Propagation of Vaccinium arboreum by Stem Cuttings for Use as a Rootstock for Commercial Bluebery Production by Jesica Rose Bowerman A thesis submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degree of Master of Science Auburn, Alabama May 6, 2012 Keywords: sparkleberry, asexual, clonal, auxin, softwood, hardwood Copyright 2012 by Jesica Rose Bowerman Approved by James Spiers, Chair, Asistant Profesor of Horticulture Eugene Blythe, Asistant Profesor of Horticulture Elina Coneva, Asociate Profesor of Horticulture Kenneth Tilt, Profesor of Horticulture ! ! ! ""! Abstract Commercial blueberries, including V. corymbosum and V. ashei, have very specific needs for optimum growth; hence, growing sites are limited. They require acidic soil (pH 4.0-5.5), good drainage, thorough aeration, and a constant moderate amount of moisture. To overcome these restrictions they could be grafted onto V. arboreum, a species adapted to les desirable growing conditions. Currently, V. arboreum plants are commercialy propagated from seds. Succesful asexual propagation techniques wil be necesary for rapid clonal propagation of selected cultivars of V. arboreum. The objective of this experiment was to identify an eficient way to propagate V. arboreum using stem cuttings. We found that IBA quick-dip concentration (0, 1000, 2500, 5000, or 7500 ppm IBA) did not influence rooting percentage of V. arboreum. The factors that influenced rooting the most were the source of the cutting and the cutting type (softwood, semi- hardwood, or hardwood). The greatest rooting succes was observed with softwood cuttings; there was also succes using semi-hardwood cuttings from plants that had been cut back in February 2010 and alowed to sprout new shoots. The results of this experiment can be used to determine the feasibility of using stem cuttings to commercialy propagate V. arboreum. ! """! Acknowledgments The author would like to thank Dr. James Spiers for taking her on as a graduate student (even though she had no experience in horticulture), and guiding her through this proces. She would like to give special thanks to Dr. Eugene Blythe for his help with the statistics and thorough explanations and aid in interpreting the results. She would also like to thank the other members of her commite, Dr. Elina Coneva and Dr. Kenneth Tilt for their help planning and refining her thesis. Additional thanks must be given to Bryan Wilkins, Jonathan Meador, Jonathan Malone, and Michael Harrison for their help collecting cuttings and seting up the experiment. The author would also like to thank Hortus USA Corporation for their generous donation of Hortus IBA Water Soluble Salts ? . Also, she would like to thank Mr. Scott Gomberg and the staf of the Robert Trent Jones Golf Trail at Grand National in Opelika, Alabama for alowing her to borrow golf carts and take cuttings from their property. The author would also like to extend thanks to her parents John Bowerman and Doris Kocher for always insisting that she maintain high quality work in school and encouraging her to perform to her maximum potential. She would also like to thank her future in-laws Brenda and Steve Drake for being loving and supportive of her educational path, even though it pulled their son six hours away! She must also thank her grandfather, Robert Burrows, for sparking her interest in plants and the outdoors at a young age. She has many fond memories of helping him in the garden. ! "#! Finaly, the author would like to thank her life partner, soul mate, best friend, cuppy-cake etc., Alex Drake, for al his love and support through the good times and the frustrating times of this proces. She is grateful that he was beside her these past two years and looks forward to FINALY geting married when they graduate! ! #! Table of Contents Abstract................................................................................................................................ii Acknowledgments..............................................................................................................ii List of Tables......................................................................................................................vi List of Abbreviations.........................................................................................................vii Chapter 1: Literature Review...............................................................................................1 Chapter 2: Propagation of Vaccinium arboreum by Stem Cuttings for Use as a Rootstock for Commercial Blueberry Production...........................................16 ! #"! List of Tables Table 1. Comparison of softwood and hardwood, subterminal and terminal rooting percentages of V. arboreum stem cuttings from two locations............................32 Table 2. Influence of IBA rate on rooting percentage, number of roots and root length of subterminal V. arboreum stem cuttings...........................................................33 Table 3. Influence of IBA rate on shoot number and total shoot length of subterminal V. arboreum stem cuttings....................................................................................34 Table 4. Influence of IBA rate on percent of cuttings with calus and calus caliper of subterminal V. arboreum stem cuttings............................................................35 Table 5. Efect of cutting source and cutting type on percent rooting, number of roots, and root length of subterminal V. arboreum stem cuttings..................................36 Table 6. Efect of cutting source and cutting type on shoot number, shoot length, percent with calus, and calus caliper of subterminal V. arboreum stem cuttings.................................................................................................................37 Table 7. Influence of IBA rate on rooting percentage, number of roots, and total root length of terminal V. arboreum cuttings..............................................................38 Table 8. Influence of IBA rate on number of shoots and shoot length of terminal V. arboreum stem cuttings .......................................................................................39 Table 9. Influence of IBA rate on percent with calus and calus caliper of terminal V. arboreum stem cuttings....................................................................................40 Table 10. Efect of cutting source and cutting type on percent rooting, number of roots and root length of terminal V. arboreum stem cuttings............................41 Table 11. Efect of cutting source and cutting type on shoot number, shoot length, percent with calus, and calus caliper of terminal V. arboreum stem cuttings...............................................................................................................42 Table 12. Correlation betwen rooting and calus presence on V. arboreum stem cuttings...............................................................................................................43 ! #""! List of Abbreviations RTJ Robert Trent Jones Golf Trail at Grand National SCMS Stone County, Misisippi IBA Indole-3-butyric acid NAA Alpha-naphthaleneacetic acid ! "! Chapter I Literature Review Genus Vaccinium The genus Vaccinium is both a large and complicated genus of the Ericaceae family. A large degree of variability can be sen throughout the genus, and some species have complicated, dificult-to-determine evolutionary histories. There are over 140 diferent species of Vaccinium in the Southeastern