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Home , Alpinia galanga, Alpinia zerumbet, Curcuma amada, Galangal, Kaempferia galanga

ENVIRONMENTAL CONDITIONS, CULTIVAR, AND PROPAGATION MATERIAL AFFECT RHIZOME YIELD AND POSTHARVEST QUALITY OF GINGER (ZINGIBER OFFICINALE ROSC.), GALANGAL (ALPINIA GALANGA LINN.), AND TURMERIC (CURCUMA SPP.)

SOFIA JESUS FLORES VIVAR

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2019

To my parents, siblings, and plant friends

ACKNOWLEDGMENTS

I would like to thank Dr. Rosanna Freyre and Dr. Paul Fisher for giving me the opportunity to work for their program initially as a Research Visiting Scholar. After that first experience, they let me continue working and guided me through my masters’ studies. I thank

Dr. Sargent for his advice and help during my postharvest evaluations.

Special thanks to present and past students from Dr. Fisher and Dr. Freyre’s labs for their help and support. Thanks to Victor Zayas, Erin Yafuso, Ulrich Adegbola, George Grant,

Jonathan Clavijo, Henry Kironde, and Nicholas Genna for being amazing friends. I thank Brian

Owens, Mark Kann and their teams for always being there to help me in the greenhouse and field. I would like to thank Dr. Pearson and his team from Mid-Florida Research and Education

Center in Apopka for letting me work in their lab and providing instruction to perform the chemical analyses of my plants. I thank Dr. Rathinasabapathi for his advice and willingness to help me with the analysis of my rhizomes. I would like to thank Dr. Gomez for her friendship and unwavering support. Thanks to James Colee from UF Agriculture Statistics for support with statistical analysis.

This research project was supported by the Floriculture Research Alliance

(FloricultureAlliance.org). I thank Hawaii Clean Seed, LLC. for supplying rhizomes in 2017,

Just Ginger growers for their donation of rhizomes in 2018, and AgriStarts for donating the micropropagated planting material.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 9

LIST OF ABBREVIATIONS ...... 11

ABSTRACT ...... 12

CHAPTER

1 INTRODUCTION ...... 14

2 ANALYSIS OF FACTORS AFFECTING SPROUTING OF GINGER (ZINGIBER OFFICINALE) AND TURMERIC (CURCUMA SPP.) RHIZOMES ...... 17

Background ...... 17 Material and Methods ...... 20 Results and Discussion ...... 23 Summary ...... 26

3 RHIZOME YIELD AND POSTHARVEST QUALITY OF GINGER (ZINGIBER OFFICINALE) AND GALANGAL (ALPINIA GALANGA) AS AFFECTED BY PROPAGATION MATERIAL, CULTIVAR, AND ENVIRONMENTAL CONDITIONS DURING PRODUCTION...... 30

Background ...... 30 Materials and Methods ...... 36 Experiment 1. Greenhouse Experiments ...... 36 Year 1 (2017-2018) ...... 36 Year 2 (2018-2019) ...... 38 Experiment 2. Field Experiment ...... 41 Results and Discussion ...... 43 Experiment 1. Greenhouse Experiments ...... 43 Year 1 (2017-2018) ...... 43 Year 2 (2018-2019) ...... 46 Postharvest Evaluations – Greenhouse Trial – Year 2 ...... 52 Experiment 2 – Field Experiment ...... 54 Postharvest Evaluations – Field Experiment ...... 59 Summary ...... 60

4 INTERACTION OF PROPAGATION MATERIAL, CULTIVAR, AND ENVIRONMENTAL FACTORS ON ORNAMENTAL PLANT PERFORMANCE, RHIZOME YIELD, AND RHIZOME QUALITY OF TURMERIC (CURCUMA SPP.). ....79

Background ...... 79 Materials and Methods ...... 83 Experiment 1. Greenhouse Experiments ...... 83 Year 1 (2017-2018) ...... 84 Year 2 (2018-2019) ...... 85 Experiment 2. Field Experiment ...... 88 Results and Discussion ...... 90 Experiment 1. Greenhouse Experiments ...... 90 Year 1 (2017-2018) ...... 90 Year 2 (2018-2019) ...... 92 Postharvest Evaluations – Greenhouse Trial – Year 1 ...... 99 Experiment 2 – Field Experiment ...... 101 Postharvest Evaluations – Field Experiment ...... 107 Summary ...... 109

5 CONCLUSIONS ...... 128

LIST OF REFERENCES ...... 132

BIOGRAPHICAL SKETCH ...... 145

LIST OF TABLES

Table page

2-1 Mean values and Tukey grouping comparison for days to sprout, number of sprouted buds after 4 and 8 weeks, shoot and root length and final weight of ginger and turmeric rhizomes from different sources in Experiment 1...... 27

2-2 Mean values and Tukey grouping comparison for days to sprout, number of sprouted buds after 4 and 8 weeks, shoot and root length and final weight of ginger and turmeric rhizomes from different sources in Experiment 2...... 28

2-3 Mean values and Tukey grouping comparison for initial weight, initial number of visible buds, initial length and diameter of rhizomes of ginger and turmeric cultivars from different sources in Experiment 1 and 2...... 29

3-1 Mean values and Tukey grouping comparison for plant type, average new shoot number, height increase, number of flowers, overall rating, and SPAD with varying photoperiods in the greenhouse in year 2 (2018-2019)...... 62

3-2 Mean values and Tukey grouping comparison for plant type, storage period, and color parameters of rhizomes grown under natural days in year 2, harvested and stored for 2 weeks...... 63

3-3 Mean values and Tukey grouping comparison for plant type, storage period, and color parameters of rhizomes grown under long days in year 2, harvested and stored for 2 weeks...... 64

3-4 Mean values and Tukey grouping comparison for plant type, average new shoot number, height increase, SPAD, overall rating, and number of flowers of plants grown in the field under full sun and 60% shade in year 2...... 65

3-5 Mean values and Tukey grouping comparison for plant type, storage period storage period, and color parameters of rhizomes harvested from the field, and stored for 2 weeks...... 66

4-1 Mean values and Tukey grouping comparison for plant type, average new shoot number, height, SPAD, overall rating, and number of flowers in the greenhouse under natural and long days in year 2...... 112

4-2 Mean values and Tukey grouping comparison for plant type, storage period, and color parameters of rhizomes grown the greenhouse under natural days in year 2, harvested and stored for 2 weeks...... 113

4-3 Mean values and Tukey grouping comparison for plant type, storage period, and color parameters of rhizomes grown the greenhouse under long days in year 2, harvested and stored for 2 weeks ...... 114

4-4 Mean values and Tukey grouping comparison for plant type, average new shoot number, height, SPAD, overall rating, and number of flowers in the field under full sun or 60% shade...... 115

4-5 Mean values and Tukey grouping comparison for plant type, storage period, and color parameters of rhizomes grown in the field under full sun and 60% shade, harvested and stored for 2 weeks ...... 116

LIST OF FIGURES

Figure page

3-1 Rhizome, shoot and root fresh mass of ginger plants grown in the greenhouse under natural or long days in year 1 ...... 67

3-2 Rhizome fresh mass from ginger plants grown in the greenhouse in year 1...... 68

3-3 Carbohydrate partitioning of ginger plants grown in the greenhouse under natural or long days in year 1...... 69

3-4 Rhizome, shoot, root, and total fresh mass of ginger and galangal plants grown in the greenhouse under natural and long days in year 2...... 70

3-5 Visual scale from 1 to 5 to measure overall aesthetic performance of ginger plants grown in the greenhouse in year 2...... 71

3-6 Rhizome fresh mass from ginger and galangal plants grown in the greenhouse under natural and long days in year 2...... 72

3-7 Carbohydrate partitioning of ginger and galangal plants grown in the greenhouse under natural or long days in year 2...... 73

3-8 Postharvest rhizome weight loss of ginger and galangal plants grown in the greenhouse in year 2, harvested and stored for 2 weeks...... 74

3-9 Decay symptoms of rhizomes harvested in 2019 after 2 weeks of postharvest storage ....75

3-10 Rhizome, shoot, root, and total fresh mass of ginger and galangal plants grown in the field under full sun and 60% shade in year 2...... 76

3-11 Carbohydrate partitioning of ginger and galangal plants grown in the field under full sun and 60% shade in year 2 ...... 77

3-12 Postharvest rhizome weight loss of ginger and galangal plants grown in the field in year 2, harvested and stored for a period of two weeks...... 78

4-1 Rhizome, shoot, and root fresh mass of turmeric plants grown in the greenhouse under natural and long days in year 1 ...... 117

4-2 Carbohydrate partitioning of turmeric plants grown in the greenhouse under natural and long days in year 1 ...... 118

4-3 Rhizome, shoot, root, and total fresh mass from turmeric plants grown in the greenhouse under natural and long days in year 2...... 119

4-4 Rhizome fresh mass from one turmeric plant type grown in 14.5 L, under natural days in the greenhouse from in year 2...... 120

4-5 Rhizome fresh mass from one turmeric plant type grown in 14.5 L, under long days in the greenhouse from in year 2...... 121

4-6 Visual scale from 1 to 5 to measure overall aesthetic performance of turmeric plants grown in the greenhouse under natural and long days in year 2...... 122

4-7 Carbohydrate partitioning from turmeric plants grown in the greenhouse under natural and long days from in year 2...... 123

4-8 Postharvest rhizome weight loss from turmeric plants grown in the greenhouse under natural and long days in year 2, harvested and stored for a period of 2 weeks...... 124

4-9 Rhizome, shoot, root, and total fresh mass of turmeric plants grown in the field under full sun and 60% shade in year 2...... 125

4-10 Carbohydrate partitioning of turmeric plants grown in the field under full sun and 60% shade in year 2...... 126

4-11 Postharvest rhizome weight loss of turmeric plants grown in the field under full sun and 60% shade in year 2, harvested and stored for a period of 2 weeks ...... 127

LIST OF ABBREVIATIONS

ANOVA Analysis of Variance

BA Benzyladenine

BAP Benzylaminopurine

CRF Controlled Release Fertilizer

DLI Daily Light Integral h Hours

HSD Honestly Significant Difference masl Meters above sea level

MAP Modified Atmosphere Packages

PAR Photosynthetically Active Radiation

PGR Plant Growth Regulator ppm Parts Per Million

PSREU Plant Science Research and Education Unit

R:FR Red/far-red ratio

RH Relative Humidity

RLI Relative Light Intensity s Seconds

UF University of Florida

U.S. United States

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

Sofia J. Flores Vivar

August 2019

Chair: Rosanna Freyre Major: Horticultural Sciences

There is economic opportunity for growers in the United States (U.S.) to diversify their current commercial production by introducing ginger (Zingiber officinale Roscoe), galangal

(Alpinia galanga Linn.) or turmeric (Curcuma spp.). These species are considered as high-value crops for Florida due to the pungent spicy taste, fragrant aroma, and medicinal attributes of their rhizomes. These species are excellent candidates as alternative “superfoods” and have gained popularity in recent years. However, due to the lack of standard production guidelines, local production is currently limited. The objectives of this study were to (a) evaluate plant material sources and cultivars for greenhouse and field production of ginger, galangal, and turmeric, and

(b) identify environmental conditions that can extend the growing season of plants by avoiding dormancy during short days and maximize high quality rhizome production.

Rhizome quality is essential for sprouting and they can be dormant at planting, thus propagation is still a challenge for growers. Additionally, storability of rhizomes is limited, affecting their quality. Therefore, a propagation experiment was performed two times to determine whether rhizome quality varies over storage time and if storage affected sprouting. In both experiments,

rhizomes of ginger ‘Bubba baba’ sprouted earlier, had more sprouted buds and grew more than turmeric cultivars (particularly ‘White Mango’ and ‘BKK’). This can be attributed to having the best initial rhizome quality (highest rhizome weight and bud number), while turmeric rhizomes had the lowest quality. When rhizomes of these cultivars and others including micropropagated ginger (ginger “tc”) and turmeric (yellow “tc”) were grown in the greenhouse under two different photoperiods, larger containers (50.5 L) promoted more growth and higher rhizome yield than in smaller containers (5.7 L). The diameter of rhizomes harvested from micropropagated plants in the first year was smaller than that obtained from rhizome-derived plants. In the second year, plants grown under night interruption lighting (long days) had higher new shoot number and shoot fresh mass than under natural daylength. Under natural days, ginger ‘Bubba baba’ and turmeric ‘Black’ had significantly lower yields than those grown under long days. Under both photoperiods, galangal “tc” had the highest rhizome yield. Ginger “tc” first and second generation (“ownrhiz”) had similar high yields, however, the former had smaller rhizome diameter. Turmeric white “tc” had the highest yield under long days, but there was no difference in yield between turmeric yellow “ownrhiz” and yellow “tc” under the two photoperiods. In the field, however, white “tc” had the lowest yield, and yellow “ownrhiz” had higher yield than yellow “tc”. There were no differences in yields in the field for any of the genera between full sun and 60% shade. Results suggest that micropropagation may be more effective to develop clean stock material for seed rhizomes of ginger and turmeric than for attempting maximum rhizome yields during the first year. Also, other shading strategies should be tested to see if rhizome yields can be improved compared to growing in full sun. Overall, research showed that differences in yield depend upon genotype, plant material, and environmental conditions, and an economic analysis is needed to identify the most efficient production conditions.

CHAPTER 1 INTRODUCTION

In the U.S. there is increasing demand for earthy spices such as ginger, galangal, and turmeric. These species belong to the family Zingiberaceae, were domesticated centuries ago and are cultivated in many tropical areas of the world due to their multiple benefits besides cooking.

The rhizomes of these species are claimed to have medicinal anti-inflamatory properties (Rao et al., 2008; Ruby et al., 1995). For instance, consumption of ginger rhizomes is recommended to improve joint health, reduce blood sugar, and treat different types of cancer (Gregory et al.,

2008; Li et al., 2016; Srinivasan et al., 2016). Meanwhile, rhizomes of galangal are widely used in Asian cuisines and often to replace ginger as they also have a pungent, spicy taste and a ginger-like odor (Huang et al., 2018; Tang et al., 2018). Turmeric or Curcuma longa is by far the most important Curcuma species, due to its worldwide use as a spice and coloring agent in cooking (Li et al., 2011; Popuri and Pagala, 2013). Moreover, due to the presence of curcuminoids, turmeric rhizomes also have high medicinal potential (Niranjan et al., 2013; Ruby et al., 1995). For this reason, these three spices are highly demanded in the U.S. Consumption of ginger per capita has increased from about 0.5 kg in 1966 to 1.7 kg in 2015 (Nguyen et al.,

2019). The import value of ginger in the U.S. in 2016 was $67.05 million (Tridge, 2019a) and for turmeric it is $31.6 million and is gradually increasing (Tridge, 2019b; Nguyen et al., 2019).

Therefore, there is a lot of potential for increasing national production of these spices in the country.

Ginger, galangal and turmeric plants are widely cultivated in tropical and subtropical regions including India, China, Nigeria, Indonesia, Bangladesh, and Australia as they grow well in warm and humid climates (Chudiwal et al., 2010; Rafie et al., 2003). In contrast, production in the U.S. has been limited to Hawaii and very few states in the southeast, represented primarily by

small growers that produce conventional and organic products (Calpito et al., 2018; Hayden et al., 2004; Hepperly et al., 2004; Huang, 2016; Hunter, 2018; Rafie et al., 2003; Snyder, 2018).

Due to the similarities in climate requirements for growth, there is an opportunity for growers in

Florida to introduce these species and commercialize them as locally-grown “superfoods”.

However, production guidelines applied to local production in Florida, including planting material for propagation and optimal conditions for field or greenhouse cultivation, are yet scarce. Research on propagation methods is limited by the use of seed rhizomes, and issues related to rhizome dormancy, contamination caused by soil borne pathogens, and rhizome storage have not been fully studied (Chung and Moon, 2011; Dohroo, 1989; Furutani, et al.,

1985; Hepperly et al., 2004; Jayakumar et al., 2001; Paull and Cheng, 2015; Sanewski, 1996). In addition, studies under greenhouse or field environmental conditions comparing growing factors such as container size, photoperiod treatments, use of shading, etc. done in the U.S. are rare and limited to Hawaii (Hepperly et al., 2004; Kratky et al., 2013; Nishina et al., 1992). Considering these limitations, plus the lack of information about the wide range of species and rhizome types of these genera in the market, numerous opportunities exist to refine production systems of these crops in Florida.

The overall objectives of this thesis were to (a) evaluate plant material sources and cultivars for greenhouse and field production of ginger, galangal and turmeric, and (b) identify environmental conditions that extend the growing season of plants by avoiding dormancy during short days and maximizing high quality rhizome production.

In Chapter 2, various cultivars of ginger and turmeric seedrhizomes from two sources were soaked in water or treated with plant growth regulators before planting to study their effect on sprouting. There was also potential to evaluate the effects of initial rhizome quality, which

varied depending on rhizome weight and size (diameter and length), and by the duration of the postharvest storage period.

Chapter 3 aimed to (a) evaluate the effect of different propagation material (rhizome- derived and micropropagated transplants), container size, and photoperiod (natural and long days) on rhizome yield of different ginger cultivars under greenhouse conditions, which was conducted in 2017, “year 1” and (b) evaluate the effect of different propagation materials and environmental conditions (photoperiod or shading treatments) on growth and rhizome yield of different cultivars of ginger and galangal under greenhouse or field conditions.

In Chapter 4, rhizome-derived and micropropagated transplants of different turmeric species and cultivars were compared. Rhizome yield from turmeric plants grown in different container sizes and photoperiods were evaluated under greenhouse conditions in 2017, “year 1”.

The effect of different propagation materials and environmental conditions (photoperiod or shading treatments) on growth and rhizome yield of turmeric plants was evaluated in the greenhouse or field.

For both Chapters 3 and 4, the “year 2” greenhouse experiment was conducted from 2018 to 2019. The photoperiod treatments evaluated were “natural days” and “long days” via night interruption . In the field, the experiment also took place from 2018 to 2019 and the shading conditions were 0% shade (“full sun”) and 60% shade. Plant growth, overall plant performance, and rhizome yield were compared for three types of ginger and one micropropagated galangal.

Separately, these variables were also compared for four rhizome-grown turmerics two micropropagated transplants (white and yellow turmeric), and one second generation yellow turmeric originally obtained from micropropagation.

CHAPTER 2 ANALYSIS OF FACTORS AFFECTING SPROUTING OF GINGER (ZINGIBER OFFICINALE) AND TURMERIC (CURCUMA SPP.) RHIZOMES

Background

Ginger (Zingiber officinale) and turmeric (Curcuma spp.) are usually propagated through vegetative rhizome pieces, with adventitious roots and lateral shoots emerging from their nodes

(Hayden et al., 2004). Rhizomes are underground storage organs that remain dormant in the soil and allow ginger and turmeric plants to survive during adverse periods (Abelenda and Prat,

2013). Along with other compounds, starch is one of the main metabolic products stored in this organ, which serves as an energy store for germination and rooting (Ravindran and Babu, 2005).

In ginger, starch can constitute up to 60% of total mass (Talele et al., 2015), while for turmeric species starch levels (dry weight basis) range from 45.2% to 48.5% (Sajitha and Sasikumar,

2014)

After harvest, undamaged and well-developed rhizomes are selected and sanitized for preserving as seed (Ravindran et al., 2007). Ginger and turmeric rhizomes are usually stored in well-ventilated containers at 12 to 14 °C with 60% to 70% relative humidity (RH). For ginger, temperature should not be lower than 12 °C as rhizomes are chilling sensitive (Paull and Cheng,

2015). Overall, rhizomes can be stored for 90 and up to 105 days in ventilated polythene bags

(Ravindran and Babu, 2005). A high percentage of healthy rhizomes was recovered, and high sprouting (99%) was observed in the field (Ravindran et al., 2007). However, storability of rhizomes is limited, and if they are stored for long periods they encounter issues related to weight loss and decreased starch content (Chung and Moon, 2011). Paull et al. (1988) observed that shriveling becomes evident after a 10% of weight loss in ginger rhizomes. Furthermore, ginger rhizomes stored at 10 and 15 °C for five months had a weight loss of about 23% and a rapid reduction in starch content after the two first months of storage (Shukor et al., 1986). In

contrast, optimum and uniform germination was observed when seed rhizomes were stored for

30 and 45 days before planting in the field (Kerala Agricultural University, 1993). It is important to consider these issues related to long-term storage of fresh rhizomes because they lose quality over time, and preservation of seed rhizomes is one of the most important aspects for rhizome successful propagation.

The size of rhizomes pieces in ginger and turmeric used for propagation can vary between growing locations and cultivars, and larger rhizomes produce higher yield

(Hailemichael and Tesfaye, 2008; Kandiannan et al., 2010; Whiley, 1990). Turmeric seed rhizome size of 30 g or above results in greater plant growth and yield (Hossain et al., 2005), and

Asian commercial producers use ginger seed rhizomes between 15 and 75 g (Ravindran and

Babu, 2005). An alternative approach to rapidly multiply turmeric plant number is via a mini-set technique, where rhizome pieces of about 7 g are used (Aswathy and Jessykutty, 2016).

However, a reduction in rhizome size is often associated with seedling mortality due to infection through the cut rhizome surfaces (Ravindran et al., 2007). Ginger rhizomes are usually cut into one or two sections so that each seed piece has two to four well-developed buds (Rafie and

Mullins, nd). Rhizomes can be cured by drying them in a clean and disease-free area for three days or more at ambient temperature and 60% RH (Hepperly et al., 2004; Gupta and Verma,

2011; Jayashree et al., 2015; Kaushal et al., 2017). Instead of treatment with fungicides, rhizomes can also be treated with hot water (50 °C) to help control fungal attack and to ensure optimum germination (Nair, 2013). After storage, rhizome pieces are usually planted in moist sawdust in plastic seedling trays at 25 ± 2 °C (Labrooy and Abdullah, 2016), because temperatures above 30 °C reportedly lead to quick germination but weak sprouts (Ravindran and

Babu, 2005). The germination process takes about 50 days for ginger (Ravindran and Babu,

2005). However, since ginger rhizome buds undergo dormancy, sprouting is poor and erratic and take even more time for younger seed pieces (Sanewski, 1996). For turmeric, germination usually starts about two weeks after planting and lasts for up to four weeks (Ravindran et al.,

2007).

Other crops such as potato (Solanum tuberosum) or yam (Dioscorea spp.) also have underground organs that undergo dormancy. Research in these crops has shown that proteins and nucleic acids regulate dormancy and sprouting, and dormancy has a genetic component because the time required for sprouting varies between potato cultivars (Visse-Mansiaux et al., 2017).

Studies in turmeric (C. longa) indicate that the stable dormant period is only 30 days. However, sprouting takes place 75 days after harvest and proteins are synthesized at the end the dormancy period, similar to potato tubers (Jayakumar et al., 2001).

Growth regulators can induce even and rapid sprouting in plant storage organs.

Cytokinins play an important function in the control of cell proliferation and bud break in many plants (Abelenda and Prat, 2013; Criley, 1988). Benzyladenine (BA) or benzylaminopurine

(BAP) and kinetin are cytokinins that can be synthetically produced, but also occur naturally at low levels in some plant species (Kieber and Schaller, 2014). Ethylene is also responsible for many processes and responses to biotic and abiotic stresses (Corbineau et al., 2014). Studies have demonstrated the effects of ethylene on breaking dormancy of seeds and sprouting of various bulbous plants via a series of complex signaling networks (Esashi and Leopold, 1969).

Within Zingiberaceae, the efficacy of plant growth regulators on rhizome sprouting has been tested under research conditions. Curcuma alismatifolia had rapid and increased shoot emergence when treated with 100 ppm of BAP or 750 ppm of Ethephon (an ethylene-releasing compound) for 30 minutes (Thohirah, et al., 2010). Similar results were obtained on Kaempferia

parviflora with a combination of 750 ppm Ethephon and 150 ppm BAP (Labrooy and Abdullah,

2016). In ginger, highest sprouting (95.6%) was recorded when rhizomes were treated with 100 ppm BAP for 24 h, and 25 and 50 ppm promoted high sprouting (>80%) compared with the control (54%) (Aswathy and Jessykutty, 2016). In the same experiment, treatments with ethephon at 125 ppm and 250 ppm for 30 min resulted in lower sprouting (<60%) compared to

BAP treatments and were not different from the control. Moreover, a hot water treatment (51 °C) in combination with ethephon (750 ppm) for 10 min increased shoot number compared to untreated ginger rhizomes (Furutani, et al., 1985). Alternatively, soaking ginger rhizomes in tap water at ambient temperature for 24 h, 10 days prior to the planting date has been recommended as a treatment to increase sprouting (Ravindran and Babu, 2005). Another study found that endodormancy of ginger rhizomes was reduced by seven days of desiccation before planting, but longer periods of storage reduced shoot growth (Sanewski, 1996).

