Biolistic Transformation of Citrullus vulgaris Schrad (Watermelon)

Abstract

Abstract:In this study, DNA-coated gold particles were used to transform cotyledon sections including axillary meristem of Citrullus vulgaris using the biolistic method for optimizing the transformation parameters and eventually regeneration of putative transgenic watermelon plants. Initially, five biolistic parameters namely helium pressure, macrocarrier to target tissue distance, explant ages, DNA concentrations and osmotic treatment were optimized using plasmid pRQ6 (carrying both gusA and hpt genes). The optimized biolistic parameters for cotyledon sections of Citrullus vulgaris explants were determined as follow: 1100 psi helium pressure, 6 cm target distance, 4-day-old explants, concentration of DNA at 1.2 µg/bombardment and osmotic treatment for 24 h on medium supplemented with 0.6 M mannitol prior to bombardment. Using the optimized parameters, transformation of watermelon cotyledons was carried out using pAHG11, pCAMBIA 1301, co-transformation (pAHG11+pCAMBIA 1301) and pCambar plasmids. Bombarded tissue's were selected on either 5 mg LG 1 hygromycin or 2 mg LG PPT. Finally, the transformants produced were subjected to GUS histochemical assay. 1Integration of the transgenes into transgenic watermelon genome was confirmed by PCR analysis.

Factors affecting genetic transformation by particle bombardment of the prickly pear cactus (O. ficus-indica)

“…In addition, distance of flight has a very important role in tissue damage and DNA penetration in plants. In our study, when we increased the distance of flight, the GUS transient expression levels were considerably lower than the ones observed when using 8 and 11 cm (Table 1 ); this may be due to the velocity decrease of the microcarriers that reduces the penetration force and DNA integration (Suratman et al 2010 ).…”

“…The type and concentration of the osmotic agent may increase the transient gene expression by reducing the turgor pressure in cells, helping to increase the survival probability of the cells by avoiding cell rupture after the blast generated from the bombardment (Rosillo et al 2003). The positive effect of using mannitol and combinations with sorbitol has already been reported in plant systems such as watermelon (Suratman et al 2010) and coffee (Gatica et al 2009). Even though the use of mannitol generated positive results in foci formation (Table 1), the sorbitol + mannitol treatment and the maltose + sorbitol + mannitol treatment did not generate any synergic effect that could enhance GUS expression.…”

“…The osmotic treatment prior to particle bombardment is used to reduce cell damage, enhance transient GUS expression, increase stable transformation, and facilitate cell membrane stabilization after transformation (Suratman et al 2010 ). The previous studies made in nopal utilized a 12–16-h period of exposition prior to particle bombardment, but the osmotic agent was not described (Llamoca-Zarate et al 1999b ).…”

Generation of transgenic watermelon resistance to Cucumber mosaic virus facilitated by an effective Agrobacterium-mediated transformation method

“…The earliest study on watermelon transformation was reported by Choi et al, they successfully obtained a transgenic watermelon plant that expresses the GUS reporter gene (Choi et al 1994). Thereafter, several delivery methods have been utilized for watermelon transformation; these methods include biolistic transformation (Suratman et al 2010) and pollen tube pathway (Chen et al 1998), with the Agrobacterium tumefaciens-mediated transformation being the predominant method. And few transgenic watermelon plants were successfully obtained, such as expression an untranslatable chimeric construct containing truncated ZYMV coat protein (CP) and PRSV W CP genes to generate ZYMV and PRSV resistence watermelon (Yu et al 2011b), expressing the yeast HAL1 gene to enhance watermelon salt tolerance (Ellul et al 2003), expressing the Cucumber green mottle mosaic virus coat protein gene (CGMMV-CP) to develop CGMMV resistance in watermelon rootstocks (Park et al 2005), and so on.…”