Histological and histochemical examination of floral initiation was conducted to determine the pattern of flowering in Rudbeckia hirta, a long-day (LD) plant. Plants were grown under 8-hour short days (SDs) until they had 14 to 16 expanded leaves. Half of the group of plants was moved to LD conditions consisting of natural daylength plus a 4-hour night interruption. Rudbeckia hirta had a pattern of differentiation in flowering similar to that reported in species requiring one inductive day for initiation. Rudbeckia hirta required 8 LDs for evocation and 18 LDs for completion of initiation. Involucral bracts initiated after 18 LDs, after which the receptacle enlarged and was capped by a meristematic mantle of cells signaling the start of development. Floret primordia did not initiate, even after 20 LDs. Increases in pyronin staining were observed in actively dividing cells of the procambium, leaf primordium, and corpus of the vegetative meristems. After 8 LDs, the pith rib meristem stained darkly, a result indicating the arrival of the floral stimulus. An increase in pyronin staining was also observed in the meristematic mantle covering the receptacle after 18 LDs, a result indicating increased RNA levels.
Richard L. Harkess and Robert E. Lyons
L.G. Buckley, E.T. Graham, and R.N. Trigiano
Zygotic and somatic embryos are purported to follow similar developmental sequences, but few investigations have thoroughly compared the two processes. Developing pods of Cercis canadensis L. (redbud) were collected from trees on the Knoxville campus of the University of Tennessee once or twice per week from 28 March to 8 August 1991. At least 10 ovules/sample date were fixed in FAA to evaluate zygotic embryo ontogeny. A minimum of 40 ovules/sample date were aseptically excised and placed on SH medium supplemented with 9.0 μM 2,4-D and 5 mM ammonium ion to initate somatic embryogenesis. Zygotic and somatic embryos were prepared for histological examination using standard paraffin techniques. Somatic embryos developed primarily from cotyledons and epicotyls of zygotic embryos mat were cultured between 6 June and 19 July. Somatic and zygotic embryos were subtended by multiseriate suspensors and progressed through recognizable globular, cordate and cotyledonary stages of development. Cotyledon morphology was similar for both embryo types. However, many somatic embryos failed to differentiate dome-shaped shoot meristems exhibited by their zygotic counterparts.
Neil O. Anderson, Peter D. Ascher, Richard E. Widmer, and James J. Luby
The generation time (0.75 to 1.5 years) in perennial, hexaploid chrysanthemums [Dendranthema grandiflora Tzvelv. (Chrysanthemum morifolium Ramat.)] impedes the rate of progress for sexual breeding programs in creating new clonal cultivars, inbred lines for hybrid seed production, and genetic studies. Modifications to the crossing environment and embryo rescue were evaluated to minimize the chrysanthemum generation cycle. One greenhouse chrysanthemum clone was outcross-pollinated using a bulk pollen source. Following emasculation, inflorescences were either left in situ or the peduncle bases were placed in styrofoam boards floating on a solution of 1% sucrose and 200 ppm 8-HQC under laboratory conditions. Embryogenesis occurred at a faster rate under laboratory conditions as tested with histological techniques; the heart stage appeared as early as the second day after pollination, compared with 11 days using in situ methods. Total embryogenic development time ranged from 25 (laboratory seed development) to 52+ days (in situ ripening). In a second test, embryo rescue (ER) significantly improved percent seed set, percent germination, and percent of progeny reaching anthesis relative to normal development. ER progeny from both garden parents were significantly earlier in total generation time than corresponding non-ER siblings. Laboratory seed development and ER were then used sequentially to obtain an average progeny generation time of =100 days, thus allowing for three generations per year. The potential impact of these two techniques on breeding chrysanthemums and other perennial crops with long generation times is discussed.