Interest in ginger and turmeric production is increasing within the U.S. (Chapman, 2016;

Olgers, 2017; Lensing, 2018), but research on the plant material available to U.S. growers has been limited in terms of the evenness of rhizome sprouting. The objective of this research was to evaluate whether forcing treatments, cultivars and rhizome source affect the uniformity and speed of sprouting of ginger and turmeric rhizomes. We conducted two experiments in a growth chamber with one cultivar of ginger and three of turmeric obtained from two different sources.

Materials and Methods

Two experiments were conducted in a growth chamber to measure the effect of three factors (forcing treatment, cultivar, and source of the rhizomes) on sprouting of seed rhizomes.

Experiment 1 was conducted from 12 June to 7 Aug. 2018, while experiment 2 was conducted 36 days later, from 17 July to 13 Sept. 2018. Experiments were conducted at the UF Environmental

Horticulture Research Greenhouse Complex in Gainesville, FL. Rhizomes of ginger (‘Bubba

baba’) and turmeric (‘Hawaiian Red’, ‘White Mango’ and ‘BKK’) were obtained from two different sources: “Commercial” rhizomes were supplied by Just Ginger (Zolfo Springs, FL), and

“UF” material was harvested from the same cultivars planted in the field or greenhouse at UF in

June 2017 and harvested in January or February, 2018 (Gainesville, FL). Rhizomes from UF were stored in a cool chamber at 14.6 ± 1.3 °C (mean ± standard deviation) and 66.0 ± 18.8%

RH until the beginning of each experiment (127 and 161 days for experiments 1 and 2, respectively). Commercial rhizomes were freshly harvested and obtained in 17 Apr. 2018 and were stored at 13.6 ± 0.5 °C and 63.3 ± 19.7% RH until the beginning of each experiment (73 and 107 days, respectively).

Rhizomes were treated either with 50 or 150 ppm of BAP (6-BA; PhytoTechnology

Laboratories, Shawnee Mission, Kansas) by soaking for 30 minutes; 250 or 750 ppm of ethephon

(Florel; Lawn and Garden Products, Inc., Fresno, CA) by soaking for 30 min; hot water (at 50

°C) for 10 min; room temperature (22°C) water for 30 min or for 24 h; or were not treated

(control). A total of 64 labeled seed pieces (8 forcing treatments x 8 cultivars) were randomly placed on moistened paper towels, spaced 5 cm apart and distributed in four black plastic flat trays (T.O. Plastics, Inc. Clearwater, MN, USA.). The experimental design was a randomized complete block design with 12 replicates per forcing treatment, cultivar, and source. Each replicate rhizome per forcing treatment, cultivar and source combination was randomly located on each of the 12 shelf sections (blocks) in the growth chamber. A fogger (Vevor Machinery

Equipment Co., City, State, USA) was placed in the center of the chamber to keep optimum moisture levels, and the paper towels were moistened with approximately 100 ml of tap water every day. Rhizomes were weighed, initial rhizome length and diameter were measured with a caliper (Fowler, Ultra-Call Mark III, Switzerland), and number of visible buds were counted.

Twice a week for eight weeks, the first bud sprouting was recorded for each seed piece. Rhizome final weight and primary root and shoot length were measured at the end of eight weeks.

At the time of harvest, rhizome quality affects storability of rhizome seeds. Thus, if rhizomes are stored for longer periods, they will encounter issues related to weight loss and decreased starch content (Chung and Moon, 2011; Shukor et al., 1986) which affects quality, and ultimately affects sprouting, growth and yield (Ravindran et al., 2007). Therefore, two identical experiments were performed to understand how rhizome quality varies over storage time and whether it affected sprouting. Experiment 1 was conducted from 12 June to 7 Aug. 2018.

Rhizomes averaged 4.1 ± 0.5 cm (mean ± standard deviation) in length and 1.7 ± 0.39 cm in diameter, contained 2.8 ± 0.9 visible buds, and weighed 9.5 ± 4.9 g. Air temperature averaged

25.5 ± 0.3 °C, RH was 89 ± 4.3%, light intensity was 19.9 µmol·m2·s-1 of photosynthetically active radiation (PAR), and the average daily light integral (DLI) was 1.14 mol·m-2·d-1. Final data were collected after 56 days. Experiment 2 was conducted 36 days later, from July 17 to

September 13, 2018. Rhizomes were approximately 3.8 ± 0.7 cm, contained 0.3 ± 0.4 visible buds, and weighed 7.8 ± 5.8 g. This difference in initial bud number and weight from experiment

1 is most likely caused by the extended storage period (36 days). The growth chamber was maintained at 25.5 ± 0.4 °C and 88.5 ± 4.3% RH, with the same light conditions as above. Final data were recorded after 58 days.

Data from experiment 1 and 2 were analyzed separately using PROC GLM in SAS (SAS

Version 9.4; SAS Institute, Cary, NC), with Tukey’s Honestly Significant Difference (HSD) at P

= 0.05 for mean separation. Multiple linear regression model and Pearson correlations were also done using SAS.

Results and Discussion

Overall, rhizomes had an average of 83.3% sprouting in experiment 1 and 61% in experiment 2. Sprouting took place after an average of 29.4 (experiment 1) and 31.7 days

(experiment 2). Rhizomes of turmeric and ginger have been reported to typically sprout after four weeks, although the majority of the sprouts emerge after 6-8 weeks (Evenson et al., 1978;

Sanewski and Fukai, 1996). Similarly, in our study there was an increase in bud number over time for most of the cultivars, from week four to week eight. There are inherent differences in shoot emergence between planting pieces from different parts of the rhizome (Sanewski and

Fukai, 1996), which may explain the variability in our data.

Forcing treatments did not have an effect on the sprouting process of ginger and turmeric rhizomes (Tables 2-1 and 2-2). There were no significant effects of the chemical and water forcing treatments on days to sprout, sprouting percentage, bud number on weeks four or eight, final rhizome weight or shoot and root length. In contrast to our study, Curcuma alismatifolia showed rapid and increased shoot emergence when treated with 100 ppm of BAP or 750 ppm of ethephon (Thohirah, et al., 2010) and in Zingiber officinale up to 95.6% sprouting was recorded when rhizomes were treated with 100 ppm BAP and even lower concentrations (25 and 50 ppm) promoted high sprouting (>80%) compared with untreated rhizomes (54%) (Aswathy and

Jessykutty, 2016). This indicates that the plant growth regulator (PGR) concentration is not the only factor that influences the sprouting process. Additionally, in other crops the response is sometimes negative. In corms of gladiolus for example, BAP delayed the sprouting process, but induced multiple shoots (Sajjad et al., 2015). In yam results of sprouting were inconsistent, indicating that PGRs do not necessarily break dormancy, but they can hasten or delay the rate of shoot apical development (Ile et al., 2006; Jaleel et al., 2007). PGRs are synthetic compounds and the majority of them are labeled as pesticides so disposal of residual solutions can be

problematic (Latimer and Whipker, 2012; Sajjad et al., 2017). Since our results indicated that

PGRs do not necessarily improve sprouting of ginger or turmeric seed rhizomes, growers would not need to spend extra money on chemical products or extra time and labor to treat their rhizomes before planting. Moreover, although water soaking treatments were not necessarily advantageous for accelerating sprouting either, soaking rhizomes in hot water is recommended as a safe sanitation procedure (Nair, 2013).

Cultivar and source and their interaction had a significant effect on sprouting and growth

(Tables 2-1 and 2-2). In both experiments, rhizomes of ginger ‘Bubba baba’ sprouted earlier

(after about 22 days), had more sprouted buds (on average 1.6), and accumulated more fresh mass after 8 weeks (on average 14.9 g) than the turmeric cultivars. Since ginger and turmeric are two different genera it is not surprising that sprouting varied considerably among cultivars.

Overall, there is a wide variability among ginger and turmeric cultivars with respect of yield attributes, and quality characters which are affected by both genetics and environmental conditions (Anandaraj et al., 2014; Ravindran and Babu, 2005). For most of the evaluated parameters there was an effect of source. The main difference between sources was that rhizomes from “UF” were stored for longer (127 and 161 days for experiments 1 and 2, respectively) than the “Commercial” rhizomes (73 and 107 days for experiments 1 and 2, respectively). Rhizomes stored for longer periods, encounter issues related to weight loss, shriveling, and reduced starch content (Chung and Moon, 2011; Paull, 1988).

The effects of cultivar and source on sprouting in part resulted from significant differences in the initial quality (bud number and weight) of rhizomes, shown in Table 2-3. In both experiments, rhizomes of ginger ‘Bubba baba’ had the highest initial weight (ranging from

16.3 to 17.9 g) and the highest initial bud number (ranging from 0.87 in experiment 2 to 4.4 in

experiment 1). In contrast, rhizomes of turmeric ‘BKK’ had the lowest initial weight (ranging from 2.1 to 4.9 g) and the lowest initial bud number (ranging from 0.03 to 2.9) along with turmeric ‘White Mango’ (ranging from 0.06 to 2.6). Since rhizomes of ginger ‘Bubba baba’ from both Commercial and UF sources weighed more at the start of each experiment they also had the highest final rhizome weight (13.9 to 15.5 g, Tables 2-1 and 2-2). This cultivar sprouted earlier and thus, produced longer shoots and roots than the other cultivars. Turmeric ‘BKK’ had a low initial weight and also the lowest final weight (1.8 to 4.1 g). In both experiments a positive correlation was found across all propagative material on initial rhizome weight and initial bud number with days to sprout (P < 0.001), bud number at week 4 (P < 0.001), bud number at week

8 (P < 0.001), and a very high correlation with final weight (P < 0.001; r = 0.92 and 0.94 for experiments 1 and 2, respectively). The formation of shoot and root meristems requires energy from the rhizome (Panneerselvam et al., 2007). Because the nutrition for germination and rooting also derives from the reserves stored in the rhizome bud, the size and nutrition of the seed rhizome have a great influence on the subsequent stages after sprouting (Ravindran and Babu,

2005). While size of rhizomes pieces used for propagation varies upon location and cultivar, the criteria is often based on yield as larger rhizomes produce higher yields (Hossain et al., 2005;

Kandiannan et al., 2010).

Higher rhizome weights are correlated with increased number of shoots per plant

(Furutani et al.,1985). For this reason, ginger growers generally use planting pieces within the 40

– 70 g size range as they produce similar high shoot numbers (Sanewski and Fukai, 1996).

Carbohydrate content plays an important role in rhizome growth. In potato, after the onset of sprouting, sugar content declines in the tubers as they are being consumed until the sprout reaches approximately 1 g of dry matter (Viola et al., 2007). Studies in ginger have demonstrated

that longer periods of storage reduce shoot growth due to a decreased of starch and sugar levels as a consequence of excessive respiration (Sanewski, 1996). In turmeric, larger rhizome seeds are less susceptible to weight loss and have enough stored food to ensure reliable germination

(Ravi et al., 2016). Studies have also shown that there is a gradual decrease of starch content in the rhizomes from the moment of harvest up to 42 days after harvest, followed by a rapid decline until sprouting (Panneerselvam et al., 2007). The same pattern has been reported in potato tubers, where the shoots control the food reserves of the tuber (Davies and Ross, 1984). However, there is limited research concerning the carbohydrate metabolism during dormancy and sprouting in turmeric rhizomes (Jaleel et al., 2007).

Summary

Propagation is one of the most important stages for optimum crop productivity. Planting material and any treatment applied will affect the vigor of the plant and, ultimately, rhizome yield. Forcing treatments did not have an effect on sprouting, but cultivar and plant source affected the uniformity and speed of sprouting of ginger and turmeric rhizomes during propagation. Rhizomes of ginger ‘Bubba baba’ sprouted as early as 19 days after planting, however, for turmeric cultivars, particularly ‘BKK’ and ‘White Mango’ sprouting was delayed up to 39 or 52 days respectively (experiment 2). Initial bud number and rhizome weight were significantly correlated with all evaluated parameters. Rhizomes in experiment 1 had better sprouting than in experiment 2, which were stored for longer (over 120 days). This suggests that initial rhizome quality has a great impact on sprouting and overall growth. Therefore, in order to enhance a uniform and rapid sprouting, high-quality (healthy, larger than 15 g, and clean) rhizomes should be carefully selected after harvest and stored under optimum conditions (12 to

14 °C with 60 - 70% RH), ideally for no longer than 100 days.

Table 2-1. Mean values and Tukey grouping comparison for days to sprout, number of sprouted buds after 4 and 8 weeks, shoot and root length and final weight of ginger and turmeric rhizomes (‘Bubba baba’, ‘Hawaiian Red’, ‘White Mango’, and ‘BKK’), from different sources (Experiment 1). Data represent the least squared means derived from a three-way ANOVA using the general linear model procedure in SAS. Different letters next to value within each column indicate significant differences according to Tukey’s honestly significant difference (HSD) test (P < 0.05). No. sprouted No. sprouted buds Final shoot length Root length (cm) Final weight Factors Days to sprout buds after 4 after 8 weeks (cm) after 8 weeks after 8 weeks (g) weeks Interaction effects Cultivar Source Ginger ‘Bubba baba’ Commercial 19.6 e 1.6 a 2.3 NS 2.1 a 1.5 a 15.5 a Ginger ‘Bubba baba’ UF 24.0 de 1.5 a 2.3 NS 1.7 ab 0.8 b 14.3 b Turmeric ‘Hawaiian Red’ Commercial 28.7 bcd 0.5 bc 1.5 NS 1.5 ab 0.4 c 7.3 c Turmeric ‘Hawaiian Red’ UF 31.1 bc 0.5 bc 1.6 NS 1.3 bc 0.3 c 8.4 c Turmeric ‘White Mango’ Commercial 33.0 ab 0.4 bc 1.3 NS 1.3 bcc 0.3 c 5.4 d Turmeric ‘White Mango’ UF 34.0 ab 0.4 c 1.4 NS 0.8 c 0.2 c 5.1 de Turmeric ‘BKK’ Commercial 27.1 cd 0.8 b 1.3 NS 1.9 ab 0.5 b 4.3 de Turmeric ‘BKK’ UF 37.9 a 0.2 c 1.1 NS 0.7 c 0.2 c 4.1 e Main effects Cultivar Ginger ‘Bubba baba’ 21.7 c 1.5 a 2.3 a 1.9 a 1.2 a 14.9 a Turmeric ‘Hawaiian Red’ 29.9 b 0.5 b 1.5 b 1.4 b 0.4 b 7.9 b Turmeric ‘White Mango’ 33.5 a 0.4 b 1.4 bc 1.1 b 0.3 b 5.2 c Turmeric ‘BKK’ 32.3 ab 0.5 b 1.2 c 1.3 b 0.3 b 4.2 d Source Commercial 27.1 bz 0.8 a 1.6 NS 1.7 a 0.7 a 8.1 a UF 31.8 a 0.7 b 1.6 NS 1.1 b 0.4 b 8.0 a ANOVA Summary Cultivar * * * * * * Forcing treatment NS NS NS NS NS NS Cultivar*Forcing treatment NS NS NS NS NS NS Source * * NS * * NS Cultivar*source * * NS * * * Forcing treatment *source NS NS NS NS NS NS Cultivar*forcing treat.*source NS NS NS NS NS NS zMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 2-2. Mean values and Tukey grouping comparison for days to sprout, number of sprouted buds after 4 and 8 weeks, shoot and root length and final weight of ginger and turmeric rhizomes (‘Bubba baba’, ‘Hawaiian Red’, ‘White Mango’, and ‘BKK’), from different sources (Experiment 2). Data represent the least squared means derived from a three-way ANOVA using the general linear model procedure in SAS. Different letters next to value within each column indicate significant differences according to Tukey’s honestly significant difference (HSD) test (P < 0.05). No. sprouted Days to No. of sprouted buds Final shoot length Root length (cm) Final weight Factors buds after 8 Sprout after 4 weeks (cm) after 8 weeks after 8 weeks (g) weeks Interaction effects Cultivar Source Ginger ‘Bubba baba’ Commercial 18.8 d 1.8 a 1.5 abc 2.1 a 1.8 NS 15.0 a Ginger ‘Bubba baba’ UF 26.5 cd 1.9 a 1.6 ab 1.4 ab 1.2 NS 13.9 a Turmeric ‘Hawaiian Red’ Commercial 24.0 cd 1.5 a 1.9 a 1.1 b 0.7 NS 6.5 b Turmeric ‘Hawaiian Red’ UF 27.4 c 1.1 bc 1.6 ab 1.6 ab 0.6 NS 5.1 c Turmeric ‘White Mango’ Commercial 36.4 b 0.9 bc 1.0 bcd 0.8 bc 0.3 NS 3.8 cd Turmeric ‘White Mango’ UF 52.4 a 0.1 d 0.3 d 0.2 c 0.1 NS 3.4 d Turmeric ‘BKK’ Commercial 35.2 b 0.7 cd 0.9 cd 0.9 bc 0.3 NS 1.8 e Turmeric ‘BKK’ UF 38.7 b 0.6 cd 1.3 abc 0.7 bc 0.2 NS 2.7 de Main effects Cultivar Ginger ‘Bubba baba’ 22.3 c 1.8 a 1.6 a 1.8 a 1.52 a 14.4 a Turmeric ‘Hawaiian Red’ 25.8 c 1.3 b 1.7 a 1.4 a 0.65 b 5.8 b Turmeric ‘White Mango’ 44.2 a 0.5 c 0.6 c 0.5 b 0.18 c 3.6 c Turmeric ‘BKK’ 37.0 b 0.7 c 1.1 b 0.8 b 0.27 bc 2.3 d Source Commercial 28.0 bz 1.2 a 1.3 a 1.2 a 0.8 a 6.8 a Source 35.4 a 0.9 b 1.2 a 1.0 a 0.5 b 6.3 b ANOVA Summary Cultivar * * * * * * Forcing treatment NS NS NS NS NS NS Cultivar*Forcing treatment NS NS NS NS NS NS Source * * NS * * * Cultivar*source * * * * NS * Forcing treatment *source * NS NS NS NS NS Cultivar*forcing treat.*source NS NS NS NS NS NS zMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 2-3. Mean values and Tukey grouping comparison for initial weight, initial number of visible buds, initial length and diameter of rhizomes of ginger and turmeric cultivars (‘Bubba baba’, ‘Hawaiian Red’, ‘White Mango’, and ‘BKK’), from different sources (Experiment 1 and 2). Data represent the least squared means derived from a three-way ANOVA using the general linear model procedure in SAS. Different letters next to value within each column indicate significant differences according to Tukey’s honestly significant difference (HSD) test (P < 0.05). Initial weight (g) Initial bud number Initial length (cm) Initial diameter (cm) Factors Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Exp.1 Exp.2 Interaction effects Cultivar Source Ginger ‘Bubba baba’ Commercial 17.9 a 16.9 a 3.2 bc 0.9 NS 4.8 b 4.7 a 2.1 a 2.1 a Ginger ‘Bubba baba’ UF 16.8 a 16.3 a 4.4 a 0.9 NS 5.1 a 4.9 a 2.0 b 2.0 a Turmeric ‘Hawaiian Red’ Commercial 8.8 b 7.7 b 1.8 d 0.2 NS 3.7 e 3.8 b 1.8 c 1.7 b Turmeric ‘Hawaiian Red’ UF 9.7 b 5.7 c 3.6 b 0.2 NS 4.4 c 4.0 b 1.8 c 1.4 c Turmeric ‘White Mango’ Commercial 7.4 c 4.6 cd 2.6 c 0.1 NS 3.9 de 3.3 c 1.6 d 1.4 c Turmeric ‘White Mango’ UF 6.1 d 4.3 de 1.8 d 0.0 NS 3.5 f 3.2 cd 1.6 d 1.4 c Turmeric ‘BKK’ Commercial 4.6 e 2.1 ef 2.9 c 0.0 NS 3.9 d 2.9 d 1.3 e 0.9 e Turmeric ‘BKK’ UF 4.9 de 3.0 f 2.6 c 0.0 NS 3.7 ef 3.3 c 1.3 e 1.2 d Main effects Cultivar Ginger ‘Bubba baba’ 17.4 az 16.6 a 3.8 a 0.9 a 4.9 a 4.8 a 2.1 a 2.0 a Turmeric ‘Hawaiian Red’ 9.3 b 6.7 b 2.7 b 0.2 b 4.1 b 3.9 b 1.8 b 1.5 b Turmeric ‘White Mango’ 6.7 c 4.4 c 2.2 c 0.1 b 3.8 c 3.2 c 1.6 c 1.4 c Turmeric ‘BKK’ 4.7 d 2.5 d 2.8 b 0.0 b 3.7 c 3.1 c 1.3 d 1.0 d Source Commercial 9.7 NS 7.9 a 2.6 b 0.3 NS 4.1 b 3.7 a 1.7 NS 1.5 a Source 9.4 NS 7.3 b 3.1 a 0.3 NS 4.2 a 3.8 b 1.7 NS 0.5 b ANOVA Summary Cultivar * * * * * * * * Source NS * * NS NS * * NS Cultivar*source * * * NS * * * * zMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

CHAPTER 3 RHIZOME YIELD AND POSTHARVEST QUALITY OF GINGER (ZINGIBER OFFICINALE) AND GALANGAL (ALPINIA GALANGA) AS AFFECTED BY PROPAGATION MATERIAL, CULTIVAR, AND ENVIRONMENTAL CONDITIONS DURING PRODUCTION

Background

The use and demand of spices in the United States (U.S.) is increasing. Per capita spice consumption increased from ~0.5 kg in 1966 to 1.7 kg in 2015, and import values reached

$1,801 million in 2016 (Nguyen et al., 2019; Tridge, 2019). Among the most commonly consumed spices in the country, ginger (Zingiber officinale) rhizomes are high-value, sought- after products that have gained popularity in recent years. Ginger rhizomes are commonly used as a fresh, dried, or processed products and are claimed to have medicinal anti-inflammatory properties. Therefore, consumption of ginger rhizomes has been recommended to improve joint health, reduce blood sugar, and treat different types of cancer (Gregory et al., 2008; Li et al.,

2016; Srinivasan et al., 2016).

Galangal (Alpinia galanga), known also as “greater galangal” or “Thai ginger”, is another edible within the Zingiberaceae family, mainly cultivated in Asia (Ecocrop-FAO, 1997).

Galangal rhizomes have similar medicinal properties as ginger. In addition, due to their similarities in taste and odor, galangal is often used to replace ginger in Asian cuisines

(Chudiwal et al., 2010; Huang et al., 2018; Tang et al., 2018).

Ginger and galangal are commonly propagated by seed rhizomes (Chudiwal et al., 2010;

Ravindran and Babu, 2005). For ginger propagation, rhizome size can vary between 15 and 75 g depending upon the growing location and cultivar (Ravindran and Babu, 2005; Whiley, 1990).

For galangal, rhizomes with at least one developed shoot are recommended to maximize yield

(Peter, 2004). Careful disinfection is an important consideration when using seed rhizomes, as they are highly susceptible to Fusarium, Pythium, Ralstonia, and other soil-borne diseases that

can lead to yield losses (Dohroo, 1989; Hepperly et al., 2004). An alternative to seed rhizome propagation is to use tissue culture-derived transplants, which can ensure pathogen-free, uniform starting material. However, transplants are more expensive than seed rhizomes and tend to result in lower yield (i.e., smaller rhizomes) during the first production cycle (Smith and Hamill, 1996;

Ravindran and Babu, 2005).

