PREPARING NICOTINE BY CULTURING A NICOTIANA STRAIN

Abstract: A process for producing nicotine which comprises cultivating a strain of Nicotiana tabacum or Nicotiana rustica in suspension culture under sub-maximal growth conditions which do not inhibit nicotine production, in the presence of an auxin and a cytokinin.

Description: PREPARING NICOTINE BY CULTURING A. NICOTIANA STRAIN This invention relates to a process for preparing nicotine by in vitro cultivation of a variety of Nicotiana tabacuni or Nicotiana rustica in a nutrient culture medium. Work shich has hitherto been published on the cultivation of Nicotiana tabacum varieties implies that some , but by no means all, of the tested varieties contain low levels of nicotine. B1uch of the work has been done n conjunction with observations on growth using batches of callus culture. Cultivation in suspension cultures is described in Nature, Vol. 201 (1964), pp. 614 and in Fiochem. Physiol. Pflanzen, " 62, 513 (19/1), pp. 503-513. In the former reference, it is stated that "the highest nicotine levels were found at the end of the growth". A conclusion¯ of the latter reference is that callus cultures start to produce alkaloid (including nicotine) after the addition of cycloheximide (which inhibits growth), whereas, in suspension cultures, alkaloi production is dependent on the conditions of growth. It appears that alkaloid production was stimulated during sub-maximal growth, alkaloids accumulating during tne initial phase of growth, e.g. within a period of five days. It was observed after the initial phase that the level of nicotine ren:ainedsubstan- tially constant. Another method of regulating growth discussed in the second reference, in order to achieve greater nicotine production, is by using the tedium proposed in Ann. Sci. Nat. Biol. Veg. 14,1 (1953) by R. Heller. It appears generally to be the case that nicotine production is at a maximum under conditions of sub-maximal growth but hitherto theiehas been no disclosure of how cultivation of Nicotiana varieties might be made a commercial proposition. The effect of hormone levels has been observed by Shiio et al, Agr. Biol. Chem. 37 (8), pp. 1857-1864, and Ohta et al, Agr. Biol. Chef. 42 (6), 978), pp. 1245-1251, using callus cultures. -The reported results, particularly of the former reference, are extremely difficult to interpret but, in general, there is no certainty that results achieved with callus cultures can be reproduced with suspension cultures. Callus cultures consist of large cell agglomerates, and nicotine may be produced even when the conditions are suitable for maximal growth, because cells within agglomerates'are not growing. Indeed, one of the foremost workers in this field, Tabata, reported in 'Frontiers of Plant Tissue Culture" (1978)ed. Thorpe, Proc. 4th Int. Cong, Plant Tissue and Cell Culture, that nicotine production may be up to 4O%4less in suspension culture than in callus culture. In suspension culture, it is likely that an optimal growth medium will stimulate cell growth and inhibit nicotine production in a substantially uniform manner. Shiio et al and Ohtz et al both used auxins in their cultivation media, and these hormones, at high concentration levels, tend to reduce cell agglomeration in liquid cultures but also to inhibit nicotine production. In particular, when Shiio et al grew Nicotiana tabacum yar. Bright Yellow on a medium containingl-naphthaleneacetic acid, they found that kinetin inhibited both growth and nicotine production. According to the present invention, a process for producing nicotine conlprises cultivaticg a strain of Nicotiana tabacur. or Nicotiana rustica in suspension culture under sb-inaximal growth conditions which do not inhibit nicotine production, in the presence of an auxin and a cytokinin. Thus it has been found that conditions can be achieved under which good nicotine yields can be obtained when both hormones are present. The nicotine can then be isolated from the system, i.e. from the cells and/or from the culture medium. Suitable auxins are indoleacetic acid (IAA), l-na- phthaleneacetic acid (NAA) and (2,4-dichlorophenoxy)acetic acid. R4A is the preferred auxin because it is heatstable and can be sterilised by autoclaving. The preferred