Zahid ALI, Wajeeha SAEED, Saadia NASEEM, Faheem AHMAD, Ahmed AKREM, Nighat YASMEEN, Hans Joerg JACOBSEN

Phenotypic evaluation of transgenic peas (Pisum sativum L.) harboring AtNHX1 demonstrates stable gene expression and conserved morphology in subsequent generations

In recent years, transgenic approaches have played a significant role in improving traits that help plants overcome abiotic stresses. Combined gene discovery and functional genomics have helped identify diversified mechanisms and gene families, which improved productivity under various abiotic stresses. We report on the genetic stability and persistent morphological features of transgenic pea (Pisum sativum L.) plants harboring the dicistronic vector construct pG0229MASnhx1/luc over five generations. In addition to salt stress tolerance, the transgenic plants showed frost tolerance compared to wild-type (WT) plants. Frost tolerance of AtNHX1 transgenic pea plants was unexpected and needs further investigation. We report morphological and molecular characteristics of transformed plants after long-term storage at 30-50 °C. The transgenic plants were morphologically stable and genetic stability of integrated genes was confirmed prior to and after transfer of plants to a glasshouse. Leaf size, shape, and color, plant height, number of tendrils, flower shape, pod shape, and grains were morphologically similar to the WT counterpart in all transgenic generations. This is the first report showing the genetic stability of transgenic pea plants harboring the salt stress tolerance gene (AtNHX1) from Arabidopsis thaliana in subsequent generations over a period of 6 years.

Keywords:

Transgenic pea, Na+/H+ antiporter, morphology, genetic stability, stress tolerance,