United States alone, and they can be divided into six unique subgenera (Radford et al., 1968). The first, Oxycoccus, is made up of cranberry species such as V. macrocarpon and V. oxycoccus. The Herpothamnus subgenus is comprised of creeping blueberries such as V. crassifolium and V. sempervirens. Commercial blueberries are located in the subgenus Cyanococcus. There are four main types of commercial blueberries. Lowbush blueberries (V. angustifolium), or wild blueberries, are dwarf, woody, deciduous shrubs found New Hampshire up through Maine and into New Brunswick and Nova Scotia (Trehane, 2004). The northern highbush blueberry (V. corymbosum) is a taler species of shrubby blueberry. They are typicaly found from North Carolina extending north into Canada and as far west as Illinois, Indiana and Michigan (Trehane, 2004). Rabbiteye blueberries (V. ashei) are erect, spreading shrubs and are more adept than northern highbush to growing conditions in the south. They require fewer chiling hours than northern highbush blueberries and are found in the southeastern United States from central Florida to eastern North Carolina ! #! and west to northern Arkansas and eastern Texas (Trehane, 2004). Southern highbush blueberry is a general term for hybrids of two, sometimes three Vaccinium species. They are early ripening, similar to northern highbush, and have a low chiling hour requirement like rabbiteye. In some hybrids, V. darrowii also provides heat and drought resistance (Trehane, 2004). There is only one species of the Oxycoccoides subgenus in the Southeast, which is V. erythrocarpum, or the Southern Mountain Cranberry. Also, there is only one species of the Batodendron subgenus in the Southeast, which is the sparkleberry, or V. arboreum. Finaly, the Polycodium subgenus consists of multiple varieties of the species V. stamineum, or deerberry (Radford et al., 1968). Vaccinium arboreum Vaccinium arboreum has multiple common names including sparkleberry, farkleberry, tree-huckleberry and winter-huckleberry (Balinger et al., 1982). It is the only species of Vaccinium that reaches tree size and can grow as tal as 10 meters (Radford et al., 1968). The breast-height trunk diameter can be as great as 35 cm (Lyrene, 1997). Vaccinium arboreum is a perennial semi-evergreen, and retains its leaves through much or al of the winter (Radford et al., 1968). The foliage is dark green, and in one western variety, V. arboreum var. glaucescens, glaucescent leaves are present. This variety, however, is not considered diferent enough from V. arboreum to be ruled a separate species (Balinger et al., 1982). The root system is made up of coarse roots with a large taproot (Lyrene, 1997). It also has an erect, single trunk growth habit and can grow to resemble a smal tree (Balinger et al., 1982). In addition to those mentioned previously, V. arboreum posseses several traits that would make it a desirable ornamental plant. The leaves are alternate and eliptic, ! $! turning a reddish-purple color in the fal (Radford et al., 1968). Vaccinium arboreum blooms in the late summer and has smal, white flowers organized in an elongated raceme inflorescence (Brooks and Lyrene, 1998). Vaccinium arboreum is a very low- maintenance species due to its drought and soil pH range tolerance. Other atractive ornamental qualities include its exfoliating bark, similar to crapemyrtle (Lagerstroemia indica L.), and the fact that it is semi-evergreen. The fruit are smal, black, shiny (Radford et al., 1968), very dry and leathery (Balinger et al., 1982), and are often described as ?grity and inedible? (Brooks and Lyrene, 1998). The fruit, however, are edible and appreciated by some. Fruit are readily eaten by birds and would therefore be useful for atracting songbirds (Stockton, 1976). Vaccinium arboreum is widespread throughout the Southeast, ranging from southern Virginia south to central Florida, and west to Texas, central Oklahoma and southeast Misouri (Brooks and Lyrene, 1998). It is capable of growing in coarse to medium textured soils with a soil pH tolerance of 4-7 (Radford et al., 1968). In addition, it is the only species of Vaccinium found in mafic or calcareous soils, and it is a shade- tolerant species (Brooks and Lyrene, 1998). Vaccinium arboreum and commercial blueberry species such as V. corymbosum and V. ashei are in two diferent subgenera, and the relationship betwen them is therefore uncertain. If anthocyanins are of taxonomic importance, V. corymbosum and V. ashei are closely related to V. arboreum because the anthocyanins in the fruit are extremely similar. They both posses twelve anthocyanins within their fruit. This suggests that these commercial blueberry species are related to V. arboreum and would therefore have a greater chance of grafting succes (Balinger et al., 1982). Other factors ! %! that influence grafting succes include the environmental conditions following grafting. The temperature can influence the rate of calus growth, with higher temperatures encouraging more rapid calus formation. This only occurs to a certain temperature, when any further increase in temperature may retard calus formation or cause cel death. The temperature tolerance depends on the species being grafted. The graft also must have high humidity for proper formation of calus. If cambium cels are exposed to drying air the cels wil be kiled and a graft union is les likely to form (Hartmann et al., 2002). Curent bluebery propagation Throughout the world, blueberry acreage is expanding rapidly. There has recently been an increase in demand for fresh blueberries available throughout the year (Isutsa et al., 1994). This increased demand has led to the need to quickly propagate elite blueberry cultivars. A common method for propagation is by using stem cuttings. Hardwood, softwood and semihardwood cuttings have al proven to be a reasonably succesful method of propagation, with greater than 50% rooting (Mainland, 2006). Another method of propagation is by tisue culture, which can lead to as many as 95% of propagules rooting (Isutsa et al., 1994). Plants propagated using micropropagation tend to have a bushier growth habit, which alows for more flower buds per plant (Miler et al., 2006). This faciltates more fruit than those propagated by cuttings, with the average berry weight being about the same (El-Shiekh et al., 1996). Although this sounds ideal, there are drawbacks to using micropropagation. First, seting up and maintaining a micropropagation facility is extremely expensive and time-consuming. The increased rooting succes may not outweigh the cost of setup. Also, in order to have succes with micropropagation, the workers must be skiled in working in vitro (Miler et al., 2006). ! &! Finaly, even though micropropagation leads to a bushier plant that produces more fruit, a larger number of fruit would be lost if they are mechanicaly harvested. Mechanical harvesting involves a machine grasping the base of the plant and shaking to remove the berries, which are caught below the plant. A plant with a large number of low branches increases the chance of blueberries mising the collection platform and faling to the ground (Miler et al., 2006). Tisue culture is a succesful way to propagate blueberries, but there are drawbacks to using this method. An increase in demand for blueberries also increases the demand for suitable growing sites. Because of V. corymbosum?s very specific needs, there is a lack of space with the required qualities available for expansion (Balington et al., 1990). Highbush blueberries