Carotenoid Concentration and Composition in Winter Squash: Variability Associated with Different Cultigens, Harvest Maturities, and Storage Times

In the present study, carotenoid concentrations and profiles of carotenoids were determined at harvest and during storage in fruit of several cultigens of winter squash, including several New Hampshire (NH) breeding lines, the cultivars Waltham Butternut and Puritan Butternut in Cucurbita moschata (Duch. and the hybrid cultivars Sunshine, Eclipse, Space Station, and Thunder in Cucurbita maxima (Duch. Cultigens were grown at research farms in either Madbury or Durham, NH, in 2007, 2008, and 2009, and fruit were harvested at either 40 or 60 days after pollination (DAP) and stored for 0, 30, or 60 days at 14 8C before sampling. Total carotenoid concentrations were determined spectrophotometrically and carotenoid profiles were determined using high performance liquid chromatography (HPLC) Carotenoid concentrations in fruit samples of C. maxima cultigens harvested at 60 DAP ranged from 146 to 320 mg*g fresh weight (FW) compared with concentrations of 274 to 623mg*g FW after storage for 60 days. Carotenoid concentrations were lower among C. moschata cultigens, ranging from 42 to 145 mg*g FW at 60 DAP, and from 84 to 239 mg*g FW after 60 day storage. The nutritionally important carotenoids, lutein, zeaxanthin, and b carotene, comprised 41% to 63% of the total carotenoid profile in theC. maxima kabocha hybrids; whereas, the carotenoids neoxanthin and flavoxanthin comprised 37% to 59% b Carotene and lutein were the major nutritionally beneficial carotenoids identified in 'Waltham Butternut' and three inbred lines of C. moschata, along with much smaller concentrations of a carotene. Neoxanthin and violaxanthin comprised between 14% and 29% of the total carotenoid profiles among the four cultigens analyzed. In the popular cultivar Waltham Butternut, the b carotene concentration was 17.3 mg*g FW at 60 DAP, but increased to a maximum of 57.8 mg*g FW after storage for 30 days; whereas, lutein concentrations increased from 27.3 mg*g FW at 60 DAP to 44.6 mg*g FW after 60 day storage. In Sunshine, a popular kabocha cultivar, b carotene increased from 26.0 mg*g FW at 60 DAP to 104 mg*g FW after 60 day storage. Lutein zeaxanthin concentrations in 'Sunshine' were 58.8 mg*g FW at 60 DAP, but increased only to 71.8 mg*g FW after 60 day storage. Total carotenoid concentrations and carotenoid profiles in 'Sunshine' over three growing seasons, and in 'Waltham Butternut' 'NH.Mo421' and 'NH.Mo851' over two growing seasons, were similar. Carotenoids are ubiquitous fat soluble, yellow orange and red pigmented compounds found in leaves as part of the chloroplast structure and in the chromoplasts ofmany root and fruit crops (Britton and Khachik, 2009) They are typically subdivided into two groups, hydrocarbons (carotenes) and themore polar xanthophylls or hydroxy containing carotenoids, the latter often esterified to fatty acids. Carotenes, chiefly a carotene and b carotene, and the xanthophyll, b cryptoxanthin, are precursors to vitamin A, which has a significant role in vision, in maintaining a healthy immune system, and in regulation of gene expression during growth and development (Britton, 2009) Two xanthophylls, lutein and zeaxanthin, are present in the retina of humans and subhuman primates, and necessary for visual acuity and as a filter for short wave light (Schalch et al. 2009) They are also two of the six most prominent carotenoids in human blood plasma and tissues (Canene Adams and Erdman, 2009) Other carotenoids found in plants, such as violaxanthin and neoxanthin, have an important function in photosynthesis and plant hormone physiology, but are largely absent in human plasma and organs, and have yet to be linked to a role in human health (Barua and Olson, 2001; Canene Adams and Erdman, 2009; Khachik, 2009; Milborrow, 2001) Mesocarp tissue of immature winter squash fruit is low in carotenoids, but carotenoid concentrations begin to progressively increase beginning 15 to 20 d after fruit set (Boiteux et al. 2007; Kon and Shimba, 1988) Variation in total carotenoid concentrations in mature squash among cultivars in the three major species of winter squash, Cucurbita pepo, C. maxima, and C. moschata, have been reported (Azevedo Meleiro and Rodriguez Amaya, 2007; Boiteux et al. 2007; Jacobo Valenzuela et al. 2011; Schaffer et al. 1986) as well as variation associated with harvest and storage times (Arima and Rodriguez Amaya, 1988; Arvayo Ortiz et al. 1994; Bycroft et al. 1999; Hopp et al. 1960) However, the diversity of germplasm analyzed is low, and in most reports, not representative of cultivars currently used in North America. Since the late 1980s, with the advancement of HPLC, the major carotenoids in the three major species of winter squash have been identified (Azevedo Meleiro and Rodriguez Amaya, 2007; Gonz alez et al. 2001; Jacobo Valenzuela et al. 2011; Khachik and Beecher, 1988) including