Protoplast Isolation and Transformation in Oil Palm.

The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection and DNA microinjection, highlighting also the critical steps associated with the method. Briefly, we will describe the efficient isolation of protoplasts from 3-month-old suspension calli collected at 14 days after cultured. Digestion of the calli with an optimal composition of enzyme solution yielded over 2 × 106 protoplasts/mL with the viability of more than 80%. The concentrations of DNA, PEG, and magnesium chloride and application of heat shock treatment are the crucial determinants for efficient PEG-mediated transfection. Using the optimal PEG transfection conditions, a transfection efficiency of more than 20% could be obtained. At the same time, protoplasts embedded in alginate layer cultured for 3 days and injected with 100 ng/µL of total DNA solution are the optimal factors for microinjection. We successfully regenerated the injected protoplasts to calli expressing green fluorescent protein (GFP) signals when cultured in optimal medium and cultivation procedures.

Ripening and genotype control stilbene accumulation in healthy grapes.

In grapes, stilbene synthesis occurs in the skin, and it is induced by biotic and abiotic stresses. To date, experimental evidence of a constitutive production of resveratrols in healthy grape is scarce and not conclusive. The aim of the present work was to investigate stilbene biosynthesis in healthy grapes both at biochemical and molecular levels. By measuring the concentration of resveratrols in ripe berries of 78 Vitis vinifera varieties for 3 years, we could identify significant differences among genotypes, providing the first tentative varietal classification based on resveratrol content. Furthermore, an increasing stilbene accumulation from veraison to ripening phase was also observed. Using real-time reverse transcription polymerase chain reaction and a berry-specific cDNA array, gene expression analysis was carried out on two distinct pools of berries belonging to the high and low resveratrol producers and on three berry developmental stages. The stilbene synthase, phenylalanine ammonia-lyase, and 4-coumarate-CoA ligase expression profiles showed an increasing concentration of these transcripts from véraison to maturity and a higher accumulation in the grape of high resveratrol producers. Macroarray data analysis revealed that high resveratrol levels are also accompanied by the up-regulation of genes involved in plant defense and the concomitant underexpression of genes related to the ripening process and to indole alkaloid synthesis.