KR900007085B1 - Methods for enhanced somatic enbryogenesis - Google Patents

Abstract

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Description

[Name of invention]

Methods and Media for Increasing Embryogenesis

Detailed description of the invention

(Related application)

This application is a continuation of patent application 496,186, filed May 19,1983.

(Technology field)

FIELD OF THE INVENTION The present invention relates generally to the culturing of embryonic plant cells and tissues, and more particularly to methods of using improved media and mediums that are particularly suitable for preserving embryos produced from somatic tissues in vitro. will be.

(Background technology)

Recent advances in genetic manipulation are recognized to allow plant breeders to avoid the delays inherent in crop growth in classical breeding techniques. However, a problem with the application of this technique is that single cell and multicellular organisms require different methods to change the overall genetic information. For microorganisms, there are studies that have attempted to change at the cellular level with the assurance that changes will be reproduced through the continuous generation of cells.

In multicellular organisms such as plants, genetic engineering is performed at the cellular level and then regenerated to allow mature plants to express new traits. The heterologous genetic material is introduced into the host cell, for example by plasmid insertion, so that certain changes or protoplast fusions can provide large scale genetic engineering. In addition, genetic engineering using conventional plant breeding techniques on mature plants can be used to introduce new properties and transform them into agriculturally useful varieties (Allard, RW, Principles of Plant Breeding, John Wiley and Sons, New York, (1960): Simmons, NW, Principles of Crop Improvement, Langman Group, Ltd, London, (1972)].

In vitro culture of plant cells and tissues needs to be preserved in a medium in which the culture provides nutrition and maintains viability. Examples of commonly used tissue media are described in the literature. Huang, L. and T. Murashige, "Plant Tissue Culture Media: Major constituents, their preparation, and some applications" Tissue Culture Association, Manual 3: 539-548, Tissue Culture Association, Rockville, M.D., (1977) and de Fossard, R.A. Tissue Culture for plant propagators, University of New England printery, Armidale, N.S.W., Australia (1976). Preservation of such cultures can be facilitated in the organ, tissue, or cell culture of the plant.

In somatic embryogenesis cultures, somatic plant cells are typically induced on nutrient medium and undergo cell division, producing a population of atypical cells known as callus. Callus can be preserved through subculture and multiplied. Callus can also be induced to mutate to produce the constituent tissues and organs of a mature plant. Somatic embryos may also be formed from other already existing embryos in culture. Parent embryos can germinate, from the immature globular stage to the maturation stage (Lupotto, E., "Propagation of an embryonic culture of Medicago sativa L.", Zeit). , Pflanzenphysiol. 111: 95-104 (1983). Thus, somatic embryos can arise from non-variable callus or from embryos already present in plant tissue culture.

In this way, genetic changes are carried out in the cell or embryonic stage and then conserved through successive growth to produce a complete crop of the same genotype. This allows plant breeders to avoid normal genetic barriers in plant reproduction to obtain more uniform and advantageous crops.

Conventional techniques can be used to generate a number of plants from 1 g of cells by somatic embryogenesis. See Evans, D.A, W.R. Sharp, and C.E. Flick, "Growth and behavior of cell cultures: embryogenesis and organogenesis", in Plant Tissue Culture-198l, T.A. Thorpe, ed., Academic Press, pp. 45-113 (1981). These germs can be germinated and then transferred to greenhouses or fields where mature crops can grow. Plant breeders will be able to use these plants to obtain useful genetic changes or to nourish varieties used in plant breeding plans.

Techniques for measuring the quantity and quality of somatic tissue obtained by culturing somatic plant parts are known. The amount of somatic embryos can be measured by determining the yield of structures associated with the stage of embryo growth [Fujimura, T., and A. Komamine, "Synchronization of somatic embryogenesis in a carrot cell suspension culture" plant Physiology, 64: 162. 164 (1979): Verma, DC And D.K. Dougall, "Influence of carbohydrates on quantitative aspects of growth and embryo formation in wild carrot suspension cultures" plant physiology 59: 81-85 (1977). This is usually measured by using a dissecting microscope to count the number of structures.

Breeding quality can be assessed in a variety of ways. Embryo growth is typically determined by visual examination of globular, heart-shaped, torpedo-like and seedling stages. Ammirato, PV, The effects of abscisic acid on the development of Somatic embryos form cells of caraway (Carum carri L.) Botanical Gazette 135: 328-337 (1974). Growth or quality of embryos can also be determined from the yield of seedlings obtained from each germination. Drew, RLW, "The development of carrot (Daucus carota L embryoids (derived from cellsuspension culture) into plantlets on a sugar-free basal medium "Horticultural Research 19:79 (1979) .However, despite the importance of determining the yield of functionally useful embryos in the use of land, Formation is rarely measured.

Techniques for improving the quantity or yield of pears have been described in the literature. For example, the formation of somatic embryos is known to require a source of ammonium in the medium for embryogenicity to occur. See Halperin, W. and DFWetherell, `` Ammonium requirement for embryogenesis in vitro ", Nature 205: 519. -520 (1965); Walker, KA, and SJ Sato, "Morphogenesis in callus tissue of Medicago sativa: the role of ammonium ion in somatic embryogenesis", Plant Cell Tissue Organ Culture 1: 109-121 (1981). Since the plant cell medium contains some ammonium, it has been important to adjust the ammonium concentration to a suitable level to obtain high yields and good quality embryos. Walker and Sato, supra; Wetherell, DF and DK Dougall. Sources of nitrogen supporting growth and embryogenesis in cultured wild carrottissue ". Physiologia Plantarum 37: 97-103 (1976)].

