Composition for regulation of gametophytic self-incompatibility, control method of gametophytic self-incompatibility of a plant and the plant self-pollinated by using said control method

This application has been filed under 35USC 371 as the national stage of international application PCT/KR01/00306, filed Feb. 28, 2001.

The present invention relates to a regulation composition for gametophytic self-incompatibility, a control method for gametophytic self-incompatibility and a plant self-pollinated by using the control method. More particularly, the present invention relates to a regulation composition for gametophytic self-incompatibility containing sulfates, especially CuSO 4 and ZnSO 4 , as an inhibitor that prevents a style-specific RNase activity regulating gametophytic self-incompatibility; a control method for gametophytic self-incompatibility of plants by using the regulation composition for gametophytic self-incompatibility; and a plant self-pollinated of which gametophytic self-incompatibility is destroyed by using the control method.

Over half of the flowering plants in this world have gametophytic self-incompatibility. The gametophytic self-incompatibility means the property that is not self-pollinated. Therefore, the gametophytic self-incompatible plants can be pollinated only by genetically different pollen. So, such plants can only bloom without any fruition in case that there is only one species.

On the other hand, natural pollination between plants belonging to different species is mediated by pollinators such as honeybee and drone fly, or by wind. However, pollinators are decreasing suddenly in recent years due to environmental pollution according to the excess use of agricultural chemicals and rapid industrialization. Although the number of pollinators and pollinizers is sufficient, it is difficult to achieve the stable rate of fruitions in case that the working of pollinators is disturbed by the hindrance factor of weather conditions such as low temperature, strong wind, and rain fall, which appear in blooming season every year.

Furthermore, the rate of natural fruition in the farm house cultivating only one species of crop for high profit gets to further decrease because pollinizers are insufficient.

Therefore, since it is difficult to acquire both fruition stability of crops and good quality of fruits under the condition of natural pollination, in recent years artificial pollination using imported pollens has been carried out, or a method that releases pollinators such as artificially bleeded Osmia ocrnifrons which is imported from foreign countries in blooming season has been used. Especially, in artificial pollination method, pollens of different pollinizers are smeared on the stigma of style by human, not by pollinators. So it is not economical because it needs many labor and high costs.

In natural fruit trees, flowering plants, medicinal plants, and vegetables of eggplant family, a style-specific RNase is secreted from a pistil, the reproductive organ RNase having the different genetic phenotype according to the species exists in a style. The mechanism is as follows. A style-specific RNase is secreted when self-pollen tube elongates from a style of a pistil to an ovary, and degrades only rRNA of self-pollen selectively (McClure et al, Nature, 1991). By the mechanism, the pollen tube doesn't elongate to the ovary, that is, the elongation of pollen tube is destroyed at a specific site, ⅓ point of a pistil, and finally fruition cannot be produced as a result of being unable to accomplish pollinatioin by self-pollen.

Furthermore, according to the research up to now, the RNase secreted from a style enters into self- or nonself-pollen unselectively, but the RNase binds to an inhibitor or a receptor within self-pollen which react with the RNase specifically. It is speculated that this reaction is related to the signal transduction pathway degrading rRNA of self-pollen selectively, and the pollination cannot be accomplished because self-pollen tube is destroyed before it reaches to the ovary in case of self-pollen.

On the other hand, nonself-pollen, which is originated from pollinizer having different genetic phenotype transferred by pollinators or wind, can elongate the pollen tube normally. This phenomenon is illustrated by the hypothesis according to the research up to now. The hypothesis is as follows. Since the structure of the receptor that binds with RNase secreted from a pistil which exists in the pollen of different phenotype, is not identical to that of the receptor of its own phenotype, signal transduction pathways are not progressed thereinafter. As a result, RNase does not attack rRNA of nonself-pollen, and the elongation of pollen tube is induced normally so that pollination can be achieved finally.

Though many researchers in the world keep studying an inhibitor or a receptor molecule of pollen now, however, they have not found clear clue about that. It was only reported that RNase secreted from a style was found in the elongation tissue of pollen tube by observing through the microscopic and immunological method.

