot induction medium further containing a selection agent and an agent for eliminating Agrobacterium.
Shoots of multiple shoots that have grown to a shoot length of 0.1 to 3 cm, preferably 0.5 to 1.5 cm, and more preferably about 1 cm are cut off and subcultured into a medium containing a selection agent, an agent for eliminating Agrobacterium, and a gelling agent, but not containing a multiple shoot inducer (selection medium containing no multiple shoot inducer). Furthermore, explants in which multiple shoots have not yet been confirmed with the naked eye are subcultured into a medium containing a multiple shoot inducer, a selection agent, and an agent for eliminating Agrobacterium (selection medium containing multiple shoot inducer). Explants that have not yet become multiple shoots can become multiple shoots during culture in the selection medium containing the multiple shoot inducer.
The cells are subcultured and cultured every 1 to 3 weeks, preferably about every 2 weeks, 1 to 20 times, preferably 1 to 10 times, more preferably 1 to 6 times, and even more preferably 1 to 3 times.
Thereafter, the shoots that have been subcultured and cultured on the selection medium not containing a multiple shoot inducer are subcultured and cultured on a medium containing an agent for eradicating Agrobacterium (a shoot elongation medium after selection (preferably immediately after selection)), and allowed to grow for 2 weeks to 2 months, preferably 3 weeks to 5 weeks, and more preferably about 1 month.
Explants in which multiple buds have not yet been confirmed with the naked eye and explants in which multiple buds have reached the limit of size at which they can be confirmed with the naked eye are subsequently subcultured and cultured on a selection medium containing a multiple bud inducer every 1 to 3 weeks, preferably about every 2 weeks, and allowed to grow for 2 weeks to 2 months, preferably 3 to 5 weeks, and more preferably about 1 month.
Among the shoots grown on the shoot elongation medium after selection (preferably immediately after selection) and the shoots grown on the selection medium containing a multiple shoot inducer, shoots that have elongated without turning white or brown are subcultured on a selection medium not containing a multiple shoot inducer and cultured for 3 days to 2 months, preferably 1 to 4 weeks, more preferably 2 to 3 weeks, and shoots that survive and grow are selected as transformants and transferred to a drug-free basic medium.

(Production of transformed seeds)
The selected shoots are grown under any conditions. The term "growing under any conditions" is not particularly limited as long as the plant body can be grown until the target gene is expressed, but it is preferably grown until buds appear, and more preferably until seeds can be harvested (grown to adulthood). In this specification, "adult" refers to quinoa that can form seeds.
The temperature conditions for growth are not particularly limited as long as the plant can survive at the temperature, but light/dark conditions of 20°C to 28°C/14°C to 22°C are preferred, 22°C to 26°C/16°C to 20°C are more preferred, and 24°C/18°C are even more preferred.
The light and dark conditions for growth are not particularly limited as long as the plant can survive. For example, 10-18 hours light (light)/14-6 hours dark (dark) conditions or 12-16 hours light (light)/12-8 hours dark (dark) conditions may be used, with 14 hours light/10 hours dark conditions being preferred.
For example, the plants are subcultured every 1 to 3 weeks, preferably every 2 weeks, in a basal medium that does not contain a selective drug (drug-free basal medium) and grown until the seeds can be harvested, thereby obtaining seeds into which the target gene has been introduced.

(Method for producing adventitious shoots, adventitious shoots and/or multiple shoots into which a gene of interest has been introduced)
Preferred methods for producing adventitious shoots, adventitious shoots and/or multiple shoots into which a target gene of quinoa has been introduced according to the present invention can be exemplified below, but are not particularly limited thereto.
(1) Explants cultured on a multiple shoot induction medium are placed in an infection solution, and preferably subjected to ultrasonic treatment and reduced pressure treatment, and then cultured on a coculture medium.
(2) After culturing (1), the culture is cultured in a sterilized medium.
(3) After the cultivation in (2), the cells are cultivated in a multiple shoot-inducing medium (which may contain a selection agent).
(4) After culturing in (3), the shoot or terminal bud is excised together with the explant and cultured on a selection medium.
(5) The culture product of (4) is cultured on a medium for shoot elongation after selection, and further grown on MS medium to obtain adventitious buds, adventitious buds and/or multiple buds.