In plant cell cultures and mature plants, ammonium and glutamine or glutamate can be readily converted by cells. See Ojima, K. and K. Ohira, "Nutritional requirements of callus and cellsuspension cultures" in Frontiers of Plant. Tissue Culture-1978, TA Thorpe, ed., University of Calgary Offset Printing Services, pg. 265-275 (1978): Miflin, B.J. And P.L.Lea, "Ammoniumassimilation" in The Biochemistry of Plants, P. Stumpf and E. Conn eds., Academic Press., Vol. pg. 169-201 1980: Dougall, D.K. "Current problems in the regulation of nitrogen metabolismin plant cell cultures", in Plant Tissue Culture and its Biotechnological Application. W. Barz, E. Reinhard, and M.H. Zenk, eds., Springer-Verlag, Berlin, pg. 76-81 (1977).

The perceived approximation of ammonium, glutamine and glutamate in plant metabolism is reflected in a protocol that systematically studies the effects of amino acids on somatic formation. Studies using carrot cells have removed the ammonium from the plant cell medium and tested the effect on somatic formation with a single amino acid (Wetherell and Dougall, supra; Kamada, H. and H. Harada, "Studies on the organogenesis in carrot tissue cultures II.Effects of amino acids and inorganic nitrogenous compounds on somatic embryogenesis," Zeit. Pflanzenphysiologie 91: 453-463 (1979). Weatherel and Dogel have shown that addition of amino acids does not improve embryogenicity over ammonium ions. On the other hand, Kamada and Harada found that alanine and glutamine act as good sources of reducing nitrogen for embryogenicity in the absence of ammonium or in the absence of ammonium and nitrate. Reinert, J. and M. Tazawa, "Wirkung von Stick-stoffverbindungen und von Auxin auf die Embrogenogenese in Gewebekulturen," Planta 87: 239 (1969), (Text in German)] add 20 mM or more amino acids. Objected.

수준은 체배형성을 자극하는 중요요인이다.내부의

수준은 외부에서 공급된

또는 아미노산으로 부터 유도되거나, 생물학적으로 질산염이

로 환원되어 유도된다. 내부에서,

는 유기 질소화합물로 전환하여 정상적인 세포가 필요로하는아미노산을 공급할 수 있다[참조:Tazawa 및 Reinert, 상기 문헌]. 따라서, 아미노산은 암모늄을 방출함으로써, 체배형성을 자극하는 작용을 하는 것으로 믿어진다.The above studies indicate equivalence between sources of reducing nitrogen such as ammonium and amino acids (Wetherell and Dougall, Kamada and Harada, supra). The metabolic equivalence of ammonium and amino acids has also been taught in studies of Tazawa and Reinert. -173 (1969)]. A study of ammonium levels in carrot cultures carrying out somatic formation revealed that the level of ammonium was related to the amount of somatic formation at the culture rate, regardless of whether the culture received ammonium, amino acids or nitrates. . In conclusion, internal

Levels are important factors in stimulating germination.

Level is externally supplied

Or derived from amino acids, or biologically

It is reduced to and induced. Internally,

Can be converted to an organic nitrogen compound to supply the amino acids needed by normal cells (Tazawa and Reinert, supra). Thus, it is believed that amino acids act by stimulating embryogenesis by releasing ammonium.

Factors that have been well known to improve pear growth are abscisic acid, zeatin, gibberellic acid, high sucrose and light. Ammirato, P.V. And F.C. Steward, "Some effects of environment on the development of embryos from cultured free cells", Botanical Gazette 132: 149-158 (1971); Ammirato, P.V., "Hormonal control of somatic embryo development from cultured cells of caraway.Interactions of abscisic acid, zeatin and gibberellic acid", Plant Physiology 59: 579-586 (1977). The action of ammonium and amino acids on the quality of embryos is not known [Ammirato, PV, "The regulation of somatic embryo development in plant cell cultures: Suspension culture techniques and hormone requirements", Bio / Technology 1: 68-74. (1983)].

In addition, to date, as a method of measuring embryo quality, conversion frequency is not recognized or systematically used to improve embryo growth. Pear maturity has been measured by visually assessing the shape of the pear, but this method does not measure the frequency of plant formation from each pear.

It is therefore an object of the present invention to provide methods and materials for increasing the quantity and quality of somatic embryos produced from plant tissues.

It is also an object of the present invention to provide an optimal source of reducing nitrogen to form a multiplication.

It is also an object of the present invention to provide a method and material capable of mass propagation of a large number of plant species through somatic embryo formation.

It is also an object of the present invention to provide methods and materials for generating a plurality of viable multiplications having the same genetic and expressive properties.

Other objects, advantages and features of the present invention will become apparent from the following description and the appended examples.

(Initiation of invention)

The present invention provides novel improvement methods and materials that produce a number of good somatic embryos from plant tissues by adding optimal amounts of amino acids and sources of reducing nitrogen. One aspect of the present invention relates to a source of ammonium ions, together with one or more amino acids selected from the group consisting of proline, alanine, arginine, glutamine, asparagine, serine, ornithine, glutamate and amides, alkylesters and dipeptidyl derivatives thereof. A plant cell medium is provided which comprises a medium added in an amount sufficient to substantially increase the number or quality of the somatic embryos produced in comparison to embryos produced in a medium without added ingredients.

Another aspect of the invention contains little ammonium ions, and at least one amino acid selected from the group consisting of proline, arginine, asparagine, ornithine, lysine and amides, alkyl esters and dipeptidyl derivatives thereof contains these components. Compared to the embryos produced in the medium without addition, a plant cell medium added is provided in an amount sufficient to substantially increase the number or quality of the resulting somatic embryos.

Also provided are methods of using the media of the invention.

Best way to carry out the invention

The present invention provides a method of increasing the quantity and quality of embryos produced from plant tissue by providing a medium for culturing said cells and tissues containing a sufficient amount of selected amino acids to stimulate somatic embryo formation.

