FORMULATIONS OF PLANT CULTURE MEDIA AND APPLICATIONS THEREFOR

Abstract: An aqueous medium for plant tissue culture is provided including a nutritively effective concentration of reduced nitrogen, preferably in the form of an amino acid such as arginine employed in an amount of from 1.0 to 5.0 mM, and having not more that 1.5 mM NH4<+>. A phosphorus compound at a concentration of at least 2.5 mM measured as phosphate is also included. A chelating agent such as EDTA, and an iron compound present in molar excess over the concentration of the chelating agent are employed to control divalent ion concentrations. Potassium ions are maintained at a concentration of below 14 mM. The use of media containing ammonium ions at less than 1.5 mM combined with a supply of reduced nitrogen in the form of arginine, with low potassium levels, has proved useful as media for organized growth, particularly for use with coniferous plants.

Description: FORMULATIONS OF PLANT CULTURE MEDIA AND APPLICATIONS THEREFOR This invention relates to new formulations of cultur media and applications of these for growth of plants an plant cells, tissues and organs. This application has particular but not exclusiv application to coniferous plants and to permitting growth an normal development of these plants in tissue culture, and fo illustrative purposes reference will be made to suc application. However, it is to be understood that thi invention could be used in other applications, such a culture media suitable for other plant species. It has been recognized that plant tissue cultur methods may be useful to clonally multiply valuable plant through micropropagation and somatic embryogenesi procedures. Culture of plant cells may be useful in othe ways, including generation of genetic diversity throug somaclonal variants, regeneration of plants from protoplasts, cells, calli, and intra- or intergenic hybrid forms of these, regeneration of genetically modified plants from any suc plant cells or structures, selection for resistance to an imposed or otherwise incurred stress, whether natural or artificial, the production of plants free from pathogens, the • rejuvenation of mature tissues, the rescue of embryos fro non-viable seeds, or to the culture of plant cells, calli, or organs for production of plant secondary products or other valuable extracts. In broad terms, tissue culture is conducted in a culture medium which is usually an aqueous nutrient composition. However, it has been found generally that few species and genotypes are adequately responsive to plant tissue-culture procedures for commercial application, and some species are recognized as particularly recalcitrant. The responses of many plant species are confined to specific genotypes. Coniferous plants are generally difficult, as are many other woody plants, and pine plants are particularly difficult. Where success is reported, this is limited to a fraction of the genotypes desired, with response frequencies within clonal populations (such as rooting frequency, regeneration frequencies generally) being relatively low with high attrition and/or slow growth characteristics. It has been determined by the present applicant that a substantial reason for the failure of species and genotypes hitherto considered to be inherently recalcitrant to tissue culture lies in the selection of formulations of the nutrient media employed, it being determined that sub-optimal media and the resulting nutrient stresses imposed on the cultured plants are a primary cause of failure. The present invention aims to alleviate deficiencies of the prior art culture media and to provide media which are reliable and efficient in use. With the foregoing and other objects in view, this invention in one aspect resides broadly in an aqueous medium for plant tissue culture and including:- a nutritively effective concentration of reduced nitrogen, wherein said reduced nitrogen includes not more that 1.5 mM NH 4 + ; a phosphorus compound at a concentration of at least 2.5 mM measured as phosphate; a chelating agent, and an iron compound present in molar excess over the concentration of said chelating agent. Preferably, the source of reduced nitrogen includes an amino acid, with it being particularly preferred to use arginine as the predominant form of reduced nitrogen. Of course, combinations of amino acids and other forms of reduced nitrogen may be employed. The preferred arginine source of reduced nitrogen is advantageously employed in an ' amount of from 1.0 to 5.0 M, with it being especially preferred to utilize an amount resulting in a concentration of about 2.5 mM. The use of concentrations of ammonium ^ ions of less than 1.5 mM in media for plant growth combined with a supply of reduced nitrogen in the form of arginine has not hitherto been reported as useful in the art of media formulation for organized growth, particularly for use with coniferous plants. Organised growth of embryos has been reported using media incorporating arginine or other organic forms o nitrogen, but in these media ammonium ions have been supplie at relatively high concentrations of at least 2.0 mM. It ha become apparent that media in accordance with the presen invention are advantageous since use of elevated ammonium io concentrations tend to inhibit normal root development an detract from optimal shoot growth. Preferably, the media in accordance with the presen invention include a relatively low level of potassium ions with it being preferred to utilize a concentration o potassium ions below 14 mM. It has been determined tha there is an inhibitory effect of high levels of potassiu ions on root development in tissue cultures such as are use in micropropagation of plants for transplantation. Media in accordance with the present inventio include a relatively high total phosphorus content of a least 2.5 mM measured as phosphate, it having been determine that high concentrations in media in accordance with th present invention provides for good root development i tissue culture specimens. Preferably, the phosphoru concentration measured as phosphate is from 2.5 mM to abou 5.0 mM. Preferably, the chelating agent is selected fro Ethylene Dia ine Tetra-acetic Acid (EDTA) and it derivatives. Where EDTA is selected as the chelating agent an iron-to-chelate ratio of 1.2 or higher is preferred wit it being particularly preferred to utilize an iron to chelat ratio of about 1.5. Of course the optimum ratio of iron t chelate will vary depending on the chelate selected and th concentrations of other transition metal ions present in th medium. In general, it has been determined that a guidin principle may be found in constructing media where the iron chelate combination is non-stoichiometric in order t manipulate the complex equilibrium which regulates th availability of divalent metals in the medium. Preferably, the total iron of the medium is betwee 120 μM and 180 μM, with the total EDTA of the medium prefera being in the range of 70 μM to 130 μM, within the requirem that the iron always be in stoichiometric excess over chelate in the medium. Preferably, the stoichiometric excess of iron over chelate is at least 20 μM. The media may include any of the additional nutrients and/or additives commonly utilized in growth media, including hormones, amino acids, vitamins, other minerals, pH buffers and the like. However, it is preferred that the sum of the total concentrations of the transition metals Zn, Cu, Ni and Co, not exceed the total level of chelate in the media. Preferably, the media are selected from compositions in accordance with the following parameters. Macroelements (mM) Range K 5-14 Mg 0.5-1.5 Ca 2-4.5 Na 0.25-0.75 NH Δ 0.5-1.5 N0 3 8-16 P0 A 2.5-5 S0 4 1.5-3.5 Cl 0.025-2.5 Arginine 2.0-3.5 Pyridoxine 0.05-0.3 Thiamine 0.3-0.8 Sucrose 25000-55000 The invention will be further described wit reference to the following Example A composition in accordance with the above embodiment is suitable for use as a general purpose culture medium for P. radiata culture processes, the medium proving efficacious for a wide range of genotypes. These processes include cell culture, callus culture, protoplast culture, shoot culture, root culture, meristem culture, micropropagation, embryo rescue, somatic embryogenesis, organogenesis, regeneration, transformation procedures, and any related methods. The present medium was assessed through measurement of growth of . radiata embryos in vitro compared with known benchmark media, namely, GD (Greshoff and Doy, 1972), LP Modified (von Arnold and Eriksson, 1981), and the LP Modified-like formulation MS (Murashige and Skoog, 1962). Embryo growth after 28 days of culture at 25 ** -C yielded the following comparative results:- Formulation Growth index (by wt. ) Present formulation 1.0 GD 0.32 LP Modified 0.48 MS 0.48 Upon dissection of the embryos, it was determined as an average that the improvement in growth was primarily directed to root development, which accounted for about 75% of relative weight gain. The described embodiment is a single medium formulation which provides the appropriate nutrient conditions for a wide range of responses. This medium will allow optimal or near optimal growth of both shoot and root apices, and will support growth of disorganised callus or cell cultures, to a moderate mass, at near-maximum rates. It is therefore particularly useful for use in supporting healthy developmental growth such as in shoot development, root development, and embryogenesis, provided appropriate hormones, as well as appropriate physical conditions and choice of tissue explant, are employed in conjunction with the nutrient medium. The composition is useful for avoiding stress-induced developmental aberrations, and stress-induced genetic variation. The composition also has use as a base which can be modified in physiological and other studies of nutrient requirements where responses to specific nutrients can be obtained without incurring other growth-limiting nutrient conditions. The medium is very suitable for most species of coniferous plants, but its use with any plant species is considered to be within the scope of the present invention. It will of course be realised that while the above medium formulation has been given by way of example of this invention, all such and other modifications and variations thereto as would be apparent to persons skilled in the art are deemed to fall within the broad scope and ambit of this invention as defined in the claims appended hereto.

