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Clonal Propagation of Pitaya (Hylocereus Sp.) Hybrids

Permanent Link: http://ufdc.ufl.edu/UFE0045104/00001

Material Information

Title: Clonal Propagation of Pitaya (Hylocereus Sp.) Hybrids
Physical Description: 1 online resource (53 p.)
Language: english
Creator: Condon, Warren P
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: cactaceae -- dragonfruit -- hybrids -- hylocereus -- micropropagation -- pgrs -- pitahaya -- pitaya
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Long overlooked outside its native range in Central America, pitaya (Hylocereus spp.) has become an increasingly popular specialty item in markets spanning the globe from Asia to Europe due to its exotic appearance, potential health benefits and possible use as a food coloring.  The creation of hybrids is one path to improving the taste of the fruit which sometimes fails to match its striking visual qualities.  Micropropagation provides an avenue for greater production of new material in a shorter period of time.  Testing was conducted on five lines of pitaya hybrid explants, using combinations of NAA (0 and 0.05 µm.L-1) and TDZ (0, 0.5, 1.0 and 2.27 µm.L-1).  Measurements were taken for callus production, number of shoots and number of roots.  While the combination of 0.05 1-naphthaleneacetic acid (NAA) and 0.5 thidiazuron (TDZ) concentrations provided the best overall shoot production, this was not consistent across the different lines established in the experiment.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Warren P Condon.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Vendrame, Wagner A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045104:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045104/00001

Material Information

Title: Clonal Propagation of Pitaya (Hylocereus Sp.) Hybrids
Physical Description: 1 online resource (53 p.)
Language: english
Creator: Condon, Warren P
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: cactaceae -- dragonfruit -- hybrids -- hylocereus -- micropropagation -- pgrs -- pitahaya -- pitaya
Environmental Horticulture -- Dissertations, Academic -- UF
Genre: Horticultural Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Long overlooked outside its native range in Central America, pitaya (Hylocereus spp.) has become an increasingly popular specialty item in markets spanning the globe from Asia to Europe due to its exotic appearance, potential health benefits and possible use as a food coloring.  The creation of hybrids is one path to improving the taste of the fruit which sometimes fails to match its striking visual qualities.  Micropropagation provides an avenue for greater production of new material in a shorter period of time.  Testing was conducted on five lines of pitaya hybrid explants, using combinations of NAA (0 and 0.05 µm.L-1) and TDZ (0, 0.5, 1.0 and 2.27 µm.L-1).  Measurements were taken for callus production, number of shoots and number of roots.  While the combination of 0.05 1-naphthaleneacetic acid (NAA) and 0.5 thidiazuron (TDZ) concentrations provided the best overall shoot production, this was not consistent across the different lines established in the experiment.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Warren P Condon.
Thesis: Thesis (M.S.)--University of Florida, 2012.
Local: Adviser: Vendrame, Wagner A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0045104:00001


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1 CLONAL PROPAGATION OF PITAYA ( HYLOCEREUS SP.) HYBRIDS By WARREN PAUL CONDON 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 2012

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2 2012 Warren Paul Condon

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3 To my loving parents whose endless encouragement provided the inspiration and motivation for me to pursue my education

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4 ACKNOWLEDGMENTS I would like to t hank my supervisor y committee, Dr. Wagner Vendrame, Dr. Kimberly Moore and Dr. Jonathan Crane for sharing their wisdom, guidance, and encouragement throughout my education at the University of Florida. Addi tionally I would like to recognize the contributions of Dr. George Fitzpatrick, who never failed to keep my attention with his wide range of knowledge on so many subjects and the stories that accompanied them. assistants, Ania, Alba, Janet and Miriam, who provided me with their support and insight over the years, helping to make going to the lab fun.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREV IATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Botanical Classification ................................ ................................ ........................... 15 Origin and Distribution ................................ ................................ ............................ 16 Environmental Factors ................................ ................................ ............................ 16 Irrigation ................................ ................................ ................................ ........... 16 Light ................................ ................................ ................................ ................. 17 Temperature ................................ ................................ ................................ ..... 17 Soil ................................ ................................ ................................ ................... 18 Fertilization ................................ ................................ ................................ ....... 18 Flowering and Pollination ................................ ................................ ........................ 18 Cultivation Techniques and Practices ................................ ................................ ..... 20 Support ................................ ................................ ................................ ............. 20 Propagation ................................ ................................ ................................ ...... 21 Off season flora l induction ................................ ................................ ................ 22 Physio chemical and Potential Future Uses ................................ ........................... 23 Pests and Diseases ................................ ................................ ................................ 24 Harvest and Post harvest ................................ ................................ ....................... 26 World Market ................................ ................................ ................................ .......... 27 3 MATERIALS AND METHODS ................................ ................................ ................ 32 Parent Material Selection and Hybridization ................................ ........................... 32 Culture establishment ................................ ................................ ............................. 32 In Vitro Treatments ................................ ................................ ................................ 33 Experimental Design and Statistical Analysis ................................ ......................... 34

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6 4 RESULTS AND DISCUSSION ................................ ................................ ............... 37 Seed Germination and Seedling Growth ................................ ................................ 37 Callus and Shoot Formation ................................ ................................ ................... 37 Root Formation ................................ ................................ ................................ ....... 39 5 SUMMARY AND CONCLUSIONS ................................ ................................ .......... 45 LIST OF REFERENCES ................................ ................................ ............................... 46 BIOGRAPHIC AL SKETCH ................................ ................................ ............................ 53

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7 LIST OF TABLES Table page 3 1 Plant growth regulator (PGR) concentrations used for clonal propagation of hybrid pit aya ................................ ................................ ................................ ....... 35 4 1 Total number of explants exhibiting sign ificant callus (amount of callus exceeds size of original explant) by treatment number ................................ ....... 40 4 2 Total number of explants exhibiting shoots ................................ ........................ 41 4 3 Total number of shoots produced per treatment separated by line ..................... 41 4 4 Total number of explants with roots ................................ ................................ .... 42 4 5 Total number of roots produced per treatment separated by line ....................... 42

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8 LIST OF FIGURES Figure page 1 1 Multiple pitaya cuttings attached to a single pole ................................ ................ 14 2 1 Flower with location of stigma high above the anthers ................................ ....... 29 2 2 Pitaya inter planted among mangosteen using the single post method ............... 29 2 3 Single post method modified with supporting structure on top ........................... 30 2 4 Trellis method of growing pitaya in early phase of establishment ....................... 31 2 5 Mature planting of pitaya using the trellis method ................................ ............... 31 3 1 Flower, growthhabit and fruit flesh characteristics of Hylocereus guatamalensis (A,C,E) and H. stenopterus (B,D,F) ................................ ............ 36 4 1 Weeks required for ten pitaya seedlings to reach desired 6 cm length when grown on Mur ashige and Skoog (1962) medium. ................................ ............... 40 4 2 In vitro culture showing single and multiple shoots being generated from callus. ................................ ................................ ................................ ................. 43 4 3 Comparison of in vitro shoot formation for hybrid pitaya lines (1 5) under different treatments (T1 T8) ................................ ................................ ............. 44

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9 LIST OF ABBREVIATION S CAM Crassulacean A cid M etabolism DA P Days A fter P ollination GAP Good Agricultural Practices PPF Photosynthetic Photon Flux TA Tissue Acidity (In CAM plants, used as a measurement of photosynthetic activity)

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10 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 CLONAL PROPAGATION OF PITAYA ( HYLOCEREUS SP.) HYBRIDS By Warren Paul Condon December 2012 Chair: Wagner Vendrame Major: Horticultural Sciences Long overlooked outside its native range in Central America, pitaya ( Hylocereus sp p .) has become an increasingly popular specialty item in markets spanning the globe from Asia to Europe due to its exotic appearance, potential health benefits and possible use as a food coloring. The creation of hybrids is one path to improving t he taste of the fruit whic h sometimes fails to match its striking visual qualities. M icropropagation provides an avenue for greater production of new material in a shorter period of time. Testing was conducted on five lines of pitaya hybrid explants, using combinations of NAA (0 and 0.05 m L 1 ) and TDZ (0, 0.5, 1.0 and 2.27 m L 1 ). Measurements were taken for callus production, number of shoots and number of roots. While the combination of 0.05 1 naphthaleneacetic acid ( NAA ) and 0.5 thidiazuron ( TDZ ) concentrations provided t he best overall shoot production, this was not consistent across the different lines established in the experiment.