The present invention also provides such increased amounts and qualities by providing a medium for culturing the cells and tissues described above containing a source of ammonium ions and selected amino acids in an amount sufficient to stimulate the amount and quality of somatic embryos. The present invention also provides a method of using the plant tissue medium.

It is already known that amino acids act as simple equivalents to the desired ammonium medium component, but surprisingly, it has been found that amino acids can be used in place of ammonium ions to increase multiplication production above the equivalent concentration of ammonium. It has also been found that surprisingly, selected aino acids with additional sources of ammonium ions can provide unexpectedly increased benefits from the simple addition of increased ammonium ion concentrations.

Medium containing proline, arginine, lysine, asparagine, ornithine and amides, alkyl esters and dipeptidyl derivatives of these amino acids, and containing little ammonium ions, media containing cultures derived from culture tissue It has been found to increase the amount and quality of.

In addition, a sufficient amount of ammonium ions to stimulate embryogenesis or reconstitution, proline, alanine, arginine, glutamine, lysine, asparagine, serine, ornithine, glutamate and amide, alkyl ester and dipeptidyl derivatives of these amino acids It has been found that media containing one or more amino acids selected from the group can provide a similar fold increase.

Surprisingly, the media of the invention have been found to increase the yield of somatic embryos from callus beyond the typical media that have been used to date for induction, regeneration and preservation of embryonic tissues. In each step of this embryogenic process, the media of the present invention can be used to obtain a much higher yield of embryonic tissue than the results already provided. In addition, through the practice of the present invention, advantageous advantages can be obtained in a variety of useful plant species, including alfalfa, celery, cotton, corn and rice.

Plant cell media that can be improved by the practice of the present invention include already known plant tissue media as described in the literature, the techniques and methods of which are incorporated herein by reference. Huang, L. And T. Murashige, supra, and Cloning agricultural plants via in vitro techniques, BV Conger, ed., CRC Press, Inc. pp.172 et seq.]. In general, the plant medium provides the plant with panaxitis, which controls the pH and osmotic balance of nutrients, sources of energy such as sugar, plant hormones and aqueous solutions.

Representative examples of such plant cell media are media known as Schenk and Hildebrand (SH) media, the techniques and methods of which are incorporated herein by reference. See Schenk, R.U. And A.C. Hildebrandt, "Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures", Can J. Bot., 50: 199 (1972)]. Herdebrandt, hormone-free SH medium used for most salts, vitamins and Medium containing sucrose but not containing 2,4-D, pCPA and kinetin.

In addition, media known as murashi and scrubbing (MS) can be applied in place of SH media. Murashige, T. And F. Skoog, "A revised medium for rapid growth and bioassays with Tobacco tissue cultures", Physiologia Planta., 15: 473-497 (1962). The techniques and methods of this document are also incorporated herein by reference. As used hereinafter, hormone-free MS medium is a medium which is described as containing mostly salts, vitamins and sucrose but not indole-3-acetic acid and kinetin.

The basic plant cell medium that can be used in the practice of the present invention is selected, in part, according to the species of the selected plant tissue, which belongs to those of ordinary skill in the art for those skilled in the practice of tissue culture and multiplication of plant cells. Is considered.

Many important crops and horticultural species can be reproduced through tissue culture and somatic formation. The varieties of Table 1 below include, but are not limited to:

TABLE 1

A more detailed list of somatic embryos can be found in the literature, and relevant sections are incorporated herein by reference. Evans, DA et al ., "Growth and Behavior of Cell Cultures: Embrogenesis and Organogenesis. in Plant Tissue Culture: Methods and Applications in Agiculture , Thorpe, ed., Academic Press, P 45 et seq (1981).

Many of the amino acids known in the prior art, with some exceptions, possess the common feature of the presence of free carboxyl groups and free unsubstituted amino groups at α-carbon atoms. Proline is an obvious exception because the α-amino group of proline is substituted and is actually α-amino acid.

Amino acids are generally divided into protein amino acids and nonprotein amino acids, with the most commonly recognized 20 protein amino acids. Such amino acids contain four subgroups: amino acids containing nonpolar or hydrophobic substituents (e.g. alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine): a non-charged polar R group Amino acids containing (eg, serine, threonine, tyrosine, asparagine, glutamine, cysteine and, speculatively, glycine); Amino acids containing negatively charged R groups (eg aspartic acid, glutamic acid): and amino acids containing positively charged R groups (eg lysine, arginine, and speculative histidine).

In addition to the twenty conventional amino acids, there are a number of other amino acids that appear little or no in protein. Hydroxylysine Hydroxyproline is rarely found and is present only in fiber proteins. In addition, 150 different amino acids are known to occur in free or bound form in different cells and tissues, as well as many common amino acids of the β, Y and Δ types, as well as most commonly the intermediates in arginine synthesis, citrulline and orni There is a teen.

In addition to the basic amino acid structures described above, amino acids can be modified in a number of ways without changing the functionality in the present invention. Among these changes, amino acid amides and amino acid alkyl esters are produced by adding amino groups and carboxy groups, respectively. Dipeptidyl derivatives of amino acids can also be produced by bridging two amino acids through an α-carboxy group and an α-amino group. It will be readily appreciated that each pair of amino acids is two potential dipeptidyl derivatives.