require acidic soil, in the range of pH 4.0-5.5, with a high amount of organic mater, Fe, and N in NH 4 + form (Darnel and His, 2006). They also need soil with good drainage, ample aeration, and a relatively consistent moderate amount of moisture (Balinger et al, 1982). V. corymbosum has a fibrous, shalow root system that is sensitive to drought and wind damage (Lyrene, 1997). Since it is limited to such specific growing habitats, the increase in blueberry production is limited unles a way to overcome some of these factors is found. Breding with V. arboreum There are many traits of V. arboreum that would be ideal to have in commercial blueberry species, and V. arboreum could be used as a gene source to incorporate these traits. V. arboreum flower late in the season, which would reduce the risk of crop loss due to spring frosts. Berries of V. arboreum ripen late in the growing season, from September to December. V. arboreum also grows in areas that are not desirable for commercial ! '! blueberries (Wenslaf and Lyrene, 2003). There are also traits of V. arboreum that would not be desirable in commercial blueberries. The berries are dark and shiny, and are barely palatable. They have a grity texture due to large seds and a low juice content. Also, the berry size is quite smal, usualy smaler than those of commercial blueberry species (Lyrene, 1997). Although it may be beneficial to have some of these traits in commercial blueberries, there are also drawbacks. V. arboreum as a rootstock One way to help increase the culture of V. corymbosum is to graft onto a plant more suited to les desirable growing conditions. A potential rootstock would be V. arboreum, which has the ability to grow in many areas that would be unsuitable for commercial blueberries. First, V. arboreum has a coarse root system with a long tap root (Lyrene, 1997). Because of this, it is a very drought-resistant species and is able to grow in areas dryer than V. corymbosum can tolerate (Balinger et al., 1982). Next, V. arboreum is one of the few Vaccinium species that can grow in calcareous soils, meaning they can survive in conditions with a higher soil pH. Vaccinium corymbosum has a very limited soil pH range that it can tolerate, about 4.0-5.5 (Darnel and His, 2006), but V. arboreum can tolerate soil pH from 4-7 (Radford et al., 1968). Also, V. arboreum has a greater capacity to survive in soils that have NO 3 - as the prominent form of nitrogen in the soil (Darnel and His, 2006). It can also tolerate soils that have limited quantities of iron, a condition that is not ideal for V. corymbosum. V. arboreum is also more eficient in acquiring nitrate than V. corymbosum (Poonnachit and Darnel, 2004). Finaly, V. arboreum has an erect growth habit consisting of a single trunk, which would minimize fruit loss when mechanical harvesting techniques are used (Balington et al., 1990). ! (! Overal, there are many advantages to using V. arboreum as a rootstock for V. corymbosum to aid in expanding the blueberry industry to met higher demands. V. corymbosum has been succesfully grafted onto V. arboreum, which shows that there is a genetic afinity and that grafting is possible (Galeta and Fish, 1971). V. ashei has also been succesfully grafted onto V. arboreum (Balington et al., 1990). Several techniques have proven succesful; these include early spring cleft, whip or side grafts, and late summer t-budding (Balington et al., 1990). Although grafting V. corymbosum or V. ashei onto V. arboreum appears to be a great idea, there are drawbacks. First, it is costly and the inconsistency of results may make grafted blueberry plants economicaly unfeasible (Balington et al., 1990). There is also a limited availability of suitable stock material, and propagating V. arboreum is a dificult task. In order to take advantage of the positive improvements that grafted blueberry plants would bring, a commercialy aceptable way of propagating V. arboreum is needed. Propagation V. corymbosum and V. ashei There are several succesful methods of propagating V. corymbosum and V. ashei that could potentialy be used to propagate V. arboreum. Cuttings- specificaly hardwood and softwood- are the most widely used method of propagation in the commercial industry (Mainland, 1993; Miller et al., 2006). Miler et al. (2006) found that southern highbush (V. corymbosum) and rabbiteye (V. ashei) blueberries had the greatest rooting succes when using softwood cuttings, and northern highbush had a lower rooting percentage but was stil succesful. Both softwood and hardwood cuttings taken from the terminal or middle position on the branch had higher rooting percentages than cuttings ! )! taken from the basal portion of the branch. While softwood cuttings usualy have a higher rooting percentage, many propagators prefer to use hardwood cuttings because propagation can be done in the slower, dormant season and cuttings can be kept in a cooler until an appropriate time for sticking (Mainland, 1993). The traditional media used for cuttings is sphagnum peat, either alone or with varying proportions of sand or perlite (Pokorny and Austin, 1982; Shelton and Moore, 1981), but there are also alternatives to using peat that may save money. Pokorny and Austin (1982), for example, found that miled pine bark could be used as an alternative to sphagnum peat and actualy increased the percentage of rooting and root quality in V. ashei. Another method of increasing rooting succes is to remove leaves from the bottom half of the cutting when working with softwood cuttings. This increased the percentage of cuttings that rooted in V. corymbosum (Mainland, 1993). Micropropagation is another method of propagating commercial blueberry plants, but it is seldom used due to the cost and the increase in genetic variance (Mainland, 1993; Miler et al., 2006). V. arboreum The current method for propagating V. arboreum is using seds. They are more dificult to germinate than commercial blueberry species from section Cyanococcus (Lyrene and Brooks, 1995). When using methods similar to those used for germinating Cyanococcus species (placed uncovered in sphagnum peat in an unheated greenhouse and watered with intermitent mist for 3 hours a day for 2 months), sparkleberry sedlings grew poorly (Lyrene and Brooks, 1995). One method for increasing germination succes was to soak the seds overnight in a gibberelic acid solution (4g per liter of water) (Lyrene and Brooks, 1995). When propagating V. arboreum to use as a rootstock, seds ! *! would not be ideal due to the genetic variance of each plant. An asexual propagation method would be needed to propagate specimens with ideal qualities to serve as a rootstock. To date, there has been litle succes in propagating V. arboreum. There has also been very litle research on the subject. Reese (1992) used semihardwood cuttings to try to propagate V. arboreum. Cuttings were taken from plants growing in their native habitat. The basal end of each cutting was cut at a slant to expose more cambium tisue. Several treatments were used including quick dips of various concentrations of IBA+NA, a 24 hour soak in wilow water, a 24 hour soak in water, wilow water plus Hormodin III, water plus Hormodin III, only Hormodin III, a five-second quick dip in 95% ethanol, and a control (water). A 1:1 (volume) miled pine bark:perlite substrate was used in rhizotrons so that rooting could be observed. Cuttings were placed under intermitent mist for ten seconds every five minutes. After three months, data was taken on the rooting. The Hormodin III had the