Although somatic embryogenesis in vitro has been carried out successfully in a number of plants, a limiting factor in many somatic embryogenic systems is that plantlet regeneration is not obtainable or restricted to low frequencies. We have developed a repetitive, high frequency somatic embryogenic system in pecan (Carya illinoensis) and have identified effective treatments for improved somatic embryo conversion. A 6 to 10 week cold treatment followed by a 5 day desiccation, promoted enhanced root germination and extension, and epicotyl elongation. Light and transmission electron microscopic evaluations of somatic embryo cotyledon development will be presented and related to conversion enhancing treatments and their possible roles in embryo maturation.
Hazel Y. Wetzstein and Choong-Suk Kim
Warner Orozco-Obando* and Hazel Y. Wetzstein
Recently, the release of Hydrangea cultivars with the capacity to produce a second flush of blooms has created a great expectation in the ornamental industry. However, the lack of fundamental information on flower development of big leaf Hydrangea does not allow a descriptive explanation of why re-blooming capacity occurs. The objectives of this study were to characterize the timing and location of flower initiation and development in several H. macrophylla cultivars throughout an annual cycle. Four cultivars were evaluated: 2 exhibiting re-flowering capacity (Penny Mac-PM and Endless Summer-ES) and 2 without (Madame Emile Mouillere-MEM and Nikko Blue-NB). Plants were managed under outdoor nursery conditions and harvested at each of four different time periods. These periods represented key developmental stages: 1) Pre-induction: late summer, after completion of shoot expansion; 2) Post-induction: late fall, following short day and cold temperature exposure; 3) Dormancy: winter, post leaf abscission; and 4) Post-dormancy: early spring, just prior to bud break. At each sampling time, bud location (terminal or lateral) and stem origin (basal, lateral, terminal, or secondary) were established. All buds >;2 mm were dissected under a stereomicroscope and the degree of floral induction was determined. Floral primordial were initiated not only in the terminal buds but also within axillary buds. The degree of induction and development varied according to the stem origin, bud location and cultivar. Cultivars with re-blooming capacity had floral primordial initiated within buds at the first sampling period prior to receiving inductive conditions. This suggests they may have minimal or no photoperiodic/temp requirements for flowering.
Seong Min Woo and Hazel Y. Wetzstein
Georgia plume, Elliottia racemosa Muhlenb. ex. Elliott, is an extremely rare small tree or shrub endemic to Georgia, which is being severely affected by habitat loss and lack of sexual recruitment. In vitro plant regeneration of Georgia plume has not been previously reported and may be a method for the conservation and propagation of this threatened species. Studies evaluated the effects of sterilization methods, explant types, medium composition, and light environment on plant regeneration. An efficient plant regeneration system was developed in which adventitious shoot buds were induced using young, expanding leaf explants placed on an induction medium supplemented with 10 μm thidiazuron and 5 μm indole-3-acetic acid with Gamborg's B5 salts. Shoot elongation was promoted on a medium with 25 μm (2-isopentenyl) adenine incorporated into Woody Plant Medium. In vitro rooting studies evaluated continuous and pulse auxin treatments and ex vitro rooting trials after KIBA (indole-3-butric acid, potassium salt) dips. A 5-day pulse treatment on 100 or 150 μm indole-3-butyric acid produced ≈90% rooting of shoots with greater shoot and root dry weight than other pulse times. High rooting frequencies were obtained under in vitro and ex vitro conditions with over 85% survival of plantlets transferred to greenhouse conditions. The culture protocol was found to be effective with material collected from mature specimens in the wild from divergent populations. Tissue culture appears to be a promising approach for the propagation and conservation of this rare and threatened plant.
Seong Min Woo and Hazel Y. Wetzstein
Georgia plume (Elliottia racemosa Muhlenb. ex. Elliott) is a rare deciduous shrub or small tree. It has sustained severe loss of habitat and its range is now restricted to a limited number of sites in the state of Georgia. Tissue culture protocols have been developed as a means to propagate and conserve this threatened species using leaf explants induced on medium supplemented with 10 μm thidiazuron (TDZ) and 5 μm indole-3-acetic acid (IAA). Bud-like clusters, elongated embryo-like protrusions, and shoot-like structures were produced from the leaf explants. Morphological and histological evaluations of cultures during induction and development were conducted using light microscopy of sectioned material and scanning electron micrography. Histology of explant tissues indicates that plant regeneration of Georgia plume occurs through a shoot organogenesis pathway that involves the formation of actively dividing meristematic regions originating in subepidermal cell layers that proliferate to form protuberances on the explant surface. Numerous well-formed shoot apical meristems with leaf primordia are produced, as well as fused shoot-like structures. Elongated, embryo-like structures had various degrees of shoot apex development. Evaluations of serial sections found that they lacked a defined root apex, and that basal portions were composed of parenchymatous files of cells with a broad point of attachment to the parent tissue. The lack of bipolarity and a root pole signifies that true somatic embryogenesis does not occur.
Hazel Y. Wetzstein and S. Edward Law
Stigma characteristics and morphology can be useful in taxonomic and phylogenetic studies, indicate relationships in stigma function and receptivity, and be valuable in evaluating pollen–stigma interactions. Problematic is that in some taxa, copious stigmatic exudate can obscure the fine structural details of the stigmatic surface. Such is the case for Citrus, which has a wet stigma type on which abundant exudate inundates surface papillae. The components of stigmatic surface compounds are highly heterogeneous and include carbohydrates, proteins, lipids, glycoproteins, and phenolic compounds. This study evaluated the efficacy of several pre-fixation wash treatments on removing surface exudate to visualize the underlying stigmatic surface. Wash treatments included various buffer solutions, surfactants, dilute acids/bases, and solvents. Stigmas prepared using conventional fixation methods in glutaraldehyde had considerable accumulations of reticulate surface deposits with stigmatic cells obscured. Pre-fixation washes containing solvents such as methanol, chloroform, and ethanol left accumulations of incompletely removed exudate and crystalline deposits. Alkaline water washes produced a crust-like deposit on stigma surfaces. Buffer washes left residues of plaque-like deposits with perforated areas. In contrast, excellent removal of stigmatic exudate was obtained with a pre-fixation wash composed of 0.2 M Tris buffer, pH 7.2, containing 0.2% Triton X-100 surfactant and allowed clear imaging of the stigma and surface papillae morphology. A central sinus and radially arranged openings on the stigmatic surface were clearly visible and shown for the first time using scanning electron microscopy (SEM).
Hazel Y. Wetzstein and Charles S. Vavrina
Tomato (Lycopersicon esculentum Mill.) transplants can be affected by an intermittent physiological problem manifested by loss of apical meristem function and retarded growth rates, referred to herein as apical meristem decline (AMD). Production losses associated with this condition can be substantial. Similar abnormal and arrested development of the shoot apex has been observed in a number of other species, and referred to as blindness, budlessness, toplessness, blindwood, and bud abortion. A developmental study using scanning electron microscopy was conducted in `Agriset' tomato during an occurrence of AMD to evaluate and compare normal and afflicted plants. The AMD condition was associated with cessation of leaf primordia development and lack of flower initiation. The shoot apex of plants with AMD remained vegetative compared to normal plants which at the same age had well-differentiated flower primordia. No evidence of abortion, die back, or necrosis of the shoot apex was observed. The effects of mineral nutrient additions on symptom development varied with year. In year 1, N fertilization reduced the incidence of both AMD and retarded bud growth (i.e., the percentage of normal plants increased from 29% to 97% with N applications). Preplant applications of P, alone or in conjunction with CaCO3 and trace elements, also ameliorated AMD. In year 2, AMD was observed only at very low levels, i.e., 4% or less, and mineral nutrition had no apparent effect on AMD or normal plant number.
Hazel Y. Wetzstein and Adriana P.M. Rodriguez
Somatic embryogenic cultures of pecan (Carya illinoinensis) were induced on medium with either naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Percent embryogenesis, embryo development, and subsequent performance were assessed. Cultures induced on medium with NAA were more zygotic-like, with a higher frequency of embryos that had well-defined shoot apices. In contrast, cultures induced with 2,4-D exhibited more extensive callusing and more fused and/or abnormal embryos. Adjustment of the auxin used during induction may be a means of obtaining higher quality embryos, that have higher rates of conversion into plants.