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11 CHAPTER 1 INTRODUCTION Pitaya, p itahaya a nd dragonfruit are common name s for many of the members of the Cactaceae family found growing throughout the Americas ( Le Bellec et al ., 2006; Jaya, 2010; Tel Zur et al ., 2011) In the context of this thesis, I will refer to the 18 closely related species of night blooming cacti in the Hylocereus genus unless otherwise specif ied (Anderson, 2001; Britton and Rose, 1963) The commercial name by which fruit is known, dragonfruit, originated in Vietnam and has been credited to the wing like structures on the fruit (Mertens, 2003) The exotic looking fruit can be eaten out of ha nd or the flesh can be used in juices, jellies, jams, preserves, candies, ice creams, yoghurts, pastries, marmalades, cocktails, wines and as an ingredient in soft drinks (Luders and McMahon, 2006 ; Gunasena et al ., 2007 ; Evans et al ., 2010 ). Pitaya are touted for their potential health benefits. Researchers have identified several components that could prove beneficial to the food additive and pharmaceutical industries, including betalains, prebiotics and m yo inositol. (Harivaindaran et al ., 2008; Rebecca et al ., 2010; Wu et al ., 2006; Wichienchot et al 2010). In addition to the nutritive value of pitaya consumption the fruit are bought for their physical appeal. M any European customers buy pitaya for use as a decoration (Mizrahi et al 2002) and frequently Asian clientele purchase the fruit for presentation as an offering to their ancestors (Le Bellec et al ., 2006 ) or as a source of good luck during Chinese New Year celebrations (Jaya, 2010) Finally t he pitaya has a n enduring use as an orna mental plant both for its showy flowers and as a rootstock for other ornamental cacti species (McCurdy, 1926; Houghton, 1930).

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12 markets, pitaya became a commodity on the international market when Viet Nam began et al ., 2000). Production has since commenced in Israel, Nicaragua, Colombia, Thailand, Indonesia, Mexico, Australia, and Malaysia (Lichtenzveig et al ., 20 00; Mertens, 2003; Jamaludin et al ., 2010). In the last decade there have been small orchards established in California and Florida (Mertens, 2003; Crane and Balerdi, 2005). Growing pitaya has several positive aspects for commercial producers. Pitaya p lantings generate a fairly quick return on investment as the fruit production can be expected to begin by the year 2 and full production in the orchard occurring in year 5 (Gunasena et al ., 2007). the high demand for pitaya and it ( Jaya, 2010; Evans and Huntley, 2011). There are several issues that any potential pitaya grower should consider before planting pitaya as a crop. Despite the current high demand for the fruit, many new producers in Viet Nam have entered the marketplace and the price received for pitaya has dropped 60% from 2000 to 2007 (Hoa, 200 8 ). Evans and Huntley (2011) warned that overproduction from local and international growers could have a detri mental impact on their net profits They also noted the high initial investment costs in South Florida associated with land prices and building of a trellis system. Labor costs should also be taken into account because hand pollination is necessary for m any of the current cultivars which either require cross pollination or only produce larger, market sized fruit when cross pollinated ( Weiss et al ., 1994 ) A further concern is the taste of

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13 the fruit. Tel Zur et al (2004) found the taste of the yellow pitaya ( H. megalanthus ) to be agreeable and sweet, but she described all of the other Hylocereus spp. as having an inferior taste. Mizrahi et al (2002) reached similar conclusions but noted that hybrids from their breeding program had improved taste. Gu nasena et al ., (2007) described the fruit as having a flavor reminiscent of Kiwi ( Actinidia deliciosa ). To establish an orchard using 3 stems per trellis, an acre of pitaya requires 1,743 cuttings (Evans and Huntley, 2011) (Figure 1 1) The benefit of utilizing tissue culture as a propagation method is the potential to create large numbers of uniform plants with identical selected genetic traits (Maximova et al ., 2002) that are disease free By selecting fruits with desirable characteristics and perfor ming crosses among them, hybrids can be generated, resulting in fruits with improved characteristics, such as fruit size, flavor, color, etc. The objective of this project w as to establish a system for the clonal propagation of pitaya hybrids using in vit ro tissue culture techniques. This will facilitate the rapid introduction of new cultivars with the desired characteristics into the commercial industry when new selections are ultimately developed

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14 Figure 1 1. Multiple pitaya cuttings attached to a single pole. Photo courtesy of W. Condon.

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15 CHAPTER 2 LITERATURE REVIEW Botanical Classification The Hylocereus genus is botanically classified as being found within the Hylocereeae tribe of the Cactoideae subfamily of the Cactaceae family. The plants of t his genus are all vining type cacti, branching freely, and typically found climbing up trees in the wild. They have an epiphytic growth habit, producing aerial roots which provide a means of both support and nutrient and water absorption. The green, phot osynthetic stems typically hav e 3 winged though occasionally 4 or 5 sided, segmented, and often glaucous ribs with margins that can be horned or undulate or both (Britton and Rose, 1963; Anderson, 2001). A reoles which are found on the ridges may contai n bristles especially when young or short spines when mature ; can vary in number from zero to eight or more ; and are the genesis point for spines, additional shoots and flowers (Nerd and Mizrahi, 1997 ) The flowers, the largest in the Cactaceae family, are typically 30 cm in length, normally white although a couple of species are red to magenta, funnelform in shape, fragrant, and bloom nocturnally for a single night. (Britton and Rose, 1963; Anderson, 2001). The exocarp of the fruit can vary i n color from yellow to pink to red, has soft scales and rarely has spines. The mesocarp color ranges from white to red to magenta to pale yellow and all colors in between, and has tiny, edible, black to brown seeds ( Wichienchot et al ., 2010) Fruit can w eigh up to 800 g ( Tel Zur et al ., 2003 ). The flowers as well as the fruit are eaten in Asian cultures (Zee et al ., 2004).

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16 Much confusion exists regarding the identification of Hylocereus species, owing to the common habit of using fruit or flesh color as the main criteria for classification rather than more traditional taxonomic traits (Grimaldo Juarez et al ., 2007). Origin and Distribution The native range for pitaya traverses the Americas from Southern Mexico to northern South America as well as the West Indies (Anderson, 2001; Oldfield, 1997; Britton and Rose, 1963). Pitaya is now cultivated th roughout tropical and subtropical America the Caribbean, S outheast Asia, Israel, Australia and New Zealand, Japan, Sri Lanka, Spain a nd the United States, i.e. Hawaii, California and Florida ( Gunasena et al ., 2007; Crane and Balerdi 2009 ). S mall commercial plantings hav e been established here in South Florida with approximately 40 0 acres currently under cultivation ( Crane, personal c ommunication ). Environmental Factors Pitaya are adaptable to a wide range of environments from rainforests to deserts and the growing conditions of these locations can influence the manner in which the crop is grown. Irrigation Zee et al (2004) recommended watering twice per week with a total annual distribution of 25 to 50 inches for good fruit production and that uneven soil water levels could cause fruit to split. E xcessive rainfall has been reported to result in flower drop and fruit rot (Ja cobs 1998) To reduce the possibility of fruit split ting cause d by uneven watering periodic micro irrigation is recommended during dry periods when the fruit are present (Le Bellec, 2006; Mertens, 2003).

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17 Light In their native habitat under the canopies of tropical rainforests, most Hylocereus sp p are grow under shaded conditions and high humidity, however some species are known to grow in exposed location s under relatively dry conditions (Anderson 2000) Local environmental conditions pla y a role in how much light, or solar radiation as measured in photosynthetic photo flux (PPF), pitaya plants can tolerate In the dry conditions of the Negev Desert where PPF can reach an intensity of 2200 mmol photons m 2 s 1 30 % to 60% shading is nece ssary to avoid stem bleaching and die back (Mizrahi and Nerd, 1999) Andrade et al (2006) used tissue acidity as a measure of photosynthetic activity and determine d that stem cuttings of pitaya grew 67% longer when cultivated under environments limited t o 36 % to 48% of daily incident PPF compared to both higher and lower levels of PPF. Temperature Under California conditions, Nobel et al (2002) reported that pitaya can survive within the temperature range of 2.5 C and 45 C, however due to their Crassulacean acid metabolism ( CAM ), their optimum temperature for CO 2 uptake is 20 C. In the dry desert conditions of Israel, Mizrah i and Nerd (1999) found that most species had a minimum temperature threshold of between 0 C and 4 C with a maximum temper ature threshold of 45 C, however they noted that flower production at an average temperature of 39 C was only 15 20% of the amount produced when the average temperature was 32 C. Nerd et al (2002) similarly concluded that pitaya should not be grown as a c rop in locations with temperatures over 34 38 C due to lower flower production.

