| Mammalian Metallothionein (MTs) are a sort of low-molecular-weight, thiol-rich and metal-binding proteins, which mainly function as detoxifying agents and in essential metals metabolism. Mouse MT-1 has different domains, a and B, which not only have independent structures and functions but also differ in metal-binding favors, a-KKS-a (aa), was obtained in our lab and it shows in vitro a stronger ability than the natural MT to bind some heavy metals such as Cd. Based on this knowledge, we overexpressed MT and aa protein in Alternanthera philoxeroides and hoped to study the function of aa in vitro. Another aim of this work was to improve the tolerance and accumulation to heavy metals of plants for phytoremediation.1.Study on tissue culture of Alternanthera PhiloxeroidesThis study examined the effects of explants, media and exogenous phytohormone on tissue culture of Alternanthera philoxeroides. The stems, leaves and roots were used as the explants to study tissue cultue. Different basic media [MS, 1/2 MS, MS (1/2), B5], 0 ~ 0.5 mg/L of indoleacetic acid (IAA), 0 ~ 0.2 mg/L of naphthyl acetic acid (NAA), 0.5 ~ 5.0 mg/L of 6-benzylaminopurine (6-BA) and 0.5 ~ 4.0 mg/L of Zeatin (ZT) as additional compositions were used for inducing, differentiating and rooting tests. Induced results showed that 1/2 MS was more suitable for calli to grow and divide than B5, MS (1/2) and MS. The stem and leaf of Alternanthera philoxeroides were suitable organs for tissue culture. Stem > leaf was observed on growth rate. The combinations of NAA and BA or IAA and ZT can induce calli of the stem and leaf of Alternanthera philoxeroides, and the rate of calli induction increases with the increase of BA concentration. The higher rate of NAA/BA, the more roots from the calli format.2. Establishment of genetic transformation system for Alternanthera PhiloxeroidesIn this study the potential of a transformation system for Alternanthera philoxeroides via meristems cocultivated with Agrobacterium was evaluated. The Phosphinothricin (PPT) and Cadmium(Cd) with different mass concentrations were respectively added into the meida, the effects of PPT and Cd on the growth of explants were observed so as to determine its minimum lethal mass concentration, which was to be used for studying genetic transformation of Alternanthera philoxeroides. The experimental results showed that the minimum lethal mass concentration of PPT for the differention of Alternanthera philoxeroides was 4.0 mg/L and Cd was 150 umol/L. The study of the amenability of Agrobacterium-mediated gene transferred to Alternanthera philoxeroides indicated that the shoot regeneration ratio from excised Agrobacterium inoculated Alternanthera philoxeroides meristems for 10 ~ 15 min got to 70 %. So it was thought that this study could provide the prerequisite for genetic transformation of Alternanthera philoxeroides.3. Expression of MT and aa genes in transgenic Alternanthera PhiloxeroidesThe plant expression vector pGPTVd35S-MT/aa gene was indemnified and introduced into Agrobacterium tumefaciens strain EHA105. A method of regenerating from the meristem was developed for Agrobacterium-mediated transformation. In this procedure, the cells of the meristem were targeted for transformation. In this present study, the shoot developed successfully on media which did not contain plant hormone, transformants of meristems were selected on 1/2 MS medium with 4.0 mg/L PPT or 150 umol/L Cd2+. However, the transformation frequency of meristem tissue proved to be extremely low. From all transformation experiments, in which a total of 1364 meristems were excised, two transformed shoots were obtained. The presence of the aimed genes in transformants genome was detected by PCR. Dot blot analysis revealed that MT and aa protein had expressed in Alternanthera Philoxeroides. Transformants displayed higher resistance to Cd than control plants. The growth of the roots and shoots of plants transformed by MT and aa genes wasunaffected by Cd up to 300 umol/L, whereas control plants showed severe inhibitio...
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