Ginger and galangal are widely cultivated in tropical and subtropical regions of the world

(Rafie et al., 2003). However, production in the U.S. has been limited to Hawaii and a very few states in the southeast. It is grown primarily by small growers that produce conventional and organic ginger products (Hayden et al., 2004; Hepperly et al., 2004; Hunter, 2018; Rafie et al.,

2003). Ginger grows well in warm, humid climates and can be cultivated in altitudes ranging from 0 to 1,500 m above sea level (Sasikumar et al., 2008). Optimum growing temperatures for ginger range between 20 to 25 °C, and lower temperatures (<15 °C) can lead to dormancy (Nair,

2013, Ravindran and Babu, 2005). Similarly, galangal is commonly grown in altitudes up to

1,200 m above sea level, with optimum temperatures ranging from 27 to 32 °C (Ecocrop-FAO,

1997; Ochatt and Jain, 2007). Given the environmental conditions that promote yield for ginger and galangal, both species could adapt well to the warm (20 to 35 °C), humid summer climates in Florida, potentially becoming viable crops for commercial growers. Additionally, among the different sub-industries in horticulture, the containerized production of vegetable and ornamental plants has increased significantly in the last few years in the U.S. (Judd et al., 2015) and Florida is one of the states with the highest national sales ($1,796 million in 2014) within the horticulture industry (USDA, 2014). Therefore, growing these species in greenhouses could be a good alternative in Florida, as it is possible to control certain aspects of the growing environment

allowing for year-round production and offering opportunities to increase productivity and maximize profit for the grower (Majsztrik et al., 2017; Suhaimi et al., 2012).

To develop successful ginger and galangal production systems in Florida, however, considerations about environmental limitations that affect plant growth and development must be considered. Ginger plants enter dormancy in early winter, when natural photoperiods decrease below a critical night length of 12 hours (Adaniya et al., 1989; Pandey et al., 1996). When they are grown in the field, rhizomes have to be harvested during this period, limiting the rhizome production to specific seasons during the year. However, some studies have demonstrated that the crop does not experience dormancy under long photoperiod (~16 h) whereas rhizome production is promoted by photoperiods of ~10 h (Adaniya et al., 1989; Pandey et al., 1996).

Day-extension or night-interruption with electric lamps are strategies commonly used by the horticulture industry to control the seasonality of flowering plants by manipulating the critical night length of photoperiodic-sensitive crops. Most photoperiodic research has focused on flowering rather than on vegetative-growth responses. However, few studies have shown the significant effect that photoperiodic control has on ginger (Adaniya et al., 1989; Pandey et al.,

1996). Therefore, by manipulating the photoperiod under controlled conditions, growth and rhizome yield could be maximized. In contrast, galangal does not seem to be sensitive to photoperiod-induced dormancy. Tang et al. (2018) reported that galangal flowers during the summer in the subtropics, but it can produce flowers and rhizomes all year in tropical areas regardless of the photoperiod. Information regarding cultivation of galangal is scarce.

Excess solar radiation is another environmental aspect that could affect ginger and galangal production in Florida. Under natural conditions, ginger and galangal have been reported to grow well under shade as excess irradiance under full sun promotes tip burn on the leaves

(Babu and Jayachandran, 1997; Chudiwal et al., 2010; Stephens, 2015). In contrast, galangal is reported to grow well both in shade and full sun conditions (Ochatt and Jain, 2007). Some studies in ginger have shown that when plants are grown in the field under shade, plant photosynthetic rate is increased due to the higher leaf area produced, high plant nutrient content which ultimately could led to high yields (Kumar et al., 2005; Ravindran and Babu, 2005).

Higher rhizome yield in ginger was found when plants were grown under low to medium levels of shade, including 20%, 25%, 40% compared to higher levels such as 60% or 75% (Ajithkumar and Jayachandran, 2003; Babu and Jayachandran, 1997). While some studies found that high levels of shade reduced ginger yield compared to no shade, plant wilting was reduced during the hottest time of the day (Kratky et al., 2013). Then, under shade, leaf temperature can be reduced, decreasing transpiration rates and enhancing overall plant growth (Kratky et al., 2013; Tew,

1962).

Regardless of the growing conditions, identifying the right maturity at the point of harvest is important for optimum color and aroma, which ultimately affects rhizome quality

(Ravindran, et al., 2007). Depending on the market, ginger can be sold as a fresh product or in a peeled, split, and dried form (FAO, 2004). For fresh market there are two kinds of ginger rhizomes, the “young” and “mature”. The young ginger is bright yellow to brown with greenish- yellow vegetative buds, but no sprouts. It is mostly offered by Asian markets and does not need to be peeled. The commonly available is the mature ginger and has a tough skin that, which makes peeling required (Masabni and King, n.d; Paull and Cheng, 2015). The marketable size of a clump of rhizomes (“hands”) varies from 150 g to 300 g (FAO, 1999). To meet the minimum standard requirements for high quality fresh ginger, whole rhizomes from similar varietal characteristics must be selected. These rhizomes have to be clean and free of any visible foreign

matter. Additionally, they must be free of deterioration and rotting, with dried cut surfaces, free of pests and any damage caused by pests affecting flesh quality, as well as free of abnormal external moisture at the skin (Hawaii Department of Agriculture, 1992). According to the

CODEX standards (FAO, 1999), ginger fresh rhizomes are classified as “Extra”, “Class I”, and

“Class II”. Rhizomes from the “Extra” Class must be of superior quality. They must be cleaned, well-shaped and free of defects, with the exception of very slight superficial defects. Rhizomes from “Class I” must be of good quality, firm and without evidence of shriveling and sprouting.

The keeping quality for this kind of rhizomes tolerates light skin defects due to rubbing as long as they are healed and dry, with total surface area affected not exceeding 10%. In contrast, rhizomes classified as “Class II” do not qualify for inclusion in the higher classes, but satisfy the minimum requirements mentioned above. Rhizomes should be reasonably firm and skin defects due to rubbing (healed and dry) cannot exceed an affected total surface area of 15%; although early signs of sprouting, slight markings caused by pests, and bruises are allowed.

For ginger, the optimum time of harvest mostly depends on the end use. For instance, between five to six months after planting (before full maturity) ginger rhizomes are harvested for fresh products and preserves due to their low pungency and fiber content (Nishina et al., 1992).

At about nine months after planting, rhizomes reach their maximum pungency and fiber content.

For planting material harvest can be further delayed (~9 months) until the leaves of ginger are completely dried (FAO, 2004). Rhizomes of ginger intended for storage need to be first cured

(held at 22 – 26 oC and 70% RH) for several days to allow the skin to thicken and the cut surfaces to dry. Curing helps reduce postharvest weight loss and decay (Paull et al., 1988). After curing, ginger rhizomes should be stored in well ventilated containers at 12 – 14 °C with 60% -

70% RH and temperature cannot be lower than 12 °C as they are chilling sensitive. Galangal is

also sold fresh, in slices as preserves, or dried in powder form. In the U.S. galangal products are usually found in Southeast Asian markets (Loha-unchit, 2000). Galangal rhizomes are paler and woodier than ginger, and the flesh is creamy white. Similar to ginger, galangal rhizomes are likely to have cut ends, then these have to be healed and dry, but should not be soft or moldy

(Smith, 2017). Rhizomes of galangal develop quickly and should be harvested approximately three months after planting, otherwise they become very fibrous. However, for essential oil extraction, rhizomes are harvested after around seven months of growth (Ochatt and Jain, 2007).

In galangal, the shelf life of rhizomes is limited by browning of the cut surface, therefore they are usually treated with anti-browning solutions prior to marketing (Chinwang et al., 2015). In addition, it is reported that for a high-quality product, fresh harvested rhizomes of ginger and galangal should be washed and sanitized with hypochlorous acid (Nair, 2013; Sasikumar et al.,

2008). For both ginger and galangal adequate postharvest handling of rhizomes is required as they are sensitive to weight loss and highly susceptible to various pathogens such as Penicillium spp., Fusarium spp., and Pythium spp. (Kaushal et al., 2017; Nepali et al., 2000; Trujillo, 1963).

Numerous opportunities exist to refine ginger and galangal production systems in

Florida. Practices related to planting material for propagation, photoperiodic control, and radiation (light and temperature) stress tolerance must be developed. Therefore, the objectives of this study were 1) to evaluate the growth and yield of ginger and galangal propagules (rhizome- derived and micropropagated transplants) grown in different container sizes and under varying photoperiods in a greenhouse environment; and 2) to measure the effect of shading conditions on plant growth and rhizome yield and quality of ginger and galangal plants grown in the field.

Materials and Methods

Experiment 1. Greenhouse Experiments

Different experiments were independently conducted over a two-year period. The experiment in year 1 was conducted at the University of Florida (UF) Environmental

Horticulture Research Greenhouse Complex in Gainesville, FL. The experiment in year 2 was conducted in the greenhouses at the Plant Science Research and Education Unit (PSREU) in

Citra, FL. In year 1, propagules used were micropropagated or tissue cultured transplants “tc” of unknown ginger obtained from Agri-Starts™ Inc. (Apopka, FL, USA) and Hawaiian-grown rhizomes of cultivar ‘Bubba baba’ (“rhiz”) obtained from Hawaii Clean Seed LLC (Pahoa, HI,

USA). In year 2, the same materials were used, and in addition, rhizomes harvested from the “tc” material (“ownrhiz”: second generation “tc”, harvested after about one year of growth) and galangal “tc” (Alpinia galanga, cultivar unknown) were also included.

Year 1 (2017-2018)

This experiment was conducted from 19 Apr. 2017 to 29 Jan. 2018 and aimed to evaluate growth and yield of ginger propagules grown in two container sizes and under two photoperiods in a greenhouse environment. Propagules of ginger included micropropagated transplants (“tc”)

[planted in two different dates: “tc early” (planted on 3 Oct. 2016) and “tc late” (planted on 27

Apr. 2017)] and Hawaiian-grown rhizomes (“rhiz”) planted on 19 Apr. 2017. Both “tc” ginger transplants were initially planted individually in 380 mL containers (Pöppelmann TEKU®,

Claremont, NC) filled with sphagnum peat substrate (Klasmann-Deilman, Miami, FL, USA), while “rhiz” plants were planted in 2.78 L containers (Nursery Supplies Inc, Kissimmee, FL,

USA) filled with a substrate consisting of sphagnum peat and perlite (Fafard®2P, Sun Gro

Horticulture Distribution Inc, 770 Silver Street Agawam, MA, USA). The “tc early” transplants

were subsequently potted into 5.7 L containers (Nursery Supplies Inc) on 9 Dec. 2016 and mounded with 5 cm of additional substrate on 17 Jan. 2017.

All plants were subsequently repotted on 17 July 2017 either into 5.7 L or 50.5 L containers (Nursery Supplies Inc) with a 1:1 (v/v) mix of coarse coconut husk chips and fine coconut fiber (Envelor Inc, Old Bridge, NJ, USA). Containers were placed in two polycarbonate- covered greenhouse compartments in Gainesville, FL with automated heating and pad-and-fan evaporative cooling. Within each compartment, containers were arranged in a RCBD with eight replicate containers per plant material type and container size, where two replicate containers per plant material type and container size combination were randomly located on each of the four benches (blocks) inside the greenhouse.

One compartment received “natural days”, and the second received “long days” provided by night interruption lighting from 10 pm to 2 am with incandescent lamps at 3.2 µmol·m2·s-1 of

PAR In the natural days greenhouse, average temperature was 23.2 ± 2.2 oC and light was 11.8 ±

5.8 moles·m-2·d-1 DLI. The long days greenhouse averaged 22.0 ± 1.0 oC, with 6.7 ± 4.5 moles·m-2·d-1 DLI and 74.1% RH. In both greenhouses, plants were hand-irrigated with 17-1.8-

14.1 blended water-soluble fertilizer (Greencare Fertilizers, Kankakee, Michigan) at 200 mg/L N with each irrigation.

Between 17 – 21 Aug. 2017 all plants in 5.7 L and 50.5 L containers were mounded with

5 cm and 10 cm of additional substrate, respectively. Plants were harvested between 22 - 29 Jan.

2018. Fresh mass of rhizomes, shoots, and roots, and percent rhizome moisture [rhizome fresh mass – (dry mass of rhizomes oven dried in a 50 oC oven for three days) / rhizome fresh mass] were measured. Postharvest evaluations were not carried out. Effects of container size and plant material were analyzed separately between the two greenhouse compartments (light treatments)

with analysis of variance (ANOVA), using RStudio version 3.3.2 (RStudio, Inc., Boston,

Massachusetts, USA), agricolae, stats, and lsmeans packages. Least-square treatment means were compared using Tukey’s honestly significant difference with P = 0.05.

Year 2 (2018-2019)

In year 1, we observed that plants grown under long days remained dark green and actively grew in the winter, whereas under natural days plants showed considerable yellowing and initiation of dormancy when harvested in late January. Thus, in year 2 we wanted to evaluate whether the growing season for ginger could be extended by keeping plants green and actively growing under long days (night interruption) during the winter.

This experiment was conducted from 18 Apr. 2018 to 9 Mar. 2019 and aimed to evaluate yield of ginger and galangal propagules produced under two photoperiods in a greenhouse environment. Propagules of ginger included micropropagated transplants (“tc”), second generation “tc” rhizomes (“ownrhiz”), or Hawaiian-grown rhizomes (“rhiz”), plus a “tc” galangal. Rhizomes were initially planted individually on 18 Apr. 2018 in black plastic flat trays

(T.O. Plastics, Inc. Clearwater, MN, USA.) filled with sphagnum peat substrate (Klasmann-

Deilman) under growth chamber conditions (25 ± 0.5 °C and 84.3 ± 4.5% RH). Micropropagated ginger and galangal were transplanted into 2.78 L containers (Nursery Supplies Inc) filled with a substrate consisting of sphagnum peat and perlite (Fafard®2P) on 4 May 2018. Sprouting of rhizomes took place between 22 May and 5 June 2018. Sprouted rhizomes were then planted into

2.78 L containers with the same substrate and grown in a polycarbonate-covered greenhouse in

Gainesville. The greenhouse had a day and night temperature of 25.6 ± 1.9 °C and 24.7 ± 1.8 °C respectively. All plants were subsequently repotted on 27 June 2018 into 14.5 L pots with a mix of pine bark, sphagnum peat, perlite, and vermiculite (Fafard®52 Mix, Sun Gro Horticulture

Distribution Inc). Pots were then placed in two polycarbonate-covered greenhouse compartments

in Citra, FL with automated heating and pad-and-fan evaporative cooling. The experimental design was a split-plot design with photoperiod as the main plot and plant type as the subplot.

Within each compartment, containers were arranged in a RCBD with six replicate pots per cultivar and propagation material, where two replicate pots per cultivar and propagation material combination were randomly located on each of three benches (blocks).

One compartment received “natural days”, with day and night temperatures of 26.6 ± 3.1

°C and 21.3 ± 2.6 °C, respectively, and 9.1 ± 3.6 moles·m-2·d-1 DLI. The second greenhouse had

“long days” provided by night interruption lighting from 10 pm to 2 am with incandescent lamps at 1.32 µmol·m-2·s-1 PAR from 6 July onwards. It had 26.8 ± 3.9 °C and 21.4 ± 2.1 °C day and night temperatures respectively, and 8.4 ± 3.8 moles·m-2·d-1 DLI. Plants were drip-irrigated with tap water and fertilized using an 8 - 9 month release 15-3.9-10 Osmocote Plus™ (ICL Specialty

Fertilizer Customer, 4950 Blazer Memorial Parkway, Dublin, Ohio) controlled release fertilizer

(CRF) at a rate of 114 g.pot-1. Plants were mounded once with new substrate in 29 Aug. 2018 to approximately a 10 cm depth.

In order to assess plant growth, number of new shoots and plant height were measured every two weeks, as well as chlorophyll index using a chlorophyll meter (SPAD-502DL, Konica

Minolta Sensing, Osaka, Japan). To evaluate the overall aesthetic performance, a scale from 1 to

5 was used, where 1 = very poor quality, not acceptable, severe leaf necrosis, tip burn or chlorosis, poor form, 2 = poor quality, not acceptable, large areas of necrosis, tip burn or chlorosis, poor form, 3 = acceptable quality, few leaves with tip burn, somewhat desirable form and color, 4 = very good quality, very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form. In ginger and galangal, excellent form was

considered when plants were well branched and full, not lodged, without tip burn or chlorotic leaves, and had stems of relatively uniform length.

Plants from the natural days treatment were harvested on 22 Jan. 2019, whereas plants from long days were harvested one month later, on 21 Feb. 2019 (after about seven and eight months of growth in the finishing pots, respectively). After harvest, fresh and dry mass of rhizomes, shoots, and roots as well as percent rhizome moisture [rhizome fresh mass – (dry mass of rhizomes oven dried in a 40 oC oven until constant weight) / rhizome fresh mass] were determined (Li et al., 2016). Additionally, rhizomes from each treatment (natural and long days) were evaluated under postharvest storage conditions. Rhizomes were cleaned, disinfected with

10% bleach and cured (held at 22 – 26 oC and 70% RH) for four days. Rhizomes from natural days were then stored in a cool room at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH for 16 days, while rhizomes from long days were stored at 12.9 ± 0.1 °C and 65.1 ± 4.4% RH for 15 days. In this case the RH was adjusted with a dehumidifier. Traits evaluated were number of infected/damaged rhizomes, and percentage of weight loss [(initial - final weight / initial weight)

× 100]. Rhizome skin and flesh color were determined using a chroma meter (model CR-400,

Konica Minolta Inc., Tokyo, Japan). After curing and after the storage period, the external skin color was measured and then one representative rhizome piece for each type was cut in half to display and measure the internal flesh color. The color system (lightness-L*, chroma-C*, and hue angle-h⁰) of this meter uses values calculated from the L*a*b* system, where L* indicates the lightness, which ranges from black (0) to white (100), C* indicates chroma or saturation, and describes the vividness or dullness of a color regardless of its luminance, and h⁰ indicates hue, and is the angle that defines the actual color of the object in the color space (McGuire, 1992;

Minolta, 1994).

Growth, yield, and postharvest data from galangal and ginger plants grown in the greenhouse under natural and long days were analyzed together, where blocks were considered as random effects and photoperiod, plant type, and its interactions were considered as fixed effects. The analysis was performed using RStudio version 3.3.2 for analysis of variance

(ANOVA), with Tukey’s Honestly Significant Difference (HSD) at P = 0.05 for mean separation.

Experiment 2. Field Experiment

This experiment was conducted at the University of Florida (UF) Environmental

Horticulture Research Greenhouse Complex in Gainesville, FL and aimed to measure the effect of two factors (propagule type and light environment) on plant growth, ornamental performance, and yield under shading conditions. Rhizomes were initially planted on 18 Apr. 2018 in black plastic flat trays (T.O. Plastics, Inc.) filled with sphagnum peat substrate (Klasmann-Deilman) under growth chamber conditions (25 ± 0.5 °C and 84.3 ± 4.5% RH). Micropropagated transplants were initially planted in 380 mL containers (Pöppelmann TEKU®) filled with peat substrate (Klasmann-Deilman) and then repotted on 4 May 2018 into 2.78 L pots filled with a substrate consisting of sphagnum peat and perlite (Fafard®2P, Sun Gro Horticulture Distribution

Inc). Sprouted rhizomes were potted on 15 May 2018 (using the same container size and substrate) and grown in the same greenhouse in Gainesville. Then all plants were planted in the field on 21 June 2018 under full sun or shade. Six shade structures were randomly set up in four field rows with aluminum poles and 60% shade cloth. Plants under shade received an average day and night temperature of 25.3 ± 7.3 °C and 19.3 ± 7.4 °C, respectively, with 10.3 ± 3.6 moles·m-2·d-1 DLI. The full sun environment had an average day and night temperature of 23.3 ±

7.0 oC and 16.4 ± 6.8 °C respectively, with 21.1 ± 10.6 moles·m-2·d-1 DLI. Plants were drip- irrigated with tap water and fertilized by using an 8 - 9 month release 15-3.9-10 Osmocote

Plus™ (ICL Specialty Fertilizer Customer) CRF at a rate of 114 g·plant-1. Plants were arranged in a split-plot design, with environment (sun and 60% shade) as the main plot, and propagules as subplots. Micropropagated “tc” transplants of unknown ginger and galangal cultivars, originally obtained from Agri-Starts™ Inc were used, as well as the harvested “tc” ginger rhizomes from year 1 (“ownrhiz”), and Hawaiian ‘Bubba baba’ rhizomes. Two environment replicates were randomly located on each of three blocks and there were a total six propagule replicates per environment. Plants were harvested on 4 Feb. 2019, after about 9 months of growth.

In order to assess plant growth, number of shoots, height, and chlorophyll index were measured every two weeks. The overall aesthetic performance was determined using the same scale used for the greenhouse experiment (year 2). Similarly, fresh mass of rhizomes, shoots, and roots, percent rhizome moisture [rhizome fresh mass – (dry mass of rhizomes oven dried in a 40 oC oven until constant weight) / rhizome fresh mass] and rhizome skin and flesh color were measured after harvest. Rhizomes harvested in year 2 were also evaluated in postharvest storage conditions. Rhizomes were cleaned and treated as described above and stored at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH for 16 days. The same traits as described for the greenhouse postharvest experiments were evaluated.

Data from plants grown in the field either under full sun or shade (environment factor) were analyzed together. Blocks were considered as random effects and environment, plant type, and its interaction were considered as fixed effects and were analyzed by ANOVA with Tukey’s

HSD at P = 0.05 (RStudio version 3.3.2).

Results and Discussion

Experiment 1. Greenhouse Experiments

Year 1 (2017-2018)

Regardless of the photoperiod, container size affected rhizome fresh mass (P < 0.001), which is the important harvestable yield. When plants where grown in larger containers (50.5 L), they had average rhizome yields of 913 g·plant-1 and 815 g·plant-1 under natural and long days, respectively. In contrast, when grown in smaller containers (5.7 L), average rhizome yields were

370 g·plant-1 under natural days and 438 g·plant-1 under long days (Figures 3-1A and B).

Additionally, when plants were grown under natural days, there was interaction between plant type and container size as all plants increased yield under larger containers compared to smaller containers (P < 0.001, Figure 3-2A). However, there was no interaction between plant type and container size when plants were grown under long days (P > 0.05, Figure 3-1B). Container size is considered an important variable influencing plant and root morphology, as it is directly related to water holding capacity and root zone humidity and porosity (Chen and Wei, 2018;

Judd et al., 2015). Most geophytes grown in large containers produce multiple, fleshy, and thick roots or rhizomes (Landis et al., 2014). Research in tuber crops like potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) has also shown that production in large containers increases tuber yield (Bandara et al., 1998).

Rhizome diameter was not measured in our study, however we observed that rhizomes harvested from micropropagated ginger (tc early or tc late) had smaller diameters than those derived from rhizomes, which would most likely affect their marketability as a fresh product

(Figure 3-2). Ginger tc early were grown for approximately seven more months than tc late, but the diameter of its rhizomes was still small. Commercially, ginger plants started from rhizomes are typically grown for six months before harvest (FAO, 2004; Jayashree et al., 2015).

Regardless of the photoperiod, when grown in large containers ginger plants propagated from rhizomes produced ~960 g of fresh rhizomes, while micropropagated gingers averaged ~818 g.

Both plant types were grown for over nine months, indicating that micropropagated transplants may require longer periods of growth to achieve yields comparable to that of rhizome-derived plants. At the moment of planting, micropropagated transplants are in their first stage of growth.

They are usually very small, weigh less than 1 g, and lack the rhizome structure (Smith and

Hamill, 1996). Rhizomes are the main source of food and reserves for the ginger plant at the beginning of the crop cycle, thus a lack of this structure in micropropagated transplants may have limited plant growth and rhizome production. In addition, in a study by Ravindran and Babu

(2005), tissue-cultured ginger transplants had similar characteristics to seedling plugs, resulting in smaller yield than that of second-generation plants derived from the harvested rhizomes.

Moreover, in some field studies, the size of ginger rhizomes increased over time and reached normal size (comparable to that of mother plants) in the third year (Ravindran and Babu, 2005).

These studies support our conclusion that differences in rhizome-derived and micropropagated plants will have significantly different yield capabilities in the first year of production.