Michael Marcotrigiano and Susan P. McGlew
A two-stage micropropagation system was devised for cranberries (Vaccinium macrocarpon Ait.). Shoot-tip explants taken from four cultivars of greenhouse-grown plants were placed on media composed of Anderson's major salts, Murashige and Skoog's (MS) minor salts and organics, plus various concentrations of 2iP, IBA, and GA3. In other experiments, explant source, salt formulations for media, and rooting treatments were studied. Optimal multiplication and shoot quality occurred when nodal explants taken from greenhouse-grown or micropropagated plants were placed on medium containing 150 μm 2iP, 1.0 μm IBA, and no GA3. Histological examination revealed that the initial response of nodes to culture is axillary bud proliferation, but adventitious shoot formation occurred after 4 to 6 weeks. Cultures that contained only axillary shoots were not evident unless low levels of 2iP were used, at which point only axillary buds present on the explants were released. Proliferated shoots could be rooted ex vitro without auxin treatment. Optimal rooting occurred under high-light conditions. Plants were transplanted to the field for comparison to conventionally propagated material. Chemical names used: gibberellic acid (GA3), N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP), 1H-indole-3-butanoic acid (IBA).
Richard P. Buchner, William H Olson, Vito S. Polito, and Katherine Pinney
Walnut Blight caused by the bacteria Xanthomonas campestris pathovar juglandis is a very destructive disease for California walnut production. Streptomycin is an effective disease control material; however, Streptomycin sprays can result in significant nut drop 3 to 5 weeks after spray application. We investigated the basis for walnut drop following applications of Streptomycin (Agrimycin) for walnut blight control. Flowers and developing nuts were collected from four treatments, plus an unsprayed control. 200 ppm Streptomycim was applied at 1) budbreak; 2) pre, full, and post-bloom; 3) postbloom; 4) budbreak and postbloom; 5) untreated control. Samples were collected regularly beginning at the first budbreak spray and extending through the period of nut drop. Samples were fixed and prepared for histological examination. In treatments with a high incidence of nut drop, the embryo failed to develop. Examination of the stigma and style in flowers from these treatments showed inhibited pollen tube growth. Results indicate that Streptomycin inhibits pollen tube growth, which precludes fertilization. This pattern of development and timing of nut drop following Streptomycin application at full bloom is similar in all ways to unpollinated walnut flowers. Nut growth and development appear normal for 3 to 5 weeks; then nuts abort. If Streptomycin became available for walnut blight control, sprays timed to coincide with pistillate bloom and pistillate flower receptivity should be avoided.
William M. Walter Jr., Betsy Randall-Schadel, and William E. Schadel
Wound healing in cucumber fruit (Cucumis sativus L., cv. Calypso) was studied using histological and degradative techniques. A thick exudate appeared at the wounded surface shortly after wounding. This material retarded water loss and possibly aided in the formation of sclerified parenchyma observed 24 hours after wounding. The sclerified material was positive to a modified Weisner stain, indicating lignification was occurring. Wound periderm (cork) was initiated directly beneath the sclerified parenchyma cells within 48 hours after wounding. The cork layers were positive to Sudan IV stain, indicating suberin was being formed. The rate of phellem development decreased by 6 days after wounding. By day 7, younger phellem cells and sclerified parenchyma cells were stained by Sudan IV. Degradation of the wound tissue by chemical procedures demonstrated that relatively large amounts of lignin and suberin were deposited during healing. Fragments from the lignin degradation Indicated that lignin was composed mainly of gualacyl and p-hydroxyphenyl residues. Suberin was found to contain mainly 1,16-hexadecane and 1,18-osctadecene decarboxylic acids detected as the silylated diol derivatives.
Yong Cheong Koh and Fred T. Davies Jr.
The leaves of vegetative stolons of greenhouse grown Cryptanthus `Marian Oppenheimer' (wide leaf clone) were cultured in modified MS media to induce adventitious shoot formation via callus formation. The best callus induction medium was basal MS medium with 10 μM NAA, IBA and BA. Pure green (843), maroon (3), striped (2) and albino plantlets were obtained. Most of the albino plantlets were stunted, tightly clumped together and impossible to score. The medium which produced the highest average number of non-albino plantlets was basal MS medium with 0.3 μM NAA, IBA and BA All non-albino plantlets were rooted in MS medium with 5.4 μM NAA and transplanted ex vitro with a survival rate of 96.7%. The maroon plantlets became green two weeks after transplanting. Histological studies revealed that C. `Marian Oppenheimer' (wide leaf clone) has two tunicas (L1 and L2) and a corpus (L3). Callus on the leaf explant arose mainly from the L2 and L3. Apparently C. `Marian Oppenheimer' (wide leaf clone) is a GWG periclinal chimera.