cytokinin is kinetin. As is inown, tlie respective functions of auxins and cytokinins are on cell wall plasticity and cell expansion, and on cell division. For any particular Nicotiana strain and any given growth nutient medium, it is usually or invariably found that there are three phases: (1) an initial or lag phase during which there is little increase in fresh weight, although cell numbers may increase substantially; (2) a growth phase; and (3) a final phase in which there is little growth as the maximum has almost or completely been reached. As is well documented, it is possible to determine conditions of sub-maximal growth and high nicotine production for a given Nicotiana strain, and we find that it is possible to prepare a suspension culture of cells of that variety in a suitable medium and allow it to reach a high level of nicotine production, per unit biomass. The highest nicotine production per unit biomass may not occur until after, say, 7 days under such conditions. Prior to the sub-maximal growth stage, it may be desirable to culture the cells under maximal growth. conditions to produce the necessary biomass. There are a number of ways in hitch conditions can be controlled in order to achieve sub-maximal growth when it is desired to start nicotine production. One is to introduce a growth inhibitor and another is to lim'it growth nutrients, i.e. to reduce or omit certain nutrients from the medium which should normally contain assinilabie sources of carbon (such as sucrose or molasses) and nitrogen and other nutrients and vitamins. A particularly suitable medium for use in this invention is based on that proposed by Murashige and Skoog in Physiologia PI. 15 (1962),473-497, which has been widely used for culturing Nicotiana tabacum varieties. The use of an increased level of thiamine, as recommended by Linsmaier and Skoog in Physiologia P1. 19 (1965), pp. 100-127, is also very satisfactory. The contents of such media can be controlled as desired in order to achieve sub-maximal growth. Alternatively, growth may be inhibited by introducing a protein synthesis inhibitor such as cycloheximide, although1this is less desirable as some proteins can be a desirable by-product of the process of this invention. A particularly preferred method of inhibiting growth and thereby increasing nicotine production is to control the hormone level. The level of such hormones is critical and should not exceed 10pS' for, say, NAA, and lout for, sy, kinetin. In general, a concentration of less than these maximum amounts down to about O.luM for the auxin and down to 0.01 uM for the cytokinin is suitable, preferred ranges being from 0.5 to 5p for the auxin and from 0.05 to 5pS1 for the cytokinin. The preferred ranges for 2,4-D and analogous auxins may be a factor of 10 lower than for NAA. It is often preferable to conduct the culture in the dark,e.g. in the substantial absence of visible light or under low intensity light. In this way, higher nicotine yields can be obtained than in light. It is usually preferred to use a Nicotiana tabacum, rather than a Nicotiana rustica.strain. Particularly suitable Nicotiana tabacum varieties are Maryland Mammoth, Speight G 28 and, especially, N.C.2512, although the Bright Yellow and Virginica varieties can also be used. It;hen it is desired to produce isotopically labelled, e.g. radioactive (e.g. C14 or N15 labellcd), nicotine, then this may be achieved by including suitably labelled precursors in the medium, e.g. if all the SH4 and NO3 components in the medium are N15 labelled. Suspcnsion cultures are obtained by transferring several grams of soft, loosely packed callus tissue into a liquid medium, followed by continuous genle-agitatiOn. Although many tissues of the appropriate plant can be used as a source of callus, easily sterilised and relative- ly undifferentiated tissues are the most suitable. Consequently, cultures are usually derived either from stem pith or from sterile young seedlings. As the cul-ture grows, single cells and small clumps of cells break way fron the callus and become dispersed in the rne'diun. After 1 to 3 weeks, depending on the growth rate of the culture, the suspended tissues are separated from the larger callus lumps by filtration or sedimentation, and