HORTSCIENCE 53(3):268–274. 2018. https://doi.org/10.21273/HORTSCI12520-17 attributes to cultivars released. These include pink flowers from wild-collected Rhododen- dron prinophyllum (Small) Millais and large, An Evaluation of Two Seedling durable, orange flowers from Rhododendron calendulaceum (Michx.) Torr. (Moe and Phenotyping Protocols to Assess Pellett, 1986; Widrlechner, 1982). Additional ornamental characteristics such as fragrance pH Adaptability in Deciduous from Rhododendron atlanticum (Ashe) Rheder and white flowers from Rhododen- Rhododendron dron viscosum (L.) Torr. were also intro- Azalea ( sect. gressed into the breeding germplasm Pentanthera (Hokanson, 2010). The combination of traits G. Don) from each of these species and their segrega- tion in seedling populations has enabled a di- Alexander Q. Susko verse series of Rhododendron cultivars to be Department of Horticultural Science, University of Minnesota, 1970 Folwell developed for northern areas of the United Avenue, St. Paul, MN 55108 States, where cultivation of the genus was historically thought to be impossible because Timothy A. Rinehart of extreme winter cold temperatures USDA-ARS Thad Cochran Southern Horticultural Research Laboratory, (Widrlechner, 1982). P.O. Box 287, Poplarville, MS 39470 The greatest systematic breeding effort has been focused on improving the cold James M. Bradeen hardiness of floral and vegetative tissues Department of Plant Pathology, The Stakman-Borlaug Center for (Moe and Pellett, 1986). Initial cold hardi- Sustainable Plant Health, 1991 Upper Buford Circle, St. Paul, MN 55108 ness evaluations required substantial screen- ing time in the field as the initial parental Stan C. Hokanson1 germplasm and progeny evaluations that led Department of Horticultural Science, University of Minnesota, 1970 Folwell to ‘Northern Lights’ spanned the years 1957– 79, a 22-year period. These initial efforts Avenue, St. Paul, MN 55108 resulted in a series of deciduous azalea Additional index words. iron deficiency chlorosis, high-throughput phenotyping, rhizosphere cultivars that are reliably cold-hardy in acidification, abiotic stress tolerance, Ericaceae USDA Zone 4 (Hokanson, 2010). As culti- vation of deciduous azaleas has spread Abstract. Deciduous azaleas are an important element of residential and commercial throughout the Midwestern United States, landscapes in the United States after substantial trait improvements to increase their tolerance to high pH or calcareous soils market appeal. Despite progress in breeding for ornamental characteristics and cold (calcium carbonate, CaCO3) that predomi- hardiness, intolerance to elevated pH and calcareous soils continues to limit their use in nate in the region has become the most managed landscapes. Therefore, we assessed the utility of in vitro and greenhouse pressing trait to improve. High-pH soils phenotyping approaches to evaluate and select for improved soil pH tolerance to increase severely limit deciduous azalea growth, pri- the efficiency of breeding for this important trait. The research presented offers an marily by restricting micronutrient availabil- example for implementing image-based phenotyping to expedite cultivar development in ity, such as iron, for uptake by roots (Brady woody ornamental crops. and Weil, 2004). Bicarbonate ions present in calcareous soils raise soil pH and keep the soil buffered, resulting in slower acidification Deciduous azalea (Rhododendron sect. Department of Agriculture (USDA) hardi- of soil, further limiting micronutrient avail- Pentanthera G. Don) cultivars and species ness Zone 4 northern landscapes (Hokanson, ability (Brady and Weil, 2004). This reduc- are highly ornamental shrubs that are valued 2010). With nearly 630,000 plants sold at tion in iron uptake on high-pH or calcareous in northern landscapes for their colorful and a combined wholesale value of $1.02 million soils leads to chlorosis and decreased photo- prolific flowers. One important group of de- between 2014 and 2016, these and other synthetic capacity, which over time reduces ciduous azalea cultivars is the Northern deciduous azalea cultivars represent a reliable vigor and increases mortality of deciduous Lights series, which since 1979 has source of revenue for woody plant growers azaleas in the landscape (Galle, 1974). Im- grown to 15 interspecific hybrids including who cater to landscape markets in cold proving adaptability to high-pH or calcareous the original ‘Northern Lights’, ‘Mandarin climates (L. Caton, Sidhu and Sons, personal soils (henceforth referred to as pH adaptabil- Lights’, and ‘Lemon Lights’ that bring vivid communication). Given the series’ popularity, ity) of cultivars would reduce plant mainte- pink, orange, and yellow colors into U.S. the University of Minnesota