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Effects of Light Regime and Iba Concentration on Adventitious Rooting of an Eastern Cottonwood (Populus Deltoides) Clone

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Effects of Light Regime and Iba Concentration on Adventitious Rooting of an Eastern Cottonwood (Populus Deltoides) Clone EFFECTS OF LIGHT REGIME AND IBA CONCENTRATION ON ADVENTITIOUS ROOTING OF AN EASTERN COTTONWOOD (POPULUS DELTOIDES) CLONE Alexander P. Hoffman, Joshua P. Adams, and Andrew Nelson1 Abstract—Eastern cottonwood (Populus deltoides) has received a substantial amount of interest from in- vitro studies within the past decade. The ability to efficiently multiply the stock of established clones such as clone 110412 is a valuable asset for forest endeavors. However, a common problem encountered is initiating adventitious rooting in new micropropagation protocols. Stem segments were collected from bud-broken 1 year old clone 110412 cuttings, sterilized, and stimulated to initiate shoots. Developed shoots (~2 cm in height) were excised and placed into one of three rooting media that included indole-3-butyric acid (IBA) concentrations (0.5 mg/L, 1 mg/L, or 2mg/L) in full strength DKW Medium, full strength Gamborg B5 vitamins, 2 percent sucrose, 0.6 percent agar, 10 mg/L AMP, 0.2 ml/L of Fungigone. In addition to IBA concentration, cuttings were randomly assigned to light rack positions to test the effects of wide spectrum fluorescent light (100 µmol m-2 s-1 photosynthetically active radiation (PAR), 16/8 hour photoperiod) and light emitting diode light (LED; 4:1 red-to-blue diodes, 250 µmol m-2 s-1 PAR, 16/8 hour photoperiod). After a month of exposure, there was limited rooting exhibited across treatments. However, fluorescents (3.58 ± 1.02) produced significantly better performing microcuttings (judged on morphology, visual vigor, and survival) than LEDs (2.7±0.86) (p<0.005). The high light intensity of the LEDs may be prompting weaker performance through unfavorably high transpiration-induced auxin uptake. While LEDs may play a role in future micropropagation protocols, results suggest that wide spectrum florescent lights produce better performing eastern cottonwood 110412 microcuttings. INTRODUCTION roots (Costa and others 2013, De Klerk 2002). Two Eastern cottonwood (Populus deltoides), in addition in-vitro microenvironmental factors that have been to other Populus species and their hybrids, has investigated for stimulating favorable adventitious become an important constituent in woody species rooting characteristics are auxin concentration and light biotechnology, in-vitro culture, and genetic engineering regime (i.e., quality, intensity, and photoperiod). (Confalonieri and others 2003). The species has been widely cultivated due to its adaptability, growth rate, While auxin has been well documented as one of the woody biomass production, wood industry uses (e.g., main phytohormones in a number of physiological paper and pulpwood), and has become established as processes such as apical dominance it has also been a model system because of its small genome size, short documented in a number of adventitious rooting rotation cycle, rapid growth rate, and ease of vegetative processes such as regulating cell division, cell propagation (Confalonieri and other 2003, Jansson and elongation, and the initiation of root apical meristems Douglas 2007). Over the past 50 years or more, the (Mironova and others 2010). Recent success of eastern identification and screening efforts of tree improvement cottonwood micropropagation and Agrobacterium programs have produced a collection of superior clones transformation studies has been documented with which could benefit from the application of vegetative two commonly used auxins, indole-3-acetic acid (IAA) multiplication techniques such as micropropagation or Indole-3-butyric acid (IBA) (Cavusoglu and others (Thorpe and others 1991). While each micropropagation 2011, Chaturvedi and others 2004, Yadav and others process encompasses complex genetic, molecular, and 2009). However, a recurrent trend in these experiments physiological relationships, one step of the process that is clone-specific responses to identical auxin has received a substantial amount of interest in recent concentrations, which has prolonged the process of years is the induction and formation of adventitious developing new protocols (John and others 2014, Yadav 1Alexander P. Hoffman, Graduate Research Assistant, University of Arkansas at Monticello, Monticello, AR, 71656; Joshua P. Adams, Assistant Professor of Silviculture and Genetics, Louisiana Tech University, Ruston, LA. 71272; Andrew Nelson, Assistant Professor of Silviculture, Arkansas Forest Resources Center, University of Arkansas Division of Agriculture, Monticello, AR 71656 Citation for proceedings: Schweitzer, Callie J.; Clatterbuck, Wayne K.; Oswalt, Christopher M., eds. 2016. Proceedings of the 18th biennial southern silvicultural research conference. e–Gen. Tech. Rep. SRS–212. Asheville, NC: U.S. Department of Agriculture, Forest Service, Southern Research Station. 