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Development of a Procedure to Maximize Production of Hardy Rootstocks of Citrus Using Stem Cuttings

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Development of a Procedure to Maximize Production of Hardy Rootstocks of Citrus Using Stem Cuttings
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Beeson Jr., R.C. and Silva, D. (2017) Development of a Procedure to Maximize Production of Hardy Rootstocks of Citrus Using Stem Cuttings. American Journal of Plant Sciences, 8, 2837-2846.
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Beeson, Richard
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American Journal of Plant Sciences
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Shoots of Citrus sp. Kuharske were used to develop protocols for rooting reportedly HLB resistance rootstocks under intermittent mist. Investigated were shoot maturity, nodes per cutting, leaves per cutting, effects of buds, auxin concentrations and auxin solvent. Shoot maturity was most influential for success, with cuttings taken below the first 30 cm of active terminal growth producing greater root generation. Use of a thickening agent (Natrosal) to dilute the commercial auxin was second most in importance for rooting success. Root mass increased with increasing number of leaves. Cutting stems between nodes or below the lowest bud were inconsequential. To produce maximum number of viable cuttings, single node-single leaf cuttings were preferred. Single bud cuttings produced one shoot after rooting. This was adventitious since multi-node cuttings usually sprouted new shoots that would need to be removed before budded. Evaluation of the best combination of auxin and cutting-related attributes were evaluated with four additional common rootstocks in June 2016. Rooting was 100% successful. A quick dip (0.5 s) in a 7500 ppm solution of Dip&Gro produced the most root generation in six weeks for all rootstocks. Root quantity varied by rootstock.
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American Journal of Plant Sciences 2017, 8, 28372846 http://www.scirp.org/journal/ a jps ISSN Online: 21582750 ISSN Print: 21582742 DOI: 10.4236/ajps.2017.811192 Oct 30, 2017 2837 American Journal of Plant Sciences Development of a Procedure to Maximize Production of Hardy Rootstocks of Citrus Using Stem Cuttings Richard C. Beeson Jr.1*, Dilma Silva2 1University of Florida Institute of Food and Agricultural Science MidFlorida Research and Education Center, Apopka, USA 2Agromillora Florida, Inc., Wildwood, USA Abstract Shoots of Citrus sp. Kuharske were used to develop protocols for rooting r eportedly HLB resistance rootstocks under intermittent mist. Investigated were shoot maturity, nodes per cutting, leaves per cutting, effects of buds, auxin concentrations and auxin solvent. Shoot maturity was most influential for success, with cuttings taken below the first 30 cm of active terminal growth producing greater root generation. Use of a thickening agent (Natrosal) to d ilute the commer cial auxin was second most in importance for rooting success. Root mass increased with increasing number of leaves. Cutting stems between nodes or below the lowest bud were inconsequential. To produce maximum number of viable cuttings, single node single l eaf cuttings were preferred. Si ngle bud cuttings produced one shoot after rooting. This was adventitious since multi node cuttings usually sprouted new shoots that would need to be r emoved before budded. Evaluation of the best combination of auxin and cu tt ing related attributes were evaluated with four additional common rootstocks in June 2016. Rooting was 100% successful. A quick dip (0.5 s) in a 7500 ppm solution of Dip&Gro produced the most root generation in six weeks for all rootstocks. Root quantity v aried by rootstock. Keywords Citrus Propagation, Auxin Concentration, Stem Maturity, Cutting Length 1. Introduction Floridas citrus industry has been devastated by Huanglongbing (HLB) disease, also known as citrus greening. Caused by the bacteria Candidatus liberibacteraHow to cite this paper: Beeson Jr., R C. and Silva D (2017 ) Development of a Pr ocedure to Maximize Production of Hardy Rootstocks of Citrus Using Stem Cuttings Amer i can Journal of Plant Sciences, 8 2837 2846 https://doi.org/10.4236/ajps.2017.811192 Received: September 30, 2017 Accepted: October 27, 2017 Published: October 30, 2017 Copyright 201 7 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0 ). http://creativecommons.org/licenses/by/4.0/ Open Access