More specifically, the following is true:
(1) Explants cultured on multiple shoot induction medium for several days are placed in an infection solution, treated with SV, and cultured on coculture medium for several days.
(2) After culturing in the coculture medium in (1), the explants are cultured in a bacteria-free medium for 3 to 10 days.
(3) After the cultivation in (2), the cells are cultivated for 1 to 3 weeks in a multiple shoot-inducing medium containing a selective agent.
(4) After culturing in (3), the shoot or terminal bud is excised together with the explant and cultured on a selection medium.
(5) The culture product of (4) is cultured for 1 to 3 weeks on a medium for shoot elongation after selection, and then further grown on a basal medium or MS medium to obtain adventitious buds, adventitious buds and/or multiple buds.

More details are as follows:
(1) Explants cultured on multiple shoot induction medium for 1 to 2 days are placed in an infection solution, treated with SV, and cultured on coculture medium for 2 days (25°C, in the dark).
(2) After the pre-culture, the explants are washed and cultured in a bacteria-free medium for 7 days.
(3) Culture for two weeks in multiple shoot induction medium (BA 2 mg/L) containing 50 mg/L G418 and 250 mg/L cefotaxime sodium salt.
(4) Shoots or terminal buds that have grown to about 1 cm are cut together with the explant and transferred to a selection medium that does not contain BA (basal medium containing 50 mg/L G418 and 250 mg/L cefotaxime sodium salt). Explants in which multiple shoots have not yet been confirmed with the naked eye are transferred to a selection medium containing BA, and then transferred to the same medium approximately every 2 weeks until shoots appear (shoots are usually induced after 1-2 times, and then proceed to (5)).
(5) Shoots or terminal buds that have finally grown to 1 cm or more are excised together with the explant, or the shoots alone are excised without the explant. The excised culture product is cultured for 2 weeks on shoot elongation medium (MS medium containing cefotaxime sodium salt) after selection (preferably immediately after selection), and then is grown on MS medium not containing cefotaxime sodium salt for subsequent subculture.

(Various media)
In the production method of the present invention, a medium known per se can be used, and examples thereof are given below.
Multiple shoot induction medium is a medium containing a multiple shoot inducer, and is not particularly limited as long as it can induce adventitious shoots, adventitious shoots, and multiple shoots in quinoa.
Coculture medium: A medium known per se that contains a nitrogen source.
- Sterilization medium A medium containing a known agent for sterilizing Agrobacterium.
Selection medium: A medium known per se in which quinoa shoots can be induced, cultured or grown.
Medium for shoot elongation after selection (preferably immediately after selection) This is MS (basal medium) that allows the selection of shoots derived from quinoa multiple shoots, and contains cefotaxime sodium salt for the purpose of removing Agrobacterium.
MS medium (basal medium)
A medium known per se in which quinoa multiple shoots and shoots derived from quinoa multiple shoots can be grown.

(Method for producing adventitious buds, adventitious buds and/or multiple buds)
The present invention also covers a method for producing adventitious shoots, adventitious shoots and/or multiple shoots of quinoa into which a target gene has not been introduced. Examples of such methods include, but are not limited to, those listed below.
(1) Explants containing the cotyledonary nodes and hypocotyls of quinoa seedlings are cultured on multiple shoot induction medium.
(2) After culturing in (1), the selected shoots are cultured on a medium for shoot elongation.
(3) After the cultivation in (2), the plants are cultivated or grown in a basal medium or an MS medium to obtain adventitious buds, adventitious buds and/or multiple buds.

The present invention will be described in detail below with specific examples, but the present invention is not limited to these examples.