Large containers resulted in more shoot fresh mass under long days (average of 1,473 g) than under natural days (average of 949 g). There was no interaction between plant type and container size under natural days, and there was more shoot growth in the tc late plants (~913 g) compared with tc early (~507 g) or ginger rhiz (~387 g,) (Figure 3-1C). Meanwhile, under long days there was interaction between container size and plant type. In the small containers tc early had more shoot fresh mass (~1,796 g) than tc late (~552 g) and ‘Bubba baba’ (~500 g), while in large containers tc late and ‘Bubba baba’ had comparable shoot fresh mass (2,092 and ~1,500 g, respectively) and tc early was lower (~829 g), Smith and Hamill (1996) also found several small

(up to a total of ~32) shoots in micropropagated ginger. Rhizomes are modified stems that also allow plants to naturally propagate and grow. During the early stages of ginger growth, the sprouted apical bud becomes the main tiller (shoot) and as it grows, its base enlarges into a rhizome developing the primary and secondary rhizomes, commonly known as fingers. As growth continues, buds on the secondary fingers can develop into tertiary shoots and tertiary fingers (Ravindran and Babu, 2005). Therefore, there is a close relationship between shoots and rhizomes. Due to this rhizome – shoot interaction and the fact that large containers promoted high rhizome yield on all plants including tc under both photoperiods, we can infer that the high number of shoots was favored by the high number of rhizome pieces produced during the growing season.

Under natural days, root fresh mass was affected by container size (P < 0.001), plant type

(P < 0.001), and by the interaction of container size and plant type (P < 0.05, Figure 3-1E).

Similar to shoot fresh mass, there was higher root fresh mass in larger containers (553 g) than in smaller containers (124 g). Ginger tc late had higher root fresh mass (~639 g) than the other gingers (<267 g) and it was the only cultivar that significantly increased root mass (by 849.3 g) when grown in larger containers (P < 0.05, Figure 3-1E). Under long days, root fresh mass was only affected by the interaction of plant type and container size (P < 0.01). Ginger tc early and late had the highest root fresh mass (164.4 g) and ginger ‘Bubba baba’ had the lowest (46.2 g,

Figure 3-1F). Micropropagated ginger plants are characterized by having a larger number of roots than rhizome-derived plants (Smith and Hamill, 1996). Under long days, for all plant types shoot mass was high while root mass was low. However, differences between shoot and root mass did not result in a difference in rhizome mass.

Total fresh mass was affected by the interaction of container size and plant type under both natural days (P < 0.05) and long days (P < 0.001) (Figures 3-1G and H). Under natural days and in small containers ginger tc early had the highest total fresh mass (1,777.1 g) compared to tc late (~706 g) and ‘Bubba baba’ (~533 g), while in larger containers tc late had higher total fresh mass (~3,383 g) compared to ‘Bubba baba’ (~2,051 g) and tc early (~1,046 g).

Meanwhile, under long days and in small containers tc early had higher total fresh mass (~2,849 g) than tc late and ‘Bubba baba’, while in larger containers tc late and ‘Bubba baba’ had higher mass than tc early (~2,542 g)

The percentage of rhizomes dry mass under natural days, was >80% of the total for

‘Bubba baba’ in the two container sizes and for tc early in the small containers, while it was <

65% for tc raly in the larger containers, and tc late in both container sizes (Figure 3-3A) As expected, shoot growth was favored under long days (>57% in all container sizes, (Figure 3-3B).

Therefore, manipulating photoperiodic lighting via night interruption is a good strategy to maximize shoot growth which ultimately can promote higher rhizome yields. In previous studies, partitioning of ginger biomass favored rhizome enlargement in plants derived from rhizomes, whereas shoots were favored in micropropagated plants (Smith and Hamill, 1996). However, in our study this was only true under natural days, as the rhizome biomass was >85% for rhizome derived plants, while it was below 40% under long days.

Year 2 (2018-2019)

Photoperiod and plant type (the combination of species and planting material) affected several aspects related to growth and yield of ginger and galangal (Table 3-1 and Figure 3-4).

Regarding overall plant growth and performance, the average number of new shoots (P < 0.01), height increase (P < 0.001), chlorophyll index (P < 0.001), total number of flowers (P < 0.05), and overall plant performance (P < 0.01) was significantly different among plant type.

Micropropagated ginger and galangal plants had the highest number of new shoots compared to rhizome-grown (rhiz and ownrhiz) ginger (0.8 and 1.1, respectively; Table 3-1). This confirms a previous study stating that in contrast to rhizome-derived plants, micropropagated ginger plants produce more, but smaller shoots (Smith and Hamill, 1996). Shoot number was also affected by photoperiod (P < 0.01). Regardless of species, plants grown under long days produced more new shoots than those grown under natural days. This was expected, as previous trials comparing photoperiods showed that ginger plants grown under 16 h produce more sprouts than plants grown under 10 h or natural days (Ravindran and Babu, 2005).

Some plants were started from rhizomes and others from tissue culture, thus the initial plant height at the beginning of each experiment was not uniform. Therefore, the increase in height was used to determine shoot growth. This was unaffected by photoperiod, and differed between plant types (Table 3-1). Galangal tc and ginger rhiz had the highest increase in plant height, as there was a growth of over 60 cm, compared to ginger tc and rhiz (≤37.1 cm). Ginger plants traditionally grown from rhizomes produce shoots that are 60 to 90 cm tall, however, they can reach up to 122 cm under commercial conditions (Nishina et al., 1992; Moghaddasi and

Kashani, 2012). The higher increase in height of rhizome-derived ginger compared to micropropagated plants can be explained by the establishment differences which allowed for rhizome-derived plants to start growing earlier and thus, reach a taller height than plantlets regenerated through callus (Babu and Jayachandran, 1997). In addition, micropropagated plants lack the rhizome structure, therefore they have less reserves for optimal plant growth (Smith and

Hamill, 1996).

SPAD values differed between plant types (P < 0.001, Table 3-1). Ginger plants either grown from rhizomes and micropropagated transplants had the highest SPAD readings (48.1 and

46.6, respectively). Both tc and ownrhiz gingers had similar SPAD values (47 and 45, respectively), so we can infer that plant quality in terms of chlorophyll content does not change for second generation tc plants, as. Galangal had significantly lower SPAD values (<39) than ginger plants. The relative chlorophyll content measured in the leaves by SPAD is another indicator of active plant growth. That is because chlorophyll, an important molecule for photosynthesis, is also a good indicator of plant nutrient status (particularly nitrogen) and leaf senescence (Xiong et al., 2015). Hence, ginger plants with SPAD values above 40 tend to indicate good plant performance, which could also translate to higher yields (Li et al., 2018;

Wang et al., 2019). The values we found are similar to those found in a study comparing the effect of soil moisture level on chlorophyll content of ginger, which found that optimum plant growth (high number of new shoots, stem diameter and plant height) was achieved under 25% soil moisture, with SPAD readings of ~49 (Li et al., 2018).

All ginger types produced flowers, however the ownrhiz had the highest total number of flowers (2.4), whereas ‘Bubba baba’ produced the fewest (0.6). Galangal produced almost no flowers during the growing period (0.1, Table 3-1). While ginger plants use their rhizome resources primarily for vegetative growth during the entire growing period, flower production in edible ginger plants (as opposed to those grown for ornamental purposes) is unpredictable.

Flowering is not common in edible ginger, but if it occurs, it might be regulated by photoperiod

(Ravindran and Babu, 2005). Studies in Japanese ginger (Zingiber mioga) have found that flower bud initiation is promoted under long days (Stirling et al., 2002). In addition, flowering and vegetative growth of adult clonal plants might compete for resources in the plant. Several authors have stated that flowering on other geophytes such as curcuma, yam bean, sugarcane negatively affects yield as this process usually takes away energy from rhizomes (Kandiannan et al., 2015;

Vimala and Nambisan, 2005). However, in our experiment this flowering event did not appear to affect rhizome yield, probably due to the low number of flowers produced through the growth cycle (Figure 3-4A).

Many species in the family Zingiberaceae are valued as ornamental foliage plants because they tend to have multiple stems with lush, green or variegated leaves, which provides a tropical illusion in the landscape. Other species such as Zingiber aromaticum, Z. zerumbet,

Alpinia zerumbet, and Hedychium coronarium have flowers and thus, as also valued as ornamental plants. (Gilman, 1999abc; Ravindran and Babu, 2005). Alternatively, they are used for the spicy flavor and medicinal properties of their rhizomes. However, some accessions may have potential as both landscape ornamental plants and as edible crops. Thus, in order to evaluate the overall aesthetic performance of these two genera, a subjective visual scale from 1 to 5 was used. This attribute was significantly affected by plant type (P < 0.01) and photoperiod (P <

0.001), (Table 3-1). All plants grown under natural days had a better aesthetic performance (an average overall rating of 4, Table 3-1) than the ones grown under long days (3.2), however all plants had “acceptable” quality, meaning that ginger and galangal plants had a few leaves showing symptoms of tip burn (Figure 3-5).

After evaluating these growth characters under both photoperiods and plant types, it is important to determine if the same trend is found for biomass production and yield data, and if there is any correlation between these growth characters and yield. Plant type (P < 0.01) and the interaction of photoperiod and plant type (P < 0.05) on rhizome fresh mass were significant

(Figure 3-4A). Regardless of the photoperiod, ownrhiz had the highest rhizome yield (1,440 g·plant-1) followed by galangal tc (1,207 g·plant-1) and ginger rhiz (817 g·plant-1). Rhizome yield can vary greatly among species, and ginger cultivars are extremely sensitive to the environment,

then rhizome yield could change depending on weather events (Ravindran and Babu, 2005). For galangal, it is reported that plants are able to grow and produce rhizomes year-round in tropical areas (Tang et al., 2018). In contrast, ginger requires long days for continuous growth and development. Otherwise, plants will become dormant and unless day length is reduced gradually

(to 10 to 11 h of light), rhizome production might be affected (Pandey et al., 1996). Studies looking at effects of photoperiod on micro-rhizome formation in vitro have reported micro tuberization of ginger in the dark (Sharma and Singh, 1995). In contrast, others have shown that micro-rhizome formation is promoted with a 16 - 24 h·d-1 photoperiod (Nayak and Naik, 2006;

Zheng et al., 2008). Therefore, managing protocols for photoperiodic induction of micro-rhizome production under in vitro conditions is unclear, and variation seems to be attributed to the different genetic makeup of the species. Differences for rhizome yield among photoperiodic treatments were not significant, but ‘Bubba baba’ had low yield under natural days (466 g) and high yield under long days (1,168 g). In contrast from the previous year, micropropagated plants had high rhizome yield under both photoperiods. However, as in year 1 rhizome diameter was very small (Figure 3-6) corroborating that micropropagated plants behave like seedlings and that rhizome size is usually very small during the first season (Ravindran and Babu, 2005).

Plant type (P < 0.001), photoperiod (P < 0.01), and the interaction of plant type and photoperiod affected shoot fresh mass (P < 0.001, Figure 3-4B). Regardless of daylength, galangal had the highest shoot fresh mass (average 610 g). Overall for all plant types, shoot fresh mass was higher under long days (370 g) than under natural days (210 g). This is an expected result, considering that previous studies have shown that ginger plants grown under 16 h·d-1 produce more sprouts compared to plants grown under 10 h·d-1 or natural days (Ravindran and

Babu, 2005). Regardless of photoperiod, galangal had the highest shoot fresh mass, followed by

‘Bubba baba’, which was not different from the other types of ginger plants, ranging from 107 to

267 g (Figure 3-4B). The difference in shoot fresh mass between galangal and ginger plants can be explained by the differences in shoot number, plant height, or both (Table 3-1). As indicated previously, micropropagated ginger plants usually have several, small shoots (Smith and Hamill,

1996). Additionally, when micro propagated plants are grown in ideal conditions with optimum light photoperiod, sugar and exogenous plant hormones such as cytokinins, they experience little physiological stress, reducing their photosynthetic net rate (Abbas et al., 2011; Mei-lan et al.,

2003). Thus, propagation of ginger and galangal under in vitro conditions result in anatomical and physiological responses that are significantly different from those of plants grown under greenhouse conditions. However, when plants are moved to greenhouse conditions, they can progressively acclimate at a slow growth rate (Preece and Sutter, 1991; Pospíšilová et al., 1999).

Root fresh mass was significantly affected by plant type (P < 0.001) and photoperiod (P

< 0.05), but not by the interaction of both factors (Figure 3-4C). There was a higher root fresh mass under long days compared to natural days. Overall, galangal and ginger tc had the highest root fresh mass (460 and 296 g, respectively), but ginger tc was not significantly different from the other ginger types, and their fresh mass varied from 100 to 183 g. As previously stated, growth of fibrous roots is beneficial for the proper absorption of water and nutrients by ginger plants, and they increase as shoot number increases (Ravindran and Babu, 2005). Galangal and ginger tc had the highest number of shoots, thus it is not surprising to find that they have large root mass. However, Smith and Hamill (1996) found slightly larger root fresh mass in micropropagated plants (up to ~87 g) compared to rhizome-derived plants (~30 g).

Total fresh mass under long days varied among plant types (P < 0.001) and was also affected by the interaction of plant type and photoperiod (P < 0.05, Figure 3-4D). Total fresh

mass of ‘Bubba baba’ was 817 g higher under long days than under natural days. However, and although not significant, an opposite trend was observed for ginger tc, as total fresh mass decreased over 300 g·plant-1 under natural days. Our findings can be attributed to the higher rhizome and shoot fresh mass measured under the long photoperiods, considering that rhizomes and shoots contributed the most to the total fresh mass (Figures 3-4 and 3-7), which is similar to the findings of year 1.

Ravindran and Babu (2005) state that many growth parameters including number of shoots, leaves, secondary and tertiary rhizome fingers, plant height, or weight of adventitious roots are usually correlated with rhizome yield. In our experiment, shoot number was positively and significantly correlated with rhizome yield (r = 0.43, P = 0.004). Moreover, Rai et al. (1999) reported that higher rhizome yields were strongly associated with chlorophyll content and carbohydrate, however we did not find such positive correlation with SPAD values (P > 0.05).

Postharvest Evaluations – Greenhouse Trial – Year 2

Galangal had the highest weight loss both under natural (35.7%, Figure 3-8A) and long days (26.3%, Figure 3-8B). There were no differences among the rest of the ginger types and the range of weight loss varied from 4.3% to 9.8%. One of the most important factors that affect the rhizome quality is the loss of internal moisture (Chung and Moon, 2011). These authors compared the changes in weight of ginger rhizomes stored in modified atmosphere packages

(MAP) with non-packed rhizomes at 12⁰ C and found over 30% weight loss after a month of storage in the control rhizomes. This is higher than our findings, where we stored the rhizomes for two weeks, however closer to the moisture loss in galangal. Additionally, due to the high humidity (~90%) maintained in the cooling chamber when rhizomes from the natural days were stored, all rhizomes suffered from decay as their skin had more mold than when rhizomes from long days were stored at lower RH (~70%). However, galangal had the highest level of decay

(Figure 3-9) as rhizomes from both environments were infected with Fusarium sp. (~83% from long days-high RH, and ~33% from natural days- lower RH).

Regarding color attributes, we hypothesized that even though these can vary among plant types, they would not significantly change with photoperiod. The purpose of extending the daylength is to promote active growth by inducing more photosynthesis that ultimately affect rhizome quantity, while maintaining the rhizome quality. According to our results, external

(surface) and internal measurements of these parameters were not affected by photoperiod after the growth period (P > 0.05). However, quality can change over time, therefore color parameters were measured before and after storage. The storage period did not affect external and internal color on rhizomes grown under long days (P > 0.05, external C* was the exception as it became more gray, decreased from 26.7 to 25), but it did affect the color when rhizomes grew under natural days (P < 0.05). External L* decreased by 4 and C* decreased by ~16, while internal C* increased by ~6 after storage (Tables 3-2 and 3-4). As any other horticultural crop, ginger and galangal are sensitive to postharvest processes as they usually lose glossiness and the skin darkens after storage (Paul and Cheng, 2015). Color changes are intensified when commodities are stored at higher temperatures (Yang, 2017). According to Chung and Moon (2011) lower L* values indicate increased browning in ginger rhizomes, then rhizomes grown under natural days became slightly brown after storage. Under long days, galangal had the highest external C* (35.6,

Table 3-3) and there were no differences among ginger species (values around 22). Based on this color system, it is expected that values do not differ within a genus, but that there is variation between genera. Similarly, under natural days, galangal had the highest C* value (44.6, Table 3-

2). There were no differences between gingers and h⁰ was not significantly different between genera.

Under both photoperiods and based on hue and chroma values, surface color of galangal rhizomes were yellow (h⁰ ~80⁰) and more vivid (C* ~40) than ginger types which had a dull (C*

<33) orange color (h⁰ ~26⁰) and were brighter than galangal (L* >63, Tables 3-2 and 3-3). In contrast, flesh color of galangal was duller than external and similar to ginger (both C* ~30) maintaining their high lightness (L* >65, Tables 3-2 and 3-3). Our results agreed with Paul and

Cheng (2015), who characterized ginger as having an intense sheen on skin.

Rhizomes from both photoperiods were harvested and stored at different times, therefore we cannot compare the postharvest performance of both together. However, even though there was some weight loss the external color of rhizomes grown under long days was not affected by the storage period compared to rhizomes from natural days (Tables 3-2 and 3-3). The loss of weight and the fungal infection impacted negatively the visual appearance of galangal rhizomes, as this resulted in rhizomes with superficial shriveling with moldy spots and less bright color.

Experiment 2 – Field Experiment

In our field experiment, only a few aspects of growth were affected by the environment

(Table 3-4). Plant height was the only parameter affected by plant type (P < 0.001) and the interaction of plant type and environment (P < 0.05, Table 3-4). There were no significant differences by environment within ginger types, however galangal grew the most under shade

(137.5 cm) and ginger ownrhiz grew the least under full sun (30.3 cm). Light is required for plant growth and development. However, not all plants have the same optimum requirements, as deficient and excessive light intensities can be injurious for the plant (Manaker, 1997). Light intensity can be modified by providing shade to plants. Studies have shown that shade improves conditions for efficient photosynthesis activity, which ultimately can increase yield (Zhao et al.,

1991). In situations with low light, under shaded conditions, plants have evolved developing two opposite strategies: shade avoidance and shade tolerance. Most plant species have the ability to

avoid shade, their phytochrome receptors can perceive the reduction in red/far-red (R:FR) ratio and stimulate elongation growth (Ruberti et al., 2012). This could be one of the reasons why we found higher growth under shade. Other studies in ginger have also reported an increase in plant height and shoot number at reduced light intensity (Wilson and Ovid, 1993; Vastrad et al., 2006).

Average shoot number was affected only by plant type (P < 0.01) and in contrast to what was found in our greenhouse experiments, ginger ownrhiz had the smallest average shoot number (0.6, Table 3-4) compared to the other gingers. As previously mentioned, micropropagated ginger usually produce several shoots compared to rhizome-derived plants

(Smith and Hamill, 1996). However, the transition from in vitro conditions (high RH and low light) to the field makes establishment complicated, and this might have limited shoot production

(Preece and Sutter, 1991; Pospíšilová et al., 1999, Rout et al., 2000).

The chlorophyll index (SPAD) also varied among plant types (P < 0.05, Table 3-4) and ginger ‘Bubba baba’ rhiz had significantly higher SPAD (47.6) than ginger ownrhiz (41.5). Even though we did not find significant differences across environments, other studies growing ginger under different shade levels (20%, 40%, and 60%) have reported high flavonoid content with increased shade levels when there is also high chlorophyll content in the leaves as they are positively correlated (Klaus, 2001; Ghasemzadeh et al., 2010). Nevertheless, all plant types had optimal chlorophyll content (>40; Li et al., 2018) leading to an acceptable plant performance

(overall rating ~3), which was also significantly different among plant types (P < 0.01, Table 3-

4). Galangal had the highest rating (3) followed by ‘Bubba baba’ rhiz, and this was not significantly different from the other gingers (2.5 to 2.7). Plants produced less flowers in the field compared to the greenhouse regardless of the genus, although there were no significant differences among plant type, environment or the interaction (P > 0.05), (Tables 3-1 and 3-4).

Rhizome fresh mass was significantly influenced by plant type (P < 0.001), the environment (P < 0.05), and the interaction of plant type and environment (P < 0.05, Figure 3-

10A). Under both environments, galangal had the highest rhizome fresh mass, and this was higher under full sun (2,836 g) than under shade (2,012 g). The three ginger types had similar yields when grown under shade (from 234 to 841g), but ginger ownrhiz had higher fresh mass under both environments (~884 g), proving that micropropagated plants can have high yields after the first year. Studies comparing different shade levels and cultivars of ginger have demonstrated that high rhizome yield and quality can be achieved with some level of shade

(20%, 25% or 40%) (Ajithkumar and Jayachandran, 2003; Ravindran and Babu, 2005).

Ghasemzadeh et al. (2010) state that under shade ginger plants increase nutrient uptake therefore, high yield and quality is increased. However, we did not see higher yields for ginger under shade. It may be that this level of shade (60%) was too high and it made plants allocate resources to vegetative aerial growth, which promoted shoot elongation instead of nutrient translocation to the rhizomes (Ruberti et al., 2012). In addition, when the amount of light is not limited plants maximize their photosynthetic capacity and increase their growth rate (Miner et al., 2005).

Studies in ginger state that higher light quantity (>1,180 μmol·m-2·s-1) is required for rhizome growth in the field (Dewan et al., 1995, Kun et al., 2002, Xianchang et al., 1996). In our study, plants growing under full sun received ~1248 µmol·m-2·s-1 whereas under shade they received

~674.4 µmol·m-2·s-1.According to Vastrad et al., (2006) this higher natural light might have promoted better nutrient uptake, leading to higher rhizome yield, compared to shade conditions.

Overall, our results agree with Ajithkumar and Jayachandran (2003) as they found reduced yields when plants grew under 60% or 80% shade.

There were no significant differences in fresh shoot mass production between environment and the interaction of plant type and environment, and plant type was the only factor that influenced this growth (P < 0.001, Figure 3-10B). Normally, under shade plants invest in high foliar biomass to optimize light capture and utilization (Yuan et al., 2016), although this was not apparent in this study. Galangal had the highest shoot fresh mass (~2,500 g) compared to the gingers (on average ~450 g) which was not significantly different (range 360

– 607 g). The high shoot mass in galangal might be explained by its higher height and shoot number compared to the gingers. There was a positive correlation between height and shoot fresh mass (r = 0.62, P < 0.001). This is particularly important as shoots photosynthesize and promote rhizome and root development (Klingeman et al., 2004). Thus, it is not surprising that galangal also had the highest root (~396 g) and total fresh (~5,300 g) mass compared to gingers (P <

0.001, Figure 3-10C and D). Root fresh mass was influenced by the environment (P < 0.05), and plants grown under full sun produced significantly more roots (176 g) than the ones growing under shade (121 g). When plants grow under full sun, this higher light intensity is used to allocate more of the biomass to roots to capture water and nutrients to sustain the high transpiration and growth rates (Yuan et al., 2016). Ginger ownrhiz significantly produced more total fresh mass than the other two gingers altering the carbohydrate partitioning (Figure 3-11).

Unlike species that avoid shade, very little is known about regulation of shade tolerance.

This kind of plants have the ability to grow and survive in very low light environments, while light-demanding species need higher light intensities for optimal growth (Ruberti et al., 2012;

Gommers et al., 2013; Laurentius et al., 2013; Yuan et al., 2016). Ginger grows well under moderate light intensity and when they grow under shade tissue temperature decreases, this also slows down photorespiration, promoting a better utilization of photosynthates. Therefore, some

studies have shown that ginger plants grow and produce higher yields under shade (Babu and

Jayachandran, 1997; Chudiwal et al., 2010), but others have not found the same response

(Ajithkumar and Jayachandran, 2003; Vastrad et al., 2006). Besides, studies in turmeric and other crops have suggested that the efficacy of shading might be affected by other factors such as daylength, humidity or rainfall (Hossain et al., 2009). Since these responses are not always consistent, they are considered plastic responses (Chmura et al., 2016). These responses are manifested by a wide diversity of organisms under particular environmental conditions. In other words, the term plasticity is defined as the production of multiple phenotypes from a single genotype, as a response to biotic and abiotic aspects of the environment (Miner et al., 2005;

Yuan et al., 2016). In ginger it is known that rhizomes can be transported long distances, therefore many ginger species might have spread throughout the tropical and subtropical areas around the world producing different phenotypes, making some of them more adapted to shade than others (Ravindran and Babu, 2005).

