Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0025—Culture media for plant cell or plant tissue culture
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/04—Plant cells or tissues

Definitions

• This invention relates generally to the culturing of embryonic plant cells and tissue and more specifically to an improved medium particularly adapted for sustaining embryos produced by induction from somatic tissue in vitro and a method of using the medium.
• somatic plant cells are typically induced to undergo repeated cell divisions on a nutritive culture medium substrate, producing an amorphous cell mass known as callus.
• the callus can be maintained through subculture to allow mass proliferation.
• the callus may also be induced to undergo differentiation, which produces the organized tissues and organs of the mature plant.
• Somatic embryos may also form in culture from other preexisting embryos. The parent embryos may range from the immature globular stage to mature, germinating embryos. Lupotto, E., "Propagation of an embryonic culture of Medicago sativa L.”, Zeit. Dephysiol. 111:95-104 (1983).
• somatic embryos may arise from undifferentiated callus or from pre-existing embryos in plant tissue culture.
• genetic changes may be affected on a cellular or embryo level and then maintained through subsequent development to produce an entire crop with identical genetic characteristics. This allows the plant breeder to bypass the normal genetic barriers in plant reproduction, and obtain a more uniform and advantageous field crop.
• Somatic embryo quality can be assessed by various methods. Embryo development is typically determined visually by searching for globular, heart, torpedo and plantlet stages. Aramirato, P.V., "The effects of abscisic acid on the development of somatic embryos from cells of caraway (Carum carri L.) "Botanical Gazette 135:323-337 (1974). Embryo development or quality can also be determined from the yield of plantlets obtained from individual somatic embryos. Drew, R.L.W., "The development of carrot (Daucus carota L. ) embryoids (derived from cell suspension culture) into plantlets on a sugar-free basal medium” Hoxticultural Research 19:79 (1979). However, plantlet formation is rarely measured despite its importance in determining the yield of functionally useful embryos for field use.
• NH 4 + is the critical factor in stimulating somatic embryogenesis.
• the internal NH 4 + level is derived from either externally supplied NH 4 + or amino acids, or by the biological reduction of nitrate to NH 4 + .
• NH 4 + can be converted to organic nitrogen compounds to supply amino acids for normal cell requirements, Tazawa and Reinert, suora. Hence amino acids are believed to act by releasing ammonium, which stimulates embryogenesis.
• This invention provides novel and improved methods and materials for producing numerous high quality somatic embryos from plant tissue by the addition of optimal amounts of amino acids and sources of reduced nitrogen.
• One aspect of the present invention provides a plant cell culture medium which comprises a medium with a source of ammonium ion, together with an addition of at least one amino acid selected from the group consisting of proline, aianine, arginine, glutamine, asparagine, serine, ornithine, glutamate and the amides, alkyl esters and dipeptidyl derivatives thereof in an amount sufficient to increase the number or quality of somatic embryos produced, compared to the embryos produced in culture media without such addition.
• Another aspect of the present invention provides a plant cell culture medium substantially free of ammonium ion, further comprising an addition to the medium of at least one amino acid selected from the group consisting of proline, arginine, asparagine, ornithine, lysine and the amides, alkyl esters and dipeptidyl derivatives thereof in an amount sufficient to substantially increase the number or quality of somatic embryos produced, comnarad to the embryos produced in culture media without such addition.
• at least one amino acid selected from the group consisting of proline, arginine, asparagine, ornithine, lysine and the amides, alkyl esters and dipeptidyl derivatives thereof in an amount sufficient to substantially increase the number or quality of somatic embryos produced, comnarad to the embryos produced in culture media without such addition.
• the single drawing is a graphic representation of the increase in number of somatic embryos produced as a function of the concentration of amino acids added to the medium.
• the present invention provides methods for enhanced quantity and quality of embryos produced from plant somatic tissue by providing a medium for culturing said cells and tissue which contains a sufficient amount of selected amino acids to stimulate somatic embr ⁇ genesis.
• the present invention also provides for such enhanced quantity and quality by providing a medium for culturing such cells and tissue which contains selected amino acids to ⁇ ether with sources of ammonium ion in amounts sufficient to stimulate the quantity and quality of somatic embryos. Also provided is a method for using such plant tissue culture medium.
• amino acids served as simple equivalents to the desired ammonium media component
• amino acids can serve as replacement for ammonium ion which enhance the production of somatic embryos over the equivalent concentrations of ammonium.
• selected amino acids together with an additional source of ammonium ion can provide substantially increased benefits which would not be predicted from a simple additive effect of increased ammonium ion concentration.
• a medium which contained an amino acid selected from the group consisting of ⁇ roline, argmine, lysine, asoaragine, ornithine, and the amidds, alkyl esters and dipeptidyl derivatives of these a ⁇ ino acids, which medium is substantially free of ammonium ion, provides enhanced quantity and quality of somatic embryos derived from the cultured somatic tissue.
