JP3577530B2 - How to create mutant plants - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for minimizing plant damage and inducing a mutant plant exhibiting various morphologies at a high rate, and more particularly to a method for producing a mutant plant by irradiation with heavy ion beams.
[0002]
[Prior art]
Irradiation of the plant with gamma rays or X-rays or treatment of the plant with an agent such as ethyl methanesulfonate (EMS) can artificially mutate the plant [Euphytica 69: 95-101 (1993) )]. Since some of such mutants have superior traits not found in conventional plants, plant breeding using mutations has been conventionally performed.
[0003]
However, it is difficult to accurately irradiate specific tissues of plants with radiation such as gamma rays and X-rays, so a great deal of work is required to fix genetic mutations that occurred in some cells as genetic traits of the entire individual And In addition, drugs such as EMS induce mutations, damage plants themselves, and reduce germination rates, so that it is difficult to obtain a large number of mutants.
[0004]
Incidentally, radiation called a heavy ion beam can be produced by ionizing and accelerating various elements such as carbon and nitrogen using a cycloton. As an example of producing a mutant of a plant by heavy ion beam irradiation, there is an example of irradiating a rice seed with a heavy ion beam to obtain a mutant strain resistant to bacterial blight of rice (RIKEN Accel. Prog. Rep. 26: 109, 1992).
[0005]
However, since this method irradiates the seeds with a heavy ion beam, the seed embryo part that grows as a plant has already differentiated into many cells, and it is difficult to cause mutation only in the cells that carry out genetic control. In addition, genes are fixed in a stable structure in dried seeds, and have a drawback such that when the state is changed by mutation, they often become lethal.
[0006]
[Problems to be solved by the invention]
As described above, artificially inducing a mutation is a very useful technique for producing a plant with excellent traits, but the conventional method still has a satisfactory frequency of mutants. Could not be induced.
The present invention has been made in view of such problems of the prior art, and it is an object of the present invention to minimize the damage to plants and to induce mutants exhibiting various morphologies at a high rate. It is to provide a method that can be performed.
[0007]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems, and as a result, by irradiating the plant ovary after pollination with a heavy ion beam, it is possible to induce mutants exhibiting various forms at extremely high frequency. And completed the present invention.
That is, the present invention provides a method for producing a mutant plant, comprising irradiating a plant ovary after pollination with a heavy ion beam and selecting a plant having a morphological abnormality from the irradiated plants. It is.
[0008]
The present invention also provides a method for producing a chlorophyll-deficient plant, which comprises irradiating a plant ovary after pollination with a heavy ion beam, and selecting a chlorophyll-deficient plant from the irradiated plants.
Hereinafter, the present invention will be described in detail.
The heavy ion beam used in the present invention is not particularly limited as long as it can induce a mutant, and for example, a nitrogen ion beam, a carbon ion beam, a neon ion beam, an argon ion beam and the like can be used. The dose of the heavy ion beam may be determined according to the type of ion beam to be used, the type of plant to be irradiated, and the like. Since the induction frequency is low, it is preferable to set the range of 5 to 100 gray. If the irradiation range of the heavy ion beam is too wide, damage to the plant is increased. Therefore, it is preferable to set the diameter to about 5 to 20 mm.
[0009]
The plant to be subjected to heavy ion beam irradiation is not particularly limited, but preferred examples include tobacco, Arabidopsis, rice, orchid, and the like. The irradiation time of the heavy ion beam is preferably to irradiate the egg cell immediately after fertilization, which is likely to cause mutation. More specifically, it is preferably 2 to 108 hours from pollination (immediately after fertilization to 80 hours).
[0010]
After irradiating the plant ovary after pollination with a heavy ion beam, seeds of the plant are sown and germinated. At the germination stage, a mutant can be obtained by selecting an individual having a morphological or physiological abnormality. The morphological abnormality here means different from the normal plant in appearance, for example, chlorophyll deficiency, needle-like leaves, gravity insensitivity, dwarfism, premature birth, etc., and physiological abnormalities include drug resistance, environmental stress resistance, etc. It is. In addition, among the deficiencies of chlorophyll referred to here, in addition to those that do not contain chlorophyll at all, those that lack so-called variegated chlorophyll and those that exhibit light green color because the amount of chlorophyll is less than that of normal plants are also included. Including.
[0011]
【Example】
[Example 1]
Nitrogen ions (7+: 135 MeV / u) were accelerated by a RIKEN cyclotron and irradiated to the ovary of tobacco variety BY-4 at a diameter of 10 mm (see FIG. 1). In the experiment, the irradiation time was divided into five stages of 36 hours, 48 to 60 hours, 72 to 84 hours, and 96 to 108 hours after pollination, and the dose was divided into five stages of 5, 10, 50, 100, and 200 gray. Was. The LET (linear energy transfer) at this time was 28.5 KeV / mm. One month after irradiation with the nitrogen ion beam, M1 seeds were harvested and the germination rate of the seeds was determined. The result is shown in FIG. As shown in FIG. 2, when the dose is 200 Gray, the germination rate is extremely low, and it is estimated that the plant body has been seriously damaged.
[0012]
