Asia AsPacPacific J. Mol. Journal Biol. Biotechnol., of MolecularVol. Biology 16 (2), and2008 Biotechnology, 2008 Vol. 16 (2) : 25-33
Efficient regeneration and transformation of recalcitrant sweet potato
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Efficient regeneration and Agrobacterium tumefaciens mediated transformation of recalcitrant sweet potato (Ipomoea batatas L.) cultivars. Rolando García González1,2*, Danalay Somonte Sánchez2, Zurima Zaldúa Guerra1, Jesús Mena Campos2, Alina López Quesada2, Rolando Morán Valdivia2, Ariel D. Arencibia2, Karla Quiroz Bravo3 and Peter DS. Caligari3 Departamento de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Chile. Avenida San Miguel, No.3605. 2 Centro de Ingeniería Genética y Biotecnología de Camagüey. A.P. 387, C.P. 70100. Camagüey, Cuba. 3 Instituto de Biología Vegetal y Biotecnología, Universidad de Talca. 2 Norte 685, Talca, Chile. 1
Received 20 June 2007 / Accepted 15 May 2008 Abstract. Sweet potato is a major world crop and its behavior under in vitro culture is genotype dependent. We studied several factors influencing the regeneration and transformation efficiency of two recalcitrant cultivars: Jewel and CEMSA 78354. Growth regulators, explant preparation and removal of apical dominance were evaluated in order to optimize the regeneration steps. At the same time, the influence of environmental conditions for the interaction between Agrobacterium tumefaciens and leaves were evaluated to obtain higher transformation efficiencies. For Jewel, the best results were obtained when intact leaf explants were cultivated on MS medium supplemented with 0.5 mgL-1 indol-3-acetic acid for four weeks (Regeneration frequency, RF= 2.02). However, for CEMSA 78354 the best results were obtained by culturing leaf explants on MS medium supplemented with 1.0 mgL-1 paclobutrazol and 1.0 mgL-1 naftalenacetic acid (RF= 0.98). Optimal transformation conditions were obtained for both cultivars by co-cultivating leaf explants with Agrobacterium tumefaciens in liquid MS medium for 24 hours at 28ºC in stationary cultures in the dark. Acetosyringone influence on the transformation efficiency was found to be dependent on the co-cultivation temperature but it did not increase the transformation efficiencies. Molecular evidences by PCR, Southern blot and Dot blot demonstrated the effectiveness of the transformation procedure. The protocol described here is currently in use to produce transgenic sweet potatoes from different cultivars with high efficiency.
Keywords. Ipomoea batatas; organogenesis; Paclobutrazol; Agrobacterium tumefaciens mediated transformation;. INTRODUCTION Sweet potato (Ipomoea batatas Lam.) is a perennial, herbaceous, dicotyledonous species of the morning glory family Convolvulaceae that is grown as an annual crop in many countries. It is one of the most versatile, and yet under-exploited, crop species in the world, its current annual production is estimated at 133 million tons, mainly growing in 100 developing countries and being placed among the five most important food crops in over 50 of these. Despite its major importance sweet potato has not received significant attention from plant biotechnologists (Midmore et al., 2003). In recent years, only a few groups have reported molecular biology manipulation results in terms of increasing its nutritional quality (López et al., 1996) and to give pest resistance (Newel et al., 1995). Other groups have reported results on sweet potato conservation (Blakesley et al., 1996), protoplast Abbreviations: PBZ, Paclobutrazol; BAP, 6- benzilaminopurine; IAA, Indol-3-acetic acid; NAA, Naftalenacetic acid; Zea, Zeatin; Btt, Bacillus thuringiensis tenebrionis.
isolation and regeneration (Sihachakr, 1987), and plant regeneration from roots, petioles, stems and leaves (Gosukonda et al., 1995). A relatively high success in developing an efficient system of embryogenic suspension cultures for a wide range of sweet potato genotypes especially for commercial cultivars was obtained but the methods was time consuming and quite difficult to establish (Liu et al., 2001). Sweet potato has been transformed using different systems including microprojectile bombardment (Prakash and Varadarajan, 1992) electroporation of protoplasts (Garcia et al., 1998), Agrobacterium rhizogenes (Otani et al., 1993) and Agrobacterium tumefaciens (Otani et al., 2001). In general, the results on sweet potato genetic transformation have shown low efficiency of a high genotype dependent response (Otani et al., 2003; Song et al., 2004; Shimada et al., 2006; Luo et al., 2006). Sweet potato is considered a highly recalcitrant species in *Author for Correspondence. Mailing address: Departamento de Ciencias Agrarias y Forestales, Universidad Católica del Maule, Chile. Avenida San Miguel, No.3605. Talca. E-mail:
[email protected]
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AsPac J. Mol. Biol. Biotechnol., Vol. 16 (2), 2008
terms of regeneration and transformation because the above mentioned cultivar responses to in vitro treatments (Aloufa, 2002) and this has limited the applications of genetic engineering technologies (Newel et al., 1995; Gosukonda et al., 1995). It also shows a high genetic diversity (Wang et al., 1998) and its major anthocyanins and polyphenolic compounds are genotype dependent (Islam et al., 2002) which could explains, along with other factors, its genotype dependent responses to in vitro manipulation. Here, is reported an efficient methodology for sweet potato regeneration and transformation. Two very important cultivars, which are both considered as very recalcitrant to be in vitro manipulated were studied, namely: Jewel and CEMSA 78354. This research was done in two steps. In the first step, different growth regulator relationships and four procedures for leaves explant manipulation were studied in order to establish an efficient medium for shoot induction. In the second step factors that affect Agrobacterium tumefaciens mediated transformation, such as induction of vir genes, time of co-cultivation, temperature and co-culture conditions, were tested.
MATERIALS AND METHODS Plant material. Sweet potato plants of Jewel and CEMSA 78354 cultivars were kindly donated from the collection of the Instituto Nacional de Investigaciones en Viandas Tropicales (INIVIT) from Santo Domingo, Cuba. Plants were propagated in glass tubes containing 7 ml of MS salts and vitamins (Murashige and Skoog, 1962) of solid medium supplemented with 3% sucrose, 0.5 mgL-1 IAA and 6 gL-1 agar-agar (Difco), with the pH adjusted to 5.6-5.7 prior to autoclaving. For all the regeneration and transformation experiments, explant donor plants were grown in the propagation medium for four weeks. Growth regulator studies and explants manipulation for organogenic regeneration. MS basal salt mixture with vitamins, supplemented with sucrose 3% and solidified with 6.0 gL-1 agar-agar (Difco), was used for all the experiments. The pH was adjusted to 5.6-5.7 before autoclaving (20 min; 121 ºC; 138 kPa), and all growth regulators were added prior autoclaving except zeatin riboside, which was filter-sterilized and added to the cooled medium. All experiments were set up in five repetitions for each growth regulator combination. Each repetition was composed of six leaves. Leaf explants were incubated at 25 ºC and in a 12/12 h photoperiod. Leaf explants were prepared by removing the four ends of the lamina, and then cultivated with the axial side of the leaf expanded and fully in contact with the medium. All the regeneration experiments were evaluated four weeks after planting in the tested media. Regeneration Frequency (RF) was used to choose the best
Efficient regeneration and transformation of recalcitrant sweet potato
regeneration media. It was calculated as: RF= (RS/TNE). Where: RS, is the total number of individual rooted shoots and TNE, is the total number of explants. Statistic analyses were performed to test the differences between treatments using ANOVA and LSD Test at p
