Half-Sib Selection for Higher Betalains Concentration and Lower Total Dissolved Solids in Table Beets (Beta Vulgaris)

AN ABSTRACT OF THE THESIS OF

Monzarath Hernandez for the degree of Master of Science in Horticulture presented on May 29, 2020.

Title: Half-Sib Selection for Higher Betalain Concentration and Lower Total Dissolved Solids in Table Beets (Beta vulgaris).

Abstract approved: ______James R. Myers

Betalains are a group of compounds that are major natural food colorants used by the food processing industry. These secondary compounds are found in only a few orders of plants with the Caryophyllales being the source of several domesticated crops. In particular, the family

Chenopodiaceae in general and table beets (Beta vulgaris) specifically are the primary source for betalains for commercial extraction. Table beets are preferred because of high pigment concentration in the enlarged root in a crop that is relatively easy to grow, harvest and process.

The primary betalains found in table beet are betacyanins (red pigments) and betaxanthins

(yellow pigments). The food colorant industry is mainly interested in the betacyanin betanin, but betanin content is highly correlated with betalains so that selection for total betalains will result in an increase in betanin. Beets are also an economic source of sugars (primarily sucrose), which resulted in the development of sugar beets that are unpigmented but have sugar content of more than 20%. Table beets with moderately high sugar content have better flavor for culinary processes, but high sugars reduce efficiency of the extraction process of betalains for food colorants. Sucrose content in table beet is highly correlated with total dissolved solids

(TDS), which can be easily measured with a refractometer whereas quantification of sucrose is more involved. Table beets with high betanin and low TDS are being sought by the food

concentrate industry. The objective of this study was to breed for high betalain pigment and low TDS in table beets. While quantification of betalains can be performed by UV-Vis spectrophotometry, the process is laborious and not suited to evaluating large numbers typically found in a breeding program. As a substitute for direct measurement of betalains, selection based on colorimeter measurements using the CIE L*a*b* color coordinate system was investigated. Parents were obtained from the USDA National Plant Germplasm System and from commercial sources. Twelve plant introduction accessions were chosen based on passport data in the GRIN database that indicated that they were pigmented and possessed low

(<5%) sucrose content. Also selected were commercial lines with strong and uniform pigmentation, but moderately high TDS content. Six rounds of half-sib mass selection were performed to select simultaneously for increased pigment and decreased TDS. The process involved growing plants in the field in a late summer – early fall trial, evaluating pigment and

TDS content of 10 roots per family and placing selected roots into cold storage (2°C) where they were vernalized for a minimum of 11-12 weeks. Roots were then transferred to the greenhouse in January and allowed to bolt, flower and random mate. Seeds were harvested in

May and June and used for the next cycle field trial. From the accessions and cultivars grown in the field in 2014 - 2019, samples of 10 roots from each accession were taken for pigment and TDS analysis. From 20 - 30% of the individuals with high pigment and low TDS were retained in each cycle. In 2019, remnant seed from previous cycles along with the current cycle

(cycle 5) were included in a replicated field trial so that all cycles could be evaluated in the same environment and heritability and gain from selection could be calculated. In conjunction with the colorimeter measurements, betalains were quantified in 2019 using UV-Vis spectrophotometer, and correlations between colorimeter and spectrophotometer data were

calculated. For betalains, family means for CIE L* had a steady decease throughout the years indicating selection for lower L* resulted in families with darker pigmentation. TDS family averages, on the other hand, showed essentially no change over cycles. When reviewing data across years, two factors that may have influenced TDS levels were year to year environmental variation and the fact that betalains are usually present as a glycoside such that any increase in the amount of betalains will be associated with additional glyosidic content. Colorimeter measurements were converted to Royal Horticultural Society colors and RGB (Red-Green-

Blue) color values. Over time, the half-sib population showed an increase in the purple-red and grey-purple groups. The population had a high narrow-sense heritability of 0.74, and also a relatively high change from selection of 0.75 per cycle for L*. TDS had low h2 and change from selection of 0.16 and 0.062% per cycle, respectively. In the case of the correlation of UV-

VIS spectrophotometer data with colorimeter measurements, evaluation of six lines in the 2019 population showed a strong negative relationship between colorimeter and spectrophotometer values. However, there was a weak regression coefficient (r2=-0.03) for L* vs. betanin among families of the entire 2019 population. Overall, selection based on L* resulted in transgressive segregation for betanin, with some families outperforming commercial cultivar. Thus, the colorimeter appears to have utility as a technique for rapid and high throughput betalain measurement.

Half-Sib Selection for Higher Betalains Concentration and Lower Total Dissolved Solids in Table Beets (Beta vulgaris).

by Monzarath Hernandez

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Presented May 29, 2020 Commencement June 2020

Master of Science thesis of Monzarath Hernandez presented on May 29, 2020

APPROVED:

Major Professor, representing Horticulture

Head of the Department of Horticulture

Dean of the Graduate School

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request.

Monzarath Hernandez, Author

ACKNOWLEDGEMENTS There is quite a large number of number of people who I am greatly appreciated for their assistance in the completion of my project. I owe my sincere appreciation to my principal investigator, Dr. Jim Myers for his creative insights, knowledge in the profession, and support during the grad school experience. I would like to show my gratitude Dr. Ryan Contreras, Dr. Yanyun Zhao for their guidance and assistance as thesis committee members. As well, Dr. Jean Hall was very sweet and kind to serve as a graduate council representative. Great thanks to Dr. Jennifer Kling with her assistance with data analysis for my research.

Special thanks to Joel Davis and Shinji Kawai for their support and assistance in my project. To all my lab mates, grad and undergrad students throughout my grad school experience thank you for being a helping hand out in the field or greenhouse, and person to lean on when school would become overwhelming. To my parents, sisters and friends, thank you for encouraging and supporting my decision to go back to graduate school to pursue my dreams.

In Deep Gratitude to Kerr Concentrates Inc. for funding this project. It was an honor to work on this project. Eugene Heuberger and Jose Guerrero, it was privilege working with you and learning from you about the food processing process for quantifying betalains from beet juice.

TABLE OF CONTENTS Chapter 1 ...... 1 Introduction ...... 1 Literature Review...... 4 Introduction ...... 4 Table Beets...... 4 TDS: Comparison of Sugar Beets and Table Beets ...... 8 Genes controlling Color ...... 9 Colorants, and their Market Value ...... 10 Colorimetry ...... 12 Objectives ...... 14 Hypothesis...... 14 Chapter 2: Selection for Lower L* Value and Lower Sugar Concentration in Table Beets ..... 15 Introduction ...... 15 Methods and Materials ...... 15 Production environments ...... 15 Germplasm ...... 15 Project initiation ...... 16 Spring: Production and harvest by half-sib family ...... 20 Summer: Field trials and evaluation ...... 21 Fall: Evaluations of CIE L*a*b* & total dissolved solids ...... 23 Winter: Greenhouse and selections ...... 25 Data Analysis ...... 27 Results ...... 29 Analysis of cycles of selection across years ...... 29 Analysis of all cycles grown in a single trial in 2019 ...... 35 Discussion and Conclusion ...... 48 Chapter 3 Correlation between Colorimeter and UV-Vis Spectrophotometer ...... 53 Introduction ...... 53 Materials and Methods ...... 53 Base population ...... 53 Cycle 5 analysis ...... 54 Betalain calculation ...... 55

Data Analysis ...... 56 Results ...... 57 Discussion and Conclusion ...... 64 Chapter 4 ...... 68 General Conclusion ...... 68 References ...... 72 Appendix A Colorimeter L* value Data on Half-Sib Families and Checks ...... 76

LIST OF FIGURES Figure Page

1.1. Zoning is the alternating colors of dark red and white in the concentric rings in Table Beets. The Image shown is the variety ‘Chioggia’ with labeled rings………..……….....……7

1.2. Table beet 'Pablo' (left) and sugar beet (right) showing differences in root shape...... 9 1.3. R and Y loci determine the pigmentation in Table Beets. Recessive r and Dominant Y produce yellow beets (Left) and Dominant R and Y loci produce red beets (Right)………………………………………………………………………………...... 10

2.1. The seed to seed method used to conduct half-sib mass selection of table beets bred for increased pigmentation and low total dissolved solids at Oregon State University Method . ……….………………...…………………………………………………………..19

2.2 Two beet roots cut in cross-section to show strong (left) and minimal zoning (right).……………………………………………………………………….………………..24

2.3. L*a*b* and TDS (% brix) from cycles 1 – 5 (2015 – 2019) of half-sib mass selection in table beets. F statistic and probabilities are for the hypothesis that cycles are not significantly different. a) Box and whisker plot for L*; b) Box and whisker plot for a*; c) Box and whisker plot for b*; and d) Box and whisker plot for TDS…………………………………………..34

2.4. L*a*b* and total dissolved solids for a table beet half-sib mass selection population with all populations grown in the field at the OSU Vegetable Research Farm in 2019. a) L*. b) a*. c) b*. d) Percent TDS…………………...... 38

2.5. Three-dimensional plots of root colors for a table beet population selected over five cycles of half-sib mass selection. L*, a* and b* are plotted on the y, x and z axes, respectively. Dots represent individual families and are shown with equivalent RGB colors. a) Cycle 0 b) Cycle 1 (2015); c) Cycle 2 (2016); d) Cycle 3 (2017); e) Cycle 4 (2018); f) Cycle 5 (2019)………………………………………………………………………………………..43

3.1. Absorbance across wavelengths from 400 – 600 nm for four table beet samples with different L* values.……………………………………………………………………..…….56

3.2. Four selected samples of table beet extract after dilution (left). Samples with lighter shade of color had a lower betanin absorbance as quantified by UV-Vis spectrophotometry as shown in the table to the right……………………………………………………………57

LIST OF FIGURES CONTINUED Figure Page

3.3. Regression of L* and betanin mg/ 100g FW of a table beet population selected over five cycles for increased betanin content. Data are means of two replicates from cycles combined with the population grown in the field at the OSU Vegetable Research farm in 2019……………………………………………………………………………….……..…. 62

3.4. Eight table beet lines with CIE L* values of 19.5 or lower and TDS of 12% shown with red labels. The mean betanin content of these lines was 0.358%. Two commercial cultivars used as checks are labeled purple.……………………………………………………….….63

LIST OF TABLES Table Page

2.1 List of the 11 accessions from NGPS, 8 commercial cultivars, and checks grown at the OSU Vegetable Farm……………………………………………………………………..…17

2.2. Number of families, number of roots and selection intensities applied during cycles of selection of a table beet population for selection of increased root pigment and lower total dissolved solids……………………………………………………………………………...22

2.3. Analysis of variance for L*a*b* and color parameters and total dissolved solids for a table beet population across cycles in the individual field trials of half-sib mass selection……………...…………………….……………………………………………...... 30

2.4 Two-tail T-test assuming unequal variances comparing means for L* and TDS between cycles of selection for a table beet population being bred for high betalain and low TDS content……………………………………………………………………………………....32

2.5. Linear regression equations and regression coefficients for L*, a*, b* and TDS across cycles (single year field trials) of a table beet population grown at the OSU Vegetable Research Farm.……..…………………………………………………………….……….....35

2.6. Analysis of variance for L*a*b*color parameters and total dissolved solids for a table beet population subjected to five cycles of half-sib mass selection; all cycles grown in a single trial in 2019. …………..………………………………………………………………..…….…..36

2.7. Linear regression equations and R2 for L*a*b* and TDS regressed on cycles of a table beet population grown in 2019 in a lattice design field trial at the OSU Vegetable Research Farm …………………………………………………………………………………..……..38

2.8. Single factor analysis for the 2019 field trial of a table beet population subjected to half-sib mass selection over five cycles of selection at the OSU Vegetable Research Farm. Shown are variances and heritability’s for L* and percent total dissolved solids. …………………………….…………………………………….……...…..…………39

2.9. Gain from selection for total dissolved solids and L* in a table beet half-sib population selected over five years……………………………………………………………………….39

2.10. The Royal Horticultural Society (RHS) and RGB color values measured in 2014 for the base population of table beet germplasm, consisting of 11 USDA-NPGS accessions and eight commercially available cultivars. ………………………..…………………………………..41

LIST OF TABLES CONTINUED Table Page

2.11. The RHS fans, groups and values, and corresponding RGB colors for a table beet population consisting of five cycles (corresponding to years 2015 – 2019) evaluated at the OSU Vegetable Research Farm in 2019. Cells for percent of roots in a particular color group are colored to visualize shifts in groups over cycles of selection. Colors range from blue (0%) through white to red (67.4%)..………………………………………….…………………….41

2.12. Analysis of variance comparing half-sib families and five commercial table beet cultivars for CIE L*a*b* and total dissolved solids (TDS). Data from a trial conducted at the OSU Vegetable Research Farm in 2019. …………………………………...... ……44

2.13. T test: Two _Sample Assuming Equal Variances on set of families and Checks on their Total dissolved Solids. …………………………………………………………...... 45

2.14. Half-sib table beet families showing root color parameters and total dissolved solids (TDS), with percent TDS of 9 or lower. Data from families and check cultivars grown in the field at the OSU Vegetable Research Farm in 2019…………………………………..….....46

3.1. Mean square and probability for Betanin and E value for Reps 1 and 2. Both had significant differences between each rep…………………………………………………....58

3.2. Mean square and probability for Betanin and E value from an ANOVA of a table beet family selected for high betanin …………...... 59

3.3 Pearson correlation coefficients for CIE L*a*b*, hue angle, chroma and betanin mg/ 100 FW from a table beet population selected for high betanin and low TDS. Data is from five cycles grown in the field at the OSU Vegetable Research farm in 2019……………………………61

3.4 Table 3.4 Pearson correlation coefficients for CIE L*a*b*, hue angle, chroma and betanin mg/ 100 FW from a table beet population selected for high betanin and low TDS for the initial germplasm…………………………………………………………………………………....61

LIST OF APPENDIX TABLES

Table Page

A.1 Colorimeter L* value Data on Half-Sib Families and Checks ……..………….75

DEDICATION

To My Father, You would work 10-12 hours a day to provide our family with the very best. You would sacrifice vacation time to make sure my sisters and I had all the best to succeed in school. Both mom and You did everything to see us thrive, you wanted us to prevail in life. You would help me pot up sunflowers, catch ladybugs, or explain all about crops you were working with, you showed the beauty and wonders of agriculture. I am dedicating you to my thesis because you made this happen. Your admiration for Agriculture was enchanting and to your perseverance to succeed in life; I would not here if It wasn’t for you. I not only share half your DNA but your love for Agriculture.

Love you, Your Daughter

Chapter 1 Introduction Have you ever gone to the grocery store and walked through the aisles and come across red-colored tortilla chips, red velvet cake, or pink cereal? One thing you may not know, that all these items have in common, is the use of natural food colorants to provide these red to pink colors. There are currently two classes of secondary plant compounds primarily used for food colorants in the food processing industry: betalains, and anthocyanins. Both are high valued natural colorants, but anthocyanins seem to get all the attention, probably due to it being widely distributed in the plant kingdom, thus making it more accessible. Betalains are only found in plants of the order Caryophyllales, which reduces accessibility to just a few cultivated plant species. Similar to anthocyanins, betalains provide pigmentation to the plant and other beneficial attributes. Betalains have stability over a wide pH range, which has made it the pigment of choice for the food industry today, in which they are widely used as natural dyes for dairy, confectionery, and meat products (Azeredo, 2009). Overall, betalain stability provides greater advantages compared to less pH-stable anthocyanins for use by the food industry.