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2838 American Journal of Plant Sciences siaticus [1] HLB is spread by Asian psyllids and progressively attacks and kills fine root. Up to 40% loss of fine roots occurs before symptoms in above ground portions of trees are noticeable. Infection leads to smaller fruit size and loss, si gnificant yield loss, and over time dying and unproductive groves. Unfortunately, common citrus rootstocks used for decades are susceptible to HLB. Controlling HLB requires advances in both tree health management and di sease resistance. Screening, propagating, and producing disease resistant root stock is fundamental to control. Fortunately, over the past several years vigorous and apparently disease free shoots have been found on declining trees in dying groves. Thes e shoots have been propagated in limited numbers, challenged r epeated with HLB inoculation, and shown few to no signs of HLB infection [2] While these selections h ave potential as rootstock, most are difficult to propagate by tissue culture and none produce viable seeds. Scions of these selections have been grafted onto common rootstocks for multiplicationvia vegetative cuttings to evaluate their potential as rootst ock. How to root common citrus cuttings has been long known; how to economically root chimeras at a commercial scale has not. Application of plant growth regulators, vegetative cutting age, and stem length ha s been shown to improve rooting, but not definitively and only on limited c itrus germplasm. Propagation of citrus stem cuttings has a long history, dating back to the late1800s [3] Since then there have been several reports on how auxin type, mixtures and concentration induce rooting of different varieties of citrus. As early as 1935 the rooting of lemon cuttings in coarse sand beds in sash covered propagation frame s inside a glass greenhouse was described [4] Te mperatures were held around 30C and cuttings were misted regularly. Cuttings of mature 15 cm long stems were trea ted with 1000, 2000 or 4000 ppm I n dole acetic acid (IAA). The 2000 ppm concentration was most effective in i n creasing roo ting when a minimum of two mature leaves were retained, produ c ing well rooted plants without stem dieback. Stems treated with 1000 ppm pr o duced little root mass and were similar to the water control. Naphthalene acetic acid (NAA) also improved root regeneration, but at half (1000 ppm), the co n centration required if using the Indo butyricacid (IBA) [5] All of these conce n trations were applied using the 24 hour dilute soak method. The use of intermittent mist for rooting citrus cuttings occurred in 1950 [5] In 1999 [6] a procedure for rooting of cuttings from rootstocks of Carrizo and sour orange taken in September was reported. Cuttings of 20 cm in length (each containing 6 to 10 nodes) were taken from 1.5 year old trees. These were wounded, treated with 2500 ppm of IBA in water and placed in vermiculite under mist for two months. Root mass, shoot mass and root length were greater for cuttings than comparable seed grown plants. This was attributed to greater leaf area of cuttings, compared to seedlings of the same age, and could have been budded sooner. Auxin concentrations of 1000 and 3000 ppm IBA and N AA were evaluated on 12 selections of different Citrus genotypes [7] Cuttings were placed

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2839 American Journal of Plant Sciences under intermittent mist for 10 sec every 5 min during daylight. Success varied with auxin source and concentrations. The cultivar Carrizo rooted at 83% when treated with 3000 ppm NAA, while similar concentrations of IBA only produced 67% success. Total root lengths six weeks after treatment initiation in late Se ptember were 36 cm with NAA and 24 cm with IBA. Six years earlier [8] trials containing IBA and NAA at 3000 ppm stimulated the greatest root production in both juvenile and matur e cuttings of Swingle citrumelo stems 15 cm in length, with three or four leaves rooted in the greatest numbers. More roots regenerated with IBA on juvenile cuttings, while mature cuttings produced more roots with NAA. In 2011 [9] node stem cuttings