Furthermore, it has been reported that gametophytic self-incompatibility of fruit trees like apple, pear, coffee and almond as well as some flowering plants, medicinal plants and eggplant family like wild-type tomato, eggplant, tobacco and potato, is regulated by pistil(style) and gene-specific RNase (II-Kyung, Chung et al., Plant Molecular Biology, 26:757-762, 1994; II-Kyung, Chung et al., Journal of Korean Breeding Science, 29(1):41-46, 1997; II-Kyung, Chung et al., Journal of Plant Physiology, 154:63-70, 1999).

In relation to the report, the present inventor previously reported a style-specific RNase isolated from wild-type tomatoes in 1992 (II-Kyung, Chung et al., Bioscience Biotechnology Biochemistry 57(7):1172-1176, 1993; Japanese patent application No. 1,262,865). Also the present inventor identified a gene related to gametophytic self-incompatibility of plants like tomato and Lillop Koreana Kakai (II-Kyung, Chung et al., Plant Molecular Biology, 26:757-762, 1994; II-Kyung, Chung et al., Plant Cell Physiology, 36(8):1621-1627, 1995; Japanese Patent No. 7-187557).

To overcome the problem of the gametophytic self-incompatible plants, the present inventor identified an inhibitor preventing a style-specific RNase activity, provided a control method for gametophytic self-incompatibility by using the inhibitor and a plant self-pollinated, and suggested a new agricultural method that is very economical as well as ensures good quality of fruits by manipulating plants of fruit trees to be pollinated or producing fruits by self-pollen.

The object of the present invention is to provide a regulation composition for gametophytic self-incompatibility containing an inhibitor, which prevents a style-specific RNase regulating gametophytic self-incompatibility.

Another object of the present invention is to provide a control method for gametophytic self-incompatibility by using the regulation composition.

Also the object of the present invention is to provide a plant self-pollinated in which gametophytic self-incompatibility is destroyed.

Hereinafter, the present invention is described in detail.

The present invention provides a regulation composition for gametophytic self-incompatibility which includes inhibitor inhibiting a style-specific RNase activity regulating gametophytic self-incompatibility.

The present inventor has tried to induce pollination by self-pollen by controlling gametophytic self-incompatibility of plants existing in the natural world, by finding out the style-specific RNase inhibitor related to gametophytic self-incompatibility.

First of all, the present inventor isolated and purified RNase from the style, the reproductive organ of Fuji apple, which plays a critical role in causing gametophytic self-incompatibility (see table 2). Afterwards, the present inventor isolated RNase respectively from a root, leaf, stalk, petal and calyx of Fuji apple, and electrophoresed the said RNases with the RNase isolated from a style of Fuji apple. Hence, the present inventor confirmed that RNase isolated from a style is a style-specific RNase by RNase activity staining (see FIG. 1 ). Furthermore, to investigate the difference between style-specific RNases according to species, the present inventor isolated and purified RNase from the style of Hongro, Hongok, Fuji, Gookwang and Sgaroo, and then, performed RNase activity staining and silver staining. As a result, it was confirmed that style-specific RNases are different according to species of apple (see FIG. 2 ).

To confirm the style-specific RNase of the Fuji apple controls gametophytic self-incompatibility, the present inventor observed the elongation pattern of pollen tube of Fuji apple by adding the style-specific RNase to the medium for pollen tube elongation (William Jahnen et al., Plant Cell , (1):501-510, 1989; Harris et al., Plant Physiology, 189:360-367, 1989), which can induce the elongation of pollen tube from pollen artificially (see FIG. 3 ).

From the result, it can be confirmed that a style-specific RNase controls gametophytic self-incompatibility by inhibiting the elongation of pollen tube.

