Many wetland plant species used for aquascaping and wetland revegetation projects are collected from donor wetland sites for planting elsewhere. Increased demand for wetland plants has lead to over-collection and subsequent environmental damage to these donor sites. Micropropagation provides an ecologically sound alternative to field collection and allows for production of under utilized wetland species and genotypes that are either slow-growing or difficult to propagate using conventional methods. Sagittaria latifolia Willd. (Duck-potato), a rhizomatous herbaceous wetland species, was established in vitro from surface-sterilized lateral and terminal rhizome shoot-tips cultured in liquid basal medium 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. Prior to multiplication, responsive Stage I cultures were indexed for cultivable bacteria and fungi. Shoot multiplication occurred in vitro through formation of multiple node rhizomes bearing terminal shoots. Duck-potato exhibited a high sensitivity to relatively low benzyladenine (BA) levels. Maximum rhizome and shoot production occurred from single shoot explants initially cultured on agar-solidified BM supplemented with 4.0 μM BA for 28 days. However, repeated subculture on BM supplemented with greater than 2.5 μM BA resulted in increased mortality, reduction in multiplication rate, or production of dormant corms. Consistent shoot multiplication (four to five shoots/explant) was possible in the presence of 1.5 μM BA. Maximum (100%) acclimatization and rooting was attained by direct sticking of Stage II microcuttings in soilless growing medium contained in 38 cell plugs. Production of salable plants bearing multiple rhizomes was possible within 6 weeks post-transplant. Preliminary observations indicate that corm formation in Sagittaria latifolia may be mediated by photoperiod.
Michael E. Kane and Charles Lane
Nancy Phiman and Michael E. Kane
Beach stabilization by replanting dune species such as Uniola paniculata L. (Sea Oats), is an accepted practice to control erosion in the southeastern United States. Increased restrictions on collection of sea oat seed and plant material for propagation is of increasing concern. Development of micropropagation protocols for establishment and production of sea oats from donor plants of known phenotype would be useful for selecting and producing plants with commercially valuable characteristics. Terminal and lateral shoot tips (3 mm wide and 4 mm high) from containerized plants were surface sterilized and established on Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine solidified with 0.8% TC® Agar. Terminal tiller shoot tips were more responsive than lateral shoot tips. Four monthly subcultures were. required for stabilized shoot multiplication from culture lines established from terminal tiller shoot tips. Shoot organogenesis frequently occurred from the cut leaf surfaces of subcultured shoot clusters. Microcuttings were established ex vitro in plug cells containing sand or vermiculite.
Edward F. Gilman and Michael E. Kane
Post-planting root development of red maple (Acer rubrum L.) on a well-drained site was compared with that on a site with a high water table. Container-grown red maple planted in 1985 were excavated in 1988 and cross-sectional root area (CSRA) calculated for roots >1 cm diameter, 5 cm beyond the edge of the original container rootball. Adventitious roots were generated in the field after planting, not in the container. Total adventitious CSRA was three times greater than CSRA of roots generated from the original container-produced root system. The number of adventitious roots (7.6) generated from the trunk and primary root after planting was greater than the number of roots originating from the existing root system (4.2). Adventitious root origin on both sites was within 5 cm of the soil surface, above the often circling, kinked, or twisted roots found within the container root ball. Four of the five largest roots were of adventitious origin. Root number, size, and growth rate were not modified by differences in cultural and environmental conditions between sites.
Myrna Stenberg, Michael E. Kane and Nancy Philman
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.
Jennifer A. Gargiulo and Michael E. Kane
The genus Cryptocoryne (Araceae) contains some of the most commercially important amphibious species used in the aquarium plant trade. However, seed production is rare and vegetative propagation by rhizome division is extremely slow. Procedures for in vitro establishment, axillary shoot proliferation and plantlet acclimatization of Cryptocoryne Becketti Thwaites ex Trimen were determined. Surface sterilized rhizomatous shoot tips were established on a medium consisting of Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine (BA) and 0.57 μM indole-3-acetic acid (IAA) solidified with 0.8% TC® Agar. Effects of medium supplementation with factorial combinations of BA (0 - 25 μM) and IAA (0 - 10 μM) on axillary shoot proliferation from single node explants were determined after 28 days. Maximum axillary shoot proliferation (`l-fold increase) occurred on medium supplemented with 25 μM BA and 1.0 μM IAA. Excellent microcutting rooting (100%) was achieved by direct sticking in Vergro Klay Mix A. Greenhouse acclimatization of rooted microcuttings was 100%.
