JPH037599A - Early detection of redifferentiating individual by gene transfer - Google Patents

Abstract

PURPOSE:To carry out early detection simply and efficiently by transferring a specific marker gene to plant tissue to form a callus. CONSTITUTION:A chimera gene derived from soil bacterium Agrobacterium rhizogenes is directly bonded to a chimera gene derived from norvaline synthase to give a marker gene (A) bonded to a promoter range. Then a plasmid transduced with the gene A is transferred to a bacterium (B) containing the gene A. Then plant tissue (C) such as stem of carrot is infected with the bacterium B and cultured in a Murashige & Skoog medium (MS medium) containing synthetic auxin to give a culture mixture (D). Then the component D is cultured in a medium for sterilizing the bacterium B to kill the bacterium B and further cultured in MS medium containing kanamycin for about 3 months to give a callus comprising a cell resistant to kanamycin, containing the above-mentioned chimera gene. Then beta-glucuronidase activity of the callus is measured to detect existence of redifferentiating ability of plant individual or progress state of redifferentiation at an early stage.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for early detection and selection of calli having regeneration ability or plant individuals that have started regeneration from calli.

[Conventional technology] Cultivation technology involves removing various organs, tissues, cells, etc. from plants and propagating and growing them in a sterile manner for the purpose of plant breeding, mass propagation, production of useful metabolites, etc. It has been widely attempted in recent years as a plant tissue culture.

Such plant tissue culture usually involves removing specific organs, tissues, cells, etc. from plants as explants, dividing and proliferating them in a medium containing plant hormones that promote cell division, and cultivating a specific morphology. After forming a dedifferentiated callus, which is a dedifferentiated cell mass, and culturing this callus under appropriate conditions (for example, in a hormone-free medium), somatic embryos, adventitious leaves, adventitious roots, etc. can be produced. redifferentiation into tissues and organs having some form of
This is then done by regenerating into a complete plant.

In that case, even if cultured under the same conditions, not all calli will regenerate and regenerate the plant body, and many calli will not regenerate. Therefore, it is necessary to select those that regenerate and those that do not. Conventionally, the methods of selecting calli that can regenerate or those that have already regenerated from calli include (1) culturing a large number of calli as they are and observing them with the naked eye. A method of selecting individuals that are clearly shown to have redifferentiated forms such as somatic embryos, adventitious leaves, and adventitious roots by observation with a microscope, etc. (2) Selection in callus with regeneration ability or cells of already redifferentiated individuals A method of selecting for the presence or absence of regeneration ability by measuring the concentration of components that are known to be taken up in high concentrations by cells (for example, in suspension culture of carrots, glucose is present in the cells of individuals with the ability to form somatic embryos). is known to be selectively taken up at high concentrations, and such selective properties are utilized for selection); A method of selection to determine the presence or absence of regeneration ability by measuring the amount of substances that are known to be produced in high concentrations in humans (for example, in suspension culture of carrots, there is no accumulation in the cells of individuals with somatic embryogenic ability). It is known that powder is selectively accumulated at high concentrations, and this property is used for selection.

However, method U (1) requires culturing a large number of calli as they are, regardless of whether they have differentiation potential, and is not an efficient method as it requires time, effort, and a large number of culture vessels and equipment. Ta. In addition, the methods (2) and (3) above are useful for detecting and measuring specific substances that indicate the presence or absence of redifferentiation ability.
This was not a desirable method because it required high-precision analytical equipment and equipment such as MR, was time-consuming, and in some cases, the callus was often damaged by the sharp measurements.

[Contents of the Invention] The present inventors have continued their research in search of a method that can quickly and easily detect and select calli that have the ability to regenerate or plant individuals that have started redifferentiation from callus. As a result, we found that there are genes that are not expressed in individuals that do not regenerate, but are expressed only in calli that have the ability to regenerate or in individuals that have started redifferentiation from callus, and that genes with this characteristic can be used as marker genes. The present invention was completed based on the discovery that callus having regeneration ability and individuals that have started redifferentiation from callus can be detected and selected very easily and efficiently.