614 p. 478 HARDWOOD REGENERATION and others 2009). In addition, there are still a number METHODS of established clones for which a micropropagation, Plant Material and therefore adventitious rooting, protocol has not The eastern cottonwood 110412 clone was originally been derived. Further research with these clones, selected for propagation by the USDA Forest Service such as the 110412, can help determine what auxin Center for Bottomland Hardwood Research due to its concentrations illicit the most favorable adventitious excellent rooting, good form, and above average growth rooting characteristics and how clone-specific on various sites during initial screening trials (Personal responses can be implemented in future protocols. communication, Dr. Randy Rousseau). The 110412 clone Another benefit to modern micropropagation research has also been cited for its phytoremediation abilities is the investigation of new technological advancements (Cardellino 2001, Minogue and others 2012, Rockwood such as light-emitting diodes (LEDs) and how they may and others 2006).Approximately 61 cm long hardwood affect adventitious root formation. cuttings of clone 110412 were collected from 1-year old, coppiced trees located on the campus of Mississippi Light quality (i.e., color), intensity, and photoperiod State University (Starkville, MS) during late winter 2013. can each be manipulated within an in-vitro system Cuttings were taken out of cold storage (4°C) during to produce a unique light regime with the intent of late fall 2014, cut to 20 cm in length, and soaked in stimulating specific physiological and morphological room-temperature water for one week. After soaking, changes, such as adventitious rooting, in microcuttings. the base of each cutting was dipped in GreenLight© Studies performed within the past 20 years have Rooting Hormone (San Antonio, TX) and placed (dipped documented adequate in-vitro Populus adventitious -2 -1 end down) into 950 ml Mini-Treepots (Stuewe & Sons, rooting using low intensity (~50-100 µmol m s PAR) Inc., Tangent, OR) containing EarthGro topsoil until only white fluorescents on a 16 hour light / 8 hour dark (i.e., ~25 percent of the cutting remained aboveground. The long-day) photoperiod (Cavusoglu and others 2011, Mini-Treepots were then placed into 3.28 L growing Gozukirmizi and others 1998, Kang and others 2009, trays, six pots per tray. The growing trays were placed Noël and others 2002, Phan and others 2004, Thakur underneath four Sylvania T12 40W wide spectrum and Srivastava 2006, Yadav and others 2009). However, fluorescent tube light bulbs emitting 102.3 ± 6.8 µmol there have been a number of studies, conducted on m-2s-1 photosynthetically active radiation (PAR) on a 16/8 both woody species and other crop species, that have (i.e., long day) photoperiod until initial bud break. All documented the potential role of far-red light (Kraepiel hardwood cutting establishment took place in a lab at and others 2001), red light (Li and others 2010), blue the School of Forest Resources, University of Arkansas light (Pinker and others 1989), and the implications of at Monticello campus. potential auxin-light quality interactions (Britz and Sager 1990, Chée 1986). In addition, recent technological Stem Collection and Shoot Initiation advancements such as LEDs have provided researchers new resources to implement within in-vitro light quality Internode and node segments were collected from research. shoots developed from hardwood cuttings in early January, 2015. These were cut into 3 cm segments LED’s compact engineering, solid-state construction, and then surface sterilized using a 25 minute agitated and especially their specific light spectrum capabilities wash in 15mL of 30 percent bleach-ddH20 solution ® has caught the attention of a number of plant tissue supplemented with ~1 mL Tween 20 and overhead culture and horticultural researchers (Bula and others ultraviolent light exposure (fig. 1). After the initial 1991, Morrow 2008). While there has been documented wash, stem segments were gently rocked ten times success, in terms of favorable adventitious rooting with sterilized ddH20, drained, and gently rocked an characteristics, in a number of annual crops such additional ten times with fresh sterilized ddH20. Each as Akihime strawberry (Fragria x ananassa Akihime) stem segment was then pressed into a BioWorld™ (Nhut and others 2003) and upland cotton (Gossypium Extra Deep Mono Petri Dishes containing ~36 mL hirsutum L.) (Li and others 2010) there is currently of shoot initiation media (SIM) to stimulate shoot a lack of woody species, and specifically eastern development. The SIM consisted of full strength cottonwood, in-vitro research utilizing LEDs. Given the McCown Woody Plant Medium (McCown
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