JPS6158598A - Sexing of dioecious plant in seedling stage - Google Patents

Abstract

PURPOSE:To enable the early and easy determination of the sex of a dioecious plant in seed proliferation, by determining the peroxidase activity or esterase activity of the plant. CONSTITUTION:The juice collected from the leaf of the plant to be examined is subjected to the electrophoretic treatment to effect the development of the enzymes of the juice. The enzymes are dyed with a specific reagent, and the sex of the plant is judged according to the pattern of the isozyme. Substances having electrical charge, e.g. enzymes, etc. migrate toward the cathode or anode under electrical field by electrophoresis. As a result of the difference of the electrophoretic behavior of isozymes of male and female plants, it has been found that peroxidase or esterase gives different patterns between the sexes independent of the stage of growing, i.e. grown plant or seedling stage. The sex of the plant can be determined by the pattern. Since the sex of the plant can be determined by this process only by collecting a piece of leaf, the seed proliferation which cannot hitherto be used can be carried out thereby.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for determining the sex of dioecious plants, and particularly to a method for determining the sex of plants at the seedling stage.

(Prior Art) In dioecious plants, only the female plants produce fruits and are often useful. However, there was a problem in that it was not possible to determine the sex of the seedlings at the seedling stage.

For example, date palms are female NE and are important as food in arid and semi-arid areas of the tropics.They also contain a large amount of sugar in their fruit, and sugar can also be obtained from the sap, making them alcoholic. It can also be used as a raw material, and 125 sentences from 1 ton of fresh fruit (1 ton of fresh fruit)
It is said that it is possible to produce ethanol of 350-400M).

For practical cultivation, it is said that one male plant for every 50 to 100 female plants is sufficient for this date palm. Seed propagation has a high multiplication rate, but in the case of these seedlings, the appearance rate of male plants is high, and it is not possible to distinguish between the sexes until the flowering period, 5 to 10 years after sowing, so there is a lot of waste. Seed propagation methods have not been put to practical use. The conventional method is to use suckers from female plants.
r), but this method has the problem of low multiplication rate.

(Problems to be solved by the invention) The present invention does not use the conventional sucker method, but uses seed propagation (seedlings) to easily distinguish between males and females in 7 stages, leaving only female plants and removing male plants. The purpose of this study is to provide a method for determining sex.

(Means for Solving the Problems) The sex discrimination method of the present invention is characterized by measuring peroxidase activity and/or esterase activity possessed by dioecious plants.

More specifically, the enzymes in the sap taken from the leaves of the plant to be discriminated are developed by electrophoresis, and the enzymes in the sap are stained with a predetermined chemical to discriminate based on the isozyme pattern.

Since the present invention aims at early identification, leaves of plants in the seedling stage are used, but it can of course be applied to adult trees as well.

In the following description, starch gel electrophoresis is used as the electrophoresis method, but other electrophoresis methods may be used.

(Function) The present invention is based on the electrophoretic phenomenon in which a substance with its own charge, such as an enzyme, moves toward an anode or a cathode in an electric field, and is the result of investigating the differences in isozymes between male and female plants. , peroxidase, or esterase, female and male plants show different patterns for both adult trees and young seedlings, so males and females are determined based on the results of these patterns.

(Example) The present invention will be explained below with reference to Examples.

Example 1: Distinguishing the sexes of date palms First, 60 g of thawed starch was added to 50 mJ of a buffer solution with a pH of 8.5.
A starch gel electrophoresis tank was prepared by placing the starch gel in water of step 1, heating it, molarizing it, and then pouring it into a migration tank to solidify it.

Next, the date palm leaves to be identified were collected, the leaf pieces were ground in a mortar, and the resulting sap was adsorbed onto Toyo Filter Paper Th, 50. The filter paper that has absorbed the leaf juice in this way is
After immersing it in the starch gel in the electrophoresis tank, place it in a cold storage at 10°C or less to prevent a decrease in activity, and apply 200 V DC.
The electrophoresis was carried out under constant voltage for 4 hours.

After the electrophoresis was completed, peroxidase and esterase isozyme staining was performed in the following manner.

a) Peroxidase = 1% orthodianisidine solution in acetone (30 m), pH 4,0) Lis-acetate buffer (30 m)
Text, water 237m text.

After mixing, a total of 300 mJl of 3% hydrogen peroxide solution was poured into the gel, reacted for 10 minutes, washed with water, and examined the enzyme zone.

b) Esterase: 0. IM-α-naphthyl acetate acetone solution 3 mJL, water 270 mJ1. pH7,
After stirring, 300 mg of Fast B Iue RR salt was added and poured into the gel while filtering through gauze. After reacting for 60 minutes, the mixture was washed with water and the enzyme zone was examined.