Michael Marcotrigiano and Susan P. McGlew
In an effort to accelerate breeding programs and to study somaclonal variation, a micropropagation system was devised for cranberries (Vaccinium macrocarpon). Using a factorial design, explants taken from greenhouse grown plants were placed on Anderson's medium containing different concentrations of 2ip' GA3, and IBA, with 4 cultivars tested over 3 subcultures. In other experiments, explant source, macro and micro salt formulations, and rooting treatments, were studied. Optimal multiplication and shoot quality occurred when single node explants taken from greenhouse grown plants were placed on Anderson's media containing 150 uM 2iP, 1.0 uM IBA and no GA3. Histological examinations indicate that initial response is axillary bud proliferation but upon subculture adventitious shoot formation may be possible. Proliferated shoots could be rooted ex vitro in plug trays under plastic tents and without hormone treatments. Optimal rooting occurred under high light conditions in a 1:1 (v:v) peat:sand mix. Plants were easily transplanted into the field in spring and will be evaluated by comparison to conventionally propagated material.
Takuya Tetsumura and Hisajiro Yukinaga
When cultured in vitro, roots of four Japanese persimmon (Diospyros kaki L.) cultivars formed adventitious shoots on MS medium with 10 μm zeatin and 0.01 μm indole-3-acetic acid, although their organogenetic capacities varied. Histological study revealed that the origin of the adventitious shoots was the pericycle. The regenerated shoots grew well on the shoot proliferation medium (MS with 5 μm zeatin). Final rooting percentages of shoots regenerated from roots of three of the four cultivars were greater than those of shoots that originated from shoot tips and that had been subcultured >50 times. Shoots regenerated from `Jiro' roots rooted 10 days earlier, had more roots than those from shoot tips, and maintained higher rooting ability over ten subcultures. Rooted `Hiratanenashi' shoots regenerated from roots survived better after acclimatization than those from shoot tips. No obvious variants were observed either in vitro or in the field. The trees regenerated from roots flowered within 4 years. These findings suggest that partial rather than true rejuvenation was responsible for both the early flowering and the juvenile characteristics, i.e., the enhanced rooting ability, observed in the regenerated plants. Chemical name used: 6-(4-hydroxy-3-methylbut-2-enylamino) purine (zeatin).
Tekalign Tsegaw, S. Hammes, and J. Robbertse
Potato (Solanum tuberosum L.) treatment with paclobutrazol resulted in short and compact plants having dark green and thicker leaves, and wider stem and root diameters. Investigating the underlying anatomical modifications in response to the treatment was the objective of the study. Plants of potato cultivar BP 1 were treated with 0, 45.0, 67.5, and 90.0 mg paclobutrazol per plant as a foliar spray. A month after treatment leaf, stem and root materials were taken from the control and plants treated with 67.5 mg paclobutrazol, and histological observations were made using light microscope. Leaves of treated plants showed an increased chlorophyll a and b contents, thicker epicuticular wax layer, elongated and thicker epidermal, palisade and spongy mesophyll cells. paclobutrazol increased stem diameter by about 58% due to induction of thicker cortex, larger vascular bundles, and wider pith diameter associated with larger pith cells. Widening the cortex and the induction of more secondary xylem vessels in response to paclobutrazol treatment increased the root diameter by about 52%. Paclobutrazol treatment remarkably increased the accumulation of starch granules in the stem pith cells and cortical cells of the stem and root. This study is similar to the other relevant studies in reporting an increased leaf thickness, and stem and root diameters; however, most of the underlying anatomical modifications described above have not been reported previously.