Table beets and Swiss chard (Beta vulgaris L.) are known for their dark red or orange pigmentation and their earthy taste. The magnificent colors that table beets possess are due to the betalains betacyanin for pink to red colors and betaxanthin for yellow to orange colors.

Table beets were originally grown for fresh market or processed food consumption, but more recently, extracts have found popularity as a natural food colorant (Goldman and Navazio,

2008). Domesticated in Europe, table beets were introduced to the United States in the seventeenth century. Original introductions were open-pollinated varieties and it wasn’t until

the second half of the 20th century that table beets were formally bred, using techniques such as mass selection and inbreeding to produce F1 hybrids (Goldman and Navazio, 2003).

Some characters have always been of interest to plant breeders of table beets, such as pigmentation, early or late bolting and sugar concentration. Sugar beets are closely related to table beets and were derived from them in the 18th century to provide a source of sugar when access to sugar cane from the tropics was cut off by British blockades of the French. Sugar beet roots lack pigment but have been bred for sucrose amounts exceeding 20% (table beets have around 10% sugars) (McGrath, and Panella 2019). Pigmentation and sugar concentration are important factors in culinary quality of table beets for consumption and for betalain extraction for the food processing industry. Wolyn and Gabelman (1990a) were able to increase the concentration of betalains in a population comprised of six U.S. and European cultivars by

45% in three cycles by recurrent mass selection. Goldman et al. (1996) reported that after eight cycles of selection, total betalain content had increased up to 20% and had not yet plateaued.

Three terms are often used in reference to sugar content in fruits and vegetables. These are brix, soluble solids and total dissolved solids. Brix is measured by light refraction of a solution, which is directly related to its soluble or total dissolved solids (TDS) content. Brix is specifically calibrated to measure sugar content, but when other compounds are present, the measurement of sugars will not be totally accurate and it is a measure of soluble or total dissolved solids. Total dissolved solids may contain organic or inorganic substances, such as sucrose, salts, metals, cation and anion dissolved in water (Bauder and Sigler 2005). The amount of TDS contained by beets has been of interest to breeders for culinary purposes. TDS in beets are composed of about 75% sucrose with the remainder being other sugars such as glucose and fructose as well as organic acids. As such, brix gives a relatively accurate estimate

of sugar content in table beets. Lower TDS allows for easier extraction of betalains, even though high TDS improves flavor and quality. Selection simultaneously for higher betalains and lower TDS can potentially provide a cultivar that is highly desirable for the food industry as a natural food colorant and quite possibly out-compete any cultivar currently in the market.

Literature Review Introduction In this section, I provide a background on table beets as a crop including origins, botanical relationships, production and economic value. I also detail what is known about betalains, their chemical structure, and importance to the food industry. Subsequently, I describe the tools used for quantifying betalains in beet including the colorimeter and spectrophotometry, the latter of which is commercially used in the food industry today.

Table Beets Table beet, (Beta vulgaris subsp. vulgaris L.), is a root crop within the Chenopodiaceae family. Beets are in a core eudicot clade in the order Caryophyllales, and are often found in marginal and stressful environments, especially those with saline soils of littoral zones near oceans (Angiosperm Phylogeny Group, 1998, 2003; Stevens, 2001).

The Chenopodiaceae family contains 34 genera and over 1,300 species; the family is also known as the Goosefoot family (Eckardt 2006). The complex family varies from crops used globally for sucrose production, food processing, and fresh market production, however, the family also contains plant species considered noxious weeds. The crops widely used for human consumption consist of leafy greens and petioles (leaf beet, spinach and Swiss chard), and root crops (table beet, mangel, and sugar beet) (Goldman and Navazio 2003). The leafy form was domesticated in the western Mediterranean and the Canary Islands and introduced to the Americas during the 17th century. It is thought that when the wild B. vulgaris subsp. maritima leafy progenitor was taken to Northern Europe, the cultivated form was selected for swollen roots that resulted in the modern table beet.

B. vulgaris is a diploid (2n = 18) with perfect flowers, however it is predominately out- crossed via wind pollination (Ford-Lloyd 1995; Poole 1937). Table beets will readily cross

with all forms of B. vulgaris, such as Swiss chard, sugar beet and mangel or fodder beet. In

Oregon, a three – mile isolation is required in seed production to prevent B. vulgaris species from cross pollinating with one another. Beets are usually self-incompatible, hence, individual plants will not self-pollinate and produce seed. While plants need to cross with a genetically different plant to produce viable seed, there has been instances of self-pollination, which have been used for hybrid breeding programs (Goldman and Navazio, 2003). Flower production in

B. vulgaris requires vernalization (chilling period) to promote bolting and flowering. To initiate flowering, beets require around 8 - 11 weeks of vernalization at temperatures below

50°F (10°C) followed by warming temperatures and the daylight lengthening during the spring.

B. vulgaris is biennial; the first year the plant grows an enlarged fleshy root, and the following year after vernalization the plant will bolt and produce seed stalks. The seed stalk can grow as tall as 60 - 120 cm (2 – 4ft.) (Benjamin et al., 1997). Beet flowers contain no petals and are organized into racemes. There are two different forms of flowering habit: two to five flower clusters that encircles an extended bract form multigerm seed, while flowers borne in axillary bracts produce monogerm seeds (Navazio, 2012). Multigerm plants are more highly desirable to growers for the high seed vigor they provide compared to monogerm types, but monogerm seed allows precision spacing at planting and does not require hand thinning to establish desired plant populations.

Beets are a cool season crop that prefer cool, wet springs and relatively dry summer days with cool nights and moderate daytime temperature between 16 - 22°C (60 - 70°F)

(Goldman and Navazio 2003). Table beets are very sensitive to heat and recommendations are to be grown where summer temperatures do not exceed 24°C (75°F). Beets are best grown in a fairly deep, well drained sandy loam, but roots can be replanted in heavier clay soil for seed

production (OSA, 2018). They are sensitive to boron deficiency, which can cause crown rot, blackening of leaf margins, cankers on the exterior of the root or heart rot within the roots.

Table beets are grown worldwide for both fresh market and processing and should not be confused with sugar beets that are only grown for processing. Table beets are mainly produced in the northern United States (USDA, 2019). Production in the United States is generally low with around 3,200 ha per year (Anon, 2000). For commercial production, beets are harvested around 5.0 – 6.5cm (2 to 2½ in.) in diameter. Roots smaller than this size have relatively low yield and roots larger than optimal have reduced quality. Commercially, roots that have a spherical shape are preferred although some cultivars have a cylindrical root shape.

The swollen taproot is caused by supernumerary cambiums, which form early in plant development. Before the supernumerary cambiums develop, a cylindrical vascular cambium forms between the primary xylem and phloem. Vascular cambium then gives rise to the supernumerary cambiums. The cambiums are visible as concentric rings in the swollen tap root. It is currently unknown what triggers the development of supernumerary cambiums, but plant hormones apparently play a role in its development (Benjamin et al., 1997). Following the development of the vascular cambiums, the root expands through cell division, and storage of carbohydrates begins. Hole et al. (1984) suggested that the storage of carbohydrates and the formation of a swollen taproot is independent of the formation of vascular cambiums.

Both table beets and sugar beets contain sucrose A B as their primary storage carbohydrate. Sucrose C concentration (measured as total dissolved solids or D TDS) is largely determined by the ratio between large

and small cells that compromise the parenchymatous

and vascular zones, respectively of each ring. Both Figure 1.1. Cross section of ‘Chioggia’ table beet root with sucrose and betalains accumulate in the vacuoles of the labeled rings. A. Phelloderm, B. Vascular bundle C. Parenchyma parenchyma cells (McGrath and Panella, 2019). tissue, D. Inner cambial core Through breeding and selection, sugar concentrations (Thajeel and Abbass 2019). are significantly higher in sugar beets compared to table beets (Benjamin et al., 1997).

When the vascular cambiums show different pigmentation from intervening storage parenchyma layers, the phenomenon is called zoning. Zoning in table beets is defined as concentric rings with different colors ranging from dark to light color in the cross-section of the root (Figure 1.1). Zoning is undesirable in commercial table beets, but it can be considered an attribute in some fresh market cultivars such as ‘Chioggia’ and others selected for their aesthetic appearance that show concentric rings alternating between dark red and white colors

(Schrader and Mayberry, 2003). Zoning has a genetic component as evidenced by cultivars such as Chioggia but the phenomenon also shows environmental plasticity with zoning more apparent when beets are grown at high temperatures or outside of their region of adaptation. To obtain roots uniformly colored throughout, beets are recommended to be grown in climates with warm days 16 to 22 o C (60-72 oF) and cool nights of 4 to 12 (40 to 55oF) (Navazio, 2012).

The storage parenchyma zones that are light in color are assumed to have minimal, if any betalains (Schrader and Mayberry, 2003), and therefore would decrease the overall betalain content of the root. For processors extracting beet pigments, selection for minimal or no zoning is criteria to maximize the amount of betalains within a beet root.

TDS: Comparison of Sugar Beets and Table Beets Table beets and sugar beets are the same species, but they vary in horticultural characteristics, and end use. Sugar beets are mainly grown for high sugar concentration, and they provide around 45% of world’s sugar (McGrath and Panella, 2019). The table beet root has a strong yellow to orange to red pigmentation and moderate levels of TDS. Betalains were selected against in sugar beets because these compounds increase viscosity and slow crystallization of sucrose (McGrath and Panella, 2019). Red beets range from three to 12%

TDS, while sugar beets have been found to more than 20% TDS; ~ 75% TDS as sucrose

(Hoffmann et al., 2005; Schiweck et al., 2007; Hoffmann, 2010). For culinary purposes, table beets with TDS in the higher end of the range are preferred whereas for pigment extraction, table beets are preferred to have low TDS because lower TDS allows for more efficient extraction of betalains. Table and sugar beets also differ in root shape and the size at which they are used. Sugar beet roots are harvested when as large as possible to maximize TDS whereas culinary quality decreases in roots greater than about 6.5 cm. Table beet roots are generally spherical or cylindrical in shape with small taproot and neck whereas sugar beets have a conical shape with large taproot and neck.(Figure 1.2)

Courtesy Jim Myers

Figure 1.2. Table beet 'Pablo' (left) and sugar beet (right) showing differences in root shape.

Genes controlling Color Betalain pigmentation in table beets is controlled by the linked R and Y loci (Keller,

1936). Three alleles at the R locus determine the ratio of betacyanin to betaxanthin in the table beet roots and leaves (Wolyn and Gabelman, 1990b). Pigment ratio is controlled by incomplete dominance in the Rt and R genotypes, and the color patterning is primarily affected by the R and Y locus. (Figure 1.3) Dark red color in table beets is a function of both dominant R and Y loci being present whereas R y genotypes have pink-purple roots (Figure 1.1). The yellow colored beets are produced by genotype r Y (Wolyn and Gabelman, 1990b). The Bl gene conditions a blotchy phenotype, and it assumed that the gene may disrupt pigmentation

synthesis (Watson and Goldman, 1997). All of these three simply inherited genes are important for pigmentation development in beets. However, pigmentation and the quantity of betalain pigment biosynthesis is believed to be controlled by an additional loci. The betalain concentration behaves like a quantitative trait in that it is affected by environment, exhibits continuous phenotypic variation and cannot be explained entirely by the segregation of one or two genes (Watson and Gabelman 1984).

Figure 1.3. R and Y loci determine the pigmentation in Table Beets. Recessive r and Dominant Y produce yellow beets (Left) and Dominant R and Y loci produce red beets (Right).

Colorants, and their Market Value Table beets are primarily grown for fresh and processed consumption but natural colorants are a growing segment of the market. Red beet extract is the sole source of betalains used as natural food colorants (Rodriguez-Amaya, 2016). The food colorant global market value was estimated at $1.79 billion dollars in 2016, with a growth rate of 5.9% (Grand View

Research, 2018). Natural food colorants are used to either restore color of food products that was lost to leaching during food processing or to add novel colors to attract consumers.

Colorants are used for a variety of products in the market, such as cereal, medicines, yogurt,

soda, cakes, and icing. Betalains are the only FDA-approved color extracted from beetroot to be used as a food colorant (FDA, 2019).

Betalains are nitrogenous pigments that produce two alkaloids groups, (betacyanins red purple) and betaxanthins (yellow orange); these compounds differ slightly in their chemical structure (Azeredo, 2009; Esquivel, 2016, Poltural & Aharoni, 2018; Rodriguez- Amaya,

2016). Betalains are derived from tyrosine via betalamic acid, and are a 1,7 diazaheptamethin chromophore with different sugar types attached. Betalamic acid is cleaved from L-DOPA between the 4- and 5- positions (Fischer and Dreiding, 1972; Impelizzeri and Piatelli, 1972;

Clement et al. 1994). Various sugars molecules may be attached to different points on the aromatic nucleus. The typical sugars found in betalain are glucose, sophrose, or glucuronosyl- glucose positioned at the C5- or C6- site of the betalain molecule. Glycosylation typically occurs before and after the condensation reaction (Sciuto et al. 1972).

Brockington et al. (2015) performed phylogenetic analyses that suggest a single origin of betalains, early in the evolution of the Caryophyllales, and at least two subsequent independent losses of betalains in conjunction with reversion to anthocyanins. Anthocyanins and betalains appear to be mutually exclusive, and have not been found in the same plant

(Kimer et al. 1970; Stafford 1994). Both anthocyanins and betalains are synthesized in the cytosol and are transported to the vacuoles in plant cells. Localization of the compound in plant organs is similar between the two pigments. For instance, fruits, leaves, stems, roots and flowers are all places where these compounds can be found (Delgado-Vargas et al., 2000).

Also, these compounds are both water soluble and can be easily leached or extracted during processing. Betalains are heat- and light-sensitive and the compound will be begin to degrade when exposed (Herbach et al., 2016). Betalains are more desirable than anthocyanins due to

higher pH stability across a range of 3 to 7, and have greater solubility in water than anthocyanins (Stinzing and Carle 2007). Also, anthocyanins at a pH over 4 will begin to lose their color (Stinzing and Carle 2007).

Colorimetry To quantify the amount of betalains in a crop, tools such as colorimeters and spectrophotometers are used by food processing industry. A spectrophotometer measures the amount of received light for each wavelength in the visible light range. UV-Vis spectrophotometers extend the range into the UV spectrum and measure the maximum light absorption of a substance in nanometers (Bauman 1962). The majority of food processing companies use UV-Vis spectrophotometers, which can be used to determine the concentrations of both betacyanin and betaxanthin. The maximum absorption for betaxanthin and betacyanin concentration are measured at 476 nm and 538 nm, respectively. To measure the concentrations of these compounds, they needed to be calculated at an absorption value of 1% or A1% (Von Elbe 1972). Absorption at 600nm is obtained to correct for any impurities in the solution. The absorptivity value is the extinction coefficient representing a 1% solution (1.0 g/100 ml) and is 1120 for betacyanin and 750 for betaxanthin (Wyler and Dreiding, 1957;

Piattelli and Minale, 1964). UV-Vis spectrophotometers require the substance to be in liquid form to measure the absorption level. The use of UV-Vis spectrophotometry is preferred for the food industry because of its simplicity and speed compared to chromatographic procedures.