Light requirement in ginger varies across the different growing stages and they require lower light intensities in the initial growing stages (germination and seedling) and higher light in the actively growing stage (Ravindran and Babu, 2005). Based on the height, shoot number and overall ratings in the first six weeks (data not shown), we observed better growth in plants grown under shade than under full sun in the initial stages. Moreover, shoot fresh mass (r = 0.87, P <

0.001) and plant height (r = 0.62, P < 0.001) were positively correlated to rhizome yield.

Therefore, shade levels could be modified during the plant growth, starting with a higher shade level and then reducing it, aiming to maximize shoot growth. This shoot-rhizome relationship was also reported in many other ginger studies (Roy and Wamanan, 1990; Abraham and Latha,

2003).

Postharvest Evaluations – Field Experiment

As previously stated, fresh rhizomes are very sensitive to decay after harvest, affecting their storability and quality. Therefore, rhizome quality after harvest was also evaluated.

Rhizome weight loss was not affected by environment (P > 0.05), however it was different among plant types (P < 0.001). Overall, rhizomes from the field lost similar moisture (~13%) comparable to rhizomes from the greenhouse (~18%). However, ‘Bubba baba’ rhizomes had high weight loss (~23%) and galangal had the highest rhizome weight loss (32.5%, Figure 3-12).

Additionally, some decay was observed on the ‘Bubba baba’ rhizomes, suggesting its higher susceptibility to shriveling and physical degradation than micropropagated plants when produced in the field.

Surface color of rhizomes was not affected by the environment (P > 0.05, Table 3-5). However, internal color differed between environments (P < 0.05). Since rhizomes from both shade and full sun environments were harvested and stored at the same time postharvest storage was evaluated. This factor did not affect most color parameters (P > 0.05, Table 3-5). Overall, all rhizomes became darker, as L* moved from ~66 to ~63.2 after the storage period. Moreover, rhizome color varied among ginger types (P < 0.001). Rhizomes of ginger ownrhiz was internally and externally brighter (L* >70). While flesh color tended to be duller (C*<22), internal color wass more vivid (C* 38) and external h⁰ was very similar among ginger types (as values ranged from 76⁰ to 78⁰), but significantly different from galangal (~71⁰). Flesh h⁰ was different depending on plant type (P < 0.001). However, according to the color space system, they all belong to the yellow group as h⁰ values ranged from 84.4⁰ to 96.2⁰ (Table 3-5).Since we were assessing different species, it was expected to find differences in color and even interaction between plant type and storage period (P < 0.05) or environment and plant type (P < 0.05) across genera. However, these values were not significantly different within genus (data not shown).

Summary

During year 1, ginger plants grown in larger containers produced more shoot, root, and rhizome mass. In addition, during this first year, natural days consistently produced higher rhizome yield, whereas long days increased yield when plants were grown in small containers.

We found that micropropagated plants had small diameters and were not be suited for marketability as a fresh product. It is likely the lack of a rhizome structure negatively affects plant growth during the initial production cycle.

In year 2, all plants actively grew under long day treatments applied during the winter.

Shoot number and shoot and rhizome fresh mass were higher under long days than under natural days. Ginger ownrhiz and galangal had the highest rhizome yield. The high yield and marketable rhizome size of these micropropagated plants indicates that the tissue culture technique may be effective as clean stock material to produce seed rhizomes for first-year (galangal) and second- year (ginger) production.

In our field experiment, galangal grew the most under shade and almost doubled the size of plants grown in the greenhouse, which allowed for high rhizome yield. However, higher yields for galangal were found under full sun, while yield of ginger plants was not affected by the shading treatments. Regardless of the shade, all plant types had optimal chlorophyll content, which led to an acceptable plant performance overall. As opposed to our greenhouse experiment, plants produced less flowers and lower rhizome yield in the field. Shoot fresh mass and plant height were positively correlated to rhizome yield. Thus, shade levels could be modified during the plant growth to maximize shoot growth and rhizome yield.

Regarding rhizome quality after harvest, external (surface) and internal color parameters were not affected by photoperiod. Although galangal had the highest rhizome yield, high quality rhizomes were difficult to maintain. Therefore, more postharvest studies on galangal should be

done, focusing on harvest time and storage conditions. In addition, field harvested ‘Bubba baba’ was more susceptible to decay and weight loss than when grown in the greenhouse. Overall, the external color of rhizomes grown under long days was not affected by the storage period compared to rhizomes from natural days. Although internal rhizome flesh became opaque, the color of field grown rhizomes did not vary across plant types.

Table 3-1. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), average new shoot number, height increase, number of flowers, overall rating, and SPAD in the greenhouse under natural (<12 h) and long days (>12 h) from 2018 to 2019 (year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. SPAD Treatment New shoot number Height increase (cm) No. of flowers Overall ratingz Chlorophyll index Main effects Plant type Galangal tc 1.4 a 65.5 a 0.1 b 3.2 ab 39.5 cb Ginger ‘Bubba baba’ rhiz 0.8 b 60.2 a 0.6 ab 3.8 a 48.1 a Ginger tc 1.5 a 37.1 b 0.8 ab 3.6 ab 46.6 ab Ginger ownrhiz 1.1 ab 16.3 b 2.4 a 2.9 b 44.9 b Photoperiod Long days 1.4 ay 48.9 NS 0.75 NS 3.2 b 44.6 NS Natural days 1.0 b 40.6 NS 1.18 NS 4.0 a 45.0 NS ANOVA Summary Plant type * * * * * Photoperiod * NS NS * NS Plant type*Photoperiod NS NS NS NS NS zA subjective visual scale from 1 to 5 was used to measure overall aesthetic performance, 1 = very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2 = poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3 = acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4 = very good quality with very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 3-2. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes grown under natural days (<12 h) and stored for two weeks at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH. Data represent the least squared means derived from a two- way ANOVA using the general linear model procedure in R. External Internal Treatment L*z C* h⁰ L* C* h⁰ Main effects Plant type Galangal tc 63.1 NS 44.6 a 74.7 NS 64.8 NS 29.7 NS 79.1 b Ginger ‘Bubba baba’ rhiz 67.5 NS 32.9 ab 80.9 NS 62.2 NS 26.6 NS 84.8 ab Ginger tc 65.1 NS 27.3 b 78.2 NS 68 NS 28.1 NS 91.7 a Ginger ownrhiz 69.4 NS 27.5 b 78.7 NS 68.7 NS 27.3 NS 88.6 b Date Initial 68.3 a 41.8 a 79.9 NS 67.6 NS 24.9 a 82.6 NS Final 64.3 b 25.5 b 76.2 NS 64.6 NS 30.7 b 88.5 NS Interaction effects

Plant type*Date Galangal tc Initial 63.5 NS 54.7 NS 72.2 NS 65.9 NS 26.8 aby 76.8 bc Galangal tc Final 62.8 NS 36.1 NS 77.1 NS 63.9 NS 32.1 ab 81.1 abc Ginger ‘Bubba baba’ rhiz Initial 72.5 NS 49.8 NS 85.0 NS 64.3 NS 24.2 ab 74.2 c Ginger ‘Bubba baba’ rhiz Final 64.2 NS 21.7 NS 76.8 NS 60.8 NS 28.2 ab 91.8 ab Ginger tc Initial 67.8 NS 32.4 NS 81.5 NS 71.2 NS 27.6 ab 94.7 a Ginger tc Final 62.3 NS 22.3 NS 74.9 NS 64.8 NS 28.7 ab 88.7 abc Ginger ownrhiz Initial 70.2 NS 33.7 NS 81.4 NS 67.6 NS 20.4 b 84.7 abc Ginger ownrhiz Final 68.7 NS 21.4 NS 76.0 NS 69.8 NS 34.2 a 92.5 ab ANOVA Summary Plant type NS * NS NS NS * Date * * NS NS * NS Plant type *Date NS NS NS NS NS * zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 3-3. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes grown under long days (>12 h) from 2018 to 2019 and stored for two weeks at 12.9 ± 0.1 °C and 65.1 ± 4.4% RH. Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. External Internal Treatment L*z C* h⁰ L* C* h⁰ Main effects Plant type Galangal tc 61.5 b 35.6 a 82.7 a 66.2 NS 30.3 b 84.3 b

Ginger ‘Bubba baba’ rhiz 65.8 ab 21.5 b 74.2 b 67.4 NS 32.9 ab 92.6 a Ginger tc 63.5 ab 21.9 b 74.5 b 66.6 NS 28.4 b 92.5 a Ginger ownrhiz 67.6 a 22.9 b 75.5 b 73.5 NS 36.9 a 94.7 a Date Initial 64.1 NS 26.7 ay 76.9 NS 68.5 NS 32.0 NS 89.9 NS Final 64.2 NS 25.0 b 76.9 NS 68.3 NS 30.9 NS 91.0 NS ANOVA Summary Plant type * * * NS * * Date NS * NS NS NS NS Plant type *Date NS NS NS NS NS NS zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 3-4. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), average new shoot number, height increase, SPAD, overall rating, and number of flowers of plants grown in the field under full sun and 60% shade. Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. New shoot Height increase SPAD-Chlorophyll Treatment No. of flowers Overall ratingz number (cm) index Main effects Plant type Galangal tc 1.1 ab 116.2 a 0.1 NS 3.0 a 44.7 ab Ginger ‘Bubba baba’ rhiz 0.9 ab 74.0 b 0.3 NS 2.7 ab 47.6 a Ginger tc 0.6 b 54.0 bc 0.2 NS 2.6 b 41.8 ab Ginger ownrhiz 1.4 a 47.8 c 0.3 NS 2.5 b 41.5 b Environment Full sun 1.1 NS 99.4 NS 0.6 NS 2.7 NS 48.4 NS Shade 1.0 NS 129.5 NS 0.8 NS 2.9 NS 57.2 NS Interaction effects Plant type*Environment Galangal tc - Full sun 1.2 NS 95.0 aby 0.1 NS 2.9 NS 43.8 NS Galangal tc - Shade 0.9 NS 137.5 a 0.0 NS 3.0 NS 45.7 NS Ginger ‘Bubba baba’ rhiz - Full sun 1.0 NS 74.7 ab 0.6 NS 2.6 NS 45.8 NS Ginger ‘Bubba baba’ rhiz - Shade 0.9 NS 73.4 bc 0.0 NS 2.7 NS 49.4 NS Ginger tc - Full sun 0.7 NS 56.2 abc 0.5 NS 2.4 NS 37.5 NS Ginger tc - Shade 0.5 NS 51.8 bc 0.0 NS 2.7 NS 46.2 NS Ginger ownrhiz - Full sun 1.3 NS 30.3 c 0.1 NS 2.5 NS 36.5 NS Ginger ownrhiz - Shade 1.5 NS 65.3 bc 0.5 NS 2.6 NS 46.4 NS ANOVA Summary Plant type * * * * Environment NS NS NS NS Plant type *Environment NS * NS NS zA subjective visual scale from 1 to 5 was used to measure overall aesthetic performance, 1 = very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2 = poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3 = acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4 = very good quality with very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Table 3-5. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes harvested from the field (full sun and 60% shade), and stored for two weeks at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH. Data represent the least squared means derived from a three-way ANOVA using the general linear model procedure in R. L*z C* h⁰ Treatment External Internal External Internal External Internal Main effects Plant type Galangal tc 57.5 c 63.1 b 36.1 a 28.3 b 70.8 b 84.4 d Ginger ‘Bubba baba’ rhiz 64.4 b 63.1 b 19.9 c 30.8 b 76.1 a 92.2 b Ginger tc 66.1 b 63.2 b 22.2 b 31.0 b 76.6 a 89.1 c Ginger ownrhiz 70.3 a 75.6 a 21.2 bc 38.0 a 78.6 a 96.2 a Environment Full sun 65.1 NS 68.9 a 25.4 NS 34.1 a 74.8 NS 92.0 a Shade 63.9 NS 64.1 b 24.9 NS 30.3 b 75.9 NS 89.1 b Date Initial 65.7 ay 67.5 NS 25.2 NS 32.0 NS 77.2 a 90.7 NS Final 63.2 b 65.0 NS 25.1 NS 31.9 NS 73.6 b 90.1 NS ANOVA Summary Plant type * * * * * * Environment NS * NS * NS * Date * NS NS NS * NS Plan type*Environment NS NS NS NS * NS Plant type*Date NS NS NS NS * NS Environment*Date NS NS NS NS NS NS Plant type*Environment NS NS NS NS NS NS Plant type*Environment*Environment NS NS NS NS NS NS zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

Figure 3-1. Mean values and Tukey grouping comparison for ginger planting materials (‘rhiz’, ‘tc early’ planting, ‘tc late’ planting), container size (5.7 L and 50.5 L), rhizome fresh mass, shoot fresh mass, and root fresh mass under natural (<12 h-A, C, E, and G) or long days (>12 h-B, D, F, and H) from 2017 to 2018 (year 1) in the greenhouse. Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of eight replicate containers ± 95% confidence intervals.

Figure 3-2. Photographs comparing ginger planting materials (‘rhiz’, ‘tc early’ planting, ‘tc late’ planting), container size (5.7 L and 50.5 L) and rhizome fresh mass, under natural (<12 h) and long days (>12 h) from 2017 to 2018 (year 1) in the greenhouse. Photographs are courtesy of Sofia Flores.

Figure 3-3. Mean values and Tukey grouping comparison for ginger planting materials (‘rhiz’, ‘tc early’ planting, ‘tc late’ planting), container size (5.7 L and 50.5 L), carbohydrate partitioning under natural (<12 h, A) or long days (>12 h, B) from 2017 to 2018 (year 1) in the greenhouse.

Figure 3-4. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), rhizome fresh mass (A), shoot fresh mass (B), root fresh mass (C), and total fresh mass (D) under natural (<12 h) and long days (>12 h) from 2018 to 2019 (year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of six replicate containers ± 95% confidence intervals.

Figure 3-5. Visual scale from 1 to 5 to measure overall aesthetic performance of ginger plants grown in the greenhouse from 2018 – 2019 (year 2) under natural (<12h) and long days (>12h). 1) very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2) poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3) acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4) very good quality with very acceptable and desirable color and form, 5) excellent quality, perfect condition, premium color and form. Photographs are courtesy of Sofia Flores.

Figure 3-6. Photographs comparing plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’) and rhizome fresh mass, under natural (<12 h) and long days (>12h) in the greenhouse from 2018 to 2019. Photographs are courtesy of Sofia Flores.

Figure 3-7. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), carbohydrate partitioning under natural (<12 h, A) or long days (>12 h, B) in the greenhouse experiment and harvested in 2019 (year 2).

Figure 3-8. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’) and rhizome weight loss from plants grown under natural (<12 h) and long days (>12 h) from 2018 to 2019 and after a storage period of 2 weeks at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH (natural days) and at 12.9 ± 0.1 °C and 65.1 ± 4.4% RH (long days). Bars represent the mean of six replicate plants ± 95% confidence intervals.

Figure 3-9. Photographs of rhizomes grown in the greenhouse under natural days (<12 h) and harvested in 2019 showing decay symptoms after 2 weeks of storage at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH. Photographs are courtesy of Sofia Flores.

Figure 3-10. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), rhizome fresh mass, shoot fresh mass, root fresh mass and total fresh mass under full sun and 60% shade (field experiment). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of six replicate containers ± 95% confidence intervals.

Figure 3-11. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’), carbohydrate partitioning for plants grown in the field under full sun (A) and 60% shade (B) and harvested in 2019 (year 2).

Figure 3-12. Mean values and Tukey grouping comparison for plant type (‘galangal tc’, ‘ginger Bubba baba rhiz’ and ‘ginger tc’ and ‘ginger ownrhiz’) and weight loss of rhizomes from plants grown in the field from 2018 to 2019, harvested and stored for a period of 2 weeks at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH. Bars represent the mean of six replicate plants ± 95% confidence intervals.

CHAPTER 4 INTERACTION OF PROPAGATION MATERIAL, CULTIVAR, AND ENVIRONMENTAL FACTORS ON ORNAMENTAL PLANT PERFORMANCE, RHIZOME YIELD, AND RHIZOME QUALITY OF TURMERIC (CURCUMA SPP.).

Background

The United States (U.S.) is a major importer of spices, and in 2017 the import values reached $1,801 million (Nguyen et al., 2019). Among the most commonly consumed spices in the country, turmeric (Curcuma spp.) is gaining popularity as its rhizomes are high-value products. Turmeric is a common name used for several species belonging to the genus Curcuma, within the family Zingiberaceae. Curcuma longa Linn. is by far the most popular species as its rhizomes are commonly used as spice in cooking and contain high amounts of bioactive compounds, such as curcuminoids, that are known to have several pharmacological properties

(Choudhary and Kumar, 2018; Popuri and Pagala, 2013; Ravindran et al., 2007; Ruby et al.,

1995).

Curcuma is large genus comprising approximately 130 edible, medicinal and ornamental species (Ravindran et al., 2007; Prasad and Aggarwal, 2011). Curcuma caesia Roxb. commonly called “Black turmeric” due to its bluish-black and odorous rhizomes, is another important medicinal plant. The rhizomes are traditionally used for the treatment of fever, epilepsy, asthma, and other diseases as well as cosmetic purposes (Pandey and Chowdhury, 2003; Sweetymol and

Thomas, 2015). Similarly, Curcuma amada Roxb and Curcuma mangga Val. & Zijp. are both commonly known as “mango ginger” because their rhizomes have a raw mango flavor and aroma that is attributed to a high content of the volatile oil ocimene (Ayodele et al., 2018;

Policegoudra et al., 2011). These two species are morphologically similar to Curcuma longa, but their rhizomes are less pungent and have a creamy-yellow color (Ravindran et al., 2007).

Traditionally their rhizomes have been used in culinary preparations and are also claimed to have

health benefits. However, little is known about growing conditions of this turmeric for high rhizome production.

Due to the presence of curcuminoids and other several bioactive compounds (such as turmerone, curcumene, curzernone, etc.) in the rhizomes of these species, they all have important medicinal properties (Ayodele et al., 2018; Chatterjee et al., 2012; Jatoi et al., 2007). Moreover, since the import values of turmeric in the U.S. have been increasing gradually in the last few years there is a high potential demand for these turmeric species in the country (Akter et al.,

2019; Das et al., 2013; Nguyen et al., 2019; Ravindran et al., 2007; Smith et al., 2017; Tridge,

2019b).

Flowering and fruit set varies greatly among Curcuma species under varying environmental conditions. There are reports of flowering and no flowering types of C. longa, but no seed set has been reported in any of them (Ravindran et al., 2007). For this reason, turmeric is traditionally propagated by seed rhizomes. Preservation of rhizomes for seed material is labor intensive as additional time and space is required (Nasirujjaman et al., 2005). In addition, rhizome-derived plants are susceptible to soil-borne diseases such as Pythium, Fusarium and bacterial wilt (Pseudomonas solanacearum), which requires preventative fungicide applications.

The risk of transmittance of these diseases from one generation to the next contributes to important losses to growers (Ravindran et al., 2007; Salvi et al., 2002). This situation makes the use of healthy rhizomes necessary. Thus, rhizomes are treated with chemical fungicides or

Trichoderma harzianum as it is estimated that a three-fold increase in rhizome yield could be possible by disease control (Ma and Gang, 2006; Ravindran et al., 2007). An alternative to seed rhizome propagation is to use tissue culture-derived transplants, which can ensure pathogen-free and uniform starting material. Currently, this technology is limited by expense, and yield is

typically lower the first year compared to rhizome-derived plants (Salvi et al., 2002; Smith and

Hamill, 1996).

Turmeric is cultivated in tropical and subtropical regions of the world (Ravindran et al.,

2007) and production in the U.S. has been limited to Hawaii and very few states in the southeast.

It is primarily grown by small growers that produce conventional and organic turmeric products

(Calpito et al., 2018; Huang, 2016; Hunter, 2018; Snyder, 2018). Turmeric grows well from sea level to 1,500 masl and optimum growing temperatures are around 20 to 35 °C. Extended periods of low temperatures (<14 °C) can lead to dormancy (Jayashree et al., 2015; Ravindran et al.,

2007). Turmeric is considered a shade crop by farmers and it is often a component of perennial crop-based cropping systems including coconut, areca nut trees, and in forest plantations providing substantial income for the farmers (Bhuiyan et al., 2012; Kittur and Sudhakara, 2016).

Even though growth parameters such as shoot biomass and curcumin production seem to be maximized when plants are grown under some level of shade (from 25% to ~50%), lower rhizome yields are obtained with higher (>50%) shade levels (Hossain et al., 2009; Ravindran et al., 2007). Therefore, to develop successful turmeric production systems in Florida, considerations about environmental limitations such as excess of radiation must be considered.

For ornamental species of Curcuma, a dormancy period is required for flowering

(Sirirugsa and Newman, 2000). In the northern hemisphere, under field conditions, turmeric plants start undergoing dormancy in the fall-winter months and the rhizomes will grow back during the summer (Panneerselvam, 1998; Ravindran et al., 2007). Therefore, under Florida conditions, dormancy of plants might limit the rhizome production to specific seasons during the year. In contrast, under controlled environments day-extension or night-interruption with electric lamps are strategies commonly used by the horticulture industry to control certain aspects of

growth such as flowering. Few studies have shown the significant effect that photoperiodic control has on vegetative growth of various crops including ginger, citrus nursery trees, or tomato (Adaniya et al., 1989; Brar and Spann, 2014; Dominique-Andre et al., 1998; Inoue, 1989;

Pandey et al., 1996). Therefore, by manipulating the photoperiod under controlled conditions, growth and rhizome yield could be maximized.

Regardless of the growing conditions, after seven to nine months the plant is ready for harvest depending on the variety (Jayashree et al., 2015). In India and Japan, rhizomes usually develop during the fall and mature in the winter, when the harvest takes place. Even though plants can be harvested earlier, winter harvests are recommended for higher dry yield (Hossain,

2010). Similar to ginger, turmeric is available in several product forms. Rhizomes can be marketed fresh or dried for use in cooking, medicine, or as planting material (seeds). Although turmeric is mostly consumed in powder form, it is commonly imported as a whole rhizome in order to be processed into powder or oleoresin later on. Fresh turmeric rhizomes are graded into fingers, bulbs, and splits. Fingers are the secondary branches from the mother rhizome, usually 2 to 8 cm long and are easier to grind than the bulbs. Bulbs and splits are the bulbs cut into halves or quarters before curing, which are more fibrous and therefore more expensive than the fingers

(FAO, 2004). Rhizome quality is judged by a clean and smooth skin, uniform skin and flesh colors, and a clean snap when broken. Turmeric cleanliness specifications for import pertain to whole rhizomes. Depending on the final market product, harvested rhizomes go through a number of postharvest processes such as boiling, drying and polishing (Ravindran et al., 2007).

For fresh market, rhizomes are washed, cleaned and sanitized with hypochlorous acid. In addition, ~20% of the harvest is retained every season for seed material (Dodamani et al., 2017).

Then, cleaned and graded rhizomes are packed generally in burlap gunny bags and stored in a

cool, dry but well-ventilated room to prevent rot or postharvest pathogens (FAO, 2004;

Jayashree et al., 2015; Pankaj, 2017). To maintain high internal phytochemical (curcuminoid levels) quality and flavor of rhizomes, temperatures of ~14 °C are recommended (Policegoudra et al., 2011). Therefore, due to the perishable nature of the rhizomes (susceptible to rotting, sprouting and shriveling), proper postharvest handling is required to keep them in healthy conditions and should be considered as an important factor for successful turmeric production.