PDF

___

Ali Z, Schumacher HM, Heine-Dobbernack E, El-Banna A, Hafeez FY, Jacobsen HJ, Kiesecker H (2010). Dicistronic binary vector system - a versatile tool for gene expression studies in cell cultures and plants. J Biotechnol 145: 9-16.
Ali Z, Ullah N, Naseem S, Haq MI, Jacobsen HJ (2015). Soil bacteria conferred a positive relationship and improved salt stress tolerance in transgenic pea ( Pisum sativum L.) harboring Na + / H + antiporter. Turk J Bot 39: 962-972.
Apse MP (1999). Salt tolerance conferred by overexpression of a vacuolar Na + /H + antiport in Arabidopsis . Science 285: 1256- 1258.
Apse MP, Sottosanto JB, Blumwald E (2003). Vacuolar cation/H + exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na + /H + antiporter. Plant J 36: 229-239.
Ashraf MI, Pervez MA, Amjad M, Ahmad R, Ayub M (2011). Qualitative and quantitative response of pea ( Pisum sativum L.) cultivars to judicious applications of irrigation with phosphorus and potassium. Pakistan Journal of Life and Social Science 9: 159-164.
Attari MIJ, Javed AY (2013). Inflation, economic growth and government expenditure of Pakistan: 1980–2010. Proc Econ Financ 5: 58-67.
Bakht T, Khan IA, Khan MI, Khan I, Khattak AM (2009). Weed control in pea ( Pisum sativum L.) through mulching. Pakistan Journal of Weed Science and Research 15: 83-89.
Bradford MM (1976). Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Casanova E, Trillas MI, Moysset L, Vainstein A (2005). Influence of rol genes in floriculture. Biotechnol Adv 23: 3-39.
Cate CH, Loonen AE, Ottaviani MP, Ennik L, van Eldik G, Stiekema WJ (1990). Frequent spontaneous deletions of Ri T-DNA in Agrobacterium rhizogenes transformed potato roots and regenerated plants. Plant Mol Biol 14: 735-741.
Chen LH, Zhang B, Xu ZQ (2007). Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na + /H + antiporter gene AtNHX1 in common buckwheat ( Fagopyrum esculentum ). Transgenic Res 17: 121-132.
Christensen B, Sriskandarajah S, Müller R (2009). Biomass distribution in Kalanchoe blossfeldiana transformed with rol- genes of Agrobacterium rhizogenes . Hortic Sci 44: 1233-1237.
Christey MC (1997). Transgenic crop plants using Agrobacterium rhizogenes -mediated transformation. In: Hairy Roots: Culture and Applications. Amsterdam, the Netherlands: Harwood Academic Publishers, pp. 99-110.
El-Banna A, Hajirezaei MR, Wissing J, Ali Z, Vaas L, Heine- Dobbernack E, Jacobsen HJ, Schumacher HM, Kiesecker H (2010). Over-expression of PR-10a leads to increased salt and osmotic tolerance in potato cell cultures. J Biotechnol 150: 277- 287.
Giovannini A, Pecchioni N, Rabaglio M, Allavena A (1997). Characterization of ornamental Datura plants transformed by Agrobacterium rhizogenes . In Vitro Cell Dev Biol-Pl 33: 101- 106.
Godfray HCJ, Beddington JR, Crute IR, Haddad L, Lawrence D, Muir JF, Pretty J, Robinson S, Thomas SM, Toulmin C (2010). Food security: the challenge of feeding 9 billion people. Science 327: 812-818.
Gopinath KA, Kumar N, Mina BL, Srivastva AK, Gupta HS (2009). Evaluation of mulching, stale seedbed, hand weeding and hoeing for weed control in organic garden pea ( Pisum sativum subsp. Hortens L.). Arch Agron Soil Sci 55: 115-123.
Graham PH (2003). Legumes: importance and constraints to greater use. Plant Physiol 131: 872-877.
Guy CL, Niemi KJ, Brambl R (1985). Altered gene expression during cold acclimation of spinach. P Natl Acad Sci USA 82: 3673- 3677.
He C (2005). Expression of an Arabidopsis vacuolar Sodium/ Proton antiporter gene in cotton improves photosynthetic performance under salt conditions and increases fiber yield in the field. Plant Cell Physiol 46: 1848-1854.
Innes NL (2008). Global status of commercialized biotech/GM crops. Exp Agr 44: 574.
Ishitani M, Xiong L, Stevenson B, Zhu JK (1997). Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis : interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell 9: 1935-1949.
James C (2010). A global overview of biotech (GM) crops: adoption, impact and future prospects. GM Crops 1: 8-12.
James C (2012). Global status of commercialized biotech/GM crops: 2012. ISAAA Brief 44.
James C (2014). ISAAA report on global status of biotech/GM crops: 2013. China Biotechnol 34: 1-8.
Jefferson-Moore KY, Traxler G (2005). Second-generation GMOs: where to from here? Agbioforum 8: 143-150.
Khan H, Subhan M, Mehmood S, Durrani MF, Abbas S, Khan S (2008). Purification and characterization of serine protease from seeds of Holarrhena antidysenterica . Biotechnology 7: 94-99.
Khan TN, Ramzan A, Jillani G, Mehmood T (2013). Morphological performance of peas ( Pisum sativum ) genotypes under rainfed conditions of Potowar region. J Agri Res 51: 51-60.
Kohli A, Miro B, Twyman RM (2010). Transgene integration, expression and stability in plants: Strategies for improvements. In: Kole C, Michler CH, Abbott AG, Hall TC, editors. Transgenic Crop Plants. Heidelberg, Germany: Springer, pp. 201-237.
Limami MA, Sun LY, Douat C, Helgeson J, Tepfer D (1998). Natural genetic transformation by Agrobacterium rhizogenes . Plant Physiol 118:543-50.
Ling Q, Huang W, Jarvis P (2010). Use of a SPAD-502 meter to measure leaf chlorophyll concentration in Arabidopsis thaliana . Photosynth Res 107: 209-214.
Mantyla E, Lang V, Palva ET (1995). Role of abscisic acid in drought- induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana . Plant Physiol 107: 141-148.
Murashige T, Skoog F (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15: 473-497.
Murtaza G, Asghar R (2013). Effects of salicylic acid on sucrose synthase activity during seed development and germination in pea ( Pisum sativum L.). Bangl J Botany 42: 83-90.
Popova LP, Maslenkova LT, Yordanova RY, Ivanova AP, Krantev AP, Szalai G, Janda T (2009). Exogenous treatment with salicylic acid attenuates cadmium toxicity in pea seedlings. Plant Physiol Bioch 47: 224-231.
Rugini E, Caricota G, Muganu M, Taratufolo C, Cavilli M, Cavilli C (1997). Genetic stability and agronomic evaluation of six-year- old transgenic kiwi plant for rolabc and rolb genes. Acta Hortic 1: 609-610.
Sakakibara KY, Saito K (2006). Review: genetically modified plants for the promotion of human health. Biotechnol Lett 28: 1983- 1991.
Sottosanto JB, Saranga Y, Blumwald E (2007). Impact of AtNHX1, a vacuolar Na + /H + antiporter, upon gene expression during short- and long-term salt stress in Arabidopsis thaliana . BMC Plant Biol 7: 1-15.
Stewart PA, McLean WP (2008). Public perceptions of benefits from and worries over plant-made industrial products and plant- made pharmaceuticals: the influence of institutional trust. Rev Policy Res 25: 333-348.
Tamirisa S, Vudem DR, Khareedu VR (2014). Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis . J Exp Bot 65: 4769-4781.
Tepfer D (19984). Transformation of several species of higher plants by Agrobacterium rhizogenes : sexual transmission of the transformed genotype and phenotype. Cell 37:959-67.
Tuteja N, Banu MSA, Huda KMK, Gill SS, Jain P, Pham XH, Tuteja R (2014). Pea p68, a DEAD-box helicase, provides salinity stress tolerance in transgenic tobacco by reducing oxidative stress and improving photosynthesis machinery. PLoS One 9:e98287.
Xiong L (1999). Interaction of osmotic stress, temperature, and abscisic acid in the regulation of gene expression in Arabidopsis . Plant Physiol 119: 205-212.
Zhang HX, Blumwald E (2001). Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat Biotechnol 19: 765-768.