Extraction of betalains from the beet juice is easy due to its water solubility.

Ideally, higher betalain concentration in table beets should be selected using the UV-

Vis spectrophotometer method because it directly measures concentration of betalains.

However, UV-Vis spectrophotometry is not a feasible tool for the breeding process where many samples have to be processed in a short amount of time. Beet roots need to be liquefied

and betalain compound is light and heat intolerant making it a labor-intensive and time- sensitive process. Stintzing et al. (2008) discovered that there is a significant correlation between CIE L* and betaxanthin/betacyanin content. They were able to predict color blends of betanin/betacyanin from yellow and red beets based on their CIE L* and chroma value at pH 4.

A potentially better way to select for higher betalain concentration would be to use a colorimeter with a CIE LAB scale. The International commission on Illumination (CIE) 1976 developed a way to express color numerically by quantifying light and color saturation. CIE

LAB parameters consist of CIE L* a*, b* values, these parameters can help determine, lightness, hue angle and saturation of color of a solid. CIE LAB color space is constructed as a three-dimensional color solid; lightness increases or decreases along the vertical axis and saturation of color across the central point. CIE L* value indicates lightness, from 0 (black) to

100 (white), and a* and b* indicate the color directions. Both a* and b* values can either be positive or negative, +a* is the red direction, -a* is the green direction, +b* is the yellow direction, and -b* is the blue direction. The a* and b* values are not independent values, and are used to calculate chroma and hue angle. Saturation of color increases as the absolute value of the a* and b* values increases (CIE 1976). Saturation is determined by the equation ((a*)2 +

(b*)2), which is classified as chroma. Hue angles are calculated as the arctan(b*/a*) and represents the color tone of the substance. Hue angle expresses color tone between the hypotenuse and 0o on the a* axis.

The use of CIE LAB colorimeter is not a common practice at present in the food industry, however the colorimeter tool would allow for faster evaluation of betalains. Selection for lower CIE L* should increase the darkness of the red table beets and be related increasing betalain content in the progeny.

Objectives The objective of this research was to breed table beets for high betalains and low TDS using half-sib mass selection. Table beets were subjected to six cycles of recurrent half-sib mass selection while selecting for darker pigmentation and lower sugar concentrations using a colorimeter to measure pigment intensity and a refractometer to determine TDS. Our goal was to lower TDS by 3-4% in the open-pollinated half-sib family population, and to increase betalain pigment as much as possible. Heritability and rate of gain for betalains (CIE L*) and

TDS were calculated for five cycles of selection. In addition the 2019 population was quantified by both colorimetric measurements and UV-Vis spectrophotometer. Correlation between CIE L*a*b* and UV-vis spectrophotometer is also reported for betalains content.

Hypothesis H0: Simultaneous selection for high betalains and low TDS would not result in any changes in these parameters over cycles of selection. Alternative hypothesis is that there would be changes, with the possibilities being both traits trended in the direction of selection, or only one trait trended in the direction of selection while the other showed no difference. The second experiment null hypothesis would be that there would be no association between pigment intensity as measured by the colorimeter and betalains content as measured by UV-Vis spectrophotometry. The alternate hypothesis would be that the two measurements are correlated.

Chapter 2: Selection for Lower L* Value and Lower Sugar Concentration in Table Beets Introduction Betalains are primarily used in the food industry as natural food colorants, and table beets

(Beta vulgaris) are the predominant source of betalains. Table beets provide a readily available source because the pigment is concentrated in the enlarged root (Rodriguez-Amaya 2016).

Spectrophotometry is commonly used to determine content of betalains in the food processing industry, however, measuring betalains using UV-Vis spectrophotometry is not feasible for large numbers of beets in breeding populations. For this project, colorimeter color data was used to estimate content of betalains. Sugars in beet roots interfere with betalains extraction and a refractometer was used to quantify sugar levels. An open-pollinated population of beet accessions were assembled, with some possessing relatively high pigment amounts and normal sugar concentrations and others with moderate pigment amounts and low sugar levels. Half-sib mass selection was practiced for increased betalains and lower sugar concentration in this population.

Methods and Materials Production environments Field work was conducted at the OSU Vegetable Research Farm located on the upper bench of the Willamette River (latitude N44.571209, longitude W123.243261) at 77 masl. Soil on the farm is a Chehalis silty clay loam soil with pH levels ranging from 5.5- 6.7. Greenhouse activities took place on the OSU campus in a 67 m2 room in the West Greenhouses. The room temperature was maintained at 18-25oC (64-77°F) during the day and 13.8-17.2oC (57-63oF) at night.

Germplasm In 2014, OSU Vegetable Breeding program obtained seed of 11 beet accessions from the USDA National Plant Germplasm System collection maintained at the Western Regional 15

Plant Introduction Station in Pullman, WA (Table 2.1). According to data in the Germplasm

Information Resource Network (GRIN) these accessions met the criteria of pigmented roots and had low (<5%) sucrose content (Table 2.1). Along with the 11 NGPS accessions, seven commercial cultivars with high pigmentation and normal TDS amounts were selected. These were grown at the OSU Vegetable Research Farm to create the base population. The germplasm for the base population remained the same until cycle 5, when the commercial hybrid ‘Akela’ was added to the population because of its low L* value. To easily identify the families retained over time, accessions and cultivars were assigned a “TB” number related to their maternal parent and location in the field during cycle 2. The sequential number represent the number of the root throughout the remaining cycles.

Project initiation We conducted five cycles of half-sib mass selection starting in summer of 2014 and continuing to spring of 2020. The seed to seed method (Navazio 2012) was used complete a single cycle in one year (Figure 2.1). Table beets are normally biennial, requiring more than a year for the crop to reach the reproductive stage and produce seed. To hasten this process, selected beet roots from trials planted mid-summer at the OSU Vegetable Research Farm were dug in the fall and were vernalized for 10 - 12 weeks at 5°C. In January, the roots were moved to greenhouse and were potted and allowed to grow. Plants were grown in an enclosed greenhouse where the wind-borne pollen could circulate in the room and would facilitate intermating. Seeds were harvested in late spring/early summer and planted out in the field again in mid-summer.

Table 2.1. List of the 11 table beet accessions from USDA-National Plant Germplasm System (NPGS), seven commercial cultivars used to create the population, and five checks grown at the OSU Vegetable Farm.

Stem color Root color 1-9 1-9 TB1 Lines Accession Name Source Description scale Description2 scale3 Root shape TB1-5 Kamayu Tozer Dk2 red w/ stripes 7 8 Red 8 Spherical TB6-8 Pablo Bejo Dk2 red 7 8 Red 8 Spherical TB9 Vulture Logan Zenner Dk2 red 8 8 Red 8 Oblong-cylindrical NPGS- TB26-47 Okrugla PI357357 Dk2 red 9 Dk red 9 Oblate NPGS- TB45-95 IDBBNR4621 PI414934 Red w/ white stripes 5 Red 7 Spherical NPGS- TB96-112 IDBBNR5250 PI164810 Dk2 red w/ stripes 7 Red 8 Slight oblate TB113- NPGS- Dk2 red w/ light Oblate w/thick 131 Choghundar PI164805 stripes 7 Red 7 taproot TB132- NPGS- Slight oblate w/ 147 Choghundur PI163182 Red w/ light strips 8 Red 7 thick taproot TB150- NPGS- Dk2 red w/ light Oblate w/ thick 155 IDBBNR5214 PI141919 stripes 7 Red 7 taproot NPGS- Dk red TB200 Avon Early (red) PI323938 Red w/ white stripes 5 orange 5 Thick taproot NPGS- Green-pink w/ red TB201 Avon Early (white) PI323938 stripes 8 White 1 Thick taproot NPGS- TB202 Good For All Rikssort PI269309 Red w/ stripes 6 Red 8 slight oblate NPGS- Dk2 red, slight TB203 Koransko (red) PI357354 striping 7 Red 7 Pointed conical NPGS- White- TB204 Koransko (colored) PI357354 Pale pink-red-orange 3 orange 3 Pointed conical NPGS- TB205 Koransko (white) PI357354 Green-pale pink 2 White 1 Pointed conical

Table 2.1. (Continued). Stem color Root color 1-9 1-9 TB Lines Accession Name Source Description scale3 Description2 scale3 Root shape TB206 Red Titan Logan Zenner Dk2 red w/ stripes 7 7 Red 8 Spherical TB207 Red Ace Chriseed Dk2 red w/ stripes 7 8 Red 8 Spherical Dk2 red w/ light TB208 Merlin Chriseed stripes 7 8 Red 7 Spherical Dk2 red w/ light TB209 Dark Red Detroit Chriseed stripes 7 9 Red 8 Spherical TBcheck1 Pablo Bejo Dk2 red 7 8 Red 8 Spherical TBcheck2 Taunus Bejo Dk2 red 8 8 Red 8 Oblong-cylindrical TBcheck3 Akela Osborne Dk2 red 8 red 8 Spherical TBcheck4 Boro Bejo Dk2 red 7 8 Red 8 Spherical Dk2 red w/ light TBcheck5 Dark Red Detroit Chriseed stripes 7 8 Red 7 Spherical 1 TB=Table beets 2Dk=dark red pigment. 3Color scale: Table Beets were rated from 1 to 9 depending on their saturation of color, with 1 being white to 9 being violet-purple:

1 2 3 4 5 6 7 8 9

Year 2014 2015 2016 2017 2018 2019 2020z Cycle C0 C1 C2 C3 C4 C5 C6

Season all

Spring Summer F Winter Spring Summer Fall Winter Spring Summer Fall Winter Spring Summer Fall Winter Spring Summer Fall Winter Spring Summer Fall Winter Spring Summer Fall Germplasm acquisition Field production Root evaluation & selection Vernalization Grow out and pollination Seed set & harvest zLight gray boxes indicate current or future activities at the time of this writing.

Figure 2.1. The seed to seed method used to conduct half-sib mass selection of table beets bred for increased pigmentation and low total dissolved solids at Oregon State University.

During the summer field trials, plots were evaluated for visual appearance

(pigmentation), root shape, and diseases (mainly powdery mildew caused by Erysiphe betae

[Vanha] Weltzien). Twenty beet roots per family were harvested that were 5 – 9 cm (2 –3½ in.) in diameter and possessed uniform dark red pigmentation. This size range was used because size of beets roots influences betalain concentration; smaller beets of 2.5 -3.5 cm (1- 1½ in.) tend to have twice as much pigmentation as beets at 7.5 - 8.5 cm (2 -3 in) in size (Nilsson 1973). Using a visual scale, the best 10 roots were selected from the 20 harvested roots. A slice of the selected root’s “cheek” was removed and placed individually in plastic Ziploc bags and were frozen at -

29°C (-20°F) for subsequent refractometer analysis. The interior surfaces exposed by the cut for refractometer analysis were used to measure CIE LAB values for each root, and the six with lowest L* value for each family were retained and vernalized. Due to the design of the mating system, families in each cycle had a common female but a random mix of males resulting in half- sib families.

Spring: Production and harvest by half-sib family Subsequent cycles followed a similar pattern of production. Bolting and flowering was conducted in the greenhouse for better control of environmental conditions and to eliminate potential for cross-pollination of table or sugar beet and chard seed production fields produced by seed companies in the Willamette Valley. Western Oregon is the location for almost all Beta spp. seed production for North America, and because the crop is wind-pollinated, requires a three mile isolation zone around each field.

During pollen shed, fans were placed in the greenhouse to facilitate wind transfer of pollen. Once flowering and pollen shed was complete, we removed fans and inflorescences were allowed to mature and dry prior to harvest. Seed formation began three to four weeks after pollen shed, around late April to early May. Seeds set in the greenhouse were harvested in the spring as

half-sib families. Seed harvest was initiated at the beginning of May and continued to the end of

June. The open-pollinated population had a mixture of monogerm and multigerm seed families, and threshing was done by hand to reduce any loss of seed. Seed was stored at 4oC (39o F) and relative humidity of around 45%. Remnant seed was retained from each cycle, half of which was provided to Kerr Concentrates, the remainder held for a trial of all cycles of selection to document gain from selection.

Summer: Field trials and evaluation Each cycle was grown in the field and evaluated for foliage and root color and other horticulture traits. Cycles 0 to 4 were planted as a single replicate with checks repeated twice.

Order of experimental families and checks was randomized in each cycle. Plots were 4.6 m (15 ft.) in length with 0.76 m (30 in) rows. The trials in each year were rotated to a new location on the farm. Seeds were not treated with a fungicide before planting. Around 600 lbs./acre of 12-10-

10 all-purpose fertilizer was applied in the field trials before sowing. A pre-emergence herbicide

Magnum (Syngenta) was applied after sowing but before emergence of the crop to minimize weeds. Herbicide control was supplemented with hand weeding. Throughout the six cycles, beets were susceptible to cucumber beetle damage and were sprayed once or twice during the early season with Sevin (Bayer). Approximately 200 seeds were planted per plot, which were thinned during week 3 to 40 - 50 plants spaced 5 – 7.6 cm (2 -3 in) apart. Beets received 2.5 cm (1 in) irrigation by solid set overhead sprinklers on a weekly basis.

In cycle 0, 11 accessions and seven cultivars were planted in the summer field trial (Table

2.2). Seeds from 54 half-sib families were planted in cycle 1 and all successfully germinated.

Cycle 2 had a total of 155 plots consisting of 150 half-sib families and five commercial check cultivars; three families had no germination. Cycle 3 had around 97 half-sib families and four commercial checks. Cycle 4 had 106 half-sib families and four checks with one plot not

germinating, and two plots having low germination of 10 or fewer roots. The Cycle 5 had 159 half-sib families seeded with five checks of commercially available cultivars. A new commercially available cultivar (Akela) was added to the field trial in cycle 5 because of its excellent betalain content.

Table 2.2. Number of families, number of roots and selection intensities applied during cycles of selection of a table beet population for selection of increased root pigment and lower total dissolved solids.

