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Carlos A. Lazcano, Fred T. Davies Jr., Sharon A. Duray, Andres Estrada-Luna, and Victor Olalde-Portugal

Mature cladodes of prickly-pear cactus (Opuntia amyclaea Tenore. cv. Reina) were treated with five wounding methods and four concentrations of potassium salt indolebutyric acid (K-IBA) to stimulate adventitious root formation. K-IBA from 4144 to 41,442 μm (1000 to 10,000 mg·L-1) increased root number and root dry weight; however, root length was decreased at 41,442 μm (10,000 mg·L-1). Root number and root dry weight were higher with wounding methods that had larger wounded surface areas. K-IBA altered rooting polarity and stimulated adventitious root formation along the wounded cladode surfaces. Treatments without suberization had a higher percentage of rotted cladodes. This research validates the commercial practice in Mexico of suberizing cladodes early in the propagation cycle. Auxin application could be of commercial benefit for enhanced rooting in the clonal regeneration of new selections for prickly-pear cactus orchards. The wounding methods and auxin treatments utilized make an excellent classroom demonstration for manipulating rooting polarity.

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Carole H. Saravitz, Frank A. Blazich, and Henry V. Amerson

Adventitious shoots developed on cotyledons of Virginia pine (Pinus virginiana Mill.) excised from seeds germinated for 3, 6, or 9 days and cultured on media containing 0.5 to 10 mg/liter benzyladenine (BA). Shoot regeneration was greatest (46 shoots per embryo) on cotyledons from seeds germinated for 6 days and placed on medium containing 10 mg/liter BA. Shoots were excised and elongated on medium lacking BA. Following elongation, shoots were placed on media containing 0 to 40 mg/liter indolebutyric acid (IBA) for 14 days followed by transfer to the same medium lacking auxin. Without IBA treatment, percent rooting was 3% and increased to 50% for concentrations of 5 to 40 mg/liter. Rooted shoots averaged 2.0 roots per shoot without auxin treatment, 3.3 roots when treated with 5 mg/liter IBA and root number increased linearly with increased IBA concentration up to 40 mg/liter (4.5 roots). Plant lets were transferred to growing medium and acclimated successfully to greenhouse conditions.

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Carole H. Saravitz, Frank A. Blazich, and Henry V. Amerson

Adventitious shoots developed on cotyledons of Virginia pine (Pinus virginiana Mill.) excised from seeds germinated for 3, 6, or 9 days and cultured on media containing 0.5 to 10 mg/liter benzyladenine (BA). Shoot regeneration was greatest (46 shoots per embryo) on cotyledons from seeds germinated for 6 days and placed on medium containing 10 mg/liter BA. Shoots were excised and elongated on medium lacking BA. Following elongation, shoots were placed on media containing 0 to 40 mg/liter indolebutyric acid (IBA) for 14 days followed by transfer to the same medium lacking auxin. Without IBA treatment, percent rooting was 3% and increased to 50% for concentrations of 5 to 40 mg/liter. Rooted shoots averaged 2.0 roots per shoot without auxin treatment, 3.3 roots when treated with 5 mg/liter IBA and root number increased linearly with increased IBA concentration up to 40 mg/liter (4.5 roots). Plant lets were transferred to growing medium and acclimated successfully to greenhouse conditions.

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Barbara M. Reed

Cultures of 49 Pyrus species and cultivars and one Pyronia (Pyrus × Cydonia hybrid) selection were screened in vitro to determine a rooting method suitable for a wide range of germplasm. Auxin treatment was required for rooting in most cases. Eighteen of the 50 accessions rooted with a 15 sec. 10 mM indole-3-butyric acid (IBA) dip followed by growth on medium with no growth regulators (NCR). Medium with 10 μM IBA for one week followed by NCR medium produced 12 rooted accessions, but NCR medium alone produced little or no rooting. A 15 sec. dip in 10 mM naphthaleneacetic acid (NAA) followed by NCR medium was tested on 29 accessions which rooted poorly on the other three treatments. Twice as many (28%) rooted on NAA as on either IBA treatment (14% each). Additional treatments combining IBA with darkness or higher temperature were also tested and were successful for some cultivars. P. calleryana, P. koehnei, P. pashia, P. hondoensis, P. ussuriensis, P. betulifolia, P. regelii, P. pyrifolia hybrid cv. Shinseiki and the Pyronia selection failed to root. Twenty two of the 32 P. communis cultivars rooted on at least one treatment.

