Deciduous azalea (Rhododendron sect. Pentanthera G. Don) cultivars and species are highly ornamental shrubs that are valued in northern landscapes for their colorful and prolific flowers. One important group of deciduous azalea cultivars is the Northern Lights series, which since 1979 has grown to 15 interspecific hybrids including the original ‘Northern Lights’, ‘Mandarin Lights’, and ‘Lemon Lights’ that bring vivid pink, orange, and yellow colors into U.S. Department of Agriculture (USDA) hardiness Zone 4 northern landscapes (Hokanson, 2010). With nearly 630,000 plants sold at a combined wholesale value of $1.02 million between 2014 and 2016, these and other deciduous azalea cultivars represent a reliable source of revenue for woody plant growers who cater to landscape markets in cold climates (L. Caton, Sidhu and Sons, personal communication). Given the series’ popularity, the University of Minnesota breeding program continues to expand the series, with the first true red ‘UMNAZ633’ Electric LightsTM Red and double-flowered ‘UMNAZ493’ Electric LightsTM Double Pink introductions bringing new ornamental characteristics to cold-hardy woody ornamental germplasm (Hokanson et al., 2015).
Progenitors of the cultivars mentioned previously primarily resulted from crosses between North American deciduous azalea species native to the southern and eastern United States with European deciduous azalea hybrids (Mollis Azalea, Exbury, and Knapp Hill hybrids) from the early 20th century (Moe and Pellett, 1986). Each of these native species contributes unique ornamental attributes to cultivars released. These include pink flowers from wild-collected Rhododendron prinophyllum (Small) Millais and large, durable, orange flowers from Rhododendron calendulaceum (Michx.) Torr. (Moe and Pellett, 1986; Widrlechner, 1982). Additional ornamental characteristics such as fragrance from Rhododendron atlanticum (Ashe) Rheder and white flowers from Rhododendron viscosum (L.) Torr. were also introgressed into the breeding germplasm (Hokanson, 2010). The combination of traits from each of these species and their segregation in seedling populations has enabled a diverse series of Rhododendron cultivars to be developed for northern areas of the United States, where cultivation of the genus was historically thought to be impossible because of extreme winter cold temperatures (Widrlechner, 1982).
The greatest systematic breeding effort has been focused on improving the cold hardiness of floral and vegetative tissues (Moe and Pellett, 1986). Initial cold hardiness evaluations required substantial screening time in the field as the initial parental germplasm and progeny evaluations that led to ‘Northern Lights’ spanned the years 1957–79, a 22-year period. These initial efforts resulted in a series of deciduous azalea cultivars that are reliably cold-hardy in USDA Zone 4 (Hokanson, 2010). As cultivation of deciduous azaleas has spread throughout the Midwestern United States, tolerance to high pH or calcareous soils (calcium carbonate, CaCO3) that predominate in the region has become the most pressing trait to improve. High-pH soils severely limit deciduous azalea growth, primarily by restricting micronutrient availability, such as iron, for uptake by roots (Brady and Weil, 2004). Bicarbonate ions present in calcareous soils raise soil pH and keep the soil buffered, resulting in slower acidification of soil, further limiting micronutrient availability (Brady and Weil, 2004). This reduction in iron uptake on high-pH or calcareous soils leads to chlorosis and decreased photosynthetic capacity, which over time reduces vigor and increases mortality of deciduous azaleas in the landscape (Galle, 1974). Improving adaptability to high-pH or calcareous soils (henceforth referred to as pH adaptability) of cultivars would reduce plant maintenance and open up new markets in continental climates characterized by calcareous or high-pH soils.
Although some systematic selection against iron deficiency chlorosis has occurred in evergreen Rhododendron, the variation is hard to phenotype consistently because of confounding nutrient deficiencies and measurement error using a qualitative (1–10) rating scale in breeding populations (Dunemann et al., 1999; Preil and Ebbinghaus, 1994). Furthermore, prospects for improving pH adaptability in deciduous azalea are slowed by long generation times and limited timeframe (at leaf flush) for observing symptoms of high pH stress such as iron deficiency chlorosis. Like many woody ornamentals, deciduous azaleas take between 8 and 10 months from crossing to seed germination, and an additional 2 years before plants reach maturity for field trials (Susko, 2016). Growing plants in containers before planting in the field requires space and labor that is often a limiting factor in determining how many seedlings can be produced for screening each season. Therefore, selection at the early seedling stage could both reduce the number of plants carried forward and expedite breeding deciduous azaleas.
Rather than waiting for the manifestation of chlorosis in whole plant experiments, characterization of the root–soil interface (rhizosphere), including localized reduction in pH (rhizosphere acidification), could more quickly indicate whether certain genotypes will successfully acquire micronutrients and mitigate symptoms of high pH stress (Guerinot and Yi, 1994). Known as Strategy 1 iron acquisition, dicot plants extrude protons into the rhizosphere to create favorable conditions for iron solubility and reduction that enable its translocation into root cells (Brady and Weil, 2004; Briat and Lobréaux, 1997). Direct acidification of the rhizosphere by proton extrusion by plasma membrane H+-ATPase is a well-documented response to iron deficiency in dicot plants (Yi and Guerinot, 1996). Indirect acidification of the rhizosphere through other nutrient exchanges such as nitrogen is also possible (Haynes, 1990). Specifically, ammonium uptake also occurs in concert with proton extrusion, and thus, results in acidification of the rhizosphere (Escobar et al., 2006). Regardless of the exact mechanism by which rhizosphere acidification is initiated, quantifying phenotypic variation in rhizosphere acidification capability could facilitate identification of selections capable of acquiring sufficient iron to promote healthy growth under calcareous soil conditions.
Given the quantitative nature of pH adaptability identified in other plants (Froechlich and Fehr, 1981; Gogorcena et al., 2001; Gonzalo et al., 2011), the use of image-based phenotyping methods could improve our ability to more quickly and more precisely identify selectable genetic variation for traits such as rhizosphere acidification that play a role in pH adaptability. Image-based phenotyping protocols are potentially well suited to ameliorating the problems (i.e., subjectivity, slow speed) inherent in phenotyping pH adaptability. Image-based screening approaches have been used in many other crops to improve quantification of challenging traits, including root architecture, water use efficiency, and nutrient deficiencies while maintaining yield goals (Berger et al., 2010; Clark et al., 2013; Shi et al., 2013). Systematic phenotyping protocols could then be used to identify superior genotypes within existing breeding germplasm or in wild populations to identify new sources of tolerance for elevated pH for deciduous azaleas.
To address the challenges of identifying pH adaptability in deciduous azaleas, we evaluated an in vitro phenotyping protocol to identify quantitative variation in pH change in the root rhizosphere. We focused our screening methods on in vitro plants in an effort to increase the efficiency of breeding elevated pH–tolerant plants. We tested seedlings from the same crosses in vitro using pH indicator dye to measure plant-induced changes to the culture medium pH in the presence of liming treatments. We also tested seedlings from the same families in the greenhouse to measure leaf area change in response to liming treatments. Leaf area was quantified using image analysis methods implemented in MATLAB (MathWorks, Inc., Natick, MA) and ImageJ (National Institutes of Health, Bethesda, MD) to detect any relationship between rhizosphere acidification in vitro and seedling size when progeny of the same family were grown in a greenhouse. Ultimately, we sought to identify seedling deciduous azaleas with improved pH adaptability for use as progenitors of future cultivars.
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