Selected Families/ roots Expected Observed Accessions Roots accessions Roots contributing selection selection Cycle or families evaluated selected selected to gene pool intensity intensity no. % 0 18 285 -- 114 114 40 1 54 306 -- 91 54 29.7 17.6 2 147 882 14 220 100 25.0 11.3 3 97 582 11 200 170 34.3 29.2 4 106 636 8 196 164 30.5 25.8 5 159 954 5 204 -- 21.4 --

Cycle 5 and remnant seed from the previous four cycles (cycles 1-4) were planted together in a lattice experimental design to evaluate heritability and gain from selection. Not all accessions had remnant seed available for this trial. A lattice design is an augmented incomplete statistical design to increase precision and produce accurate estimates of treatment effects. The lattice experimental design consisted of 16 sub-blocks in three reps with each block having 16 entries. In total there were 256 plots in each rep each being three meters (10 ft.) long and planted with approximately 200 seeds. Overall, there were 768 plots surrounded by border rows of commercially available cultivars. Two lines from 2015 did not germinate possibly due to the age of the seed and Akela and 12 lines from Cycles 2 - 4 had low stand counts of 50 or fewer plants

in each rep. Remnant seed that was low only supplied around 70 seeds in the 2019 trial, however, each plot consisted of 50 plants.

Fall: Evaluations of CIE L*a*b* & total dissolved solids During the fall, beets were harvested and evaluated for pigmentation, shape and diseases.

Twenty roots were harvested from the field with uniform shape, size around 5 – 9 cm (2- 3 ½ in) and small necks. During cycles 1-2, the fall protocol consisted of harvesting 20 roots of 5 – 10 cm (2 ½ - 4 in) diameters, and selecting for minimal orange striping in the petioles. Orange striping was reported to have a correlation with zoning (OSA 2008), so was implemented as a selection criterion for the population. For cycles 3 - 4, 20 roots were harvested when the majority of the plots reached 5 – 9 cm root diameters. Cycle 5, employed a different protocol to reduce the chance of the roots exceeding 9 cm, which consisted of harvesting 20 roots from all 768 plots over a 3 week period and storing these in a walk-in cooler maintained at 0 - 4oC (32 - 40 o F) at

OSU Lewis Brown Farm until they could be processed.

Once harvest was completed, we began data collection for color and TDS% parameters.

Petioles were cut five cm (two in.) from the beet neck, and were washed until free of any visible soil. Each root was given a vertical cut at the cheek and the portion removed to expose the interior of the beet was saved for TDS analysis. The best 10 beets with dark pigmentation and no or minimal zoning (Figure 2.2) were saved and the remaining ten were discarded. A BC-10 colorimeter (Konica) was used to quantify colors using the CIE L*a*b* scale for all samples from cycles 1 - 5. Two readings per root were taken from the center of exposed interior of the root and the values were averaged. L*a*b* values were recorded for all 10 roots, and the 6 roots with lowest L* were retained. These were allowed to suberize for 24 h before being placed in cold storage to vernalize. Data was generally collected over a 2 month span, from the end of

September to November.

The portion cut from the cheek of the root was saved in a

Ziploc bag, labeled corresponding to the half-sib family and root number, and were frozen at -34 °C (-29 °F) Figure 2.2. Two beet roots cut in cross-section to for up to two months, until they could show strong (left) and minimal zoning (right). be processed for TDS measurements.

Samples were frozen to reduce the possibility of rot while colorimeter data was taken. Around 30 to 40 half-sib families were processed per day with the entire analysis taking a month. Frozen samples were allowed to thaw for 24 hours at room temperature and samples were lightly macerated to release beet juice for analysis. TDS was measured using a digital refractometer

(Pal-1, ATAGO Co.) that had a TDS range of 0-53%. We recorded two readings per sample, which were averaged.

Each half-sib family was placed in a bin and were vernalized for 10 weeks at 3 - 4°C (34

- 37°F) for the first 2 cycles. The protocol for vernalization was modified during cycles 3 - 5.

During these cycles, after a dip in 5% sodium hypochlorite solution (Clorox®), beets were allowed to suberize for 24-48 hours. In cycle 3, storage bins were filled with cedar shavings to minimize moisture and reduce beet loss to mold formation (I. Goldman, personal communication). A slight reduction was noticed in cycle 3 after this change and large reductions in infection were observed in the two subsequent cycles. Roots of selected beet families were placed in #16 brown paper grocery bags with holes punched to allow air ventilation. A small layer of cedar shaving covered the bottom of the bag; beets were then placed in the bag and

covered with more cedar shavings. In the three final cycles, beet vernalization was extended to

11 - 12 weeks.

Winter: Greenhouse and selections While beets were being vernalized, L*a*b* and TDS values were analyzed, and families with low L* and low TDS were selected. Selection varied from year to year, but we aimed to have around 200 roots or 30% of the population to cross-pollinate during the spring. However, loss of roots during vernalization created more intense selection with retention ranging from 11 –

25% of the population. In the first cycle, 15 accessions from the base populations were vernalized for 10 weeks, and went on to cross pollinate in the spring (Table 2.2). In cycle 1, 306 roots from 51 half-sib families were vernalized and 91 roots were selected with L* ranging from

18 - 21, and TDS of 8 - 11%, however only 54 roots survived to flowering. From 147 half-sib families in Cycle 2, we vernalized 882 roots and selected 25% of the population with L* < 20 and TDS < 10%. We lost the majority of the roots due to grey mold during vernalization and were left with 100 roots from the 25% selected. In Cycle 3, we had 97 half-sib families and we vernalized 582 roots; 34% were selected with L* of 16 - 19.9 and TDS < 10. We lost about 14% of the 34% selected beets because of grey mold in cold storage and the greenhouse. Cycle 4 had a total of 106 half-sib families, and 636 roots that were vernalized, with 30% of the population selected. The selected beets had L* < 19.9 and TDS <10%. About 5% of the population was lost during vernalization. In cycle 5 we also used the L*/brix ratio as suggested by our food processing partner, selecting for a range of 1.25-2.01.

For cycle 5, we intensified selection pressure by retaining beets with L* <19 and TDS <

10%. The 2019 population was also analyzed by UV-Vis spectrophotometer to quantify the amount of betanin in each half-sib family. Beets with an average total betanin of 20 - 25% and

L* < 19 were selected from reps 1 and 2. The combined reps provided 47 half-sib families with

L*of 16 - 19 and TDS of 8 - 10%. Within each rep, we selected around 14% of the beet root population. Seventeen of the 47 half-sib families were the same in each rep because they produced similar results in both reps for L* and TDS. This constituted 204 roots that were planted in the greenhouse from 954 roots that were vernalized. Probably as a result of a second sample removed from roots for spectral analysis, the majority of the roots were infected with gray mold. Only three out of 204 roots had signs of shoot formation, and were saved in the greenhouse, while the rest were discarded. The three roots were from two different half-sib families. To obtain sufficient root numbers, we took roots from rep 3 families that had vernalized in the field. Overall, we had 24 half-sib families, 14 of the 24 were the same families we selected from reps 1 or 2. The remaining 10 half-sib families, had desirable L* and TDS values. All families had average L* of 17 - 19 and TDS of 9 - 14%. One-hundred and forty-four beet roots were planted in the greenhouse in March, 2020. From rep 3, we selected around 10% of the population which constitutes the most intense selection to date.

Following vernalization for each of the six cycles, selected roots were relocated to the greenhouse in early to mid-February. They were placed in 2.4L pots containing Sungro professional growing mix (Sun Gro Horticulture). Each pot was given ~11g slow release fertilizer (Osmocote Classic Controlled Release; 14-14-14). Beets were watered as needed and after 6 weeks, fertilizer was reapplied. Beets were arranged in the greenhouse in a randomized design to increase the chances of intermating with individuals from different families. Bolting began about seven weeks after planting. The one meter flower stalks were staked to prevent breakage with about 5cm of the flower heads left lose to allow the flowers to shed pollen when tapped or agitated by wind. Once pollen shed began in 20% of the population, fans were placed in the greenhouse to stimulate wind to facilitate pollen transfer. Fans were run continuously for 3

– 4 weeks. In addition to fans, every morning, flower stalk were tapped to release pollen. A biocontrol program using Cucumeris and Hypoaspis spp. mites and Orius spp. bugs was implemented for thrips control. A few pots were affected by Rhizoctonia halfway through flowering in cycles 3 - 5. Affected beets were drenched with Chipco 26019 fungicide (OHP).

Beets in cycle 5 were drenched twice with Chipco 26019 (OHP) to control gray mold that had infected in cold storage. Seeds were harvested in late May to June.

Data Analysis

Experimental design

Cycles 0 and 4 were in non-replicated field trials. Excel was used for cycles 2-4, to randomize entries. Cycle 5 was organized as a square lattice design because of the large number of plots and required the use of the α-design program Gendex DOE Tool kit 8.0 (Patterson &

Williams, 1976). An α -design allows for treatments to be replicated within blocks and sub- blocks while minimizing the chances of experimental units grouped in proximity, as well as accounting for increased variability within reps, blocks and sub blocks in the square lattice design.

Analysis of Variance

PROC GLM in SAS (v. 9.4, Cary NC) was used to analyze on a yearly basis for cycles 1-

5. Variables were analyzed for any differences between the cycles. Least square means (lsmeans) and significant differences were calculated between cycles for all the variables. We also determined if the check lsmeans were significantly different from half-sib line lsmeans. The lattice design required R.3.6.1 to generate ANOVA output for all the parameters to evaluate for significance among cycles, families, blocks and reps.

Mean comparisons

For Cycles 1 to 4 grown individually in years, two-sample T-tests were performed in

Excel between previous and current cycles to determine whether there were significant changes each year for L* and TDS. L*a*b* values and TDS for all samples were also plotted to visualize population variation.

Heritability and gain from selection

Narrow-sense heritability (h2) was calculated in R using the “eemeans” package and constructing a cross side analysis with each year being a block. Narrow-sense heritability was calculated for L*a*b* values and TDS. Also, gain from selection was calculated from heritability

푖(1/4)휎2 using the formula 퐴 where i is the standard deviations from the means, 휎2 is the additive 휎 퐴 푃ℎ푠

genetic variance and 휎푃ℎ푠 is the phenotypic variance among half-sibs, to determine expected gain per cycle for L*a*b* and TDS.

Color analysis

The L*a*b* values were converted to RGB (Red-Green-Blue) color values and translated to Royal Horticultural Society color charts using an Excel macro developed by Lattier and

Contreras (2020). Three-dimensional plots of L*a*b* values were generated in MATLAB 9.7

R2019b (Natick, Mass). We also, calculated the percentages for every RHS color group within each cycle.

Results Analysis of cycles of selection across years

Analysis of Variance

There were significant differences among cycles for L* (P < 0.0001) and L* varied significantly (P < 0.0001) among families. There was a significant difference (P < 0.01) for family by cycle (GxE) interaction (Table 2.3). The families had variation for L* indicating that the L* has changed throughout the five cycles of selection.

Similar to L*, a* had significant differences (P < 0.001) among all five cycles. Families were also significantly differentiated (P < 0.001) for a*. Family by cycle interaction had a smaller but significant F statistic (P<0.01) for a* (Table 2.3); b* was only significantly different

(P < 0.001) for family and cycle main effects. This can be attributed to the selection for L* and dark red-purple color throughout the cycles.

Throughout the five cycles, TDS varied significantly (P < 0.001). TDS showed no significantly differences among families, or for the family by cycle interaction.

Table 2.3. Analysis of variance for L*a*b*color parameters and total dissolved solids for a table beet population subjected to five cycles of half-sib mass selection.

Sum Mean F Source df Pr(>F)z Sq. Sq. Value L* Cycle 4 51.96 51.96 49.82 0.00 *** Family 64 157.27 2.46 2.36 0.00 *** Entry 170 268.50 1.58 1.51 0.00 ** Cycle x Family 28 47.46 1.70 1.63 0.03 * Residuals 280 292.01 1.04 --- a* Cycle 4 201.20 201.18 16.52 0.00 *** Family 64 1326.30 20.72 1.70 0.00 ** Entry 170 2743.90 16.14 1.33 0.02 * Cycle x Family 28 590.30 21.08 1.73 0.01 * Residuals 280 3409.90 12.18 --- b* Cycle 4 86.88 86.88 93.48 0.00 *** Family 64 228.97 3.58 3.85 0.00 *** Entry 170 197.35 1.16 1.25 0.05 * Cycle x Family 28 18.50 0.66 0.71 0.86 Residuals 279 259.30 0.93 --- TDS Cycle 4 55.28 55.28 29.80 0.00 *** Family 64 102.87 1.61 0.87 0.75 Entry 169 210.20 1.24 0.67 1.00 Cycle x Family 28 25.80 0.92 0.50 0.99 Residuals 266 493.37 1.86 --- zStatistically significant at: *** P<0.001, ** P<0.01, * P<0.05.

Mean comparisons across cycles of selection

Each cycle was independently evaluated for color parameters and TDS, and results were compared with the previous cycle using a two-tailed t-test (Table 2.4). Table beets were selected for low L* and TDS from cycles 1 – 5 with the percentile of selected beets varied throughout the cycles (Table 2.4). The means for L* and TDS of the base population and Cycle 1 were significantly different with L* decreasing while TDS increased approximately 1%. Cycles 2-5 had similar results with significant differences in t-tests for L*a*b* and TDS (P < 0.0001) between each sequential cycle (Table 2.4; Figure 2.3). L* trended in the desired direction of smaller values across cycles (Table 2.5), TDS was significantly different (P < 0.0001) for every comparison of cycles, and trended towards an increase in % TDS. R2 from linear regression was small but the slope of the line was significantly different from zero (Table 2.5). This is in spite of selecting for TDS of 10% or lower. Across the cycles, a* had a trend of decreasing until the final cycle and b* had a trend of increasing, but dropped in the final cycle (Figure 2.3c & d). For linear regression, the parameters a* and b* had small R2 and statistically significant but slopes near zero (Table 2.5).

The population in 2015 (cycle 1) had more outliers above the maximum quantile than in other years, reflecting the fact that some half-sib families had L* > 22. Fewer such outliers were observed in subsequent years, with the cycle 5 population having a more compressed data set for

L*. While the Cycle 5 population showed a slight increase (L* = 19) compared to Cycle 4 (L* =

18.3), the cycle 5 population variances were smaller, implying a decrease in variability in the population.

Table 2.4. Two-tail T-test assuming unequal variances comparing means for L*a*b* and TDS between cycles of selection for a table beet population being bred for high betalain and low TDS content.