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18 Soil Under the canopy of the tropical and subtropical rainforests of the Americas, pitaya grow in topsoil that is rich in organic matter, however as long as drainage is not a problem, they can be found growing under a range of soils from sand y loam found in net houses of plants grown in Israel to the calcareous soils of South Florida ( Raveh et al ., 1998; Crane and Balerdi, 2005 ) Some literature suggest s that Hylocereus sp. are salt tolerant (Black, 2003) Cavalcante et al (2008) determined that with increasing salinity, the following effects were recorded: reduced amount of stems, lower st em height and width and shorter roots. After 90 days, 50% of the seedlings irrigated with saline water with an electrical conductivity of EC w 4.0 dS m 1 were dead. Fertilization Fertilization recommendations vary by location, climate and soil component composition. In Taiwan, the recommended fertilization regimen is 9 lbs of steer manure combined with 3.5 oz of 13 13 13 inorganic fertilizer s tarting in April and every 4 months th ereafter (Zee, 2004). In the arid conditions of the Negev desert in Israel, Kaimov and Mizrahi (2006) utilized a fertigation solution 70 mg l 1 NPK containing minor elements. This was applied twice per week in the summer months and once per week in the winter months. Some growers are producing fruit using only manures for fertilizer so they can compete in the organic market (Gunasena, 2007). Flowering and Pollination Pitaya are long day plants, producing flowers in a series of waves during the summer a nd fall months in the Northern Hemisphere and in the winter and spring months in the Southern Hemisph ere One exception is H. polyrhizus which can fruit nearly all year in Australia (Luders and McMahon, 2006.) A dry period prior to floral

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19 induction is beneficial for increased flower production (Crane and Balerdi 200 5 ). The number of flowers produced is directly related to the number of resulting fruits in self fertile species Hylocereus spp. flowers are nocturnal, starting to open in the late afternoon from 1 to 1.5 h before sunset, becoming completely open at sunse t and start to close about 1.5 h after daybreak ; being completely closed by noon. Nectar used to attract pollinators is secreted at the base of the flower tube (Weiss et al 1994 ). The flower buds emerge from the areoles (Luders and McMahon, 2006). Natural pollinators are thought to include moths bats bees and a butterfly in the Maduca genus ( Zee et al ., 2004; Le Bellec et al ., 2006 ; Luders et al ., 2006 Valiente Banuet et al. 2006 ) Pollination within 24 hours of anthesis optimizes fruit set (Weiss et al ., 1994) Flowers will abort within one week if fertilization is unsuccessful (Martinez, et al ., 2005) The re are both self pollinating (self compatible) and self infertile (self incompatible) species and cultivars (Raveh et al ., 1993 ) Self compatible types are characterized by having anthers at the same height as the stigma, and pollination occurred when the flowers closed and pushed the two parts together. Fertilization with a flower or between flowers of the same clone result in fruit set. However t hese autogamous varieties usually produce larger fruit when cross pollinated with other varieties or species then if they self pollinate ( Weiss et al ., 1994; Nerd and Mizrahi 1997; Lichtenzvieg et al 2000). Self infertile varieties ha ve a minimum 2 cm gap between the anthers and the stigma and require pollination from another clone or species (Weiss et al ., 1994) (Figure 2 1) Self infertile varieties require cross pollination by either another species or a different cultivar (Mertens, 2003). If the plants are compatible, this type of pollination can be expected to result in a nearly 100% fruit set

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20 ( Lichtenzvieg et al, 2000 ). Fruit size is influenced by the s ource of pollen (Weiss et al ., 1994). Knowing the optimum source for pollination of a commercial clone or cultivar may enhance not only fruit yields but also fruit quality. Hylocereus spp. pollen may be stored at subfreezing temperatures (<0 C) and 5% to 10% moisture content. S ubsequent pollination with stored pollen 3 to 9 months later resulted in 100% fruit set However, p ollen stored at 4 C was 30% to 40% less effective and produced smaller fruit (Metz et al ., 2000). The ease of intra and inter spec ific crosses of Hylocereus spp. facilitates the production of hybrids and consequently the development of new cultivars. Cultivation Techniques and Practices Support The natural vining growth habit and aerial roots of pitaya allow it to attach itself to many different surfaces, including stone, living or dead wood and cement. While they can grow and fruit directly on the ground, from a commercial aspect this is discouraged as weeding, pollination and harvesting of the fruit is difficult ( Zee, 2004; Le B ellec et al ., 2006). For these reasons, pitaya are usually grown on a support syste m either as individual poles (Figure 2 2), single poles modified with a support system on top (Figure 2 3), or as part of a trellis system (Figure s 2 4 and 2 5) Strong s upport systems, consisting of wood or concrete poles are preferred as a mature pitaya plant can weigh over 91 kg ( 200 lbs ) (Crane and Balerdi, 2009) In a comparison of different support types, Yus of f et al (2008) found that the individual pole system p roduced signific antly larger numbers of flowers, fruits and total fruit weight than either the V shape system or the T bar trellis. Spacing within and between rows varies greatly, from 1.5 m x 1.5 m to 1.5 m x 2.5 m to 1 m x 3 .1 m (Weiss et al ., 1994; Nerd et al ., 1999; Yusoff et al ., 2008).

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21 Gunasena et al (2007) reported distances up to 3.7 m between plant s and nearly 5 m between rows, noting that variations were related to trellis type and that the increased distances had a positive influen ce on disease reduction. Periodic pruning is recommended to further reduce disease occurrence as well as encourage flowering and fruit production Propagation The progeny of Hylocereus species may be propagated by seed and a wide variety of asexual prop agation methods. The propagation of pitaya by seed is essential for breeding programs (ElObeidy, 2006). The hybrid seeds that result from cross pollination provide a valuable source for new cultivars (Cisneros et al ., 2011). A single fruit can contain o ver 7,000 seeds ( Tel Zur et al ., 2011 ) Seed coat color is linked to germination potential: seeds with hard black coats have greater germination rates than seeds with soft brown coats which are nearly always aborted (Cisneros, et al ., 2011). Seedlings displayed a better survival rate and faster development when grown in 65 ml pots compared to 10 ml to 15 ml containers ( Aparecida de Andrade et al ., 2008). Germination rates were highest at 20 C to 24 C (ElObeidy, 2006). One drawback to producing pit aya from seedlings is the length of time prior to fruit production. Le Bellec et al (2006) reported that seedlings may bear fruit in as little as 3 years, whereas Crane and Balerdi (2009) indicated that it may take up to 7 years for seedlings t o produce fruit. A sexual reproduction by cuttings is quite common and cuttings may produce fruit in as little as 10 to 15 months (Crane and Balerdi, 2009). Grafting is possible but rarely used outside of the ornamental industry since cuttings are simple and have high success rates (Gunasena et al ., 2006). In vitro micropropagation of members of the Hylocereus genus has previously been successfully accomplished by multiple

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22 researchers, using a variety of different plant growth regulators (PGRs) Drew (2002) examined the effects of the cytokin i n 6 (gamma,gamma Dimethylallylamino)purine ( 2ip ) the influence of the size of the explant and the number of explants in the containers and their corresponding effects on shoot proliferation. Mohammed Yas seen (2002) investigated the effects of NAA and various levels of TDZ on decapitated and longitudinally divided explants. Pelah et al ., (2002) worked on H. megalanthus and found that once calli were established, shoot production and elongation were better on MS medium (Murashige and Skoog, 1962) alone than when the same medium was augmented with 6 benzyladenine or gibberellic acid ( GA 3 ) Giusti (2002) compare d the responses of basal, lateral and apical explants of three endangered species in the Cactaceae family ( Mammillaria pectinifera, Pelecyphora aselliformis and Escobaria minima ) using mixtures of the auxin NAA combined with th e cytokinins 6 Benzylaminopurine (BA), TDZ and kinetin. Androgenesis (anther culture) and gynogenesis (egg cell parthenogenes is) have both proven successful as methods of plant regeneration, however they have been species specific, limited almost entirely to H. megalanthus ( Benega Garcia et al ., 2009a; Benega Garcia et al ., 2009b). Off season floral induction In order to increa se the value of a crop it is beneficial to produce the fruit in the off season. Hylocereus species are susceptible to off season floral induction through the use of KNO 3 and by employing artificial lighting the latter of which is more efficient a nd has shown fewer side effects ( Luders and McMahon, 2006; Hoa, 2008) Hoa (2008) describes th e lighting technique as hanging 75 watt incandescent light bulbs approximately 1 m high between rows, ensuring that the light strikes all stem surfaces from 8 pm to 10 pm for 15 to 20 consecutive nights. If successful, floral induction will