• a medium containing ammonium ion and at least one amino acid selected from the group consisting of proline, aianine, arginine, glutamine, lysine, asparagine, serine, ornithine, gluatamate and the amides, alkyl esthers and dipeptidyl derivatives of these amino acids in an amount sufficient to stimulate embryogenesis or embryo conversion can provide similar embryo enhancements. It has been surprisingly discovered that the medium of the present invention provides increased yield of somatic embryos from callus tissue over the typical media used heretofore in the induction, regeneration and maintenance of embryonic tissue.
• plant culture media provide plant nutrients, sources of energy such as sugar, plant hormones and buffered salts to control the pH and osmotic balance of an aqueous solution.
• SH Schenk and Hildebrandt
• Schenk, R.U. and A.C. Hildebrandt "Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant ceil cultures".
• Can. J. Bot., 50:199 (1972) the disclosures and formulae of which are incorporated herein by reference.
• hormone-free SH medium is the medium disclosed therein including the major salts, vitamins and sucrose, but without the 2,4-D, pCPA and kinetin.
• hormone-free MS medium is the medium disclosed therein including the major salts, vitamins and sucrose, but without the indole-3-acetic acid and kinetin.
• the selection of the basic plant cell culture medium to be utilized in the practice of the present invention will be dictated, in part, by the species of plant somatic tissue selected, and is considered to be within the ordinary skill of one experienced in the tissue culture of plant cells and the practice of somatic embryogenesis.
• Amino acids can be divided generally into protein and nonprctein amino acids wherein protein amino acids include the 20 most commonly recognized. These amino acids include four subgroups: Those with nonpolar or hyrophobic substitutions, including alanine, leucine, isoleucine, valine, proline, phenylalanme, tryptophan and methionine; amino acids with uncharged polar R groups including serine, threonine, tyrosine, asparagine, glutamine, cysteine and, presumably, glycine; amino acids with negatively charged R groups including aspartic acid and glutamic acid; and amino acids with positively charged R groups including lysine, arginine, and, presumably, histidine.
• amino acids can be modified in numerous ways without altering their ability to function in the present invention. Among these alterations include the formation of amino acid amides and amino acid alkyl esters by the addition of amino and carboxy groups respectively.
• dipeptidyl derivatives of the amino acids can be formed by linking two amino acids through the ⁇ -carboxy group and ⁇ -amino group. It will be readily appreciated that each pair of amino acids will have two potential dipeotidyl derivatives. Also of importance to the present invention is the provision of a source of ammonium ion (NH 4 + ) to supplement the amino acid-containing media of the present invention. Sources of ammonium ion are also well known in the art of plant tissue culture.
• ammonium ion is provided by way of the inclusion in the medium of a quantity of non-toxic salt of ammonium, formed with an anion which balances the ammonium ion charge, e.g., ammonium chloride, ammonium phosphate or ammonium sulphate.
• a quantity of non-toxic salt of ammonium formed with an anion which balances the ammonium ion charge
• ammonium chloride e.g., ammonium chloride, ammonium phosphate or ammonium sulphate.
• Other sources of ammonium ion are disclosed in Walker, K. A. and S. J. Sato, "Plant Cell Tissue Organ Culture” 1: 109-121 (1981), a the relevant portions of which are incorporated herein by this reference.
• the following examples are provided in order to illustrate various aspects of the present invention. The examples should not be taken as implying any limitation to the scope of the present invention, which is defined exclusively the claims appended hereto.
• alfalfa embryogenesis can be routinely induced in the Regen S line of Saunders and Bingham, "Production of Alfalfa Plants from Callus Tissue,” Crop Sci., 12:804-803 (1972).
• Plants of Medicago sativa cultivar Regen S derived from the second cycle recurrent selection for regeneration from the cross of the varieties Vernal and Saranac were used. Callus was initiated by surface sterilizing petioles with 50% Clorox R for five minutes, washing with H 2 O and plating on hormone-free SH medium, containing the salts, vitamins and sucrose of Schenk- Hildebrandt medium (Schenk, R.U. and A. C. Hildebrandt, supra, (1972)). The medium contained 25 ⁇ M ⁇ - naphthyleneacetic acid and 10 ⁇ M kinetin and 0.8% (w/v) agar (termed maintenance medium). Callus which formed on the explant tissue was separated from the remaining uncallused tissue and repeatedly subcultured on maintenance medium. Callus was subcultured at 3 week intervals and grown under indirect light at 27° C.
• Induced ceils were asceptically sized on a series of column sieves (Fisher Scientific) under gentle vacuum. Cell clumps either fell or were forced through a 35 mesh (480 ⁇ m) and collected on a 60 mesh (230 um) through stainless steel screen. Cells retained on the 60 mesh screen were washed with 500 ml of SK minus hormone medium for every 100 ml of induction culture volume. The washing medium was removed by vacuum. The fresh weight of the cell clumps was taken and cells were resuspended in SH medium without hormones at 150mg fresh weight per ml.