Through the above procedures, we investigated the possibility of determining the sex of seedlings from the isozymes of peroxidase and esterase.

The results are shown in FIGS. 1 to 4.

Figure 1 shows the electrophoretic pattern of peroxidase zymodalum in leaves of adult trees, and Figure 2 shows the pattern of peroxidase zymograms in leaves of seedlings.

Furthermore, FIGS. 3 and 4 are diagrams showing the patterns of esterase zymograms, with FIG. 3 showing the results for leaves of adult trees, and FIG. 4 showing the results for leaves of seedlings.

As can be seen from the results in Figures 1 to 4, there is no difference in both adult trees and seedlings between male and female plants in the esterase zymogram pattern, but there are clearly different patterns between the male and female plants in the peroxidase zymogram. Since male plants show the same pattern in both adult trees and seedlings, it was confirmed that it is possible to distinguish between the sexes of date palms at the young seedling stage.

In addition, in Figures 1 to 4, the numerical values are at the origin (Or
igin), + indicates the anode side, - indicates the cathode side.

For mature trees, the Ibusuki Experimental Site of the Faculty of Agriculture, Kagoshima University (Ibusuki City, Kagoshima Prefecture), the Greenhouse of the Tropical Agriculture Research Center of the Ministry of Agriculture, Forestry and Fisheries (Yatabe Town, Tsukuba District, Ibaraki Prefecture), and the entrance of New Fuji Hotel in Atami City, Shizuoka Prefecture, and the same city. Leaves were collected from a total of 10 plants, 1 to 2 of each sex, planted at four locations on the coast, and seeds were collected from the Ibusuki Experimental Site of the Faculty of Agriculture, Kagoshima University and the Tropical Agriculture Research Center of the Ministry of Agriculture, Forestry and Fisheries in Tsukuba. The tests were conducted using leaves (juvenile leaves and pinnate compound tJ) collected from 30 plants that were sown and cultivated in the University Agriculture and Forestry Technology Center greenhouse and the Biological Research Institute greenhouse. As a result, all showed similar results. This shows that there are no differences depending on the breeding area.

Example 2: Determining the sex of jojoba at the seedling stage using zymograms Jojoba is grown in tropical areas near the border between the United States and Mexico.
The wax contained in the fruit of a shrub belonging to the boxwood family that grows in semi-arid subtropical regions is considered to be a useful resource.

However, jojoba is also a dioecious plant and it is difficult to tell the sexes until the flowering stage. Therefore, as in the date palm shown in Example 1, when the isozymes of peroxidase and esterase were analyzed by electrophoresis, differences between males and females were observed in both the peroxidase zymogram and the esterase zymogram, and it was determined that it was possible to distinguish between the sexes at the seedling stage. Ta.

The results are shown in FIGS. 5 and 6. Regarding the peroxidase Zymodalum shown in Figure 5, in the leaves of the female plant, O is at the cathode.
A light blue color at a distance of 8 to ioam from rigin, 14 to
A warm brown band was observed at 16 mm, and a warm brown band was observed at 15 to 18 mm from the origin of the cathode in the male plant.

In the esterase zymogram shown in Figure 6, a band was observed only in the female plant, and a 15 mm wide deep red band was observed from the origin of the anode.

As can be seen from the results in FIGS. 5 and 6, in the case of jojoba, there are differences in patterns between male and female plants in both peroxidase zymodalum and esterase zymograms, and male and female plants can be easily distinguished.

(Effects) According to the present invention, male and female plants can be easily distinguished just by collecting leaf pieces, so seed propagation, which has not been done in the past, can be carried out. Therefore, the multiplication rate can be increased, and unnecessary male or female plants can be removed during the seedling stage, so cultivation efficiency can be increased.

The present invention is not limited to the above embodiments, but can be applied to dioecious plants in general.

[Brief explanation of drawings]

Figure 1 shows the pattern of peroxidase zymodalum in the leaves of an adult date palm tree. Figure 2 shows the peroxidase zymogram pattern in the leaves of the same seedling. Figure 3 shows the pattern of peroxidase zymodalum in the leaves of the same seedling. Figure 4 shows the pattern of esterase zymogram in the leaves of the same seedling. Figure 5 shows the pattern of peroxidase zymodalum in the leaves of jojoba. Figure 6 shows the pattern of esterase zymogram in the leaves of the same seedling. FIG. 3 is a diagram showing a zymogram pattern.

Claims (2)

[Claims] (1) A method for determining the sex of dioecious plants at the seedling stage, which comprises measuring the peroxidase activity and/or esterase activity of the plants. (2) The determination method according to claim 1, characterized in that the peroxidase activity and/or esterase activity of a sap obtained from a plant body such as a leaf piece of a plant is measured by starch gel electrophoresis.