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23 commence 3 to 5 days afterwards. While Khaimov and Mizrahi (2006) had contradictory results when attempting to manipulate the flowering season by altering the photoperiod th ey did find success in other methods Those methods involved the use of the plant growth regulators [N (2 chloro 4 pyridinyl) N phenylurea (CPPU) and gibberellic acid (GA 3 ). The former induced an earlier flowering period and the latter delayed flowering. They further found that a later season crop could be produced by removing flower buds early in the reproductive cycle. Physio chemical and Potential Future Uses In pitaya m oisture content is greater than 80% and the fruit is high in vitamin C, phosphorus and calcium (Morton, 1987) The stems contain higher concentrations of ascorbic acid than the fruit (Jaafar, et al 2009). Total soluble solids have been reported to range from 13% to 19% (Zee et al ., 2 004) While pitaya has broken into the world market as an edible fruit in the past 3 0 years, and has been used as a rootstock for ornamental cacti for decades, new research is leading to the discovery of other possible uses for the fruit. One recent study has shown that pitaya peels have potential as a viable source of pectin. Experiments conducted using three different extraction methods (ammonium oxalate/oxalic acid, deionized water and acid) yielded between 15% and 20% pectin from dried alcohol insolub le residues. These totals were similar to that acquired from apples and citrus (Nazaruddin et al ., 2011). O ther investigations use d both the peel and the pulp of the red and purple fleshed varieties, which are rich in the pigment betalain, and found fav orable results for their use as a natural dye and food colorant (Harivaindaran et al ., 2008 ; Rebecca et al ., 2010 ). Rebecca et al (2010) demonstrated that the specific chromophore of betalain contained in red fleshed pitaya betacyanin,

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24 has strong antiox idative properties Pitaya may have anti cancer properties. An analysis by Wu et al (2006) found that the peel of the pitaya was effective in inhibit ing the growth of B16F10 melanoma cells. Myo inositol which when deficient in the human body h as been associated with maladies ranging from diabetes to multiple sclerosis, was found in significant amounts in Hylocereus polyrhizus lending further credibility to the potential for pitaya et al ., 2012). In a similar vein, pitaya has been identified as a possible source of prebiotics, which can encourage the activity of bacteria that promote colon health (Wichienchot et al 2010). Pests and Diseases There are several disease pathogens which can affect pitaya Curvularia lunata was reported in Malaysia ( Masratul Hawa et al ., 2009), with symptoms that began as small, round necrotic lesions, light pink to beige in color, that eventually enlarged and joined together. Anthracnose ( Colletotrichum gloeosporioides ) has been reported by in Japan Florida and Malaysia, (Taba et al ., 2006; Palmateer et al ., 2007; Awaya et a l 2009), and is characterized by chlorotic haloes surrounding reddish brown lesions. Awaya et al. (2009) further described decreased titrateable acidity (TA) and reduced fruit soluble solid contents (SSC) when infections of anthracnose as well as another pathogen, brown rot ( Monilinia fructicol a) were present. The degree of infection was related to the amount of ino culum present with higher concentrations affecting fruit firmness as well as TA and SSC. Bipolaris cactivora was determined to be the cause of pitaya fruit rot in Japan (Taba et al. 2007) where brown spots developed into water soaked lesions with olive g reen to black powdery spots in the center. Similarly stem rot and fruit blotch were linked to B cactivora in Israel and Florida (Ben et al 2011 ;

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25 Tarnowski e t al 2010) Stem canker disease ( Neoscytalidium dimidiatum ) was detected in Taiwan (Chuang, et al., 2012), expressed as diminutive orange depressions that developed into cankers. Also in Taiwan, Cactus Virus X was discovered after tests on minor mottling appeared systemically on pitaya stems (Liou et al ., 2001) Fusarim oxysporum was identified in Argentina as the cause of basal stem rot on pitaya used as rootstocks for the ornamental industry. Symptoms started at the soil line and moved distally, however the roots showed no sign of infection (Wright et al ., 2007). Two variations of Fusarium semitectum were associated with Hylocereus polyrhizus in Malaysia ( Masratul Hawa et al ., 2010). Yellow to brown lesions affecting the stems and young developing fruit of pitaya were identified as Gilbertella stem rot ( Gi lbertella persicari ) by Taba et al (2010). ( Dothiorella sp.) and the bacterium Xanthomonas campestris were problematic for pitaya. The presence of the former was identified by brown spots (1 to 3 mm in diameter) appearing on the surface of the stems that if present in sufficient quantities and t he latter was characterized as a rot that could eventually reduce the stem to its core. A wide variety of animals and insects have been reported to affect pitaya stems, flowers and ultimately fruit production. Included in this grouping are birds, rodents, iguanas, possums, ants beetles, borers, caterpillars, slugs, mealybugs, scale s, thrips mites and fruit flies ( Barbeau 1990 ; Luders and McMahon, 2006; Hoa, 2008; Crane and Balerdi, 2009). The consequences of insect pest attack s reduced harvests due to injuries to the lea ves flowers and fruit, including scarification punctures aborted fl owering and damaged fruit (Tepora and Rint, 2007). Since pitaya play an important

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26 ruit flies are of primary co ncern and recommended controls include protein bait traps containing either the insecticide fipronil or a comb ination of methyl eugenol and electricity (Hoa, 2008). Harvest and Post harvest Pitaya are non climacteric and therefore should be harvested when the skin color is almost fully developed (Nerd et al ., 1999; Tonetto de Freitas et al ., 2012). A study that measured 44 Hylocereus spp. accessions determined that the minimum time required before fruit were ripe was between 28 and 41 days after pollination (DA P ). One exception was H. megalanthus which required between 90 and160 DAF before the fruit was harvest able (Tel Zur et al 2011) Once harvested, one of the challenges remaining is to ship fruit to the market at the proper stage of ripeness (Mizrahi and Nerd, 1999). Since Viet Nam produces fr uit for both the local and export market, harvesting time vari es. For the export market, pitaya are picked 25 28 DA P whereas for the local market, harvesting is not completed until 29 30 DA P The difference in harvest time was attributed to international consumers preferring pitaya that was less sweet ( To et al ., 2 000). Zee et al (2004) suggested that allowing the fruit to mature for 50 DA P allowed them to attain greater weight and sweetness. Yield estimates range from approximately 16 t ha 1 in the second year for an arid environment in Israel to 30 t ha 1 for the tropical climates of Viet Nam ( Mizrahi et al 1997 ; Raveh et al 1998). Postharvest quarantine treatments and storage and shipping conditions for pitaya have been investigated. At room temperature, the fruit begin to show signs of deterioration in 10 days or less ( Mizrahi and Nerd, 1999; Zee et al ., 2004). As pitaya are potential hosts for fruit fly ( Bactrocera spp.), hot air treatments were tested and found to be an effective method of addressing biosecurity concerns. Four weeks after

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27 fruit were su bjected to short duration treatments (20 min at 46.5C), internal fruit quality was not significantly different than non treated fruit, however the same could not be said for the external appearance ; specifically the bract s and stem s had become dehydrated (Hoa et al ., 2006). Mizrahi et al (2002) identified the shriveling of the scales on the fruit as the main reason for fruit value loss. Kowitcharoen et al (2010) found that with a 2 kJ/m 2 application of UV C irradiation, several of the storage related problems could be addressed; the loss of fruit weight could be decreased the change in color of the bracts could be delayed and the chlorophyll degradation could be repressed. Nerd et al ( 1999) reporte d that when fruits were harvested at a nearly full color stage, they could be stored at 14C for 2 weeks or 20C for 1 week and still maintain a commercial quality However while they found that fruit could be stor ed for up to 3 weeks at 6C fruit quality deteriorated when the fruit was returned to room temperature. When film wrap was applied to the individua l fruits at 3 different stages of ripeness, Chandran (2010) found no reduction in appearance, firmness or TSS during stor age at 6C ove r a period of 15 days. Zahid et al (2012) found that that the application of 600 nm droplets containing 1.0% chitosan was an effective method of delaying the appearance of symptoms of C. gloeosporioides (anthracnose) on pitaya kept in cold storage for up to 4 weeks. World Market Hylocereus spp. are often found in the markets in the areas where they are grown ( Le Bellec et al 2006 ; Gunasena et al ., 2007 ) Europe and Asia are the largest importers of fruit with Thailand and Viet Nam being the main suppliers of the Asian market and Israel the lead provider of fruit for the European market (Jaya, 2010). Europe, North America and Japan were identified as high value markets for Vietnamese

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28 exporters (Hoa, 2008.) Antioxidants and other health benefits attributed to the fruit are driving an emerging market in the United States (Evans and Huntley, 2011). C onsumer acceptance of new fruit crops, confusion caused by the use of multiple names for the same fru it and having the fruit reach the market in a prime condition are some of the hurdles identified in creating a greater market presence for Hylocer e us spp (Mizrahi and Nerd, 1999.) The latter two issues are important factors related to consumer acceptance. Studies have shown that consumers are not willing to compromise on taste in order to acquire reputed health benefits of fruit (Tuorila and Cardello, 2002; Sabbe et al ., 2009). I n Viet Nam, fruit quality and food safety has been a roadblock to the high value export markets identified above To address those concerns, new varieties have been developed and Good Agricultural Practices (GAP) have been adopted (Hoa, 2008). Le Bellec et al (2006) identified falling retail prices combined with increasing airfreight costs to the European market as limiting factors for Asian suppliers.

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29 Figure 2 1 Flower with location of stigma high above the anthers. (Flower type is generally self incompatible.) Photo courtesy of W Condon. Figure 2 2. Pitaya interplanted among mangosteen using the single post method. Photo courtesy of W. Condon.

