Tobacco cell cultures callus tissue

Biotests for the detection and quantitative measurement of C. measure one of the following 1. stimulation of cell division in tissue cultures, e.g. in tobacco or soybean callus tissue 2. enlargement of cells in the leaf disc test, often with bean or radish leaves 3. inhibition of the loss of chlorophyll from detached leaves 4. promotion of seed germination in the dark. 

Mixed Cytokinins. The first cytokinin, kinetin [525-79-1] (3), was isolated from stale herring sperm (8) but, like so many biologically active natural products, it was later found in the vascular system of tobacco stems and leaves (9). Yeast also proved to have a very high titre of kinetin (see Yeasts) (8). The compound is very active in increasing cell division in tobacco wound callus tissue that has been cultured on White s agar medium supplemented with 2 mg/L of indole-3-acetic acid (IAA) [87-51 -4], The presence of IAA is mandatory to induce cell division in the presence of kinetin. 

The chimeric tissue problem has been solved by co-cultivating plant protoplasts with A. tumefaciens (Figure 14), then treating these protoplast suspensions with an antibiotic that selectively kills the A. tumefaciens. The protoplasts are plated onto a nurse layer of tobacco cells which feed the individual protoplasts and aid them in regenerating into pure colonies. The colonies ultimately form callus, and this allows for the production of pure cultures of transformed cells. These cultures can then be regenerated into plants. 

Tobacco is one of the major model systems in plant cell and tissue culture studies. Particularly in developing techniques for genetic engineering has it played a major role. Consequently numerous papers have described various aspects of tobacco plant biotechnology. For example, almost 200 entries for plant cell culture media for tobacco are given in the tabulation by George et al. 189). Some of the most commonly used media for plant cell cultures were, in fact, developed for tobacco callus cultures 190,191). Indeed, root cultures of several Nicotiana species were described as early as 1938 by White 192). Collins and Legg 193) reviewed the use of cell and tissue culture methods for the improvement of tobacco. 

The interaction of lAA with another PGR, kinetin, which is a cytokin, has been observed by growing tobacco stem pith callus in tissue culture. Jablonski and Skoog (29) have reported that with a low cytokinin to auxin ratio in the tissue culture, the result is a mass of loosely arranged, undifferentiated cells or callus, but with a high cytokinin to auxin ratio, the result is the growth of cultured plantlets with stems and leaves. 

Furthermore, SAM not only plays a pivotal role in PA and ethylene biosynthesis, but also in the formation of putrescine-derived alkaloids. Thus, SAM acts as a methyl donor in the conversion of putrescine into A-methylputresdne, an intermediate in the biosynthesis of nicotine (Mizusaki et al., 197 lb). Therefore, it would be interesting to explore the auxin-ethylene-PAs interaction on PA conjugates and putrescine-derived alkaloids metabolism in the above-mentioned tobacco callus tissue system. Indeed, it has been reported that inhibition of SAM decarboxylase by MGBG resulted in an increase of nicotine production in tobacco root cell suspension cultures (Blume, 1985). Unfortunately, nothing about PA conjugates was indicated in this report. 

A tremendous upsurge in synthetic activity in this field is evident. A stereoselective synthesis of cis-zeatin (22), whose geometrical isomer was first isolated from Zea mays and shown to be a stimulant of cell division in plant tissue cultures, has been reported (Scheme 2). The Diels-Alder reaction of 1-chloro-l-nitro-socyclohexane (19) with isoprene gave the dihydro-l,2-oxazine hydrochloride (20) in moderate yield. Liberation of the base of (20) followed by zinc-acetic acid reduction gave the required amino-alcohol (21), which upon treatment with 6-chloropurine provided a separable mixture of cis-zeatin (22) and trans-zeatin. Standard tobacco callus bioassay for cytokinin activity showed that the natural trans-zeatin is at least 50 times more active than the synthetic cis-isomer (22). 

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