|Abstract or Summary
- Red raspberry, Rubus idaeus L., is a valuable crop for the U.S. Pacific Northwest and clonal propagation is required to produce disease-free plants and for germplasm preservation. One challenge of red raspberry micropropagation is the wide variation in growth response among the cultivars. The studies described in this dissertation were designed to provide more information on the mineral nutrient requirements of in vitro cultures.
Mineral nutrient studies using response surface methodology (RSM) were designed to determine which mineral nutrients significantly improved the growth and development of shoot cultures. Mineral nutrients of Murashige and Skoog (MS) medium were used as factors: NH₄NO₃, KNO₃, mesos (CaCl₂, MgSO₄ and KH₂PO₄), minor nutrients (B, Cu, Mn, Mo, and Zn) and Iron. Models indicated that shoot growth and proliferation were significantly influenced by mesos and nitrogen. The effects varied by cultivar for some characteristics, but all cultivars had improved growth or appearance on some experimental treatments when compared to MS medium. Increased mesos was the most significant factor associated with plant quality, multiplication and shoot length in all cultivars.
Individual mesos components were optimized using a 3-dimensional RSM experimental design. High concentrations of CaCl₂ and KH₂PO₄ significantly increased shoot length for most cultivars. All cultivars required significantly higher concentrations (p ≤0.05) of all three mesos components (2.5-3.0×) for improved growth and shoot quality compared to MS medium. Mineral status of the shoots reflected the amount of minerals in the growth medium. A mixture component design was applied to optimizing the nitrogen salts (NH₄NO₃ and KNO₃) on MS medium with 2.5× mesos. Eight red raspberry cultivars and one R. odoratus L. were evaluated followed by a screen of three additional species. Increased NO₃⁻ concentration was the most significant factor for improved shoot quality. The effects of the NH₄⁺ and K⁺ mixture and interactions between the components in the mixture and NO₃⁻ affected most cultivars. The standard MS nitrogen level combined with the 2.5× mesos concentration was optimal for most cultivars, but three other modified nitrogen levels also improved overall quality, shoot elongation, multiplication for many of the cultivars.
Since the largest effects on growth were from increased mesos, plant metabolism was studied using 'Indian Summer' as a model cultivar. Shoots grown on standard MS medium (1.0× MS mesos) were compared to shoots grown on 1.5× and 2.5× MS mesos. After 9 weeks growth, shoots were evaluated for quality factors and metabolic changes. Metabolic changes were determined by liquid chromatography electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) coupled to liquid chromatography. Metabolic analysis indicated that shoots grown on increased mesos had reduced amino acids (glutamine, arginine histidine, tryptophan and proline) and the secondary metabolites: proanthocyanidin (epi-catechin), quercetin, and ellagic acid compared to those on standard MS.
This study determined changes in the growth medium mineral content required to greatly improve the growth of 21 red raspberry cultivars and several Rubus species. Increasing the mesos concentration was the most important mineral factor for improved growth response. Four combinations of nitrogen compounds were effective for improving growth of most cultivars. The production of red raspberries by micropropagation is a multimillion dollar business and these improvements would increase productivity that could positively impact that industry.