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James F. Harbage and Dennis P. Stimart

Many physiological responses in plants are influenced by pH. The present chemiosmotic hypothesis suggests that auxin uptake into plant cells is governed by pH. Since auxin is used widely to enhance rooting, the influence of pH on 1H-indole-3-butyric acid (IBA) induced adventitious root formation was examined. Roots were initiated aseptically in 5 node apical shoot cuttings of micropropagated Malus domestica 'Gala'. Initiation was induced using a four day pulse in IBA and 15 g/L sucrose at pH 5.6 and 30C in the dark. Observations showed pH rose to 7.0 or greater within 1 to 2 days from microcutting placement in unbuffered initiation medium. Root numbers from shoots in media containing 1.5 μM IBA buffered with 10 mM 2[N-morpholino] ethanesulfonic acid (MES) to pH 5.5, 6.0, 6.5 or 7.0 with KOH resulted in average root numbers of 14.2, 10.9, 8.7, and 7.1, respectively, while unbuffered medium yielded 7,6 roots per shoot. Comparison of MES buffered medium at pH 5.5, 6.25 or 7.0 in factorial combination with IBA at 0, 0.15, 1.5, 15.0, and 150.0 μM resulted in a significant pH by IBA interaction for root number. At 0, 0.15 and 1.5 μM IBA root numbers were greatest at pH 5.5. At 15.0 μM IBA, pH 6.25 was optimal and at 150.0 μM IBA all three pH levels produced equivalent root numbers. A calorimetric assay to measure IBA removal from the initiation medium by microcuttings of `Gala' and `Triple Red Delicious' showed more IBA removal at pH 5.5 than at pH 7.0. Possible reasons for the effect of pH on adventitious root formation will be discussed.

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A.D. Bryan, Z. Pesic-VanEsbroeck, J.R. Schultheis, K.V. Pecota, W.H. Swallow, and G.C. Yencho

Decline in sweetpotato yield and storage root quality has been attributed to the accumulation of viruses, pathogens and mutations. To document the effects of decline on yield and storage root quality, two micropropagated, virus-indexed, greenhouse produced G1 `Beauregard' meristem-tip cultured clones, B94-14 and B94-34, were compared with 1) micropropagated B94-14 and B94-34 clones propagated adventitiously up to five years in the field (G2, G3, G4, G5); and 2) nonmicropropagated, unimproved stock of `Beauregard' seed in field trials during 1997 to 2001. At least three trials were located each year in sweetpotato producing regions in North Carolina. In 2000 and 2001, two trials were monitored weekly for foliar symptoms of Sweet potato feathery mottle virus (SPFMV) and other potyviruses, and virus-indexed for selected viruses using Ipomoea setosa and nitrocellulose enzyme linked immunosorbant assays (NCM-ELISA). Only SPFMV was detected in field samples using NCM-ELISA, but this does not rule out the presence of newly described viruses infecting sweetpotato for which tests were unavailable. Monitoring indicated that all G1 plants became infected with SPFMV by the end of the growing season, and that G2 to G5 plants were probably infected in their initial growing season. G1 plants consistently produced higher total yield, total marketable yield (TMY), U.S. No. 1 root yield and percent No. 1 yield than G2 to G5 plants. G1 plants also produced storage roots with more uniform shapes and better overall appearance than storage roots produced from G2 to G5 plants. Also, G2 to G5 storage roots tended to be longer than G1 storage roots. Rank mean yield and storage root quality measurements of each location were consistent with means averaged over locations per year and suggested a decrease in yield and storage root quality with successive seasons of adventitious propagation. Linear regression analysis used to model yield and storage root quality measurements of seed generations G1 to G5 indicated that total yield, TMY, No. 1 yield, percent No. 1 yield, shape uniformity, and overall appearance decreased gradually, and that length/diameter ratios increased gradually with generation. The rate of decline in No. 1 yield was greater for B94-34 compared to B94-14. Both viruses and mutations of adventitious sprouts arising from storage roots probably contribute to cultivar decline in sweetpotato, but further studies are needed to determine their relative importance. A simple profitability analysis for G1 vs. G2-G4 planting material conducted to facilitate better understanding of the economics of using micropropagated planting material to produce a crop in North Carolina revealed that growers have a potential net return of $2203/ha for G1 plants, $5030/ha for G2 plants, and $4394/ha for G5 plants. Thus, while G1 plants generally produce higher No. 1 yields, a greater monetary return can be achieved using G2 planting materials because of the high costs associated with producing G1 plants. Based on this analysis, the best returns are accrued when growers plant their crop using G2 and/or G3 seed.