)의 공급원을 제공한다는점에서 중요하다. 암모늄 이온의 공급원은 또한 식물 조직배양 기술 분야에 잘 공지되어 있다. 전형적으로,이러한 암모늄 이온은 암모늄 이온의 하전과 균형을 이루는 음이온에 의해 생성된, 많은 비-독성 암모늄염(예:암모늄 클로라이드, 암모늄 포스페이트 또는 암모늄 설페이트)의 배지에 포함되어 제공된다. 다른 암모늄 이온의 공급원은 문헌에 기술되어 있으며, 관련 부분은 본 명세서에 참고문헌으로 인용된다[참조:walker, K.A. 및 S.J. Sato, "Plant Cell Tissue Organ Culture"1.109-121(1981)].In addition, the present invention is an ammonium ion supplementing the amino acid-containing medium of the present invention (

It is important in that it provides a source of Sources of ammonium ions are also well known in the plant tissue culture art. Typically, such ammonium ions are provided included in the medium of many non-toxic ammonium salts (eg ammonium chloride, ammonium phosphate or ammonium sulfate) produced by anions that balance the charge of ammonium ions. Other sources of ammonium ions are described in the literature, and relevant portions are incorporated herein by reference (walker, KA and SJ Sato, "Plant Cell Tissue Organ Culture" 1.109-121 (1981)).

The following examples are intended to illustrate various aspects of the invention. The examples do not limit the scope of the invention which defines the scope of the claims appended hereto.

[Experiment]

In general, methods useful for carrying out somatic formation with plant cells and tissue culture are well known and only slight modifications suitable for the selected plant species are required. Such examples are described in the literature, and relevant portions are incorporated herein by reference. Plant Tissue Culture : Methods and Applications in Agriculture, Thorpe, ed., Supra (1981).

For example, alfalfa embryogenesis can generally be derived from Regen S Line in Shanders and Bingham (Saunders and Bingham, "Production of Alfalfa Plants from Callus Tissue", Crop Sci., 12: 804-808 (1972).

Medigo Sativa

Variety Regen S plants are derived from Second cycle recurrent Selection, which regenerates by crossing Vernal and Saranac varieties. Callus is a petiole (Petiole) 50% Clorox ^R (Clorox ^R) a surface sterilized, washed with water for 5 minutes, then swengkeu-Hildebrand medium [see: Schenk, RU and AC Hildebrandt, supra, (1972); It is initiated by application on a hormone-free SH medium containing salts, vitamins and sucrose. The medium contains 25 μM α-naphthylene acetate 100 μM kinetin and 0.8% (W / V) agar (named Conservation Medium). Callus generated in in vitro culture tissue is isolated from residual non-callus tissue and preserved in medium Sub-culture by repeating in phase. Callus are subcultured at 3 week intervals and then matured under indirect light at 27 ° C.

Collect 3-9 g of callus subcultured from a plate of preservation medium for 17-24 days, then 100 ml liquid containing 50 μM of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM kinetin (B). Transfer to SH and induce [walker: kA, ML Wendeln and E.G. Jaworski, Plant Sci. Lett. 16: 23-30 (1979)]. Cells are incubated for 3 days under indirect light in a 500 ml flask on an orbital shaker of 100 R.P.M. (at 27 ° C.).

Induced cells are aseptically sorted by size as a series of column sieves (fisher scientific) under mild agitation. The cell populations are sedimented or passed through 35 mesh (480 μm) and then collected on 60 mesh (230 μm) through a stainless steel screen. The cells retained on the 60 mesh screen are drawn to 500 ml of hormone-free SH medium for every 100 ml of induction culture volume. Wash. The washed medium is removed in vacuo. The fresh weght of the cell population is measured and resuspended in hormone free SH medium at a biomass weight of 150 mg per ml. 75 mg (0.5 ml) of resuspended cells are dispensed on about 10 ml of agar solidification medium in a 60 mm x 15 mm Petri dish.

는 2.6mM이다)를 포함한다. 암모늄 이온 비함유 배지는 SH의 NH₄H₂PO

대신에 동량의 NaH₂PO₄를 사용하여 형성할 수 있다.25mM

비교용 배지는 12.5mm(NH₄)₂SO₄가 보충된 암모늄 비함유 배지로 이루어진다. 환원 질소의 모든 유기 및 무기 공급원은 O.2μm 여과하여 멸균한 다음, 새로운 오트클레이브 배지에 연속적으로 가한다.In addition, somatic embryogenesis in suspension culture would occur when 300 mg (2 ml) of resuspended cells were added to 8 ml of hormone-free liquid SH medium contained in a 50 ml Erlenmeyer flask. Embryogenic medium was SH medium containing 3% (W / V) sucrose without hormone (

Is 2.6mM). Ammonium ion free medium is NH ₄ H ₂ PO of SH

Instead, an equivalent amount of NaH ₂ PO ₄ can be used to form 25 mM.

The comparative medium consisted of an ammonium free medium supplemented with 12.5 mm (NH ₄ ) ₂ SO ₄ . All organic and inorganic sources of reducing nitrogen are sterilized by 0.2 μm filtration and subsequently added to fresh oatclave medium.

(Saran Wrap

)으로 밀봉한 다음100rpm의 오비탈 진탕기상에서 14일 동안 배양한다. 고체화 배양물로부터 28cm 또는 현탁 배양물로부터200cm 떨어진 차가운 백색 형광 튜브로부터 27℃에서 12시간 조사하면서 배양한다.Each treatment is generally applied to 10 replicates. The dish is wrapped with parafilm and incubated for 21 days. Suspension flasks with foam stoppers and Saran wrap

Sara Wrap

) And incubate for 14 days on an orbital shaker at 100 rpm. Incubate for 12 hours at 27 ° C. in a cold white fluorescent tube 28 cm from the solidification culture or 200 cm from the suspension culture.

Embryogenesis is visually determined by counting the number of green centers formed on the callus with a stereoscopic microscope at 10 × magnification after incubation. The size of the ship is measured using an eyepiece corrected at 10 × magnification. The shape of the ship is determined by visual inspection. Conversion to complete plants with embryonic roots and young shoots (first herbaceous leaves) supplemented with 25 μM gibberelic acid and 0.25 μM α-naphthyleneacetic acid solidified to 0.8% agar from amino acid treatment in 21 days of initial culture. By aseptic transfer to a 1 / 2-century hormone-free SH medium.