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X. Wang, J.T.A. Proctor, S. Krishna Raj, and P.K. Saxena

Ginseng is a very valuable agricultural species grown for its root, which contains pharmacologically active constituents. One limiting factor for expansion of ginseng production is an efficient method for mass propagation. Currently, seeding is the principal method of propagating ginseng, but the embryo of ginseng seeds at harvest is immature. A stratification schedule consisting of a cool-warm-cool temperature treatment over 18-22 months is required for embryo development and seed germination. An alternative for the efficient production of ginseng is mass propagation through the use of in vitro culture techniques. The objective of this work was to develop a highly efficient system for regeneration of ginseng. The efficacy of three auxins, viz. 2,4-D, NAA and dicamba, were compared for the induction of somatic embryogenesis in American ginseng. Somatic embryos formed on ginseng cotyledonary, zygotic embryo, and shoot explants after 8 weeks of induction by the auxins. Significantly more somatic embryos were induced by culture of any of the ginseng explants on media supplemented with 5 μmol·L-1 2,4-D than any other auxin treatment. Histological and SEM studies confirmed that the regenerants were somatic embryos. Somatic embryos germinated and developed into normal plants in 3-6 months. The development of a regeneration system for ginseng using somatic embryogenesis is a necessary first step for mass propagation and the improvement of American ginseng.

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Barbara M. Reed

Micropropagated shoots of 49 Pyrus species and cultivars and one selection of Pyronia veitchii (Trabut) Guillaumin were evaluated to test their responses to several in vitro rooting techniques. Auxin treatment was required for rooting in most cases. Eighteen of 50 accessions rooted ≥50% with a 15-second, 10-mm IBA dip followed by growth on medium with no growth regulators (NGR). Twelve accessions rooted on a medium with 10 μm IBA applied for 1 week followed by NGR medium for 3 weeks; NGR medium alone was effective for only two accessions. Twenty-eight accessions rooted poorly with IBA treatments; an additional treatment of a 15-second dip in 10 mm NAA followed by NGR medium produced ≥50% rooting for eight genotypes. Root production increased for 10 of 19 especially recalcitrant genotypes by 10 μm IAA treatments in darkness or at 30C and NAA dip treatments. Of rooted shoots, 73% survived acclimation in the greenhouse. Selections of Pyrus betulifolia Bunge, P. calleryana Decne., P. hondoensis Kikuchi and Nakai, P. koehnei C. Schneider, P. pashia Buch.-Ham. ex D. Don, P. pyrifolia (Burm.f.) Nakai cv. Shinseiki, P. regelii Rheder, P. ussuriensis Maxim., and the Pyronia veitchii selection failed to root in any of the treatments. Twenty-five of 32 P. communis L. cultivars and three other species rooted on at least one of the treatments. Chemical names used: 1-naphthaleneacetic acid (NAA), 1H-indole-3-butyric acid (IBA), 1H-indole-3-acetic acid (IAA).

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April L. Warner and R. Daniel Lineberger

Cotyledon explants and zygotic embryos of Lycopersicon esculentum H132, OH8442, and OH2253 were cultured on Murashige and Skoog medium containing varying concentrations of 2,4-D and NAA with and without 10-7 M zeatin. NAA above 10-5 M and 2,4-D above 10-6 M inhibited root formation from cotyledons. Zygotic embryos were removed from developing ovules at the globular, heart, and torpedo stages and later germinated on hormone-free medium. Globular structures that resembled immature zygotic embryos were produced at NAA concentrations between 10-4 and 10-3 M and 2,4-D concentrations between 10-5 and 10-4 M. Treatments reported to enhance maturation and germination of somatic embryos of other species, including subculture to a hormone-free medium with and without activated charcoal, the addition of an ABA treatment subsequent to the auxin treatment, isolation of individual structures from the explant, and a liquid medium rinse containing activated charcoal, have not been successful in stimulating further development of the globular structures.

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Takuro Suyama, Kunio Yamada, Hitoshi Mori, Kiyotoshi Takeno, and Shohei Yamaki

A cDNA library was constructed from poly(A)+RNA extracted from pollinated fruit of `PMR-142' cucumber (Cucumis sativus L.). Subtraction hybridization was made between the cDNAs and poly(A)+RNA from unpollinated fruit to isolate cDNA clones that corresponded to the genes preferentially expressed in the pollinated fruit. We isolated three cDNAs, which were 756, 826, and 998 nucleotides long and designated Csf1, Csf2, and Csf3, respectively. When fruit growth was triggered by pollination, auxin treatment and natural parthenocarpy, Csf2 was always expressed. Time course of expression of the Csf2 gene was nearly parallel to that of the fruit growth. Nucleotide sequences of the Csf cDNAs were fully determined. Homology of the deduced amino acid sequence for Csf1 showed 75% identity with a pea extensin. Only 37%, 33%, and 26% homology was found between Csf2 and bell pepper CaSn-2, tobacco FB7-4, and opium poppy gMLP15, respectively. The Csf3 sequence showed 68% identity with the large subunit of 60S ribosomal protein L3 of Arabidopsis thaliana.

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William M. Proebsting

Douglas-fir clones have a wide range of rooting potential, but the species is generally considered difficult to root. We have reported previously that NAA is approximately 14-times more active than IBA in the clones tested, with an optimum of about 5 to 10 mM NAA. In contrast, other programs routinely use about 25 mM IBA to propagate Douglas-fir cuttings, a concentration that is relatively inactive in our clones. To address questions raised by these observations, we have incorporated auxin treatments into our long term program to select Douglas-fir clones with high rooting potential. We collect 20 cuttings of each clone identified in Christmas tree plantations, and retain clones rooting ≥ 80%. Beginning in 1991, we treated 10 cuttings of each clone with 5 mM NAA, the other 10 cuttings with 25 mM IBA. Over three years, 1158 clones received the split treatments. Of 222 clones rooting ≥ 80%) approximately half rooted ≥ 80% in response to NAA only. The remainder either responded to IBA or to both NAA and IBA. These results support our previous observations that NAA stimulates rooting of Douglas-fir better than IBA. However, they also suggest that there may be clones sensitive to IBA or to both NAA and IBA.

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Stephen B. Ryu and Jiwan P. Palta

Lipids have been thought to be important largely in membrane structure and energy reserve. It is now evident that lipids and lipid-derived metabolites play a role in many critical cellular processes. Recent studies have shown that membrane lipid-based signaling mediated by phospholipases such as phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD) constitutes a crucial step in plant responses to abiotic and biotic stresses. Phospholipases and their products also play a role during plant growth and development. For example, PLA2-derived lysophospholipids acted as growth regulators that retard senescence of plant tissues. Interestingly, the PLA2 products inhibited the activity of PLD, which has been suggested to be a key enzyme responsible for membrane lipid breakdown leading to plant senescence. Endogenous levels of lysophospholipids, such as lysophosphatidylethanolamine (LPE), could be increased in castor bean leaf discs by the treatment of auxin (50 μM), which is known to be a activator of PLA2. Pretreatment of leaf discs with a PLA2 inhibitor before auxin treatment nullified the auxin effect and rather resulted in accelerated senescence even compared to the nontreated control. Our recent results suggest a potential role of PLA2 products as biologically active molecules mediating hormonal regulation of growth and senescence. One such product LPE is being commercially exploited for retarding senescence and improving shelf life of fruits, vegetables, and cut flowers.