JP3319475B2 - Basil hybrid test method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for testing a hybrid of basil which can objectively determine the difference in genetic characteristics between the hybrid of the basil and its parents. Using the true leaves except for the basil petiole,
A hybrid assay for basil, characterized in that the extract obtained from the true leaves is subjected to isoelectric focusing and the genetic characteristics are determined based on the difference in isozyme pattern obtained by staining the developed isozyme active site. It is about the method.

[0002]

2. Description of the Related Art Heretofore, studies on isozymes of plants have already been conducted on some plants, for example, peroxidase activity and / or esterase of sap obtained from plants such as leaf pieces of plants. A gender discrimination method for dioecious plants, particularly a method for gender identification at the seedling stage, comprising measuring the activity by starch gel electrophoresis has been proposed (JP-A-61-58598).

In this method, a sap taken from a leaf of a plant to be discriminated is developed by starch gel electrophoresis to develop an enzyme in the sap, stained with a predetermined drug, and the sex of the sap obtained is determined by the isozyme pattern obtained. More specifically, for dioecious plants such as dates, the fact that the female and male strains have different isozyme patterns to determine their gender. Although the document discloses a method for discriminating the sexes of dioecious plants, it does not disclose any knowledge or the like relating to a hybrid test of plants in general.

[0004] As relates to hybrid purity of the plant, for example, as a method for determining the hybrid purity of F1 hybrids of tomatoes, after allelic variants ADH-1 "and ADH-1 ¹ separation, alcohol dehydrogenase activity and visual detection A method for coloring for use, comprising ADH-1 ″ and ADH-
1 ¹ separation method of determining the hybrid purity of F1 hybrids of tomatoes, characterized in that it is made by focusing isoelectric has been proposed (JP-A-4-248995).

[0005] Specifically, this method uses tomato seeds, examines the electrophoresis pattern obtained by isoelectric focusing of the extract, and compares the extract with the characteristic band of both parents to obtain F1 seeds. Although the method is to check whether or not the purity is hybrid, the method is generally based on the degree of the hybrid quality, that is, the purity of the seed varieties, because the seed price of the tomato is primarily determined. This is based on the premise that it is necessary to test the hybrid purity of seeds, and the document does not disclose the use of tissues other than plant seeds.

On the other hand, basil (Ocimum basil)
icum) is an aromatic annual plant, which has good fragrance in whole plants, especially leaves and stems, is used as a flavoring agent, a medicinal ingredient, etc., and there are many types, but it is extremely easy to cross and has a flavor. Varieties management is extremely important because the components are easily changed. Even if hybrids are produced by crossing, it may be difficult to distinguish them from their appearance.In order to investigate leaf shapes, flower shapes, etc., and to investigate flavor components, plants must be A fairly large-scale facility and acquisition are required, for example, a certain period of cultivation is required, and many plants are required.

[0007] As described above, isozyme surveys on plant seeds are used in other plant species such as tomatoes. In many cases, such a method is not effective when the seeds have variations, because the determination accuracy is poor. On the other hand, it is known in other plant species that the use of leaves depends on the position and age of the leaves, but there has been no study on basil. No knowledge has been obtained.

[0008] As described above, conventionally, enzymes are extracted from a sap extracted from a plant such as a leaf piece of a plant, developed by starch gel electrophoresis, and discriminated by isozyme patterns obtained by staining with a predetermined agent. Although this method has been proposed, the method is limited to discriminating the gender of the dioecious plant at the seedling stage, and the purity of the hybrid of the F1 hybrid of tomato is determined by gel electrophoresis pattern. Has been proposed, the method uses seeds of F1 hybrid of tomato, the method of using the seeds is not effective when there is variation in seeds, Its use is limited to seeds of F1 hybrids of tomatoes, and it has been conventionally used for other plant species, especially hybrid hybrids of basil. A method of performing the hybrid test easily, and with a high degree of accuracy is not known at all, its development has been strongly demanded.

[0009]

SUMMARY OF THE INVENTION In view of such circumstances, the present inventors have conducted a quick test on hybrids of basil hybrids, for which an effective hybrid test method has not been established. As a result of intensive studies with the aim of developing a new method for easy implementation, using the true leaves excluding the basil petiole and using the enzyme component, that is, the extract obtained from the true leaves As a result, the inventors have found that it is possible to objectively, simply and highly accurately discriminate the difference in genetic characteristics from their parents, and have completed the present invention.

