Regeneration of oil palm plantlets introduced by P5CS gene using Agrobacterium-mediated transformation

Asmini Budiani, Imam Bagus Nugroho, Hayati Minarsih, Imron Riyadi

Abstract


Abstrak

Cekaman kekeringan dapat mempengaruhi produktivitas tanaman perkebunan. Rekayasa genetika merupakan salah satu cara untuk meningkatkan produktivitas tanaman perkebunan penting seperti kelapa sawit. Tujuan dari penelitian ini adalah melakukan perekayasaan kelapa sawit melalui introduksi gen P5CS dengan transformasi berbasis Agrobacterium untuk meningkatkan ketahanan tanaman terhadap cekaman kekeringan. Pada penelitian ini perakitan kelapa sawit transgenik yang tahan terhadap cekaman kekeringan dilakukan melalui transformasi gen P5CS (Δ1-pyrroline-5-carboxylate synthetase) ke dalam kalus embriogenik (embryogenic calli – EC) menggunakan Agrobacterium. Plasmid pBI_P5CS yang membawa gen P5CS ditransfer dari Escherichia coli XL1 Blue ke Agrobacterium tumefaciens AGL1 melalui konjugasi. Selanjutnya klon Agrobacterium yang membawa plasmid pBI_P5CS digunakan untuk menginfeksi kalus embriogenik kelapa sawit dengan perlakuan 100 ppm asetosiringon. Kalus transforman diregenerasi pada media de Fossard (DF) yang ditambahkan 50 ppm kanamisin dan 250 ppm sefotaksim. Kalus transforman diseleksi melalui uji GUS dan metode PCR menggunakan primer NPTII dan P5CS1. Uji GUS dilakukan untuk menyeleksi kalus transforman yang ditunjukkan dengan reaksi positif pembentukan warna biru pada kalus yang berhasil ditransformasi dengan konstruk pBI_P5CS. Pengujian dengan menggunakan PCR memberikan hasil positif dengan adanya profil pita PCR pada visualisasi menggunakan pewarnaan SYBR Green, yang menunjukkan amplikon berukuran ~ 0,7 kb untuk gen NPTII dan ~ 0,4 kb untuk gen P5CS pada elektroforesis dengan gel agarosa. Hasil dari penelitian ini adalah diperolehnya kalus transforman terseleksi yang telah diregenerasi dan tumbuh menjadi planlet.

[Kata kunci: cekaman kekeringan, Elaeis guineensis Jacq., rekayasa genetika, planlet]

Abstract

      Environmental abiotic stressors particularly drought has detrimental effects upon the productivity of estate crops. Increasing the crop tolerance towards drought stress through genetic engineering is one of the strategies employed to maintain steady productivity of valuable crop, i.e. oil palm. The aim of this study was to engineer oil palm with a better tolerance towards drought by introducing P5CS1-pyrroline-5-carboxylate synthetase) gene via Agrobacterium–mediated transformation into embryogenic calli (EC). The pBI_P5CS plasmid harboring P5CS gene was transferred from Escherichia coli XL1 Blue to Agrobacterium tumefaciens AGL1 by conjugation. The positive clone of transformed Agrobacterium was then used to infect oil palm EC by the addition of 100 ppm acetosyringone. The transformed ECs were regenerated in the de Fossard (DF) media supplemented by 50 ppm kanamycin and 250 ppm cefotaxime followed by GUS assay and PCR-based screening using NPTII and P5CS1 primers. The positive EC clones were confirmed by GUS assay, which produced blue coloration on positive transformed oil palm EC. A positive result of PCR screenings was depicted by PCR products in SYBR Green staining gel agarose electrophoresis with the expected band size of ~ 0.7 kb for the NPTII gene and ~ 0.4 kb for the P5CS gene. This study has successfully selected and regenerated pBI_P5CS transformed oil palm embryogenic calli into plantlets.

[Keywords: drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets]


Keywords


drought tolerance, Elaeis guineensis Jacq., genetic engineering, plantlets

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References


Albert B, F Le Caherec, MF Niogret, P Faes, JC Avice, L Leport & A Bouchereau. (2012). Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions. Planta 236, 659–676.

