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.
Carole H. Saravitz, Frank A. Blazich, and Henry V. Amerson
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.
Veronique Declerck and Schuyler S. Korban
Leaf segments of Prunus persica L. (peach) collected from greenhouse-grown plants and from micropropagated shoots were cultured on a basal medium containing half-strength Murashige and Skoog (MS), Staba vitamins, sucrose (30 g/1) and agar (6.5 g/l); medium adjusted to pH 5.6. The influence of 6 different growth regulators at 3 concentrations (5, 10, 15 μM) were investigated using leaf explants from proliferating shoots of 'Elberta Queen' peach. With thidiazuron (TDZ), compact and multiple green calli were obtained; with benzyladenine and zeatin, lower numbers of small sized calli were obtained; with kinetin, no callus development was observed. Among auxin treatments, both Dicamba and 2,4-D resulted in friable white and yellow calli. Most of the calli produced in all treatments were formed along the cut margins of the explants. In an another experiment, leaf explants of' Bellaire' (greenhouse) and `Elberta Queen' (in vitro shoots) were used to determine the influence of a large scale concentration of TDZ (3 to 23 |iM). Explants from greenhouse and in vitro leaves resulted in higher levels of callus development at TDZ concentrations of 8-13 μM. Higher TDZ levels resulted in necrosis of leaf explants. The-influence of different carbon sources on callogenesis was investigated. We observed more green and compact calli with glucose than with sucrose and fructose at 100 mM. The influence of the glucose at 10 different concentrations (30 to 300 mM) was also investigated.
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).
Jason J. Griffin and F. Todd Lasseigne
The snowbells (Styrax L.) are a group of flowering shrubs and trees distributed throughout the warm-temperate regions of the northern hemisphere. In all, there are about 120 species, of which only Styrax japonicus Sieb. & Zucc. (Japanese snowbell) and its cultivars are currently of commercial significance. Other species may also posses desirable horticultural traits that could be valuable on their own merit, or used in plant improvement programs. Currently there is little information regarding asexual propagation of the lesser known species. The results herein show that propagation of a diverse collection of Styrax(15 taxa) is possible by stem cuttings. However, species and cultivars within a species do not respond to auxin treatment similarly. The percentage of rooting of many taxa was improved when cuttings were treated with 3000 or 8000 ppm (0.3% or 0.8%) of the potassium salt of indolebutyric acid (K-IBA). However, rooting was unaffected by K-IBA treatment in some taxa, while rooting was negatively affected by K-IBA in others. Additionally, the number of roots produced per rooted cutting were affected by K-IBA treatment. In some instances, K-IBA increased the number of roots per rooted cutting. However, in most of the taxa, root number was unaffected.
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.
Sunghee Guak, Norman E. Looney, and Leslie H. Fuchigami
We propose that return flowering of `Fuji' apple can be improved if sufficient flower clusters are removed during or shortly after bloom. In this study conducted at Corvallis, Ore., we evaluated two synthetic auxins, MCPB-ethyl and the Na salt of NAA, each at 0, 4, 8 and 16 ppm, as blossom cluster thinners. Each auxin treatment was applied alone or with 100 ppm ethephon as a tank mix. Six-year-old `Fuji'/M.26 trees were sprayed at full bloom of the king flowers (≈85% of whole-tree full bloom). A follow-up treatment of Sevin XLR (800 ppm carbaryl) was made at 11-mm fruit diameter to determine if carbaryl's known effectiveness as a fruitlet thinner was influenced by the bloom-time auxin or auxin + ethephon treatments. MCPB-ethyl proved ineffective as a bloom-time thinner, whereas the NAA effect on cluster removal was linear with concentration, 16 ppm NAA completely defruiting 33% of initial flower clusters. On control trees fewer than 12% of flowering clusters failed to set fruit. Ethephon alone defruited 25% of the clusters and NAA+ethephon defruited 51% of clusters. It is notable that the NAA and ethephon + NAA treatments did not reduce fruit set on the remaining clusters, resulting in considerable need for hand-thinning. Carbaryl effectively reduced total crop load by increasing the number of defruited clusters and reducing the incidence of doubles and triples. There was evidence to suggest that its effectiveness was compromised by the bloom-time NAA and/or ethephon sprays.
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.
Carlos A. Lazcano, Fred T. Davies Jr., Andrés A. Estrada-Luna, Sharon A. Duray, and Victor Olalde-Portugal
Mature cladodes of prickly-pear cactus (Opuntia amyclaea Tenore. `Reina') were treated with five wounding methods and four concentrations of potassium salt indole-3-butyric acid (K-IBA) to stimulate adventitious root formation. The wounding method and K-IBA had highly significant effects on root number and root dry mass of cladodes. Interaction between K-IBA and wounding methods showed that greater root number was obtained at the higher auxin concentrations and with wounding methods that had the greatest cut surface area. K-IBA concentrations from 4,144 to 41,442 μm (1,000 to 10,000 mg·L-1) increased root dry mass. Only the wounding method affected rotting of cladodes. Treatments allowing suberization had a higher percentage of nonrotted cladodes. This research validates the commercial practice of allowing cladodes to suberize early in the propagation cycle. K-IBA altered rooting polarity and stimulated adventitious root formation along the wounded cladode surfaces. The vertical nonsuberized wounding methods and auxin treatments are an excellent classroom demonstration for manipulating rooting polarity. Auxin application and wounding could be of commercial benefit for enhanced rooting in the clonal regeneration of new selections for prickly-pear cactus orchards.
Ramon Dolcet-Sanjuan and Elisabet Claveria
Micropropagation of Pistacia vera `Mateur' was improved by adding MeJA to the multiplication and rooting media. Shoot-tip cultures established from grafted trees were maintained on a modified Murashige and Skoog medium containing 5 μm BA and 0.05 μm IBA. Adding 0.3, 1, or 3.2 μm MeJA improved shoot multiplication rates 2.5, 3.0, and 2.3, respectively. There was a significant interaction between the effects of auxin and temperature on the percentage of shoots forming roots. At 25C, the percentage of shoots forming roots was higher in the presence of NAA than IAA or IBA, whereas, at 28C, there was no difference among the auxins. Adding MeJA to the best auxin treatments-31.6 μm NAA at 25C and 31.6 μm IAA at 28C-increased the percentage of shoots forming roots and number of roots per shoot but decreased root length. More than 80% of the shoots rooted at 25C when 1 μM MeJA was added to the root induction medium, which contained 31.6 μm NAA, and the root elongation medium, without auxin. The large number of short roots induced by MeJA facilitated plantlet transfer to soil and acclimation. Chemical names used: methyl jasmonate (MeJA); N6-benzyladenine (BA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), indole3-acetic acid (IAA).