Materials and Methods
(material)
〇Plant Quinoa (Chenopodium quinoa)
Strain
Agrobacterium tumefaciens LBA4404
Agrobacterium rhizogenes A13
Agrobacterium tumefaciens C58
Binary vector: The binary vector pIG121-Hm was used, which has a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence (see OHTA, Shozo, et al. Construction and expression intobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant and cell physiology, 1990, 31.6: 805-813).

(MS (Murashige and Skoog) medium: basal medium)
MS Plant Salt mixture (Murashige and Skoog medium mixed salts, Nippon Pharmaceutical) for 1L
Vitamin for MS (MurashigeandSkoog Vitamin Solution 1000 ×, liquid, plant cell culture tested, SigmaAldrich) 1mL
Sucrose 30.0 g
After autoclaving (121°C, 15 min), appropriate amounts were dispensed into sterile petri dishes.

(AB medium) (Agrobacterium culture medium)
Sucrose 5 g/L
Bacteriological Agar (Agar) 15 g/L
*After autoclave sterilization at 121℃ for 15 minutes, the following was added aseptically.
20×AB salt 50 ml/L
20×AB Buffer 50 ml/L
+50 mg/L Kanamycin Hydrochloride (Km)
*Km was prepared at a concentration of 5 mg/mL, filter sterilized, and stored at -20°C.
(20xAB salt)
20 g/L NH4Cl
6 g/L _MgSO4 · _7H2O
3 g/L KCl
0.268 g/L _CaCl2 · _2H2O
0.05 g/L _FeSO4 · _7H2O
* After autoclaving at 121℃ for 15 minutes, the product was stored at room temperature.
(20xAB Buffer)
60 _g / _LK2HPO4
26 g/L _{NaH2PO4 · 2H2O}
* After autoclaving at 121℃ for 15 minutes, the product was stored at room temperature.

(YEP medium (liquid))
Bacto polypeptone 10 g/L
Bacto Yeast Extract 10 g/L
NaCl 5 g/L
pH 7.2
*After adjusting the pH, the solution was sterilized in an autoclave at 121℃ for 15 minutes and then stored at room temperature.

(Multiple shoot induction medium)
The following was added to the MS medium to adjust the pH:
BA 2 mg/L
pH 5.8
*After adjusting the pH, 3.0 g/L of gellan gum was added and the solution was sterilized in an autoclave (121°C, 15 minutes).

(Solution for preparing infectious fluid)
The following was added to the MS medium to adjust the pH:
BA 2 mg/L
0.01% silver
pH 5.8
*After adjusting the pH, the solution was sterilized in an autoclave at 121°C for 15 minutes.

(Coculture medium)
The following was added to the MS medium to adjust the pH:
BA 2 mg/L
Casamino Acids, Bacto 10 g/L
pH 5.8
*After adjusting the pH, 3.0 g/L of gellan gum was added, sterilized in an autoclave (121°C, 15 minutes), cooled slightly, and acetosyringone was added to a final concentration of 100 μM, mixed well, and an appropriate amount was dispensed into sterile petri dishes.

(Sterilization medium)
The following was added to the MS medium to adjust the pH:
BA 2 mg/L
pH 5.8
*After adjusting the pH, add 3.0 g/L of gellan gum, sterilize in an autoclave (121°C, 15 minutes), allow to cool slightly, add cefotaxime sodium salt to a final solution of 250 mg/L, mix well, and dispense appropriate amounts into sterile petri dishes.

(Selection medium)
The following was added to the MS medium to adjust the pH:
BA 2 mg/L (optional, no BA)
pH 5.8
*After adjusting the pH, add 3.0 g/L of gellan gum, autoclave (121°C, 15 minutes), allow to cool slightly, add 250 mg/L of cefotaxime sodium salt and 50 mg/L of geneticin G418 final, mix well, and dispense appropriate amounts into sterile petri dishes.