That is, the present invention introduces into plant tissue a marker gene that is expressed only in calli having regeneration ability or in individuals that have started redifferentiation from callus, forms callus, and then performs callus generation during callus propagation culture or regeneration from callus. This is a method for detecting individuals in which the marker gene is expressed at the early stage of differentiation.

In the present invention, the plant tissue into which a marker gene is introduced includes the plant itself, various organs constituting the plant, parts thereof, and free plant cells, and refers to those capable of forming callus. Plant tissues that can be used in the present invention include, for example, shoot tips (growing points at the tips of plant buds), pollen-containing rot, pollen, roots, stems, leaves, seeds, embryos, ovules, ovaries, and free cells. (particularly protoplasts, which are naked cells whose surrounding cell walls have been removed); more specifically, carrot stems and their protoplasts; tobacco leaf sections and their protoplasts; rice, wheat, Cereal seeds such as corn and barley and their protoplasts;
Fruits 11 (pear, apple, citrus, grape, blueberry, etc.), flowers (carnation, clematis, rose, geranium, poinsettia, begonia, etc.), ornamental plants (Benjamina, rubber tree, etc.), vegetables (strawberries, tomatoes, etc.) , wasabi, Japanese yam, tiger lily, garlic, taro, butterbur, sweet potato, potato, etc.), trees (eucalyptus, poplar, cedar, birch, etc.),
Medicinal plants (many of which include nebula, crow's-eye, red palm, poplar, rhubarb, Japanese cabbage, etc.), plantation crops (banana, pineapple, hemp, sugarcane, etc.)
Examples include the shoot apex of plants (such as cotton) and their protoplasts, and such plant tissues may be obtained and prepared by conventional methods employed in plant tissue culture.

In the present invention, the marker gene is introduced into the plant tissue before callus formation.
It is usually carried out on plant tissue pieces taken out from plants or on protoplasts separated therefrom, and the introduced marker gene is incorporated into the cells of the plant tissue, particularly into its chromosomes.

The expression of the marker gene is controlled by the promoter region, etc. that exists upstream of the marker gene, but such selective expression of the marker gene in callus that has regeneration ability or in individuals that have started redifferentiation from callus. is caused by this promoter region (i.e., the promoter region exhibits its promoting effect specifically only in individuals with redifferentiation ability or those that have already redifferentiated) or by the marker gene itself (i.e., the promoter region is There are cases in which the marker gene has a promoting effect regardless of the presence or absence of differentiation, and is expressed only in calli capable of redifferentiation or in individuals that have started redifferentiation from callus.

As a marker gene, its expression may cause, for example, the generation of fluorescence, the formation of a specific pigment, antibacterial properties against specific bacteria, drug resistance to specific drugs, unique sensitivity to specific plant hormones, or forms that are not present in native plants. It is preferable that the expression thereof be easily detected and that it does not inhibit redifferentiated individuals.

An example of a marker gene that combines 70 motor regions is the soil bacterium Agrobacterium rhizogenes.
bacterium rhizogenes)
-Plasmid 0pen Reading Frame
The promoter region of the e12 (ORF12) gene was converted into β-glucuronidase (β-glucuronidase) of Escherichia coli.
idase; hereinafter referred to as rGUSj) gene, and a chimeric gene in which NPTII (kanamycin resistance gene) is linked to the promoter region of tubalin synthase, etc., and the Agrobacterium rhizogenes. It has been experimentally confirmed that the promoter region of the ORF 1 2 gene of the Ri-plasmid exhibits its promoting effect specifically only in callus having redifferentiation ability or in individuals that have started redifferentiation from callus.

To create a chimeric gene incorporating a marker gene, any of the known methods for introducing a chimeric gene into plant tissue cells or protoplasts can be employed. For example, the chimeric gene can be introduced into cells by directly introducing the chimeric gene itself into plant cells, by inserting the chimeric gene into a vector obtained by improving a plasmid with self-replication ability or by improving 77-di DNA, The vector can also be used in plant tissue cells. This can be carried out by directly introducing the vector into protoplasts, by introducing the vector into bacteria that have a high ability to infect plants, and infecting individual plants with the bacteria.