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Paula M. Pijut

Butternut (Juglans cinerea L.), a native hardwood to the northeastern United States, is a valuable species for its wood and edible nuts. Butternut is becoming endangered in its native range as a result of a virulent fungal (perennial canker) pathogen, Sirococcus clavigignenti - juglandacearum. Micropropagation techniques are being developed to clone disease-resistant specimens. Axillary buds, obtained from 2-3-month old seedlings, were induced to break buds in vitro and form a single shoot when cultured on Murashige and Skoog (MS) medium supplemented with 200 mg/l casein hydrolysate, 3% sucrose, and 2 mg/l 6-benzylaminopurine. Roots were initiated on microshoots when cultured on half-strength MS medium containing 100 mg/l casein hydrolysate, 1.5% sucrose, and 0.5 mg/l indole-3-butyric acid for seven days in the dark. Adventitious roots elongated when shoots were placed in the light on the same medium, but with 2% sucrose, and no growth regulators. Rooted plantlets were successfully acclimated ex vitro. These results provide a basis for the development of techniques to micropropagate selected, mature, disease-resistant butternut germ plasm.

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Christopher L. Rosier, John Frampton, Barry Goldfarb, Farrell C. Wise, and Frank A. Blazich

Seven concentrations of indole-3-butyric acid (IBA), seven concentrations of 1-naphthaleneacetic acid (NAA), and a nonauxin control were tested over three growth stages to determine their ability to promote adventitious rooting of stem cuttings from 3- and 4-year-old stock plants of virginia pine (Pinus virginiana Mill.). Cuttings were harvested September 2000 (semi-hardwood), February 2001 (hardwood), June 2001 (softwood), and October 2001 (semi-hardwood), treated with auxin concentrations ranging from 0 to 64 mm and placed under intermittent mist in a greenhouse. Rooting percentage, percent mortality, number of primary roots, total root length, root symmetry, root angle, and root diameter were assessed following 16 weeks. Growth stage affected every rooting trait measured except root symmetry and diameter. Auxin type affected total root length and root diameter, while auxin concentration affected every rooting trait except root angle. The highest predicted rooting percentages (46%) occurred when semi-hardwood cuttings were collected in September 2000 and treated with 7 mm auxin. Cuttings collected within the same growing season (2001) exhibited the highest predicted rooting percentage (33%) when softwood cuttings were treated with 6 mm auxin. Semi-hardwood cuttings rooted in 2001 produced the greatest number of roots and root lengths. Root diameter was significantly greater when NAA rather than IBA was applied, especially at higher concentrations.

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Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne

A new in vitro protocol was developed for multiple bud induction and plant regeneration from embryonic axis explants of four common bean (Phaseolus vulgaris L.) and two tepary bean (P. acutifolius A. Gray) lines. The explants were prepared from two embryo sizes, 3 to 4 mm and 5 to 7 mm, corresponding to pods collected after 15 and 25 days from flowering, respectively. The embryonic axis was cultured on Gamborg's B5 basal medium with 0, 5, 10, or 20 μm BA in combinations with 0, 1, or 2 μm NAA. The cultures were maintained at 24 to 25C under continuous light or incubated in darkness for 2 weeks followed by continuous light before transfer to the secondary B5 medium (0 or 2 μm BA or 2 μm BA plus 4 μm GA3). Adventitious roots or a single shoot with roots formed on the explants cultured on media without plant growth regulators. Multiple buds were induced on all BA media, but more were produced with 5 or 10 μm for most lines. Dark incubation greatly enhanced multiple bud initiation. Shoot buds were not produced on media containing NAA alone or in combinations with BA. On the secondary medium, six to eight shoots per explant for common bean and up to 20 shoots per explant from tepary bean were observed after 3 weeks. Mature, fertile plants were produced from these shoots. Chemical names used: benzyladenine (BA); 1-naphthaleneacetic acid (NAA); gibberellic acid (GA3).