(Medium for shoot elongation immediately after selection)
MS medium
pH 5.8
*After adjusting the pH, 3.0 g/L of gellan gum was added, autoclaved (121°C, 15 min), cooled slightly, and cefotaxime sodium salt was added to the mixture at 250 mg/L (a drug for eliminating Agrobacterium. Multiple shoots were induced in a medium containing the drug, and shoots that had grown to a certain extent were grown in MS medium without cefotaxime sodium salt). The mixture was mixed well and appropriate amounts were dispensed into sterile petri dishes.

(Sterile sowing of quinoa seeds)
1) Approximately 50 quinoa seeds were placed in a 1.5 mL Eppendorf tube and rinsed with tap water three times.
2) Next, rinse with 70% ethanol for 20 seconds.
3) Place in 5% NaOCl for 10 minutes with occasional mixing.
4) Using a pipette, wash the tube with sterile water 4 to 5 times.
5) The seeds were soaked up with sterile water and sown sparsely in MS medium dispensed into sterile dishes, about 30 seeds per dish. The tip of the pipette used for sowing was cut off with scissors.

[Consideration of drug (antibiotic) concentration used for selection]
In this example, the type and concentration of antibiotics to be used for drug resistance selection in quinoa were determined.

The concentrations of Km (kanamycin): 50, 100, 200, 300 mg/L, G418 (geneticin): 10, 20, 30, 40, 50, 60 mg/L, and Hg (hygromycin): 10, 20, 50 mg/L were used. Cefotaxim sodium salt (250 mg/L) for sterilization had almost no effect on the growth of the plant body, so it was added to the medium used from the sterilization medium to the selection of shoots.
1) Leaf disks (approximately 8 mm in total length) were taken from the cultured quinoa plants and cultured on callus induction medium (MS medium containing 1 mg/L 2,4-D and 0.1 mg/L BA) containing each antibiotic.

[result]
Regarding Km, at a high concentration (300 mg/L), although the leaf discs turned yellow to some extent and callus formation was inhibited, it was difficult for them to completely die, and it was determined that there was a high possibility that this would lead to the escape phenomenon, in which non-transformed cells redifferentiate.
It was confirmed that G418 has the same effect as Km. The selection concentration was set at 50 mg/L, at which almost no callus formation was observed (Table 1). 50 mg/L is a concentration that exceeds the 20-30 mg/L used in the selection of chrysanthemum plants.
Regarding Hg, browning occurred early even at low concentrations (10 mg/L), and at 20 mg/L 100% of the plants died (turned white), so 20 mg/L was selected as the selection concentration (Table 1).
In the actual selection, multiple shoots were initially induced on a medium containing G418 (50 mg/L), and then individual shoots were selected on a medium containing Hg (20 mg/L), which has a stronger selection effect. However, in the selection after transformation, the number of surviving shoots could not be secured with Hg (20 mg/L), so the Hg concentration was lowered to 10 mg/L or selection was performed only with G418 (50 mg/L).

[Selection of quinoa lines with high multiple shoot induction]
In this example, a quinoa line that actively induces multiple shoots was selected. Note that the present inventors have confirmed that regeneration after callus formation is difficult in quinoa.
Therefore, we established a system to induce multiple shoots by using seedlings approximately one week after aseptic sowing and culturing the area from the tip of the cotyledon including the cotyledon node to 2-5 mm from the hypocotyl (the state where the true leaves are barely visible, hereafter sometimes referred to as the CT (cotyledon tip)) in a medium containing cytokinin (Tables 2 and 3).
In Table 2, the explants were cultured for 2 weeks or more, and the number of explants which induced two or more adventitious buds was counted.
When two or more adventitious buds were induced per explant, it was judged that the explant had become a multiple bud body (MBB).
Callus was induced on MS medium containing 1 mg/L 2,4-D and 0.1 mg/L BA, and MBB was induced on MS medium containing 2 mg/L BA.
In Table 3, the number of MBBs/explants: 20 individuals were planted, and the average number of MBBs per explant was calculated, excluding dead individuals. In Table 3, the number of MBBs/explants = 1 indicates that MBB formation was not achieved. Since all values were greater than 1, MBB formation was confirmed.
The accession numbers in each table are serial numbers for each quinoa line provided by the Agricultural Research Service (ARS) of the United States Department of Agriculture (USDA) in this study.
Among 18 quinoa accessions, accession numbers #79 (US National Plant Germplasm System accession number: PI 614882) and #80 (US National Plant Germplasm System accession number: PI 614883), which showed the most abundant multiple shoot formation, were used for transformation (Tables 2 and 3).

