JPH0347021A - Method for proliferating melon seedling by tissue culture - Google Patents

Abstract

PURPOSE:To efficiently mass proliferate seeds and seedlings of a melon by culturing the shoot apex part of a melon in a culture medium containing a carbohydrate in a high concentration, forming adventitious embryo-forming calli and then culturing the resultant calli in a culture medium in a reduced concentration of the carbohydrate. CONSTITUTION:The shoot apex part of a melon is cultured in a culture medium containing 5-12%, preferably 6-9% carbohydrate, inorganic salts, vitamins and phytohormones to induce calli having the ability to form adventitious embryos. The resultant calli are then transplanted into a culture medium containing 1-4% carbohydrate, inorganic salts and vitamins, e.g. preferably gelan gum solid culture medium, and cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for propagating cloned seedlings by producing somatic embryos from melon stem apices.

(Prior technology) The inheritance of traits in melons is complex, and many of the traits targeted for breeding are genetically recessive or quantitatively inherited, and combining and fixing these traits has not been done in the past. It is said that cross-breeding is extremely difficult. Therefore,
It is desired to breed varieties with superior characteristics that can be obtained through hybridization or mutation.

However, there is no other way to propagate individual plants obtained by mutation or the like other than vegetative propagation, and if the purpose is to cultivate them, young seedlings must be vegetatively propagated quickly and efficiently. The most efficient method is the application of tissue culture.

For melons, regeneration of plants derived from adventitious buds from callus has already been reported (Hideyoshi Toyoda, 19117).
: Plant tissue culture 4(1), 8, Suemzlsu, N.
,, l(,01suka, 1986:Japan
1. B+eed,,36(suppl,l),22
, Bouxbdallah, L., M., Branch.
trd 1986: z, pHxnsenxuc
hl 96.82, Na1lo, T,, M,
511o, 1985japan I, Bree
d,, 35(suppl, 2), 3θ). However, the above method, after inducing shoots from callus,
Because it requires two steps: rooting the shoots and obtaining plants, it is inefficient for mass propagation.

Another method has been reported in which somatic embryos are produced from cotyledons in ripe seeds and hypocotyls of seedlings grown by aseptic seeding (Orid!lc, T,, K.

0oszv1. 1986: Ixpxn J, Br
eed,,36. 424). However, with this method,
Normally, for mass propagation of clones with superior traits or desired traits, plant tissues such as mutant lines and superior hybrids must be used as material, so it is difficult to form somatic embryos from cotyledons derived from seeds. Even if this is approved, mass proliferation of cloned seedlings cannot be expected.

In addition, Japanese Patent Application Laid-open No. 63-248320 proposes a method for producing somatic embryos by liquid culture from shoot apical tissues and cotyledons, but somatic embryos are produced directly from shoot apical tissues as materials. Since the number of plants is limited, it is difficult to obtain a large number of plants using this method.

(Problems to be Solved by the Invention) As mentioned above, conventional methods have limitations on materials such as somatic embryos derived from seeds, and quantitative limitations such as somatic embryos directly derived from shoot apical tissue. Disadvantages such as limitations and insufficient suitability for mass proliferation such as redifferentiation of adventitious buds from callus are observed. Therefore, the present invention proposes a novel propagation method that can produce somatic embryos and efficiently propagate large quantities of melon seedlings.

(Means for Solving the Problems) The present invention forms somatic embryos by forming a part of a melon trophozoite into an explant, placing it in a plant tissue culture medium containing a high concentration of carbohydrates, and culturing it. Callus (somxli)
c enb+7o (enic egllus) can be obtained, and the somatic embryogenic callus thus obtained can be subcultured in a genetically stable state without losing the ability to form somatic embryos. This method is based on the knowledge that a large number of somatic embryos are formed by culturing callus in a solid medium with a reduced carbohydrate concentration, and that young seedlings can be easily obtained from somatic embryos.

That is, in the method for propagating melon seedlings according to the present invention, the top of the melon stem is mixed with 5 to 12% of carbohydrates, inorganic salts, vitamins,
After culturing in a medium containing plant hormones to induce a callus with somatic embryogenic ability, this callus or a callus obtained by subculturing this callus is further cultured in 1 to 4
% of carbohydrates, inorganic salts, and vitamins to promote the development of somatic embryos to obtain melon seedlings, and when forming somatic embryos from the callus, as a medium. Claim 2 is characterized in that a gellan gum solid medium is used.

Hereinafter, the method for producing somatic embryos and propagating melons according to the present invention will be explained in detail.

Induction of somatic embryogenic callus: Sterilize the melon shoot apex with an appropriate concentration of sodium hypochlorite, wash with sterile water, and remove the shoot apex aseptically under a stereomicroscope. Furthermore, in the case of melons grown aseptically, the stem apex is removed in the same manner without sterilization. The shoot apex is placed on an artificial agar solid medium containing carbohydrates, an inorganic salt composition, vitamins, and plant hormones. This agar solid medium has a high carbohydrate concentration of 5 to 12%, and its pH is usually adjusted to 5 to 6. Further, as the inorganic salt composition, compositions such as Murashige-Skoog medium and Linsmeyer-Skoog medium can be used according to the specified concentration or diluted as appropriate.

