Transformation of a Plant with Wheat-Salt Tolerance - Assignment Example

Name Institutional Affiliation Transformation of a Plant: Wheat-Salt Tolerance This is a protocol for the generation of wheat plant that has undergone transformation and conferred with ability to tolerate high saline conditions. Plant Material 1. What is the explant? Immature wheat embryos As explants, immature embryos are used in callusing, plant regeneration and somatic embryogenesis (Ahloowalia, 1982). 2. Is the explants transformed directly or must callus be generated first? Callus must be generated first. Callus generation depends with medium used and it is important for genetic variation and green plant formation (Carman et al., 1988). 3. What media is used to induce the callus? i. Callus initiation is enabled on MS (Murashige and Skoog, 1962) supplemented with 100mg/l myoinositol, 0.4 mg/l thiamine-HCl, 30g/l Sucrose, 2.5 mg/l of 2, 4-dichlorophenoxyacetic acid (2, 4-D), and 0.5 mg/l of kinetin. ii. Callus proliferation was enabled by supplementing the MS with 50mg/l ascorbic acid under various salt stress conditions of 2.5, 5, 10, or 15g/l NaCl. iii. The plantlet regeneration medium entails MS2X made up of MS salts and macronutrients. 4. What do the various media consist of? MS supplemented with 2, 4-D, kinetin, sugar, and agar as base in callus formation. 2, 4-D is a growth regulator that has been implicated in tissue culture-induced variability (Shoemaker et al., 1991). MS2X composed of MS salts, double strength macronutrients, but without 2, 4-D in shoot generation MS/2 (half-strength) MS without 2, 4-D in root regeneration 5. Is the media modified? e.g. MS media with half strength macronutrients The media is modified by adjusting pH to 5.8 prior to adding 1% agar, and autoclaved at 120 degree Celsius for 20 minutes. For culture initiation and growth, cultures are maintained 5 weeks in continuous darkness at 25 degree Celsius day temperature and 18 degree Celsius night temperature. Low temperature treatment has been associated with a fast rate of differentiation in wheat (Hau & Teng, 1994). For regeneration, cultures are passed to MS media with double strength macronutrients and after formation of shoots, cultures are passed to MS/2, being MS medium with half strength macro-nutrients, without 2,4-D for stimulating vigorous root formation. Expression Vector(s) 6. In most cases there will be two genes: Promoter-GOI-terminator, Promoter-selectable marker-terminator. Promoter genes confer the desired trait of the plant. In this case, the gene of interest is one that is one that confers the trait of salt tolerance in wheat. TaNHX genes in wheat do this by the synthesis of sodium/hydrogen antiport protein located on the vacuole membrane (Zhu, 2003). Selectable-marker genes confer selective growth on the plant cells so that transformed cell can outgrow the non-transformed cells. The selectable marker should not induce broad phenotypic effects. In this case, the selectable-marker gene will help identify cells that have taken up the gene of interest (GOI). 7. Cite the promoter, gene and terminator for both the GOI and selectable marker In the GOI, the promoter is UBi 1 (Ubiquitin 1); the gene is TaNHX1; and the terminator is NOS (Vasil et al., 1993). In the selectable marker, the promoter is 35S, the gene is PMI (manA) from E. coli, and the terminator is NOS (Vasil et al., 1993). 8. Choice of promoter-monocot or dicot, tissue specific (seed, leaf, roots) or constitutive. The choice of the promoter is a constitutive one such that there will always be direct expression of salt tolerance in all tissues without the promoters having to depend on environmental and developmental factors (Xiao, et al., 2005). Transformation 9. What method? Agrobacterium-mediated or biolositcis The transformation method used for inducing salt tolerance trait in wheat is Agrobacterium-mediated. Agrobacterium species is one of the vectors that have been used for the genetic manipulation of plants (Patnaik, Vishnudasan, & Khurana, 2006). Agrobacterium tumefaciens is a soil-borne, Gram-negative bacterium first reported by Smith and Townsend (1907) as being the causative agent of crown gall tumour. Agrobacterium-mediated transient expression protocol was then adopted for quick analysis of gene activities in plants. 10. What is the target tissue? The target tissue is leaves from seedlings of green wheat seedlings. The leaves contain the vacuoles, in which the gene for conferring the salt tolerance trait is found (Munns, James & Lauchli, 2006). 