Accession numbers #101, 109, and 110 showed poor germination.

[Agrobacterium strain study]
In this example, an Agrobacterium strain optimal for inducing multiple shoots in quinoa was examined.
Among the Agrobacterium strains LBA4404, A13, and C58, LBA4404 and C58, which vigorously induced multiple shoots and had high survival rates thereafter, were used in the following examples.

[Conditions for SV treatment (damaging treatment by ultrasonic treatment and reduced pressure penetration treatment)]
In this example, the optimal conditions for SV treatment were examined.
Sonication (QSONICA) treatment (0, 20, 30, 40 Amplitude) was followed by vacuum application (-800 psi for 5 min or none) to promote infection of Agrobacterium into Keanu.
To establish the conditions for SV (sonication and vacuum infiltration) treatment, explants (CT) were placed in an infection preparation solution that did not contain Agrobacterium, and subjected to sonication and vacuum treatment, respectively, and the number of multiple shoots induced was measured.
The results are shown in Tables 4 to 6. As shown in Tables 4 and 5, in #79 and #80, even with SV treatment, multiple shoot induction was confirmed to be equal to or greater than that without SV treatment (0 AMP). This confirmed that the SV treatment of the present invention can contribute to improving transformation efficiency without adversely affecting multiple shoot induction. In terms of treatment time, the highest multiple shoot induction was observed at 10 seconds with 20 AMP (Table 6).
To promote bacterial infection while minimizing damage to the explants (CT), the explants were wounded by placing them in the Agrobacterium infection solution (ultrasonic treatment (20 or 30 AMP, 10 to 15 sec)) and then immediately applying reduced pressure (-800 psi, 5 min) to perform the infection treatment.
When the plants were infected with the Agrobacterium infection solution after ultrasonic treatment, no surviving shoots were observed among the shoots induced by the subsequent selective culture. However, when ultrasonic treatment was performed in the presence of the Agrobacterium infection solution, surviving shoots were confirmed (Tables 7 to 9).

SV: Sonication and vacuum infiltration
CT was cultured in MS medium containing 2 mg/L BA for 2 days before SV treatment.

Amplitude 0 is for decompression processing only.
Those that did not become MBB were counted as having an MBB number of 1.
Adventitious shoots/explants: total number of adventitious shoots/number of explants examined, excluding shoots that had not yet grown at the time of counting.
#79 had a slightly higher MBB count at 30 AMP/10sec, and #80 had a slightly higher MBB count at 40 AMP/10sec.

[Preparation of transformants]
In this example, transformants were prepared by SV treatment based on the conditions set in Examples 1 to 5 above.
(Plant material)
The seedlings were used about 5 days after sowing in a sterile environment. The CTs were grown from the tip of the cotyledon including the cotyledonary node to 2-5 mm from the hypocotyl (when the primary leaves were barely visible) on multiple shoot induction medium for 1-2 days and then used for transformation (infection with Agrobacterium). They were then placed at 4°C for several hours to one day prior to infection with Agrobacterium.