Amini S, C Ghobadi & A Yamchi (2015). Proline accumulation and osmotic stress: an overview of P5CS gene in plants. Journal of Plant Molecular Breeding 3(2), 44–55.

Azevedo RA, RF Carvalho, MC Cia & PL Gratão (2011). Sugarcane under pressure: An overview of biochemical and physiological studies of abiotic stress. Tropical Plant Biology 4(1), 42–51.

Ben Rejeb K, C Abdelly & A Savouré (2014). How reactive oxygen species and proline face stress together. Plant Physiol Biochem 80, 278–84.

Carr MKV (2011). The water relations and irrigation requirements of oil palm (Elaeis guineensis): a review. Exp Agric 47, 629–652.

Carrer H, TN Hockenberry, Z Svab, & P Maliga (1993). Kanamycin resistance as a selectable marker for plastid transformation in tobacco. Molecular and General Genetics 241(1–2), 49–56.

Conte S, D Stevenson, I Furner & A Lloyd (2009). Multiple antibiotic resistance in Arabidopsis is conferred by mutations in a chloroplast-localized transport protein. Plant Physiol 151(2), 559–573.

Corley RHV & PBH Tinker (2015). The oil palm. 5th ed. Oxford, UK, Wiley-Blackwell.

Dislich C, AC Keyel, J Salecker, Y Kisel, KM Meyer, M Auliya, AD Barnes, MD Corre, K Darras, H Faust, B Hess, S Klasen, A Knohl, H Kreft, A Meijide, F Nurdiansyah, F Otten, G Pe’er, S Steinebach, S Tarigan, MH Tolle, T Tscharntke & K Wiegand (2017). A review of the ecosystem functions in oil palm plantations using forests as a reference system. Biological Reviews 92(3), 1539–1569.

Eisenhut M, A Bräutigam, S Timm, A Florian, T Tohge, AR Fernie, H Bauwe H & APM Weber (2017). Photorespiration is crucial for dynamic response of photosynthetic metabolism and stomatal movement to altered CO2 availability. Mol Plant 10(1), 47–61.

Fitranty N, F Nurilmala, D Santoso & H Minarsih (2003). Effectivity of Agrobacterium to transfer P5CS gene into sugarcane callus PS 851 clone. Menara Perkebunan 71 (1), 16–27.

Flügel F, S Timm, S Arrivault, A Florian, M Stitt, AR Fernie & H Bauwe (2017). The photorespiratory metabolite 2-phosphoglycolate regulates photosynthesis and starch accumulation in Arabidopsis. The Plant Cell 29, 2537–2551.

Hayat S, Q Hayat, MN Alyemeni, AS Wani, J Pichtel & A Ahmad (2012). Role of proline under changing enviromental. Plant Signaling & Behavior 7(11), 1456–1466.

Haworth M, D Killi, A Materassi, A Raschi, & M Centritto (2016). Impaired stomatal control is associated with reduced photosynthetic physiology in crop species grown at elevated [CO2]. Front Plant Sci 7, 1568.

Izawati AMD, GKA Parveez & MYA Masani (2012). Transformation of oil palm using Agrobacterium tumefaciens. In JM Dunwell & AC Wetten (eds.), Transgenic Plants: Methods and Protocols. Totowa, NJ, Humana Press. p. 177–188.

Jazayeri SM, YD Rivera, JE Camperos-Reyes & HM Romero (2015). Physiological effects of water deficit on two oil palm (Elaeis guineensis Jacq.) genotypes. Agron Colomb 33(2), 164–173.

Kavi-Kishor PB & N Sreenivasulu (2014). Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell and Environment 37(2), 300–311.

Lee JH & Schöffl F (1997). GUS activity staining – a powerful tool in plant molecular biology. In SB Gelvin & RA Schilperoort (eds.), Plant Molecular Biology Manual Dordrecht, Springer Netherlands. p. 23–32.

Lestari R, M Rio, F Martin, J Leclercq, N Woraathasin, S Roques, F Dessailly, A Clément‐Vidal, C Sanier, D Fabre, S Melliti, S Suharsono, & P Montoro (2017). Overexpression of Hevea brasiliensis ethylene response factor HbERF-IXc5 enhances growth and tolerance to abiotic stress and affects laticifer differentiation. Plant Biotechnol J 16(1), 322–336.