highest rooting percentage at 12.5% and the control (water) had the lowest rooting percentage with 0%. Al of the treatments were statisticaly similar, and it was concluded that this would not be a commercialy feasible way to propagate V. arboreum (Rese, 1992). A second experiment was performed using hardwood cuttings. A similar set up was used, but the treatments were diferent. They included Hormodin III, mechanical wounding, and Hormodin III in addition to mechanical wounding, plus various combinations of IBA+NA and wounding. When the cuttings were examined for rooting, only two in the entire experiment had rooted (Reese, 1992). This is also not a commercialy feasible way to propagate V. arboreum. ! "+! Stockton (1976) tried to propagate V. arboreum using softwood stem cuttings. Four diferent concentrations of K-IBA disolved in water were used (0, 10000, 15000, 20000) for a quick dip. A 2:2:1 (volume) peat:perlite:sand substrate was used. The cuttings were placed under intermitent mist for 8 seconds every 10 minutes during daylight hours. After 60 days, the cuttings were checked for rooting. Minimal to no succes was sen in al of the treatments (Stockton, 1976), and therefore this is not an eficient way to root V. arboreum. Stockton (1976) also tried to achieve rooting using rhizome cuttings. The rhizomes were taken later in the year, from July until November. The caliper of the rhizomes varied from 0.5 to 3 cm in diameter and 10-30 cm in length. The rhizomes were placed verticaly with either the distal end up or the proximal end up and were placed at least 3 cm below the surface of the substrate. There was some succes rooting, but this would not be a feasible way to propagate V. arboreum because the succes rate is not high enough to make it worthwhile; furthermore, harvesting rhizomes results in the death of the stock plant (Stockton, 1976). Deciduous azaleas and other hard to root species Since propagating V. arboreum has been dificult, a review of rooting techniques for another hard to root species may be helpful to gain insight on how to root V. arboreum. One group of plants to consider would be deciduous azaleas, which are also in the Ericaceae family and are acid-loving plants. The succes rate of azalea propagation by stem cuttings depends on many factors such as the time of year, the specific cultivar, substrate, irrigation levels, bottom heat, and amount of light. Knuttel and Addison (1984) used ?Royal Lodge?, ?Visco Sepala?, ! ""! ?Sunset Boulevard?, ?Satan?, ?Crimson Tide?, and ?Pink Jolly? stock plants that were kept in a controlled temperature overwintering structure. This way, the temperature can be gradualy raised to alow the plants to come out of dormancy early and cuttings, therefore, can be taken earlier in the year, around April-May (Knuttel and Addison, 1984). Young, herbaceous growth that is slightly firm was considered ideal cutting material (Knuttel and Addison, 1984). Cuttings should be slightly hairy and fel like they are about to snap when bent double (Nienhuys, 1980). Various concentrations of IBA and NA, depending on what is recommended for the specific cultivar, can also be used to achieve a higher rooting percentage. Another way to aid in rooting is to provide bottom heat at 21.1-22.8?C to keep the medium warm (Knuttel, 1984; Mylin, 1982; Nienhuys, 1980). Light intensity is another factor that influences the rooting succes of azalea cuttings (Read and Economou, 1983); an increase in rooting percentage was observed as light intensity decreased. Cuttings rooted with lower intensity light (10 ?Em -2 s -1 ) had a rooting percentage of 88.3%, compared to 65.8% when using high intensity light (75 ?Em -2 s -1 ) (Read and Economou, 1983). Using these techniques, alone or together, can result in higher rooting percentages for deciduous azalea cuttings. Another hard-to-root, Ericaceous species is Kalmia latifolia. Wiliams and Bilderback (1980) used K. latifolia cuttings taken in September, October, and November. Hormone treatments included 0.1% fenoprop + talc, 1.0% K-IBA + Benomyle + talc, and a 0.5% K-IBA 10 s quick dip. Cuttings remained in a 1:1 peat:perlite substrate for 165 days. The month the cuttings were taken influenced the rooting percentage, with the cuttings in September having a significantly higher rooting percentage than those taken in October and November, which had statisticaly similar rooting percentages in al three ! "#! treatments. The highest rooting percentage was observed in cuttings taken in September and treated with 0.1% fenoprop + talc with 55%. Rooting percentages observed overal ranged from 12-55% (Wiliams and Bilderback, 1980), which is not feasible for commercial production. Plant Hormones and Plant Growth Regulators Plant hormones and plant growth regulators (PGRs) are used in propagation of many species. A plant hormone is a naturaly occurring chemical that is synthesized within the plant and is involved in the growth and development of that plant. The five major plant hormones are auxins, cytokinins, gibberelins, abscisic acid, and ethylene. In addition to naturaly occurring hormones, other chemicals, both naturaly occurring and synthetic, can induce a response in a plant. These chemicals are grouped together and are known as PGRs (Hartmann et. al., 2002). Auxin is the most widely used plant hormone for the induction of adventitious roots in cuttings. It is naturaly produced in leaf primordial, young leaves, and developing seds in the form of the chemical indole-3-acetic acid (IAA). Auxin is important in the phenomenon of apical dominance by inhibiting lateral bud break. Another naturaly occurring form of auxin is indole-3-butyric acid (IBA). There are also several synthetic forms of auxin, including indole-3-butyric acid-potasium salt (K-IBA). Usualy, IBA is found in salt form, which is water-soluble; otherwise it is only soluble in alcohol, which could burn sensitive cuttings. Other synthetic forms of IBA include alpha- naphthaleneacetic acid (NA), 2,4-dichloro-phenoxy-acetic acid (2,4D), and 2,4,5-tri- chloro-phenoxy-acetic acid (2,4,5T) (Hartmann et. al., 2002). By using an auxin application the rooting succes may be increased. ! "$! Conclusion The alure of the positive changes that using V. arboreum as a rootstock could bring is the driving force behind further investigating methods of propagating V. arboreum. In addition, V. arboreum could also be used as an ornamental plant because of its atractive bark (similar to crapemyrtle), atractive fal foliage colors, semi-evergreen to evergreen habit, fruit desirable to birds, and tree-like growth habit. There are still a number of factors that could be explored, including semihardwood cuttings, rooting substrate, diferent hormone treatments or wounding, and other environmental factors such as the amount of light and moisture, that justify continuing forward with research on productive and commercialy useful methods of propagating V. arboreum. ! ! "%! Literature Cited Balinger, W. E., E. P. Manes, and J. R. Balington. 1982. Anthocyanins in ripe fruit of the sparkleberry, Vaccinium arboreum MARSH. Can. J. Plant Sci. 62:683-687. Balington, J. R., B. W. Foushee, and F. Wiliams-Rutkosky. 1990. Potential of chip- budding, stub-grafting or hot-calusing following saddle-grafting on the production of grafted blueberry plants. Proc. N. Amer. Blueberry Res.-Ext. Workers Conf. 114-120. Brooks, S. J., and P. M. Lyrene. 