Methods for introducing the chimeric gene itself or a vector into which the chimeric gene has been inserted into plant cells include methods for introducing it by electrical stimulation (electroporation method), microinjection method, polyethylene glycol method, and direct application method. It will be done. In addition, when introducing a vector into which a chimeric gene has been inserted into a bacterium that is highly capable of infecting plants and infecting individual plants with the bacterium, Agrobacterium chumen, a type of Durum-negative soil bacterium, is used. Fasciens (Agrobac-terium tum)
sfaciens), Agrobacterium rhizogenes
es), various viruses, etc. In particular, Agrobacterium tumefaciens is a suitable bacterium for introducing the above-mentioned marker genes into plant cells. A part of the T-DNA region of this plasmid is integrated into the chromosomal DNA of plant cells.
This is due to the fact that it can be introduced into A to transform cells.

In this case, in addition to the above-mentioned marker gene that indicates the ability to differentiate or differentiate, other characteristics (for example, antibacterial properties against specific bacteria, the ability to form specific active ingredients, resistance to specific drugs, specific By simultaneously incorporating genes expressing plant hormone sensitivity, morphological characteristics different from those of the original plant, etc. into a chimeric gene or vector, it is possible to obtain differentiated individuals that express these characteristics together.

In the present invention, callus is formed from the plant tissue into which the marker gene has been introduced as described above, and the formed callus is further propagated and cultured. Formation of callus and its proliferation can be carried out by any commonly known method, such as inorganic elements, vitamins, carbon sources, VNs as an energy source, plant growth regulators (auxin, cytokinin, etc.). Plant tissue or protoplasts collected aseptically are cultured in a sterilized medium with the addition of hormones, etc., and dedifferentiated to proliferate amorphously; callus is formed (
(hereinafter referred to as "callus induction"), the formed callus is transferred to a new medium containing a plant growth regulator such as auxin and further propagated (subcultured). In this case, plant growth regulators such as auxin and cytokinin are essential for callus induction and subculture. Furthermore, if callus induction is carried out using a solid medium such as agar, and subculture is carried out using liquid culture, each of these processes is performed satisfactorily.

Next, the callus grown through the above subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as "morphogenesis"), and ultimately to regenerate a complete plant. Such morphogenesis can be achieved by appropriately setting the types and amounts of plant growth regulators such as orkinin and cytokinin, various components such as carbon sources, light, temperature, etc. Embryo, adventitious roots, adventitious buds, adventitious stems, leaves, etc. are formed, and the plant grows into a complete plant. In the present invention, morphogenetic culture can be performed by any known method.

In the present invention, the detection and selection of callus having regeneration potential or plant individuals that have started redifferentiation from callus is carried out by examining the expression state of marker genes in the above-mentioned subcultured callus, and/or; This is done by examining the expression of marker genes in individual plants at the beginning of redifferentiation from callus. Detection and selection of marker gene expression may be carried out depending on the expression content of the marker gene introduced into plant cells. For example, if the marker gene produces a specific dye or fluorescence, the detection and selection of the marker gene expression may be carried out depending on the expression content of the marker gene introduced into the plant cell. By testing and selecting for presence/absence and strength, or if the marker gene confers antibacterial properties against specific bacteria or resistance to specific drugs, subcultured plant individuals or plants at the early stage of morphogenesis can be selected. This is carried out by exposing individual plants to the bacteria or drug and collecting those that survive without being attacked by the bacteria or drug.

Then, the callus selected according to the present invention has the ability to regenerate and redifferentiation from the callus is initiated; the individual plant can be grown into a complete plant by a method commonly employed in plant tissue culture, or conditions before becoming a complete plant (e.g. encapsulated artificial seeds, drying pressure,
Freeze-dried cells and tissues, etc.) can be stored, sold, etc.

