Molecular Identification of Fungal Contamination in Date Palm Tissue Cultures.

Fungal contamination of in vitro cultures of date palm (Phoenix dactylifera L.) is the major constraint to their initiation and maintenance. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer (ITS) products of the most common fungal contaminants of date palm tissue culture. To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. Polymerase chain reactions were accomplished by ITS primers (ITS1 and ITS4) for each fungal contaminant as well as for sequencing. Subsequently, they are analyzed by Basic Local Alignment Search Tool (BLAST) search of ITS sequence to reveal the identity of each individual fungal contaminant species. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment.

Genotoxicity assessment of high concentrations of 2,4-D, NAA and Dicamba on date palm callus (Phoenix dactylifera L.) using protein profile and RAPD markers.

Genetic stability and uniformity of in vitro-derived date palm plants has a major importance to ascertain true-to-typeness of produced plants. The goal of present study was to evaluate the genetic toxicity of different plant growth regulators on date palm callus at initiation stages using protein patterns and RAPD analysis. Date palm offshoots of Hillawii cultivar were dissected, apical meristems were divided into four segments and cultured on callus induction medium containing the plant growth regulators as 2,4-D at 50 and 100 mg/L; NAA at 30 mg/L and Dicamba at 10 mg/L. The changes occurred in protein profile of callus when treated with high concentration of 2,4-D (100 mg/L), including loss of normal fragments (19 and 66 KDa polypeptides in control), as well as, appearance of new fragments, while at low concentration of 2,4-D (50 mg/L) and Dicamba treatment, the protein patterns showed no changes compared to control profile. Similar trends of polymorphisms were obtained with RAPD marker. The high concentration of 2,4-D produced more polymorphic fragments in comparison to control treatment. The DNA profile was identical between 2,4-D at low concentration and control. Dendrograms were generated using similarity indices of protein and RAPD results, and revealed that genetic similarity index was high between 2,4-D treatment at low concentration and control, as separated in one subcluster, followed by Dicamba and NAA, while, the highest genetic distance was obtained between 2,4-D at high concentration and control treatment and separated alone in one cluster.