treated with 2500 ppm IAA in water pr oduced more roots than lower concentrations (500 to 2000 ppm) and had higher success rates and growth. Here are presented the results of employing a co mmer cial auxin mixture it interacts with stem maturity for large scale rooting of citrus stem cuttings. This was undertaken to enhance rooting the limited nu mber of available potential HLB resistant plant materials for evaluation in co mm ercial groves. The research reported here evaluates a commercial mixture of IBA and NAA that has worked very well for rooting a wide range of woody o rnamental plants [10] but has not been assessed for rooting of citrus. The obje ctives of this study were to: 1 ) Determine effect of shoot maturity on rooting of citrus stem cuttings. 2 ) Determine the optimum auxin carrier and auxin concentration for max imum rooting success. 3 ) Evaluate the best combination of stem maturity, auxin concentration on other common citrus rootstocks. 2. Materials and Method 2.1. Effect of Stem Maturity on Rooting Two experiments were conducted to identify the most promising combinations of auxin concentrations, stem maturity and stem length using the citrus root stock cultivar Kuharske. Kuharske originated China and Japan and is prized for its excellent control of burring nematodes. A third experiment was conducted to evaluate the best auxin treatment using other common rootstocks. In Exper iment I, 60 cm shoots of young Kuharske rootstocks were harvested on 5 May 2015. These were cut into single node stems with one bud at either end and one leaf at the distal end. Six cuttings were made from each 30 cm stem. The pro ximal end of each cutting was quick dipped (0.5 sec) into one of eight auxin sol utions ( Table 2 ) before placement an in moistened cell tray (IP110, 45 cell trays, Stueve & Sons, Tangent, OR) in a c ommercial substrate of 60% Canadian peat moss: 40% perlite (Fafard 2P, SunGro Horticulture, Agawam, MA). Auxin sol utions of 0, 4000, 6000 and 8000 ppm were derived by dilutions of a 15% co mmercial auxin source (Dip n Gro, Clackamas, OR, USA). Solutions wer e diluted either with de ionized water or a solution (8 mg/liter) of Natrosol (Natrosol 250HBR PA, Ashland, Wilmington, DE). Natrosol is a food grade powder

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2840 American Journal of Plant Sciences thickening agent. This was similar to the successful application of using Cell u wett (Hort Specia lties Inc., Pinckney MI) for the rooting of western he mlock [11] Cell u wett was not locatable when this experiment was initiated. Each treatment was replicated with 19 similar cuttings Trays were placed under an overhead mist system consisting of two mi st no zzles (Dramm mist 360NW Green; Dramm Corp., Netherlands) spaced to u niformly cover 3.5 m2 of rooting trays. Mist timing and duration was controlled by a Sterling Controller 30 (Superior Controls, Torrance, CA). Mist was pulsed 15 seconds every 10 minu tes from 6 am to 10 pm EST from May to September. Five weeks after initiation, all cuttings were drenched a 150 ppm N of a co mmercial liquid fertilizer (20 10 20 Peters, Everris, Dublin, OH). Trays were left in the mist for 41 days, then harvested for measurements of new root and shoot mass. Dry mass was weighed to 0.1 mg (MettlerAE100, Mettler Toledo, Colu mbus, OH). Shoot growth was measured to the nearest mm using a ruler when available. 2.2. Second Experiment In the second experiment, there were six cuttings classes, each treated with four levels of auxin concentrations. Cuttings were placed under mist as described above on 18 May 2015. The six classes of cuttings consisted to of two types of cuttings ( Table 1 ), one type retain buds at both the proximal and distal ends of a cutting, retaining one bud more than the number of leaves. The other type had the proximal bud removed, such that leaf and bud number were the same. All cuttings were taken from Kuharske rootstocks below the first 30 cm of stem u nder shoot tips. Each cutting class was quick dipped (0.5 sec.) in one of four auxin solutions, 0, 2500, 5000 and 7500 ppm auxin. All auxin concentrations were prepared as described previously and diluted with the Natrosol solution. After trea ting the proximal end, each cutting was inserted into 45 cell seedling trays as in the first experiment and used the same substrate described above. Each