Statistic Year 2014 2015 2016 2017 2018 L* value Mean 27.7 19.9 20.4 19.4 18.3 Variance 239.8 1.5 0.7 0.8 1.3 No. obs. 18 54 155 104 106 df 17 70 208 208 t statistic 2.13 2.75 9.06 5.77 P(T ≤ |t|) 0.05 0.01 0.00 0.00 T critical 2.11 1.99 1.97 1.97 a* Value Mean 36.9 33.2 32.3 30.8 30.5 Variance 78.8 15.8 11.7 12.8 19.2 No. obs. 18 54 155 104 106 df 31 84 206 208 t statistic 2.72 1.6 2.62 0.312 P(T ≤ |t|) 0.00 0.00 0.00 0.37 T critical 1.66 1.99 1.65 1.65 b* Value Mean 7.1 4.2 5.3 5.1 4.9 Variance 17.3 2.7 0.6 0.8 1.3 No. obs. 18 54 155 104 106 df 31 84 162 208 t statistic 4.5 -5.54 2.1 0.9 P(T ≤ |t|) 0.00 0.00 0.00 0.02 T critical 1.66 1.99 2.0 2.0 TDS% Mean 9.9 10.8 10.0 11.1 11.8 Variance 0.7 0.8 0.6 1.5 1.7 No. obs. 18 54 155 104 106 df 31 84 162 208 t statistic 3.90 -5.54 7.84 -4.24 P(T ≤ |t|) 0.00 0.00 0.00 0.00 T critical 2.04 1.99 1.97 1.97

TDS had no extreme outliers throughout the years, nonetheless, the 2017 population had greater variation among half-sib families with a maximum of 14%. Following 2017, TDS increased in subsequent years. Considerable variation in TDS was observed in 2016, 2017, and 2019 half-sib families with some with TDS as low as 7.5%. These results imply that a few families may potentially have lower TDS than the majority of families. However, environmental factors may contribute to the variability in sugar production in table beets considering the fact that TDS of

~7.5%, was not observed in the 2018. In that cycle, half-sib populations, ranged from 9.4 – 15% for TDS.

a) b)

c) d)

Figure 2.3. CIE L*a*b* and TDS (% brix) from cycles 1 – 5 (2015 – 2019) of half-sib mass selection in table beets. F statistic and probabilities are for the hypothesis that cycles are not significantly different. a) Box and whisker plot for L*; b) Box and whisker plot for a*; c) Box and whisker plot for b*; and d) Box and whisker plot for TDS.

Table 2.5. Linear regression equations and regression coefficients for L*, a*, b* and TDS across cycles (single year field trials) of a table beet population grown at the OSU Vegetable Research Farm.

Parameter R-Square Linear Equation L 0.26*** y = 957.09 + (-0.46) x a 0.01*** y = -563.06 + 0.29 x b 0.09*** y = 557.37+ (-0.27) x TDS 0.06*** y = -497.01 + 0.25 x zStatistically significant at: *** P<0.001.

Analysis of all cycles grown in a single trial in 2019

Analysis of Variance

In 2019, all 5 cycles were grown in the same environment for a better estimate of the genetic variance associated with these traits. Half-sib families were significantly different (P ≤ 0.001) for

L* in the 2019 field trial. There were significant differences (P ≤ 0.001) for cycle and family x cycle interaction (Table 2.6). The field trial showed significant differences (P ≤ 0.001) between reps, likely due to environmental factors, but not for blocks nested in reps.

Similarly, a* was significantly different (P ≤ 0.01) among the reps, families and cycles

(Table 2.6) b* value, in the other hand was significantly different (P ≤ 0.01) only among families. There was no significant for b* value in any other parameter.

There was no significant difference among half-sib families, for percent TDS (Table 2.6).

Reps showed significant differences (P ≤ 0.001), and sugar concentration variation perhaps can be attributed to variation across the field. Significant differences (P ≤ 0.001), in TDS were observed for cycles. There is variation of TDS in each cycle, however, the family x cycle interaction was not significant for TDS.

Table 2.6. Analysis of variance for L*a*b*color parameters and total dissolved solids for a table beet population subjected to five cycles of half-sib mass selection; all cycles grown in a single trial in 2019.

Source df Sum Sq. Mean Sq. F Value Pr(>F) L* Family 30 523.2485 17.44162 6.09 <.0001 Block(Rep) 45 119.8067 2.662371 0.93 0.6064 Rep 2 64.65339 32.3267 11.28 <.0001 Cycle 4 788.3417 197.0854 68.77 <.0001 Family x Cycle 55 688.2976 12.5145 4.37 <.0001 --- a* Family 30 1853.082 61.7694 5.12 <.0001 Block(Rep) 45 493.5447 10.96766 0.91 0.6438 Rep 2 287.473 143.7365 11.91 <.0001 Cycle 4 2073.856 518.464 42.96 <.0001 Family x Cycle 55 1376.403 25.02551 2.07 <.0001 --- b* Family 30 308.0406 10.26802 6.62 <.0001 Block(Rep) 45 68.30516 1.517892 0.98 0.5145 Rep 2 7.942042 3.971021 2.56 0.0782 Cycle 4 161.6079 40.40198 26.04 <.0001 Family x Cycle 55 517.4462 9.408113 6.06 <.0001 --- TDS Family 30 105.0866 3.502886 1.53 0.0369 Block(Rep) 45 139.575 3.101667 1.35 0.0658 Rep 2 141.7283 70.86415 30.94 <.0001 Cycle 4 56.99988 14.24997 6.22 <.0001 Family x Cycle 55 143.0537 2.600977 1.14 0.2412 ---

Mean comparisons – 2019 lattice design trial

L* had a 5 - 22% decrease as half-sib families shifted to darker pigmentation, although, in the final cycle, the L* value increased by 3%. There were significant differences (P ≤ 0.0001) in L* across cycles in the lattice experiment conducted in 2019 (Figure 2.4). L* was consistently lower in each succeeding year; the selection for lower L* appears to have shifted the population to darker pigment. Overall, L* decreased from 24.0 to 19.9. R2 for L* was a relatively small at

0.27, however the slope was significantly different from zero (Table 2.7).

The a* and b* values also had highly significant trends across cycles for both analyses

(Figure 2.4b & 2.4c). The trends for a* and b* were not a linear from cycle to cycle, but overall a* decreased (became less red) and b* increased (trending from blue towards yellow). As well, a* and b* had small R2 but regression lines had slopes significantly different from zero. (Table

2.7).

Total Dissolved solids had a positive slope away from zero indicating that we reject the null hypothesis, there were differences between the means in each cycle. Even though the means for TDS changed throughout cycles, it was in the opposite direction from what we intended. The

TDS means increased despite constant selections of lower TDS. The parameter for TDS had a small R2, indicating there was no trend, but much random variation.

a) b)

c) d)

Figure 2.4. L*a*b* and total dissolved solids for a table beet half-sib mass selection population with all populations grown in the field at the OSU Vegetable Research Farm in 2019. a) L*. b) a*. c) b*. d) Percent TDS

Table 2.7. Linear regression equations and R2 for L*a*b* and TDS regressed on cycles of a table beet population grown in 2019 in a lattice design field trial at the OSU Vegetable Research Farm.

Parameter R-Squarez Linear Equation L 0.23*** y = 2327.19 + (-1.14) x a 0.15*** y = 3406.08+ (-1.67) x b 0.11*** y = -1105.21 + 0.54 x TDS 0.01*** y = -355.04 + 0.18 x zStatistically significant at: *** P<0.001. 38

Heritability and gain from selection

Based on the trial of all cycles in 2019, we calculated the narrow-sense heritability (h2) of both L*and TDS. Under single factor analysis of the lattice design, L* had an h2 of 0.74. Not only did L* have a high h2, it also had a relatively high decrease in gain from selection of 0.75 units per cycle (Tables 2.8 & 2.9). TDS % had a relatively low h2 of 0.16, and gain from selection of 0.06% per cycle (Table 2.8 & 2.9). The a* parameter had relatively high h2 of 0.62, and gain per selection of 0.58, while b* had a low narrow-sense heritability of 0.18 and low gain from selection (0.20). (Tables 2.8 & 2.9)

Table 2.8. Single factor analysis for the 2019 field trial of a table beet population subjected to half-sib mass selection over five cycles of selection at the OSU Vegetable Research Farm. Shown are variances and heritabilities for L* and percent total dissolved solids.

Single-Factor L* a* b* TDS% Analysis V (Genetic variance among half-sib G 3.23 4.12 0.09 0.15 families) Ve (Error variance) 2.16 7.33 1.34 2.32

VE (Environnent variance) 2.20 5.27 0.67 1.16 V (Phenotypic variance among P 4.31 9.39 0.76 1.31 half-sib families) VA (Additive genetic variance) 12.90 16.40 0.39 0.59 h2 (Narrow-sense heritability for 0.74 0.62 0.18 0.16 half-sib family means)

Table 2.9. Gain from selection for total dissolved solids and L*a*b* in a table beet half-sib population selected over five cycles.

Gain from selection for half-sib Gain Per cycle families TDS% 0.06 L* Value 0.75 a* 0.58 b* 0.20

Color analysis

The pigmentation observed in the beet population was measured using the CIE L*a*b* scale, then converted to RGB colors and matched to the Royal Horticultural Society (RHS) color scale. The RHS colors were found in Fans 2 and 4 and ranged from the greyed-purple (187B,

185A,) red-purple (59A, 59B & 61A), to red (53A) groups throughout cycles 1 - 5 in 2019. The initial germplasm had some RHS greyed-white, greyed-orange greyed-red, greyed-purple as well as red and re-purple groups colored beets (Table 2.10). The greyed-white and greyed-orange were selected out and not allowed to open-pollinate with the remaining population. Cycle 1

(2015) had a narrower range of color than the base population, ranging from light purple to dark purple. The remaining cycles showed an increase of the darker toned red-purple groups in the population (Table 2.11). The red-purple group 61A decreased throughout the cycles with this color making up only 2.2% of the population in cycle 4 and being completely lacking in cycle 5.

Greyed-purple (185A) colored half-sib families were selected out of the population by cycle 3.

The two colors that increased over time were greyed-purple (187B) and red-purple (59A) with the latter predominating (Table 2.11). Selection for lower L* without constraints on a* and b* shifted the population to darker red-purple or grey-purple colored groups. As shown in the three- dimensional plots, the half-sib populations have become more concentrated around L* of 20 or lower and positive a* and b* in the range of 0-50, and 0-20, respectively (Figure 2.5).

Table 2.10. The Royal Horticultural Society (RHS) and RGB color values measured in 2014 for the base population of table beet germplasm, consisting of 11 USDA-NPGS accessions and seven commercially available cultivars.

RHS Fan RHS Group RHS Value Fan 4 Greyed-White 156A Fan 4 Greyed-White 156A Fan 4 Greyed-Orange 177D Fan 4 Greyed-Orange 177D Fan 4 Greyed-Red 178B Fan 4 Greyed-Purple 185A Fan 4 Greyed-Purple 187B Fan 2 Red-Purple 59A Fan 2 Red-Purple 59B Fan 2 Red-Purple 59C Fan 2 Red-Purple 61A Fan 1 Red 53A

Table 2.11. The Royal Horticultural Society (RHS) fans, groups and values, and corresponding RGB colors for a table beet population consisting of five cycles (corresponding to years 2015 – 2019) evaluated at the OSU Vegetable Research Farm in 2019. Cells for percent of roots in a particular color group are colored to visualize shifts in groups over cycles of selection. Colors range from blue (0%) through white to red (67.4%).

2015 2016 2017 2018 2019 RHS Fan RHS Group RHS Value % Fan 4 Greyed-Purple 187B 2.0 4.0 8.7 11.6 22.1 Fan 2 Red-Purple 59A 39.0 48.7 56.8 62.7 67.4 Fan 2 Red-Purple 59B 21.0 19.0 17.0 13.9 8.2 Fan 1 Red 53A 13.0 14.2 13.0 9.3 1.9 Fan 4 Greyed-Purple 185A 6.0 0.0 Fan 2 Red-Purple 61A 19.0 14.2 4.3 2.3

Figure 2.5: Three-dimensional plots of root colors for a table beet population selected over five cycles of half-sib mass selection. L*, a* and b* are plotted on the y, x and z axes, respectively. Dots represent individual families and are shown with equivalent RGB colors. a) Cycle 0 b) Cycle 1 (2015); c) Cycle 2 (2016); d) Cycle 3 (2017); e) Cycle 4 (2018); f) Cycle 5 (2019).

Cycle 5 half -sib families and five commercial table beet cultivars used as checks were compared for L*a*b* and TDS (Table 2.12). The color parameters all showed highly significant differences P ≤ 0.0001) whereas there were no significant differences between families and checks for TDS. Overall, 31 lines within families had promising L* values that were significantly lower than the checks. The remaining lines were not significantly different from the check cultivar mean (Appendix A). Selection for L* has resulted in desirable half-sib families with L* less than or equal too commercial lines currently used in the food processing.

While in general, sugar production (TDS) varied throughout the years and may have had a steady increase in the population, there were a few half-sib families with the desired low TDS, suggesting that lowering the sugar concentration below the average of 9-10% might be possible.

In the 2019 field trial, the checks had lsmeans for TDS ranging from 11.1 to 12.1 while the half- sib families had a mean of 10.76% and the range of the 2019 field trial was 6.1-15%. The TDS varied among families, but a few had consistently low TDS of around 9%, ranging from 6.1 –

8.9% (Table 2.15). The comparison of families vs. checks had a t statistic of 10.8 and was highly significant (P ≤ 0.0001) (Table 2.13). The families included TB18, TB24 TB33, TB45, TB66,

TB86, TB103 and TB87, with the line TB24-3-6-4 in the 2019 environment with a TDS of 6.1%

(Table 2.14).

Table 2.12. Analysis of variance comparing eight half-sib families from cycle 5 and five commercial table beet cultivars for CIE L*a*b* and total dissolved solids (TDS). Data from a trial conducted at the OSU Vegetable Research Farm in 2019.

Mean Squarez Source df L* a* b* TDS Families vs 13.1*** 60.1*** 13.1*** 3.2 checks 30 Rep 2 0.06 4.3 0.46 1.5 Error 28 15.7 7.16 0.47 2.88 zStatistically significant at: *** P<0.001.

Table 2.13. Two-sample T test assuming equal variances on set of families and checks on their total dissolved solids.

Checks Families Mean 11.34 8.29 Variance 0.78 0.26 df 50 t Stat 10.86 P(T<=t) two-tail 0.00 t Critical two-tail 2.01

Table 2.14. Half-sib table beet families showing root color parameters and total dissolved solids (TDS), with percent TDS of 9 or lower. Data from families and check cultivars grown in the field at the OSU Vegetable Research Farm in 2019.