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30 Figure 2 3 Single post method modified with supporting structure on top. Photo courtesy of JHCrane.

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31 Figure 2 4. Trellis method of growing pitaya in early phase of establishment. Photo courtesy of W. Condon. Figure 2 5. Mature planting of pitaya u sing the trellis method. Photo courtesy of W. Condon.

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32 CHAPTER 3 MATERIALS AND METHOD S Parent M aterial S election and H ybridization Cuttings of two different pitaya species, Hylocereus guatemalensis and Hylocereus stenopterus were provided by a grower in C alifornia and grown to maturity in Florida. Plants of H. guatemalensis produce 4 inch long fruits with a crimson red exterior and dark red flesh, and large white flowers. Plants of H. stenopterus are characterized by a green fruit that develops undertones of pink as it matures, a white flesh, and brilliant pink flowers (Figure 3 1 ) Prior to performing crosses, H. guatemalensis was selected as the fema le parent and two days prior to anthesis, th e flower bud was covered with a plastic bag to prevent possible cross contamination. Pollen was collected from H. stenopterus on the night of flower opening Pollen was applied to H. guatemalensis After pollination, flowers were covered with a plastic bag. The cross between both plants was performed on July 21, 2009 and the resulting fruits were harvested on August 26, 2009. Culture Establishment Hybrid seeds (300 400) were separated from the fruit pulp using a strainer under running water, and subse quently placed in a 250 ml beaker with distilled water and 2 drops of Tween 20 (Cayman Chemical Company, Ann Arbor, MI) stirred for 5 minutes, and then air dried for 15 20 min. Under a laminar flow hood, the seeds were subject to surface sterilization by placing them in a 70% ethanol solution for 5 minutes under agitation, followed by 5 minutes in a 3% sodium hypochlorite (The Clorox Company, Oakland, CA) solution, and three separate 1 minute rinses in autoclaved distilled water.

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33 Fifty sterilized seeds we re then placed in Magenta GA 7 boxes (Magenta Corporation, Chicago, IL) containing MS medium (Murashige and Skoog, 1962) supplemented with 3 0 .0 g L 1 sucrose and solidified with 7 g L 1 agar (Bacto Agar, Fischer Chicago, IL, USA), with pH adjusted to 5. 7. Five seeds were placed per magenta box with a total of ten boxes. Boxes were sealed wi t h Parafilm M (Bemis, Neehah, WI, USA). Culture establishment was performed on May 21, 2011. Cultures were maintained in a Percival E30B incubator (Percival Scient ific, Inc., Perry, IA, USA) at 26 2 C; 320 mol m 2 s 1 ; 18/6 light/dark; under 6 x 9A Philips fluorescent bulbs. Germination was observed within 1 4 weeks after culture establishment. After 6 months, the 5 most vigorous seedlings (a minimum of 6 c m in length ) were selected as lines and used as explant sources for in vitro clonal propagation. The selected seedlings were removed from the magenta boxes and placed on autoclaved petri dishes under the laminar flow hood, where they were initially dissected into sections of 2 mm, and then subdivided into 3 sections of about 0.5 0.7 mm each. A total of 144 explants were utilized per line. The explants were then placed in magenta boxes containing the same MS medium used for germination. Three explants were placed per magenta box. In Vitro Treatments Magenta boxes containing explants were distributed among seven treatments containing different combinations of plant growth regulators (PGRs) and a control (no PGRs) (Table 3 1). Plant growth regulators used were NAA at 0.05 m and TDZ at 0.5 m, 1.0 m or 2.27 m added to a basal MS medium. The basal MS medium (no PGRs) was used as a control. Cultures were maintained under the same environmental conditions as described previously. Cultures were monitored on a

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34 weekly basis for four months For eac h treatment evaluations were performed for callus presence, culture development, contamination rate, shoot formation, number of shoots per explant, number of shoots with roots, and number of root s per explant. Experimental Design and Statistical Analysis The experimental design was completely randomized and consisted of five hybrid lines submitted to seven treatments, plus a control in a factorial design, with 6 replications of 3 explants per treatment; 5 hybrid lines x 8 treatments (Table 3 1) x 18 replic ations. Data was analyzed using analysis of variance (ANOVA), and means were compared using s range test at = 0.05. The entire experiment was repeated.

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35 Table 3 1 Plant growth regulator (PGR) concentrations used for clonal propagation of hybr id pitaya. Two PGRs were used, 1 naphthaleneacetic acid (NAA) and thidiazuron (TDZ) Treatments (T1 T8) were comprised of different concentrations of PGRs, including NAA at 0.05 m and TDZ at 0.5, 1.0, and 2.27 m, plus controls (no PGRs). PGR concent rations PGR T1 T2 T3 T4 T5 T6 T7 T8 NAA 0.0 0.05 0.0 0.05 0.0 0.05 0.0 0.05 TDZ 0.0 0.0 0.5 0.5 1.0 1.0 2.27 2.27 A B C D

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36 Figure 3 1 Flower, growth habit and fruit flesh characteristics of Hylocereus guatamalensis (A,C,E) and H. stenopterus (B,D,F). Photos courtesy of W. Condon. E F

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37 CHAPTER 4 RESULTS AND DISCUSSI ON Seed Germination and Seedling Growth Of the fifty seeds initially placed on the MS medium for germination, 34 germinated within the three week s in culture, with a total of 3 8 seeds germinated (76%) after six weeks (data not shown) This is similar to what ElObeidy (2006) found for pitaya when germinated at 24 C (83%) and 28 C (73%) Contamination was observed within a single culture, representi ng a loss of 10%. Seedlings started to reach the desirable size (about 6 cm in length) for explant selection after nine weeks in culture. However, 2 2 24 weeks were necessary for 10 seedlings to reach the desired length ( Figure 4 1 ) Similarly, Aparecida de Oliveira et al (1995) reported that seedlings of Cereus peruvianus required 8 weeks to reach 2 to 3 cm in length. Callus and Shoot Formation Callus was initiated at the basal part of the explants where they contact ed t he medi a and along cut surfaces All explants produced varyi ng degrees of callus formation within 1 week which initially appeared light green and granular Some calli turned greenish red to purple or brown at the higher concentration levels of TDZ (1.0 m L 1 and 2.27 m L 1 ) within 3 weeks after initial PGR introduction For treatments without TDZ, callus growth was limited for all lines, except for line 4 (Table 4 1) Treatments containing a combination of TDZ and NAA (T4, T6 and T8) produced more callus than treatments wit h TDZ alone (T3, T5 and T7) This is in agreement with Balch et al (1998) who found that benzyladenine (BA) treatments including NAA at different concentrations produced greater callus than those with BA alone. The treatment containing only NAA (T2) produced less significant callus th a n the control in our

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38 experiment, which suggest s that NAA is either unnecessary to produce callus or that it promotes callus growth only when used in conjunction with other PGRs. In contrast Mammillaria san angelensis h a d higher rates of shoot multiplication per explant as compared to the control as NAA in concentrations increased from 10.74 m L 1 to 32.22 m L 1 (Rubluo et al ., 2002) All explants treated with TDZ (T3 T8) produced higher number s of shoots than treatme nts without TDZ (control [T1] and NAA only [T2] ). Both the number of explants producing shoots and the overall number of shoots produced per treatment were highest for T4 (Tables 4 2 and 4 3) The control (T1) produced the least number of shoot s and the lowest number of explants with shoots. This is indicative that overall the combination of 0.5 m L 1 TDZ and 0.05 m L 1 NAA w as the most effective treatment for the hybrids generated in this experiment and that across all lines, the addition of PGRs had a positive effect on shoot production (Figure 4 2) Looking at the statistical analysis of mean shoots per treatment per line, the T4 combination of PGRs did not provide the maximum number of shoots for each line (Figure 4 3 ). Both T3 and T5 out performed T4 as applied to Line 2. This supports the conclusion reached by Balch et al (1998) that different cactus species require different PGR treatments to obtain optimum shoot generation, and goes one step further and suggests that the same requirem ent for different treatments applies to lines of the same species. While Giusti et al (2002) also had good results generating axillary shoots across 3 species of cacti when using a combination of NAA and TDZ (0.05 m L 1 and 2.27 m L 1 respectively), the best results in that study were achieved when the NAA was not included in that combination. In contrast to the current experiment, Guisti combined NAA with different cytokin i ns at

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39 varying levels and many of these treatments did not produce any shoots for the 3 species tested Axillary shoots are preferred over those from callus due to possible somaclonal variation This is more important for fruit crops where uniformity is a consideration (Drew and Azimi, 2002). Root Formation The treatment which contained NAA alone (T2) produced more explant s with roots and more total roots than all of the other treatments combined (Tables 4 4 and 4 5) This did not hold true for each line however, as Line 1 had a greater number of explants generating roots with T4 and more total roots in the control group (T1). This is further evidence of genotypic reaction variation to the PGRs In a study involving Hylocereus undatus Drew and Azimi (2002) noted that roots can be induced on a medium free of PRGs, regardl ess o f explant size, however exposure to 10 m L 1 of the auxin indole 3 butyric acid (IBA) for up to 5 days did produce longer roots. In preliminary tests (data not reported), there was no difference in root formation from the control medium to those with NAA and TDZ. All plants that were mov ed to the greenhouse acclimated, however those with the humidity tent covering them initiated growth quicker. These plants will be transferred to the field and evaluated for desirable fruit, flower, physiochemical and g rowth pattern characteristics.