That is, an object of the present invention is to provide a method for objectively discriminating a difference in genetic characteristics between a parent and a parent of a hybrid hybrid of basil.

[0011] The present invention also provides a hybrid hybrid of basil, which does not require a period for cultivating a plant to some extent as in the past, and uses only the true leaves to obtain the genetic characteristics of the hybrid with its parents. It is an object of the present invention to provide a method for objectively and early discriminating a difference.

Further, the present invention provides a simple and highly accurate genetic hybrid of basil hybrids with their parents regardless of the leaf position, environment, leaf age, etc. of the plant. It is an object of the present invention to provide a method that can objectively determine the state of the object.

[0013]

The structure of the present invention for achieving the above object comprises the following technical means (1) to ( 3 ). (1) After isolating the enzyme components of basil leaves by isoelectric focusing, the isozyme activity was measured using PGM (pho
sphoglucomutase), PGI (phos
phoglucose isomerase), PPO
(Polyphenol oxidase), POD
A method for detecting a hybrid of basil, wherein the method is measured using the activity of (peroxidase) .

(2) An extract obtained from basil leaves is subjected to isoelectric focusing to stain the developed isozyme active site, and then to obtain the obtained PGM, PGI, POD, PPO,
Combine appropriate ACP, MDH and GDH band patterns
In addition, a method for testing a hybrid of basil, characterized in that its genetic characteristics are determined based on the difference in its isozyme pattern.

(3) The method for testing hybrid of basil according to the above (1) or (2), wherein the basil leaves are true leaves excluding the basil petiole.

[0016]

Next, the present invention will be described in more detail. The present invention, as described above, for the hybrid hybrid of basil, the enzyme component of the true leaf except the petiole of basil, that is, the isozyme active site developed by subjecting the extract obtained from the true leaf to isoelectric focusing and developing The present invention relates to a method for testing a hybrid of basil, which objectively discriminates a difference in genetic characteristics from its parents based on a difference in an obtained isozyme pattern after staining.

Basil (Ocimum basilicu)
m) is an annual of Labiatae,
Widely distributed in the tropics, height is about 60cm, leaves are oval, flowers are white or reddish, 6 varieties grow, bloom from midsummer to mid-autumn, fruits are small and black, whole plants, especially leaves There is a good scent on the stalk and it is widely cultivated as a culinary herb.
The leaves are dried and used as flavoring agents, medicinal ingredients and the like.

In the present invention, among the basil plants, a part of the true leaves collected so as not to include the stem and petiole as much as possible is used. The true leaves up to three nodes below the nodes where the leaves are considered to be completely expanded, that is, the upper true leaves, the middle true leaves,
It is preferable to use young leaves of axillary bud leaves and extract and use the active enzyme component from the true leaves using an extraction buffer or the like.

As the extraction buffer, for example,
0.1 M Tris-HCl, ascorbic acid, PVP (polyvinylpyrrolidon)
e) K-30, Triton X-100,
2-mercaptoethanol (Mercaptoetha)
No.) is preferably used, but the present invention is not limited thereto. Any buffer having the same effect can be used. Further, the extraction operation of the enzyme is performed by a method in which the material is ground by a conventional method, and an extraction process is performed with an extraction buffer.

The obtained extract is placed in a cooler at 10 ° C. or lower in order to prevent a decrease in activity.
m) and the like, and subjected to isoelectric focusing using a polyacrylamide gel, for example, pH gradient 3-9, 4-6.5.
Then, electrophoresis is carried out by a standard method under electrophoresis conditions under a constant voltage of 2000 V DC.

After the completion of the electrophoresis, isozyme activity staining is performed. In particular, the following methods are exemplified as preferable isozyme stains which are suitable as indicators. 1) PGM (phosphoglucommutase) Using 0.1 M Tris-HCl buffer (pH 7.5), MgCl ₂ .6H ₂ O, glucose-1-P (Na salt), NADP ⁺ , MTT, PMS, A mixture of glucose-6-PDH is used, poured into a gel, and electrophoresed in the dark at 35 ° C. for about 60 minutes at pH 4-6.5, and the spot position is examined.

2) PGI (phosphogludos)
e isomerase) 0.1M Tris-HCl buffer (pH 7.5) was used, and as reagents, MgCl ₂ .6H ₂ O, fructose-6-P (Na) ₂ , NADP ⁺ , MTT, PMS,
Use a mixture of glucose-6-PDH, pour it into the gel, and in the dark at 35 ° C. for about 120 minutes, pH 4-6.5.
After electrophoresis, the spot position is investigated.

3) POD (peroxidase) First, O-dianisidine (dianisidin) was used as solution I.
dine), acetic acid, distilled water, and a reagent consisting of 0.2 M acetate buffer (pH 4.9) are poured onto the gel, and the solution
After reacting at 25 ° C. within 25 minutes,
0.2 M acetate buffer (pH 6.5), 30% H ₂
After pouring a reagent consisting of O ₂ and allowing it to migrate at room temperature for about 10 minutes at pH 3-9 in the dark, the spot position is examined.

4) PPO (polyphenol oxidase) Using 0.2 M acetate buffer (pH 5.8), a reagent I containing P-phenylenediamine and catechol (pyrocatechol) as a reagent is poured into the gel, and the mixture is placed in the dark. 30
After reacting for about 20 minutes at room temperature, Reagent II containing ascorbic acid was further poured, and pH 3-
After migration in step 9, the position of the spot is investigated.

5) ACP (acid-phosphatase) A reaction solution using 0.5 M acetate buffer (pH 4.8) and containing α-naphthyl acid sodium phosphate and Fast Garnet GBC as reagents Was poured into a gel and the pH was 4-6.
After running in step 5, the position of the spot is investigated.

6) MDH (male dehydrr)
ogenase) 0.05M Tris-hydrochloric acid (pH 7.0) was used, and D, L-malate (malate), NA
A reagent containing D ⁺ , NBT, and PMS was poured into the gel, and the mixture was electrophoresed at 35 ° C. for about 120 minutes at pH 3-9 in the dark.
Investigate the spot position.

7) GDH (glutamate deh)
ydogenase) 0.125 M sodium phosphate (pH 6.0) buffer was used, and L-glutamate, NA
A reagent containing D ⁺ , NBT, and PMS is poured on the gel, and the gel is electrophoresed at 35 ° C. for 60 minutes in the dark at pH 3-9.

In addition to these isozymes, ADH (a
lcohol dehydrogenase), EST
(Estrace), AMY (amylase), LA
P (leucine aminopeptides)
e), GOT (glutamic-oxaloacet)
ic transamine, XDH (xant
ine dehydrogenase), LDH (la
Cate dehydrogenase), 6-PG
D (6-phosphogluconate dehy
(dogenase), the isozyme activity was stained in the same manner.

In this way, the extract collected from the true leaves of basil is subjected to isoelectric focusing to develop the enzyme in the extract, stained with a predetermined reagent as described above, and then subjected to isozyme purification. Analyze the pattern.

As the isoelectric focusing method, an isoelectric focusing method using a polyacrylamide gel is exemplified as a preferable one, but the present invention is not limited to this, and any isoelectric focusing method having the same effect can be used. It can be used as appropriate.

The present inventors have proposed the following method.
As a result of examining the band patterns of 15 kinds of enzymes in the extract of basil true leaves, there were isozymes showing a clear band pattern that were effective for phylogenetic classification of the hybrids of basil. Succeeded in finding something to do. Among the 15 types of enzymes, PGM, PGI, POD, PPO, ACP,
It was found that seven kinds of MDH and GDH showed effective band patterns, and that the isozyme patterns of the seven kinds of enzymes did not vary between the same strains. In addition, as an isozyme, these may be used alone or appropriately in combination.
By analyzing the isozyme pattern, it is possible to objectively discriminate the difference in genetic characteristics from its parents with ease and high accuracy, confirming that it can be used effectively for hybrid testing of basil. Was done.

As described above, such an isozyme pattern does not fluctuate between the same strains of the hybrid hybrid of basil, and thus it has been confirmed that the isozyme pattern is particularly useful for assaying F1 hybrid of basil and the like. .

[0034]

EXAMPLE Next, as an example of the present invention, an example in which the isozyme pattern was actually investigated for 16 basil strains and statistical analysis was performed based on the data will be described.