The present inventor observed the elongation patterns of pollen tube of Fuji apple by adding a style-specific RNase of Fuji apple and various chemicals to the medium for pollen tube elongation, and by using the creative experimental method provided in the present invention (See FIGS. 4 and 5 ). As a result, RNase activity inhibiting the elongation of pollen tube was inhibited effectively by a sulfate and the pollen tube elongated normally. From the result, it is confirmed that metal ion-bound sulfate is an inhibitor of the RNase. Hence, the sulfate functions as a regulation composition for gametophytic self-incompatibility, particularly, among sulfates, CuSO 4 , MgSO 4 , ZnSO 4 and MnSO 4 are preferred to the regulation composition for gametophytic self-incompatibility, and CuSO 4 and ZnSO 4 are more preferable.

Further, the present invention provides a control method for gametophytic self-incompatibility by using the regulation composition for gametophytic self-incompatibility.

Upon the basis of the experimental results performed by using the medium for pollen tube elongation, the present inventor put into practice the inhibition effect of gametophytic self-incompatibility by regulation composition for gametophytic self-incompatibility of the present invention in wild-type tomato and Fuji apple, which are gametophytic self-incompatible crop growing in the practical cultivation environment.

First of all, to determine the period of treating the regulation composition for gametophytic self-incompatibility of the present invention, the present inventor treated wild-type tomato with 1 mM CuSO 4 and ZnSO 4 from 7 days before blooming to 2 days after full blooming, and Fuji apple with 1 mM CuSO 4 and ZnSO 4 from 5 days before blooming to 2 days after full blooming to investigate the rate of self-pollination. When wild-type tomato was treated with the regulation composition for gametophytic self-incompatibility of the present invention at the early blooming period, from 7 days before blooming to 4 days before blooming, the fruition rate was over 70%. And when Fuji apple was treated with the regulation composition for gametophytic self-incompatibility of the present invention at the early blooming period, from 5 days before blooming to 3 days before blooming, the fruition rate was also over 70%. Therefore, gametophytic self-incompatibility in plants can be controlled by the method of spraying the regulation composition for gametophytic self-incompatibility including an inhibitor of style-specific RNase activities during a specific period of plant growth, and it is preferred that treatment period of the regulation composition for gametophytic self-incompatibility is from budding formation period, a specific period of plant growth to the early blooming period, before full blooming.

Moreover, to investigate preferable concentration of treatment of the regulation composition for gametophytic self-incompatibility of the present invention, 0 or 1,500 ppm CuSO 4 and ZnSO 4 were treated to wild-type tomato and Fuji apple at the early blooming period and the harmful effects of a medicine and fruition rates were investigated (see FIGS. 6 and 7 ). Since absorption rate of the regulation composition for gametophytic self-incompatibility of the present invention can be increased by treating it with a spreader in cultivating fruit trees, the CuSO 4 and ZnSO 4 were treated with a spreader by a method of spraying on the fruit trees at a specific period, that is, the early blooming period.

Thus, it was shown that fruits and plants that are pollinated by self-pollen could be acquired by the treatment of a specific concentration of CuSO 4 and ZnSO 4 in wild-type tomato and Fuji apple without harmful effects of the medicine. From the results, it can be concluded that an inhibitor composition of a style-specific RNase of the present invention is effective in inhibition of a style-specific RNase without respect to species, and preferable concentration of CuSO 4 , which is treated at the early blooming period, is at 100˜700 ppm and preferable concentration of ZnSO 4 is at 100˜800 ppm. Furthermore, very high pollination rate, without harmful effects of a medicine, could be achieved by spraying the inhibitor composition for gametophytic self-incompatibility of the present invention, which was mixed with the desirable amount of spreader according to the manufacturers' instruction. Among the spreaders, the spreader of hexaconazole class, siloxane class and alkylaryl polyethoxylate class is preferable to achieve high fruition rate.

The preferable concentration of the inhibitor composition for gametophytic self-incompatibility of the present invention was shown in table 1.