Germinated plants contained individuals exhibiting morphological abnormalities. These appearances are shown in FIGS. FIG. 3 shows an individual irradiated with a nitrogen ion beam at a dose of 10 Grays 96 to 108 hours after pollination, in a state without any chlorophyll (albino). FIG. 4 shows an individual irradiated with a nitrogen ion beam at a dose of 10 Grays 72 to 84 hours after pollination, showing a small amount of chlorophyll and a pale green color. FIG. 5 shows an individual irradiated with a nitrogen ion beam at a dose of 10 Grays 72 to 84 hours after pollination, and had needle-like leaves. FIG. 6 shows an individual irradiated with a nitrogen ion beam at a dose of 200 Gray 96 to 108 days after pollination, and had needle-like leaves. FIG. 7 shows an individual irradiated with a nitrogen ion beam at a dose of 10 Grays 72 to 84 hours after pollination, and showed a normal form.
[0013]
Plants that germinated as described above exhibited various morphological abnormalities, but by focusing on chlorophyll deficiency among these morphological abnormalities, the proportion of chlorophyll-deficient individuals in all germinated individuals was determined. The result is shown in FIG. As shown in FIG. 8, when irradiation was performed 48 to 60 hours and 96 to 108 hours after pollination, the proportion of chlorophyll-deficient individuals was highest when the dose was 100 Gray, but 72 to 84 hours after pollination. When the irradiation was performed later, the proportion of chlorophyll-deficient individuals was highest when the dose was 10 Gray. When irradiation was performed 32 hours after pollination, the proportion of chlorophyll-deficient individuals was low at all doses.
[Comparative Example 1]
As test plants, two tobacco varieties (Xanthi and BY-4) were used.
[0014]
The ovary from which the style was removed was covered with absorbent cotton impregnated with 0.1% EMS, and left for 1 day. At this time, the treated portion was covered with a microtube to prevent drying. This treatment by EMS was performed in five stages 36, 48, 54 and 60 hours after pollination. One month after the treatment with the mutagen, M1 seeds were harvested and the germination rate was determined. The result is shown in FIG. As shown in FIG. 9, no reduction in the germination rate was observed at any stage in Xanthi, but in BY-4, when the treatment was performed 36 hours and 48 hours after pollination, the germination rate was reduced. Was observed.
[0015]
The germinated individuals included individuals showing various morphological abnormalities as in Example 1, but no chlorophyll-deficient individuals were found.
The percentage of morphologically abnormal individuals in all germinated individuals was determined. Further, the morphological abnormalities were classified into A) one-lobe type, B) three-lobe type, C) and other three types, and the ratios indicated for each were determined. The results are shown in FIGS. As shown in FIG. 109, the morphologically abnormal individuals were the most frequent when the processing was performed 36 hours in BY-4 and 48 hours in Xanthi. Among the observed morphological abnormalities, a one-lobe type morphological abnormality was frequently observed in Xanthi, and a three-lobe type morphological abnormality was frequently observed in BY-4.
[0016]
【The invention's effect】
The present invention provides a novel method for producing a mutant plant. This method has the following features.
(1) Since the ovary is irradiated with a heavy ion beam, a large amount of mutant seeds can be reliably produced.
Therefore, new functions can be confirmed in plant solids.
(2) Since the heavy ion beam is irradiated during the gene growth phase after fertilization, lethal mutation can be reduced.
(3) A heavy ion beam is limited to the ovary and is irradiated instantaneously, so that lethal mutation can be reduced.
(4) Albino mutant plants can be efficiently produced.
(5) Since the primordial (egg) cells are irradiated with a heavy ion beam, it is easy to obtain homozygotes having a uniform genetic trait.
[0017]
As described above, the present production method is capable of minimizing plant damage and inducing mutant plants exhibiting various morphologies at a high rate, and is an extremely useful technique in the field of plant breeding.
[Brief description of the drawings]
FIG. 1 is a diagram showing a method of irradiating a plant ovary after pollination with a heavy ion beam.
FIG. 2 is a diagram showing a relationship between a dose of a nitrogen ion beam and a germination rate.
FIG. 3 is a photograph showing the morphology of a morphologically abnormal individual.
FIG. 4 is a photograph showing the morphology of a morphologically abnormal individual.
FIG. 5 is a photograph showing a morphology of a morphologically abnormal individual.
FIG. 6 is a photograph showing the morphology of a morphologically abnormal individual.
FIG. 7 is a photograph showing the form of a normal individual organism.
FIG. 8 is a diagram showing the relationship between the dose of a nitrogen ion beam and the frequency of occurrence of chlorophyll-deficient individuals.
FIG. 9 is a diagram showing the relationship between EMS treatment time and germination rate.
FIG. 10 is a diagram showing the relationship between the processing time of EMS and the frequency of occurrence of abnormal morphological individuals.
FIG. 11 is a view showing the ratio of single-lobe abnormal morphological individuals and three-lobe abnormal morphological individuals to all abnormal morphological individuals.

Claims (2)

Irradiating the plant ovary after pollination with a heavy ion beam, and selecting plants having morphological abnormalities from plants obtained by germinating seeds derived from the irradiated plant ovary. A method for producing a mutant plant. A chlorophyll-deficient plant, wherein a chlorophyll-deficient plant is selected from plants obtained by irradiating a plant ovary after pollination with a heavy ion beam and germinating seeds derived from the irradiated plant ovary. How to create.

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