Identifier Name L* a* b* TDS% TB024 TB024-3-6-4 18.4 32.4 3.9 6.1 TB103 T103-4-5-1 19.18 26.0 3.3 6.7 TB086 TB086-4-5-4 18.9 29.7 3.7 7.2 TB128 TB128-1-6-3 19.77 36.7 5.2 7.3 TB063 TB063-01-4-6 18.95 34.5 4.7 7.4 TB024 TB024-3-6-3 20.87 39.7 4.8 7.5 TB066 TB066-1-4-3 21.25 35.9 3.5 7.7 TB073 TB073-02-6 (14) 21.12 35.7 4.9 7.8 TB066 TB066-1-2-6 21.88 38.5 6.7 7.8 TB073 TB073-02-6 (13) 21.55 39.9 4.3 7.9 TB033 TB033-2-1-1 18.54 33.0 3.9 7.9 TB018 Tb018-3-6-4 18.75 31.5 4.1 8.0 TB024 TB024-3-6-2 20.18 35.0 3.2 8.0 TB045 TB045-3-3-5 20.66 38.0 5.7 8.0 TB045 TB045-2-6-4 22.96 40.3 1.4 8.1 TB033 TB033-2-1-1 18.22 32.5 3.6 8.1 TB029 TB029-2-3-3 18.77 35.3 4.5 8.1 TB045 TB045-2-1-2 19.35 33.3 3.6 8.1 TB022 TB022-2-5-2 19.52 36.4 5.0 8.2 TB018 Tb018-3-6-6 18.96 34.2 4.2 8.2 TB018 TB018-4-5-2 18.87 33.4 4.3 8.2 TB045 TB045-2-1-1 19.15 33.8 4.4 8.2 TB082 TB082-05-5-5 20.61 38.9 4.9 8.3 TB073 TB073-02-1-4 19.05 37.7 5.1 8.3 TB155 TB155-4-4-6 18.94 33.5 3.8 8.3 TB087 TB087-4-5 -(15) 19.50 32.9 4.3 8.3

Table 2.14. (continued). Identifier Name L a b TDS% TB073 TB073-02-1-4 20.1 40.8 5.3 8.4 TB018 TB018-4-2-2 23.9 34.2 5.1 8.4 TB020 TB020-1-2 19.53 33.4 4.5 8.4 TB087 TB087-2-1-1 20.8 36.4 4.8 8.4 TB073 TB073-02-4-1 19.83 31.4 4.3 8.4 TB087 TB087-4-5 -(16) 22.08 36.0 0.0 8.4 TB020 TB020-1-3-4 22.08 41.8 5.5 8.4 TB029 TB029-2-3-1 19.09 36.4 4.7 8.5 TB066 TB066-1-2-4 19.51 31.8 4.2 8.6 TB033 TB033-2-4-2 19.45 34.5 3.3 8.6 TB082 TB082-05-5-6 19.17 34.5 3.8 8.7 TB018 TB018-3-6-2 19.96 37.5 5.2 8.7 TB087 TB087-2-4-1 20.00 31.2 3.8 8.7 TB066 TB066-1-2-3 20.05 39.7 5.6 8.7 TB055 TB055-5-2-4 19.18 31.5 3.6 8.8 TB087 TB087-2-3-4 20.60 37.5 4.6 8.8 TB087 TB087-2-3-5 21.60 37.0 2.3 8.8 TB018 TB018-4-1-1-5 21.84 38.8 4.3 8.8 TB155 TB155-4-4-4 20.69 36.1 4.7 8.8 TB029 TB029-2-1-2 19.70 36.1 4.1 8.8 TB087 TB087-2-3-4 21.24 34.8 3.8 8.9 TB018 TB018-4-5-2 18.90 36.3 3.8 8.9 TB045 TB045-2-5-6 20.41 37.3 5.0 8.9 TB029 TB029-2-3-3 19.85 37.6 4.5 8.9 TB045 TB045-2-6-1 19.76 33.4 4.5 8.9 TBcheck1 Pablo 18.40 27.5 3.7 11.8 TBcheck5 Dark Red Detroit 20.06 29.9 4.6 12.1 TBcheck3 Akela 19.16 31.0 3.5 11.4 TBcheck4 Boro 19.39 31.4 4.3 10.1

Discussion and Conclusion Selection for lower L* using half-sib mass selection over five cycles has resulted in a darker toned table beet population. Over 30 half-sib families with a significantly darker purple color compared to commercial cultivars were obtained. Prior to this study, previous measurement and selection work in table beet had been carried out using UV-Vis spectrophotometer measurements of betalain content. This work shows it is possible to conduct successful selection based on direct pigment evaluation using a colorimeter. Because breeding programs need to evaluate thousands of samples, breeders need techniques that are relatively simple, rapid and efficient to measure traits of interest. The colorimeter is a tool that speeds the process of pigment quantification and has distinct advantages in speed and efficiency over spectrophotometry.

Selection for pigmentation and total dissolved solids are dependent on genetic variation being present for both traits as well as having favorable genetic correlations (Ng and Lee, 1978;

Nilsson, 1973; Von Elbe et al., 1972 Watson and Gabelman, 1982). We began the project with variability for both of these traits although the initial variation for pigmentation was in the direction of lighter color than existing commercial cultivars, and we were unsure if there would be a negative correlation between pigment and TDS. For pigment, we saw transgressive segregation where some individuals in the final cycle had significantly higher pigment levels than anything observed either in commercial cultivars or in earlier cycles. The population is a work in progress in that it is a mix of half-sib families with pigmentation equal or superior to existing cultivars. This suggests that there is room for increased pigment concentration with further cycles of selection.

Half-sib recurrent selection has been used previously in table beets to increase pigment levels (Wolyn and Gabelman, 1990a, Gaertner and Goldman, 2005, Goldman et al., 1996).

Wolyn and Gabelman (1990a) were able to increase the concentration of betalains in a

population of six U.S and European cultivars by 45% in three cycles of recurrent mass selection.

The beet breeding program at University of Wisconsin-Madison continued the process for 16 cycles of half-sib recurrent selection for pigmentation in table beets. Pigmentation and TDS was evaluated from five tissue sections (outer, middle, center, leaf and petioles of each beet in two different environments. Pigment concentration increased at an average rate of 14.5%, 19.8%,

19.6%, 8.2% and 5% per cycle between C2-12 for outer, middle, center petiole, and leaf tissue, respectively (Gaertner and Goldman 2005).

When means per cycle from individual years are compared with all cycles grown in a common trial in 2019, the trends for color parameters were similar, but greater variation from cycle to cycle was observed in the former analysis. This implies that there was significant year to year variation due to environmental influences on pigment content. Pigment content of red table beets is known to be affected by both temperature and light (Wolyn and Gabelman, 1986;

Magruder et al., 1940). Table beets grown in hot dry summers during the day with temperatures >25oC show decreases in production of betalains (Bradley and Dyck, 1967).

Average monthly temperature in our trials never exceeded 25oC, but there was some year to year variation with 2017 being particularly warm (Agsi 2020). Even though, the half-sib population had lower L* in the independent cycles field trials than 2019 lattice field trial; there was steady decrease in L* from cycle 2 to cycle 5, the half-sib population color pigmentation has become darker.

The narrow-sense heritability for pigmentation was high at 0.74 indicating that gain from selection should be relatively easy on a single plant basis. Wolyn and Gabelman (1990a) selecting for higher pigment in beets using direct measurement of betalain obtained h2 of 0.81 in a population selected for greater pigmentation and 0. 82 in a population selected for low

pigmentation. Gain from selection for high pigmentation was also high relatively high for of both our results (0.75), and for Wolyn and Gabelman (1990a) (0.71). Although different selection techniques were used, the outcomes have been quite similar. Wolyn and Gabelman (1990b) stated that breeding in an open-pollinated population proved to be quite feasible for a wind- pollinated biennial where selection among and within families could be practiced, and we also found this to be the case. Being able to control the contributions of both male and female parents during mating doubles the expected gain per cycle (Wolyn and Gabelman 199b).

A consequence of selection for low L* was the likely fixation of the R gene controlling betanin production in table beets. Wolyn and Gabelman, demonstrated that three alleles (Rt, Rh,

RP) at the R locus dictate the ratio of betacyanin to betaxanthins. They discovered that Rt and R genotypes had incomplete dominance at the R locus (Wolyn and Gabelman 1990a). In the first generation, accessions with light- or white-colored roots were eliminated. Subsequent gain in color would then be through fixation of alleles at the R locus. Once R is fixed, it is possible that the rate of gain would plateau as suggested by Wolyn and Gabelman (1990b). However,

Goldman et al. (1996) saw continued gain for pigment over eight cycles of half-sib selection, which suggests that other genes in the population may influence betalain production. We saw a color shift in the population to primarily Royal Horticultural Society color groups red-purple and greyed-purple, which may represent selection for modifying genes. In addition to the color shift, the pigmentation of the half-sib population fell into a narrower range of L* means.

Total dissolved solids in the half-sib population varied throughout cycles with the overall trend being one of no change in sugar concentration. Selection for a decrease in TDS in our population was not successful. Previous efforts to select for lower TDS was also ineffective in table beet, while simultaneously selecting for higher pigmentation (Wolyn and Gabelman 1990a;

Goldman et al 1996). Goldman et al. (1996) in particular witnessed results similar to ours over eight cycles of selection for low total dissolved solids and highly pigmented beets; although in their population, each cycle had an increase in sugar concentration. The simultaneous selection for both high pigmentation and low total dissolved solids may be at odds because of betalain chemical structure. Betalains are formed by glycosylation of thalamic acid, thus a certain number of glucose molecules will be present as long as betalains are present (Sciuto et al. 1972). This may be why we saw little change in TDS associated with the concentration in pigment in the half-sib population. Metabolically there are some limitations to select for dark colored pigmented and low total dissolved solids in table beets at the same time. The h2 and gain per cycle of selection were low for TDS (0.15 and 0.06, respectively). These results were lower than those of

Wolyn and Goldman (1990b) with heritabilities of 0.25 and 0.27 in their two-half-sib table beet populations for TDS.

The half-sib population showed no significant differences in TDS among families in the individual field trials ANOVA, while the 2019 field trial did have significant differences. Sugar concentration increases in beets with longer duration of growing period (Scott et al., 1973) and may be affected by other environmental factors. The 2019 field trial was grown in the same environment and was harvested within a two week window unlike the trials in individual years where environment and harvest conditions varied. This probably accounts for the difference in variability in TDS in the half-sib families between the two analyses.

A decrease in TDS is possible as shown by a few lines from our 2019 trial that had TDS of 6 - 8%. In prior studies, root weight and TDS in table beets have been negatively correlated

(Gaertner and Goldman, 2005). Harvesting roots at an earlier maturity and smaller size might account for the low TDS observed. Half-sib populations varied in size from 2 - 3 ½ in., and this

variation could have contributed to the range of TDS observed in the population. Root weights and sizes were not recorded but should be a part of future studies. Replicated trials are needed for selection for low TDS % lines to determine this is repeatable in this material.

Chapter 3 Correlation between Colorimeter and UV-Vis Spectrophotometer Introduction The food processing industry normally uses UV-Vis spectrophotometry to quantify the betalains in beet roots. The tool is highly effective in determining the concentration of both betalain alkaloid groups - betacyanins and betaxanthins the red and yellow pigments of table beets. The predominant betacyanin in table beet is betanin while the main betaxanthin is vulgaxanthin (Von Elbe, 1972). UV-Vis spectrophotometry requires that the substance to be evaluated be in a liquid form. A colorimeter permits measurement of overall intensity, color hue and saturation of a solid or liquid material. These tools provide different approaches to measuring parameters of interest. In this table beet population, we assume that darker pigmentation as measured by colorimeter or spectrophotometer is associated with a higher betalain concentration. To examine the relationship and determine if a correlation between

Spectral data with CIE L*a*b* data exists, we analyzed the base population before selection commenced as well as Cycle 5 half-sib families using both tools on the same samples. Stintzing et al. (2008) determined that betanin/betacyanin hues can predicted in table beets from L* and chroma values. Confirming the relationship between these two parameters could promote the use of the simpler and more rapid CIE L*a*b* approach in the food processing industry.

Materials and Methods Base population The initial population was analyzed using the UV-VIS spectrophotometer and percent betanin was calculated. Data was gathered prior to the start of this study using a proprietary protocol. Ten beets were selected by their size from each of the 17 accessions and cultivars to represent size distribution. Around five grams of tissue extracted from each root were combined to reach a final total of 50g of sample for each line. Samples were then placed in a blender with

150 ml of distilled water and samples were blended for 1 minute or until liquefied. For each line within each replicate, duplicate aliquots of the beet root mixture were used. The liquefied samples were placed in 15ml centrifuge tubes and were centrifuged for 10 minutes at 4,185g

RCF for 10 minutes. Following centrifugation, samples were tested for their absorbance with a

UV-Vis spectrophotometer. One mL of the samples were diluted with 100 mL with DI water in a

100 ml volumetric flask. Once the samples were diluted, their absorbance were recorded for both wavelengths for betanin and vulgaxanthin.

Cycle 5 analysis CIE L*a*b* values were obtained during the fall of 2019 using a BC-10 colorimeter

(Konica), from beets freshly picked from the field. The values used in analysis were the mean

L*a*b* data obtained from each half-sib family that were selected for vernalization in the fall of

2019. Ten roots from each of the 159 half-sib families were quantified by colorimeter of which only six roots were retained and vernalized in cold storage. Data consisted of the L*a*b* values of the ten roots from all 159 half-sib families in three reps.

The six roots from each half-sib family in reps 1 and 2 were placed in brown paper bags with cedar shavings and stored at 4°C until processed for the spectrophotometer analysis. We performed spectral data analysis using a Genesys 20 UV-Vis Spectrophotometer (Thermo

Scientific) from January -February 2020 at Oregon State University. We analyzed 318 half-sib families and 5 commercial cultivars used as checks. The protocol used for extraction of betalains was that of Von Elbe (1980) with some minor adjustments. From each of six roots within a half- sib family, tissue was cut from the exposed cheek of each root, weighed to obtain 50g of sample, then chopped into cubes and samples from the six roots were mixed. Samples were then placed in a blender with 200ml of distilled water and were macerated for 1 min or until liquefied. For each family within each replicate, duplicate aliquots of the beet root mixture were used. The

liquid was transferred into 1.5 ml centrifuge tubes and was centrifuged for 15 mins at 1,950g

RCF in an Eppendorf 5417C with a 30 slot rotor. When not actively being used, liquid samples were kept in a cooler and covered to reduce the heat- and light-mediated degradation of betalains. After centrifugation, samples were transferred into 50 ml volumetric flasks, and the mass was recorded. Samples ranged 0.48-0.52g in weight. Samples were diluted with 50 ml of purified water and were shaken to mix thoroughly. The diluted solution was placed into cuvettes and their absorbance at 476 - 485nm for vulgaxanthin and 530 - 538nm for betanin was measured.

Betalain calculation For betanin we obtained the mean of the duplicate samples. Betanin % is obtained by dividing the absorbance at the wavelength (530-538) with amount concentration of the solution multiplied by extinction coefficient of 1% solution of betanin in a 1cm cell (Wyler and Dreiding,

1957; Piattelli and Minale, 1964). To obtain a visual reference of light absorption at wavelengths from 400 to 600nm, we analyzed four samples with L* of 18 to 21 by spectrophotometry (Figure

3.1).

The calculation of absorption for betaxanthins and betacyanins can be determined by the following equations (Wyler and Dreiding, 1957; Piattelli and Minale, 1964). We also calculated betanin %, E value and color shade for each beet sample using the following equations:

Betanin or vulgaxanthin = 1Ab* A1%/ c 1Ab= light absorbance at nm c= concentration A1%= extinction coefficient of 1% solution (1g/100ml) betanin- A1%= 1120 vulgaxathin- A1% 750.

1 Color shade = ( Ab538/Ab476)

E value was calculated using the Beer-Lambert law discovered Pierre Bouguer in 1729

(Swinehart, 1962):

E=Molar absorptivity E=1Ab/c*L c=Concentration L=Path length

Calculations were performed in Excel.

Data Analysis We examined the correlation between the half-sib family means of L* and betanin % for all families. We converted the betanin % to mg/100g fresh weight (FW).

Person’s multiple correlation (SAS v9.4) was used to compare L* and betanin % for the two reps of Cycle 5 and the initial cycle 1 population. The comparison between cycle 1 and cycle

5 correlation coefficient provided insight into the strength of the correlation between L* and betanin, and positive or negative sign provided insight as to whether the relationship was positively or negatively correlated.

