Relationship among growth curve, nutrient consumption and genetic transformation efficiency of 'Albariño' (<i>Vitis vinifera</i>) cell suspensions
Embryogenic cell suspensions of grapevine (Vitis sp.) have been proposed as the best target to approach genetic transformation challenges. However, optimal phase and growth period of cell suspensions for successful gene transfer have not been investigated. Here, a step by step protocol to initiate and establish cell suspensions of 'Albariño' (V. vinifera) in only 4 weeks is presented. Growth kinetics, cell viability and nutrient consumption (phosphates and nitrates) as well as the number of transient transgenic events (using the uidA reporter gene) were studied in 'Albariño' cell suspensions grown for an 18-days period. Based on biomass growth, the exponential phase of cell suspensions was reached between days 3 to 6. Nutrient uptake results point to the exhaustion of phosphate in the culture medium at day 6. Moreover, the highest number of transgenic events after biolistic bombardment was obtained from cell suspensions grown for 6 days (4032 ± 695 blue spots), compared to 12 and 18 days of continuous culture. Plant regeneration percentage varied depending on the age of the culture and the selected embryo type. In conclusion, this paper shows for the first time the relationship between growth curve and nutrient consumption of embryogenic cell suspension with efficiency in genetic transformation and plant regeneration of grapevine.
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