of the 24 treatment combinations were replicated with three blocks of 12 cuttings per treatment. Misti ng periods and duration were the same as described for Exper iment I. Cuttings in this experiment were harvested on 16 July 2015, 41 days after initiation. All cuttings were gently removed from the seedling trays, then washed Table 1. Description of types of cuttings. Class Description 1 1 node, with 1 leaf cut between nodes at the bottom of a stem 2 2 nodes, with 2 leaves, cut between nodes at the bottom 3 3 nodes, with 3 leaves, cut between nodes at the bottom 4 2 nodes, with 2 leaves, cut just below the bottom node 5 3 nodes, with 3 leaves, cut just below the bottom node 6 4 nodes, with 4 leaves, cut just below the bottom node

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2841 American Journal of Plant Sciences and dried as described in the first experiment. 2.3. Third Experiment The third experiment focused on replicating previous results with different of citrus rootstock cultivars, focusing on the two auxin concentrations of 4000 and 7500 ppm Dip&Gro. In February 2016, rootstocks of Kuharski, Swindle 13, C 35, C54 and USDA 813 were obtained and transplanted into 27 L containers us ing the same commercial blend of 60:40 Canadian peat: perlite (SunGro Ho rticulture). Trees were fertilized, irrigated and pruned lightly as needed to pr omote multiple shoots for cuttings. On 10 June 2016, 33 shoots of mature wood from each cultivar, compr ised of three replications of 11 cuttings of each root stock, were collected across several plants per rootstock. Shoots were converted to single node cuttings as described previously and treated with either 4000 or 7500 ppm Dip&Gro and placed under mist. C uttings were single node with one leaf and one bud at the distal end. This experiment began on 10 June 2016, with harvest on 21 July 2016, lasting 41 days. These experiments were conducted in a sealed polycarbonate greenhouse with a double layer polyethylene roof from early May to late July in Florida. Two green house fans activated at 27C, normally shortly after sunrise, and ran cont inuously until several hours after sunset. The greenhouse was equipped with a fine mesh screen (16 m2) on the north end for air movement and exclusion of small insects. Maximum temperatures were commonly 38C or higher most of the day. During this experimental period, daylight to dusk was around 14 hours. 2.4. Statistical Analysis All data was analyzed using SAS 9.1. Experiment I The experiment was arranged as a 2 4 2 factorial design with 2 diluents as the main factor; deionized water and a solution of Natrosol, with 4 auxin co ncentrations as the sub factor (0, 4000, 6000 and 8000 ppm) and 2 levels of stem maturity (immature and mature stems) as the sub sub factor, with a total of 320 experimental units. Statistical analyses root dry mass, shoot length and rooting percentage were analyzed using GLM. Experiment II. Data was analyzed as a 6 4 factorial design with three blocks of 13 replic ations per treatment. There were 6combinations of number of buds and leaves retained on a cutting. Sub factors consisted of 4auxin concentrations (0, 2500, 5000 and 7500 ppm), with a total of 23 experimental units. Data collected co nsist ed of root dry mass, new shoot elongation and number of new shoots. Experiment III Data was analyzed separately by cultivar as a one way ANOVA with two 2 concentrations of auxin with 3 blocks of 10 replications each. Each rootstock analyzed separately. Data consisted of both root and shoot dry mass.

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2842 American Journal of Plant Sciences 3. Results 3.1. Auxin Carriers (Experiment I) Root dry mass was recovered from all 16 sets of cuttings for Kuharske ( Table 1 ). With distilled water as the solvent, root mass was greatest (P < 0.05) with 4000 ppm auxin with cuttings taken from the oldest or lower 30 cm of stem (30 to 60 cm). Cuttings made from the upper or youngest 30 cm of stem (0 to 30 cm) produced greatest root mass at 6000 ppm. Younger stems produced less root mass (P < 0. 