(Pre-cultivation of Agrobacterium)
1) Three days before infection, Agrobacterium (LBA4404 and C58 into which a binary vector had been introduced by electroporation) that had been frozen and stored in glycerol (-80°C) was scraped off with a toothpick and applied in a straight line onto AB medium containing 50 mg/L final kanamycin hydrochloride, and cultured in an incubator (28°C, dark).
2) One day before infection, Agrobacterium that had completed cultivation was gently scraped off with a toothpick and suspended in YEP liquid medium (20 mL) containing 10 mg/L hygromycin and 50 mg/L kanamycin hydrochloride, and cultured overnight with shaking at 28°C (in the dark, at 70 rpm) to obtain cells in the logarithmic growth phase.

(Agrobacterium infection)
1) The tips of the cotyledons of quinoa plants that had been aseptically sown 5 days earlier were cut to lengths of 2 to 5 mm (sometimes referred to as explants) and cultured on multiple shoot induction medium for 1 to 2 days.
2) Just before infection, the OD ₆₀₀ was adjusted to 0.1 with the infection preparation solution to prepare the infection solution (15 mL of the infection preparation solution added to 1 mL of cultured Agrobacterium solution results in an OD ₆₀₀ of approximately 0.1. In addition, acetosyringone was added to a final concentration of 100 μM for co-culture, which is known to increase transformation efficiency).
3) Explants were removed from the multiple shoot induction medium after 1-2 days of culture (cell membranes were strengthened by culturing at 4°C for several hours immediately prior to the incubation), and 20-27 explants were placed in an infection solution (16 mL in total: 15 mL of infection solution preparation solution and 1 mL of Agrobacterium solution), subjected to SV treatment (ultrasonication at 20 AMP or 30 AMP for 10 or 15 seconds, followed by vacuum reduction at -800 psi, 5 minutes), lightly dried on a sterilized towel, and then cultured in coculture medium for 2 days (25°C, in the dark).
4) After co-cultivation, the explants were washed three times with the infection preparation solution, lightly dried with a sterile towel, and then transferred to a sterilized medium and cultured for seven days to sterilize the Agrobacterium.

(Selection of transformants and induction of multiple shoots)
1) Explants cultured in the bacteria-free medium (in some explants, induction of multiple shoots was visible to the naked eye) were transferred to multiple shoot induction medium (BA 2 mg/L) further supplemented with 50 mg/L G418 and 250 mg/L cefotaxime sodium salt, and cultured for 2 weeks. Culture in steps 1) to 4) was performed under a 14-hour photoperiod (24°C/18°C, light/dark).
2) Shoots with multiple shoots (shoot length) of about 1 cm were cut and transferred to a selection medium not containing BA, and explants in which multiple shoots could not be seen with the naked eye were transferred to a selection medium containing BA (see Figure 1). Transfers were performed approximately every two weeks (Tables 7 and 8: up to six times over a period of about three months, Table 9, which used G418: a total of about one month). Explants that had become brittle and blackened were removed at the time of transfer.
3) The shoots that were cultured for two weeks on a selection medium not containing BA in step 2) were then cultured every two weeks on a shoot elongation medium (MS medium containing cefotaxime sodium salt) immediately after selection and grown for approximately one month.
Explants in which multiple shoots had not yet been visible to the naked eye and explants that were the minimum size at which multiple shoots could be visible to the naked eye were subcultured on selection medium containing BA every two weeks, as in step 2), and grown for approximately one month.
4) Among the shoots grown on the shoot elongation medium immediately after selection in step 3) and the shoots grown on the selection medium containing BA, shoots that had elongated without turning white or brown were transferred to the selection medium not containing BA and cultured for 2 to 3 weeks, and shoots that survived were selected and transferred to drug-free MS medium (basal medium). The results are shown in Table 9.