Michael E. Kane and Craig K. Chandler
Many horticultural crops are infected with bacterial, fungal, or viral pathogens that reduce yield and/or quality. Recovery and maintenance of pathogen eradicated crops, such as strawberry (Fragaria × ananassa Duch.), have been possible following the isolation and culture of apical meristems or meristem-tips in vitro. A laboratory exercise has been developed to provide experience in the procedures required for the isolation, surface disinfection, and in vitro establishment of meristem-tip explants excised from strawberry stolons. Stolons are obtained from greenhouse-grown strawberries (`Sweet Charlie') maintained in hanging baskets under a 14-h photoperiod. Stolons are cut into single-node segments and terminal tips. The leaf blades are removed and the nodal sections are rinsed and then surface-disinfected by successive agitation in 70% ethanol and 1.05% sodium hypochlorite, followed by three rinses in sterile deionized water. In the transfer hoods, each student attempts to isolate meristem-tips and shoot tips of various sizes under high magnification provided by a stereomicroscope. Explants are inoculated onto Murashige and Skoog basal medium (Murashige and Skoog, 1962) supplemented with 30 g/liter sucrose, 80 mg/liter adenine sulfate, 1.0 mg/liter benzyladenine, 1.0 mg/liter indole-3-acetic acid, and 0.01 mg/liter gibberellic acid (GA3) and solidified as 45°slants with 1.25 g/liter Phytagel and 3.0 g/liter TC agar. Growth responses are monitored weekly. After 6 weeks, students record the percentage of visibly contaminated cultures and number shoots produced per explant. The relationship between initial explant size and in vitro growth is also determined. Students index their cultures for the presence of cultivable bacteria and fungi using sterility test media.
Edward F. Gilman and Michael E. Kane
Shoot and root growth were measured on Chinese juniper (Juniperus chinensis L. `Torulosa', `Sylvestris', `Pfitzeriana', and `Hetzii') 1, 2, and 3 years after planting from 1l-liter black plastic containers. Mean diameter of the root system expanded quadratically, whereas mean branch spread increased linearly. Three years after planting, root spread was 2.75 times branch spread, and roots covered an area 5.5 times that covered by the branches. Percentage of total root length located within the dripline of the plants remained fairly constant for each cultivar during the 3 years following planting. Root length density increased over time but decreased with distance from the trunk. During the first 2 years after planting, shoot mass increased faster than root mass. In the 3rd year, the root system increased in mass at a faster rate than the shoots. Root length was correlated with root weight. Root spread and root area were correlated with trunk cross-sectional area, branch spread, and crown area.
Murdock Ray Gillis and Michael E. Kane
Stewartia malacodendron L. (silky camellia), a small deciduous tree bearing showy flowers, has potential as a landscape plant. Propagation problems, limited availability and consequent high unit cost have slowed its acceptance as a landscape plant. Given its potential value as a landscape plant, studies were initiated to define a micropropagation protocol. Surface sterilized shoot tips and nodal explants from two-year-old container grown seedlings were established on Woody Plant Medium supplemented with 4.44 μM benzyladenine (BA) and solidified with 0.8% TC® Agar. Sustained growth of subcultured shoot tips and nodal segments required the addition of 8-15 μM gibberellic acid (GA3) to the medium. Regenerated shoots were 3 - 5 cm long, unbranched and typically consisted of three subdivisions. Effects of cytokinin type (BA, 2iP or kinetin) and concentration (0-25 μM) with factorial combinations of GA3 (0-30 μM) on shoot multiplication, elongation and diameter were determined after a 28 day culture period. Moderate GA, levels (10 & 20 μM) in combination with 2.5 μM BA yielded the highest quality microcuttings.
Cynthia Zurinsky, Michael E. Kane and Nancy Philman
Studies were completed to optimize Stage II production efficiency of Pontederia cordata, a native wetland plant. Basal shoot tips from established cultures were subcultured into 60 ml glass culture tubes, 155 ml glass baby food jars, 350 ml GA7 polypropylene vessels or 500 ml clear polypropylene tissue culture containers containing full strength Linsmaier and Skoog mineral salts and organics supplemented with 3.0% sucrose, 2.0 mg/liter benzyladenine, 1.0 mg/liter indole-3-acetic acid, 50 mg/liter citric and ascorbic acids solidified with 8 g/liter TC® agar. Shoot tip to medium volume (ml) ratio was maintained 1:10 in each culture vessel. Vessel type had no significant effect on either shoot quality or multiplication rate (9.5 shoots/shoot tip/28 days). A maximum production efficiency of 1216 shoots/ft2/28 days was achieved using GA7 vessels. Stage II shoot multiplication rate significantly decreased when the interval between subculture exceeded 28 days.
Edward F. Gilman and Michael E. Kane
Shoot and root growth were measured on Chinese juniper (Juniperus chinensis L.) Var. `Torulosa', `Sylvestris', `Pfitzeriana' and `Hetzii' 1, 2 and 3 years after planting into a simulated landscape from 10-liter black plastic containers. Mean diameter of the root system increased quadratically averaging 1, 2 m/year; whereas, mean branch spread increased at 0, 33 m/year, Three years after planting, root spread was 2, 75 times branch spread and roots covered an area 5.5 times that covered by the branches. Percentage of total root length located within the dripline of the plants remained fairly constant (71-77%) during the first 3 years following planting. Root length density per unit area increased over time but decreased with distance from the trunk. In the first 2 years after planting shoot weight increased faster than root `weight. However, during the third year after planting, the root system increased in mass and size at a faster rate than the shoots. Root length was correlated with root weight within root-diameter classes, Root spread and root area were correlated with trunk area, branch spread and crown area.