- TBcheck1

- TB66-1-4

- TBCheck3

- TBcheck5

Figure 3.1. Absorbance across wavelengths from 400 – 600 nm for four table beet samples with different L* values

Results Two reps of the 2019 population consisting of 159 samples were analyzed by UV-Vis spectrophotometry to quantify the betanin content and evaluate correlation between spectral data and colorimetric data. Samples from different families produced various shades of red during the dilution of water and beet sample due to different amount of betanin (Figure 3.2). Half-sib families with higher concentration of betanin produced darker shaded colored samples. The sample of the TB 45-2-5-6 half-sib family had the darkest tint of red compared to the other three half-sib families, and had the highest betanin mean concentration of 0.35% (Figure 3.2). Beets with lighter tones can be distinguished visually and the visual appearance is associated with light absorbance at the 530-538nm wavelength and concentration of betanin.

20A 21A 22A 23A Sample Name Betanin % 20A TB18-4-1-5 0.28 21A TB20-1-5-3 0.25 22A TB45-2-6-5 0.35 23A TB82-5-5-3 0.24 Mean 0.29

Figure 3.2: Four selected samples of table beet extract after dilution (left). Samples with lighter shade of color had a lower betanin absorbance as quantified by UV-Vis spectrophotometry as shown in the table to the right.

Both betaxanthins (vulgaxanthin) and betacyanins (betanin) were quantified using the duplicate samples and averaging for every half sib lines for all the parameters. For reps one and two, there appeared to be significant differences (P ≤ 0.001) in absorbance of betanin (Table

3.2). Vulgaxanthin at 476-485nm had absorbance equivalent to 251 mg/ 100g FW for rep one and 238 mg/ 100g FW for rep two (Table 3.1). Betanin and vulgaxanthin were expressed as mg/100g FW to correlate findings with findings with other articles relating to the topic. The reps

of 2019 population for betanin % were significantly different (P ≤ 0.001); rep one betanin equivalent of 370 mg/ 100g FW and rep two at 388 mg/ 100g FW (Table 3.2). Ideally, one would want a higher absorbance at each wavelength for betanin compared to vulgaxanthin, and betanin was higher in this case. Such a betanin:vulgaxanthin ratio would be expected to produce higher concentration of red-purple colored betalains whereas a low betanin:vulgaxanthin ratio would be produced by roots with an orange-red color (Nilsson, 1970). Both reps had a higher betanin:vulgaxanthin ratio. The difference between betanin and vulgaxanthin absorbance equivalents was 119 mg/ 100g FW for rep one and 150 mg/ 100g FW for rep two. Betanin had significant differences (P ≤ 0.001) among families (Table 3.2). Rep two beets had a higher value for betanin, a darker red pigmentation and lower L* than rep one (L* = 18.7 for rep two, 19.2 for rep one). There appeared to be no significant differences for vulgaxanthin concentration between reps and families were not significantly different in vulgaxanthin content.

Table 3.1. UV- Vis spectrophotometer analysis of betalains from a table beet population selected for high betanin and low TDS.

Rep1 Rep2 L* 19.2 18.7 Betanin range 70-496* 094-517* Betanin mean 237* 272* 1 Betaxanthins (vulgaxanthin) A476-485 251* 238* Betacyanins (betanin) A530-538 370* 388* E value 1.460 3.050 Color shade 0.619 0.615 *mg/100g FW 1A- absorption

Table 3.2. Mean square and probability for betanin and E value from an ANOVA of a table beet family selected for high betanin and low TDS.

z Mean Square Source df E value Betanin Vulgaxanthin Color shade Rep 1 1.28*** 82578*** 11931.9 0.48*** Family 18 1.15*** 28643*** 7909.2 0.03 Error 253 0.61 4613 4491.2 0.04 zStatistically significant at: *** P<0.001.

We used the Beer-Lambert Law to determine the E value, the molar absorption coefficient, which indicates how strong the absorption of the concentration is at a particular wavelength (Swinehart, 1962) E values were calculated to determine how strong the absorption of betanin is at the wavelength of 530-538nm. The average of the E values for the rep main effect varied significantly, with E values of 1.46 and 3.05, respectively. The absorption was stronger in rep two for betanin, and aligns with other findings of the difference between reps.

The color shade is the ratio of absorbance of betanin to vulgaxanthin; the two reps had similar color shades (0.619 and 0.615). The color shade of the beets indicates the hue of the substances.

There was no significant difference (P ≤ 0.001) among families of the cycle 5 population for color shade. Both the reps are producing beets of a similar dark red-purple color, but the absorbency of betanin differs.

Betanin concentration increased as the L* decreased. At L* of 22 -24, the betanin concentration was at its lowest levels of 0.070 - 0.025%. To verify that betanin content and L* concentration were inversely proportional, the CIE L*a*b* values and the spectral betanin data were subjected to correlation analysis. The correlation of L* and betanin was -0.19, and there were significant differences (P ≤ 0.01) (Table 3.3). As L* decreases (darker pigmentation), 59

betanin increases. In addition, a* and betanin were also inversely proportional; as the population shifted to a lower a* (red trending towards green) betanin concentration increased. The variable b* (blue - yellow) was directly proportional to betanin concentration. The a* and b* values converted to chroma and hue angle to understand the color and saturation of the color. Both chroma and hue angle for the beet population demonstrated a mean purple-red color; as the chroma and hue angle increase, the purple-red saturation and betanin concentration both increase. The correlation coefficients for both hue angle and chroma were significant at P ≤

0.001. The chroma and hue angle of the 2019 population confirms that betanin concentration was highly associated with purple-red color. CIE L*a*b* values are a group of parameters that should be valued as a whole, and not on their own to interpret color. Together, the CIE L*a*b* of cycle 5, displayed a dark-shaded purple-red group in both reps. One can infer that betanin concentration is higher when CIE L*a*b* moves towards darker purple-red color in colorimetric scale.

Linear regression revealed that betanin and L* had a weak association (R2 = 0.035) but the regression line was not significantly different from zero rejecting the null hypothesis. (Figure

3.3). The initial germplasm had a stronger association of L* and betanin, having a correlation coefficient of -0.69 (Table 3.4). The difference between cycles 1 and 5 might have been due to sampling procedures.

Table 3.3. Pearson correlation coefficients for CIE L*a*b*, hue angle, chroma and betanin % from a table beet population selected for high betanin and low TDS. Data is from five cycles grown in the field at the OSU Vegetable Research farm in 2019. Prob > |r| under H0: Rho=0.

L a b Hue angle Chroma Betanin % a 0.67***z 1 b -0.36*** -0.03 1 Hue angle -0.55*** -0.3*** 0.94*** 1 Chroma 0.66*** 0.99*** -0.00 -0.32*** 1 Betanin % -0.19** -0.16** 0.18** 0.22*** -0.16** 1 TDS 0.03 -0.07 -0.13* -0.09 -0.08 -0.01 -zStatistically significant at: *** P<0.001, ** P<0.01, * P<0.05

Table 3.4 Pearson correlation coefficients for CIE L*a*b*, TDS, pH and betanin % from a table beet base-population grown in 2014 and selected for high betanin and low TDS for the initial germplasm. Prob > |r| under H0: Rho=0. L* a* b* TDS % pH Betanin % a* -0.76***z 1 b* 0.26 -0.60** 1 TDS % 0.47* -0.20 -0.25 1 pH 0.14 -0.38 0.50 0.10 1 Betanin % -0.69** 0.29 0.19 -0.63** -0.03 1 Color Shade 0.63** -0.45 0.45 0.24 0.13 -0.49* zStatistically significant at: *** P<0.001, ** P<0.01, * P<0.05

600.0

500.0

400.0

300.0

200.0 R = 0.035 Betanin mg/100g Betaninmg/100g FW 100.0

0.0 15.00 17.00 19.00 21.00 23.00 25.00 27.00 L*

Figure 3.3 Regression of L* and betanin mg/ 100g FW of a table beet population selected over five cycles for increased betanin content. Data are means of two replicates from cycles combined with the population grown in the field at the OSU Vegetable Research farm in 2019.

Betanin concentration was an important variable for selection for the final cycle of mass selection. Eight half-sib families had greater betanin concentration compared to two F1 hybrid commercial cultivars used by the food processing industry. Pablo had a mean betanin content of

122 mg/100g FW, and Akela with 115 mg/100g FW, while eight half-sib families had mean betanin of 358 mg/100g FW. The half-sib families with the lowest betanin concentration were related to their L* and total dissolved solids (TDS) in a biplot of L* and TDS (Figure 3.4). The eight half-sib families had L* of 17 - 19 meeting the range of the L* values parameter designated for selection. The remaining 38 half sib lines either had equivalent or even lower betanin concentration compared to the F1 hybrid commercial cultivars. The half sib lines had means of around 46.6 mg/100g FW betaxanthins and 201 mg/100g FW betacyanin.

15 Family averages - 2019

14 Check TB1 13 TB45-2-1-1 TB63-1-4-2 12 TB66-1-6-2 TB18-4-2 11 TB82-5-5-1

10 TB63-1-4-1 Brix (%) 9 TB55-5-2 8 TB33-2-4-5 Check TB5 7 6 17 17.5 18 18.5 19 19.5 CIE L*

Figure 3.4 Eight table beet lines with CIE L* values of 19.5 or lower and TDS of 12% shown with red labels. The mean betanin content of these lines was 358 mg/100g FW. Two commercial cultivars used as checks are labeled in purple.

Discussion and Conclusion Data from the UV-Vis spectrophotometry and CIE L* colorimetric suggest that there is weak negative association between L* and betanin content. Similar studies have found CIE L* and betanin having a negative partial regression coefficient of 0.95 a for commercial beet powders (Cohen and Saguy, 1982).Others have found a high correlation between colorimetric data and secondary metabolic compounds (Watuda, 1975; Eagerman et al., 1973; Eagerman,

1978).

The weak correlation in our half-sib population could be due to the samples being in cold storage for around 6-8 weeks with the cut on roots potentially allowing oxidation of betalains before spectral analysis. Betalains can be oxidized by hydrogen peroxide and polyphenol oxidase. Pedreno and Escribano (2000) examined how oxidation affects betalains, and found that betacyanins decreased in absorbance by three times compared to betaxanthins. Both betalains showed a decrease in absorbance level, with the rates differing due to their chemical structure.

Colorimeter data was taken from the fresh incision made on the same day, while UV-Vis spectrophotometry was done 6 - 8 weeks after the incision, potentially allowing for oxidation of betalains. The fact that colorimetry and spectral data were taken at different times from the beet roots may have weakened the correlation between L* and betanin. Studies have also shown that betacyanin becomes decarboxylated and dehydrogenated as the beet tissues age (Sawicki et al.

2016). Temperature and storage affects the stability of betalains; at room temperature, betalains degrades at 13 days, however, the two betalain alkaloids degrade at different rates, betacyanins begin to degrade in 10 days (Caldas-Cueva et al., 2015; Jagannath et al., 2015). Perhaps, if the beet samples were analyzed on the same day for both UV-Vis spectrophotometry and colorimetry, the correlation between and betanin and L* may have been stronger. Even though the correlation was weak compared to Cohen and Saguey (1982) experiment, the same trend of a

negative correlation between L* and betanin was observed, indicating that as L* decreases, betanin concentration increases in table beets.

The difference between cycles 0 and 5 might have been due to sampling procedures.

While both cycles were grown under the same environmental conditions, cycle 5 roots were stored in the cooler for 8-9 weeks with a vertical cut that may have allowed oxidation of the betalains to penetrate into the root and alter the color. Cycle 0 roots were harvested and analyzed soon after, which would have limited oxidation. This may have contributed to the weak association of the L* and betanin observed in the 2019 data.

L* value only consider lightness color, which could cause a slight limitation to consider for an association between UV-Vis Spectrophotometer and L* value. Chroma the other hand take in consideration the color saturation of the half sib families. Betanin and chroma had a high correlation coefficient of r2 0f 0.66. Because chroma may have a high correlation coefficient with betanin, more research on chroma and betanin should be studied.

The two reps had significant differences between the concentrations of betanin.

Betacyanins and particularly betanin development is dependent on the length of the growing period. Betacyanin concentration is at its maximum in late summer and then remains constant

(Nilsson, 1973). Both of these replicates were harvested around the first 2½ weeks of September.

The first rep was harvested first and perhaps the earlier harvest of this rep resulted in having a lower concentration of betanin compared to rep two. The extra week of betacyanin development of betanin in rep two may have contributed to the higher absorbency of betanin in this rep.

Overall, the population had similar ratios of betanin/vulgaxanthin, resulted in significant differences between the reps, however not among families. The stable color shade in the half-sib families is perhaps due to the fact that all roots were around the same age and experienced the

same temperature at the OSU Vegetable Farm. The alkaloid group of betalains can be influenced by range of seasonal change, growing period, and the age of the plant (Watson and Gabelman,

1982; Von Elbe, 1978; Nilsson, 1973; Sapers and Hornstein, 1979). In both colorimetry and spectrophotometry, color shade and Royal Horticultural Society groups indicated that the population had red- purple colored table beets suggesting that both of these parameters had a consistent interpretation of beet root color. Cycle 5 half-sib families had greater absorbency for betanin, indicating that this cycle had higher levels than the initial germplasm.

Vulgaxanthin concentration in the 2019 population were around 250mg/100g FW, and if vulgaxanthin is increasing, it may explain our paradoxical shift in a* and b*; the beets have become less red and less blue. To confirm a shift to higher vulgaxanthin concentration, all cycles should have been evaluated. For this research only cycle 6 was evaluated for vulgaxanthin, and one cannot say with certainty that there has been increase or shift of betalain composition.

However, in the Royal Horticultural Society color analysis, the populations have shifted to a grey group, and there was shift to our a* value decreasing and b* value increasing. As a* and b* shifted they caused the hue angle and chroma to shift as well for these parameters are dependent on a* and b* values. This may have caused the population to have a greyed group Red purple color tone. Table beet color is controlled by the R and Y locus (Wolyn and Gabelman, 1990a).

Dominant R produces the red color, but Y allele controls the intensity of the pigmentation, RRyy are a pale red compared to those with RRYY. The Y locus also codes for the yellow color in table beets, as our a* and b* have shifted to less red and less blue, we may unknowingly selected for dominant Y at the Y locus through the 5 cycles of recurrent selection.

As a food colorant the optimal time to harvest the table beets would be as late as possible

(Nilsson, 1973) when betanin is at a maximum in the late summer. Recurrent selection for lower

L* has resulted in a few lines of interest to continue future breeding for higher betalains for the food industry.

Chapter 4 General Conclusion Through the five cycles of half sib mass selection, the base population has shifted to a darker red-purple pigmentation. Pigmentation in beets is correlated with the secondary compounds betalains, which contain two 2 alkaloid groups: betaxanthin produces yellow and betacyanin produces red color. Betalains are primarily used as natural food colorants in the food processing industry. Table beets are the only FDA approved crop for the use of betalains. Table beets with higher concentration of betalain would be beneficial for the food processing industry.