1998. Derivatives of Vaccinium arboreum ! Vaccinium Section Cyanococcus: I. Morphological Characteristics. J. Amer. Soc. Hort. Sci. 123:273-277. Darnel, R. L., and S. A. His. 2006. Uptake and asimilation of nitrate and iron in two Vaccinium species as afected by external nitrate concentration. J. Amer. Soc. Hort. Sci. 131:5-10. El-Shiekh, A., D. K. Wildung, J. J. Luby, K. L. Sargent, and P. E. Read. 1996. Long- term efects of propagation by tisue culture or softwood single-node cuttings on growth habit, yield, and berry weight of ?Northblue? blueberry. J. Amer. Soc. Hort. Sci. 121:339-342. Galeta, G. J., and A. S. Fish. 1971. Interspecific blueberry grafting, a way to extend Vaccinium culture to diferent soils. J. Amer. Soc. Hort. Sci. 96:294-298. Hartmann, H. T., D. E. Kester, F. T. Davies, Jr., and R. L. Geneve. 2002. Hartmann and Kester?s Plant propagation: principles and practices. 7th ed. Prentice Hal, Englewood Clifs, N.J. Isutsa, D. K., M. P. Prits, and K. W. Mudge. 1994. Rapid propagation of blueberry plants using ex vitro rooting and controlled aclimatization of micropropagules. HortScience. 29:1124-1126. Knuttel, A. J., and C. Addison. 1984. Deciduous azalea propagation: an overview of old and new techniques. Comb. Proc. Intl. Plant Prop. Soc. 34:517-520. Lyrene, P. M. 1997. Value of various taxa in breeding tetraploid blueberries in Florida. Euphytica. 94:15-22. Lyrene, P. M. and S. J. Brooks. 1995. Use of sparkleberry in breeding highbush blueberry cultivars. J. of Smal Fruit and Viticult. 3:29-38. Mainland, C. M. 1993. Efects of media, growth stage and removal of lower leaves on rooting of highbush, southern highbush and rabbiteye softwood or hardwood cuttings. Acta Hort. 346:133-140. ! "&! Mainland, C. M. 2006. Propagation of Blueberries, p. 49-55. In: N. F. Childers and P. M. Lyrene (eds.). Blueberries: for growers, gardeners, promoters. E. O. Painter Printing Company, Inc., DeLeon Springs, Fla. Miler, S., E. Rawnsley, J. George, and N. Patel. 2006. A comparison of blueberry propagation techniques used in New Zealand. Acta Hort. 715:397-401. Mylin, D. 1982. Propagation of deciduous azaleas. Comb. Proc. Intl. Plant Prop. Soc. 32:418-420. Nienhuys, H. C. 1980. Propagation of deciduous azaleas. Proc. Inter. Plant Prop. Soc. 30:457-459. Pokorny, F. A., and M. E. Austin. 1982. Propagation of blueberry by softwood terminal cuttings in pine bark and peat media. HortScience. 17:640-642. Poonnachit, U., and R. Darnel. 2004. Efect of amonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species. Ann. Bot. 93:399-404. Radford, A. E., H. E. Ahles, and C. R. Bel. 1968. Manual of the vascular flora of the Carolinas. The University of North Carolina Pres, Chapel Hil, NC. Read, P. E., and A. S. Economou. 1983. Supplemental lighting in the propagation of deciduous azaleas. Comb. Proc. Intl. Plant Prop. Soc. 32:639-645. Reese, J. C. 1992. Propagation of Farkleberry (Vaccinium arboreum) for use as a blueberry rootstock. Mis. State Univ., Starkvile, M. S. Thesis. Shelton, L. L., and J. N. Moore. 1981. Highbush blueberry propagation under southern U.S. climatic conditions. HortScience. 16:320-321. Stockton, L. A. 1976. Propagation and autoecology of Vaccinium arboreum and its graft compatibility with Vaccinium ashei. Texas A&M Univ., College Station, M.S. thesis. ,-./01.2!34!#+%4!567.8.-9.:2!;-018.-9.:!01/.-!?0@9197A:4!,9A8.-!B-.::2!! BC->601<2!=D!01.-06!@-C:!@CAN0>98969>M!91!"#$%$&'( )**$+,$$(!-("./+.)&'2!01!91>.-:.@>9C106!867.8.--M!/M8-9<4!O7N/M>9@04!"$"P#&&Q! #&)4! ! H9690A:2!D4!J42!01C-:!0II.@>91E!-CC>91E!CI!01+2+2)%2.+%( '"3$'&'(01.A!@7>91E:4!SC->:@9.1@.4!"&P)#(Q)#)4! ! ! "'! Chapter II Propagation of Vaccinium arboreum by Stem Cuttings for Use as a Rootstock for Commercial Bluebery Production Introduction In recent years, there has been an increase in consumer demand for fresh blueberries throughout the year, which also increases the demand for sites suitable for growing blueberries. Commercial blueberries, including highbush (Vaccinium corymbosum L.) and rabbiteye (Vaccinium ashei Reade), have specific requirements for optimal growth. As part of the Ericaceae family, commercial blueberries favor acidic soil, in the range of pH 4.0-5.5 (Trehane, 2004). Blueberries also need high amounts of organic mater within the soil, as wel as iron and nitrogen in the NH 4 + form (Darnel and His, 2006). Other soil characteristics include good drainage, aeration, and a relatively consistent moderate moisture content (Balinger et al., 1982). In addition to having specific growing needs, commercial blueberries also have fibrous, shalow root systems that are sensitive to drought and wind damage (Lyrene, 1997). Because of al these limitations, suitable growing sites are in short supply unles the soil is adapted using costly amendments. One way to overcome a poor growing environment is to use a rootstock that has the capability to grow where others cannot. A potential rootstock for commercial blueberries is the sparkleberry (Vaccinium arboreum Marsh), which has many desirable qualities that give it the ability to grow in many areas that would be unsuitable for ! "(! commercial blueberries. V. arboreum is one of the few Vaccinium species that can tolerate calcareous soils, meaning they survive in conditions with higher soil pH levels, from pH 4 to pH 7 (Radford et al., 1968). V. arboreum is also able to grow in conditions where the prominent form of nitrogen in the soil is nitrate (NO 3 - ) (Darnel and His, 2006). It can tolerate soils with limited quantities of iron and is more eficient at acquiring nitrate than commercial blueberries (Poonnachit and Darnel, 2004). V. arboreum has a coarse root system with a long taproot (Lyrene, 1997), making it a very drought-resistant species and les likely to be uprooted due to wind (Balinger et al., 1982). V. arboreum can grow wel in soils with les than 2% organic mater (Lyrene, 1998). Finaly, V. arboreum has an erect growth habit consisting of a single trunk that would minimize fruit loss when using mechanical harvesting techniques (Balington et al., 1990). With the increased demand for blueberries as a healthy snack, V. arboreum could be used to increase blueberry production to met the growing demands. In the past, V. corymbosum has been succesfully grafted onto V. arboreum, which shows a genetic afinity, therefore making grafting possible (Galeta and Fish, 1971). V. ashei has also been succesfully grafted onto V. arboreum (Balington et al., 1990), again showing a genetic afinity. Techniques that have proven succesful include early spring cleft, whip or side grafts, and late summer t-budding (Balington et al., 1990). One problem encountered was the production of suckers from the rootstock; these increased with the age of the plant (Eck, 1988). The current method for propagating V. arboreum is using seds. They are more dificult to germinate than commercial blueberry species from section Cyanococcus (Lyrene and Brooks, 1995). When using methods similar to those used for germinating ! ")! Cyanococcus species (placed uncovered in sphagnum peat in an unheated greenhouse and watered with intermitent mist for three hours a day for two months), sparkleberry sedlings grew poorly (Lyrene and Brooks, 1995). One method for increasing germination succes was to soak the seds overnight in a gibberelic acid solution (4g per liter of water) (Lyrene and Brooks, 1995). When propagating V. arboreum to use as a rootstock, seds would not be ideal due to the genetic variance of each plant. An asexual propagation method would be needed to propagate specimens with ideal qualities to serve as a rootstock. To use V. arboreum succesfully as a rootstock, protocols for clonal