Micropropagation is a commercially viable and ecologically sound method for producing native herbaceous wetland plants used for wetland revegetation projects. The ability to rapidly screen, select and store germplasm of wetland species genotypes with desirable characteristics of growth rate and habit, nutrient uptake capacity, and/or substrate preference would significantly impact how micropropagated wetland plants are marketed. Early screening of plantlet growth ex vitro may provide an efficient method to select for specific characteristics of growth rate and habit. Five micropropagated lines of Pontederia cordata of differing phenotype were established in vitro from Florida populations. Rooted microcuttings were established ex vitro in a shallow outdoor tank. Growth and development were monitored over a 9 week period. Significant differences in shoot growth and number, leaf area and number, flowering and dry weights were observed between the different Pontederia cordata varieties.
Myrna Stenberg, Michael E. Kane, and Nancy Philman
Dennis P. Stimart and James F. Harbage
The role of the number of adventitious roots of Malus domestics Borkh. `Gala' microcuttings in vitro on ex vitro root and shoot growth was investigated. Root initiation treatments consisted of IBA at 0, 0.15, 1.5, 15, and 150 μm in factorial combination with media at pH 5.5, 6.3, and 7.0. IBA concentrations significantly influenced final root count and shoot fresh and dry weights, but not plant height, leaf count, or root fresh and dry weights at 116 days. Between 0 and 0.15 μm IBA, final root counts were similar, but at 1.5, 15, and 150 μm IBA, root counts increased by 45%, 141%, and 159%, respectively, over the control. The pH levels did not affect observed characteristics significantly. There was no significant interaction between main effects. A significant positive linear relationship was found between initial and final root count. The results suggest a limited association between high initial adventitious root count and subsequent growth. Chemical name used: 1 H -indole-3-butyric acid (IBA).
Dorcas K. Isutsa, Marvin P. Pritts, and Kenneth W. Mudge
A protocol is presented that enables a propagator to produce field-sized blueberry transplants within 6 months of obtaining microshoots from tissue culture. The protocol involves subjecting microshoots to ex vitro rooting in a fog chamber under 100 μmol·m–2·s–1 photosynthetic photon flux for 7 weeks, transferring plants to a fog tunnel for 2 weeks, then to a greenhouse for 7 more weeks. Plant survival and rooting of cultivars Berkeley (Vaccinium corymbosum L.) and Northsky (Vaccinium angustifolium ×corymbosum) were near 100% under these conditions. Plantlets in fog chambers receiving 100 μmol·m–2·s–1 grew rapidly, while those at lower irradiance levels grew more slowly, and supplemental CO2 enhanced growth only at 50 μmol·m–2·s–1. Growth rates slowed when plants were moved into the fog tunnel; but by the end of 16 weeks, plants that were under high irradiance in the fog chamber had root systems that were 15 to 30 times larger than plants under low irradiance. Within 6 months, these plants were 30 to 60 cm tall and suitable for field planting.
Charlotte R. Chan and Robert D. Marquard
Hamamelis cultivars are typically propagated by grafting onto H. virginiana rootstock. Grafting is labor-intensive and the understock frequently suckers which can lead to the loss of the scion. A cultivar growing on its own root system would eliminate this problem. Our research was undertaken to develop a successful method of rooting micropropagules. The source material was established cultures of H. × intermedia `Diane,' H. virginiana, and a H. vernalis selection. The rooting treatments consisted of four concentrations of K-IBA (0, 5, 10, and 20 μM) in 0.02% Tween 80. Three replicates of eight cuttings each were taken from the three sources for each of the four treatments. The cuttings were placed in 50-mL beakers, cut-end down, with 10 mL of the treatment solution. The beakers were sealed with Parafilm, and cuttings were soaked for 24 h. After treatment, the cuttings were randomly stuck into Kadon flats prepared in advance with a sterile mix of 1 peat: 1 perlite and were watered-in. Cuttings were misted, and flats were covered with plastic and Remay. They were kept in a warm (19-24 °C) greenhouse. Cuttings rooted in 3 to 4 weeks and were subsequently fertilized weekly with Peter's 20N-20P-20K at 150 ppm. At 12 weeks, data were collected for the rate of survival, height, branching, number of nodes, and root mass, and the plants were transplanted to quart pots. Ninety percent of the cuttings rooted; the most favorable response was with 10 μM K-IBA, although all treatments produced >80% rooting. This method was time and labor efficient. Moreover, micropropagation is not dependent on the season, and production of new plants could proceed on a continuous basis, making this a viable alternative to commercial grafting.
R.M. Skirvin and S. Sriskandarajah
Acclimatization and growth of in vitro-derived apple shoots of two apple scion apple cultivars were compared under fogged conditions in a greenhouse and in a commercial growth cabinet (Phototron). Plant survival rates of microcuttings of `Royal Gala' and `Jonagold' were significantly better when maintained in the Phototron units than when grown in a greenhouse under fog. The number and length of roots on microcuttings was significantly higher in the Phototron than under fog. In the present study, we demonstrated that the Phototron environment was better than a fogged greenhouse for establishing apple shoots ex vitro. However, the Phototron units are so small that they hold no more than 100 to 120 plants at a time. Therefore, the units will be of most value to growers or individuals in laboratories who do not have a constant need for acclimatization facilities. Growers who acclimatize many plants should continue to use fogging or misting facilities.