The feature of the present invention lies in the concentration of this carbohydrate,
In the present invention, the concentration is usually 1 to 4%, but it is 5%.
The content is adjusted to 12%, more preferably 6 to 9%. Below 4% and above 13%, the yield of somatic embryos and plantlets is low. Carbohydrates include sucrose, glucose,
Fructose, maltose, galactose, etc. are used, and these may be used in combination.

Components of the medium other than carbohydrates may be at normal concentrations, and vitamins include thiamine, pyridoxine, nicotinic acid,
Biotin etc. are used, and as plant hormones, indole acetic acid (IAA), indole butyric acid (IBA), 2
．． 4-dichlorophenoxyacetic acid (2,4-D), 2.
4. 5-) Lichlorophenoxyacetic acid (2,4,5-
Auxins such as T), naphthaleneacetic acid (NAA), and cytokinins such as benzyladenine (BA), kinetin, and zeatin can be used.

These plant hormones can be used in combination as appropriate, and combination use of 2.4-D and benzyladenine is particularly preferred.

In addition, the temperature at which the shoot apex is cultured is preferably 20 to 30°C in order to achieve good growth.

When the shoot apex is cultured for 1 to 2 months, it grows as a pale yellow soft callus. When this callus is observed under a stereomicroscope, a large number of early somatic embryos are observed, which is clearly different from a normal completely dedifferentiated callus. The somatic embryogenic callus thus derived from a medium containing a high concentration of carbohydrates can be subcultured on gellan gum or agar solid medium with the above-mentioned composition without changing its properties. , subculture and proliferation are also possible by inoculating a liquid medium of the same composition and culturing with gentle shaking at 20 to 200+ pm.

Formation of somatic embryos, regeneration of individuals, and seedling formation: In the present invention, the callus that has the ability to form somatic embryos, which has been propagated as described above, is transplanted to a medium different from the medium in which the callus was induced, and the callus is grown as somatic embryos. Form an embryo. The medium used for somatic embryogenesis has a carbohydrate concentration of 1 to
It is characterized by a low rate of 4%.

Other compositions may be basically the same as the medium in which callus was induced. However, regarding plant hormones, it is preferable not to add them to the medium for forming somatic embryos, as is already done in tissue culture of many plants other than melons, and even if they are added, After somatic embryo formation, it is desirable to keep the amount extremely small and to continue culturing in a medium without plant hormones. This medium may be an agar medium or a liquid medium, but from the standpoint of efficient culturing of seedlings, gellan gum solid medium is most suitable, since the surface of the somatic embryos that are formed will not be soft, and healthy young plants are obtained.

When the transplanted callus is cultured under normal temperature and light conditions, a large number of somatic embryos are produced, followed by minute shoots. Suitable conditions include a temperature of 20-30°C; 2.
The illuminance is between 000 and 5,000 lux.

If you continue culturing in this state, the growth will be poor due to the high density of the shoots, so if you divide them into individual melon plants and continue culturing them on a solid medium under the same conditions, they will root and form a complete melon plant. Become. In the case of a liquid medium, after culturing with shaking at 20 to 1,100 rpm to form somatic embryos and subsequent minute shoots, the embryos are removed from the liquid medium, divided into individual parts, and cultured on a solid medium to form a complete plant. I can do it. By transplanting the plants thus obtained into vermiculite or seedling-raising soil and acclimatizing them using a conventional method, melon seedlings that can be planted in greenhouses or fields can be obtained.

The melon that can be grown in the present invention is Cuculs melo.
Those belonging to the L group include, but are not limited to, Aars type, Cantalbe type, and Hanedeu type.

(Example) Hereinafter, the present invention will be specifically explained with reference to Examples.

[Example 1] Ten shoot apical tissues of 0.2 to 0.5 mm removed from aseptically germinated greenhouse melon "Nankatsu Earls" were made to contain sucrose at a high concentration of 7%, and -ipp D
Murashige containing m and benzyladenine O, ippm.
When placed on a Skoog medium and continuously irradiated at a temperature of 25°C and an illuminance of 3000 lux for 2 months, an average of 0.6 g of yellow-white callus was induced from the shoot apical tissue. When the induced callus was observed under a microscope, many early somatic embryos were observed. Next, this callus was transplanted to Murashige-Skoog gellan gum solid medium containing sucrose at a low concentration of 3% and containing no plant hormones, and incubated at 25°C.
When cultured for 20 days under 3000 lux conditions, a large number of somatic embryos were produced. After culturing for 30 days, the somatic embryos and seedlings are separated from the tissue, transferred to a medium with the same composition, and cultured for an additional 30 to 60 days, making it possible to obtain healthy seedlings of about 3 cm with 2 to 4 leaves. Ta. - The average number of somatic embryos obtained from shoot tips was 325, and the average number of seedlings was 111.