Selection for lower CIE L* value resulted in a steady decrease throughout the cycles of selection, resulting in dark pigmented red color in the table beet population. The finding of a high narrow- sense heritability for pigmentation supports the finding of a steady increase of betalain with constant selection of pigmentation. Selection for low CIE L* value has proven to be effective in developing half-sib families with desirable colors for the food processing Industry. Selection for darker pigmentation may continue, for we have not seen color plateauing indicating. Using the colorimeter is a feasible approach to develop lines with the objective of color enhancement in a crop because of its speed and ease to use.

Sugars reduce efficiency of the extraction process and those wanting high pigment beets for food colorant extraction desire low total dissolved solids (TDS). Selecting simultaneously for lower TDS and higher betalains was not successful. When analyzed by year, the half-sib population appeared to have a steady increase over the five cycles, however, in the 2019 field trials where all cycles were grown together in the same environment, we determined that all cycles had around the same average TDS. Thus, TDS seems not to have changed during selection. Sugar concentration is influenced by the production practices such as length of the growing period being positively correlated with sugar production in table beets. Size of the beet

root is also correlated in part to length of growing season. During the cycles of production, roots may have been harvested at different sizes and different durations of growth, which may explain why TDS was variable on a per year basis, but every cycle had similar TDS means in the 2019 field trial. In addition, betalains are glycosylated and Wolyn & Gabelman (1990) suggested that there is a certain minimum level below which TDS was metabolically impossible to reach. The maintenance of TDS despite selection for reduced levels may be a response to simultaneous selection for higher concentration of betalains. However, there were a few lines with significantly lower TDS percent, suggesting that it might be possible to lower TDS. Low hereditability of TDS indicates that the trait may lack genetic variability and/or have a strong environmental component. Because of low heritability, further research conducted on selection for reduced sugar concentration in table beets should be conducted in replicated trials.

Lastly, we examined colorimeter and UV-Vis spectrophotometer data to verify correlation between L* and betanin, but L* and betanin had only a weak correlation for the 2019 half-sib population. This is at odds with what others have found. There may be a discrepancy in our data due to the colorimeter and spectral data being acquired at different times. The spectral data may have been obtained as betanin was degrading during storage. The degradation of betanin in the half-sib families might have caused a reduction of the concentration and a weakening of the correlation between L* and betanin %. Perhaps if the UV-vis spectrophotometer and colorimeter data were gathered on the same day for half-sib families, the correlation might have been stronger. The evaluation of six selected lines from the 2019 population did show a strong relationship between colorimeter and spectrophotometer data.

Selection for low L* has proven to result in desirable lines with higher concentrations of betalains than existing commercial cultivars. The use of the colorimeter for selection can readily

increase productivity in color selection in plant breeding. The colorimeter tool saves time and resources compared to the UV-Vis spectrophotometer, making it an ideal tool to quantify color parameters in crops.

The OSU half sib population produced desirable L* value and TDS through open- pollination recurrent selection. Recurrent selection in half sib families has proven to be an effective approach to select for pigmentation in table beets. Selection for pigmentation using the

OSU half sib population could possibly lower their L* values and produce darker red pigmented beets after more cycles using the same protocol of selection. The population may go through quite more cycles of recurrent selection until R locus becomes fixed. As mentioned, Goldman et al (1996) performed 8 cycles of recurrent selection for Table Beets and 20% increase of pigmentation, and the pigmentation has not plateaued.

Even though a few lines developed low TDS in the OSU half sib population, the protocol used may not be a good fit for selecting for sugar concentration in table beets. Sugar production appears to be a more quantitative trait, and the current structure of selection was not suitable for making gain from selection. Selecting for sugar concentration may need a more sophisticated replicated structure to evaluate for environmental factors and show significance of sugar concentration in table beets.

Another approach would be to use the best lines discovered during this process, and create F1 hybrids or synthetic lines. The best lines, being those with lowest L*, highest betanin concentration will need to go through additional rounds of self-pollination to stabilize the genes and either cross two parents make an F1 hybrid or use multiple partially inbred parents to eventually develop a synthetic population. Creating F1 hybrids or synthetic lines would be

preferred by the food processing Industry as these cultivars will be advantageous in their uniformity and vigor in the desired trait of higher betalain concentration.

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Appendix A Colorimeter L* value Data on Half-Sib Families and Checks

Table A.1 Colorimeter CIE L* value Ls means for 2019 Half-Sib lines P values for tests of Ls means equal to Ls means of check varieties.

TB01 TBCheck TBCheck TBCheck TBcheck i/j 0 TB018 TB020 4 5 1 2 TB010 0.89 0.44 0.00 0.56 0.13 0.57 TB018 0.89 0.25 0.00 0.46 0.08 0.47 TB020 0.44 0.25 0.01 0.26 0.28 0.27 TBCheck4 0.00 0.00 0.01 0.00 0.11 TBCheck5 0.56 0.46 0.26 0.00 0.10 0.99 TBCheck1 0.13 0.08 0.28 0.11 0.10 TBCheck2 0.57 0.47 0.27 0.00 0.99 0.10 TBCheck3 0.97 0.97 0.66 0.02 0.61 0.28 0.62 TB022 0.42 0.29 0.90 0.01 0.25 0.35 0.26 TB024 0.53 0.38 0.85 0.01 0.30 0.23 0.31 TB029 0.09 0.00 0.20 0.03 0.09 0.69 0.10 TB033 0.27 0.07 0.65 0.01 0.18 0.41 0.19 TB034 0.00 <.0001 0.00 0.47 0.00 0.17 TB045 0.09 0.00 0.19 0.03 0.09 0.67 0.10 TB055 0.16 0.03 0.36 0.03 0.13 0.61 0.13 TB063 0.89 0.98 0.34 0.00 0.47 0.09 0.47 TB066 0.51 0.24 0.79 0.00 0.29 0.20 0.30 TB073 0.86 0.92 0.33 0.00 0.45 0.09 0.46 TB082 0.04 0.00 0.08 0.06 0.06 0.99 0.06 TB086 0.96 0.74 0.23 0.00 0.55 0.07 0.55 TB087 0.09 0.00 0.18 0.02 0.09 0.66 0.10 TB088 0.36 0.20 0.80 0.01 0.22 0.39 0.23 TB100 0.15 0.04 0.33 0.04 0.12 0.71 0.12 TB101 0.99 0.93 0.62 0.02 0.64 0.26 0.65 TB102 0.59 0.49 0.28 0.01 0.97 0.10 0.98 TB103 0.80 0.71 0.44 0.01 0.79 0.18 0.80 TB104 0.99 0.93 0.62 0.02 0.64 0.26 0.65 TB105 0.85 0.77 0.49 0.01 0.75 0.20 0.76 TB128 0.06 0.00 0.12 0.07 0.06 0.99 0.06 TB155 0.56 0.42 0.81 0.01 0.32 0.22 0.32

Table A.1 continued. Colorimeter CIE L* value Ls means for 2019 Half-Sib lines P values for tests. of Ls means equal to Ls means of check varieties i/j TBCheck3 TB022 TB024 TB029 TB033 TB034 TB045 TB055 TB010 0.97 0.42 0.53 0.09 0.27 0.00 0.09 0.16 TB018 0.97 0.29 0.38 0.00 0.07 <.0001 0.00 0.03 TB020 0.66 0.90 0.85 0.20 0.65 0.00 0.19 0.36 TBCheck4 0.02 0.01 0.01 0.03 0.01 0.47 0.03 0.03 TBCheck5 0.61 0.25 0.30 0.09 0.18 0.00 0.09 0.13 TBCheck1 0.28 0.35 0.23 0.69 0.41 0.17 0.67 0.61 TBCheck2 0.62 0.26 0.31 0.10 0.19 0.00 0.10 0.13 TBCheck3 0.62 0.72 0.32 0.52 0.02 0.33 0.39 TB022 0.62 0.78 0.35 0.79 0.00 0.35 0.51 TB024 0.72 0.78 0.15 0.52 0.00 0.13 0.28 TB029 0.32 0.35 0.15 0.41 0.01 0.95 0.82 TB033 0.52 0.79 0.52 0.41 0.00 0.41 0.62 TB034 0.02 0.00 0.00 0.01 0.00 0.00 0.01 TB045 0.33 0.35 0.13 0.95 0.41 0.00 0.84 TB055 0.39 0.51 0.28 0.82 0.62 0.01 0.84 TB063 0.97 0.35 0.46 0.01 0.13 <.0001 0.01 0.06 TB066 0.72 0.73 0.97 0.06 0.41 <.0001 0.04 0.19 TB073 0.95 0.35 0.47 0.01 0.12 <.0001 0.00 0.05 TB082 0.22 0.17 0.06 0.51 0.18 0.04 0.45 0.43 TB086 0.94 0.25 0.32 0.01 0.08 <.0001 0.00 0.03 TB087 0.33 0.35 0.13 0.94 0.40 0.00 0.99 0.85 TB088 0.57 0.91 0.67 0.41 0.89 0.00 0.41 0.58 TB100 0.35 0.45 0.26 0.99 0.55 0.02 0.98 0.87 TB101 0.97 0.59 0.68 0.30 0.48 0.02 0.30 0.36 TB102 0.64 0.27 0.33 0.10 0.20 0.00 0.10 0.14 TB103 0.81 0.42 0.50 0.19 0.33 0.01 0.19 0.24 TB104 0.97 0.59 0.68 0.30 0.48 0.02 0.30 0.36 TB105 0.85 0.47 0.55 0.21 0.37 0.01 0.22 0.27 TB128 0.23 0.21 0.09 0.56 0.23 0.06 0.51 0.48 TB155 0.74 0.74 0.96 0.13 0.48 0.00 0.12 0.26

Table A.1 continued. Colorimeter CIE L* value Ls means for 2019 Half-Sib lines P values for tests of Ls means equal to Ls means of check varieties. i/j TB063 TB066 TB073 TB082 TB086 TB087 TB088 TB100 TB010 0.89 0.51 0.86 0.04 0.96 0.09 0.36 0.15 TB018 0.98 0.24 0.92 0.00 0.74 0.00 0.20 0.04 TB020 0.34 0.79 0.33 0.08 0.23 0.18 0.80 0.33 TBCheck4 0.00 0.00 0.00 0.06 0.00 0.02 0.01 0.04 TBCheck5 0.47 0.29 0.45 0.06 0.55 0.09 0.22 0.12 TBCheck1 0.09 0.20 0.09 0.99 0.07 0.66 0.39 0.71 TBCheck2 0.47 0.30 0.46 0.06 0.55 0.10 0.23 0.12 TBCheck3 0.97 0.72 0.95 0.22 0.94 0.33 0.57 0.35 TB022 0.35 0.73 0.35 0.17 0.25 0.35 0.91 0.45 TB024 0.46 0.97 0.47 0.06 0.32 0.13 0.67 0.26 TB029 0.01 0.06 0.01 0.51 0.01 0.94 0.41 0.99 TB033 0.13 0.41 0.12 0.18 0.08 0.40 0.89 0.55 TB034 <.0001 <.0001 <.0001 0.04 <.0001 0.00 0.00 0.02 TB045 0.01 0.04 0.00 0.45 0.00 0.99 0.41 0.98 TB055 0.06 0.19 0.05 0.43 0.03 0.85 0.58 0.87 TB063 0.38 0.96 0.00 0.76 0.01 0.27 0.07 TB066 0.38 0.37 0.02 0.24 0.04 0.61 0.19 TB073 0.96 0.37 0.00 0.71 0.00 0.26 0.06 TB082 0.00 0.02 0.00 0.00 0.43 0.19 0.60 TB086 0.76 0.24 0.71 0.00 0.00 0.18 0.04 TB087 0.01 0.04 0.00 0.43 0.00 0.41 0.97 TB088 0.27 0.61 0.26 0.19 0.18 0.41 0.51 TB100 0.07 0.19 0.06 0.60 0.04 0.97 0.51 TB101 0.92 0.68 0.91 0.20 0.98 0.30 0.54 0.33 TB102 0.49 0.32 0.48 0.06 0.58 0.10 0.24 0.13 TB103 0.71 0.49 0.69 0.12 0.80 0.19 0.38 0.22 TB104 0.92 0.68 0.90 0.19 0.98 0.30 0.54 0.33 TB105 0.77 0.54 0.75 0.14 0.86 0.22 0.42 0.24 TB128 0.01 0.05 0.01 0.99 0.01 0.50 0.24 0.63 TB155 0.50 0.98 0.50 0.05 0.35 0.11 0.64 0.24

Table A.1 continued. Colorimeter CIE L* value Ls means for 2019 Half-Sib lines P values for tests of Ls means equal to Ls means of check varieties. i/j TB101 TB102 TB103 TB104 TB105 TB128 TB155 TB010 0.99 0.59 0.80 0.99 0.85 0.06 0.56 TB018 0.93 0.49 0.71 0.93 0.77 0.00 0.42 TB020 0.62 0.28 0.44 0.62 0.49 0.12 0.81 TBCheck4 0.02 0.01 0.01 0.02 0.01 0.07 0.01 TBCheck5 0.64 0.97 0.79 0.64 0.75 0.06 0.32 TBCheck1 0.26 0.10 0.18 0.26 0.20 0.99 0.22 TBCheck2 0.65 0.98 0.80 0.65 0.76 0.06 0.32 TBCheck3 0.97 0.64 0.81 0.97 0.85 0.23 0.74 TB022 0.59 0.27 0.42 0.59 0.47 0.21 0.74 TB024 0.68 0.33 0.50 0.68 0.55 0.09 0.96 TB029 0.30 0.10 0.19 0.30 0.21 0.56 0.13 TB033 0.48 0.20 0.33 0.48 0.37 0.23 0.48 TB034 0.02 0.00 0.01 0.02 0.01 0.06 0.00 TB045 0.30 0.10 0.19 0.30 0.22 0.51 0.12 TB055 0.36 0.14 0.24 0.36 0.27 0.48 0.26 TB063 0.92 0.49 0.71 0.92 0.77 0.01 0.50 TB066 0.68 0.32 0.49 0.68 0.54 0.05 0.98 TB073 0.91 0.48 0.69 0.90 0.75 0.01 0.50 TB082 0.20 0.06 0.12 0.19 0.14 0.99 0.05 TB086 0.98 0.58 0.80 0.98 0.86 0.01 0.35 TB087 0.30 0.10 0.19 0.30 0.22 0.50 0.11 TB088 0.54 0.24 0.38 0.54 0.42 0.24 0.64 TB100 0.33 0.13 0.22 0.33 0.24 0.63 0.24 TB101 0.67 0.84 1.00 0.88 0.21 0.70 TB102 0.67 0.82 0.67 0.78 0.07 0.34 TB103 0.84 0.82 0.84 0.96 0.13 0.51 TB104 1.00 0.67 0.84 0.89 0.21 0.70 TB105 0.88 0.78 0.96 0.89 0.15 0.56 TB128 0.21 0.07 0.13 0.21 0.15 0.08 TB155 0.70 0.34 0.51 0.70 0.56 0.08