1. Somatic Embryogenesis in Medium Containing Ammonium

A. Leguminosa Somatic Cell Culture

)와 비교하여, 배형성을 성장 또는 억제하는 독성이있는 것으로 밝혀졌다. 이러한 아미노산은 황과 방향족 및 대부분의 측쇄족을 포함한다. 이러한 아미노산중어떤 것도 배지 비교용 이상으로 배형성을 자극하지 않으며 모두 독성이 있어,1 또는 10mM에서 캘러스의성장을 억제하거나 갈변(browning)을 야기한다.As a representative example with Leguminosae, alfalfa tissue, Medicago sativa, Regen S, are cultured as described above and analyzed for germination in a medium containing 2.6 mM ammonium according to the present invention. All protein amino acids are tested at concentrations of 1-100 mM. Two responsive forms emerge from this initial screen and based on these results, another test is performed with the sieve treated cells. Table 2 excludes amino acids of the first reaction type, which is SH-medium (2.6 mM)

Compared to), which has been shown to be toxic to grow or inhibit embryogenicity. Such amino acids include sulfur and aromatics and most of the side chains. None of these amino acids stimulate embryogenicity beyond medium comparisons and are all toxic, inhibiting callus growth or causing browning at 1 or 10 mM.

비교용보다 거의 3-배 더 많은 배를 수득하게 하며 알팔파(D)에서의 최적 암모늄 농도인 25mM

의 두배의 효과이다[참조:walker, et a1., 상기 문헌]. 알라닌, 아르기닌, 글루타민 및 리신은 모두 효과가 작지만 약25mM

수준으로 배형성을 자극한다. 세린 및 아스파라긴은 SH 비교용에 비하여 배형성의 자극은 약하지만, 배의 크기는 증가시키는 것으로 나타났다.The second responsive form from the initial screen stimulates embryogenesis or increases the size of the embryo compared to the SH comparison (see Table 2). Detailed studies of concentration dependence have been performed on these amino acids and the results are shown in FIG. The most effective amino acid for stimulating germination is proline, which is 2.6 mM

25 mM, which is the optimal ammonium concentration in alfalfa (D), giving almost 3-fold more folds than comparable

Double the effect of (walker, et al., Supra). Alanine, arginine, glutamine and lysine are all small but about 25 mM

Level stimulates embryogenesis. Serine and asparagine have been shown to have a weaker stimulation of embryogenicity than SH comparisons, but increased embryonic size.

Table 2 summarizes the amino acids and other nitrogen sources found to stimulate somatic formation in alfalfa. The ester and amide forms of proline are extremely active in stimulating the number and vagina of the embryo, as in the dipeptide, prolyl alanine. It should be noted. It should be noted that the nonprotein amino acid ornithine is also active.

TABLE 2

Effect of Reducing Nitrogen Sources on Somatic Embryogenesis in Alfalfa

Stimulus source

ammonium

Proline

Alanine

Glutamine

Arginine

Asparagine

Ornithine

Serine

Lee Sin

L-proline amide

L-Prolyl-L-Alanine

L-proline methyl ester

Using the above technique, the size of the embryo is measured by visually examining the size of the embryo. Data is shown in Table 3.

TABLE 3

Effect of Reduction Nitrogen Treatment on the Size of the Soybean Germ

Based on the data in Tables 2 and 3, the effect of amino acid addition to enhance the size of the embryos can be shown in the following order.

Arginine ≥ Glutamine ＞ Alanine ＞ Proline ＞

Using the above technique, the table was observed by observing the conversion of the pear into a complete plant with the root and the side of the young shoot (the first herbaceous leaf). The results are as follows.

TABLE 4

Conversion of Breeding to Alfalfa Seedlings

Based on the data in Table 4, the additive effects of converting embryos into seedlings are as follows:

Glutamine ＞ Alanine ≥ Arginine ＞ Proline ＞

From the correlation between the above data, the size of the embryo is a good indicator of the embryo converting into seedlings, and thus a good indication of the quality of the embryo produced by the provided technique.

The optimal amount of amino acid addition that stimulates somatic embryogenesis in agar solidified cultures and liquid suspension cultures is determined.

These data are listed in Table 5 and Table 6 as follows:

TABLE 5

의 존재하에 한천 고체화 배양물에의 아미노산 첨가에 의한 알팔파 체 배형성의 자극2.6 mM

Stimulation of Alfalfa Body Embryogenesis by Amino Acid Addition to Agar Solid Cultures in the Presence of

TABLE 6

의 존재하에 액체 현탁 배양물에의 아미노산 첨가에 의한 알팔파 체 배형성의 자극2.6 mM

Stimulation of Alfalfa Body Embryogenesis by Amino Acid Addition to Liquid Suspension Cultures in the Presence of

B. Umbelifera Somatic Cell Culture

As a representative example of the family Umbelliferae, celery seed, Apium graveolens (Calmario cultivar) is germinated for 1-2 weeks. The resulting germinated seeds are sterilized with 10% chlorox ^R solution for 20 minutes. Cotyledon or hypocotyl is removed and the in vitro culture is placed in 0.8% agar solidifying hormone-free SH medium containing 25 μM 2,4-D and 5 μM benzyladenin. After callus is initiated (3-4 weeks), callus is transferred to SH medium containing 2.5 μM 2,4-D and 0.5 μM kinetin. Additives that are easy to heat are filtered and sterilized and added to warm medium. If desired, a specific amount of inoculum tissue is obtained by filling in a uniform volume using a modified spatula instrument. Subsequently callus is subcultured in SH medium containing 1 μM Picloram and 0.5 μM benzyl adenine. For somatic embryogenesis, 75 mg of callus cells are transferred to 0.8% agar solidifying hormone-free SH medium containing filtered sterile additives and incubated for 18 to 30 days at 24 ° C. under the same conditions as in alfalfa.