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40 Figure 4 1. Weeks required for ten pitaya seedlings to reach desired 6 cm length when grown on Murashige and Skoog (1962) medium. 0 0 0 0 1 1 3 5 6 6 7 8 8 8 8 8 9 10 10 10 0 2 4 6 8 10 12 3 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Number of seedlings exceeding 6 cm in length Weeks after placement on MS medium Table 4 1. Total number of explants exhibiting significant callus (amount of callus exceeds size of original explant) by treatment number. Paired treatments (1,2 ; 3,4; 5,6; 7,8) have equal concentrations of TDZ. Even numbered pair member contains 0.05 m concentration of NAA. T1 T2 T3 T4 T5 T6 T7 T8 LINE 1 0 0 1 12 6 12 8 17 LINE 2 0 0 9 16 14 17 13 17 LINE 3 1 0 2 11 5 7 7 13 LINE 4 12 3 11 18 5 6 12 7 LINE 5 1 0 6 17 6 13 10 13 TOTAL 14 3 29 74 36 55 50 67

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41 Table 4 2 Total number of explants exhibiting shoots. Paired treatments ( T 1, T 2; T 3, T 4; T 5, T 6; T 7, T 8) have equal concentrations of TDZ. Even numbered pair member contains 0.05 m concentration of NAA. T1 T2 T3 T4 T5 T6 T7 T8 LINE 1 5 4 11 17 1 12 12 9 LINE 2 3 8 9 7 15 5 5 10 LINE 3 0 1 1 1 1 3 5 3 LINE 4 0 2 3 5 1 2 4 4 LINE 5 0 0 2 5 2 8 3 2 TOTAL 8 15 26 35 30 30 29 28 Table 4 3. Total number of shoots produced per treatment separated by line Paired treatments (T1,T2 ; T3,T4; T5,T6; T7,T8) have equal concentrations of TDZ. Even numbered pair member contains 0.05 m concentration of NAA. T1 T2 T3 T4 T5 T6 T7 T8 LINE 1 16 11 12 22 17 14 17 15 LINE 2 4 10 24 11 16 8 8 11 LINE 3 0 1 1 1 5 5 5 3 LINE 4 0 2 3 16 1 5 4 5 LINE 5 0 0 2 6 3 10 3 2 TOTAL 20 24 42 56 42 42 37 36

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42 Table 4 4 Total number of explants with roots. Paired treatments (T1,T2; T3,T4; T5,T6; T7,T8) have equal concentrations of TDZ. Even numbered pair member contains 0.05 m concentration of NAA. T1 T2 T3 T4 T5 T6 T7 T8 LINE 1 6 6 2 11 6 3 3 5 LINE 2 4 8 0 1 2 0 0 1 LINE 3 6 6 1 0 0 1 0 1 LINE 4 4 11 0 0 0 0 0 0 LINE 5 2 11 0 0 1 0 0 0 TOTAL 22 42 3 12 9 4 3 7 Table 4 5 Total number of roots produced per treatment separated by line. Paired treatments (T1,T2; T3,T4; T5,T6; T7,T8) have equal concentrations of TDZ. Even numbered pair member contains 0.05 m concentration of NAA. T1 T2 T3 T4 T5 T6 T7 T8 LINE 1 88 60 11 47 8 4 14 13 LINE 2 5 113 0 1 2 0 0 1 LINE 3 6 7 1 0 0 1 0 1 LINE 4 4 24 0 0 0 0 0 0 LINE 5 2 14 0 0 0 0 0 0 TOTAL 105 218 12 48 10 5 14 15

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43 Figure 4 2. In vitro culture showing single and multiple shoots being generated from callus. Photo courtesy of W. Condon.

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44 Figure 4 3. Comparison of in vitro shoot formation for hybrid pitaya lines (1 5) under different treatments (T1 T8). Lowercase letters across columns represent mean comparisons between treatments for each line. Uppercase letters across rows represent mean comparisons between lines for each treatment. Treatments are d escribed in Table 3 1. 0.05. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1 2 3 4 5 Mean Number of Shoots Line Number In vitro dragonfruit shoot formation T1 T2 T3 T4 T5 T6 T7 T8 Line 1 Line 2 Line 3 Line 4 Line 5 T1 0.39 cA 0.17 bAB 0.00 aB 0.00 bB 0.00 bB T2 0.28 cAB 0.44 abA 0.06 aBC 0.11 bBC 0.00 bC T3 0.50 cAB 1.06 aA 0.07 aB 0.17 bAB 0.11 abAB 1.06 aA 0.44 abABC 0.06 aC 0.89 aABC 0.33 aBC T5 0.72 abcAB 0.78 abA 0.33 aABC 0.00 bC 0.17 abBC T6 0.61 abcA 0.39 bA 0.27 aA 0.56 abA 0.33 aA 0.89 abA 0.40 bB 0.22 aB 0.22 abB 0.17 abB T8 0.67 abcA 0.50 abAB 0.17 aB 0.42 abAB 0.11 abB

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45 CHAPTER 5 SUMMARY AND CONCLUSI ONS There still remains work to be done on identifying the appropriate parameters for optimal growth of pitaya in tissue culture Photoperiod, temperature and l ight intensity may affect germin ation and growth rates for d ifferent cactus species As shown by this study there are various combinations of NAA and TDZ that produce growth different results for different lines of a single hybrid combination This indi cates that for commercial propagation of hybrid pitaya specific combinations must be determined for each line in order to maximize clonal propagation efficiency. In this experiment, p lantlets were regenerated with fully developed shoots and roots, showi ng no abnormalities. Regenerated plantlets were successfully acclimatized and transferred to the greenhouse. Further studies will be performed to evaluate field growth of these regenerated hybrids. Future work should be directed towards obtain ing the op timum pitaya fruit: large size (800 g or more); attractive combination of flesh and skin color; spineless plant and fruit; disease resistant ; attractive colorful flowers; self pollinating; and full of ingredients that will be attractive to the health conscious consumer. This thesis suggests a protocol for the generation and micropropagation of such pitaya hybrids varies

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46 LIST OF REFERENCES Anderson, E F. 2001 The Cactaceae Fami ly Timber Press, Portland, OR USA 377 381. Andrade, J L E Rengifo, M.F Ricalde, J.L Sim J. C Cervera and G Vargas Soto 2006. Microambientes de luz, Crecimiento y Fotos ntesis de la Pi tahaya ( Hylocereus undatus ) en un Agrosistema de Yucat n, M xico. Agrociencia. 40:687 697. Aparecida de Andrade, R., A.B.G. Martins and M.T.H. Silva. 2008. Development of seedlings of red pitaya (Hylocereus undatus Haw) in different substrate volumes. Acta Sci Agron, Maring 30 ( supl ): 697 700. Aparecida de Oliveira, S., M. F. Pires da Silva Machado, A.J. Prioli and C.Aparecida Mangolin. 1994. In vitro propagation of Cereus peruvianus Mill. (Cactaceae). In Vitro Cellular & Developmental Biology. 31:47 50. Awanga, Y. M.A.A. Ghani and K. Sijam 2009. Effects of Colletotrichum gloeosporioides and Monilinia fructicola on Quality of Red Flesh Dragon Fruit ( Hylocereus polyrhizus ) Proc. IS on Postharvest Pacifica 431 438. Balch, E.P.M., M.E.P. Reyes, E.V. Amador, E.M. Rangel, L.D.R.M. Ruiz and H.J.L. Vi ramontes. 1998. Micropropagation of 21 species of Mexican cacti by axillary proliferation. In Vitro Cellular & Developmental Biology Plant. 34:131 135. Barbeau, G. 1990. La Pitahaya rouge, un nouveau fruit exotique. Fruits. 45(2) :141 147. Bauer, R. 2003. A synopsis of the tribe Hylocereeae F. Buxb. Cactaceae Systematics Initiatives 17: 3 63. Ben I S. I Assouline E Levy and G Elkind 2011 First report of Bipolaris cactivora causing fruit blotch and stem rot of dragon fruit (pitaya) in Israel Phytoparasitica 39:195 197 Benega Garcia, R., B. Schneider and N. Tel Zur. 2009 a Androgenesis in the vine cacti Selenicereus and Hylocereus (Cactaceae). Plant Cell, Tissue and Organ Culture. 96:191 199. Benega Garcia, R., A. Cisneros, B. S chneider and N. Tel Zur. 2009 b Gynogenesis in the vine cacti Selenicereus and Hylocereus (Cactaceae). Plant Cell Reports 28:719 726. Black, R.J 200 3 Salt Tolerant Plants for Florida University of Florida, IFAS, Extension. Publication #ENH26.