(Selection process)
(1) Explants cultured on multiple shoot induction medium for 1 to 2 days were placed in an infection solution, treated with SV, and cultured on coculture medium for 2 days (25°C, in the dark).
(2) After the coculture, the explants were washed and cultured in a bacteria-free medium for 7 days.
(3) The cells were cultured for two weeks in a multiple shoot induction medium (BA 2 mg/L) containing 50 mg/L G418 and 250 mg/L cefotaxime sodium salt.
(4) Shoots or terminal buds that had grown to about 1 cm were cut together with the explants and transferred to a selection medium not containing BA (basal medium containing 50 mg/L G418 and 250 mg/L cefotaxime sodium salt). Explants in which multiple shoots had not yet been confirmed with the naked eye were transferred to a selection medium containing BA, and then transferred to the same medium approximately every 2 weeks until shoots appeared (shoots were usually induced after 1-2 times, and the process was moved to (5)).
(5) Shoots or terminal buds that eventually grew to 1 cm or more were excised together with the explant, or the shoots alone were excised without the explant. The excised culture products were cultured for 2 weeks on shoot elongation medium (MS medium containing cefotaxime sodium) immediately after selection, and then were grown on MS medium without cefotaxime sodium for subsequent subcultures.

#80 was used in the experiment in Table 7. The target gene was confirmed by PCR in 12 of the 20 drug-resistant individuals (Table 8).
In the experiment shown in Table 9, the same selection was carried out once for MBBs that had drug resistance (selection with G418 alone, but containing cefotaxime sodium salt), and thereafter they were grown without the drug. This is thought to be why many MBBs with drug resistance were obtained.

In the experiment in Table 9, #79 was used.
*The percentage of MBB individuals that grew with drug resistance among MBBs induced from individuals with drug resistance (CT). Of these, transformation was confirmed by PCR for all confirmed individuals (84 individuals).
This confirmed that the method of the present invention is a quinoa transformation method that allows the introduction of a target gene without using a callus formation/regeneration system.

(Growth of transformants)
For shoots that were confirmed to be transgenic (drug resistant), multiple shoots were further induced from the remaining shoot tips to ensure their numbers, and the shoots from the extended multiple shoots were then sub-planted and grown on MS medium (medium for shoot elongation immediately after selection) approximately every two weeks.
This confirmed that the method of the present invention is capable of multiplying transformants (multiple shoots) into which a gene of interest has been introduced.

(Growth of the plant body)
The shoots selected in step 4) above were subcultured and grown on drug-free MS medium (basal medium) every two weeks. The isolated shoots naturally took root, formed flower buds, and transformed seeds were harvested.
Since in vitro conditions are somewhat stressful, there is some variation between shoots, but it is believed that flower buds appear at a very early stage (at about 1 cm to 10 cm), and then flower and produce seeds.
From step 3) above, flowers will appear and seeds can be harvested within one month at the earliest, or within two months at the latest.
This confirmed that the method of the present invention can obtain seeds into which a target gene has been introduced.

(Confirmation of introduction of target gene)
In this example, it was confirmed that the target gene, GUS, was introduced into multiple shoots.
Using the method described in the above Example, hypocotyls 2 days after infection with Agrobacterium (C58 strain) were stained for GUS. The GUS staining solution used was 1 mg/ml X-Gluc cyclohexylammonium salt, 50 mM NaPi, 0.3% TritonX-100, pH 7.0. As is clear from the results in Figure 2, it was confirmed that the target gene, GUS, was expressed in the hypocotyl.
Using the above method, multiple shoots 20 days after infection with Agrobacterium (strain C58) were stained for GUS. As is clear from the results in Figure 3, it was confirmed that the target gene, GUS, was expressed in the cotyledons.
As described above, in the method for inducing multiple shoots of the present invention, a target gene can be introduced into a plant and expressed therein.