When CuSO 4 is used as an effective component of the style-specific RNase inhibitor as shown in the table 1, if the concentration of CuSO 4 is below 100 ppm, it is difficult to achieve a stable fruition rate over 70% because it cannot inhibit RNase activity appropriately, on the other hand, if the concentration of CuSO4 is over 700 ppm, it is not economical because it can induce harmful effects of a medicine. Furthermore, when ZnSO 4 is used as an effective component of the style-specific RNase inhibitor, if the concentration of ZnSO 4 is below 100 ppm, it is difficult to achieve a stable fruition rate over 70% because it cannot inhibit RNase activity appropriately, on the other hand, if the concentration of ZnSO 4 is over 800 ppm, it is not economical because it can induce harmful effects of a medicine.

Moreover, the present invention provides a plant self-pollinated by destroying the gametophytic self-incompatibility using the control method.

As the results mentioned above, a self-pollinated plant in which gametophytic self-incompatibility is destroyed can be acquired by using the regulation composition for gametophytic self-incompatibility of the present invention. In examples of plants which can be applied to the control method for gametophytic self-incompatibility of the present invention, there are fruit trees such as apple, pear, coffee and almond; flowering plants; medicinal plants; and vegetables of eggplant family such as tomatoes, eggplants, tobaccos and potatoes in addition to Fuji apple and wild-type tomato, which were used in the preferred embodiment of the present invention.

FIG. 1 shows the result of activity staining of RNase which is isolated from the root, leaf, stalk, petal, calyx and style of Fuji apple and degrades ribosomal RNA of yeast.

→: a band showing a style-specific RNase activity

FIG. 2 shows the result of silver staining of the total protein extract isolated from the style of Hongro, Hongok, Fuji, Gookwang and Sgaroo, which are agricultural apple species.

→: a band showing a genetic phenotype-specific RNase activity of Fuji apple

M: Standard Molecular Marker

FIG. 3 represents microscopic photography showing the result that the elongation of pollen tube of Fuji apple is inhibited in the medium for pollen tube elongation including style-specific RNase.

FIG. 4 represents a graph showing the inhibition degrees of chemical components inhibiting style-specific S1 and S2 RNase activities, which are isolated and purified from the style of Fuji apple.

FIG. 5 represents microscopic photography showing the result that the pollen tube of Fuji apple elongates in the medium for pollen tube elongation containing a style-specific RNase and inhibitor composition thereof.

A: addition of 1 mM ZnSO 4 and 10 units of RNase

B: addition of 2 mM ZnSO 4 and 10 units of RNase

C: addition of 5 mM ZnSO 4 and 10 units of RNase

D: addition of 1 mM CuSO 4 and 10 units of RNase

E: addition of 2 mM CuSO 4 and 10 units of RNase

F: addition of 5 mM CuSO 4 and 10 units of RNase

FIG. 6 a is a picture showing wild-type tomato at the early blooming period before it is treated with a style-specific RNase inhibitor composition of the present invention.

FIG. 6 b is a picture showing wild-type tomato pollinated by self-pollen by destroying the garnetophytic self-incompatibility after it is treated with a style-specific RNase inhibitor composition of the present invention.

FIG. 7 a is a picture showing Fuji apple at the early blooming period before it is treated with a style-specific RNase inhibitor composition of the present invention.

FIG. 7 b is a picture showing Fuji apple pollinated by self-pollen by destroying the gametophytic self-incompatibility after it is treated with a style-specific RNase inhibitor composition of the present invention.

The present invention is more specifically illustrated by the following examples.

However, it should be understood that these examples are provided only for illustration of the present invention, but not intended to limit the present invention in any manner.

As shown in the above results, gametophytic self incompatibility was destroyed by treating with sulfates, especially CuSO 4 and ZnSO 4 , the style-specific RNase inhibitor of the present invention, at the early blooming period (budding formation period˜prior to full blooming), and the pollination and fruition by self-pollen was induced stably. Therefore, the fruit and plant pollinated by self-pollen can be obtained by treating a composition, which comprises a style-specific RNase inhibitor of the present invention as a effective component, to gametophytic self-incompatible fruit tree such as apple, pear, coffee, and almond etc. with optimal concentration, not by cultivating another pollinizer. As well, the present invention provides an innovative cultivating method capable of maximizing the yield per the unit area, since high fruition rate can be achieved without assistance of pollinators.