Li J, TT Phan, YR Li, YX Xing & LT Yang (2018). Isolation, transformation and overexpression of sugarcane SoP5CS gene for drought tolerance improvement. Sugar Tech 20(4), 464–473.

Maghsoudi K, Y Emam, A Niazi, M Pessarakli & MJ Arvin (2018). P5CS expression level and proline accumulation in the sensitive and tolerant wheat cultivars under control and drought stress conditions in the presence/absence of silicon and salicylic acid. Journal of Plant Interactions 13(1), 461–471.

Marbun CLM, NT Mathius, Reflini, C Utomo & T Liwang (2015). Micropropagation of embryogenic callus of oil palm (Elaeis Guineensis Jacq.) using temporary immersion system. Procedia Chemistry 14, 122–129.

Minarsih H, D Santoso & N Fitranty (2001). Identification of P5CS gene on sugarcane by PCR using heterologous primer. Menara Perkebunan 69(1), 1–9.

Minarsih H, D Subiyarti, I Riyadi, SM Putra & L Ambarsari (2015). Evaluation of varieties, explant sources, and Agrobacterium strains for successful sugarcane transformation using P5CS gene. Menara Perkebunan 83(1), 1–9.

Noctor G, A Mhamdi & CH Foyer (2014). The roles of reactive

oxygen metabolism in drought: Not so cut and dried. Plant Physiology 164, 1636–1648.

Oettli P, SK Behera & T Yamagata (2018). Climate based predictability of oil palm tree yield in malaysia. Sci Rep 8(1), 2271.

Pandey P, V Irulappan, MV Bagavathiannan & M Senthil-Kumar (2017). Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits. Front Plant Sci 8, 537.

Paterson RRM, L Kumar, F Shabani & N Lima (2017). World climate suitability projections to 2050 and 2100 for growing oil palm. The Journal of Agricultural Science 155, 659–702.

Sinta MM, I Riyadi &Sumaryono (2011). Effect of different culture vessel closures on the growth of oil palm (Elaeis guineensis Jacq.) plantlets. Menara Perkebunan 79(1), 15–22.

Soleimani V, J Ahmadi, S Golkari, & B Sadeghzadeh (2015). Expression profiling of PAP3, BZIP, and P5CS genes in soybean under drought stress conditions. Turk J Bot 39, 952–961.

Sumaryono & I Riyadi (2011). Ex vitro rooting of oil palm (Elaeis guineensis Jacq.) plantlets derived from tissue culture. Indonesian Journal of Agricultural Science 12(2), 57 – 62.

Syarovy M, I Pradiko, E Listia, NH Darlan, F Hidayat, W Winarna & S Rahutomo (2018). Drought and haze effects on oil palm ecophysiology and productivity in south sumatra. Jurnal Penelitian Kelapa Sawit 25(3), 137–146.

Turchetto-Zolet AC, M Margis-Pinheiro & R Margis (2009). The evolution of pyrroline-5-carboxylate synthase in plants: A key enzyme in proline synthesis. Molecular Genetics and Genomics 281(1), 87–97.

Urban J, MW Ingwers, MA McGuire, & RO Teskey (2017). Increase in leaf temperature opens stomata and decouples net photosynthesis from stomatal conductance in Pinus taeda and Populus deltoides x nigra. J Exp Bot. 68(7), 1757–1767.

Voss I, B Sunil, R Scheibe & AS Raghavendra (2013). Emerging concept for the role of photorespiration as an important part of abiotic stress response. Plant Biol (Stuttg) 15(4), 713–22.

Wei C, Q Cui, XQ Zhang, YQ Zhao & GX Jia (2016). Three P5CS genes including a novel one from Lilium regale play distinct roles in osmotic, drought and salt stress tolerance. Journal of Plant Biology 59(5), 456–466.

Wu HH, YN Zou, MM Rahman, QD Ni & QS Wu (2017). Mycorrhizas alter sucrose and proline metabolism in trifoliate orange exposed to drought stress. Sci Rep 7, 42389.

Zhang L & DF Becker (2015). Connecting proline metabolism and signaling pathways in plant senescence. Front Plant Sci 6, 552.




DOI: http://dx.doi.org/10.22302/iribb.jur.mp.v87i2.336

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