[Comparative Example 1] For comparison, melon seedlings were obtained under exactly the same conditions as in Example 1, except that the callus induction medium was changed to 3% sucrose, which is commonly used. The average number of somatic embryos was 24 per shoot apex, and the average number of seedlings was 5, which were significantly lower than callus induced with 7% sucrose.

[Examples 2 and 3] Melon seedlings were obtained in exactly the same manner as in Example 1, except that the concentration of sucrose in the callus-inducing medium was changed to 5% and 9%. When sucrose is 5%, the average number of somatic embryos is 162 per shoot apex, and the average number of seedlings is 36.
When the sucrose content was 9%, the average number of somatic embryos was 182, and the average number of seedlings was 48.

[Comparative Example 2] When culturing was carried out in exactly the same manner as in Example 2 except that the sucrose in the medium for inducing callus was changed to 15%, the amount of induced callus was extremely small and was not suitable for experiments.

[Example 4] In Example 1, the composition of the medium for inducing callus was 11% sucrose and 0.2 ppm 2.4-D.
Melon seedlings were obtained in exactly the same manner except that a Murashige-Skoog medium containing O.1 ppn of benzyladenine was used. The average number of somatic embryos was 85 per shoot apex, and the average number of seedlings was 36.

[Example 5] In the same manner as in Example 1, somatic embryogenic callus induced in a medium containing 7% sucrose was subcultured. Subculture was performed by taking 0.3 g of the proliferated callus and sub-planting it in the same medium every two months. The callus growth rate was 10.4 times the fresh weight ratio. Somatic embryogenic callus that had been subcultured five times was cultured for 60 days on Murashige-Skoog gellan gum solid medium containing no plant hormones and a sucrose concentration of 3%.
00 somatic embryos and 540 seedlings were obtained.

This example shows that callus induced in a medium containing sucrose at a high concentration of 7% can be subcultured without losing its ability to form somatic embryos.

[Example 6] The same procedure as in Example 5 was carried out, except that the callus was subcultured four times in a medium containing 7% sucrose, and then the fifth subculture was carried out in a medium containing 3% sucrose. did. The results were extremely excellent, with 2080 somatic embryos and 920 seedlings per gram of callus.

[Comparative Example 3] For comparison, a 3% sucrose medium was used in place of the 7% sucrose medium in Example 5.
When subculture was attempted in the same manner as in Example 5, the ability to form somatic embryos disappeared after two subcultures.

[Example 7] Somatic embryogenic callus induced in a medium containing 7% sucrose in the same manner as in Example 1 was subcultured by liquid culture. As a method, first, a cell suspension of somatic embryogenic callus was prepared in a Murashige-Skoog liquid medium containing 7% sucrose, 211911% 2.4-D, and O, ippm of benzyladenine. This cell suspension was sieved to collect callus masses with a size of 125 to 1000 μI, and 0.4 g of the callus mass was inoculated into 20 ml of a medium having the same composition as the medium in which the cell suspension was prepared, and the mixture was incubated in the dark at 25°C. , 3 at 60rll11
The culture was carried out with shaking for 0 days.

The proliferated cells were sieved in the same manner to obtain 1 g of cells with a size of 125 to 1000 μm, 0.6 g of which was inoculated into 10 hl of a medium of the same composition, and the culture was repeated. After culturing for 30 days, it was sieved and 25
1.72 g of cell clusters with a size of 0-100 θμm were obtained.

Next, 0.1 g of the obtained cell mass was taken and placed on a 1/2 diluted Murashige-Skoog agar medium containing 1.5% sucrose and no plant hormones, and kept at 25°C and 3000 lux for 60 days. By culturing, 320 somatic embryos and 50 seedlings could be obtained per gram of callus, and it was confirmed that subculture was possible without losing the ability to form somatic embryos even by liquid culture.

[Example 8] When culturing the cell mass obtained by liquid culture in Example 7, somatic embryos were developed under the same conditions using a gellan gum solid medium instead of the agar solid medium. 286 somatic embryos per gram of callus
0 and 870 seedlings were obtained. It has been revealed that the use of gellan gum solid medium is effective for the development of somatic embryos.

(Effects of the Invention) According to the method of the present invention, melon clone seedlings can be propagated extremely efficiently simply by adjusting the carbohydrate concentration of the medium. In particular, the present invention has made it possible to subculture somatic embryogenic callus derived from the shoot apex of melon with simple operations, making it possible to carry out mass propagation of cloned seedlings.

Claims (2)

[Claims] (1) After culturing the melon shoot apex in a medium containing 5 to 12% carbohydrates and containing inorganic salts, vitamins, and plant hormones to induce a callus with the ability to form somatic embryos, Callus or callus obtained by subculturing this callus is transplanted to a medium containing 1 to 4% carbohydrates, inorganic salts, and vitamins to promote the development of somatic embryos and obtain seedlings. A method for propagating melon seedlings by tissue culture, characterized by: (2) The method according to claim 1, wherein a gellan gum solid medium is used as a medium when forming somatic embryos from the callus.