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115 9516334; phone +44 115 9513057 Key words: Proto plasts, ge netic ma nip u la tion, nuclear and organelle trans for ma tion, phys i o log i cal in ves ti ga tions, protoplast-to-plant sys tems, so matic hy bridi sa tion ACTA PHYSIOLOGIAE PLANTARUM Vol. 27. No. 1. 2005: 117-129re view liv ing cell con tents into a hypertonic so lu tion. Several years later, what may now be con sid ered as early 'bio tech nol ogy' ex per i ments em a nated from in ves ti ga tions of Kuster (1909) andMichel (1937). These work ers dem on strated that the plasma membranes of iso lated proto plasts (wall-less 'na ked cells') could be in duced to fuse, per mit ting co alescence of their cytoplasms. How ever, such in ves tiga tions passed vir tu ally un no ticed for many years un til the com mer cial avail abil ity, dur ing the 1960s, of wall de grad ing pectinases and cellulases. Such en zymes, when used ei ther se quen tially or in combi na tion, fa cil i tated the re pro duc ible iso la tion of large, work able pop u la tions (>1 x 10 6 ) of viable protoplasts from primary tissues of a range of plant species.

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P SAG12-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P SAG12-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P SAG12-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C(19)-GAs, including the bioactive GAs GA(1 )and GA(4), by 2beta-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA(1) and GA(4). Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds.