Gene expression of Anx3 in Pb-treated Nicotiana tabacum using a real-time RT-PCR

Lead (Pb) is one of the highly persistent, toxic, and widely distributed heavy metal pollutants in the environment. This heavy metal has a tendency to enter human food chain, thus affecting public health. One effective way to remove heavy metals pollutant is by using plants, a technology known as phytoremediation. One such plant that is routinely employed as an experimental model for such studies is Nicotiana tabacum. As tobacco plants are not generally consumed by herbivores, it minimizes the possibility of Pb from entering food chain. A number of studies suggest that annexins, a calcium-binding protein, does play a role in plant stress response. The expression of annexin gene in plants appeared to be regulated by tissue-specific developmental and environmental signal. A vacuole-associated annexin from N. tabacum, Anx3, was investigated, to observe the involvement of this gene in Pb-induced stress. Reverse transcription following quantitative real-time 0olymerase chain reaction (qRT-PCR) is a useful analysis to study gene expression. In this analysis, a reference gene that acts as internal control (housekeeping gene) is routinely employed for normalization of qRT-PCR against the target gene (Anx3). The candidate reference genes, L25, EF-1α, and Ntubc2, were evaluated using suitable primer pairs in order to select the most stable reference gene for normalization of qRT-PCR in this study. Using geNorm, NormFinder, and BestKeeper programs, the most suitable reference gene identified in this study was L25. The relative quantification of Anx3 gene expression normalizing against L25 was accomplished by REST software. The expression level of Anx3 in Pb-treated N. tabacum was upregulated by 2.2-fold (p < 0.05). The experimental methods used and the participation of Anx3 in defense against Pb stress will be discussed

The supervirulence plasmid pToK47 from Agrobacterium tumefaciens A281 Improves Transformation efficiency of Hevea brasiliensis

Problem statement: The present study investigates the ability of the pTok47 supervirulence plasmid form Agrobacterium tumefaciens A281 to enhance genetic transformation in Hevea brasiliensis. Approach: Hevea anther callus was transformed via Agrobacterium-mediated genetic transformation using two strains of Agrobacterium (GV2260 and GV3850) harboring the human serum albumin cDNA and the supervirulent plasmid pToK47 from Agrobacterium tumefaciens A281. The transformed callus was selected using kanamycin as the selection agent Results: The Agrobacterium strain GV2260 benefited from the presence of the supervirulence plasmid in giving a higher frequency of 7.4% transformed callus, 344.8% embryogenesis and 11.6% plantlet production compared to the corresponding strain on its own giving 0.9% transformed callus, 204.5% embryogenesis and 4.4% plantlet production. Similarly, Agrobacterium strain GV3850 conferred a higher frequency using the supervirulent plasmid, resulting in 3.5% transformed callus, 138.5% embryogenesis and 3.5% plantlet production compared to the corresponding strain on its own giving 0.7% transformed callus, 137.5% embryogenesis and 9.0% plantlet production. These findings were confirmed by the Wilcoxon Signed Rank Test that compared the effectiveness of the supervirulence plasmid in increasing the rate of genetic transformation in the calli surviving in kanamycin growth medium for GV2260 (p<0.001) and for GV3850 (p<0.05). Conclusion: This study showed that both Agrobacterium strains benefited from the presence of the supervirulence plasmid in giving a higher frequency of transformed callus, embryoids and plantlets. These results suggest that a highly virulent binary vector system might prove especially useful in generating high frequency transformation of Hevea

Regeneration of Jatropha curcas using leaf explants

A tissue culture protocol for regeneration of Jatropha curcas using leaf explants via somatic embryogenesis was established. The best response for callus induction from leaf explants was on Murashige and Skoog (MS) medium containing BA (1.0 and 2.0 mg l-1) and IBA (1.0 mg l-1). Callus was maintained in light and dark on MS medium supplemented with 0.5 mg l-1 BA and 0.5 mg l-1 IBA. The best response for shoot induction and shoot multiplication was on treatments containing (1.0 – 2.0 mg l-1) BA. The shoots were elongated and rooted on ½ strength MS medium containing 1.0 mg l-1 NAA (Napthalene Acetic Acid)

Direct regeneration and characterization of plant derived from leaf in vitro culture Patchouli ( Pogostemon cablin, Benth)

Patchouli has huge potential to serve the pharmaceutical industries due to its fragrance and aromatic essential oils. To serve the needs of the pharmaceutical and perfume industries, there is an urgent need to mass propagate clonal copies of the desired patchouli plantlets. In order to generate genetically identical clonal copies of the desired cultivar, direct regeneration of patchouli plantlets by tissue culture using leaf explants was successful. The tissue culture formulation for direct regeneration employed Murashige and Skoog medium with 0,5 mg/l 2,4 D (2,4Dichlorofenoksiacetat) in combination with (0, 0.25, 0.5, 0.75, 1 mg/l) BAP (Benzyl Aminopurine). The best response for direct shoot regeneration was observed after 35 days occurred in media containing 0.75 mg/l of BAP without 2,4 D and this gave the highest efficiency of regeneration frequency of leaf (88%) and highest number of shoot per explant (102). For elongation of shoots, MS basal medium gave a better response compared to ½ MS Medium. The average shoot elongation was about 3.3 cm and this gave an average of 7 leaflets per shoot. Elongated shoots were rooted and leaves growth using MS basal liquid medium and MS basal solid medium with or without 1 mg/l NAA (1-Naphthalene acetic acid). The best response for root induction and leaves growth was observed using MS basal liquid medium without NAA and this gave a frequency of 12 roots per shoot with an average length of 2.75 cm. The regenerated plantlets were acclimatized and hardened by transferring the test-tube plantlets into sterile water for 2 days. The plantlets were successfully established in soil containing compost and top soil (1:1) and the frequency of establishment was 90%. The clonal plantlets were verified using RAPD analysis. The results showed a very high frequency of true-to-type plantlets