비교용-처리 배와 비교한다.50mM 알라닌을 함유한 처리 배양물의 결과는 다른 모든 처리보다 더 높은 빈도의 배가 형성될 뿐만 아니라 이러한 배는 다른 배양물보다 우수한 자엽, 뿌리 및 초생엽의 성장을 보인다. 생성된 배의 총 수효는 다음과 같은 순서이다:20 내지 100mM 알라닌＞50mM 프롤린＞25mM 글루타민-N＞25mM

Amino acids proline, alanine and glutamine

Compared to the comparison-treated embryos, the results of treated cultures containing 50 mM alanine not only resulted in the formation of higher frequency embryos than all other treatments, but also the growth of cotyledons, roots and herbaceous leaves superior to other cultures. . The total number of pears produced is in the following order: 20 to 100 mM Alanine> 50 mM Proline> 25 mM Glutamine-N> 25 mM

-처리물질과 비교하여 셀러리에서의 배 수효를 자극한다. 상기 농도의 알라닌, 프롤린, 글루타민 및 글루타메이트는

처리 배와 비교하여 셀러리 배가모종으로 전환하는 것을 향상시킨다.Proline stimulates embryogenicity more than glutamine, but glutamine results in good germination of seed-phase embryos. Ammonium-treated cultures produce smaller and smaller embryos than all other treatments. 25 mM when glutamic acid is added to celery regeneration medium alone, 30 mM

Stimulates fold count in celery compared to treated material. At this concentration alanine, proline, glutamine and glutamate

Improved conversion to celery doubling seedlings compared to treated embryos.

C. Graminea somatic cell culture

As a representative species of the family Gramineae, Zea mays embryogenesis is carried out by applying the medium according to the present invention.

Ears of corn 10 days after fertilization are harvested and the immature pears are sterilely divided. Pears were N-6 inorganic salt medium containing 3% sucrose and 5μ'M 2,4-D [Chu, cc, wang, c, c., Sun, c, s., Hsu, C., Yin, K, C. And Chu, C. Y.1975

Establisnment of an efficient medium for anther culture of rice through comparative experimentson the nitrogen sources.Sci.Sin.16,659-688]. 21 days after incubation of the callus, each produced in the presence or absence of L-proline Record the pear population formed from the callus. The results are listed in Table 7, and the% response is the average of the embryonic frequency of 287-1165 replicate embryonic in vitro cultures.

TABLE 7

As another example of somatic embryogenesis, including Graminea, rice seed Oryza Sative is regenerated in accordance with the practice of the present invention.

Peel the Orissa sativa seeds, surface sterilize, then 4% sucrose, 0.26 mM tritopane, 5 μM 2,4-D, 1 μM kinetin, 2.5 g / ι (pH) of Gelite as gelling agent 6.2) Mura-shi and containing (MS) salts [Murashige, T. And F. Skoog. (1962), supra. 15-21 days later, each embryo is recorded under an anatomical microscope in the embryo formed in the scute-har region of the seed. The results for and without treatment with L-proline are shown in Table 8.

TABLE 8

D. Malvaceia somatic cell culture

As an example of the practice of the invention in plant body tissues of the family Malvaaceae, cultures of two cotton species are regenerated in a medium prepared according to the techniques of the present invention.

Gosiumum Hystherum. Cultures initiated from surface sterile seeds of Gossypium hirsutum were run for 4 weeks at 0.8% agar medium in Murashiki and Scock salt containing 3% sucrose and 0.5 μM NAA, 5 μM 2-isopentanyl adenine. Subculture. Embryos are induced by incubating for 10 days in medium containing 0.5 μM 2,4-D and 0.2 μM kinetin or with 1 μM NAA and 0.5 μM kinetin, with or without proline in liquid suspension culture. Cells are then sieved and regenerated with hormone-free SH medium containing 3% sucrose and 10 mM L-glutamine. After 4 weeks, formation of embryos with mature cotyledons is measured. The results are shown in Table 9.

TABLE 9

Effect of Proline Addition on Regeneration of Cotyledon Gossypium hirsutum Pear in Suspension Cultures

Gossypium Klotzschionum. Cultures initiated from surface sterile seeds are subcultured in murashi and scruck salts, 3% sucrose, 0.5 μM NAA and 5 μM 2-isopentanyladenin. Callus was suspended for 10 days in MS salt, 3% sucrose and 0.2 μM picloram and then regenerated for 21 days in hormone-free medium with L-glutamine. The results are shown in Table 10.

TABLE 10

Effect of L-Glutamine on Embryogenesis in Gosipiung Clotscyanum

2. Interactions of Amino Acids with Ammonium Ion Sources

농도를 변화시켜 하나의 첨가물에 대한 최적 농도가 첨가된 물질의 존재에 의하여 영향받는지의 여부를 측정한다.Cells are induced, sieve treated and applied as in the above experiment. Proline or arginine and

The concentration is varied to determine whether the optimal concentration for one additive is affected by the presence of the added substance.

의 첨가량이 0 내지 25mM 사이에서 변화하는 30mM 내지 300mM 범위 이상에서 시험한다. 결과는 표 11에 기재한다.1.Proline: Proline

The addition amount of is tested in the range of 30 mM to 300 mM or more, varying between 0 and 25 mM. The results are shown in Table 11.

의 농도 외에 아르기닌의 농도를 변화시켜 유사한 실험을 수행한다. 결과는 표 11에 기재한다.2. Arginine: Added to the medium

Similar experiments are performed by changing the concentration of arginine in addition to the concentration of. The results are shown in Table 11.