propagation of the species in large quantities are needed. To date, there has been litle research on the propagation of V. arboreum. Stockton (1976) tried to propagate V. arboreum using softwood stem cuttings and K-IBA quick-dips. Four diferent concentrations of K-IBA disolved in water were used (0, 10000, 15000, and 20000 ppm). A 2:2:1 (volume) peat:perlite:sand substrate was used. Cuttings were placed under intermitent mist for 8 s every 10 m during daylight hours. After 60 days, cuttings were checked for rooting and minimal to no succes was observed in al of the treatments (Stockton, 1976). Reese (1992) used semihardwood stem cuttings and diferent treatments to try to enhance rooting, including various levels of IBA+NA, wilow water, and Hormodin III. Cuttings were stuck in a 1:1 pine bark:peat substrate and placed in a rhizotron under intermitent mist. Similar to the previous study, litle rooting was observed, with only a 0-12.5% rooting percentage recorded among the treatments. The control treatment had 0% rooting and al of the treatments were statisticaly similar, suggesting that none of the treatments influenced rooting succes (Reese, 1992). A subsequent study using hardwood cuttings ! "*! and diferent combinations of wounding and hormones resulted in only two rooted cuttings for the entire experiment. Hence, previous research suggests V. arboreum is a very hard-to-root species, with no indication of viable treatments to enhance rooting of stem cuttings. In addition to stem cuttings, rhizome cuttings have been evaluated (Stockton, 1976). Rhizome sections were taken from July until November. The caliper of the rhizomes varied from 0.5-3 cm in diameter and 10-30 cm in length. Rhizomes were placed verticaly with either the distal end up or the proximal end up and at least 3 cm below the surface of the substrate. Though percent rooting was not reported, some root formation did occur. Stockton (1976) concluded that this would not be an ideal way to propagate V. arboreum because of the low succes rate and the likelihood of harvesting rhizomes resulting in the death of the stock plant. Determining a viable way to propagate V. arboreum would benefit commercial blueberry production as a potential rootstock, as wel as the selecting and marketing of V. arboreum as a landscape plant. Partly due to the dificulty of propagation, V. arboreum is seldom marketed as a landscape plant. However, V. arboreum can grow to be an aestheticaly pleasing semi-evergreen smal tree, with atractive fal color, exfoliating bark, and edible fruit. Since V. arboreum tolerates drought and a range of soil types, it is a good selection as an atractive woodland shrub/smal tree for xeriscaping and native plant landscaping. A viable way to clonaly propagate V. arboreum is necesary to alow for selection of plants with desirable ornamental and rootstock characteristics. The objectives of this study were to determine whether cutting type (softwood, semihardwood, or hardwood), cutting position (terminal or subterminal), IBA ! #+! concentration, or the interaction of these treatments influence rooting of V. arboreum stem cuttings. Previous experiments did not specify if the cuttings were taken from juvenile or mature wood. Only juvenile wood was used in this study, as juvenile wood is typicaly easier to root than mature wood for most species (Hartmann et al., 2002). Materials and Methods Cutting propagation material of V. arboreum was collected from two locations. Water sprouts from native, mature plants were collected from the Robert Trent Jones Golf Trail at Grand National (RTJ) in Opelika, Alabama (lat. 32?69?N, long. 85?44?W, USDA hardines zone 8a). Juvenile cuttings arising from latent buds on mature plants that had been cut back to approximately 1 m in height in February 2010 were collected from Stone County, Misisippi (SCMS) (lat. 30?80?N, long. 89?17?W, USDA hardines zone 8b). Softwood, semihardwood, and hardwood cuttings were collected, as wel as subterminal and terminal cuttings. The softwood cuttings were lignified enough to stay upright when stems were stuck in substrate, but stil flexible. Softwood cuttings from SCMS were taken the same day as semihardwood cuttings. The terminal cutting and that imediately below were used as softwood because they were stil mostly flexible. The more lignified basal cuttings, at least 30 cm from the terminal end, were used for semihardwood cuttings.. Similar softwood and semihardwood cuttings were collected from RTJ, but the semihardwood cuttings were taken 47 days after softwood cuttings were collected. Hardwood cuttings were collected while the plants were dormant, before bud break. Cuttings were trimed to 10-12 cm long. Caliper of the softwood cuttings from RTJ and SCMS averaged 2.96 and 3.81 cm, respectively. Caliper of the semihardwood ! #"! cuttings from RTJ and SCMS averaged 2.99 and 3.25 cm, respectively. Caliper of the hardwood cuttings from RTJ and SCMS averaged 2.82 and 3.07, respectively. Auxin solutions were prepared using Hortus IBA Water Soluble Salts? (Hortus USA Corp.) and deionized water. The basal end of each cutting was cut at a 45? angle and received a 10-s basal quick-dip to a depth of 3 cm in either water (control) or a solution of 1000, 2500, 5000, or 7500 ppm IBA. Cuttings were then inserted to a depth of 3 cm into a cel in a 48-cel tray (Landmark Plastic Corporation, C-T1240, cel dimensions 5.7cm!3.7cm!6.6cm). A 1:1 peat:perlite substrate was used. Hardwood cuttings from RTJ were taken and inserted on March 1, 2011. Hardwood cuttings from SCMS were taken on March 6, 2011 and inserted on March 8, 2011. Softwood cuttings from RTJ were collected and inserted on May 20, 2011. Softwood cuttings from SCMS were collected on June 19, 2011 and inserted on June 20, 2011. Semihardwood cuttings from RTJ were collected and inserted on July 6, 2011. Semihardwood cuttings from SCMS were collected on June 19, 2011 and inserted on June 21, 2011. After they were inserted, the cuttings were placed on a greenhouse bench in a 1.2 m wide by 2.4 m long by 0.9 m tal polyethylene covered enclosure at the Paterson Greenhouse Complex at Auburn University to ensure the relative humidity stayed at an appropriate level. Overhead mist was provided within the enclosure for 2 sec every 10 min. Data was collected for RTJ and SCMS hardwood cuttings on August 19, 2011. Data was collected for RTJ softwood on September 15, 2011. Data was collected for SCMS softwood on October 14, 2011. Data for RTJ and SCMS semihardwood was collected on November 11, 2011. After collecting initial data, cuttings that had formed ! ##! calus and not roots were re-inserted and checked again four weks later for rooting. Only 3 cuttings out of al the cuttings with calus but no roots formed roots in that time period. A completely randomized design was used with 30 cuttings (replications) per treatment. Rooting response (rooted or unrooted) was recorded for al cuttings, with a cutting considered rooted when any sign of adventitious roots were sen emerging from the stem. Additional data collected include number of primary roots emerging from the stem of each rooted cutting, total length of primary roots on each rooted cutting, number of rooted cuttings with new shoots, total shoot length on each rooted cutting, number of cuttings that formed calus, and calus caliper of cuttings with calus. The treatment design was a 2 ! 2 ! 3 ! 