Claims: CLAIMS 1. An aqueous medium for plant tissue culture and including:- a nutritively effective concentration of reduced nitrogen, wherein said reduced nitrogen includes not more that 1.5 mM NH 4 + ; a phosphorus compound at a concentration of at least 2.5 mM measured as phosphate; a chelating agent, and an iron compound present in molar excess over the concentration of said chelating agent. 2. An aqueous medium according to Claim 1, wherein said source of reduced nitrogen includes an amino acid. • 3. An aqueous medium according to Claim 2, wherein said amino acid is arginine employed in an amount of from 1.0 to 5.0 mM. 4. An aqueous medium according to any one of the preceding Claims, wherein the concentration of potassium ions is below 14 mM. 5. An aqueous medium according to any one of the preceding Claims, wherein said chelating agent is selected from Ethylene Diamine Tetra-acetic Acid (EDTA) and its derivatives. 6. An aqueous medium according to Claim 5, wherein a iron-to-chelate stoichiometric ratio of at least 1.2 i employed. 7. An aqueous medium according to Claim 6, wherein th total iron of the medium is between 120 μM and 180 μM, with total EDTA of the medium being in the range of 70 μM to 130 and wherein the stoichiometric excess of iron over chelate i at least 20 μM. 8. An aqueous medium for plant tissue culture an including:- Macroelements (mM) Range K 5-14 Mg 0.5-1.5 Ca 2-4.5 Na 0.25-0.75 NH 4 0.5-1.5 N0 3 8-16 P0 4 2.5-5 S0 4 1.5-3.5 Cl 0.025-2.5 Arginine 2.0-3.5 Microelements (μM) Fe 120-180 EDTA 80-130 Zn 10-40 9. Aqueous media for plant tissue culture substantially as hereinbefore defined with reference to the Example.

Inventors: TEASDALE ROBERT D, TEASDALE, ROBERT, D.
Applicants: FORBIO PTY LTD, FORBIO PTY. LTD.
Application year: 1991
Main classification: A01G7
IPC codes: A01H4, C12N5