05) than the older stems. Using only Natrosol and no auxin doubled root dry mass compared to water alone, but absolute success was very low. The addition of auxin with the thickening agent had no effect on generated root mass, independent of auxin concentration. Shoot growth occurring 41 days after initiation was exceedingly small for most treatment combinations ( Table 2 ). The greatest growth always occurred from cuttings collected from the lower 30 cm of stem. Of these, the addition of N atrosol alone or Natrosol with 8000 ppm auxin, averaged shoots of 1.5 cm or longer. Other combinations averaged 0.3 cm to no shoot growth. 3. 2 Leaf Node and Bud Quantity Effect on Root and Shoot Growth (Experiment II) The second experiment evaluated the effect of the number of nodes with leaves, and their impact on rooting relating to whether cuttings should be cut with leaf Table 2. Effects of auxin diluent, auxin concentration and stem maturity on root growth and shoot growth of Citrus sp. Kuharske. Each mean is represents 19 replications. Mean with the letter are not significantly diffident at the 0.05 percent level. Thickening agent Auxin conc. (ppm) Stem maturity Root dry mass (g) Shoot length (cm) Rooting percent H2O 0 0 12" 46.4 f 0 f 0 H2O 0 12 24" 58.3 f 0.54 def 10 H2O 4000 0 12" 88.8 cde 0.09 ef 70 H2O 4000 12 24" 133.0 a 0.86 cd 100 H2O 6000 0 12" 101.7 bcd 0.15 ef 50 H2O 6000 12 24" 112.0 abcd 0.44 def 70 H2O 8000 0 12" 68.4 ef 0.30 ef 70 H2O 8000 12 24" 121.7 ab 1.55 ab 100 Natrosal 0 0 12" 101.9 bcd 0.22 ef 90 Natrosal 0 12 24" 128.4 a 1.79 a 100 Natrosal 4000 0 12" 97.2 bcd 0.20 ef 90 Natrosal 4000 12 24" 110.7 abcd 1.14 bc 90 Natrosal 6000 0 12" 96.3 bcd 0.07 ef 90 Natrosal 6000 12 24" 118.9 ab 0.59 de 100 Natrosal 8000 0 12" 87.4 de 0.31 def 100 Natrosal 8000 12 24" 113.9 abc 1.55 ab 100

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2843 American Journal of Plant Sciences buds at either ends or just the distal end ( Table 1 ). All treatments, with exce ption of single node, single bud cuttings not treated with auxin, produced 100% rooting ( Table 3 ). It is worth noting that some untreated cuttings also produced roots. Root dry mass generally increased as auxin concentrations increased within node treatments, and incre ased with an increase in the number buds. The greatest root dry mass change occurred when three leaves and four buds were quick dipped in 7500 ppm auxin. Reducing auxin concentration to 5000 ppm with the same number of leaves and buds produced similar resu lts. However, reducing auxin concentrations to 2500 ppm reduced root dry weight by 11% when compared with the 7500 ppm auxin. All other combinations produced root dry weight that was at least 26% lower than the most prolific node and bud combination Table 3. The effects of leaves and nodes on root dry mass, shoot growth and shoot number on cuttings of Citrus sp. Kuharske. Each mean represents 38 replications. Means with the same letter are not significantly diffident at the 0.05 percent level. Treatment Auxin conc. Root dry mass (g) New shoot length (mm) Number of shoots 1 node cut at stem 0 0.166 gh 23.8 lm 0.21 h 1 node cut at stem 2500 0.174 fgh 70.8 efgh 0.89 def 1 node cut at stem 5000 0.182 fgh 12.6 m 0.21 h 1 node cut at stem 7500 0.140 gh 30.2 klm 0.53 g 2 node cut at stem 0 0.150 gh 38.9 jklm 0.58 g 2 node cut at stem 2500 0.288 d 61.6 fghij 0.79 efg 2 node cut at stem 5000 0252 de 86.3 cdef 0.95 def 2 node cut at stem 7500 0.262 de 80.9 cdefg 1.03 cde 3 node cut at stem 0 0.295 cd 100.6 abc 1.52 a 3 node cut at stem 2500 0.374 b 68.6 efgh 1.16 bcd 3 node cut at stem 5000 0.373 b 109.6 ab 1.5 a 3 node cut at stem 7500 0.367 b 99.8 abc 1.29 abc 1 node cut at node 0 0.172 fgh 54.7 hijk 0.71 fg 1 node cut at node 2500 0.131 h 36.1 jklm 0.64 fg 1 node cut at node 5000 0.203 efg 46 ijkl 0.64 fg 1 node cut at node 7500 0.232 def 61.9 efghij 0.71 fg 2 node cut at node 0 0.273 cd 79.7 cdefg 1.16bcd 2 node cut at node 2500 0.365 b 113.7 a 1.38 ab 2 node cut at node 5000 0.377 b 87.6 bcdef 1.11 bcd 2 node cut at node 7500 0.356 cb 65.8 efghi 1.05 cde 3 node cut at node 0 0.271 d 48.9 hijk 1.13 bcd 3 node cut at node 2500 0.453 a 97.1 abcd 1.47 a 3 node cut at node 5000 0.479 a 92.4 abcd 1.16 bcd 3 node cut at node 7500 0.510 a 100.7 abc 1.16 bcd