(General remarks)
From the above results, the following was confirmed.
1. The most dominant MBB-converted lines were #79 (US National Plant Germplasm System accession number: PI 614882) and #80 (US National Plant Germplasm System accession number: PI 614883).
2. In quinoa, which cannot be divided into small pieces, multiple shoots could be induced in a short period of time by using CT (2-5 mm from the tip of the cotyledonary part including the cotyledonary node to the hypocotyl) as an explant. This resulted in a large number of transformed shoots (MBBs) with little variation in the material, and a feasible transformation system was established in a short period of time.
3. Drug selection using G418 involved inducing MBBs on a medium containing BA (a plant hormone used to induce shoots of multiple shoots) (step 1), and then selecting the MBB shoots again on a medium not containing BA (step 2). This two-step selection, followed by growth on a drug-free basal medium, enabled us to obtain a large number of transformed individuals.
4. In the multiple shoot induction method of the present invention, a target gene can be introduced into a plant (particularly, cotyledons and hypocotyls) and expressed therein.

The present invention provides a novel method for transforming quinoa that does not require callus formation.

Claims (9)

A method for producing adventitious shoots, adventitious shoots and/or multiple shoots of quinoa into which a gene of interest has been introduced , comprising:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest; and (2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds,
Does not include a callus formation step and/or a regeneration step.
method.
The method according to claim 1 , wherein the step (1) comprises ultrasonic treatment and/or vacuum treatment.
The method according to claim 1 or 2, wherein the step (1) comprises ultrasonic treatment followed by vacuum treatment.
The method according to any one of claims 1 to 3, wherein the culturing in the step (2) comprises a selection step.
5. The method of claim 1, wherein the quinoa is US National Plant Germplasm System Accession No. PI 614882 or PI 614883.
A method for producing an adult quinoa plant containing a gene of interest , comprising the steps of:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest;
(2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds; and (3) culturing or growing the adventitious buds, adventitious buds and/or multiple buds to obtain adults. Or,
(1) A step of culturing or growing adventitious buds, adventitious buds and/or multiple buds obtained by the production method according to any one of claims 1 to 5 to obtain adults;
A method comprising:
Does not include a callus formation step and/or a regeneration step.
method.
A method for producing quinoa seeds into which a gene of interest has been introduced , comprising the steps of:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest;
(2) culturing the explant to obtain adventitious buds, adventitious buds and/or multiple buds;
(3) culturing or growing the adventitious buds, adventitious buds and/or multiple buds to obtain adults; and
(4) growing the adults until the seeds can be harvested to obtain seeds;
Or,
(1) A step of culturing or growing adventitious buds, adventitious buds and/or multiple buds obtained by the method according to any one of claims 1 to 5 to obtain adults; and
(2) growing the adult plant until the seeds can be harvested to obtain the seeds; or
(1) A step of growing the adult obtained by the method according to claim 6 until the seeds can be harvested to obtain seeds;
A method comprising:
Does not include a callus formation step and/or a regeneration step.
method.
The method according to any one of claims 1 to 5, comprising the steps of:
(1) placing the explants cultured on a multiple shoot induction medium in an infection solution, subjecting them to ultrasonic treatment and decompression treatment, and then culturing them on a coculture medium;
(2) culturing the culture in a sterilized medium after the culture in step (1);
(3) culturing the culture in step (2) on a multiple shoot induction medium;
(4) culturing the culture in a selection medium after the culture in step (3); and (5) culturing the culture in step (4) in a selection medium for shoot elongation to obtain adventitious buds, adventitious buds and/or multiple shoots, or culturing in a selection medium for shoot elongation and then growing in a basal medium or MS medium to obtain adventitious buds, adventitious buds and/or multiple shoots.
A method for producing adventitious shoots, adventitious shoots and/or multiple shoots of quinoa into which a gene of interest has been introduced , comprising:
(1) immersing an explant containing a cotyledonary node and hypocotyl of a quinoa seedling in an Agrobacterium infection solution containing a gene of interest;
(2) after step (1), culturing in a multiple shoot induction medium;
(3) a step of culturing in a medium for shoot elongation after the selection after the culture in step (2), and (4) a step of culturing or growing in a basal medium or MS medium after the culture in step (3) to obtain adventitious shoots, adventitious shoots and/or multiple shoots into which a gene of interest has been introduced ,
Does not include a callus formation step and/or a regeneration step.
method.