Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).
Srini C. Perera and Peggy Ozias-Akins
Sarah E. Bruce and D. Bradley Rowe
Propagation failures of Taxus are often attributed to cutting collection from stock plants of poor quality. If a quick, reliable method of determining the potential rooting of cuttings based on the condition of a specific stock plant was available for propagators, rooting success could be predicted before investing time, labor, and resources. Our studies examined chlorophyll fluorescence (Fv/Fm) as a potential tool for stock plant selection, assessment of storage conditions, and measurement of stress over the course of propagation. Ten cultivars of Taxus × media (Taxus baccata L. × T. cuspidata Sieb. & Zucc.) were used: Brownii, Dark Green Pyramidalis, Dark Green Spreader, Densiformis, Densiformis Gem, Hicksii, L.C. Bobbink, Runyan, Tauntoni, and Wardii. Storage condition treatments consisted of desiccation (low, medium, and high), duration (34, 70, and 107 days), and temperature (–30, –2.5, 0, 2.5, 5, 10, and 20 °C). Cultivars differed in Fv/Fm initially as well as over time. Correlations were not found between initial stock plant Fv/Fm and rooting percentage, number of roots, root dry weight, or root length, indicating that Fv/Fm is not a reliable indicator of stock plant propagation potential. Short storage duration at –2.5 to 2.5 °C was found to be ideal. Fv/Fm could detect substandard storage conditions only at temperature and desiccation extremes. Although chlorophyll fluorescence measurements do not appear to be a practical method of predicting adventitious rooting, there is a potential for assessing cutting or plant quality before shipping.
Brian K. Maynard and Nina L. Bassuk
Three experiments were undertaken to examine the effect of stockplant etiolation, shading, and stem banding, prior to cutting propagation, on the auxin dose-response of rooting in stem cuttings of Carpinus betulus `fastigiata'. A 2 × 2 factorial of etiolation and banding utilized stockplants forced in a greenhouse, etiolated for 1 week and banded with Velcro™ for 1 month. In a separate study shading was applied up the time of harvesting cuttings. IBA was applied to cuttings as an aqueous ethanol quick dip in concentrations ranging from 0 to 80 mM. Rooting percentage and number were best described, up to a peak response, by a linear function proportional to the logarithm of applied IBA. The inhibition of rooting by supra-optimal IBA was directly proportional to IBA concentration. Cuttings prepared from shoots which had been etiolated or banded rooted better at low IBA and at their respective optimal IBA levels. Cuttings from shoots receiving both etiolation and banding yielded higher rooting percentages and more roots per rooted cutting on average. Etiolation and banding served to increase both initial and maximum rooting capacities, and to reduce the sensitivity of cuttings to supraoptimal auxin-induced inhibition of adventitious root initiation. The auxin dose-response interacted with shading to yield the best rooting at 95% shade and 3.7 mM IBA.
Francis H. Witham, Charles W. Heuser, and Jun Chen
Ethidium bromide (EB), at 10-5 to 10-4 M, progressively inhibits NAA-induced rooting of mung bean cuttings. Cycloheximide (CH), 6-methylpurine (6-MP) and kinetin (KIN) also inhibited rooting at the same concentrations, although CH and 6-MP were more effective.
At 70 and up to 130 hours of incubation, after cuttings received a 1-ml pulse of NAA (10-4 M), they exhibited a progressive increase in the number of observed adventitious roots. The addition of one of the inhibitors, 6-MP, EB or KIN to cuttings, pulsed 48 hours earlier with NAA, showed an initial slight inhibition with increased inhibition over time. CH, however, inhibited rooting immediately after addition. From these and other similar kinetic studies, it appears that 6-MP, EB and KIN operate at the transcriptional level and that CH inhibits translation.
Lineweaver-Burk plot analysis of NAA-induced rooting inhibition showed that EB may act as a competitive inhibitor of NAA. Since EB is a known intercalating agent and competitively inhibits NAA-induced rooting, NAA may influence gene expression by ultimately binding to DNA. Studies with space-filling and computer-generated models show that both NAA and EB can bind to certain dinucleotides by an intercalation mechanism.
S. Guzman, H. Alejandro, J. Farias, A. Michel, and G. Lopez
Watermelon (Citrullus vulgaris Schrad.) is a widely grown crop throughout the tropics and subtropics. In Mexico, it is an economically important crop. In vitro adventitious shoot regeneration of watermelon has been reported from shoot tip culture, leaf, hypocotyl, and cotyledons. Hence, the objective of this study was to evaluate in vitro plant regeneration from axillary buds of triploid watermelon. Axillary buds explants were prepared from shoot of commercial cultivar in field of 60 old day plants. Explants of 2 to 3 mm were incubated 2 weeks on Murashige and Skoog (MS) shoot regeneration medium containing 2.5 mg/L kinetin (KT) or indole-3-butyric acid (IBA), or gibberellic acid (GA3), followed by 3 weeks on shoot elongation medium supplemented with different combinations of the same phytohormones. The percentage of explants (83% to 90%) that produced shoots, expansion in size of explant (0.81–1 cm) and shoot length (6 mm) were highest in MS medium containing KT or IBA. In the shoot elongation step, shoot length (0.9–1 cm) and leaves number (6–7) were highest in MS medium supplemented with 2.5 mg/L of KT or GA3 and 0.2 mg/L IBA, but the better induction of roots in elongated shoot occurred on MS medium with 2.5 mg/L KT and 0.2 mg/L IBA. The results show that axillary buds from watermelon is an alternative for the micropropagation of this crop.
William B. Thompson, Jonathan R. Schultheis, Sushila Chaudhari, David W. Monks, Katherine M. Jennings, and Garry L. Grabow
is thought by many North Carolina growers to allow for transplants to produce adventitious roots before planting in the production field (J. Jones, personal communication). These growers believe that transplants that have been held to initiate
Mohamed S. Elmongy, Xiuyun Wang, Hong Zhou, and Yiping Xia
root formation ( Davies et al., 1994 ). The roles of exogenously applied auxins and HA in the formation of adventitious roots have been previously described ( Zandonadi et al., 2007 ). In our previous study, we demonstrated the effectiveness of HA and
Takahiro Tezuka, Masashi Harada, Masahumi Johkan, Satoshi Yamasaki, Hideyuki Tanaka, and Masayuki Oda
’ developed in Fukui Prefecture ( Sato et al., 2009 ). However, the efficiency of vegetative propagation by traditional methods is insufficient for mass propagation of plantlets. Adventitious shoots can be regenerated from cut surfaces of hypocotyls or stems
J. Naalamle Amissah, Dominick J. Paolillo Jr, and Nina Bassuk
sclerenchyma ring as a barrier to the emergence of adventitious roots, it is a known fact that where extensive sclerification takes place within the cortex, rooting generally does not occur ( Maynard and Bassuk, 1996 ; White and Lovell, 1984 ). However, it is
L. Xu, G.F. Liu, and M.Z. Bao
microshoots after culture for 8 weeks (bar, 5 mm). ( E ) Plant of L. formosana , 40 d after being transplanted to soil (bar, 1 cm). For P6, no adventitious shoots were obtained on WPM without PGR, but a few roots developed (data not shown