can be subcultured by the addition of fresh medium, e.g. at 1:7 dilution. It has been observed that, if a given Ni'cotiana strain in culture is removed fror the medium at or towards thc end of the growth phase (the 'secnd Cf the three phases discussed above) and suspended in fresh medium, the three phases can occur again. Using this observation, the process of this invention can be adapted to operate store continuously than with batch cultures, in the following manner: Cells are grown under maximal growth conditions in a first vessel into which fresh medium is continuously 6r intermittently introduced. The cells in the substantially exhausted medium are decanted or flow from this first. vessel and are allowed to age in one or, preferably, a series of second vessels which overflow from one to the next as medium is introduced into the first vessel. The overflow of aged cells from the or the final second vessel is then diluted, in a third vessel or series of vessels, e.g. at 'I 10 dilution, with a medium in which tic cells grew, in a second growth phase, under sub-maximal growth conditions in the presence of the essential hormones, according to the invention. Nicotine can then be isolated from the system in the third vessel or vessels. It will be appreciated that the maximal and sub-maximal growth media may contair; the sane hormones, but at different concentrations. The following Example illustrates the invention. Example Batch suspension cultures of Nicotiana tabacum var. NC 2512 ere grown on a medium based on that of Murashige and Skoog, but with an increased level of thiamine as recommended by Linsmaier and Skoog. For convenience the medium was made up from a number of stock solutions: Stock Solutions tRgt Added Final Concentration (g/1) (mg/l) A. NH4N03 82.5 1650 B. KNO3 95.0 1900 C. H3B03 1.24 6.2 KH3P04 34.0 170 KI 0.166 0.83 Na2MoO4.2H2O 0.05 0.25 CoCl2 0.005 0.025 D. CaCl2.2H2O 88.0 440 E. MgSO4.7H2O 74.0 370 MnS04.4H20 4.46 22.3 ZnSO4.7H2O 1.72 8.6 CuS04.SH20 0.005 0.025 F. a2EDTA 7.45 37.3 FeSO4.7H2O 5.57 27.8 G. Glycine 0.4 2.0 Nicotinic Acid 0.1 0.5 Pyridoxine flCl 0.1 0.5 H. Thiamine HCL O.OS 0.4 J. Meso-Inositol 20 100 In addition, the medium contained 30 g/l sucrose, 0.2 mg/l NAA and 0.02 mg/l kinetin. The pil of the medium was adjusted to 5.7 - 5.8, and it was sterilised by autoclaving at 15 lb/in2 (121 C) for 15 minutes. The batch.cultures were grown in 250 ml. wide- necked Erlenmeyer flasks, sealed with aluminium foil caps, containing 75 ml of liquid nutrient medium, and were continuously agitated on orbital shakers in continuous low intensity light at 25 C. The results were as follows: Culture Age Fresh Weight Nicotine (days) (g/l) tg/ml culture) 0 35 0 7 62 7 10 68 16 15 79 67 20 108 99 24 112 152 Equally successful results have been obtained by scaling up the preceding experiment using suspension cultures grown in five-litre fermenters.

Claims: CLAIMS i. A process for producing nicotine, which comprises cultivating a strain of Nicotiana tabacum or Nicotiana rustica in suspension culture under sub-maximal growth conditions which do not inhibit nicotine production, in the presence of an auxin and a cytokinin. 2. A process according to claim 1 wherein the auxin is indoleacetic acid, naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid. 3. A process according to claim 2 wherein the auxin is I-naphthaleneacetic acid. 4. A process according to claim 3 wherein the concentration of l-naphthaleneacetic acid is from 0.5 to SuM. 5. A process according to any preceding claim wherein the cytokinin is kinetin. 6. A process according to claim 5 wherein the concentration of kinetin is from 0.5 to 5pM. 7. A process according to any preceding claim wherein the strain is Nicotiana tabacum var. Maryland Mammoth, Nicotiana tabacum var. Speight G 28 or Nicotiana tabacum var. N.C. 2512. 8. A process according to any preceding claim wherein the stra-in -is cultured under maximal growth conditions and, at or towards the end of the maximal growth phase, resuspended in a fresh medium and cultivated under sub-maximal growth conditions.

Inventors: PEARSON, DAVID WILLIAM, SMITH, HARRY
Applicants: GALLAHER LIMITED
Application year: 1979
Main classification: C12N5
IPC codes: C12N5, C12P17, C12R1