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.
Florida’s citrus industry has been devastated by Huanglongbing (HLB) disease, also known as citrus greening. Caused by the bacteria Candidatus liberibacterasiaticus [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, significant 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 disease resistance. Screening, propagating, and producing disease resistant rootstock 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. These shoots have been propagated in limited numbers, challenged repeated with HLB inoculation, and shown few to no signs of HLB infection [2] . While these selections have 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 rootstock. 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 has been shown to improve rooting, but not definitively and only on limited citrus germplasm. Propagation of citrus stem cuttings has a long history, dating back to the late1800’s [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 frames inside a glass greenhouse was described [4] . Temperatures were held around 30˚C and cuttings were misted regularly. Cuttings of mature 15 cm long stems were treated with 1000, 2000 or 4000 ppm Indole-acetic acid (IAA). The 2000 ppm concentration was most effective in increasing rooting when a minimum of two mature leaves were retained, producing well-rooted plants without stem dieback. Stems treated with 1000 ppm produced little root mass and were similar to the water control. Naphthalene acetic acid (NAA) also improved root regeneration, but at half (1000 ppm), the concentration required if using the Indo-butyricacid (IBA) [5] . All of these concentrations 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 NAA were evaluated on 12 selections of different Citrus genotypes [7] . Cuttings were placed 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 September 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 mature 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 produced more roots than lower concentrations (500 to 2000 ppm) and had higher success rates and growth. Here are presented the results of employing a commercial auxin mixture, it interacts with stem maturity for large-scale rooting of citrus stem cuttings. This was undertaken to enhance rooting the limited number of available potential HLB-resistant plant materials for evaluation in commercial 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 ornamental plants [10] , but has not been assessed for rooting of citrus. The objectives 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 maximum rooting success.
3) Evaluate the best combination of stem maturity, auxin concentration on other common citrus rootstocks.
2. Materials and Method2.1. 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 rootstock 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 Experiment 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 proximal end of each cutting was quick-dipped (0.5 sec) into one of eight auxin solutions (Table 2) before placement an in moistened cell tray (IP110, 45 cell trays, Stueve & Sons, Tangent, OR) in a commercial substrate of 60% Canadian peat moss: 40% perlite (Fafard 2P, SunGro Horticulture, Agawam, MA). Auxin solutions of 0, 4000, 6000 and 8000 ppm were derived by dilutions of a 15% commercial auxin source (Dip-n-Gro, Clackamas, OR, USA). Solutions were 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 thickening agent. This was similar to the successful application of using Cell-u-wett (Hort Specialties Inc., Pinckney MI) for the rooting of western hemlock [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 mist nozzles (Dramm mist 360NW Green; Dramm Corp., Netherlands) spaced to uniformly 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 minutes 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 commercial 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, Columbus, 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 under 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 treating 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. Misting periods and duration were the same as described for Experiment 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
Description of types of cuttings
ReferencesJohnson, E.G., Wu, J., Bright, D.B. and Graham, J.H.(2014) Association of “Candidatus Liberibacter Asiaticus” Root Infection, but not Phloem Plugging with Root Loss on Huaglongbing-Affected Trees Prior to Appearance of Foliar Symptoms. Plant Pathology, 63, 290-298. https://doi.org/10.1111/ppa.12109Grosser, J.W. (2013) Personal Communication, 20 June 2013. Verbal Communication of Non-Published Results.Conger, O.H. (1889) Lemon Culture. Annual Report of the Secretary of the State Board of Michigan, 361-365.Cooper W.C. ,et al. (1940)Rooting Citrus Cuttings with Synthetic Growth-Substances 53, 174-177.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, 248-251.Rieger M. ,et al. (1992)Growth, Gas Exchange, Water Uptake, and Drought Response of Seedling-and-Cutting Propagated Peach and Citrus Rootstocks 117, 834-840.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.Ferguson, J., Young, M. and Halvorson, J. (1985) The Propagation of Citrus Rootstocks by Stem Cuttings. Proceedings of the Florida State Horticulture Society, 98, 39-42.Seran, T.H. and Umadevi, T. (2011) Influence of Indole Acetic Acid (IAA) on the Establishment of Stem Cuttings in Lemon (Citrus limon L.). Journal of Agricultural Research, 49, 517-524.Beeson Jr. R.C. ,et al. (2000)Putting the Speed Back in Quick-dip Auxin Application 45, 298-302.Foster, G.S., Campbell, R.K. and Adams, W.T. (1984) Heritability, Gain, and C Effects in Rooting of Western Hemlock Cuttings. Canadian Journal of Forest Research, 14, 628-638. https://doi.org/10.1139/x84-114Macht, 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