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47 Britton, N.L. and J.N. Rose 1963. The Cactaceae Descriptions and Illustrations of plants of the cactus family. Vol II. Dover Publications, New York, NY. 183 195. Cavalcante, I.H.L., M.Z. Beckmann, A.B.G. Martins, J.A. Gabiatti and L. Ferreira. 2 008. Water Salinity and Initial Development of Pit aya ( Hylocereus undatus ). International Journal of Fruit Science 7 ( 3 ): 81 92 Chandran, S. 2010. Effect of Film Packaging in Extending Shelf Life of Dragon Fruit, Hylocereus undatus and Hylocereus polyrh izus. Proceedings Southeast Asia Symposium on Quality and Safety of Fresh and Fresh Cut Produce. Acta Hort. 875, ISHS 2010. 105 110. Chuang, M.F., H.F. Ni, H.R. Yang, S.L. Shu, and S.Y. Lai. 2012. First Report of Stem Canker Disease of Pitaya ( Hylocereu s undatus and H. polyrhizus ) Caused by Neoscytalidium dimidiatum in Taiwan Plant Disease. 96(6):906. Cisneros, A., R.B. Garcia and N.Tel Zur. 2011. Ovule morphology, embriogenesis and seed development in three Hylocereus species (Cactaceae). Flora. 206:1076 1084. Crane, J.H., and C.F. Balerdi. 2005. The Pitaya ( Hylocereus undatus and other spp.) in Florida. University of Florida, IFAS, Extension Crane, J.H. and C.F. Balerdi. 200 9 Pitaya g rowing in the Florida home land scape. University of Florida IFAS, Extension. Publication #HS1068 Drew, R.A. and M. Azimi. 2002. Micropropagation of Red Pitaya ( Hylocereus undatus ). Acta horticulturae. 575(1):93 98. ElObeidy, A.A. 2006. Mass propagation of pitaya ( dragon fruit). Fruits. 61(5):313 319. Eva ns, E A., J Huntley, J Crane and A F. Wysocki. 2010 Cost Estima t es of Establishing and Producing Pitaya (Dragon Fruit) in South Florida Electronic Data Information Source (EDIS) FE888 UF/IFAS, Gainesville, FL. Evans, E.A. and J. Huntley. 2011. Economics of Establishing and Producing Pitaya in Southern Florida: A Stochastic Budget Analysis. HortTechnology. 21(2):246 251. Fawcett, J.A., S. Maere, Y. Van der Peer and M.C.E. Van Montagu. 2009. Plans with Double Genomes Might Have Had a Better Chance to Survive the Cretaceous Tertiary Extinction Event. Proceedings of the National Academy of Sciences of the United States of America. 106(14):5737 5742. Giusti, P., D. Vitti, F. Fiocchetti, G. Colla, F. Saccardo, and M. Tucci (2002). In v itro propagation of three endangered cactus species. Scientia Horticulturae 95. 319 332.

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48 Grimaldo Juarez, O., T. Terrazas, A. Garcia Velasquez, M. Cruz Villagas and J.F. Ponce Medina. 2007. Morphometric Analysis of 21 Pitahaya ( Hylocereus undatus ) Genot ypes. Journal of Professional Association for Cactus Development. 9 :9 9 117 G roszmann M. I K. Greaves, N Albert, R. Fujimoto, C A. Helliwell, E .S. Dennis and W.J Peacock 2011. Epigenetics in plants vernalisation and hybrid vigour Biochimica et Biophysica Acta (BBA) Gene Regulatory Mechanisms 1809 ( 8 ): 427 437 Gunasena, H.P.M. D.K.N.G Pushpakumara and M. Kariyawasam 2007. Chapter 4: Dragon fruit ( Hylocerus undatus (Haw.) Britton and Rose). In: Pushpakumara, D.K.N.G., Gunasena, H.P.M. and Singh, V.P. (2007) (eds) Underutilized fruit trees in Sri Lanka. World Agroforestry Centre, South Asia Office, New Delhi, India. 110 142. Harivaindaran, K.V., O.P.S. Rebecca and S. Chandran. 2008. Study of Optimal Temperature, pH and Stability of Dragon Fruit ( Hylocereus polyrhizus ) Peel for Use a Potential Natural Colorant. Pakistan Journal of Biological Sciences. 11(18):2259 2263. Hoa, N.V. 2008. Current Research Activities and the De velopment of Good Agricultural Practice (GAP) for Pitaya in Viet Nam. Southern Horticultural Research Institute (SOFRI), Longdihn, Chauthanh, Tiengiang, Vietnam Hoa, T.T., C.J. Clark, B.C. Waddell and A.B. Woolf. 2006. Postharvest quality of Dragon frui t ( Hylocereus undatus ) following disinfecting hot air treatments. Postharvest Biology and Technology. 41:62 69. Houghton, A.D. 1930. The Cactus Book. The MacMillan Company, New York NY, USA. 45 53. Jacobs, D 1998. Pitaya, Hylocereus undatus ( Haw) A Potential New Crop o f Australia. Western Australia Nut and Tree Crop Association Yearbook 22:11 19. Jaafar, R.A., N.Z.C. Mahmod, A.R.B.A. Rahman and R. Vasudevan. 2009. Proximate analysis of dragon fruit ( Hylecereus polyrhizus ). American Journ al of Applied Sciences. 6(7):1341. Jaya, I.K.D. 2010. Morphology and Physiology of Pitaya and It Future Prospects in Indonesia. Crop Agronomy. 3(1):44 50. Kowitcharoen, L., L. Kammapana and V. Srilaong. 2010. UV C Treatment Delays Chlorphyll Degradat Proceedings Southeast Asia Symposium on Quality and Safety of Fresh and Fresh Cut Produce. Acta Hort. 875, ISHS 2010. 105 110.

PAGE 49

49 Le Bellec, F F Vaillant and E Imbert 2006. Pitahaya (Hylocereus sp p.): a new fruit crop, a market with a future. Fruits 61:237 250. Liou, M.R., C.L. Hung and R.F. Liou 2001. First Report of Cactus Virus X on Hylocereus undatus (Cactaceae) in Tawain. Plant Disease. 85(2): 292. Luders, L. and G. McMahon. 2006. The Pitaya or Dragon Fruit ( Hylocereus undatus ). Department of Primary Industry, Fisheries and Mines, Northern Territory Government. Agnote No. D42. Martinez, R.C., M.L. Muoz and G.J.M. Guzmn. 2005. Caracterizacin morfolgica y compatibilidad sexual de cinco genotips de pitahaya ( Hylocereus undatus ). Agrociencia. 39(2):183 194. Masratul Hawa, M., B. Salleh and Z. Latiffah 2010. Characterization and intraspecific variation of Fusarium semitectum (Berkeley and Ravenel) associated with red f leshed dragon fruit ( Hylocereus polyrhizus [Weber] Britton and Rose) in Malaysia. African Journal of Biotechnology 9(3) : 273 284. Masratul H awa M., B. Salleh and Z. Latiffah 2009. First Report of Curvularia lu n ata on Red Fleshed Dragon Fruit ( Hylocereus polyrhizus ) in Malaysia. Plant Diseas e. 93(9):971. Maximova, S.N., L. Alemanno, A. Young, N. Ferriere, A.Traore and M.J. Guiltinan. 2002. Efficiency, Genotypic Variability and Cellular Origin of Primary and Secondary Somatic Embryogenesis o f the Theobroma cacao L. In Vitro Cellular & Developmental Biology Plant. 38:252 259. McCurdy, R.M. 1926. Garden Flowers. The County Life Press, Garden City, NJ. 282 Mertens, S 2003. A review of Hylocereus production in the United States J ourn al of Prof essional Assoc iation for Cactus Dev elopment. 5:98 105. Metz, C., A. Nerd and Y. Mizrahi. 2000. Viability of Pollen of Two Fruit Crop Cacti of the Genus Hylocereus Is Affected by Temperature and Duration of Storage. HortScience. 35(2):199 201. Mizrahi, Y., A. Nerd and P.S. Nobel. 1997. Cacti as Crops. Horticultural Reviews. 18:291 319. Mizrahi, Y. and A. Nerd. 1999. Climbing and Columnar Cacti: New Arid Land Fruit Crops in : Janick J.(Ed.), Perspectives on new crops and new use ASHS Press, Alexandria, VA, USA 358 366. Mizrahi, Y., A. Nerd and Y. Sitrit. 2002. New fruits for Arid Climates in: Janic J. and A. Whipkey (Eds.), Trends in new crops and new uses. ASHS Press, Alexandria, VA, USA 3 7 8 3 84

