E. C. Cocking

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.

Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose

Abstract A procedure is described for the reproducible establishment of rice cell suspension cultures from callus of embryo origin. Protoplasts were readily isolated from cell suspension of four rice cultivars, including japonica and indica types, when maintained in an amino acid-based culture medium. Sustained protoplast division from two japonica genotypes has been obtained in agarose solidified culture medium. An increase in the agarose concentration from 0.6% to 1.2% (w/v) produced a marked improvement in protoplast survival, division and plating efficiency. Protoplast division and plating efficiency frequencies of up to 26% and 0.5%, respectively, were obtained at the higher agarose level. The protoplast-derived calli were similar in appearance to explant-derived morphogenic callus and produced distinct embryo-like structures.

Endophytic colonization of plant roots by nitrogen-fixing bacteria

Nitrogen-fixing bacteria are able to enter into roots from the rhizosphere, particularly at the base of emerging lateral roots, between epidermal cells and through root hairs. In the rhizosphere growing root hairs play an important role in symbiotic recognition in legume crops. Nodulated legumes in endosymbiosis with rhizobia are amongst the most prominent nitrogen-fixing systems in agriculture. The inoculation of non-legumes, especially cereals, with various non-rhizobial diazotrophic bacteria has been undertaken with the expectation that they would establish themselves intercellularly within the root system, fixing nitrogen endophytically and providing combined nitrogen for enhanced crop production. However, in most instances bacteria colonize only the surface of the roots and remain vulnerable to competition from other rhizosphere micro-organisms, even when the nitrogen-fixing bacteria are endophytic, benefits to the plant may result from better uptake of soil nutrients rather than from endophytic nitrogen fixation. Azorhizobium caulinodans is known to enter the root system of cereals, other non-legume crops and Arabidopsis, by intercellular invasion between epidermal cells and to internally colonize the plant intercellularly, including the xylem. This raises the possibility that xylem colonization might provide a non-nodular niche for endosymbiotic nitrogen fixation in rice, wheat, maize, sorghum and other non-legume crops. A particularly interesting, naturally occurring, non-nodular xylem colonising endophytic diazotrophic interaction with evidence for endophytic nitrogen fixation is that of Gluconacetobacter diazotrophicus in sugarcane. Could this beneficial endophytic colonization of sugarcane by G. diazotrophicus be extended to other members of the Gramineae, including the major cereals, and to other major non-legume crops of the World?

Transformation of Petunia protoplasts by isolated Agrobacterium plasmids

Abstract Plasmids isolated from Agrobacterium have been used to transform Petunia suspension cell protoplasts. Transformed cells, selected by their hormone independence, possessed features characteristic of crown gall tumours, i.e. continued proliferation on hormone-free media, overgrowth formation when grafted onto host plants, octopine synthesis, and lysopine dehydrogenase activity. These results are discussed in relation to the use of Agrobacterium plasmids in plant genetic manipulation.

Organogenesis and somatic embryogenesis in tissues derived from leaf protoplasts and leaf expiants of Medicago sativa

Summary Leaf protoplasts isolated from 30–35 days old seedlings of Medicago sativa (Alfalfa, Lucerne) divided to form cell colonies when cultured on a filter paper substratum over agar-solidified medium. The tissue produced formed shoots and somatic embryoids which developed into plants. This developmental pattern was similar to that of callus of seedling leaf expiant origin. Callus initiated from leaves of axenic regenerated plants also retained its totipotency. These results are discussed in relation to genetic manipulations in forage legumes.

Plant regeneration from root protoplasts of Brassica

Abstract Protoplasts isolated enzymatically from roots of Brassica alba (White Mustard), B. campestris (Turnip), B. napus (Rape) and B. oleracea (Cabbage), divided to form callus. Plant regeneration was obtained from protoplast derived tissues of B. napus and B. oleracea , but only rhizogenesis was observed with B. campestris . Tissues of B. alba remained undifferentiated. The suitability of root protoplasts for genetic manipulations in the genus Brassica is discussed.

Gene Transfer in Cereals

Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.

Strategies for signal amplification in nucleic acid detection.

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.

Interactions of rhizobia with rice and wheat

Recently, evidence has been obtained that naturally occurring rhizobia, isolated from the nodules of non-legume Parasponia species and from some tropical legumes, are able to enter the roots of rice, wheat and maize at emerging lateral roots by crack entry. We have now investigated whether Azorhizobium caulinodans strain ORS571, which induces root and stem nodules on the tropical legume Sesbania rostrata as a result of crack entry invasion of emerging lateral roots, might also enter rice and wheat by a similar route. Following inoculation with ORS571 carrying a lacZ reporter gene, azorhizobia were observed microscopically within the cracks associated with emerging lateral roots of rice and wheat. A high proportion of inoculated rice and wheat plants had colonized lateral root cracks. The flavanone naringenin at 10 and 10 M stimulated significantly the colonization of lateral root cracks and also intercellular colonization of wheat roots. Naringenin does not appear to be acting as a carbon source and may act as a signal molecule for intercellular colonization of rice and wheat by ORS571 by a mechanism which is nod gene-independent, unlike nodule formation in Sesbania rostrata. The opportunity now arises to compare and to contrast the ability of Azorhizobium caulinodans with that of other rhizobia, such as Parasponia rhizobia, to intercellularly colonize the roots of non-legume crops.

Endophytic establishment of Azorhizobium caulinodans in wheat

Nitrogen fixing nodules are formed on the roots and stems of the tropical legume Sesbania rostrata by Azorhizobium caulinodans as a result of crack entry invasion of emerging lateral roots. Advantage was taken of this invasion capability of A. caulinodans to determine whether inoculation of the non–legume wheat with A. caulinodans would result in the endophytic establishment of azorhizobia within wheat roots. Advantage was also taken of the oxygen tolerance of the nitrogenase of free–living azorhizobia to assess the extent to which the endophytic establishment of azorhizobia in wheat roots would provide a niche for nitrogen fixation of benefit to the plant. Wheat was inoculated with A. caulinodans and grown in pots under controlled conditions, without added growth reglators and without addition of fixed nitrogen. Microscopic examination of the short lateral roots of inocluated wheat showed invasion of azorhizobia between cells of the cortex, within the xylem and the root meristem Acetylene reduction assays combined with analysis of tissue nitrogen levels indicated the likelihood that colonization led to nitrogenase activity. Inoculated wheat showed significant increases in dry weight and nitrogen content as compared with uninoculated controls. We discuss the extent to which this nitrogen fixation is likely to involve symbiotic nitrogen fixation, and we indicate the need for field trials to determine the extent to which inolculation of wheat with A. caulinodans will reduce the requirement for inputs of nitrogenous fertilizers.