Embodiment 11 1. Basil test system As the basil test system, the following 16 systems of basil were used. [16 Basil Lines] 1. Suite [Sweet (S-1)]; 2. Suite [Sweet (S-2)]; Dark red [Dark
Opal (R)], 4. Dark muscle red [Dark Opa
l (LR)], 5. Dark green [Dark Opal
(G)], 6. Bush [Bush (S)], 7. 7. Greek, Green Raffle
n Ruffles), 9. Purple Raffle (Pur
ple Ruffles), 10. Lettuce (Lettu
ce), 11. Mutation (giant leaf), 12. Anise
se), 13. 13. Spice, 14. Cinnamon, Holly
y), 16. Lemon.

2. Enzymes The following 15 kinds of enzymes of basil true leaves were investigated. PGM (phosphoglucomutas)
e), PGI (phosphoglucose isis)
omelase), POD (peroxidase),
MDH (male dehydrogenas)
e), GDH (glutamate dehydrolog)
enzyme), PPO (polyphenol oxi)
dase), ACP (acid-phosphatas)
e), ADH (alcohol dehydrogen)
ASE), EST (estrace), AMY (amy)
case), LAP (leucine amine)
ptidase), GOT (glutamic-oxa)
loacetic transamine), XD
H (xantine dehydrogenase),
LDH (lactate dehydrogenas)
e), 6-PGD (6-phosphoglucona)
te dehydrogenase)

3. Extraction conditions of plant material 1) Extract The extract having the following composition was used as the extract (buffer). Tris-HCl (pH 7.2) 0.1M 100ml ascorbic acid 0.05M 880mg PVP (Polyvinylpyrolidone) K-30 5g Triton X-100 1ml 2-mercaptoethanol (Mercaptoethanol) 1ml

2) Plant part The plant part is visually observed from the shoot apex (axillary bud) and is located below the node where the leaves are considered to be completely developed.
Young leaves up to the knot were used. The sample of one cultivar was from the same strain.

4. Preparation of sample 1) The above-mentioned young true leaves were collected as true leaves of the above-mentioned 16 lines of basil, placed in a mortar, and frozen at −30 ° C. for 30 to 60 minutes. The frozen leaves in a mortar were ground with a pestle to form a powder. As an extraction buffer, 100 ml of 0.1 M Tris-hydrochloric acid (pH 7.2), 880 mg of 0.05 M ascorbic acid, PVP (polyvinyl lp)
yrolidone) K-30, 5 g, Triton (Tr
Iton) A buffer containing 1 ml of X-100 and 1 ml of 2-mercaptoethanol was used.
0.5 ml was added to 5 g. After the extraction treatment, the triturated liquid was centrifuged at 15,000 rpm and 4 ° C. for 20 minutes, and 400 μl of the supernatant was subjected to a centrifugal filtration tube (0.22 μm) containing 50 mg to 60 mg of a biological solution sample concentrate (“Mizubutori-kun” manufactured by ATTO). ).

Next, 5 μl of extraction buffer was added to the bottom of the centrifugal filtration tube, and the mixture was added at 5,000 rpm at 4 ° C. for 15 minutes.
The mixture was centrifuged for minutes, and the filtrate was used as a sample. 1 μl of the sample was used for one enzyme assay. The above operation was performed under low temperature conditions, and the solution was stored in ice water.

5. Basic conditions in isoelectric focusing of basil true leaf extract For the sample, a fast system (Past S)
ysystem (Pharmacia) under the following electrophoresis conditions. 1) pH gradient 3-9 pre-electrophoresis 2000V 2.5mA 3.5W
5 ° C 75Vh Sample addition 200V 2.5mA 3.5W
5 ° C 15Vh Main electrophoresis 2000V 2.5mA 3.5W
5 ° C. 410 Vh 2) pH gradient 4-6.5 Pre-migration 2000 V 2.0 mA 3.5 W
5 ° C 75Vh Sample addition 200V 2.0mA 3.5W
5 ° C 15Vh Main electrophoresis 2000V 5.0mA 3.5W
5 ℃ 410Vh