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2844 American Journal of Plant Sciences Mean shoot growth ranged from 12 mm for single node, single bud cuttings treated with 5000 ppm auxin, to 113 mm for two node, three bud cuttings treated with 2500 ppm auxin. Of the 24 combinations of nodes and auxin co ncentrations, seven treatments produced similar shoot growth in late summer. All had at least three buds and all but one (three nodes, three buds) were treated with 2500 ppm auxin. Shoot growth, however, was not uniform. Cuttings from both single nodes and two nodes cut between nodes, all averaged less than one shoot per cutting ( Table 3 ). Three buds were required to average at least one new shoot per cutting within 41 days. This was satisfied by two nodes cut with three buds, or three no des cut with three or four buds. 3. 3 Rootstock Growth Comparison (Experiment III) Mean root growth was higher with 7500 ppm auxin for all cultivars, but was si gnificantly different (P < 0.05) from 4000 ppm for Kuharske, C35, and USDA 812. For C 54 and Swingle 13, root dry weight was similar among concentrations. Greatest root growth occurred with Swingle 13 at over 120 mg of dry root mass, independent of auxin concentration. Cuttings of C 54 registered the lowest root growth at around 50 milligrams. Sho ot dry mass consisted of the stem and leaf of the original cutting and any gain in mass not associated with roots over the 41 days of rooting. Kuharske had the largest mass at harvest for both auxin conce ntrations, followed by C 35. The rest were generally 100 mg lighter at harvest. New shoots generated during the short rooting period were small, with greatest measured for C 54 at nearly 24 mg dry weight. In contrast there was no new shoot growth for Swingle 13 when treated with 7500 pm auxin, and less than 1 mg with 4000 ppm auxin. Though not significant (P < 0.05), there was a weak trend of greater new shoot growth occurring with cuttings treated with 4000 ppm auxin compared to 7500 ppm. Root to shoot ratios trended be higher when 7500 ppm was used. 4. Dis cussion The most prominent factor affecting root growth was stem maturity. Within each auxin concentration and carrier, cuttings taken below the first 30 cm of actively shoots produced more root and more shoot dry mass than those from the upper 30 centimet ers. The exception was when cuttings were not treated with auxin and dipped only in distilled water, where some root generation occurred, but there were no differences in root mass. Beyond stem maturity, effects of auxin carrier (distilled water or Natroso l) and auxin concentrations were entangled ( Table 2 ). Overall greatest root mass occurred when using water as the carrier at 4000 ppm auxin. This was similar to (P > 0.05) treating cuttings with no auxin, but using Natrosol as the carri er. However overall highest root masses were produced with 6000 or 8000 ppm i ndependent of carrier. Using Natrosol as the carrier improved rooting percentage from 90 to 100 percent. This contrasted with using water as the carrier, which

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2845 American Journal of Plant Sciences only produced rooti ng success above 70% with cuttings taken from the older, lower 30 cm stem. Auxin type has been shown to have significant effect on rooting of citrus. IAA has rarely been used for citrus since it is rapidly metabolized [11] In 1985 [8] it was reported that 3000 ppm IBA was best for rooting juvenile cuttings, whereas mature cutting rooted better with 3000 ppm NAA. In most cases, using NAA for citrus has increased root mass c ompared to IAA or IBA and required lower concentrations [7] Dip&Gro contains a mixture of 1 part NAA and 2 part IBA. IBA is chemically close the natural IAA and acts quickly to promote cell div ision, whereas NAA is chemically dissimilar, promoting longer cellular activity [12] While almost always some roots generated regar dless of treatment, this was not the case for shoot growth ( Table 2 ). Treatment with water and no auxin produced no shoot growth on cuttings. All other combinations produced some new shoot growth during the six week rooting period. As with root generation, new shoot growth was also greater from the lower 30 cm of stem than the upper 30 cm. Shoot growth was greatest with the 8000 and 4000 ppm auxins trea tments. Even with the lower 30 stem segment, shoot growth was reduced for both water and Natrosol with the 6000 ppm concentration. In Experiment II, rooting success was near 100% for all cuttings. However, cutting type had significant effects on root mass, shoot length and number of shoots ( Table 3 ). Auxin concentratio ns had little effect on root mass ( Table 3 ). Except for Treatment 4, there were no differences (P > 