11. What are the conditions of transformation? E.g Agrobacterium concentration, co-cultivation period, and temperature, biolistic conditions (type of gun, pressure, distance) The efficiency of transformation can be increased by the manipulation of either the target tissue of the Agrobacterium to enhance both the competency of the plant tissue and the gene expressions (Ding, et al., 2009). Required seedling age for this case is 8 to 10 day old plantlets, cultured from immature embryo which produce high rate of genotype variability. The Agrobacterium tumefaciens strain is grown in medium supplemented with 100mg/L carbenicillin and 100mg/Lspectinomycin, placed in shaking incubator at 250 rpm until the cells reach a desired concentration. The cells are centrifuged and re-suspended in half volume of inoculation medium and the suspension incubated at 28 degree Celsius for 1 hour with continuous shaking at 180rpm. These factors will enhance the bacterial pili formation needed for gene transfer between bacteria and the tissue (Ding et al., 2009). After incubation, target tissues are inoculated with the Agrobacterium on a petri plate for 1 to 2 hours in dark condition by pouring the bacteria over the explants. The wheat tissues are transferred to a dark incubation box for 3 days co-cultivation period (Ding et al., 2009). Generally, all incubations during transformations are carried out in tissue culture room at 25 ± 1 ºC, 8h/16h photoperiod and PAR of 350 µmol m/s. 12. Are there any pre-treatments or post-treatments to the target tissue? Ten-day old wheat seedlings are incubated in a growth chamber at 25 degree Celsiuls with 16hr/8hr photoperiod. The plantlets are then subjected to 200mM sodium chloride for 3 days to enable cloning of the TaNHX1) Triticum aestivum Sodium Hydrogen antiporter) gene. The leaf samples are then collected and stored in -80 degree Celsius freezer for RNA isolation (Jones, Doherty, & Wu, 2005). 13. How long after transformation are target tissues transferred to the selection? After co-cultivation period which takes 3 days, transformed wheat tissues are washed with liquid MS medium supplemented with 160mg/l Timentin for 1 hour, and then transferred to the selection process (Jones, 2005). Regeneration 14. What is the selection agent-what concentration? The selection agent is Mannose at a concentration of 10g/L (Bhalla, 2006). 15. Are their multiple steps in the selection and regeneration process? Does each step have different media? Growing shoots are transferred to rooting medium supplemented with selection agent for further selection and rooting (Bhalla, 2006). Transformed immature embryos are placed onto callus induction medium maintained in dark at 25 degree Celsius for 21 days (Bhalla, 2006). For regeneration, embryogenic calli are transferred to germination medium RDZ in light condition. After three weeks in the germination medium, they are transferred to regeneration medium supplemented with selection agents (Bhalla, 2006). References: Ahloowalia, B. (1982). Plant regeneration from callus culture in wheat. Crops Science, 22(2), 405-410. Bhalla, P.L. 92006). Genetic engineering of wheat: Current challenges and opportunities. Trends in Biotechnology, 24, 305–311. Carman, J., Jeferson, N., & Campbell, W. (1988). Induction of embryogenic Triticum aestivum L. calli: Quantification of genotype and culture medium effects. Plant Cell Tissue and Organ Culture, 12: 83-95. Ding, L., Li, S., Gao, J., Wang , Y., Yang , G., & He, G. (2009). Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat. Molecular Biology Reports, 36, 29-36. Hau, B., & Teng, S. (1994). Effect of low temperature treatment on differentiation of long term culture calluses of wheat. Plant Physiology Communications 30, 108-110. Jones, H. (2005). Wheat transformation: Current technology and applications to grain development and composition. Journal of Cereal Science 41, 137-147. Jones H., Doherty, A, and Wu, H. (2005). Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods,1, 5. Munns, R., James, R., & Lauchli, A. (2006). Approaches to increasing the salt tolerance of wheat and other cereals. Journal of Experimental Botany, 57(5), 1025-1043. Murashige, T., and Skoog, F.(1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology of Plant, 15(3), 473-497. Patnaik, D., Vishnudasan, D., & Khurana, P. (2006). Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum. Current Science, 307- 317. Shoemaker, R., Amberger, K., Palmer, R., Oglesby, L. & Ranch, J. (1991). Effect of 2, 4-D concentration on somatic embryogenesis and heritable variation in soybean (Glycine max L. Mer R.) In vitro Cellular Development Biology27, 84-88. Smith, E., F. & Townsend, C. (1907). A Plant-tumour of bacterial origin. Science 25 (643), 671–673. Vasil, V., Srivastava, V., Castillo, A.M., Fromm, M.E. and Vasil, I.K. (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. BioTechnology, 11, 1553-8. Xiao, K., Zhang, C., Harrison, M., & Wang, Z. (2005). Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants. Molecular Breeding, 15, 221-231. Zhu, J., K. (2003). Regulation of ion homeostasis under salt stress. Current Opinion in Plant Biology, 6, 441-445. Read More