6. Isozyme (Isozyme) activity staining conditions (within petri dish) Each isozyme developed by isoelectric focusing was stained with a reagent. Among the above enzymes, particularly, the conditions for isozyme activity staining of seven enzymes useful as indicators are as follows. 1) PGM (phosphoglucomutase) Buffer Tris-HCl (pH 7.5) 0.1 M 50 ml 25 ml Reagent MgCl ₂ .6H ₂ O 1 M solution 0.5 ml 0.25 ml qualose-1-P (Na salt) 75.0 mg 37.5 mg NADP ⁺ 7.5 mg 3.75 mg MTT 10.0 mg 5.0 mg PMS 2.0 mg 1.0 mg coulose-6-PDH 20 units 10 units Conditions 35 ° C Dark for about 60 minutes Electrophoresis pH 4-6.5 Spot position Alkaline side

2) PGI (phosphoglucose isomerase) Buffer Tris-HCl (pH 7.5) 0.1 M 50 ml 25 ml Reagent MgCl ₂ .6H ₂ O 1 M solution 0.5 ml 0.25 ml fructose-6-P (Na) ₂ 40.0 mg 20.0 mg NADP ⁺ 10.0 mg 5.0 mg MTT 10.0 mg 5.0 mg PMS 2.0 mg 1.0 mg Coolose-6-PDH 10 units 5 units Conditions 35 ° C Dark for about 120 minutes Electrophoresis pH 4-6.5 Spot position Alkaline side

3) POD (peroxidase) I solution O-dianisidine (dianisidine) 50 mg 25 mg acetic acid 0.42 ml 0.21 ml distilled water 35.0 ml 17.5 ml 0.2M acetic acid buffer (pH 4.9) 15.0 ml 7.5 ml II liquid 0.2M acetic acid buffer (pH 6.5) 49.0 ml 24.5 ml 30% H ₂ O ₂ 0.1 ml 0.05 ml Conditions I. 4 ° C Dark within 25 minutes II. Room temperature Dark for about 10 minutes Electrophoresis pH 3-9 Spot position Acid side

4) MDH (malate dehydrogenase) Buffer Tris-HCl (pH 7.0) 0.05 M 50 ml 25 ml Reagent D, L-malate 0.2 M 1180 mg 590 mg NAD ⁺ 1.0 mM 35.7 mg 17.85 mg NBT 0.43 mM 17.6 mg 8.8 mg PMS 0.163 mM 2.5 mg 1.25 mg (Buffer and malate were mixed and the pH was adjusted to 7.0 with NaOH) Conditions 35 ° C Dark for about 120 minutes Electrophoresis pH 3-9 Spot position Alkaline side

5) GDH (glutamate dehydrogenase) Buffer Sodium phosphate (pH 9.0) 0.125 M 50 ml 25 ml Reagent L-quartamate 0.25 M 1180 mg 919.6 mg NAD ⁺ 1.5 mM 53.8 mg 26.9 mg NBT 0.43 mM 17.6 mg 8.8 mg PMS 0.163 mM 2.5 mg 1.25 mg (Buffer and glutamate were mixed and the pH was adjusted to 9.0 with NaOH.) Conditions 35 ° C. Dark about 60 minutes Electrophoresis pH 3-9 Spot position Alkaline side

6) PPO (polyphenol oxidase) Buffer Buffer Acetate (pH 5.8) 0.2 M 50 ml 25 ml Reagent I. p-Phenylenediamine 0.05% 25 mg 12.5 mg Catechol (Hirocatechol) 10 mM 55.06 mg 27.53 mg II Ascorbic acid 10 mM 88.07 mg 44.03 mg Condition I. 30 ° C. Dark for about 20 minutes II. Room temperature Dark for about 5 minutes Electrophoresis pH 3-9 Spot position Alkaline side

7) ACP (acid-phosphatase) Buffer acetate (pH 4.8) 0.5 M 50 ml 25 ml Reagent α-naphthyl acid sodium phosphate 50 mg 25 mg Fast Garnet GBC salt 50 mg 25 mg (Adjust the pH with NaOH before adding FastCarnet.) Conditions 35 ° C Dark electrophoresis pH4-6.5 Spot position Alkaline side

Under the conditions and operations described above, the possibility of hybrid testing of 16 basil strains was examined from the isozyme pattern of each enzyme. The results are shown in FIGS.
As is clear from the results of the dendrograms of FIGS.
The isozyme pattern of each of the enzymes PGI, PPO, PGM, POD, ACP, MDH and GDH showed the same pattern for all strains, but it was found that some showed different patterns depending on the strain.