Functional analysis of a latex-specific promoter (hevein) of Hevea for enhanced recombinant protein production in Hevea brasiliensis

Any protein, either naturally occurring or designed synthetically, could be produced safely, inexpensively and in large quantities using the power of the rubber trees for the production of recombinant proteins on a plantation scale with sunlight and water. In the past, the Rubber Research Institute of Malaysia has generated transgenic Hevea that expressed recombinant human serum albumin, scFv antibody in latex serum. However, the production is still below the 1.0 % level for commercial feasibility. This study evaluates the promoter activity of hevein gene, which encodes the most abundant soluble protein in Hevea latex. Genetic constructs were prepared with the test hevein promoter fragments Hev P1 (0.35kb), Hev P2 (0.45kb) and Hev P3 (0.73kb) that were inserted 5’ to the reporter gene uidA in pGPTV-KAN expression vector. The expression vectors containing the hevein promoter fused to uidA in Agrobacterium tumefaciens GV2260 were then used to infect Hevea anther callus. Putative transgenic rubber plantlets generated via somatic embryogenesis tested for gene insertion, gave signals for single and mutiple copies for uidA gene and this was similarly reflected in the positive control (plants with 35S CaMV promoter) by Southern analysis. The recombinat protein levels for β-glucuronidase (GUS) in the latex of Hevea transformants and controls was quantified using ELISA technique. The levels of GUS in latex serum from positive controls (B6 plants) ranged from 0.063 to 0.079 µg/ml of latex serum. The levels of GUS protein (µg/ml of latex serum) in Hevea transformants P1 ranged from 0.0052 to 1.55; P2 ranged from 0.005 to 0.0012; P3 ranged from 0.0017 to 0.0027. The highest expressing transgenic plants was derived from B6 using the 35S CaMV promoter for uidA gene and P1 containing the smallest fragment of the hevein promoter for uidA gene. This study paves the way to sucessfully produce recombinant proteins of commercial value in transgenic rubber trees

Response of Nicotiana tabacum grown under Pb stress: A preliminary study

Heavy metal contaminations in soil, water and air has become a major problem since the onset the industrial revolution. Among the heavy metals that are being studied, lead (Pb) is one of the highly persistent and widely distributed in the environment. This heavy metal contaminant can be transferred into the food chain, causing the bioaccumulation of heavy metals in the organism tissues, and thus affecting public health. Phytoremediation emerges as a remediation tool based plant to clean up heavy metal contaminated sites. Hence, a better understanding of the physiological, biochemical and molecular responses in plant could facilitate the success of this plant-based technology. Nicotiana tabacum is used in the present study because it poses the important characteristics to study gene expression in plants. Furthermore, it is repulsive to herbivores, and thus minimizes the possibility of Pb from entering food chain. In this preliminary study, the physiological response of N.tabacum exposed to Pb was investigated in a laboratory condition. The growth and morphological changes of Pb-treated N.tabacum were compared with those without Pb treatment (control). Pb concentrations of up to 2.0 mM were used and symptoms of Pb toxicity were observed

Hevea genetic transformation for enhanced recombinant pharmaceutical production by the use of hevein promoter

Pharmaceuticals produced in the latex cytosol of genetically transformed Hevea can be harvested nondestructively by conventional tapping. This study evaluates the promoter activity of the 5’-untranslated upstream regulating region of hevein gene, which encodes the most abundant soluble protein in Hevea latex. Constructs were prepared with the test hevein promoter fragments Hev P1 (0.35kb), Hev P2 (0.45kb) and Hev P3 (0.73kb) that were inserted 5’ to the pharmaceutical genes i.e. human protamine 1 (HP1) and human atrial natriur tic factor (HANF), in pGPTV-Kan expression vector. The expression vectors containing HP1 and HANF were electroporated into Agrobacterium tumefaciens GV2260 containing supervirulent plasmid pToK47, which were then used to infect Hevea anther callus. The growth of the putative transformed Hevea callus was monitored on kanamycin selection media. The presence of the pharmaceutical genes (HP1 and HANF), the hevein promoter fragments, and nptII selection marker were verified by PCR on sampled putative transformed callus. The remaining callus tissues will be sub-cultured and transferred into differentiation media for embryoids formation and plantlet regeneration

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: Effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicyclate on enzyme activity

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a Km value of 4.02 ± 0.64 μM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress