of a 12-year-old tree. Amer. J. Bet. 75:767– (Engl. trans.) Plantlet formation from embryonic tissue. New 777. Owens, J.N. and M. Molder. 1979. Bud de- Zealand J. For. Sci. 7:199–206. Momot, T.S. and A.M. Smirnov. 1978. Or- velopment in Larix occidentals. 1. Growth Thompson, D.G. and J.B. Zaerr. 1981. In- ganogenesis from various organs of the Si- and development of vegetative long shoot and duction of adventitious buds on cultured shoot berian and Dahur larch (Larix sibirica Maxim. vegetative short shoot buds. Can. J. Bet. tips of Douglas-fir (Pseudotsuga menziesii and L. dahurica Turcz.) and yellow pine (Pi- 57(7):687-700. (Mirb.)Franco). Colloque Intl. sur la Culture nus ponderosa Dougl.) cultured in vitro. Izv. Reilly, K. and J. Washer. 1977. Vegetative “in vitro” des essences Forestières. Foun- Acad. Nauk. SSSR. Ser. B. 5(6):758-761. propagation of radiata pine by tissue culture: tainbleau, France. HORTSCIENCE 25(1):116-118. 1990. Objectives of this study were to: 1) deter- mine if unsuberized roots (Sutton and Tinus, Use of Dyes to Facilitate Measurement 1983) encompassed all the newly regener- ated roots within the first 15 to 30 days after of New Root Growth of Apple planting; 2) evaluate the effectiveness of se- 1 2 lected dyes for distinguishing between newly Michael A. Arnold and Eric Young regenerated roots and those present at plant- Department of Horticultural Science, North Carolina State University, ing; and 3) determine if the dyes had any Raleigh, NC 27695-7609 effects on plant growth and development. Fully chilled, dormant, l-year-old ‘Star- Additional index words. Malus domestics, safranin-O, methylene blue, neutral red, krimson’ on seedling rootstock were root- crystal violet, alizarin red S, root regeneration potential, root regeneration capacity pruned to 20 cm below the root collar and Abstract. The root systems of dormant l-year-old apple (Malus domestics Borkh. dipped for 15 sec in one of six dye treatments ‘Starkrimson’) grafted on seedling rootstock were dipped for 15 seconds in one of six or a water control. Dye treatments included dye treatments or a water control. Trees were destructively harvested at 15, 30, and safranin-O (basic red 2), methylene blue (basic 90 days after treatment to monitor new root and shoot growth and effectiveness of dyes blue 9), neutral red (toluylene red), crystal for distinguishing between regenerated roots and those present at planting. Neutral violet (basic violet 3, gentian violet, methyl red and methylene blue allowed effective separation of new and old roots through 90 violet), alizarin red S (all obtained from days after treatment, and had few adverse effects on root or shoot growth, with a Sigma, St. Louis) and Hantover meat brand- water control being the standard. Crystal violet and safranin-O allowed effective sep- ing ink (Hantover, Kansas City, Me. ) dis- aration between regenerated and previously existing roots, but these dyes adversely solved in water at 1% (w/v) [as used by affected new root and shoot growth. Alizarin red S and Hantover meat branding ink Kaufmann, (1968)]. Safranin-O and crystal were ineffective for distinguishing between regenerated and previously existing roots violet (methyl violet) were chosen based on by 15 days after treatment. Kaufmann’s (1968) reported success and neutral red based on Schumacher et al. (1983). Methylene blue and alizarin red S were in- In studies involving determination of root indistinguishable from older existing roots. cluded because they can stain cutinized cell regeneration potential (RRP), or root regen- In apple, this change may occur within a walls and they have high volubility in water, eration capacity (RRC), it is important to week under certain environmental conditions respectively (Johansen, 1940). Hantover meat distinguish between regenerated and previ- (Rogers, 1939). branding ink was selected because it was in- ously existing roots. RRP or RRC consists Schumacher et al. (1983) found that neu- expensive and, based on its use as a meat of two components, elongation “of existing tral red dye at several concentrations was an dye, nontoxic to humans. roots, and/or initiation of new adventitious effective and non-toxic root dye for Phas- Root systems were placed between moist roots (Sutton and Tinus, 1983). RRP or RRC eolus vulgaris L. and Glycine max L. How- paper towels for 10 min to allow excess dye is usually estimated by measuring the white ever, their studies were brief (6 to 12 days) to drain from the root system. On 21 Apr. root tips (number, length, weight) appearing and involved only recently germinated her- 1988, 15 seedlings of each dye treatment and under specified environmental conditions over baceous seedlings. Carman (1982) devel- the control were potted in Turface (calcined a certain period (Sutton and Tinus, 1983). oped a nondestructive