Since demand for spices continues to rise in the U.S. and due to the wide variety within the Curcuma genus, numerous opportunities exist to optimize turmeric production systems in

Florida. Practices related to planting material for propagation, photoperiodic control, and radiation (light and temperature) stress tolerance must be developed. Moreover, evaluating the growth and production of this alternative crop started from different planting materials and grown in different container sizes can help us determine the best growing conditions for high quality rhizome production in a greenhouse. Therefore, the objectives of this study were 1) to evaluate the growth and yield of turmeric propagules (rhizome-derived and micropropagated transplants) grown in different container sizes and under varying photoperiods in a greenhouse environment; and 2) to measure the effect of shading conditions on plant growth and rhizome yield and quality of turmeric plants grown in the field.

Materials and Methods

Experiment 1. Greenhouse Experiments

Two experiments were conducted over a two-year period. The experiment in year 1 was conducted at the University of Florida (UF) Environmental Horticulture Research Greenhouse

Complex in Gainesville, FL. The experiment in year 2 was conducted at the Plant Science

Research and Education Unit (PSREU) in Citra, FL. In year 1, propagules used were

micropropagated or tissue cultured transplants “tc” of unknown yellow turmeric obtained from

Agri-Starts Inc. (Apopka, FL, USA) and Hawaiian-grown rhizomes of cultivar ‘Black’, ‘BKK’,

‘Hawaiian Red’, and ‘White Mango’ (“rhiz”) obtained from Hawaii Clean Seed LLC. (Pahoa,

HI, USA). In year 2, the same materials were used, and in addition, rhizomes harvested from the

“tc” material (“ownrhiz”: second generation “tc”, harvested after about a year of growth) and

“tc” of an unknown white cultivar were also included.

Year 1 (2017-2018)

This experiment was conducted from 19 Apr. 2017 to 29 Jan. 2018 and aimed to evaluate growth and yield of turmeric propagules grown in two container sizes and under two photoperiods in a greenhouse environment. Propagules of turmeric included Hawaiian-grown rhizomes (“rhiz”) planted on 19 Apr. 2017 and micropropagated transplants (“tc”) planted on 27

Apr. 2017. Micropropagated transplants were initially planted individually in 380 mL containers

(Pöppelmann TEKU®, Claremont, NC) filled with sphagnum peat substrate (Klasmann-

Deilman, Miami, FL, USA), while “rhiz” rhizomes were planted individually in 2.78 L containers (Nursery Supplies Inc, Kissimmee, FL, USA) filled with a substrate consisting of sphagnum peat and perlite (Fafard®2P, Sun Gro Horticulture Distribution Inc, 770 Silver Street

Agawam, MA, USA).

All plants were subsequently repotted on 17 July 2017 either into 5.7 L or 50.5 L containers (Nursery Supplies Inc) with a 1:1 (v/v) mix of coarse coconut husk chips and fine coconut fiber (Envelor Inc, Old Bridge, NJ, USA). Containers were placed in two polycarbonate- covered greenhouse compartments in Gainesville with automated heating and pad-and-fan evaporative cooling. Within each compartment, containers were arranged in a randomized complete block design with eight replicate containers per plant material type and container size,

where two replicate containers per plant material type and container size combination were randomly located on each of the four benches (blocks) inside the greenhouse.

One compartment received “natural days”, and the second received “long days” provided by night interruption lighting from 10 pm to 2 am with incandescent lamps at 3.2 µmol·m2·s-1 of

PAR. In the natural days greenhouse, average temperature was 23.2 ± 2.2 oC and light was 11.8 ±

5.8 moles·m-2·d-1 DLI. The long days greenhouse averaged 22.0 ± 1.0 oC, with 6.7 ± 4.5 moles·m-2·d-1 DLI and 74.1% RH. In both greenhouses, plants were hand-irrigated with 17-1.8-

14.1 blended water-soluble fertilizer (Greencare Fertilizers, Kankakee, Michigan) at 200 mg/L N with each irrigation.

Between 17 – 21 Aug. 2017 all plants in 5.7 L and 50.5 L containers were mounded with

5 cm and 10 cm of additional substrate, respectively. Plants were harvested between 22 - 29 Jan.

2018. Fresh mass of rhizomes, shoots, and roots, and percent rhizome moisture [rhizome fresh mass – (dry mass of rhizomes oven dried in a 50 oC oven for three days) / rhizome fresh mass] were measured. Effects of container size and plant material were analyzed separately between the two greenhouse compartments (light treatments) with analysis of variance (ANOVA), using

RStudio version 3.3.2 (RStudio, Inc., Boston, Massachusetts, USA) and Agricolae, Stats, and lsmeans packages. Least-square treatment means were compared using Tukey’s honestly significant difference with P = 0.05.

Year 2 (2018-2019)

This experiment was conducted from 18 Apr. 2018 to 9 Mar. 2019 and aimed to evaluate yield of turmeric propagules produced under two photoperiods in a greenhouse environment.

Propagules of turmeric included micropropagated transplants (“tc”, including a white cultivar), second generation “tc” rhizomes (“ownrhiz”), or Hawaiian-grown rhizomes (“rhiz”). Rhizomes were initially planted on 18 Apr. 2018 in black plastic flat trays (T.O. Plastics, Inc. Clearwater,

MN, USA.) filled with sphagnum peat substrate (Klasmann-Deilman) under growth chamber conditions with 25 ± 0.5 °C and 84.3 ± 4.5% RH. Micropropagated propagules were transplanted into 2.78 L containers (Nursery Supplies Inc) filled with a substrate consisting of sphagnum peat and perlite (Fafard®2P) on 4 May 2018. Sprouting of rhizomes took place between 22 May and

5 June 2018. Sprouted rhizomes were then planted into 2.78 L containers with the same substrate and grown in a polycarbonate-covered greenhouse in Gainesville, FL. The greenhouse had a day and night temperature of 25.6 ± 1.9 °C and 24.7 ± 1.8 °C, respectively. All plants were subsequently repotted on 27 June 2018 into 14.5 L pots with a mix of pine bark, sphagnum peat, perlite, and vermiculite (Fafard®52 Mix, Sun Gro Horticulture Distribution Inc). Pots were then placed in two polycarbonate-covered greenhouse compartments in Citra, FL, with automated heating and pad-and-fan evaporative cooling. The experimental design was a split-plot design with photoperiod as the main plot and plant type as the subplot. Each greenhouse compartment had a different photoperiod. Within each compartment, containers were arranged in a randomized complete block design with six replicate pots per cultivar and propagation material, where two replicate pots per cultivar and propagation material combination were randomly located on each of three benches (blocks).

One compartment received “natural days”, with day and night temperatures of 26.6 ± 3.1

°C and 21.3 ± 2.6 °C respectively, and 9.1 ± 3.6 moles·m-2·d-1 DLI. The second greenhouse had

“long days” provided by night interruption lighting from 10 pm to 2 am with incandescent lamps at an average of 1.32 µmol·m-2·s-1 PAR from 6 July onwards. It had 26.8 ± 3.9 °C and 21.4 ± 2.1

°C day and night temperatures respectively, and 8.4 ± 3.8 moles·m-2·d-1 DLI. Plants were drip- irrigated with tap water and fertilized using an 8 - 9 month release 15-3.9-10 Osmocote Plus™

(ICL Specialty Fertilizer Customer, 4950 Blazer Memorial Parkway, Dublin, Ohio) controlled

release fertilizer (CRF) at a rate of 114 g.pot-1. Plants were mounded once with new substrate in

29 Aug. 2018 to approximately a 10 cm depth.

In order to assess plant growth, number of shoots and height were measured every two weeks, as well as chlorophyll index using a chlorophyll meter (SPAD-502DL, Konica Minolta

Sensing, Osaka, Japan). To evaluate the overall aesthetic performance a scale from 1 to 5 was used, where 1 = very poor quality, not acceptable, severe leaf necrosis, tip burn or chlorosis, poor form, 2 = poor quality, not acceptable, large areas of necrosis, tip burn or chlorosis, poor form, 3

= acceptable quality, few leaves with tip burn, somewhat desirable form and color, 4 = very good quality, very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form (Figure 4-1). In turmeric, excellent form was considered when plants were well branched and full, not lodged, without tip burn or chlorotic leaves, and had stems of relatively uniform length.

To demonstrate that the growing season could be extended, plants from the natural days treatment were harvested on 22 Jan. 2019, whereas plants from long days were harvested one month later, on 21 Feb. 2019 (after about seven and eight months of growth in the finishing pots, respectively). After harvest, fresh and dry mass of rhizomes, shoots, and roots as well as percent rhizome moisture [rhizome fresh mass – (dry mass of rhizomes oven dried in a 40 oC oven until constant weight) / rhizome fresh mass] were determined (Li et al., 2016). Additionally, rhizomes from each treatment (natural and long days) were evaluated under postharvest storage conditions.

Rhizomes were cleaned, disinfected with 10% bleach and cured (held at 22 – 26 oC and 70%

RH) for four days. Rhizomes from natural days were then stored in a cool room at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH for 16 days, while rhizomes from long days were stored at 12.9 ± 0.1 °C and 65.1 ± 4.4% RH for 15 days, after adjusting the RH with a dehumidifier. Traits evaluated

were number of infected/damaged rhizomes, percentage of weight loss [(initial -final weight/ initial weight) ×100] and internal and external color determined using a chroma meter (model

CR-400, Konica Minolta Inc., Tokyo, Japan). After curing and after the storage period, the external skin color was measured and then one representative rhizome piece for each type was cut in half to display and measure the internal flesh color. The color system (lightness-L*, chroma-C*, and hue angle-h⁰) uses values calculated from the L*a*b* system, where L* indicates the lightness, which ranges from black (0) to white (100), C* indicates chroma or saturation, and describes the vividness or dullness of a color regardless of its luminance, and h⁰ indicates hue, and is the angle that defines the actual color of the object in the color space (McGuire, 1992;

Minolta, 1994).

Growth, yield, and postharvest data from turmeric plants grown in the greenhouse under natural and long days were analyzed together, where blocks were considered as random effects and photoperiod, plant type and its interaction were considered as fixed effects and were analyzed using RStudio version 3.3.2 for analysis of variance (ANOVA), with Tukey’s Honestly

Significant Difference (HSD) at P = 0.05 for mean separation.

Experiment 2. Field Experiment

This experiment was conducted at the University of Florida (UF) Environmental

Plastics, Inc.) filled with sphagnum peat substrate (Klasmann-Deilman) under growth chamber conditions (25 ± 0.5 °C and 84.3 ± 4.5% RH). Micropropagated transplants were initially planted in 380 mL containers (Pöppelmann TEKU®) filled with peat substrate (Klasmann-Deilman) and then potted individually on 4 May 2018 into 2.78 L pots filled with a substrate consisting of

sphagnum peat and perlite (Fafard®2P, Sun Gro Horticulture Distribution Inc), while sprouted rhizomes were potted individually on 15 May 2018 (using the same container size and substrate) and grown in the same greenhouse in Gainesville. Plants were transplanted in the field on 21

June 2018 under full sun or shade. Six shade structures were randomly set up in four field rows with aluminum poles and 60% shade cloth. Plants under shade received an average day and night temperature of 25.3 ± 7.3 °C and 19.3 ± 7.4 °C, respectively, with 10.3 ± 3.6 moles·m-2·d-1 DLI.

The full sun environment had an average day and night temperature of 23.3 ± 7.0 oC and 16.4 ±

6.8 °C respectively, with 21.1 ± 10.6 moles·m-2·d-1 DLI. Plants were drip-irrigated with tap water and fertilized using an 8 - 9 month release 15-3.9-10 Osmocote Plus™ (ICL Specialty

Fertilizer Customer) CRF at a rate of 114 g·plant-1. Plants were arranged in a split-plot design, with environment (sun and 60% shade) as the main plot and propagules or plant type as subplots.

Micropropagated transplants “tc” of unknown white and yellow turmeric cultivars, originally obtained from Agri-Starts Inc. were used, as well as the harvested “tc” turmeric rhizomes from year 1 (“ownrhiz”), and Hawaiian rhizomes of ‘Black’, ‘BKK’, ‘Hawaiian Red’, and ‘White

Mango’ were also included. Two environment replicates were randomly located on each of the three blocks and there were a total six propagule replicates per environment. Plants were harvested on 4 Feb. 20189, after about 9 months of growth. In order to assess plant growth, number of new shoots, height, and chlorophyll index were measured every two weeks. As in the greenhouse experiment (year 2), the same rating scale was used to evaluate the overall aesthetic performance. Similarly, fresh mass of rhizomes, shoots, and roots, percent rhizome moisture

[rhizome fresh mass – (dry mass of rhizomes oven dried in a 40 °C oven until constant weight) / rhizome fresh mass], and rhizome skin and flesh color were measured after harvest. Rhizomes were also evaluated in postharvest storage conditions. Rhizomes were cleaned and treated as

described above and stored at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH for 16 days. The same traits as described for the greenhouse postharvest experiments were evaluated.

Data from turmeric plants grown in the field either under full sun or shade (environment factor) were analyzed together. Blocks were considered as random effects and environment, plant type, and its interaction were considered as fixed effects and were analyzed by ANOVA with Tukey’s HSD at P = 0.05 (RStudio version 3.3.2).

Results and Discussion

Experiment 1. Greenhouse Experiments

Year 1 (2017-2018)

Under both natural and long days, plant type, container size, and the interaction of plant type and container size had a significant effect on rhizome fresh mass (P < 0.001, Figures 4-1A and B). Regardless of the day length, plants grown in larger containers (50.5 L) consistently had higher rhizome yield, by ~314 and ~427 g·plant-1 in natural and long days, respectively (P <

0.001). This was an expected response as usually large containers favor the growth of plants with multiple, fleshy and thick roots or rhizomes without any physical restriction (Landis et al., 2014).

Under natural days ‘Hawaiian Red’ was the cultivar with the highest rhizome yield (672.4 g, P <

0.001). Under long days ‘Hawaiian Red’ (689.7 g) and ‘White Mango’ (626.7 g) had the highest yields. In contrast, yellow tc turmeric had the lowest yield under both environments (~200 g). All rhizome-derived plants have high yields comparable to other studies (Waman et al., 2018). This might be explained by the large rhizome seed used in this experiment (~33 g; data not shown) as previous studies have reported that large seed rhizomes (~20 g) have large buds, which provide vigorous seedlings that potentially contribute to higher yields. On the other hand, other studies have found higher total weight of rhizomes per plant when plants were grown from rhizomes compared with micropropagated plants (Salvi et al., 2002). Ravindran et al. (2007), state that

tissue-cultured plants of turmeric behave like seedlings and required at least two crop seasons to develop rhizomes of normal size. Similarly, Nayak and Kumar (2006) found higher yields on rhizome-derived plants over micropropagated turmeric and they attributed it to the presence of the pre-formed rhizome in the conventionally propagated plants. Like in ginger, micropropagated plants are usually very small, weighing less than 1 g, and lack the rhizome structure, which is the main source of reserves for plant growth (Smith and Hamill, 1996). Therefore, this response can be explained by the clear morphological differences between rhizomes and micropropagated plants.

Regardless of the environment, the use of large containers resulted in higher shoot

(~1,140 g) and root (~308 g) fresh mass (P < 0.001) as values were ~339 g and 107 g for shoot and root fresh mass respectively, when plants grew in smaller containers (Figures 4-1C to F).

Moreover, under both environments, there was also an effect of plant type (P < 0.001) on shoot fresh mass. ‘Hawaiian Red’ had the greatest shoot fresh mass (724 g under natural days and

1,551 g under long days, compared with other turmeric types (Figure 4-1). During the first stages of growth, the primary fingers of turmeric rhizomes grow in different directions, they can grow up from the ground level producing aerial shoots or undergo further branching (Ravindran et al.,

2007). Thus, this rhizome – shoot interaction, especially when grown in large containers, can explain the high number of shoots found in ‘Hawaiian Red’ since it also had high rhizome fresh mass. In contrast, tc plants did not stand out as their shoot fresh mass was low, contradicting the report by Ravindran et al. (2007) that micropropagated plants of turmeric present multiple but small shoots. Under natural days, turmeric ‘Black’ had the highest root fresh mass when grown in 50.5 L containers (806.7 g), while ‘Hawaiian Red’ had the lowest root fresh mass (92.6 g,

Figure 4-1E). Under long days, root fresh mass was not significantly different among turmeric

types, and the interaction of plant type and container size was not significative (P > 0.05, Figure

4-1F). However, root mass of turmeric ‘Black’ was not high and overall, all turmeric plants had low root fresh mass (<160 g, Figure 4-1E).

Overall, container size, plant type and the interaction of these two factors affected total fresh mass under both natural and long days (P < 0.01). Higher fresh mass (2,488.3 g) was found under long days and larger containers than in smaller containers and under natural days (524 g).

‘Hawaiian Red’ showed the highest growth, regardless of the environment. In contrast, the micropropagated turmeric (yellow tc) had the lowest total fresh mass under natural days (~740 g), however it had higher mass under long days (~1,000 g, Figures 4-1G and H). As a percentage of total biomass, therefore, plants of ‘Hawaiian Red’ allocated the greatest amount of growth towards rhizome growth under long days, while the cultivar ‘Black’ allocated more growth towards rhizome and roots than shoots. This high root fresh mass in ‘Black’ was expected as this turmeric is characterized for presenting roots tubers containing mostly starch (Ravindran et al.,

2007). Moreover, as expected, shoot growth was promoted under long days (Figures 4-2A and

B) suggesting that the growth season could be extended to favor rhizome enlargement in plants derived from micropropagation and growth in general for all turmeric plants.

Year 2 (2018-2019)

Photoperiod and plant type (the combination of species and planting material) affected some parameters related to growth and yield of turmeric (Table 4-1 and Figure 4-3). Regarding overall plant growth and performance, photoperiod affected the average new shoot number (P <

0.01), total number of flowers (P < 0.05) and overall plant performance rating (P < 0.001).

Regardless of the plant type, plants grown under long days produced more new shoots and flowers than under natural days. High shoot number under long days was expected as this characteristic was noticeable in our first experiment. Moreover, other studies have reported that

ginger plants growing under 16 h produce more sprouts than plants grown under 10 h or natural days (Ravindran and Babu, 2005). Ravindran et al. (2007) state that flowering takes place after

~4 months of growth, when days were getting shorter in our conditions (mid-October). Thus, the long days allowed for more flower production. On the other hand, even though overall plant performance was lower under long days (2.7) than under natural days (3.4), overall plant quality was still acceptable (~3, Table 4-1).

Shoot number (P < 0.001), height increase (P < 0.001), chlorophyll index (P < 0.001), total number of flowers (P < 0.01), and overall rating (P < 0.001) were different among plant types. The yellow turmeric species started from micropropagation had the highest number of new shoots (on average 1) compared to their equivalent rhizome-derived (yellow ownrhiz, 0.6) and

‘BKK’ which had the lowest number of new shoots (0.2, Table 4-1). This response agreed with

Salvi et al. (2002), Nayak and Kumar (2006) and Ravindran et al. (2007) because micropropagated turmeric plants produced multiple and higher number of shoots compared to rhizome-derived plants. In addition, Salvi et al. (2002), Nayak and Kumar (2006) state that this high shoot formation maybe due to the carry over effect of benzyladenine supplied to the medium under in vitro conditions. Starting plants from rhizomes could be disadvantageous in this aspect, as after planting primary rhizome fingers initially produce shoots and after some time, they undergo branching producing secondary and tertiary rhizomes. However, these secondary or tertiary rhizomes branches usually do not produce aerial shoots because of their positive geotropic growth (or obliquely downward growth), limiting their shoot production. This characteristic varies by turmeric types and the longa types have more sideward growth whereas the aromatica types have more downward growth (Ravindran et al., 2007). This may be the reason for the variation in shoot number observed within the genus.

Plant height was not affected by photoperiod but by plant type only (P < 0.001).

Rhizome-derived cultivars ‘White Mango’ and ‘BKK’ had the highest increase in plant height as they grew over 100 cm height, compared to micropropagated yellow and white turmerics (<10 cm, Table 4-1). While in other studies no difference was found among turmeric tc or rhizome derived (Nayak and Kumar, 2006), Curcuma plants are characterized by having a height that ranges from 1 to 2 m and growth parameters such as plant height can significantly vary among cultivars (Ravindran et al., 2007). Some Indian cultivars were reported to have maximum and minimum heights of 70 and 44 cm, respectively and there are reports of mango turmeric (C. amada) with leafy shoots up to 100 cm tall (Ravindran et al., 2007; Prasath et al., 2017). Similar to ginger, higher increase in height of rhizome-derived plants can be explained by the greater success on establishment after transplanting compared to micropropagated plants, allowing rhizome-derived plants to grow earlier and faster, getting taller than micropropagated transplants, which are lacking the reserves from the rhizome.

Another indicator of active growth is the relative chlorophyll content in the leaves of turmeric measured by the SPAD meter. Chlorophyll, an important molecule for photosynthesis, is a good indicator of plant nutrient status (particularly nitrogen) and leaf senescence (Xiong et al., 2015). Hence, turmeric plants with high SPAD values (>40 - Lakshmi and John, 2015), indicate good plant performance, which could also translate in high yield (Hossain et al., 2009;

Wang et al., 2019). In this experiment SPAD values were significantly different only among plant types and rhizome-derived ‘Black’ turmeric had the highest SPAD reading (46.5) under both photoperiods (P < 0.001, Table 4-1). This high SPAD in turmeric ‘Black’, might have accounted for its high overall performance, and it has potential as an ornamental plant. Even though we had lower SPAD values compared to other studies (~43 - Lakshmi and John, 2015),

according to our results we can infer that plant quality in terms of chlorophyll content does not change after a second generation from tc plants, as both yellow (tc and rhiz) turmeric had similar

SPAD values (~34).

Turmeric species can be dual purpose because in addition to their rhizome production, they are characterized by having flowers with large and brightly colored top bracts that are the main attraction as ornamental plants. However, not all species have distinctive bracts, or flower consistently every year (Ravindran et al., 2007; Kandiannan et al., 2015). In our experiment, we found distinct responses in respect to flowering of micropropagated turmeric. The yellow tc did not have flowers at all during the whole growing period, but the white tc had the highest number of flowers (on average 2.3) under both photoperiods (Table 4-1). Other studies have shown that flowering on turmeric plants is very rare and fluctuates considerably among species and cultivars. Even when it can take a few years for the plant to start flowering for the first time, in some cases this is still erratic and unpredictable (Nasirujjaman et al., 2005; Kandiannan et al.,

2015). This suggests that long term studies are required to understand the flowering behavior of turmeric plants.

Along with their flower production, many species in the family Zingiberaceae have multiple stems with lush, green or variegated leaves providing a tropical effect in the landscape.

Therefore, they have great potential for use as landscape and patio plants (Kuehny et al., 2005) and as mentioned previously, considerable variation can occur within the genus, thus some accessions may have potential as both landscape ornamental and edible crops. In order to evaluate their overall aesthetic performance, a scale from 1 to 5 was used. A rating of 1 for instance, means that the plant exhibits very poor quality and would not be accepted for an ornamental market, while a 5 means excellent quality for the market (uniform green leaf color

and without damages; Figure 4-6). As stated above, this attribute was significantly affected by plant type (P < 0.01, Table 4-1). The rhizome-derived cultivars had high ratings (≥3.1), while the tc turmerics had low ratings (<3). Therefore, conventionally propagated plants can be of very good quality, with desirable color and form, acceptable for both edible and ornamental market.