TABLE 11

Effect of interaction of amino acids with ammonium ions in alfalfa (average number of folds generated in seven or more tests)

를 첨가할 때 상승효과가 생기는 것으로나타났다.In each case, the optimal amount of arginine or proline and

It was shown that synergistic effect occurs when adding.

와 L-프롤린에 대하여 체 배의 양과 모종으로의 효과를 시험한다.Some of the examples listed in Table 11 were repeated to obtain various concentrations.

Test the effect of seedlings and their effects on seedlings on and L-proline.

TABLE 12

Effect of Ammonium and Proline on the Amount and Quality of Somatic Germination

Medium containing proline and ammonium increases the amount of pear and proline improves the quality of the pear in the presence of high or low concentrations of ammonium.

3. Amino Acid Addition to Medium Containing Almost No Ammonium Ion

를 제외시켜 상기 실시예에서와 같이 알팔파 세포를 유도하여 도포한다. 체배형성에대한 아미노산 농도 범위의 효과를 시험한 결과를 표 13에 요약한다.In badge formation

Except for inducing and applying alfalfa cells as in the above example. The results of testing the effect of the amino acid concentration range on somatic embryo formation are summarized in Table 13.

TABLE 13

을 거의 함유하지 않는 배지의 한천 고체화 배양물에의 아미노산 첨가에 의한 알팔파 체배형성의 자극

Stimulation of Alfalfa Embryogenesis by Addition of Amino Acids to Agar Solidified Cultures Containing Almost No Containing Medium

가 존재하지 않는 경우, 상기의 아미노산이 배지중의 유연한 환원 질소 공급원으로서 체배형성을 자극하는 것으로 나타났다. 또한, 오르니틴의 제외된 경우에도, 전술한 농도 범위로 상기의 첨가를 함으로써 크기, 형태 및 성숙도로서 측정한, 생성된 배의 질이 실질적으로 향상된다.realistically

Is absent, it has been shown that these amino acids stimulate somatic embryonic formation as a source of flexible reducing nitrogen in the medium. In addition, even in the case of the exclusion of ornithine, the addition of the above-mentioned concentration range substantially improves the quality of the resulting embryo, measured as size, shape and maturity.

4. Mixing of Amino Acids

부재하의 알팔파 배양물에 혼합 아미노산의 첨가 효과를 나타낸다. 혼합 아미노산은배의 수효에 대하여 상승효과를 제공한다.The table below

The effect of addition of the mixed amino acid to the alfalfa culture in the absence is shown. Mixed amino acids provide synergistic effects on the number of embryos.

TABLE 14

Large, high quality embryos are observed in treatments containing a mixture of proline and other amino acids.

While the invention has been described in some detail above by way of illustration and example, changes and modifications that can be made within the scope of the appended claims will be apparent to those skilled in the art.

[Brief Description of Drawings]

FIG. 1 is a graph showing that the resulting multiples increase as a function of amino acid concentration added to the medium.

Claims (25)

