Agrobacterium tumefaciens Mediated Transformation of Symbiodinium spp.

Symbiodinium spp conducts symbiosis mutualism within a wide phyletic range of marine invertebrate hosts including corals and anemones. The present study investigates the transformation of foreign genes into the free living cultured Symbiodinium spp by co-cultured Symbiodinium cells with A. tumefaciens. Ti-plasmidbased binary vector harboring the GUS and GFP genes were transformed into Symbiodinium cells by co-cultivation. GUS histochemical assay was monitored in Symbiodinium cells under light microscopy. Putative GFP in transformed Symbiodinium cells was detected by immunoassaying with antibodies against GFP protein. These results suggest that A. tumefaciens could provide efficient tools for gene transformation of Symbiodinium cells.

Anatomical Structure and Terpenoid Content of Zodia (Evodia suaveolens Scheff) Leaves

Zodia (Evodia suaveolens Scheff) is a member of Rutaceae contain terpenoids, triterpenoids, alkaloids, flavonoids, and xanthones which have anti-mosquito activity. This research aimed to observe the anatomical structure, the location, and distribution of terpenoid based on the leaves' age. Anatomical slides preparation of leaves were made using the paraffin embedding method with safranin staining. The distribution of terpenoid was analyzed by the histochemical assay. Leaf anatomical structure shows that the 3rd and 6th leaf bifacial (dorsiventral) consisted of the upper epidermis, mesophyll (palisade and sponge), collateral vascular bundle, parenchyma midrib, abaxial epidermis and oil glands in mesophyll that is underneath both epidermises. The diameter of oil glands with larger sizes was on the 6th leaf, whereas the density is not different in the 3rd and 6th leaves. The histochemical test showed that terpenoid was observed in the leaf vascular bundles, oil glands, and epidermis.

Transient Transformation of Artemisinic Aldehyde ∆ 11 (13) Double Bond Reductase (dbr2) Gene into Artemisia annua L.

Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens-mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303-dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA-dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA-dbr2 construct and transformation into A. annua have been successfully performed.

In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)