Transposon tagging is the direct way of gene identification and cloning in living organisms. In plants, well characterized transposable elements are available from Zea mays and Antirrhinum majus. These have been used as simple insertion mutagens to clone genes in both native and several heterologous plant species. Transposon mediated techniques are also increasingly being used to study the pattern and regulation of gene expression in plants. Recently, transposons have been used in ingenious ways to bring about deletion and inversion of chromosomal segments. The transposon-based reverse genetics and its potentials in assigning biological functions to the known DNA sequences makes it useful in functional genomics.
This review traces the developments in the use of plant transposons, from a simple technique for insertional mutagenesis to a powerful tool for gene discovery and study of gene function.

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Metallothioneins (MTs) are a family of cysteine (Cys)-rich, low-molecular-weight proteins ubiquitously presented in animals, fungi, cyanobacteria and plants. There have been a number of reports concerning the molecular cloning and characterization of genes encoding metallothionein (MT) in plants. The precise function of plant MTs is not yet clear. Nevertheless, the evidence suggests that MTs may be related to metal detoxification and other environmental stresses. Recent developments in studies on plant MTs will be discussed.

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Transgenic plants of Asparagus officinalis cv. Mary Washington 500W were generated by Agrobacterium-mediated transformation. After induction of flowers by treatment with atrazine, calli were induced from male and female plants. Calli of male and female origin were co-cultivated with Agrobacterium EHA101 (pIG121Hm) and EHA101 (pARK5), respectively. About thirty shoots developed from female calli and three shoots developed from male calli within six weeks on selective medium supplemented with 100mg/l kanamycin and 500mg/l claforan. Most of the shoots did not form roots. Seven shoots from female calli formed roots and were successfully transferred to soil in a greenhouse. But shoots of male calli did not form any roots. Stable integration and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R₀ and R₁ generations. In an analysis using the polymerase chain reaction (PCR), fragments of both the NPT II gene and the bar gene were amplified from five R₀ plant fragments. Southern analysis showed that both NPT II and bar probes hybridized to products of PCR from five R₀ plants. One of the five R₀ plants was crossed with a non-transgenic male plant. The segregation ratio of transformed to non-transformed R₁ plants was about 1:1. These results indicated that drug-resistant genes were transferred to Asparagus by Agrobacterium-mediated transformation.

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Protocorm like bodies (PLBs) derived from callus of Phalaenopsis utilized sucrose, maltose and sorbitol for their growth in vitro. These carbon sources affected differently and could control PLB growth. On sucrose supplemented medium a few PLBs produced plantlets and most others regenerated yellowish or greenish callus like body (CLB). Almost 80% of unrooted and 58% of rooted plantlets developed yellowish green CLB at the base of plantlets. On maltose supplemented medium, PLBs regenerated PLBs and a few plantlets. In subsequent culture, about 44% of unrooted and 24% of rooted plantlets initiated green PLBs at the base of cultured plantlets. On sorbitol supplemented medium, most of the PLBs developed plantlets and a few additional PLBs. Among the carbon sources tested, sorbitol supported plantlet development the best in vitro and proved to be the most suitable carbon source for plantlet initiation and development from PLB.

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Hypocotyl explants of chilli pepper (Capsicum frutescens) were inoculated with Agrobacterium rhizogenes strain A13 harbouring the binary vector pBI121, which contains the CaMV 35S promoter linked to the GUS reporter gene. After 4 weeks of culture, the hairy roots produced were transferred onto Schenk and Hildebrandt (SH) medium supplemented with 250mgl^-1 Cefotaxime. Subsequently they were selected in liquid SH medium containing 20mgl^-1 kanamycin. When treated with X-glucuronide, vascular and root tip tissues were predominantly stained. A phenylalanine ammonia-lyase (PAL) cDNA from parsley (Petroselinum) was subsequently linked to the 35S promoter and transferred into hairy roots using the same A. rhizogenes strain. Successful transformation was confirmed by Southern hybridisation. Hairy roots containing the PAL transgene showed different PAL activity, slow growth and altered morphology.

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A hybrid Bt toxin gene was made from cryIA (b) and cryIA (c) after optimization of their codon usage to meet high G+C content in rice and placed under control of the rice Actin I promoter along with its first intron. This hybrid Bt toxin gene was introduced into the genome of an elite indica rice cultivar IR72 through biolistic method. Five independent transformants were identified out of 27 transgenic plants obtained. The level of the hybrid Bt toxin was estimated to be 0.01%-0.20% of the total soluble protein in leaf or stem tissue of the transgenic plants. This considerable range of expression levels was studied for a strong insecticidal effect. As demonstrated by insect bioassay, neonate larva mortality rates of yellow stem borer (Scirpophaga incertulas) (YSB) for selected T₀ transgenic rice plants as well as T₂ homozygous lines were 100%. This is the first report of a hybrid Bt toxin gene [cryIA (b) and cryIA (c)] driven by rice Actin I promoter in any cereal crop including rice conferring resistance to insect pest. The present hybrid Bt toxin gene deployment strategy will provide sustainable insect pest resistance in rice.

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A novel glutelin gene was cloned from a cDNA library of maturing rice seeds (Oryza sativa L. cv Nipponbare). The 2.0kbp insert contained an open reading frame encoding a 510 amino acid polypeptide (M^r 57, 116). This novel glutelin shares 46 to 49% amino acid identity with previously identified rice glutelins. A phylogenetic analysis of cloned glutelins indicates that this gene constitutes a new, fifth class of glutelin gene families. The Asn-Gly processing sequence which is highly conserved in 11S seed storage proteins is replaced by Asn-Val in the sequence of the novel glutelin. The amino acid composition extending 50 residues both upstream and downstream of the Asn-Val site was less hydrophilic than in other glutelins. The N-terminal half corresponding to the acidic domains of other glutelins possesses a higher pI value (7.46) than found in other glutelins. Expression of the gene was detected in maturing seeds, but not in roots or leaves.

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To study transcriptional regulators, an in vivo functional assay is indispensable. Here we report a new convenient vector set for a transactivation assay in plants. The system consists of a luciferase reporter controlled by a synthetic promoter with GAL4-binding sites and an effector to express any fusion protein with the GAL4 DNA binding domain. Co-transfection of the two plasmids causes transactivation of the reporter by the expressed GAL4-effector fusion if the effector exhibits transcriptional activation activity. A multicloning site was introduced into the effector vector which should facilitate the construction of GAL4-effector fusions. In tobacco transient assay by microprojectile bombardment, a 27-fold transactivation for the GAL4-B17 fusion as an effector was demonstrated with this system.

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We constructed new λTI phage vectors, λTI3, λTI4, λTI5 and λTI6, which were automatically convertible into Ti-plasmid binary vectors by using the Cre-lox site-specific recombination system of bacteriophage P1. Hygromycin B phosphotransferase (HPT) gene was inserted in the T-DNA region of these λTI vectors as a plant selectable maker. Tobacco plants, which were transformed by Agrobacterium containing the Tiplasmids resulted from the automatic conversion of the λTI vectors, could be tightly selected by hygromycin B. This λTI system will certainly provide a powerful tool for genetic complementation test of candidate genes obtained by positional cloning.

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