Table 1 shows the band sharing ratios of the 16 enzymes of the basil in the isozymes of the above 7 enzymes (1 to 16 in Table 1 indicate 1 of the basil test lines used in Example 1).
~ 16). As is clear from Table 1, it was found that there was a difference in band sharing ratio among the 16 basil strains. Therefore, it is clear that the basil strain can be reliably identified by selecting and combining enzymes having different isozyme patterns depending on the strain.

[0051]

[Table 1]

Next, among the 16 basil lines, Basil Spice, Basil Sweet (S-1), and F1 hybrids thereof were PGM, PGI, PPO and POD.
The results of the isozyme patterns obtained by developing the four kinds of enzymes by isoelectric focusing are shown in (a) to (d) of FIGS.

8 to 9, among the 16 basil lines, Basil Sweet (S-2), Basil Cinnamon, and F1 hybrids thereof, as in FIGS.
The results of the isozyme patterns obtained by developing the four kinds of enzymes GM, PGI, PPO and POD by isoelectric focusing are shown in (a) to (d) of FIGS.

The basil cinnamon (Basil Cinnamon) among the 16 basil strains was identified by the PGM,
FIGS. 12 to 15 show the results of investigation on four kinds of enzymes, PGI, PPO and POD. Furthermore, the stability of the isozyme pattern of the hybrid hybrid between basil cinnamon (Basil Cinnamon) and basil sweet (Basil Sweet, S-2) was determined using PGM, PGI, PPO.
FIG. 16 shows the results of an investigation on the four enzymes POD and POD.
To 19.

As is clear from the isozyme patterns (a) to (d) in FIGS. 8 and 9, according to the PGI isozyme pattern, basil spice and F1 show the same pattern, but PGM, PPO, and POD have the same pattern. According to each isozyme pattern, F1 showed a pattern different from that of the parents, and it was found that the difference in genetic characteristics from the parents could be objectively determined based on the patterns.

As is clear from the isozyme patterns (a) to (d) in FIGS.
According to the isozyme pattern of I and PPO, basil sweet (S-2) and F1 show the same pattern, but according to the isozyme pattern of POD, F1 is
It shows a pattern different from that of the parental lines, indicating that POD alone is effective, and by combining the patterns of each isozyme obtained by developing these enzymes, it is possible to reduce the difference in genetic characteristics from the parents. objective,
And it turned out that it can be distinguished easily.

As is clear from the isozyme patterns due to the difference in the positions of the leaves in FIGS. 12 to 15, at least the upper leaves (upper true leaves), the middle leaves (central true leaves) and the axillary buds were subjected to the hybrid test. It has been found that a clear pattern that can be suitably used is obtained. Further, FIG.
As is clear from the 19 isozyme patterns, the isozyme pattern of the hybrid hybrid was found to be highly stable without fluctuation between the same lines. These were similarly examined using other lines, and similar results were obtained.

As is clear from the results of these isozyme patterns, the isozyme patterns obtained by developing PGM, PGI, PPO and POD are useful for testing hybrid hybrids of basil. It has been confirmed that when combined and determined, hybrids can be determined with high accuracy, and the accuracy of the determination can be increased.

According to the study by the present inventors, the isozyme patterns of the above four enzymes are, inter alia,
It turned out to be useful, but it was also found that the isozyme patterns of other enzymes can be used effectively similarly by appropriately combining them.

[0060]

According to the present invention, it is possible to objectively determine the difference in genetic characteristics between a parent and a parent of a hybrid of basil simply and simply by collecting a true leaf of basil. it can. Therefore, crossing is likely to occur, the variation is large even in commercially available seeds, and it is difficult to discriminate only by the phenotype due to its morphological characteristics, and the systematic classification of a hybrid hybrid of basil that is easy and highly accurate Can be assayed and at the seedling stage of the basil plant,
Hybrids can be determined, and an efficient cultivation plan can be constructed.

Further, hybrids of basil, for which it is difficult to distinguish varieties from the appearance, can be objectively distinguished from genetic characteristics. In addition, the conventional method of discriminating from leaf and flower shapes and flavor components required a certain period of plant cultivation, but the present technology requires at least one leaf, and therefore specifies the plant at an early stage. It is possible to implement the present invention without depending on the position, environment, and leaf age of the leaves.

