Agrobacterium-Mediated Transformation of Three Freshwater Microalgal Strains

Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp.

Biolistic transformation of Eucalyptus grandis x E. urophylla callus

A procedure for genetic transformation of the hybrid Eucalyptus grandis × E. urophylla using particle bombardment is described. Cotyledon- and hypocotyl-derived calli growing on SP medium supplemented with 2�m�thidiazuron or on MS modified (MSM) medium supplemented with 10 m 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.5�m�6-benzylaminopurine (BAP), were used as target material for bombardment assays. Multiple preincubation and bombardment conditions were tested. Tungsten particles were coated with the plasmid pBI426 harbouring a β-glucuronidase (gus) and neomycin phosphotransferase II (npt II) gene fusion controlled by a double 35S cauliflower mosaic virus (CaMV) promoter. Four days after bombardment, the transient transformation efficiency was determined by expression of the gus gene. Fully GUS-positive calli were then obtained after 105 d in MSM medium supplemented with 2,4-D, BAP, and the selective agent kanamycin at 200 mg L-1. The presence of the gus gene in these kanamycin-resistant calli was confirmed by polymerase chain reaction analysis. Extensive experiments were performed aiming to identify conditions for the regeneration of these GUS-expressing calli. However, they were unable to regenerate transgenic shoots, suggesting that conditions suitable for regeneration are unsuitable for transformation and vice versa.

Transfer of Lycopersicon chilense Chitinase Gene to Cultivated Tomato (L. esculentum)

We have isolated a drought-induced chitinase gene from L. chilense, a wild tomato species. Owing to our interests in genetic improvement of cultivated tomato, we have transferred the L. chilense chitinase gene to this species. The transformation plasmid constructed contained the coding sequence of L. chilense chitinase gene linked to CaMVS35 promoter as well as the NPT II gene linked to nopaline synthetase promoter. The leaf disk transformation regeneration technique was applied to one commercial tomato cultivar and four inbred lines. Shoots were produced on the selection medium through direct or indirect organogenesis. Plantlets that have been rooted on kanamycin-containing medium were transferred to soil where they grew to maturity and produced flowers and fruit. The transgenic nature of some of the analyzed plants was confirmed by polymerase chain reaction. Research is continuing to evaluate transgenic plants with regard to their level of tolerance to phytogenic fungi.

Transient chimeric gene expression in pollen of five conifer species following microparticle bombardment

Mature pollen of lodgepole pine (Pinusconcorta Dougl.), yellow cypress (Chamaecyparisnootkatensis (D. Don) Spach), western hemlock (Tsugaheterophylla (Raf.) Sarg.), jack pine (Pinusbanksiana Lamb.), and black spruce (Piceamariana (Mill.) B.S.P.) was bombarded with gold particles coated with four different plasmid constructions, pRT99GUS, pBM113Kp, pAct1-D, and pGA984, using the biolistic PDS-1000/He device. A protocol was devised for efficient gene transfer and gene expression assay in pollen. False positive results for expression of the β-glucuronidase (GUS) gene assayed with the substrate X-glucuronide were observed with pollen of yellow cypress, western hemlock, and lodgepole pine. The highest levels of transient GUS gene expression were obtained with plasmid pBM113Kp, which carried the GUS gene under the control of the wheat abscisic acid inducible early methionine promoter. The plasmids pRT99GUS (35S promoter) and pAct1-D (rice actin promoter) yielded similar intermediate levels of transient GUS gene expression. The pollen-specific promoter of the α-tubulin gene from Arabidopsisthaliana (pGA984) yielded the lowest levels of gene expression in pollen. Of the four species, yellow cypress showed the lowest levels of transient GUS gene expression and black spruce yielded the highest levels. The neomycin phosphotransferase II (NPT II) gene was also tested as a reporter gene for pollen transformation and was easily assayed via ELISA. The fusion gene between NPT II and GUS genes was detected at a lower level than the nonfused NPT II gene when under the control of the same 35S promoter. The method devised here could be used for the study of tissue-specific gene expression in conifer pollen.