Node position influences viability and contamination in hazelnut shoot

Authors: HAND, CHARLES - Oregon State University; WADA, NAMI - Oregon State University; Stockwell, Virginia; Reed, Barbara

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary: The purpose of this study was to improve micropropagation of hazelnut. Micropropagation is a widely-used tissue culture method to produce numerous identical plants from a single 'mother' plant with desirable traits. For example, conventional breeding has resulted in new cultivars of hazelnut with resistance to a devastating fungal disease called eastern filbert blight. To ensure that the resistance is present in the plants in nurseries or growers orchards, the new plantlets are grown from buds collected from the resistant 'mother' plants, rather than from nuts. The seedlings from buds will be identical to the 'mother' plant, whereas nuts may differ depending on the genetic material in the pollen from the 'father' plants. There are two major challenges to micropropagation of hazelnut. One is the presence of microorganisms on trees. The microorganisms may be benign on the tree, but can grow rapidly in the tissue culture medium and kill the buds. The second challenge is that some buds do not grow when placed in tissue culture media. These two challenges were addressed in this study. We found that growing 'mother' plant shoots in greenhouses decreased the number of buds that were contaminated. We found that the bacterial contaminants common on hazelnuts belonged to the pseudomonas-complex, which are common on plants and in the environment and thus difficult to avoid on plant tissues. We showed that hazelnut buds could be treated with dilute bleach, then submerged in liquid culture media to detect surviving microorganisms for a week before transferring non-contaminated buds onto solid tissue culture media. This 'indexing' method decreased the number of buds that were killed in tissue culture by microbial contaminants. Finally, we found that buds closer to the tip of branches had improved viability in tissue culture and decreased incidence of microbial contamination. Altogether, these easy-to-adopt advancements in micropropagation of hazelnut may improve the efficiency in generating large numbers of clonal hazelnut plants.

Technical Abstract: Initiation of shoot cultures is difficult in many woody plants due to internal microbial contaminants and general lack of juvenility in material from the source plants. Hazelnuts (Corylus avellana L.) are generally difficult to initiate into culture for these same reasons. This study was designed to determine the effect of nodal position and collection techniques on the viability and contamination of shoot explants. In addition, we identified culturable bacteria sampled from surface-disinfested explants. Explants were collected from scion wood grafted onto seedling rootstocks and grown in a greenhouse. Single-node explants, excluding the shoot tip, were collected and node location documented. After surface disinfestation, explants were held in liquid contaminant-detection/indexing medium for one week and the effect of this treatment on explant viability was evaluated. Node position was important for obtaining viable contaminant-free explants. Bacterial contamination increased with the distance from the shoot tip. The use of contaminant-detection medium as a part of the initiation procedure did not affect viability. Explant derived bacteria were identified as belonging to Brevundimonas sp., Pseudomonas sp., through 16S rRNA sequence and API® tests. The best procedure for collecting axenic, viable hazelnut explants was to collect from the first three apical nodes, excluding the shoot tip, of actively-growing greenhouse plants and use contaminant-detection/indexing techniques to identify contaminant-free cultures at initiation.