[Brief description of the drawings]

FIG. 1 is a dendrogram showing the isoelectric focusing pattern of PGI isozymes in the true leaves of 16 basil lines.

FIG. 2 is a dendrogram showing isoelectric focusing patterns of isozymes of PPO in the true leaves of Basil 16 lines.

FIG. 3 is a dendrogram showing the isoelectric focusing pattern of the isozyme of PGM in the true leaves of 16 basil lines.

FIG. 4 is a dendrogram showing the isoelectric focusing pattern of POD isozymes in the true leaves of 16 basil lines.

FIG. 5 is a dendrogram showing an isoelectric focusing pattern of ACP isozymes in true leaves of 16 basil lines.

FIG. 6 is a dendrogram showing isoelectric focusing patterns of MDH isozymes in true leaves of 16 basil lines.

FIG. 7 is a dendrogram showing the isoelectric focusing pattern of GDH isozymes in the true leaves of 16 basil lines.

FIG. 8 shows isozyme patterns by isoelectric focusing of Basil F1 hybrids and parents [(a)-(b)].

FIG. 9 shows isozyme patterns by isoelectric focusing of F1 hybrids of basil and parents [(c) to (d)].

FIG. 10 shows isozyme patterns of basil F1 hybrids and their parents by isoelectric focusing [(a)-
(B)].

FIG. 11 shows isozyme patterns of basil F1 hybrids and their parents by isoelectric focusing [(c)-
(D)].

FIG. 12 is an isozyme pattern (PGM) of basil cinnamon at each plant site.

FIG. 13 is an isozyme pattern (PGI) of basil cinnamon at each plant site.

FIG. 14 is an isozyme pattern (PPO) of basil cinnamon plant by site.

FIG. 15 shows isozyme patterns (POD) of basil cinnamon plants at different sites.

FIG. 16 is an isozyme pattern (PGM) of a hybrid hybrid of cinnamon and basil sweet.

FIG. 17 is an isozyme pattern (PGI) of a hybrid hybrid of cinnamon and basil sweet.

FIG. 18 is an isozyme pattern (PPO) of a hybrid hybrid of cinnamon and basil sweet.

FIG. 19 is an isozyme pattern (POD) of a hybrid hybrid of cinnamon and basil sweet.

[Explanation of symbols]

1. 1. Suite [Sweet (S-1)] Sweet [Sweet (S-2)] 3. 3. Dark red [Dark Opal (R)] 4. Dark muscle red [Dark Opal (LR)] 5. Dark green [Dark Opal (G)] Bush [Bush (S)] 7. Greek 8. 8. Green Raffles Purple Ruffle
s) 10. Lettuce 11. Mutation (giant leaf) Anise 13. Spice 14. 14. Cinnamon Holly 16. Lemon 17. Red perilla (reference) 18. Blue perilla (reference)

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Kazue Yasuda 1-5-7 Mikitei Sakaecho, Higashiosaka-shi, Osaka House Foods Industry Co., Ltd. (56) References Can. J. Plant Sci. , Vol. 62, No. 3 (1982) p. 621-630 Bull. Akita Pref. Coll. Agr. , No. 13 (1987) p. 51-57 JPN. J. Breed. Vol. 1 (1991) p. 73-83 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/00-1/70 BIOSIS (DIALOG) WPI (DIALOG) MEDLINE (STN)

Claims (3)

(57) [Claims] After isolating the enzyme components of basil leaves by isoelectric focusing, the isozyme activity was determined by PGM.
(Phosphoglucomutase), PGI
(Phosphoglucose isomeras
e), PPO (polyphenol oxididas)
e), utilizing the activity of POD (peroxidase)
Basil method of hybrid assays and measuring Te. 2. Extracts obtained from basil leaves are subjected to isoelectric focusing to stain the developed isozyme active sites, and then obtained PGM, PGI, POD, PPO, AC
Combine appropriate band patterns of P, MDH, GDH
A method for testing a hybrid of basil, wherein the genetic characteristics are determined based on the difference in the isozyme pattern. 3. The method for testing a hybrid of basil according to claim 1, wherein the basil leaves are true leaves excluding the petiole of basil.