nutritionally important carotenes, a carotene and b carotene, and the xanthophyll lutein. In two of the most recent studies involving North American cultivars, the complete carotenoid profiles were not analyzed (Itle and Kabelka, 2009; Murkovic et al. 2002) and in the latter study, samples were not saponified. In squash, saponification is considered an important analytical procedure for HPLC analysis to allow for separation and quantification of xanthophylls, which are often esterified with fatty acids (Khachik, 2009; Khachik and Beecher, 1988; Rodriguez Amaya, 2001) Carotenoids such as neoxanthin and violaxanthin may be abundant in winter squash (Azevedo Meleiro and Rodriguez Amaya, 2007) and for squash improvement through breeding, data on the proportion of these in the total carotenoid profile are important. There have been few studies on postharvest changes in carotenoid composition (Kon and Shimba, 1988; Zhang et al. 2014) but such changes could be important for assessing the nutritional status of winter squash. 472 HORTSCIENCE VOL. 51(5) MAY 2016 The objectives of this study were to determine the effects of year, differences in maturity at harvest, and storage time on total carotenoid concentrations and carotenoid profiles in fruit mesocarp tissue of several cultigens of C. maxima and C. moschata winter squash. In the present work, we included both a few popular commercial cultivars and also some NH breeding lines with diverse genetic parentage, most of which had been selected visually for intense orange pigmentation in fruit.  In C. moschata, fruits from four F5 (2007) and F6 (2008 and 2009) breeding lines and three related, openpollinated cultivars of butternut squash were used in this study. The open pollinated cultivars were Waltham Butternut (WBN) Puritan Butternut (PBN) and New Hampshire Baby Butternut (NHBBN) Seed of these cultivars were obtained from two generations of self pollinations to improve uniformity. NHBBN was derived from the original Butternut cultivar crossed to Tsurukubi, a Korean cultivar, to eliminate the problem of crooknecking in butternut squash (Mutschler and Pearson, 1987; Yeager and Meader, 1957) Waltham Butternut, a widely grown cultivar, was derived from a cross between NHBBN and an African C. moschata accession, and PBN was a selection out of WBN, but with slightly smaller fruit (Mutschler and Pearson, 1987) Four University of New Hampshire (UNH) breeding lines, NH.Mo421, NH.Mo851, NH.Mo910, and NH.Mo2521, were derived from a cross of 'Bugle' a butternut release from Cornell University with intermediate powdery mildew resistance, to a breeding line [NH.(PxB) 118 24 21] with oblate fruit shape. The breeding line was derived from an heirloom strain from Maine with oblate fruit, crossed to a breeding line derived from an open pollinated Peruvian accession with high carotenoid concentration. In C. maxima, four kabocha UNH breeding lines bred for intense flesh color, NH.Max33 12, NH.Max6331, NH264, and NH.Max5273, were evaluated for total carotenoid concentrations. Carotenoid profiles were examined in four commercial kabocha hybrids, 'Eclipse' 'Thunder' 'Space Station' (Rupp Seeds,Waseon,OH) and 'Sunshine' (Johnny's Selected Seeds, Albion, ME) Fruit of the NH kabocha hybrids are dark green at maturity and similar in appearance to the original Buttercup cultivar, but with small blossom scars andmore rounded shoulders. The parents of 'Eclipse' are a bush inbred line, NH.Max6331, with a complex pedigree, and NH.Max5273, a vining inbred line derived from the F1 hybrid Kurijiman ( The parents of 'Thunder' are NH.Max5273 and a bush line, NH.Max2 12 11, developed before 1995. NH.Max6331 is one of the parents of 'Space Station' the other being a semibush line, NH.81110 2, developed from a complex pedigree. Three of the cultigens, NH.Max33 12 (F7) NH.Max264 (F5) and 'Sunshine' have orange rinds and carry the B gene (Paris and Brown, 2005) for precocious orange pigmentation of the fruit. Plant culture. Plots were established at the Woodman Research Farm in Durham, NH (lat. 43 N) or at the nearby Kingman Research Farm in Madbury, NH, in 2007, 2008, and 2009, using randomized complete block designs with three (2007 and 2009) or four (2008) replications and eight plants per plot. In 2008, the C. moschata cultigens and C. maxima breeding lines were grown in adjacent plots, and the C. maxima hybrids were grown in a separate field, and included additional hybrids not used in the present study. Plants were started from seed in 50 plug trays with Promix media (Premier Tech Horticulture, Quakertown, PA) and transplanted at the two true leaf stage during the first week in June. Plants were grown on raised beds 15 cm high 60 cm wide) covered with 31.8 mm black polyethylene mulch, and with drip tape (203.2 mm T tape with 30 cm emitter spacing; T Systems International, San Diego, CA) laid at a depth of 3 cm slightly off the bed center. Bed centers were 2.1 m apart. Before bed formation, fertilizer was a