[Effects of the Invention] In the present invention, a marker gene expressed in a callus having regeneration ability or a plant individual that has started redifferentiation from a callus is introduced into the plant tissue and tissue culture of the plant is performed. It is possible to detect and select calli that have the ability to regenerate and plant individuals that have started redifferentiation from callus at the callus stage (i.e., the callus stage or the early stage of the start of redifferentiation from callus). Plant tissue culture can be performed without requiring space.

In addition, by selecting a marker gene that is easy to detect in the present invention, regeneration from callus or callus that has regeneration ability can be started extremely easily and efficiently without requiring expensive analytical equipment, equipment, or effort. ; Individuals can be detected and selected at an early stage.

EXAMPLES The present invention will be explained below using Examples, but the present invention is not limited thereto.

Example 1 Soil bacterium Agrobacterium rhizogenes (Agr.
Ri of obacterium rhizogenes)
- GUS gene, which is a marker gene of E. coli, was ligated to the promoter region of the 0RF12 gene of the plasmid to form a conjugate 1. Conjugate 2 was also formed by ligating the kanamycin resistance gene NPTn (neomain 7m7o transferase) of Escherichia coli to the 355 promoter gene of cauliflower mosaic virus. Conjugate 2 was ligated downstream of conjugate 1 to create a chimeric gene. This chimeric gene was returned to an E. coli plasmid, and the triparental mating method (Tripar
Agrobacterium tumefaciens, a type of Durham-negative soil bacterium, was
faciens).

Approximately 1 cm of 7-day-old carrot stems grown aseptically
: Cut v'J and treat it with 25μ of the antibiotic kanamycin.
Agrobacterium tumefaciens into which the chimeric gene had been inserted was infected by immersing the above Agrobacterium in a culture solution containing L-broi containing s/mQ and rifampicin 1100 AL/mQ for 2 minutes. Next, 1 mg of 2,4-dichlorophenoxyacetic acid (hereinafter referred to as "2,4-DJ"), which is a synthetic auxin, was added.
/mQ containing Murashige-Sko
og medium (hereinafter referred to as 7MS medium) at 25°C for 2 hours.
Cultured for 1 day.

Add this to the antibiotic Kura7 Oranwo 500μg/m12t7
) and cultured at 25°C for about 1 week to kill Agrobacterium tumefaneens. Further, the cells were cultured for 3 months in MS medium containing kanamycin at a ratio of 100 μg/m (2 parts) to form a callus composed of kanamycin-resistant transformed cells into which the above chimeric gene had been introduced.

The CUS activity of this callus was measured by the following method.

[GUS activity measurement method 1 Lysis buffer based on sodium phosphate
Substrate methyl umbelliferyl g
A mixture containing lucronide (hereinafter referred to as rMUG) is prepared. Add Lysis buffer to the callus in the Ebbendorf tube and homogenize with a glass rod. After centrifuging this, a new Ly
Add sis buffer. Furthermore, create it with the above?
=Add Lysisbuffer containing MUG and heat to 37℃
The time in the incubator is set as 0 minutes, and 30 minutes later, the reaction is stopped by adding 0.2M sodium carbonate.

This was applied to a fluorescence spectrophotometer to measure absorbance at a wavelength of 455 nm, and GUS activity was measured using a calibration curve prepared in advance.

As a result, no expression of characteristics based on the GtlS gene was detected in the callus.

This callus was divided into pieces with an average size of 100 μmm and transferred to a liquid MS medium containing no plant hormones at 25°C.
The cells were cultured for 3 days; however, on the 3rd day, early somatic embryos that could be barely observed with the naked eye were formed for the first time, and the number of such somatic embryos gradually increased. The number of early somatic embryos formed during this period was counted over time and GUS activity was examined using the method described above, and the results shown in I in the figure were obtained.