PAGE 50

50 Mohamed Yasseen, Y. 2002. Microp ropagation of Pitaya ( Hylocereus undatus Britton et Rose). In Vitro Cellular & Developmental Biology Plant. 38:427 429. Morton, J. 1987. Strawberry Pear. pgs. 347 348. In: Fruits of warm climates. Julia F. Morton, Miami, FL. Murashige, T. and F. Skoo g. 1962.A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 15:473 497. Murthy B.N.S., S.J. Murch and P.K. Saxena. Thidiazuron: A Potent Regulator of in vitro Plant Morphogenesis. In Vitro Cellular & D evelopmental Biology Plant. 34(4):267 275. Nazaruddin, R., S.M.I. Norazelina, M.H. Norziah and M. Zainudin. 2011. Pectins from dragon fruit ( Hylocereus polyrhizus ) peel. Malaysian Applied Biology. 40(1):19 23. Nerd, A Y Sitrit, R A Kaushik and Y Mizrahi 2002. High summer temperatures i nhibit flowering in vine pitaya crops ( Hylocereus spp.). Scientia Horticulturae 96:343 350. Nerd, A., F. Gutman and Y. Mizrahi. 1999. Ripening and postharvest behavior of fruits of two Hylocereus species ( Cactaceae). Postharvest Biology and Technology. 17:39 45. Nerd, A. and Y. Mizrahi. 1997. Reproductive Biology of Cactus Fruit Crops. Horticultural Reviews. 18:321 346. Nobel, P S., E De La Barrera, D W. Beilman, J H. Doherty, and B R. Zutta 2002. Temperature Limitations for Cultivation of Edible Cacti in California. Madro o. 49(4):228 236. Oldfield, S 1997. Status Survey and Conservation Action Plan Cactus and Succulant Plants. International Union for Conservation of Nature and Natural Resources (ICUN) Publication Services Unit, Cambridge, UK. Palmateer, A. J. and R. C.Ploetz 2007. First Occurrence of Anthracnose Caused by Colletotrichum gloeosporioides on Pitahaya. Plant Disease 91(5):631. Pelah, D., R.A. Kaushik, Y. Mizrahi and Y Sitrit. 2002. Organogenesis in the vine cactus Selencerius megalanthus using thidiazuron. Plant Cell, Tissue and Organ Culture. 71:81 84. Raveh, E J Weiss, A Nerd and Y Mizrahi 199 3 Pitayas (Genus Hylocereus): a new fruit crop for the Negev Desert of Israel. New crops: exploration, research and commercialization : proceedings of the second National Symposium New Crops Indianapolis, Indiana, October 6 9, 1991. 491 495.

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51 Rebecca, O.P.S., K.V. Harivaindaran, A.N. Boyce and S. Chandran. 2010. Potential Natural Dye with Antioxidant Properties from Red Dragon Fruit ( Hylocereus polyrhizus ). Proceedings Southeast Asia Symposium on Quality and Safety of Fresh and Fresh Cut Produce. Acta Hort. 875, ISHS 2010. 477 486. Rebecca, O.P.S, A.N. Boyce and C. Somasundram. 2012. Isolation and Identification of Myo Inositol Crystals from Dragon Fruit ( Hylocereus polyrhizus ). Molecules. 17(4):4583 4594. Rojas Ar chiga, M. and C. V zquez Yanes. 2000. Cactus seed germination: a review. Journal of Arid En vironments. 44:85 104. Rubluo, A., T. Mar n Hern ndez, K. Duval, A. Vargas and J. M rquez Guzm n. 2002. Auxin induced morphogenetic responses in long term in vitro subcultured Mammillaria san angelensis S nchez Mejorada (Cactaceae). Scientia Horticultur ae. 95:341 349. Sabbe, S., W. Verbeke and P. Van Damme. 2009. Confirmation/disconfirmation of International Journal of Food Science and Technology. 44:539 551. Taba, S., M. Nak azato, K. Nasu, T. Takushi, and Z. Moromizato (2011). Gilbertella stem rot of pitaya ( Hylocereus undatus ), a new disease caused by Gilbertella persicari J a p a n ese J ournal of Phytopathol ogy 77:291 294. Taba, S., D. Mikami, K. Takaesu, A. Ooshiro, Z. Moromizato, S. Nakasone and S. Kawano. 2006 Anthracnose disease of pitaya ( Hylocereus undatus ) by Colletotrichum gloeosporioides Japanese Journal of Phytopathology 72:25 27. Taba S N Miyahira K Nasu T Takushi and Z Moromizato 2007. Fru it rot of S trawberry pear (pitaya) caused by Bipolaris cactivora J ournal of Gen eral Plant Pathology. 73:374 376 Tel Zur, N., S. Abbo, D. Bar Zvi and Y. Mizrahi. 2003. Chromosome Doubling in Vine Cacti Hybrids. Journal of Heredity. 94(4):329 333. Tel Zur, N., S. Abbo, D. Bar Zvi and Y. Mizrahi. 2004. Genetic Relationships among Hylocereus and Selenicereus Vine Cacti (Cactaceae): Evidence from Hybridization and Cytological Studies. Annals of Botany. 94:527 534. Tel Zur N., Y. Mizrahi, A. Cisneros, J. Mouyal, B. Schneider and J.J. Doyle. 2011. Phenotypic and genomic characterization of vine cactus collection (Cactaceae). Genetic Resource Crop Evolution. 58:1075 1085. Tepora, T. and R.J. Rint. 2007. Notes on the inc idence of insect pests and diseases of dragon fruits. Phillipine Entomologist. 21(2):196.

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52 To, L.V., N. Ngu, N.D. Duc, D.T.K. Trinh, N.C. Thanh, D.V.H. Mien, C.N. Hai and T.N. Long 2000. Quality Assurance in Agricultural Produce. ACIAR Proceedings 100, 101 114. Tonetto de Freitas, S., N.T. Nham and E.J. Mitcham. 2011. Pitaya (Pitahaya, Dragon Fruit) Recommendations for Maintaining Postharvest Quality. University of California Davis. Tuorila, H. and A.V. Cardello. 2002. Consumer responses to an of f flavor in juice in the presence of specific health claims. Food Quality and Preference. 13:561 569. Valiente Banuet, A., R. Santos Gally, M.C. Arizmendi and A. Casas. 2006. Pollination biology of the hemiepiphytic cactus Hylocereus undatus in the Teh uac n Valley, Mexico. Journal of Arid Environments. 68:1 8. W eiss, J. A Nerd, and Y Mizrahi. 1994. Flowering Behavior and Pollination in C limbing Cacti with Fruit Crop Potential. HortScience 29(12):1487 1492. Wichienchot, S., M. Jatupornpipat and R.A Rastall. 2010. Oligosaccharides of pitaya (dragon fruit) flesh and their prebiotic properties. Food Chemistry. 1 20(3):850 857. Wright E.R., M. C. Rivera and A. Ghirlanda 2007. Basal Rot of Hylocereus undatus Caused by Fusarium oxysporum in Buenos Aires, Argentina Plant Disease 91(3): 323. Wu, L. C., H. W. Hsu, Y. C. Chen, C. C. Chiu, Y. I. Lin and J.A. Ho. 2006. Antioxidant and antiproliferative activities of red pitaya. Food Chemistry. 95:319 327. Yusoff, M M., R A. Halim, M T.M. Mohamed, S O S. Rastan and Z Meon. 2008. G rowth, yield and fruit quality of red dragon ( Hylocereus polyrhizus ) fruit as affected by plant support system and intercropping with long bean ( Vigna sinensis ). Journal of Food, Agriculture & Environment. 6(3&4):305 311. Zahid, N., A. Ali, S. Manisckam, Y. Siddiqui and M. Maqbool. 2012. Potential of chitosan loaded nanoemulsions to control different Collectotrichum spp. and maintain quality of tropical fruits during cold storage. Journal of Applied Microbiology 113:925 939. Zee, F C. R. Yen and M Nishina. 2004 Pitaya (dragon fruit, strawberry pear). Honolulu (HI): University of Hawaii. 3 p. (Fruits and Nuts; FN 9).

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53 BIOGRAPHICAL SKETCH Warren Paul Condon was born in Miami and has been a life long resident. His degrees include a B.A. in h ospitality m anagement from Florida International University (1989), and a B.S in e nvironmental h orticulture from the University of Florida (2009) He has volunteered for the past 15 years at Fairchild Tropical Gardens and the Fruit and Spice Park, and served as Vice President (2001 2005) President (2005 2006) and as a member of the Board of Directors (2001 2012) of the Tropical Fruit and Vegetable Society of the Redland His love of exotic fruit has taken him on forays to the Caribbean, Asia, Australia, and throughout the Americas, and will no doubt continue to do so.