staining technique to clay) medium in 3.5-liter containers and Such measurements may bean unambiguous measure new root growth over time using placed in a completely randomized design on means of estimating root regeneration over individual soil drenches with red, blue, and a greenhouse bench. Plants were watered as short periods. However, over longer periods, yellow chlorotiazinyl dyes. However, this needed and fertilized weekly with 125 ppm regenerated roots may undergo metacutiza- technique is limited to container studies using N from 20N–8.7P-16.6K Peters water-sol- tion and root browning, therefore becoming porous media and mild staining conditions. uble fertilizer (W.R. Grace, Fogekville, Pa.). The effectiveness of Carman’s technique for Temperatures were set at 24/18C day/night studying woody plant root growth is un- with natural photoperiods. Received for publication 28 Nov. 1988. Paper no. 11924 of the Journal Series of the North Carolina known, as the study was also limited to re- Five seedlings of each treatment and con- Agricultural Research Service, Raleigh, NC 27695_ cently germinated herbaceous seedlings of trol were destructively harvested after 15, 7601. This study will be included as part of a Sorghum bicolor (L.) Moench. Kaufmann 30, and 90 days. Shoot characteristics mea- dissertation to be written in partial fulfillment of (1968) tested neutral red, orange H, India sured included new and old total shoot lengths, the requirements for the PhD degree by M.A.A. black ink, safranin-O, and methyl violet as new and old shoot fresh weights, new shoot The use of trade names in this publication does root dyes on Pinus echinata Mill. Only saf- dry weights, total number of vegetative buds, not imply endorsement by the NCARS of the ranin-O and methyl violet were considered number of buds showing any green (bud- products named, nor criticism of similar ones not effective for separating old from new roots. break), and number of buds elongating ( ³ 3 mentioned. The cost of publishing this paper was While Kaufmann observed no apparent tox- mm). Measured root characteristics were old defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must icity problems, he cautioned that further root fresh weight, new root fresh and dry be hereby marked advertisement solely to indicate evaluation was needed before making large- weights, number and total length of roots this fact. scale use of this technique. Additionally, no ³ 0.6 mm in diameter (large roots, days 15 ‘Graduate Research Assistant. data were presented on growth effects of the and 30 only), number of roots < 0.6 mm in ‘Professor. dyes. diameter (small roots, days 15 and 30 only). 116 HORTSCIENCE, VOL. 25(1), JANUARY 1990 Table 1. Root and shoot growth at 15 days after dye treatment of the root system of dormant ‘Starkrimson’ on seedling rootstock in year 1. z zValues are means of five observations. yDiameter; small, < 0.6 mm; large, ³ 0.6 mm. *,**,***Significantly different from the control at P £ 0.20, 0.10, or 0.05, respectively. A random, sample of 10 diameters of large surface, 1 cm behind the pruned surface, the scribed previously. After 15 days, growth roots for each plant was measured under a midpoint of the root, and at the graft union. characteristics were measured and analyzed dissecting microscope for calculations of large The bark was stripped from the remaining as described previously. root surface area. New root : new shoot ra- primary root sections to visually determine Unsuberized, white, and-light brown roots tios, percent budbreak (number of buds bro- the extent of dye uptake and staining. and roots originating from pruned surfaces ken/total number of buds), and large root To achieve greater precision in estimating were assumed to be newly regenerated tis- surface area [3.14 r (r 2 + new root the effects of the two most promising dyes sue. Using this premise, new roots on the length 2) ½; days 15 and 30 only] were cal- on initial root and shoot growth, the growth controls were distinguished easily from old culated. Means were separated using a least- portions of the experiment were repeated (5 roots on the basis of degree of suberization, squares means procedure (SAS Institute, May 1989) for the control, methylene blue, browning, and position at 15 and 30 days 1985) because one plant at day 90 was miss- and neutral red treatments using 15 plants after planting. Use of the above criteria to ing. per treatment. The plants were placed in a distinguish new root growth for controls dur- Following each harvest, cross-sections of randomized complete-block design in the ing the first 30 days after treatment showed the primary root were made at the pruned greenhouse with growth conditions as de- no under- or over-estimation of new root tis- sue compared to the less-ambiguous dye treatment criteria (Tables 1-3).