0.05) in root dry mass among auxin concentrations of 2500 to 7500 ppm within cutting types. Where no auxin was applied, root mass was lo wer (P < 0.05) than similar auxin treated cutting, except for Treatments 1 and 4. Greatest root dry mass occurred from cuttings with four remaining leaves treated with any auxin concentration. The second highest root masses occurred on cuttings with three nodes either cut b elow the third node or between nodes. Both these cutting types retained three leaves. Previous research reported greater root generation when cutting retained a minimum of two mature leaves [4] [5] [8] Root growth from cuttings with two nodes were larger (P < 0.05) if cut between nodes, leaving several centimeters of stem tissue below the lowest bud. Cuttings of single nodes, cut between nodes, generally had the lowest root mass but roo ting success was 100 percent. Experiment III focused on two auxin concentrations and five rootstocks. Root gener ation was strong for all rootstocks. However relationships between cutting dry mass and new shoot elongation were poor. Amounts of root generation a ppear to be rootstock specific. 5. Conclusion All citrus rootstocks evaluated here produced an abundance of new roots within six weeks when treated with 7500 ppm Dip&Gro diluted with a thickening agent dur ing the spring to fall period. Natrosol, a commercial thickening agent used

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R C. Beeson Jr. D Silva DOI: 10.4236/ajps.2017.811192 2846 American Journal of Plant Sciences for human consumption, functioned well as the diluent, but is not commercially available for propagation. Over the past 20 years Cell u wett (ethylellulose), once marked by Grifflin Corp., has been used as a thickening agent for successful propagation of over 130 woody plant species for research and in plant propag ation classes [10] In 2016, a source of Cell u wett was discovered and is available (Hort Specialties Inc., Pinckney MI). Acknowledgement s The authors of this paper would like t o thank the Citrus Research and Develo pment Foundation for their support of our research. References [1] Johnson, E.G., Wu, J., Bright, D.B. and Graham, J.H.(2014) Association of Cand idatus Liberibacter Asiaticus Root Infection, but not Phloem Plugging with Root Loss on HuaglongbingAffected Trees Prior to Appearance of Foliar Symptoms. Plant Pathology, 63 290 298. https://doi.org/10.1111/ppa.12109 [2] Grosser, J.W. ( 2 013 ) Personal Communication 20 June 2013. Verbal Communic ation of Non Published Results [3] Conger, O.H. (1889) Lemon Culture Annual Report of the Secretary of the State Board of Michigan, 361 365. [4] Cooper, W.C. (1940) Rooting Citrus Cuttings with Synthetic Growth Substances. Proceedings of the Florida State Horticulture Society, 53 174 177. [5] Ochse, J.J. and Reark, J.B. (1950) The Propagation of Sub Tropical Fruit Plants by Cuttings, A Progress Report Proceedings of the Florida State Horticulture Society, 63 248251. [6] Rieger, M. (1992) Growth, Gas Exchange, Water Uptake, and Drought Response of Seedling and Cutting Propagated Peach and Citrus Rootstocks. Journal of the American Society for Horticultural Science, 117 834 840. [7] Sabbah, S.M., Grosser, J.W., Chandler, J.L. and Louzada, E. S. (1991) The Effect of Growth Regulators on the Rooting of Stem Cuttings of Citrus, Related Genera and Intergeneric Somatic Hybrids. Proceedings of the Florida State Horticulture Society, 104 188 191. [8] Ferguson, J., Young, M. and Halvorson, J. (1985) The Propagation of Citrus Root stocks by Stem Cuttings. Proceedings of the Florida State Horticulture Society, 98 3942. [9] Seran, T.H. and Umadevi, T. (2011) Influenc e of Indole Acetic Acid (IAA) on the Establishment of Stem Cutt ings in Lemon (Citrus limon L.) Journal of Agricultural Research, 49 517 524. [10] Beeson Jr., R.C. (2000) Putting the Speed Back in Quick dip Auxin Application. SNA Research Conference, 45 298 302. [11] Foster, G.S., Campbell, R.K. and Adams, W.T. (1984) Heritability, Gain, and C E ffects in Rooting of Western Hemlock Cuttings. Canadian Journal of Forest R esearch, 14 628638. https://doi.o rg/10.1139/x84114 [12] Macht, D.I. and Grumbein, M.L. (1937) Influence of Indole Acetic, Indole Butyric, and Naphthalene Acetic Acids on Roots of Lupinus Albus Seedlings. American Journal of Botany, 24 457 460. https://doi.org/10.2307/2436433