5 complete factorial design with four factors: 1) source (water sprouts from mature plants or sprouts arising from latent buds on cut back plants), 2) cutting position on the stock plant (terminal and subterminal), 3) cutting maturity (softwood, semihardwood and hardwood) and 4) IBA rate (0, 1000, 2500, 5000, and 7500 ppm), for a total of 60 treatment combinations. The experimental design was a completely randomized design. Data were analyzed using generalized linear models with the GLIMMIX procedure of SAS (version 9.2; SAS Institute Inc., Cary, NC). Rooting was analyzed using the binomial distribution and a logit link function, count data were analyzed using the Poison distribution and a log link function, and measurement data were analyzed using the normal distribution and the identity function. Comparisons of least squares means were conducted using the Schafer-Simulated adjustment for multiple comparisons. Correlations betwen calus and rooting were run using the Pearson Correlation test. ! #$! Results In al the results, source refers to whether the cuttings came from water sprouts of mature, wild plants from RTJ, or from wild plants that had been cut back and alowed to sprout shoots from latent buds in SCMS. Type refers to whether the cutting is softwood, semihardwood, or hardwood. Position refers to whether the cutting is subterminal or terminal. IBA refers to the 10 s IBA quick-dip rate (ppm) to which the cutting was subjected. In the original model, only softwood and hardwood cuttings were used. This was due to the fact that semihardwood, terminal cuttings were unavailable at one of the sources. Using only the softwood and hardwood cuttings alows for a 4-factor analysis. After sequentialy removing nonsignificant four-way and three-way interactions, the only three-way interaction term that was significant was source!type!position; therefore, the way that source and type afect a cutting could vary depending on the position. Rooting percentages of subterminal cuttings were similar for softwood cuttings from both RTJ and SCMS (38.5% and 34.3%), but terminal softwood cuttings from SCMS had a lower rooting percentage (29.2%) than RTJ cuttings (43.3%) (Table 1). For hardwood cuttings, rooting percentages were statisticaly similar when using terminal cuttings, but when using subterminal cuttings RTJ cuttings had a higher rooting percentage than SCMS cuttings (Table 1). Because the efects of source and type could vary due to the position of the cutting, separate analyses were run for subterminal and terminal cuttings. Subterminal cuttings There were no significant efects of IBA rate on rooting percentage (Table 2) or any of the parameters (root number, root length, shoot number, shoot length, calus ! #%! presence, and calus caliper) measured on subterminal cuttings (Tables 2-4). The source and type of cutting did influence the rooting percentage. The highest rooting percentage occurred when using softwood cuttings from RTJ with a rooting percentage of 38.6%. Similar rooting was observed in SCMS softwood and semihardwood with rooting percentages of 34.6% and 28.5% respectively (Table 5). None of the treatments significantly influenced the number of primary roots per rooted cutting (Table 5). Type of cutting influenced the length of primary roots. The longest roots were found on SCMS semihardwood cuttings with an average total root length of 23.3 cm (Table 5). SCMS softwood and hardwood and RTJ semihardwood cuttings had statisticaly similar root lengths, with average total root lengths of 18.3, 17.0, and 17.1 cm respectively (Table 5). The source and type of cutting both influenced the number of new shoots on rooted cuttings (Table 6). Hardwood cuttings from both SCMS and RTJ had more new shoots, 1.5 and 2.4 respectively, than softwood and semihardwood cuttings from either source (Table 6). The type of cutting and the source!type interaction both significantly influenced the length of new shoots. The longest shoots were observed on the RTJ hardwood cuttings with an average total combined shoot length of 5.3 cm (Table 6). Source, type, and source!type interaction al significantly influenced the percent of cuttings that formed calus. The RTJ softwood cuttings had the highest percent of cuttings with calus formation with 82.4% (Table 6). Cutting source and type also influenced the calus caliper. RTJ hardwood cuttings had the greatest calus caliper with an average diameter of 7.1 m. Statisticaly similar calipers were observed in RTJ softwood and SCMS hardwood cuttings with average diameters of 6.5 and 6.8 m, respectively (Table 6). ! #&! Terminal cuttings There were no significant efects of IBA rate on rooting percentage (Table 7) or any of the parameters measured on terminal cuttings (Table 7-9). The source and type of cutting both significantly influenced rooting percentage. The highest rooting percentage was observed on the RTJ softwood cuttings with 43.3% rooting. Softwood cuttings from SCMS had 29.2% rooting, which was greater than hardwood cuttings from both sources (2.0%) (Table 10). None of the treatments influenced the number of primary roots on rooted cuttings or the total combined length of primary roots (Table 10). Shoot number was significantly influenced by the source and type of cutting. Softwood and hardwood cuttings from both RTJ and SCMS al had les than one shoot per cutting (Table 11). None of the factors significantly influenced shoot length (Table 11). The source and type of cutting significantly influenced the percent of cuttings that formed calus. The highest percentage of cuttings with calus was observed in the softwood cuttings from both RTJ and SCMS with 73.4% and 33.9% of cuttings forming calus, respectively (Table 11). The calus caliper was not significantly influenced by any of the treatments (Table 11). Correlations There were no strong correlations betwen calus and rooting when the data were sorted by IBA treatment rate alone (Table 12). The data were also sorted by IBA rate and position, IBA rate and source, and IBA rate and cutting type. The strongest correlation betwen rooting and calus was sen in the cuttings from SCMS treated with 7500ppm ! #'! IBA. The correlation coeficient was 0.88. Overal the correlation coeficients ranged from 0.24 to 0.88, with most betwen 0.40 and 0.69 (Table 12). Discusion The IBA rate did not have a significant influence on the rooting percentage of any of the cuttings. This conclusion was also observed in previous research by Stockton (1976) with only IBA quick-dips. Reese (1992) found that combinations of IBA+NA quick-dips also did not influence the rooting percentage, nor did treatments with Hormodin III. Some other Ericaceous plants, such as Leucothoe racemeosa ?Rainbow?, are also are not afected by IBA rate (Scagel, 2005). No diference in rooting percentage was observed when treating cuttings to a 5 s quick dip of control water versus a 5 s quick dip in a 1.03% IBA, 0.66% NA solution (Scagel, 2005). The fact that IBA rate did not afect rooting percentage of V. arboreum may be a characteristic of Ericaceous species. Based on the statistical analysis, position (subterminal or terminal) alone did not influence rooting percentage. However, the source!type!position interaction term was significant, meaning that the efects of source and type on the rooting of a cutting could change based on the position of that cutting. For example, subterminal softwood cuttings from RTJ and SCMS had statisticaly similar rooting percentages, but terminal softwood cuttings from SCMS did not root as wel as those from RTJ. Similarly, terminal hardwood cuttings from RTJ and SCMS had statisticaly similar rooting percentages, but subterminal hardwood cuttings