In regenerating somatic tissue from induced cells in plant cell nutrient culture medium to form embryonic tissue, a medium containing a source of ammonium ions together with all the inorganic salts, vitamins and nutrients needed to maintain tissue growth, and L-proline Plant cell nutrition culture medium containing an additive of at least one amino acid component selected from the group consisting of L-arginine, L-lysine, L-asparagine, L-serine, L-ornithine and amides, alkyl esters and dipeptidyl derivatives thereof To differentiate the somatic embryos from the induced cells and to expose undifferentiated plant body tissue selected from the group consisting of Gramineae, Umbelliferae and Malvaceae body tissues to the culture medium, By inducing embryo embryogenesis to form a embryo, and then maintaining the embryo on the culture medium. A method of producing embryonic tissues from gravemia, umbelifera, and malmsea body tissues, characterized in that the embryos develop into seedlings. The medium of claim 1 wherein the medium additive is selected from the group consisting of L-proline, amides, alkyl esters and dipeptidyl derivatives thereof, wherein the final concentration of L-proline in the medium is from 1 mM to about 300 mM. Embryonic tissue production method added to the medium. The medium of claim 1 wherein the medium additive is selected from the group consisting of L-arginine, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-arginine in the medium is sufficient to provide about 3 mM to 75 mM. Embryonic tissue production method added to the medium. The medium of claim 1, wherein the medium additive is selected from the group consisting of L-lysine, amides, alkyl esters, and dipeptidyl derivatives thereof, and the final concentration of L-lysine in the medium is sufficient to provide about 1 mM to about 10 mM. Embryonic tissue production method added to the medium. The medium of claim 1 wherein the medium additive is selected from the group consisting of L-asparagine, amides, alkyl esters and dipeptidyl derivatives thereof, with a final concentration of L-asparagine in the medium in an amount sufficient to provide 3 mM within about 0.5. Method for generating embryo tissue added during the treatment. The medium of claim 1 wherein the medium additive is selected from the group consisting of L-serine, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-serine in the medium is sufficient to provide about 0.5 to 2 mM. Embryonic tissue production method added to the medium. The medium of claim 1 wherein the medium additive is selected from the group consisting of L-ornithine, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-ornithine in the medium is sufficient to provide about 1 to 3 mM. Embryonic tissue production method which is added to the medium in Hanyang. The medium of claim 1, wherein the medium additive is selected from the group consisting of L-proline amide and dipeptidyl derivatives thereof containing L-proline amide, wherein the final concentration of L-prolineamide in the medium provides about 1 to 200 mM. Embryonic tissue production method added to the medium in an amount sufficient to. The medium additive of claim 1, wherein the medium additive is selected from the group consisting of L-proline methyl ester and dipeptidyl derivatives thereof containing L-proline methyl ester, and the final concentration of L-proline methyl ester in the medium is about 0.3 to 40 mM. Embryonic tissue production method is added to the medium in an amount sufficient to provide. The method of claim 1 wherein the source of ammonium ions is selected from the group of ammonium containing compounds consisting of ammonium chloride, ammonium nitrate, ammonium carbonate, ammonium sulfate, ammonium phosphate and ammonium citrate. The method of claim 10 wherein the source of ammonium ions is provided in an amount sufficient to provide a final concentration of ammonium ions in the medium to provide about 0.5 to 50 mM. The method of claim 1, wherein the medium is selected from the group consisting of Schenk-Hildebrandt medium and Murashige and Skoog medium. In regenerating somatic tissue from induced cells in plant cell nutrient culture media to form embryonic tissues, the ions of ammonium ions are substantially liberated and necessary to maintain the growth of L-glutamine, all inorganic salts, vitamins and tissues. Nutrients and additives of at least one amino acid component selected from the group consisting of L-proline, L-arginine, L-lysine, L-asparagine, L-serine, L-ornithine, its amides, alkyl esters and dipeptidyl derivatives It provides a plant cell nutrient culture medium containing to differentiate the somatic embryos from the induced cells, the undifferentiated plant body tissue selected from the group consisting of Gramineae, Umbellliferae and Malvaceae body tissues Exposure to the culture medium induces embryogenesis of plant tissues to form a germline, and then the germline is maintained on the culture medium Characterized in that it is generated as an exhausted seedling, thereby producing an embryonic tissue from Graminea, Umbelifera and Malvaea body tissues. The method of claim 13, wherein L-glutamine is provided in an amount that provides a final concentration of L-glutamine in the medium of about 10-50 mM. The medium of claim 13 wherein the medium additive is selected from the group consisting of L-proline, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-proline in the medium is sufficient to provide from 1 mM to about 300 mM. Embryonic tissue production method added to the medium. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-arginine, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-arginine in the medium is sufficient to provide about 3 to 75 mM. Embryonic tissue production method added to the medium. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-lysine, its amide, alkyl esder and dipeptidyl derivatives, and the final concentration of L-lysine in the medium is sufficient to provide about 1-10 mM. A method for producing embryonic tissue, which is added to the medium in an amount. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-asparagine, amides, alkyl esters and dipeptidyl derivatives thereof, and the final concentration of L-asparagine in the medium is sufficient to provide about 0.5 to 3 mMol. Embryonic tissue production method added to the medium. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-serine, amides, alkyl esters and dipeptidyl derivatives thereof, in an amount sufficient to provide a final concentration of L-serine in the medium of about 0.5 to 2 mM. Embryonic tissue production method added to the medium. The medium of claim 13 wherein the medium additive is selected from the group consisting of L-ornithine, amides, alkyl esters and dipeptidyl derivatives thereof, and an amount sufficient to provide a final concentration of L-proline in the medium of about 1 to 3 mM. Embryonic tissue production method added to the medium. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-prolineamide and its dipeptidyl derivatives containing L-proline amide, wherein the final concentration of L-proline amide in the medium provides about 1 to 200 mM. A method for producing embryonic tissue, which is added to the medium in a sufficient amount. 14. The medium according to claim 13, wherein the medium additive is selected from the group consisting of L-proline methyl ester and dipeptidyl derivatives thereof containing L-proline methyl ester, and the final concentration of L-proline methyl ester in the medium is about 0.3 to 40 mM. Embryonic tissue production method is added to the medium in an amount sufficient to provide. The method of claim 13, wherein the medium is selected from the group consisting of Shank-Hildebrand medium, Murashigi and Scout medium. In regenerating somatic tissue from induced cells in plant cell nutrient culture medium to form embryonic tissue, all mineral salts, vitamins and nutrients necessary to maintain tissue growth, and together with (a) the final L-proline in the medium L-proline, amide, alkyl ester and dipeptidyl derivatives thereof, added in an amount sufficient to provide a concentration of 10 mM to 300 mM, and (b) a final concentration of L-arginine in the medium to provide from about 15 mM to about l00 mM. L-arginine, amide, alkyl ester and dipeptidyl derivatives thereof added in sufficient amounts, (c) L-lysine added in an amount sufficient to provide a final concentration of L-lysine in the medium of about 1 mM to about 10 mM, Its eids, alkylesters and dipeptidyl derivatives, (d) L-asparagine, amides thereof, added in an amount sufficient to provide a final concentration of L-asparagine in the medium from about 30 mM to about l00 mM; Alkyl esters and dipeptidyl derivatives, (e) ornithine added in an amount sufficient to provide a final concentration of L-ornithine in the medium from about 0.3 mM to about 3 mM, amides, alkyl esters and dipeptidyl derivatives thereof, ( f) difabtidyl derivatives thereof containing L-proline amide and L-proline amide added in an amount sufficient to provide a final concentration of L-proline amide in the medium to provide from about 1 mM to about 200 mM, and (g) in the medium At least one selected from the group consisting of L-proline methyl ester and dipeptidyl derivatives thereof containing L-proline methyl ester added in an amount sufficient to provide a final concentration of about 0.3 mM to about 40 mM of L-proline methyl ester of Plant cell culture media containing additives of amino acid components are provided to differentiate somatic embryos from induced cells, Gramineae, Umbelliferae ) And malvaceae (Malvaceae) body tissue selected from the group consisting of body tissues exposed to the culture medium to induce embryonic formation of the plant body tissues to form a somatic embryo, the somatic embryos are maintained on the culture medium to develop embryos as seedlings Characterized in that it comprises the steps of producing embryonic tissue from gravemia, umbelifera and malvaea body tissues. 25. The method of claim 24, wherein the medium is selected from the group consisting of Shank-Hildebrand medium and Murashigi and Scout medium.

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