In addition, early somatic embryos exhibiting GUS activity that were formed during the 3-day culture were separated and selected from undifferentiated individuals that did not exhibit GUs activity, and these were further cultured in hormone-free MS medium for 25 minutes. ℃ for 3 days to produce somatic embryos that can be easily observed with the naked eye, and coat the somatic embryos with sodium alginate and drop each embryo into a calcium chloride aqueous solution. encapsulated artificial seeds were obtained.

For comparison, transformed callus into which the GUS gene had been introduced was formed from carrot stems in the same manner as above, and after dividing it into pieces of about 100 μm on average, 2.4-D
The embryos were cultured in a liquid MS medium containing 1 mg/mQ at 25°C in the same manner as above, but no somatic embryos were formed even after 12 days, and the GUS activity of these was measured over time in the same manner as above. However, the results shown by ■ in the figure were obtained.

In the figure, 1 unit on the vertical axis (GUS activity) refers to the amount of 1 pumole of 4-MU produced per 1 minute per 1 mg of protein.

From the above results and the results shown in the figure, CUS activity is expressed with somatic embryogenesis (redifferentiation) and increases as somatic embryogenesis progresses, and therefore the marker gene G
It can be seen that by examining the l: cu s activity expressed by US, the presence or absence of regeneration ability of an individual plant or the progress state of regeneration can be detected at an early stage of culture.

Example 2 Aseptic, 1. : A 0.5 cm square section was cut from a cultivated tobacco leaf.

This tobacco collection section was treated with the antibiotic kanamycin 25 μg/m
Agrobacterium tumefaciens into which the same chimeric gene as that created in Example 1 above was inserted by immersing Agrobacterium in L-medium containing Q and rifampicin 100 μg/m Q for 2 minutes. Infect the person.

MS containing this at a rate of 1 mg/mQ of 2.4-D
The cells were cultured on a medium at 25°C for 2 days.

The antibiotic Tarafuoran uses this at 500μs/mQ
Agrobacterium tumefaciens was killed by transferring to MS medium containing a proportion of Further, the cells were cultured for about one month in MS medium containing kanamycin at a ratio of 100 mg/m+2 to form a callus composed of transformed cells resistant to kanamycin, in which the chimeric gene was introduced.

This callus was divided into pieces of about 100 μm on average, transferred to MS medium containing 1 ppm of cytokinin BA (benzyladenine), and cultured at 25°C. At the 149th day, early adventitious buds that could be clearly observed with the naked eye were formed for the first time. The number of such adventitious buds increased. As a result of microscopic observation, blue staining was observed only in the unique tissue of the early adventitious buds formed during that time, which indicated the expression of GUS.

Since GUS activity increases in the tissues of adventitious buds that regenerate into plants, it is clear that the presence or absence of differentiation can be examined and screened by simple microscopic evaluation.

Example 3 Both a plasmid containing the same chimeric gene as prepared in Example 1 and a plasmid (pCH) containing a gene resistant to the antibiotic hygromycin were introduced into rice protoplasts by electroporation.

M containing 2.4-D at a ratio of 1 mg/α
The cells were cultured on S medium at 25°C. 3 more hygromycin
The cells were cultured for one month in MS medium containing 0 μg/m Q to form a callus composed of λ igromycin-resistant transformed cells into which the chimeric gene was introduced.

This callus was mixed with M containing 100 μg/rnQ of kanamycin.
When transferred to S medium and cultured at 25°C, early adventitious buds that could be barely observed with the naked eye were formed for the first time on day 35, and the number of such adventitious buds increased sequentially.

The number of early adventitious buds formed during that time was counted over time, and the GUS activity was examined by the method of Example 2; however, the same results were obtained.

[Brief explanation of drawings]

The figure shows the relationship between the differentiation of plant individuals and the activity of the marker gene GUS in tissue culture of carrot stems.

Claims (1)

[Claims] A marker gene expressed in callus having regeneration ability or in an individual that has started redifferentiation from callus is introduced into plant tissue to form callus, and the marker gene is introduced into the plant tissue during the propagation culture of callus or at the early stage of the start of regeneration from callus. A method to detect individuals in which the gene is expressed.