breeding program nance and open up new markets in continental continues to expand the series, with the first climates characterized by calcareous or high- true red ‘UMNAZ633’ Electric LightsTM Red pH soils. and double-flowered ‘UMNAZ493’ Electric Although some systematic selection Received for publication 26 Sept. 2017. Accepted TM for publication 21 Nov. 2017. Lights Double Pink introductions bringing against iron deficiency chlorosis has occurred We thank Alan Smith for permission to use tissue new ornamental characteristics to cold-hardy in evergreen Rhododendron, the variation is culture lab space and equipment. We also thank woody ornamental germplasm (Hokanson hard to phenotype consistently because of Elsa Eschenaur and Mitchal Peterson for their et al., 2015). confounding nutrient deficiencies and measure- assistance in subculturing and help with other Progenitors of the cultivars mentioned ment error using a qualitative (1–10) rating tissue culture lab techniques presented in this previously primarily resulted from crosses scale in breeding populations (Dunemann et al., research. Mention of trade names or commercial between North American deciduous azalea 1999; Preil and Ebbinghaus, 1994). Further- products in this publication is solely for the purpose species native to the southern and eastern more, prospects for improving pH adaptability of providing specific information and does not imply recommendation or endorsement by the United States with European deciduous aza- in deciduous azalea are slowed by long gener- U.S. Department of Agriculture. USDA is an equal lea hybrids (Mollis Azalea, Exbury, and ation times and limited timeframe (at leaf flush) opportunity provider and employer. Knapp Hill hybrids) from the early 20th for observing symptoms of high pH stress 1Corresponding author. E-mail: hokan017@umn. century (Moe and Pellett, 1986). Each of these such as iron deficiency chlorosis. Like many edu. native species contributes unique ornamental woody ornamentals, deciduous azaleas take 268 HORTSCIENCE VOL. 53(3) MARCH 2018 | BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES between 8 and 10 months from crossing to seed change in the root rhizosphere. We focused initial germination when seedlings were 1 germination, and an additional 2 years before our screening methods on in vitro plants in an cm in height. The screening medium com- plants reach maturity for field trials (Susko, effort to increase the efficiency of breeding prised half-strength Woody Plant Medium 2016). Growing plants in containers before elevated pH–tolerant plants. We tested seed- adjusted to pH 7.8 with sodium hydroxide planting in the field requires space and labor lings from the same crosses in vitro using pH and supplemented with 0, 310, or 610 mg·L–1 that is often a limiting factor in determining indicator dye to measure plant-induced CaCO3 in the medium to assess the effect how many seedlings can be produced for changes to the culture medium pH in the of lime concentration on rhizosphere pH screening each season. Therefore, selection at presence of liming treatments. We also tested changes. the early seedling stage could both reduce the seedlings from the same families in the To visualize pH change, the pH indicator number of plants carried forward and expedite greenhouse to measure leaf area change in phenolsulfonphthalein (phenol red) was in- breeding deciduous azaleas. response to liming treatments. Leaf area was corporated into the tissue culture medium at Rather than waiting for the manifestation quantified using image analysis methods a final concentration of 90 mM before auto- of chlorosis in whole plant experiments, implemented in MATLAB (MathWorks, claving. Ten milliliters of the previously characterization of the root–soil interface Inc., Natick, MA) and ImageJ (National In- described medium, including the phenol (rhizosphere), including localized reduction stitutes of Health, Bethesda, MD) to detect red, was poured into 100-mL Pyrex glass in pH (rhizosphere acidification), could more any relationship between rhizosphere acidi- tubes. Individual plants from each family quickly indicate whether certain genotypes fication in vitro and seedling size when were transferred into these tubes that were will successfully acquire micronutrients and progeny of the same family were grown in then sealed with plastic caps and parafilm mitigate symptoms of high pH stress a greenhouse. Ultimately, we sought to iden- (Bemis NA, Neenah, WI). The tubes were (Guerinot and Yi, 1994). Known as Strategy tify seedling deciduous azaleas with organized in a completely randomized design 1 iron acquisition, dicot plants extrude pro- improved pH adaptability for use as pro- under fluorescent lighting with a 16-h photo- tons into the rhizosphere to create