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United States Department of Agriculture

Agricultural Research Service

Research Project: MANAGEMENT OF TEMPERATE FRUIT NUT AND SPECIALTY CROP GENETIC RESOURCES

Location: National Clonal Germplasm Repository (Corvallis, Oregon)

Title: The Effect of Explant Node Position on the Amount and Type of Bacterial Contamination in Hazelnut Shoot Cultures

Authors
item Hand, Charles -
item Reed, Barbara

Submitted to: Society for In Vitro Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2011
Publication Date: June 2, 2011
Citation: Hand, C.P., Reed, B.M. 2011. The Effect of Explant Node Position on the Amount and Type of Bacterial Contamination in Hazelnut Shoot Cultures. Society for In Vitro Biology Proceedings. 47:S67.

Interpretive Summary: New types of hazelnut resistant to eastern filbert blight are in demand by growers and micropropagation is used to rapidly increase plant availability. Hazelnut trees contain many internal bacteria, making it difficult to initiate clean cultures. This study was designed to determine which part of the plant is best for producing clean cultures and what types of bacteria are present in the plants. Cuttings from new shoots of greenhouse grown plants were sampled in late spring. Branches were cut into six single bud sections. Two types of tests were used to detect bacteria in the buds. Bacterial contamination increased with distance from the branch tip. After 6 months the first two sections had 85% and 68% bacteria-free cultures, but fewer than a third of those from the fourth to sixth section were clean. Overall about half of all the buds collected were free of bacteria and most were from the first two sections. Pure bacterial cultures were isolated and identified using DNA. Many of the same bacteria were found in each of the plants sampled. The best procedure for starting bacteria-free hazelnut cultures is to collect from the first three buds of fast-growing protected source plants and test monthly to identify those with bacteria.

Technical Abstract: New hazelnut (Corylus avellana L.) cultivars resistant to eastern filbert blight are in demand and micropropagation is used to rapidly increase plant availability. Hazelnut trees contain many endogenous microorganisms, making it difficult to initiate axenic cultures. This study was designed to determine the effect of explant location on bacterial contamination of cultures and what types of bacteria are present as contaminants. Plants of three genotypes were grown in the greenhouse to reduce external contaminants and explants were taken from new growth in late spring. Single-node explants were collected from each of five branches, from the first node below the apical meristem to the sixth node. Standard surface sterilization techniques were used. The explants were placed in pH 6.9 half-strength liquid Murashige and Skoog medium for one week, followed streaking on nutrient agar (NA) at each transfer to detect bacteria. Bacterial contamination increased with distance from the apex. After 6 months the node one had 85% bacteria-free explants and node two 68%, but <33% of explants from the fourth to sixth nodes were axenic. About half of all the explants were free of bacteria and most were from the first two nodes. Pure bacterial cultures were isolated from the explants and identified through 16S ribosomal DNA sequences. Many of the same bacteria were found in each of the three genotypes. The best procedure for collecting axenic hazelnut explants is to collect from the first three nodes of fast-growing protected source plants and use indexing techniques to identify bacteria contaminated plant cultures.

Last Modified: 7/24/2014
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