Letitia Quay, Jen A. McComb, and Kingsley W. Dixon
Seeds of two Australian terrestrial orchid species (Caladenia latifolia R.Br. and Diuris magnifica D. Jones) were germinated in a potting mix of Allocasuarina fraseriana (Miq.) L. Johnson leaf mulch and perlite (1:1). The potting mix was irradiated (7 Gy for 14 hours), steam pasteurized (70C for 30 minutes) or nontreated, and inoculated with the appropriate mycorrhizal fungus for each species, a sterile red fungus (SRF), or both. Protocorm formation and green shoots were evident at 8 and 10 weeks, respectively, after seed sowing. The highest mean number of seedlings was 84 for C. latifolia and 234 for D. magnifica per 270-ml container in pasteurized potting mix inoculated with mycorrhizal fungi and SRF. Shoots were longest after 20 weeks (28 mm for C. latifolia and 52 mm for D. magnifica, respectively) in pasteurized potting mix inoculated with mycorrhizal fungi only. Germination was absent in control treatments without mycorrhizal fungi; with SRF only; or in nonsterile potting mix with mycorrhizal fungi, SRF, or both.
Nancy Philman, Murdock Ray Gillis, and Michael E. Kane
Commercial micropropagation of wetland plants used for habitat restoration provides an alternative to field collection and facilitates production of difficult-to-propagate species and possibly selection of ecotypes that are physiologically adapted to specific habitat conditions. Knowledge of the degree of ecotypic variation within and between wetland populations is very limited. The feasibility of screening ecotypic differences in growth of micropropagated wetland plants, following acclimatization, was examined using Sagittaria latifolia Willd. (Duck-potato), a highly variable rhizomatous herbaceous wetland species that is widely distributed in southeastern Canada and the eastern United States. Plants were obtained from populations in Rhode Island, North Carolina, South Carolina, and Florida. Stage I cultures of each Sagittaria latifolia ecotype were established from surface-sterilized rhizome shoot-tips cultured in a liquid basal medium (BM) consisting of half-strength Murashige and Skoog mineral salts, 0.56 mM myo-inositol and 1.2 μM thiamine supplemented with 87.6 mM sucrose. Stage I cultures were indexed for cultivable bacteria prior to clonal multiplication of each ecotype by rhizome production on agar-solidified BM supplemented with 1.1 μM benzyladenine (BA). At 4-week intervals for 24 months, Stage II microcuttings of each ecotype were acclimatized and rooted in soilless growing medium under intermittent mist for 10 days. Plantlets were transferred to a shadehouse (50% sunlight reduction) and maintained under prevailing environmental conditions. Plant height, leaf length and number, rhizome number, corm number and weight, and flowering were determined 6 weeks post-transplant. Significant seasonal differences in leaf growth, rhizome production, corm formation and flowering were observed between ecotypes. During the growing season, induction of corm formation occurred progressively earlier in the more northern ecotypes.
R.C. Apter, F.T. Davies Jr., and E.L. McWilliams
In vitro tissue-cultured (TC) and macropropagated (MACRO) 18-day old adventitious roots of Asian jasmine [Trachelospermum asiaticum (Siebold & Zucc.) Nakai] were compared for their ability to absorb and translocate radiolabelled P from a nutrient solution. Samples were taken at 1, 2, 4, 8, 12, and 24 hours after the initial dosage of the nutrient solution with 7.4 × 10-2 MBq KH 32 2PO4/liter. TC roots were capable of absorbing P, but at significantly lower levels than MACRO roots. Greater P absorption occurred in MACRO roots within the first hour and continued for the duration of the experiment. However, there was no significant difference in the rate of P translocation from roots to shoots between treatments. Root systems formed in vitro survived acclimation and had developed into well-branched root systems after 13 weeks. Reduced P absorption by TC roots did not limit either P translocation or survivability during and after acclimation.
R.C. Apter, E.L. McWilliams, and F.T. Davies Jr.
One-node explants and one-node stem cuttings of Asian jasmine [Trachelospermum asiaticum (Siebold & Zucc.) Nakai] were rooted, respectively, in vitro [tissue culture (TC)] or by conventional macropropagation (MACRO). The TC and MACRO stem bases were then analyzed for differences in the time-course sequence of 1) root primordia initiation and development and 2) adventitious root xylem development and root-to-shoot xylem connections. Early root primordia were observed at Day 3, and, by Day 7, root-to-shoot xylem connections were equally developed in TC and MACRO systems. Continued development and emergence of adventitious roots were observed at Days 8 to 10. At Days 13 and 18, when viewed using scanning electron microscopy, TC root hairs were morphologically thicker and one-third to one-half the length of MACRO root hairs. There was no apparent difference in root-hair density. Inferior TC root-hair length may be a factor in the acclimation of TC-generated plantlets.
Marvin Pritts and Dorcas Isuta
Previous findings reveal that rooting and acclimatization of apple and blueberry plants is often difficult, inconsistent and inefficient. This experiment was set up in a fog chamber lo investigate the effects of CO2 enrichment (CDE) and irradiance on unrooted stage II microshoots. Two CO2 and 3 light levels tested were: 1350 +/- 150 (+ CDE), and 450 +/- 50 (- CDE) ppm; 30 +/- 5 (low), 55 + 10 (medium), and 100 + 20 (high) umolm-2s-1 respectively. Cultivars assessed were Berkeley and Northsky for blueberry. G65 and NY30 for apple. Blueberry microshoots acclimatized successfully and gave between 90 to 100% rooting and survival rate. Apple microshoots acclimatized and rooted slowly, exhibited great sensitivity to in vivo conditions and gave between 40 to 100% rooting and survival rate. High light induced photo-inhibition which disappeared after complete acclimatization. There was a significant difference between low light and the other two light levels. The effect of CDE was dependent on cultivar. In most cases, high light (-) CDE gave the most vigorous growth (highest plant dry weight and leaf area). There was a significant difference between (+) CDE and (-) CDE at low and medium light, but none at high light. Low light (-) CDE and medium light (+) CDE were superior over low light (+) CDE and medium light (-) CDE. respectively. Stalling out in apple microshoots was corrected by GA sprays.