After evaluating the growth characteristics for the different plant typess under both photoperiods, it is important to determine if these parameters have any influence on biomass production and yield data, and whether there is any correlation with yield. There were significant effects of plant type (P < 0.01) and the interaction of photoperiod and plant type (P < 0.05) on rhizome fresh mass (Figure 4-3A). Regardless of the photoperiod, white tc and ‘Hawaiian Red’ rhiz had the highest yield (~900 g·plant-1) whereas the micropropagated yellow (first and second generation) had the lowest rhizome yield (~600 g) along with turmeric ‘Black’ (526 g). Great variability among existing cultivars accounted for yield attributes and quality characters (Prasath et al., 2017). There is little research done on photoperiod requirements for turmeric rhizome production. However, studies looking at effects of photoperiod on micro-rhizome formation in vitro, have shown that it is promoted by relatively shorter photoperiods of 4 to 8 hours (Nayak and Kumar, 2006). While significant differences for rhizome yield among daylengths were not found in this experiment, and in contrast to what was found in our ginger experiment (very low rhizome yield of micropropagated plants), micropropagated white tc had the greatest rhizome production (over 1 kg·plant-1 under long days) without altering the size or diameter of individual rhizomes (Figures 4-4 and 4-5). Since rhizome development depends on the translocation of photosynthates and the growth of turmeric is affected by deficiencies of Fe and Zn (Dixit and

Srivastava, 2000), chlorophyll index could have influenced the growth and yield of micropropagated turmeric plants. As shown in Tables 4-1 and 4-2, overall yellow tc had the least

growth and the lowest SPAD under long days, therefore had the lowest yield. While yellow tc and white tc were both micropropagated, it appears that the white tc is genetically different and more vigorous than yellow tc, as it accumulated more chlorophyll in its leaves and produced higher yields. Moreover, it is interesting to note than even when higher levels SPAD (37.3) were recorded in yellow ownrhiz (second generation tc) also under long days, the yield was still very low and no different from yellow tc (Figure 4-3A). According to Dixi and Srivastava (2000), high SPAD does not translate in high photosynthetic efficiency as photosynthates are not always able to translocate to rhizomes and they remain accumulated in the leaves, because this process is energy consuming. In addition, we could observe that even when plants were growing in larger containers (50.5 L pots) in year 1, rhizome yield was still lower (< 500g; with ‘Hawaiian Red’ being the only exception with ~1 kg) than what we found in year 2 in smaller containers (14.5 L pots) as yields were 600 g·plant-1, confirming the great variability of this crop.

In contrast to rhizome yield, , turmeric types can have very low shoot growth. Regardless of the daylength, white tc had the lowest shoot fresh mass (150 g, P < 0.05) compared to

‘Hawaiian Red’, which was significantly greater (286 g, Figure 4-3B). There were also significant effects of photoperiod (P < 0.001), but no interactions between plant type and photoperiod (P > 0.05) on shoot fresh mass. Overall, for all plant types, shoot fresh mass was higher under long days (286 g) than under natural days (145 g). Shoot fresh mass can be explained by number of shoots, plant height or both, and since more shoots were produced under long days this might be an expected response. It is important to clarify that in contrast to year 1, this time plants were allowed to dry out (by turning off the irrigation) for one month before harvesting. Therefore, fresh mass is lower (on average 215 g) than in year 1 (on average 739 g), as leaves lost water and thus fresh weight. Similarly, other studies found reduced shoot biomass

when harvesting of turmeric was delayed from November or December to January (Hossain,

2010).

Number of shoots or leaves and plant height are often found to be highly intercorrelated among themselves (Hossain et al., 2009; Ravindran et al., 2007). In this experiment, we found in general a positive relationship between height and shoot fresh mass and shoot number. However, these values were very small, mainly because each species behaved differently. For ‘White

Mango’ the high shoot mass can be explained by its high increase in height (110.1 cm) rather than shoot number, whereas for other turmerics such as ‘Hawaiian Red’ or yellow ownrhiz it is explained by their high shoot number (on average 0.6). In contrast, the lowest shoot fresh mass found in white tc is mainly due to its low height increase (9.5 cm, Table 4-1). Moreover, shoot fresh mass is not significantly different between ‘BKK’ and yellow tc, because ‘BKK’ had a high increase in height (>100 cm) but almost no shoots (on average 0.2) while yellow tc had a higher shoot number (on average 1) but a small height increase (<10 cm, Table 4-1).

Root fresh mass was affected by plant type (P < 0.001) and photoperiod (P < 0.05), but not by the interaction of both factors (Figure 4-3C). There was a higher root fresh mass under natural days compared to long days. As noted in year 1, ‘Black’ had the highest root fresh mass

(618 g·plant-1) due to their fleshy and ellipsoid root tubers (Ravindran et al., 2007). There were no significant differences between the other turmerics (including tc types) as their fresh mass varied from 142 to 255 g. Overall, the growth of fibrous roots is necessary for the absorption of water and nutrients by the plant and they increase as the shoot number increases (Ravindran and

Babu, 2005). Yellow tc had high shoot and root fresh mass comparable to rhizome-derived plants, therefore it seems that micropropagated plants grow fast once shoots are formed. Various authors state that when plants are propagated in vitro, shoot multiplication is accompanied by

simultaneous rooting of the shoots mainly due to the relatively high concentration of plant growth regulators in the medium used for multiplication (Salvi et al., 2002; Nasirujjaman et al.,

2005).

Even though total fresh mass was not significantly affected by plant type, photoperiod or their interaction (P > 0.05, Figure 4-3D), under long days only total fresh mass varied among plant types (P < 0.05) and white turmeric types had opposite responses. Micropropagated white turmeric had higher total fresh mass (~1,300 g) compared to the rhizome-derived ‘White Mango’ as total fresh mass was ~1,000 g (data not shown). Regardless of the photoperiod, all plants had a total fresh mass of ~1,200 g. In contrast to year 1, less total fresh mass was recorded due to low shoot fresh mass of year 2 plants. Since no difference in rhizome yield was found across photoperiods, it was observed that ~52% of the growth was allocated towards rhizome biomass

(Figure 4-7). However, under long days very little growth was allocated towards root biomass

(<10%) compared to natural days (~23%), confirming the results reported by Dixi and Srivastava

(2000) that higher translocation of primary photosynthates was reported in freshly developing rhizomes than in roots. Similar to our experiment in year 1, long days promoted more shoot biomass (~37%) than the natural daylength (~23%). It is important to note that under both photoperiods the rhizome-derived ‘Black’ turmeric allocated the most on root biomass (~35%) due to its dissimilar root morphology, presenting tuberous roots.

Postharvest Evaluations – Greenhouse Trial – Year 1

Rhizome weight loss was significantly different among plant types (P < 0.001). Overall weight loss was ~10% for all turmeric types and ‘Black’ rhizomes had the lowest weight loss

(4.6%, Figure 4-8). Since rhizomes were stored separately by photoperiod (they were harvested in different dates), we cannot compare them together. However, we observed that rhizomes from long days had higher (9.5%) weight loss than the ones from natural days (7%), this maybe be due

to the lower RH set in the postharvest room or because rhizomes of ‘White Mango’ from the long days treatment had the highest weight loss (17.4%).

After harvest, rhizomes are perishable due to their susceptibility to shriveling, therefore pre-storage treatments such as curing are recommended. For instance, significantly lower weight loss (23.15%) was found when rhizomes were treated with Trichoderma before storage

(Dodamani et al., 2017). On the other hand, in our ginger field experiment, the stored rhizomes lost up to 25% of their initial weight after their storage period. Hence, it seems that turmeric is not only less sensitive than ginger, but also the types evaluated in this experiment, might have had better overall performance than other turmeric types. Micropropagated turmeric white, one of our most productive turmerics, lost little weight (5.7%) regardless of the photoperiod under which it was grown. High rhizome quality in turmeric is determined by a clean and smooth skin and flesh colors with a clean snap when broken (FAO, 2004). Therefore, if rhizomes are stored for longer periods weight loss can be manifested by superficial shriveling and less bright color, impacting their visual appearance negatively.

Color parameters were also measured before and after the storage period. For plants grown under natural days, the storage period affected the external and internal color of rhizomes

(P < 0.001), but the external color was not significantly different among turmeric types or affected by the interaction of plant type and storage period (P > 0.05, Table 4-2). All color parameters decreased after storage of rhizomes regardless of the species or cultivar. After the storage period, skin of rhizomes looked darker (L* decreased by ~6) and color shifted from pale yellow to dull orange (C* decreased by 12.4 and h⁰ went from 89⁰ to ~65⁰). In contrast, internal color was affected by all factors; plant type (P < 0.001), date [(P < 0.001), L* the exception as they all lost lightness after storage], and by the interaction of plant type and date (P < 0.001),

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regardless of the photoperiod treatment. After storage, therefore, flesh color became more vivid and tended to be more yellow (Table 4-2).

Under long days, the storage treatment did not affect external or internal color, but these varied among turmeric types (P < 0.001). The rhizome-derived cultivars ‘BKK’ and ‘Hawaiian

Red’ and micropropagated tc had similar high chroma (~31 external and ~60 internal C*) compared to turmeric ‘Black’ as values were ≤17.1 (Table 4-3). While the external color was also affected by the interaction date-plant type (P < 0.05), internal color did not vary (P > 0.05,

Table 4-3). However, if we compare them by turmeric type, we can see that L* and C* did not vary within each type. It is expected that values do not differ within a species, but that there is variation between genus.

Under both photoperiods L* and C* were very similar, meaning that flesh color was more vivid than the outer skin. Based on C* and h⁰ values, we confirmed that the internal color of ‘Hawaiian Red’, ‘BKK’, and micropropagated yellow rhizomes is brighter and orange-yellow

(C* ~41 and h⁰ ~65⁰), ‘White Mango’ and micropropagated white rhizomes are more gray yellow (C* ~31 and h⁰ ~93⁰), and ‘Black’ is more opaque, with a green-blue color (C* ~18 and h⁰ ~176⁰, Tables 4-2 and 4-3).

Experiment 2 – Field Experiment

Even when plant growth and productivity can be maximized under greenhouse conditions, field production can also be advantageous as Curcuma species are considered tropical plants and several authors have indicated that they perform very well under shade (Ravindran et al., 2007). Shade provides improved conditions for efficient photosynthetic activity promoting plant height and higher shoot biomass, which can potentially increase yield (Zhao et al., 1991;

Hossain et al., 2009). Hence, the objective of this experiment was to compare the growth and yield of various turmeric types growing under full sun or 60% shade. Additionally, as previously

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emphasized, micropropagation techniques represent an ideal alternative to produce disease-free plants, however field establishment of these plants is still a challenge as they often require more care and time for adaptation to this new environment (Rout et al., 2000).

Even though only a few aspects of growth were affected by the different environments, these growth parameters varied greatly by plant type. New shoot number (P < 0.05), plant height

(P < 0.001), chlorophyll index (P < 0.001), and overall plant performance (P < 0.001) were different among turmeric types (Table 4-4). In contrast to our greenhouse experiment, turmeric plants grown in the field produced very few shoots during their whole growth period (on average

≤ 0.3). Regardless of the light condition, rhizome-derived cultivars ‘White Mango’ and yellow ownrhiz had the highest number of shoots (on average 0.3) while micropropagated white turmeric had the lowest shoot number, although not significantly different from the rest of turmerics (0.1, Table 4-4). Other studies comparing the growth of turmeric under different light conditions have reported higher number of shoots under reduced RLI (52% or 48%) than under full sun conditions after five months of growth. However, no differences were found when measurements were taken months earlier (Hossain et al., 2009). Regarding plant height increase, some studies have also found increased plant height under increased levels of shade in comparison to full sun (Hossain et al.., 2009; Bhuiyan et al., 2012). However, in this experiment no significant differences were found between environments (P > 0.05). For optimal growth and development, not all plants have the same requirements of light intensity. Deficient and excessive light intensities can be injurious for the plant as it could lead to photoinhibition and loss of plant performance (Manaker, 1997; Takahashi et al., 2010). In situations with low light, under shaded conditions, plants have evolved developing two opposite strategies: shade avoidance and shade tolerance. Most plant species have the ability to avoid shade as their

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phytochrome receptors can perceive the reduction in red/far-red (R:FR) ratio and stimulate elongation growth (Ruberti et al., 2012). Since plant growth was not affected by shading, turmeric plants can be considered as shade tolerant plants. Plant height was significantly different depending of the type of turmeric (P < 0.001, Table 4-4). ‘White Mango’ grew the most

(113.1 cm) and white tc and yellow tc grew the least (21.3 cm). This response followed exactly the same trend as in the greenhouse, demonstrating again that plants were not affected by environmental conditions in the field and some rhizome-derived turmeric types (including yellow ownrhiz) are more vigorous than micropropagated plants.

Chlorophyll index (SPAD) of leaves was significantly affected by the environment (P <

0.01) as plants grown under shade had higher SPAD (40.2) compared to plants grown under full sun (34.1). Hossain et al. (2009) found reduced levels of SPAD when plants grew under some level of shade, but values did not change when plants were grown for another season. Even though we had positive responses, this suggests that the efficacy of shading might be affected by other factors such as daylength, humidity or rainfall. As mentioned, SPAD also varied among plant types (P < 0.001, Table 4-4) and similarly to other growth parameters, turmeric ‘White

Mango’ (and ‘Black’) had significantly higher SPAD (~44) than the rest of turmerics (~35).

Optimal chlorophyll content for turmeric has been reported as > 40 (Lakshmi and John, 2015)

Overall plant performance was acceptable (overall rating ~3). In contrast, micropropagated turmerics and yellow ownrhiz had low SPAD values (~33) and therefore they had also a poor performance (overall rating, ≤2). Leaves had large areas of necrosis, tip burn or chlorosis, and due to this poor quality, these cultivars would not be acceptable for the ornamental market. The transition of plants from in vitro conditions to the field makes initial establishment complicated, affecting initial growth and performance (Preece and Sutter, 1991; Pospíšilová et al., 1999, Rout

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et al., 2000). It was interesting to note that although ‘Hawaiian Red’ had also low SPAD (32.8) its performance was still acceptable (~3, Table 4-4).

Regardless of the genus, plants produced less flowers in the field than in the greenhouse.

Studies in other turmeric such as Curcuma alismatifolia, showed that under 60% shade plants had a delayed second flowering (Kuehny et al., 2005). However, in our field experiment, there were no significant differences among plant type, environment or the interaction (P > 0.05). As previously mentioned, studies have shown that flowering of turmeric is very rare, varying with turmeric type and can even take a few years for flowering the first time (Nasirujjaman et al.,

2005; Kandiannan et al., 2015). We observed that the turmerics white tc, ‘White Mango’ and

‘Black’ showed some flowers during their growth period (~0.3), suggesting their potential as ornamental landscape or patio plants, while the other types never produced flowers (Tables 4-1 and 4-4).

Rhizome fresh mass was influenced only by plant type (P < 0.001, Figure 4-9A) and although the environmental factor did not affect rhizome production (P > 0.05), under full sun plants yielded an average of 475 g·plant-1 and around 446 g·plant-1 under shade. Based on some studies done in turmeric, light intensity under 60% shade might be too low to promote rhizome growth. Studies have shown that low light levels promote optimal vegetative growth and increase rhizome yield, but extremely low intensity light can decrease yield (Sharma et al., 2006,

Ravindran et al., 2007, and Hossain et al., 2009). Sivaraman (1992) reported that under shade most of the photosynthates were utilized for shoot biomass affecting rhizome growth. However, in other experiments higher yields and leaf area were found under 50% shade than under 70% shade, but no trials were done under 60% shade (Ferreira et al., 2016). Regardless of the environment, ‘White Mango’ had the highest rhizome fresh mass (968 g) although not

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significantly different from yellow ownrhiz and ‘Hawaiian Red’ (~685 g, Figure 4-9A).

Turmeric ‘White Mango’ had a high number of shoots and high SPAD, it grew taller and overall had high plant performance, thusit is not surprising that it also had the highest rhizome yield.

This turmeric also produced some flowers during its growth period without altering the yield, which agrees with Kandiannan et al. (2015) as they found that yield and curcumin content were not different between flowered and non-flowered plants. In contrast to our greenhouse experiment, yellow tc had very low yield (159 g) in comparison to yellow ownrhiz (second generation tc), which yielded about four times more (685 g). This proves that micropropagated plants can have high yields after the first year, even under field conditions. However, it is noteworthy that white tc had reduced yields than when grown in the greenhouse, which may be attributed to its poor performance in the field.

There were no differences in fresh shoot mass production between environments but plant type (P < 0.001) and the interaction of plant type and environment influenced this growth

(P < 0.01, Figure 4-9B). Normally under shade plants invest in high foliar biomass to optimize light capture and utilization (Yuan et al., 2016). While under excess of irradiance (in full sun), plants can suffer due to the destruction of the photosynthetic pigments, reducing their growth

(Ferreira et al., 2016). However, this was not apparent in this study. We found that under shade

‘Hawaiian Red’ had the highest shoot fresh mass (~583 g), whereas ‘Black’ had the lowest shoot mass (62.2 g), followed by white and yellow micropropagated turmeric (<100 g). Even though these values were not significantly different when they grew under full sun, for the tc cultivars there is a slight increase on mass under shade. Opposite to number of shoots, there was a positive and significant correlation between height and shoot fresh mass (r = 0.4, P < 0.001), which is logical as shoot number was very low across turmeric types (P > 0.05). Therefore, high shoot

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mass in ‘Hawaiian Red’ might be explained by its higher height compared to the micropropagated turmerics. This is particularly important as shoots photosynthesize and promote rhizome and root development (Klingeman et al., 2004). Hossain et al. (2005) also found that shoot biomass increased with increased plant height. When plants grow under full sun, this higher light intensity is often used to allocate more of the biomass to roots to capture water and nutrients to sustain the high transpiration and growth rates (Yuan et al., 2016). Then, as expected, plants produced higher root fresh mass under full sun (200 g) than under shade (106 g, P <

0.001) and turmeric ‘Black’ had the highest root mass (~377 g) in comparison to the rest of turmerics (P < 0.001, Figure 4-9C).

Overall, total fresh mass was not affected by the environment (P > 0.05), but it was influenced by plant type (P < 0.001) and the interaction of plant type and environment (P <

0.001). Among rhizome-derived turmerics, ‘White Mango’ and ‘Hawaiian Red’ and the second generation tc (yellow ownrhiz) had the highest total fresh mass (values ranged from 1,266 g to

1,424 g) while tc turmerics had the lowest total fresh mass (359 g, Figure 4-9D). Although carbohydrate partitioning showed similar growth patterns under both full sun and shade conditions (Figure 4-10), we observed that under full sun most of the growth in rhizome-grown

‘BKK’ was allocated to the shoots (~28%) instead of rhizomes (<9%), whereas this was the case for micropropagated yellow turmeric under shade (~30% roots and ~25% rhizomes). In other studies, yield tended to reduce when RLI was less than 59% due to reduced shoot biomass

(Hossain et al., 2009). However, for yellow ownrhiz this did not occur. Rhizome biomass was

>60% and shoot biomass was around 33% under both shade and full sun environments.

Comparable to our greenhouse experiment, turmeric ‘Black’ allocated about 44% of growth to root biomass (due to its tuberous roots) compared to other turmeric types. In a trial with five

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cultivars of turmeric, significant differences in harvest index were found, reflecting the physiological variations of turmeric types (Ravindran et al., 2007).

Turmeric tolerates shade and unlike species that avoid shade, very little is known about regulation of shade tolerance. This kind of plants have the ability to grow and survive in very low light environments, while light-demanding species need higher light intensities for optimal growth (Ruberti et al., 2012; Gommers et al., 2013; Yuan et al., 2016). Shade seems to negatively affect nutrient uptake of plants, which might be another reason for low rhizome yields. However, a 2-year study in Kerala found that increasing shade levels (0%, 25%, 50%, and

75% shade) resulted in a steady decrease in nutrients uptake, but during the subsequent year, the trend was reversed (Padmapriya, 2004). Moreover, when different cultivars of turmeric were evaluated under open and intercropping systems, some cultivars had high dry matter production under the open system, due to their high relative leaf growth rate. However, in other cultivars such differences did not exist (Ravindran et al., 2007). Therefore, there is not enough research to confirm this negative production pattern. In addition, other studies have resulted in taller plants when higher shade levels (75%) are used during the initial stages of growth and 50% shade in later stages (Sheela, 1992). This suggests that light requirement in turmeric varies across the different growing stages and they require lower light intensities in the initial phases of growth.

Based on the height, shoot number and overall ratings in the first six weeks, we observed better growth in plants grown under shade than under full sun in the initial stages (data not shown).

Therefore, shade levels could be modified during the plant growth, starting with a higher shade level and then reducing it, aiming to maximize shoot growth and rhizome development.

Postharvest Evaluations – Field Experiment

As in our greenhouse experiment, these postharvest evaluations were carried out to determine whether internal or external quality of the rhizomes vary over time. Rhizome weight

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loss was not significantly affected by plant type, environment, storage period or by their interaction (P < 0.05). Overall, weight loss was slightly higher (~10%) than in plants from the greenhouse (~8%). However, ‘Black’ rhizomes had higher weight loss (9%) and white tc had the highest rhizome weight loss (16.1%, Figure 4-11), although not significantly different from the others. Harvesting rhizomes in the right moment is important, as shriveling and physical degradation of rhizomes were found when rhizomes were harvested earlier in the season, with leaves still fresh and green (Hossain, 2010). Considering that our harvest was done when shoots withered completely, this may explain the low weight loss of our rhizomes.

Color parameters on rhizomes were not affected by the environment (P > 0.05, Table 4-

5), except for external hue (P < 0.05). Since rhizomes from both shade and full sun environments were harvested and stored at the same time, postharvest storage was evaluated. It was positive to find that the light factor did not affect the color (P > 0.05). However, color varied among turmeric types (P < 0.001). Rhizomes of white turmeric types (tc and ‘White Mango’) had higher internal and external L* (≥ 69), whereas external L* of the rest of turmerics were similar (~61) and rhizomes of turmeric ‘Hawaiian Red’ and ‘Black’ were internally darker (~57) than the other types. While internal chroma was higher than the external, the trend was similar, as C* of

‘Hawaiian Red’, ‘BKK’, yellow (tc and ownrhiz), white tc and ‘White Mango’ were very similar

(values ranged from ~31 to ~47), but significantly different from turmeric ‘Black’ (< ~14).

Therefore, turmeric ‘Black’ was duller then the rest of turmerics, which had more vivid color.

Moreover, even when surface h⁰ changed across plant type (P < 0.01) it is important to highlight how different internal color of these rhizomes was. According to the color space system, turmeric ‘Black’ was considered green-blue (h⁰ ~241⁰, Table 4-5). White turmerics had a h⁰ of

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~97⁰, so they were yellow-green, whereas turmeric yellow (tc and ownrhiz), ‘Hawaiian Red’ and

‘BKK’ had h⁰ values of ~65⁰ and a yellow-orange color.

Since we were assessing different turmeric lines, as in our greenhouse experiment it was expected that we would find significant differences in color and even interactions between plant type and storage period (P < 0.05) or between environment and storage period (P < 0.05) across turmeric types. However, these values were not significantly different within the genus (data not shown).

Summary

During year 1, container size significantly affected the plant growth of turmeric plants.

Plants had higher shoot, root and rhizome mass in larger containers. Turmeric is a tropical crop that requires warm temperatures, rainfall and shade when grown in the field. However, in the northern hemisphere, turmeric plants start undergoing dormancy in the fall-winter with short days. Some authors have previously stated that flowering and growth of some species of

Curcuma can be affected by photoperiod, but there is little literature regarding rhizome growth under long or short days. During our first-year trial, we found that under long days plants had higher rhizome yield (~670 g) than under natural days (~530 g) when grown in larger containers.

Micropropagated plants started very small and due to the lack of the rhizome structure they produced very low yields. For all plant types, shoot mass was greater under long days while root mass was smaller, but these differences did not result in a difference in rhizome mass.

As observed in year 1, in year 2 all plants actively grew under long days during the winter with cold and short days. Shoot number, flower number and shoot fresh mass was higher under long days than under natural days. Under both environments, rhizome yield was increased

(by 108 g) in this second year with smaller pots (14.5 L) compared to the larger pots (50.5 L) used in year one. As in year 1, ‘Hawaiian Red’ had the highest rhizome yield (~870 g) in year 2,

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but under long days the micropropagated white tc also had high yield (~960 g). In contrast, micropropagated yellow had the lowest yield and it was no different from the second generation yellow ownrhiz (~600 g) under both photoperiods. This indicates that there is great variability on rhizome yield from micropropagated plants depending upon the cultivar. Higher shoot biomass was found under long days, although less (<300 g) than in year 1 (~740 g), due to the drying period before harvest. Carbohydrate partitioning followed the same trends as in year 1.

Under field conditions, growth and productivity of turmeric can be maximized when the appropriate level of shade is provided in the right moment. As in the greenhouse, ‘White Mango’ and ‘Hawaiian Red’ had high number of shoots, high SPAD, grew taller and had overall high plant performance, which allowed for high rhizome yield. Meanwhile, the poor performance of white tc explained the lower yield obtained in the field in comparison to the greenhouse.