Hazel Y. Wetzstein, Zibin Zhang, Nadav Ravid and Michael E. Wetzstein
Pomegranate fruit is valued for its juice-containing arils and is consumed and marketed as whole fresh fruit, extracted arils, juice, syrup (grenadine), wine, teas, seed oil, and other products. Recent consumption has rapidly increased attributable in part to reported health benefits that include efficacy against coronary heart disease, atherosclerosis, cancer, hypertension, and infectious diseases. Within commercial orchards, the size of fruits produced can be quite variable even with trees of the same genotype grown under similar cultivation practices. Although pomegranates have been cultivated since antiquity, fruit attributes, particularly those related to size, are poorly defined. In this study, compositional changes in pomegranate fruits of the Wonderful cultivar, including volume and weight, aril weight and number, pericarp weight, seed weight, and juice/pulp content, were evaluated in fruits of variable sizes. Correlations between fruit characteristics were determined, and factor analysis established fruit and aril indices. Results indicated that because fruit volume, fruit weight, and total aril weight are closely correlated, any of these characteristics can be used as an indicator of fruit size. The number of arils per fruit was highly correlated with fruit size with larger fruit containing greater numbers of arils. This is in contrast to individual average aril weight, which showed no significant relationship to fruit size. Crop production strategies aimed at increasing aril numbers may be a means for obtaining larger fruit in pomegranate.
Weiguang Yi, S. Edward Law and Hazel Y. Wetzstein
In almond [Prunis dulcis (Mill.) D.A. Webb.], fungicide sprays are required to prevent blossom blight, which can infect open flowers. Numerous studies have reported detrimental effects of agrochemical sprays on pollination, fruit set, and yield in tree fruit crops. However, effects of fungicides on pollen germination and growth in almond are little known, particularly those from recently developed active ingredients. In this study we evaluated the effects of commercial formulations of 10 fungicides on pollen germination and tube growth in almond using in vitro assays. Assays conducted at 1/100 recommended field rates (RFR) were effective in delineating differences in almond pollen sensitivity to different fungicides. Captan and azoxystrobin were the most inhibitory, with germination percentages of less than 1% of the no-fungicide control. Germination was not significantly affected by propiconazole and benomyl. Intermediate inhibitory effects on pollen germination were observed with ziram, cyprodinil, maneb, thiophanate-methyl, iprodione, and myclobutanil. In contrast to germination, tube growth was less affected by the presence of fungicide. In pollen that germinated, tube elongation was the same as in controls in five of 10 of the fungicides evaluated. Nonetheless, azoxystrobin and captan reduced tube elongation by ≈90%. Some fungicide treatments also influenced tube morphology. In the absence of field evaluation studies, in vitro germination data may provide insight on how specific chemicals may impact pollination processes and further guide in vivo studies, particularly in the case of new chemical formulations.
Carrie A. Radcliffe, James M. Affolter and Hazel Y. Wetzstein
Georgia plume (Elliottia racemosa, Ericaceae) is a threatened, woody plant endemic to Georgia's Coastal Plain region in the southeastern United States. Populations of the plant have a fragmented distribution within a restricted range and are characterized by low genetic diversity and a lack of sexual recruitment. Georgia plume cannot be effectively propagated using conventional methods. We have developed an in vitro shoot regeneration system that is effective with explants obtained from mature plants in the wild. The objective of this study was to determine the efficacy of using this in vitro protocol to regenerate proliferating shoot cultures from 34 georgia plume genotypes obtained from divergent populations. Young expanding leaves were cultured on Gamborg's media supplemented with 10 μM thidiazuron and 5 μM indole-3-acetic acid. After 8 weeks, tissues were transferred to a shoot elongation medium with 25 μM 2-isopentenyl adenine. Of the 34 genotypes tested, 91% formed shoot primordia and 85% regenerated shoots within 6 months of inoculation. This study verifies that tissue culture can be used to produce adventitious shoots from a wide range of georgia plume genotypes. Within a coordinated conservation program, tissue culture is a feasible system to use for safeguarding and reintroduction of genetically diverse plant material, which may be critical to the survival of this rare species.