• the 25mM NH 4 + control medium consisted of ammonium free medium supplemented with 12.5mM (NH 4 ) 2 SO 4 . All organic and inorganic sources of reduced nitrogen were sterilized by 0.2 ⁇ m filtration and subsequently added to freshly autoclaved medium.
• Embryogenesis was visually measured after incubation by counting green centers of organization on the callus using a stereo microscope at a magnification of 10X. Embryo size was measured using a calibrated ocular scale at 10X magnification. Embryo shape was determined by visual examination.
• Conversion of embryos to whole plants with root and shoot axis was done by aseptically transferring embryos from amino acid treatments at 21 days of initial culture to half-strength hormone-free SH medium supplemented with 25 ⁇ M gibberelic acid and 0.25 uM ⁇ - naphthyleneacetic acid solidified with 0.3% agar.
• the second response type from the initial screen either stimulated embryogenesis or caused an increase in embryo size when compared to the SH control. See Table 2. Detailed concentration dependence studies were performed on these amino acids and the results are shown in Figure 1.
• the amino acid most effective in stimulating somatic embryo formation was proline, which yielded nearly 3-fold more embryos than the 2.6 mM NH 4 + control and was twice as effective as 25 mM NH 4 + , the optimal ammonium concentration in alfalfa (D).
• Table 2 summarizes the amino acids and other nitrogen sources which have been found to be stimulatory to somatic embryogenesis in alfalfa. It is imocrtant to note that the ester and amide forms of proline are highly active in stimulating embryo numbers and quality as is the dipeptide, prolyl alanine. It is interesting to note that the nonprotein amino acid ornithine is also active.
• Umbelliferae somatic cell culture As a representative of the Umbelliferae family, seeds of celery, Apium graveolens (variety Calmario) were germinated for one to two weeks. The resulting seedlings were sterilized with a solution of 10% Cloro ⁇ R for 20 minutes. Cotyledons or hypocotyls were removed and explants were placed on 0.3% agar solidified hormone-free SH medium containing 25 ⁇ M 2,4-D and 5 ⁇ M benzyladenine. After initiation of callus (3-4 weeks), callus was transferred to SH medium with 2.5 ⁇ M 2,4-D and 0.5 ⁇ M kinetin. Heat labile additives were filter sterilized and added to warm medium.
• proline stimulated embryogenesis better than glutamine, the latter resulted in better development of seedling-like embryos.
• Ammonium treated cultures developed smaller and fewer embryos than all other treatments.
• Glutamic acid when added singly to celery regeneration medium at 30 mM, stimulates embryo number in celery compared to 25 mM NH 4 + - treated inaterial. Alanine, proline, glutamine and glutamate at the above concentrations improve celery embryo conversion to plantlets compared to NH 4 + treated embryos.
• Gramineae Somatic Cell Culture As a species representative of the Gramineae family, Zea mays somatic embryogenesis was performed employing media in accordance with the present invention.
• Ears of corn at ten days post fertilization were harvested and immature embryos were dissected from these asepticallv. Embryos were placed onto N-6 mineral salt medium (Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C. , Yin, K.C. and Chu, C.Y., 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin. 16 , 659-688) plus 3% sucrose and 5 ⁇ M 2,4-D for 21 days. After incubation callus was scored for formation of embryo masses on each callus formed while in the presence or absence of L-proline. The results were as indicated in Table 7, where, the percent response is the average frequency of embryo formation of between 287 and 1165 replicate embryo explants.
• Gossypium hirsutum Cultures initiated from surface sterilized seed of Gossypium hirsutum were subcultured on Murashige and Skoog salts plus 3% sucrose and 0.5 ⁇ M NAA, 5 ⁇ M 2-isopentanyIadenine with 0.3% agar medium for four week subcultures. Cultures were induced for 10 days to form embryos on medium containing either 0.5 ⁇ M 2,4-D plus 0.2 ⁇ M kinetin or 1 ⁇ M MAA and 0.5 uM kinetin with or without proline in liquid suspension culture. Cells were then transferred to hormone-free SH medium with 3% sucrose and 10 mM L- glutamine for regeneration. After four weeks cultures were evaluated for formation of embryos with mature cotyledons. The results were as indicated in Table 9.
• Proline was tested over a range of
• Arginine A similar experiment where the concentration of arginine was varied in addition to the concentration of NH 4 + added to the medium. The results are shown in Table 11.
• proline plus ammonium media improve embryo quantity and that proline improves embryo quality in the presence of high or low ammonium.

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• 1986
  • 1986-10-21 KR KR1019870700528A patent/KR900007085B1/ko not_active Expired
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