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Fan Cao, Xinwang Wang, Zhuangzhuang Liu, Yongrong Li, and Fangren Peng

; Fouad et al., 1992 ; Smith and Chiu, 1980 ; Wolstenholme and Allan, 1975 ) to induce roots for pecan stem cuttings. To understand adventitious root formation, the anatomy of roots for pecan stem cuttings has been studied ( Brutsch et al., 1977 ; Fayek

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E.K. Gubrium, D.G. Clark, H.J. Klee, T.A. Nell, and J.E. Barrett

We are studying the horticultural performance of two model plant systems that carry a mutant gene that confers ethylene-insensitivity: Never Ripe tomatoes and petunia plants transformed with the mutant etr1-1 gene isolated from Arabidopsis thaliana. Having two model systems to compare side-by-side allows us to determine with greater certainty ethylene's role at different developmental stages. Presence of the mutant etr1-1 gene in transgenic petunias was determined using three techniques: PCR analysis, the seedling triple response assay (inhibition of stem elongation, radial swelling of stem and roots, and an exaggerated apical hook when grown in the dark and in the presence of ethylene), and the flower wilting response to pollination, which is known to be induced by ethylene. Flowers from ethylene-insensitive petunias took almost four times as long to wilt after pollination as wild-type plants. It is well known that fruit ripening in Never Ripe tomato is inhibited, and a similar delayed fruit ripening phenotype is observed in petunia plants transformed with etr1-1. In an effort to maintain ethylene-insensitive petunia plants by vegetative propagation, we observed that the rate of adventitious root formation was much lower with transgenic plants than in wild-type plants. In subsequent experiments on adventitious root formation in Never Ripe tomato, we observed the same result. Therefore, while ethylene-insensitive tomato and petunia plants appear phenotypically normal for many characters, other factors are altered by the presence of this mutation. The fact that these changes are present in two model systems helps to define the role of ethylene perception in plant growth and reproduction.

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Tian Gong, Xin Zhao, Ashwin Sharma, Jeffrey K. Brecht, and James Colee

determine how low RH healing conditions impact the growth and development of grafted plants compared with grafts healed under standard healing conditions. An extended period under high humidity also promotes the formation of adventitious roots from the

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Zhen Zhang and Zong-Ming Cheng

Jasmonates are a group of native plant bioregulators that occur widely in the plant kingdom and exert various physiological activities when applied exogenously to plants. We investigated the effect of free jasmonic acid (JA) on stem and root growth and tuberization of potato in vitro nodal culture. Nodal cuttings of three potato cultivars, Norchip, Red Pontiac, and Russet Burbank, were cultured in 2.5 × 15 cm test tubes containing either nodal culture (MS with 2% sucrose) or tuber-inducing (MS with 8% sucrose and 11.5 μm kinetin) medium. The media were supplemented with JA at 0, 0.1, 0.5 1.0, 5.0, 10.0 and 50 m. The cultures were maintained under a 16-hour photoperiod at 24°C for 6 weeks. Potato cultivars showed different sensitivities to JA in stem growth. Norchip is the most and Red Pontiac the least sensitive cultivar. On the nodal culture medium, stem length of Norchip was promoted at 0.1–5 μm, and inhibited at 10–50 μm of JA, but that of Red Pontiac was promoted by JA at all concentrations tested. The number of nodes increased significantly on media with JA than that on medium without JA. The number of adventitious roots did not, but the lateral roots increased significantly when JA was added to the medium. On tuber-inducing media, stem length and node number did not appear to be affected by addition of JA to the medium. The number of axillary shoots increased significantly on the media with low concentrations of JA (0.1–5 μm). No microtubers formed on both media from all three cultivars in 6 weeks.

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James F. Harbage, Dennis P. Stimart, and Ray F. Evert

Anatomical events of adventitious root formation in response to root induction medium, observing changes during induction and post-induction stages, were made with microcuttings of `Gala' apples. Shoot explants on root induction medium containing water, 1.5 μm IBA, 44 mm sucrose, or 1.5 μm IBA + 44 mm sucrose after 4 days of treatment averaged 0, 0.2, 2.2, and 11.9 meristemoids per microcutting, respectively. Meristemoids formed in response to sucrose were confined to leaf gaps and traces. Time-course analysis of root induction with 1.5 μm IBA + 44 mm sucrose over 4 days revealed that some phloem parenchyma cells became densely cytoplasmic, having nuclei with prominent nucleoli within 1 day; meristematic activity in the phloem was widespread by 2 days; continued division of phloem parenchyma cells advanced into the cortex by 3 days; and that identifiable root primordia were present by 4 days. Cell division of pith, vascular cambium, and cortex did not lead to primordia formation. Meristematic activity was confined to the basal 1 mm of microcuttings. Time-course analysis of post-induction treatment revealed differentiation of distinct cell layers at the distal end of primordia by 1 day; primordia with a conical shape and several cell layers at the distal end by 2 to 3 days; roots with organized tissue systems emerging from the stem by 4 days; and numerous emerged roots by 6 days. Root initiation was detectable within 24 hours and completed by day 4 of the root induction treatment and involved only phloem parenchyma cells. Chemical names used: 1 H -indole3-butryic acid (IBA).