Moreover, yellow ownrhiz (second generation tc) yielded about four times as much as yellow tc

(~611 g compared to 159 g), proving that micropropagated plants can have high yields after the first year. However, in the greenhouse its response was completely opposite. This suggests that it might take more than two years to see consistent increased yields when using second generation rhizomes of micropropagated plants. Although we did not find great differences regarding overall growth and yield under shade or full sun, we observed better growth when plants grew under shade than under full sun in the initial stages and plant heights were positively correlated to rhizome yield. Thus, shade levels could be modified during the plant growth to maximize shoot growth and rhizome yield.

Regarding rhizome quality after harvest, color of rhizomes is affected by photoperiod to some extent. Skin of rhizomes looked brighter and more vivid under natural days, while internally they became more opaque under the same conditions. The storage period did not affect

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color of rhizomes from long days but rhizomes from natural days were affected by storage as they lost lightness and vividness. Moreover, hue values confirm the actual color of the types of turmeric used in this experiment. Regarding loss of rhizome weight during the storage period, greenhouse- grown plants had a better postharvest performance as rhizomes lost ~8 % of weight while for field grown plants weight loss was slightly higher (~11%). However, this weight loss was still low compared to other postharvest studies and no visual decay was found. Due to their high yield and good postharvest performance in the greenhouse, ‘Hawaiian Red’ and ‘White

Mango’ are the recommended rhizome-derived turmeric types. Micropropagated white tc is a new type of turmeric that should also be considered as a potential greenhouse crop. Similarly, turmeric ‘Black’ shows great potential as an ornamental landscape and patio plant, besides its valuable medicinal properties.

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Table 4-1. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, and ‘Black’), average new shoot number, height increase, number of flowers, overall rating and SPAD in the greenhouse under natural (<12 h) and long days (>12 h) from 2018 to 2019 (year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. SPAD New shoot Height increase No. of Overall Treatment Chlorophyll number (cm) flowers ratingz index

Main effects Plant type Hawaiian Red 0.6 b 53.2 c 0.1 b 3.1 c 32.2 d BKK 0.2 c 108.8 a 0.6 ab 3.5 ab 37.7 bc Yellow tc 1.0 a 8.5 da 0.0 b 2.5 e 32.3 d Yellow ownrhiz 0.6 b 53.6 c 0.7 ab 2.9 cd 35.7 bcd White tc 0.5 bc 9.5 da 2.3 a 2.6 de 35.4 cd White Mango 0.4 bc 110.1 a 0.1 b 3.2 bc 40.3 b Black 0.4 bc 81.1 b 0.3 b 3.7 a 46.5 a Photoperiod Long days 0.7 ay 65.3 NS 0.9 a 2.73 b 36.7 NS Natural days 0.3 b 56.1 NS 0.2 b 3.4 a 37.6 NS ANOVA Summary Plant type * * * * * Photoperiod * NS * * NS Plant type*Photoperiod NS NS NS NS NS zA subjective visual scale from 1 to 5 was used to measure overall aesthetic performance, 1 = very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2 = poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3 = acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4 = very good quality with very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form.. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

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Table 4-2. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White Mango’, ‘White tc’, and ‘Black’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes grown the greenhouse under natural (<12 h) days from 2018 to 2019 (year 2) harvested and stored for two weeks at 12.8 ± 0.6 °C and 89.6 ± 6.6% RH. Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. External Internal Treatment L* C* h⁰ L* C* h⁰ Main effects Plant type Hawaiian Red 62.4 NS 34.4 NS 82.6 NS 58.7 c 47.5 abc 66.5 c BKK 61.6 NS 36.1 NS 85.0 NS 60.6 c 56.0 ab 62.5 c Yellow tc 62.4 NS 44.9 NS 69.7 NS 63.3 bc 48.3 abc 66.3 c Yellow ownrhiz 60.0 NS 32.5 NS 83.3 NS 64.0 bc 57.1 a 68.2 c White tc 62.9 NS 41.9 NS 68.1 NS 69.1 ab 41.7 bc 79.6 bc White Mango 70.0 NS 28.3 NS 77.3 NS 75.0 a 34.0 c 103.3 b Black 61.2 NS 29.9 NS 73.3 NS 58.2 c 18.0 d 144.6 a Date Initial 66.1 a 41.7 a 89.0 a 63.5 NS 33.3 a 76.9 b Final 59.7 b 29.3 b 64.9 b 64.6 NS 53.1 b 92.0 a Interaction effects Plant type*Date Hawaiian Red Initial 68.0 NS 35.4 NS 108.5 NS 60.8 bcdy 27.1 ef 82.4 bc Hawaiian Red Final 56.8 NS 33.5 NS 56.7 NS 56.5 cd 67.9 ab 50.6 c BKK Initial 62.3 NS 39.4 NS 117.9 NS 61.5 bcd 35.5 de 68.5 bc BKK Final 61.1 NS 33.4 NS 57.5 NS 59.9 bcd 73.1 a 57.5 bc Yellow tc Initial 68.2 NS 56.6 NS 72.9 NS 61.9 bcd 35.5 de 65.4 bc Yellow tc Final 56.7 NS 33.1 NS 66.6 NS 64.7 bcd 61.0 abc 67.1 bc Yellow ownrhiz Initial 59.6 NS 36.2 NS 100.6 NS 65.6 bc 46.3 bcde 71.9 bc Yellow ownrhiz Final 60.5 NS 28.8 NS 66.0 NS 62.4 bcd 67.8 ab 64.4 bc White tc Initial 66.8 NS 53.6 NS 68.8 NS 61.5 bcd 30.6 def 71.3 bc White tc Final 59.0 NS 30.2 NS 67.5 NS 76.8 a 52.7 abcd 88.0 bc White Mango Initial 70.7 NS 30.1 NS 80.6 NS 70.4 ab 30.3 def 105.7 b White Mango Final 69.0 NS 26.1 NS 73.3 NS 80.4 a 38.4 cde 100.5 bc Black Initial 66.3 NS 40.4 NS 78.3 NS 62.2 bcd 27.9 ef 71.9 bc Black Final 56.2 NS 19.4 NS 68.3 NS 54.2 d 8.1 f 217.2 a ANOVA Summary Plant type NS NS NS * * * Date * * * NS * * Plant type*Date NS NS NS * * * zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

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Table 4-3. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White Mango’, ‘White tc’, and ‘Black’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes grown the greenhouse under long (>12 h) days from 2018 to 2019 (year 2), harvested and stored for two weeks at 12.9 ± 0.1 °C and 65.1 ± 4.4% RH. Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. External Internal Treatment L*z C* h⁰ L* C* h⁰ Main effects Plant type Hawaiian Red 60.6 ab 30.2 ab 60.3 NS 59.6 bcd 61.8 a 56.2 c BKK 57.3 abc 34.8 a 59.9 NS 58.6 cd 57.8 a 59.6 bc Yellow tc 60.9 ab 28.3 ab 66.7 NS 65.6 b 61.3 a 70.0 bc Yellow ownrhiz 59.8 abc 26.4 b 66.7 NS 65.1 bc 55.1 ab 69.7 bc White tc 62.3 a 26.6 b 70.4 NS 75.5 a 44.2 bc 89.3 b White Mango 62.7 a 23.5 bc 73.7 NS 77.8 a 30.2 c 97.9 b Black 55.4 ac 17.1 c 76.9 NS 56.9 d 6.0 d 206.9 a Date Initial 59.7 NS 27.0 NS 64.7 NS 65.2 NS 46.2 NS 92.1 NS Final 59.3 NS 27.1 NS 69.8 NS 63.1 NS 47.4 NS 93.2 NS Interaction effects Plant type*Date Hawaiian Red Initial 60.5 aby 27.7 bc 62.6 NS 59.2 NS 63.9 NS 55.6 NS Hawaiian Red Final 60.6 ab 33.1 ab 57.6 NS 60.1 NS 59.3 NS 56.9 NS BKK Initial 56.0 b 39.5 a 58.7 NS 59.8 NS 57.4 NS 61.2 NS BKK Final 58.6 ab 30.2 abc 61.0 NS 57.3 NS 58.3 NS 58.1 NS Yellow tc Initial 59.0 ab 29.3 abc 66.1 NS 66.0 NS 62.1 NS 69.7 NS Yellow tc Final 62.8 ab 27.3 bc 67.3 NS 65.2 NS 60.6 NS 70.3 NS Yellow Initial 62.0 ab 25.9 67.2 NS 67.0 NS 54.7 NS 70.0 NS ownrhiz bcd Yellow Final 57.6 ab 27.0 bc 66.3 NS 63.2 NS 55.5 NS 69.4 NS ownrhiz White tc Initial 64.5 a 26.1 bc 71.7 NS 75.6 NS 40.5 NS 90.1 NS White tc Final 60.1 ab 27.1 bc 69.1 NS 75.5 NS 47.8 NS 88.5 NS White Mango Initial 64.3 ab 24.2 bcd 74.6 NS 79.0 NS 29.9 NS 98.6 NS White Mango Final 59.3 ab 21.9 bcd 72.0 NS 75.4 NS 30.8 NS 96.4 NS Black Initial 54.4 b 14.6 d 59.0 NS 59.1 NS 4.3 NS 204.2 NS Black Final 56.3 b 19.7 cd 94.9 NS 54.8 NS 7.7 NS 209.6 NS ANOVA Summary Plant type * * NS * * * Date NS NS NS NS NS NS Plant type*Date * * NS NS NS NS zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

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Table 4-4. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White Mango’, ‘White tc’, and ‘Black’), average new shoot number, height increase, SPAD, overall rating and number of flowers under full sun or shade (field experiment). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. No. of SPAD- Height No. of Treatments new Overall ratingz Chlorophyll increase (cm) flowers shoots index Main effects Plant type Hawaiian Red 0.2 ab 94.6 ab 0.0 NS 2.7 a 32.8 c BKK 0.1 ab 96.2 ab 0.0 NS 2.4 b 38.8 b Yellow tc 0.2 ab 22.7 c 0.0 NS 1.9 c 31.6 c Yellow ownrhiz 0.3 a 74.2 b 0.0 NS 2.5 ab 33.7 c White tc 0.1 b 19.9 c 0.3 NS 2.0 c 34.9 c White Mango 0.3 a 113 a 0.3 NS 2.6 ab 44.1 a Black 0.2 ab 86.3 b 0.3 NS 2.6 ab 43.8 a Environment Full sun 0.2 NS 65.1 NS 0.1 NS 2.27 NS 34.1 b Shade 0.2 NS 79.7 NS 0.2 NS 2.48 NS 40.2 a Interaction effects Plant type*Environment Hawaiian Red Full sun 0.2 NS 99.8 NS 0.0 NS 2.7 abdy 30.1 NS Hawaiian Red Shade 0.2 NS 89.5 NS 0.0 NS 2.8 ab 35.6 NS BKK Full sun 0.1 NS 78.9 NS 0.0 NS 2.2 cefg 35.8 NS BKK Shade 0.1 NS 113.5 NS 0.0 NS 2.5 abcde 41.9 NS Yellow tc Full sun 0.2 NS 15.3 NS 0.0 NS 1.7 g 28.6 NS Yellow tc Shade 0.2 NS 30.1 NS 0.0 NS 2 fg 34.7 NS Yellow ownrhiz Full sun 0.3 NS 64.8 NS 0.0 NS 2.3 bcdef 30.6 NS Yellow ownrhiz Shade 0.4 NS 83.6 NS 0.0 NS 2.7 abc 36.8 NS White tc Full sun 0.1 NS 10.9 NS 0.0 NS 1.8 g 31.7 NS White tc Shade 0.1 NS 28.9 NS 0.7 NS 2.2 defg 38.1 NS White Mango Full sun 0.3 NS 112.3 NS 0.0 NS 2.8 a 40.7 NS White Mango Shade 0.4 NS 113.8 NS 0.5 NS 2.5 abcde 47.5 NS Black Full sun 0.2 NS 73.9 NS 0.5 NS 2.4 abcdef 41.0 NS Black Shade 0.2 NS 98.7 NS 0.2 NS 2.8 abc 46.6 NS ANOVA Summary Plant type * * NS * * Environment NS NS NS NS * Plant type*Environment NS NS NS * NS zA subjective visual scale from 1 to 5 was used to measure overall aesthetic performance, 1 = very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2 = poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3 = acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4 = very good quality with very acceptable and desirable color and form, 5 = excellent quality, perfect condition, premium color and form. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

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Table 4-5. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, and ‘Black’), storage period (date), and color parameters (L*, C*, h⁰) of rhizomes grown under full sun and 60% shade and stored for two weeks at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH. Data represent the least squared means derived from a three-way ANOVA using the general linear model procedure in R. L*z C* h⁰ Treatments External Internal External Internal External Internal Main effects Plant type Hawaiian Red 61.6 b 58.4 c 30.4 a 63.5 ab 58.5 d 53.5 d BKK 60.4 b 63.3 b 30.1 a 67.8 a 62.5 cd 67.5 c Yellow tc 60.9 b 66.9 b 27.2 ab 59.2 b 70.6 b 72.7 c Yellow ownrhiz 61.1 b 64.3 b 31.2 a 65.4 ab 64.7 c 64.5 cd White tc 61.0 b 78.8 a 27.7 ab 41.5 c 73.1 ab 93.1 b White Mango 69.0 a 78.7 a 25.2 b 38.1 c 77.0 a 100.9 b Black 60.8 b 55.5 c 19.4 c 9.0 d 73.8 ab 241.8 a

Environment Full sun 62.8 NS 67.1 NS 28.0 NS 48.2 NS 69.2 ay 100.9 NS Shade 61.2 NS 65.2 NS 26.7 NS 50.4 NS 67.4 b 98.6 NS

Date Initial 61.9 NS 66.3 NS 27.4 NS 48.4 NS 68.1 NS 102.1 NS Final 62.1 NS 66.0 NS 27.3 NS 50.1 NS 68.6 NS 97.6 NS ANOVA Summary Plant type * * * * * * Environment NS NS NS NS * NS Date NS NS NS NS NS NS Plant type*Environment NS NS NS NS NS NS Plant type*Date NS NS NS NS * NS Environment*Date NS * NS NS NS NS Plant type*Environment*Date NS NS NS NS NS NS zColor system: L*=lightness, C*= chroma, and h⁰= hue. yMeans separation in columns by Tukey’s multiple range test at P ≤ 0.05. NS, *, Nonsignificant or significant at P < 0.05, respectively.

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Figure 4-1. Mean values and Tukey grouping comparison for turmeric planting materials (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘White Mango’, and ‘Black’), container size (5.7 L and 50.5 L), rhizome fresh mass, shoot fresh mass, and root fresh mass under natural (< 12 h) and long days (>12 h) from 2017 – 2018 (year 1). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of 8 replicate containers ± 95% confidence intervals.

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Figure 4-2. Mean values and Tukey grouping comparison for turmeric planting materials (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘White Mango’, and ‘Black’), container size (5.7 L and 50.5 L), carbohydrate partitioning from plant grown in the greenhouse under natural (A) and long days (B) from 2017 - 2018 (year 1).

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Figure 4-3. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), rhizome fresh mass, shoot fresh mass, root fresh mass and total fresh mass from plants grown in the greenhouse under natural (<12 h) and long days (>12 h) from 2018 - 2019 (year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of six replicate containers ± 95% confidence intervals.

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Figure 4-4. Photographs comparing turmeric planting materials (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), and rhizome fresh mass from one plant grown in 14.5 L, under natural days (<12h) in the greenhouse from 2018 – 2019 (year 2). Photographs are courtesy of Sofia Flores.

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Figure 4-5. Photographs comparing turmeric planting materials (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), and rhizome fresh mass from one plant grown in 14.5 L, under long days (>12h) in the greenhouse from 2018 – 2019 (year 2). Photographs are courtesy of Sofia Flores.

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Figure 4-6. Visual scale from 1 to 5 to measure overall aesthetic performance of turmeric plants grown in the greenhouse from 2018 – 2019 (year 2) under natural (<12h) and long days (>12h). 1) very poor quality, not acceptable with severe leaf necrosis, tip burn or chlorosis, poor form (well branched plants and do not lodge); 2) poor quality, not acceptable with large areas of necrosis, tip burn or chlorosis, poor form; 3) acceptable quality with few leaves with tip burn, somewhat desirable form and color; 4) very good quality with very acceptable and desirable color and form, 5) excellent quality, perfect condition, premium color and form. Photographs are courtesy of Sofia Flores.

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Figure 4-7. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), carbohydrate partitioning from plants grown in the greenhouse under natural (<12 h) and long days (> 12 h) from 2018 – 2019 (year 2).

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Figure 4-8. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’) and rhizome weight loss under natural and long days (postharvest evaluation- year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of six replicate containers ± 95% confidence intervals.

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Figure 4-9. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), rhizome fresh mass, shoot fresh mass, root fresh mass and total fresh mass from plants grown in the field under full sun and 60% shade from 2018 – 2019 (year 2). Data represent the least squared means derived from a two-way ANOVA using the general linear model procedure in R. Bars represent the mean of six replicate containers ± 95% confidence intervals.

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Figure 4-10. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’), carbohydrate partitioning from plants grown in the field under full sun and 60% shade from 2018 – 2019 (year 2).

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Figure 4-11. Mean values and Tukey grouping comparison for plant type (‘Hawaiian Red’, ‘BKK’, ‘Yellow tc’, ‘Yellow ownrhiz’, ‘White tc’, ‘White Mango’, and ‘Black’) and rhizome weight loss from plants grown in the field under full sun and 60% shade from 2018 – 2019 (year 2), harvested and stored for a period of 2 weeks at 12.8 ± 0.3 °C and 70.9 ± 12.4% RH. Bars represent the mean of six replicate plants ± 95% confidence intervals.

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CHAPTER 5 CONCLUSIONS

This thesis evaluated the growth and productivity of various ginger, galangal and turmeric species and cultivars under varying environmental conditions with an emphasis on promoting sprouting, rhizome production and overall growth and performance. Several studies in

Asia have demonstrated that traditional cultivation using rhizomes as starting material results in high rhizome yield particularly under field conditions. However, propagation is not very efficient, and rhizomes are highly susceptible to soil-borne diseases. In contrast, micropropagated transplants represent a great alternative as they provide pathogen-free plantlets and uniform start of the crop, although their productivity is still poorly understood. Therefore, experiments were designed to evaluate sprouting performance of rhizomes under a series of forcing treatments including soaking in water and growth regulators. Additionally, greenhouse and field experiments aimed to evaluate growth and productivity of micropropagated transplants compared to rhizome-derived plants.

In Chapter 2, regardless of plant type and forcing treatment, rhizomes successfully sprouted as early as ~20 days in both growth chamber experiments. The forcing treatments used did not have a direct effect on sprouting, however the cultivar and plant source affected the uniformity and speed of sprouting. Rhizomes of ginger ‘Bubba baba’ had the best sprouting in

~23 days and rhizomes had a final weight of 15 g, whereas turmeric ‘BKK’ and ‘White Mango’ had the poorest sprouting after 33 days with a maximum final weight of ~5.3 g. The rhizomes in the first experiment had better sprouting than in the second experiment where they were stored for a longer time (over 120 days) and this led to rhizome weight loss and decay. Therefore, initial rhizome quality is of utmost importance for uniform and faster rhizome sprouting. Future work

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should evaluate the effect of different rhizome sizes for turmeric cultivars with poor sprouting such as ‘White Mango’ and ‘BKK’ when rhizomes are fresh after harvest.

In Chapters 3 and 4, the first year of the greenhouse experiment (2017-2018) demonstrated that container size positively influenced plant growth and yield. Plants of ginger and turmeric had higher shoot, root and rhizome fresh mass in larger containers compared to smaller containers. In the U.S. these species start undergoing dormancy in the fall-winter with short days and low temperatures (<15 °C). Even though there is little research on factors driving rhizome production of galangal and turmeric plants under these conditions, previous literature reported that ginger requires long days for continuous overall growth and short days for rhizome growth. During the first year, rhizome production in large containers was very similar under the natural daylength and long days photoperiods evaluated. Turmeric plants yielded ~670 g when grown under long days and ~500 g under natural days. Meanwhile, ginger had higher yields (an average ~900 g) under both long and natural days. Since micropropagated plants often start very small and lack the rhizome structure, yields can be very low on the first year, or rhizomes might not meet fresh market standards due to their small diameter. In the second year experiment

(2018-2019) ginger, galangal, and turmeric grew actively under long days, producing more shoots and higher shoot fresh mass than under natural days. Rhizome yield of all species was increased in this second year compared to year 1, and even when grown in smaller containers, but there were no differences in yield between natural or long days. There was no consistency for the different genera. on whether micropropagated plants may be most effective as clean stock material to produce seed rhizomes for second-year production. Ginger ownrhiz had the highest yield, galangal tc grew more vigorously than ginger in terms of higher shoot number and height, and produced high yields. As in the first year rhizome-grown turmeric ‘Hawaiian Red’ and

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‘White Mango’ had the highest yield. Meanwhile the micropropagated turmeric white tc also had high yield, but the micropropagated turmeric yellow tc and yellow ownrhiz had lower yields.

Therefore, it might take more than two years to obtain consistent high yields when the starting material are micropropagated transplants. Regarding overall growth and performance, gingers consistently had high chlorophyll index and more flowers were observed under long days than under natural days, with no negative effects on rhizome yield. Turmeric ‘Black’ had high chlorophyll index and was rated as a very good quality plant due its dark green leaf color with few damages, and also showing some flowers.

In the field experiment of Chapters 3 and 4 rhizome yield of ginger and turmeric was not different when plants were grown under full sun or 60% shade. However, galangal appeared to be sensitive to shade since rhizome yield was negatively affected (a ~800 g·plant-1 reduction).

Similar to the greenhouse, galangal tc and turmeric ‘Hawaiian Red’ and ‘White Mango’ grew the most in the field. This growth and acceptable plant performance accounted for their high rhizome yield, whereas turmeric white tc had poor field performance and therefore lower yield regardless of the shading condition. Contrary to results in the greenhouse, ginger yellow ownrhiz yielded more than yellow tc, confirming that micropropagated plants potentially can have higher yields after the first year. For all plants and regardless of the final yield, better growth and performance were observed during the first stages of growth. This indicates that shade may be beneficial for transplant and field establishment and particularly for micropropagated plants. Therefore, future field studies should focus on subjecting plants to shade during the first stages of growth to ensure field establishment and shoot growth and then expose them to full sun, so that enough light is available to support rhizome production. In addition, lower shading levels should also be evaluated.

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Regarding rhizome quality, color of ginger rhizomes was not affected by photoperiod, while turmeric rhizomes were slightly affected. Furthermore, the two-week storage period had an effect on color as most rhizomes lost lightness and became opaque, although maintaining their inherent color. Rhizomes from all species grown in the field were more susceptible to weight loss than the greenhouse grown, probably due to contamination with soil borne pathogens.

Galangal was the most sensitive species to rhizome weight loss and decay. Further postharvest studies evaluating storing conditions of galangal rhizomes is necessary to address these findings.

Further investigation of the cost-effectiveness of using either micropropagated plants versus seed rhizomes as well as growing either in the greenhouse or the field is required. Even though galangal grew and yielded more than the gingers, there is still scarce information about the optimal conditions and requirements for its growth and productivity, especially for field cultivation as growing under 60% shade reduced its yield. As the demand for these three spice crops increases in the country, optimizing their growth and rhizome production under different environmental conditions should be further investigated.

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BIOGRAPHICAL SKETCH

Sofia Flores was born in Lima, Peru to Tomas Flores and Mery Vivar. She grew up and studied in Lima where she graduated from the Primer Colegio Nacional de Mujeres “Rosa de

Santa Maria” (High School) in 2005. Sofia attended the National Agrarian University-La Molina and graduated with a B.S. in agronomy in 2012. After working for the industry and the Peruvian government, Sofia continued working at the National Agrarian University-La Molina as an instructor from 2013 to 2016. Sofia lectured various courses within the Horticulture Department such as Plant Propagation, Postharvest Physiology and Handling of Fruits and Vegetables, and

Introduction to Agronomy. Sofia began her studies toward a Master of Science degree at the

University of Florida in 2018 majoring in horticultural sciences. Sofia plans to continue to work in plant sciences.

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