from RTJ had a higher rooting percentage than those from SCMS (Table 1). As previously mentioned, this was also the basis for running separate analyses on subterminal and terminal cuttings. ! #(! Type of cutting greatly afected the rooting succes of V. arboreum, with softwood cuttings rooting more readily than hardwood cuttings. In previous research there was virtualy no succes rooting V. arboreum using softwood, semihardwood or hardwood cuttings (Reese, 1992; Stockton, 1976). For this experiment, the greatest rooting percentage occurred using softwood cuttings. The percent rooting of softwood cuttings ranged from 29.2-43.2%, which is a great improvement compared to any previous research. The source (RTJ or SCMS) of the cutting influenced the rooting percentage of semihardwood cuttings. Cuttings from SCMS had a similar rooting percentage to softwood cuttings, and the cuttings from RTJ had a low rooting percentage similar to hardwood cuttings (Table 5). This may be due to the quality of the cuttings obtained. The greater number of sprouts from the plants that had been cut back at the SCMS location alowed us to be more selective, and the cuttings may have been closer to softwood cuttings than the cuttings from RTJ. The cutting material from RTJ was limited, forcing the use of some les than ideal cuttings, and the cuttings had a wider range of lignification. As in previous studies, the hardwood cuttings had very litle succes. Hardwood cuttings for many species are generaly les succesful than softwood and semihardwood cuttings (Hartmann et al., 2002). Other factors important in a rooted cutting include the number or primary roots. In this experiment there were no factors that significantly influenced the number of primary roots that a rooted cutting produced. In nearly al of the combinations looked at, there were no strong correlations betwen the formation of calus and the formation of roots. The highest correlation ! #)! coeficient was observed in SCMS cuttings treated with 7500 ppm IBA with a coeficient of 0.88 (Table 12). This could suggest that this treatment combination produces the most cuttings with calus and roots. Even though the correlation is strong, however, it does not confirm that the increase in calus directly caused the increase in rooting observed. There may be other factors that influence this relationship and played a part in the increased calus and rooting. Overal, most of the correlation coeficients were betwen 0.40 and 0.69 (Table 12). Al of the coeficients were positive, meaning that calus and rooting increase as the other increases. Again, this does not mean that one necesarily directly influenced the other. Since virtualy no rooting succes of V. arboreum was observed in previous research (Reese, 1992; Stockton, 1976), the fact that we observed 29.2-43.2% rooting in softwood cuttings was encouraging. Although this may not be a commercialy feasible way to propagate V. arboreum, it demonstrates that rooting is possible and that the methods could potentialy be improved. Previous research did not mention whether juvenile or mature cuttings were used in the experiments (Reese, 1992; Stockton, 1976). Only juvenile wood was used in this study, which may explain the greater rooting succes. The next steps would be to discover ways to increase rooting to a commercialy aceptable succes rate. Some ideas that could be explored include the use of bottom heat, which is helpful when rooting similar hard-to-root species like deciduous azaleas. Bottom heat of 21.1-22.8?C has been used to succesfully improve rooting percentage of deciduous azaleas (Knuttel, 1984; Mylin, 1982; Nienhuys, 1980). Also, a substrate other than peat:perlite may prove to be beneficial. One of the most dificult parts of this experiment was keeping the cuttings from drying out without over saturating the ! #*! substrate. The use of pine bark or sand, or a combination of many components, could help with drainage and could decrease water logging. Light intensity is another factor that influences the rooting suces of azalea cuttings (Read and Economou, 1983). An increase in rooting percentage was observed as light intensity decreased. Cuttings rooted with lower intensity light (10 ?Em -2 s -1 ) had a rooting percentage of 88.3%, compared to 65.8% when using high intensity light (75 ?Em -2 s -1 ) (Read and Economou, 1983). Another technique to increase rooting succes may be to use a wounding technique that would expose more cambial tisue and encourage rooting. In addition to cuttings, other propagation methods could be explored such as mound layering. Overal, the rooting succes observed in this experiment was encouraging and demonstrates the need for the further research of V. arboreum asexual propagation. ! $+! Literature Cited Balinger, W. E., E. P. Manes, and J. R. Balington. 1982. Anthocyanins in ripe fruit of the sparkleberry, Vaccinium arboreum MARSH. Can. J. Plant Sci. 62:683-687. Balington, J. R., B. W. Foushee, and F. Wiliams-Rutkosky. 1990. Potential of chip- budding, stub-grafting or hot-calusing following saddle-grafting on the production of grafted blueberry plants. Proc. N. Amer. Blueberry Res.-Ext. Workers Conf. 114-120. Darnel, R. L., and S. A. His. 2006. Uptake and asimilation of nitrate and iron in two Vaccinium species as afected by external nitrate concentration. J. Amer. Soc. Hort. Sci. 131:5-10. Eck, P. 1988. Blueberry Science. Rutgers University Pres, New Brunswick, NJ and London, UK. Galeta, G. J., and A. S. Fish. 1971. Interspecific blueberry grafting, a way to extend Vaccinium culture to diferent soils. J. Amer. Soc. Hort. Sci. 96:294-298. Hartmann, H. T., D. E. Kester, F. T. Davies, Jr., and R. L. Geneve. 2002. Hartmann and Kester?s Plant propagation: principles and practices. 7th ed. Prentice Hal, Englewood Clifs, N.J. Knuttel, A. J., and C. Addison. 1984. Deciduous azalea propagation: an overview of old and new techniques. Comb. Proc. Intl. Plant Prop. Soc. 34:517-520. Lyrene, P. M. 1997. Value of various taxa in breeding tetraploid blueberries in Florida. Euphytica. 94:15-22. Lyrene, P. M. 1998. Germination and growth of sparkleberry sedlings (Vaccinium arboreum Marsh). Fruit Var. J. 52:171-178. Lyrene, P. M. and S. J. Brooks. 1995. Use of sparkleberry in breeding highbush blueberry cultivars. J. of Smal Fruit and Viticult. 3:29-38. Mylin, D. 1982. Propagation of deciduous azaleas. Comb. Proc. Intl. Plant Prop. Soc. 32:418-420. Nienhuys, H. C. 1980. Propagation of deciduous azaleas. Proc. Inter. Plant Prop. Soc. 30:457-459. Poonnachit, U., and R. Darnel. 2004. Efect of amonium and nitrate on ferric chelate reductase and nitrate reductase in Vaccinium species. Ann. Bot. 93:399-404. ! $"! Radford, A. E., H. E. Ahles, and C. R. Bel. 1968. Manual of the vascular flora of the Carolinas. The University of North Carolina Pres, Chapel Hil, NC. Read, P. E., and A. S. Economou. 1983. Supplemental lighting in the propagation of deciduous azaleas. Comb. Proc. Intl. Plant Prop. Soc. 32:639-645. Reese, J. C. 1992. Propagation of Farkleberry (Vaccinium arboreum) for use as a blueberry rootstock. Mis. State Univ., Starkvile, M. S. Thesis. Scagel, C. F. Isolate-specific rooting responses of Leucothoe fontanesiana cuttings to innculation with ericoid mycorrhizal fungi. J. Hort. Sci. Biotechnol. 80:254-262. Stockton, L. A. 1976. Propagation and autoecology of Vaccinium arboreum and its graft compatibility with Vaccinium ashei. Texas A&M Univ., College Station, M.S. thesis. ,-./01.2!34!#++%4!567.8.--9.:2!;-018.--9.:!01/.-!?0@@9197A:4!,9A8.-!B-.:2! BC->601<2!=D!01