The effects of genotype, basal medium, plant growth regulator (PGR), dark treatment, and antibiotics on shoot regeneration of two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were investigated. In vitro shoots were regenerated from leaf tissues in either Murashige and Skoog (MS) or woody plant medium (WPM) media supplemented with benzyladenine (BA). In general, more shoots were regenerated in WPM medium than in MS medium. Dark treatment for 3 to 5 weeks dramatically increased shoot regeneration. Addition of indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) significantly enhanced the regeneration rate and shoots of each explant. The maximum regeneration rate (100%) of B. davidii ‘Potters Purple’ was achieved when cultured in WPM containing 5 μm BA plus 5 μm IBA. The maximum regeneration rate (98.4%) of Buddleia ‘Lochinch’ was found in WPM supplemented with 20 μm BA plus 4 μm IBA. Carbenicillin at 250 to 500 mg·L−1 and cefotaxime at 125 to 250 mg·L−1, individually or combined, promoted shoot regeneration. Interactions between genotype and medium or PGRs were found. In vitro shoots were easily rooted in half-strength MS medium with or without NAA. Rooted plants were transferred to potting mix and grown in the greenhouse. This research will facilitate genetic improvement and fast propagation of Buddleia species using biotechnology.
Wenhao Dai and Cielo Castillo
Wenhao Dai and Victoria Jacques
Periwinkle, a perennial commonly used as a summer bedding plant, is known as the source of vinca alkaloids used to treat lymphocytic leukemia and Hodgkin's disease. It is also one of the natural hosts of many phytoplasma diseases, such as X-disease of major Prunus species, aster yellows, and ash yellows diseases. Therefore, periwinkle is an ideal plant species for phytoplasma disease research, such as disease transmission, species resistance, and resistant gene screening. Periwinkle tissue culture was established by incubating sterile seeds in hormone-free Murashige and Skoog (MS) medium. Plants were successfully regenerated from in vitro leaf tissues of periwinkle. Adventitious shoots were induced when leaf tissues were cultured on Murashige and Skoog (MS) medium or woody plant medium (WPM) supplemented with benzyladenine (BA) and naphthaleneacetic acid (NAA). Nearly 75% of leaf explants produced shoots in both media with 10–20 μm BA and 1 μm NAA. A mean of 4.3 shoots was produced from each explant cultured on WPM, whereas only 2 shoots were produced on MS medium under 16-h photoperiod. Leaf explants under dark treatment for 2 weeks produced big callus only, indicating that light is necessary for shoot formation. Most adventitious shoots were induced from the joint of leaf blade and petiole. In vitro shoots (>1.5 cm) were easily rooted in half-strength MS medium. Addition of NAA dramatically increased root number, with more than 20 roots being induced in 5 μm NAA medium. Rooted plants were transferred to potting medium and grown in a greenhouse.
Wenhao Dai and Victoria Magnusson
Buddleia species is commonly used as a perennial for landscaping as a result of its heavy flowers and long bloom period. However, this species has a few concerns such as lack of flower color, excessive growth, and an invasive nature. Attempts to improve Buddleia using conventional breeding methods have resulted in limited success. In this study, mutagenesis by gamma ray irradiation was used to induce genetic variations. In vitro shoot tips of two Buddleia cultivars, B. davidii ‘Potters Purple’ and Buddleia ‘Lochinch’, were exposed to 0 to 150 Gy gamma rays and then recovered in Murashige and Skoog (MS) medium supplemented with 2.5 μM benzyladenine (BA). Shoots that recovered from the gamma ray treatment were rooted in half-strength MS medium with 0.5 μM naphthalene acetic acid (NAA) and grown in the greenhouse. The growth of shoot tips was inhibited after they were exposed to gamma rays. An average of 50.8% of shoots treated with 50 Gy gamma rays were recovered, whereas only 9.7% and 6.5% of shoots recovered when exposed to 100 and 150 Gy gamma rays, respectively. After transfer to the greenhouse, a few plants showed reduced growth with some dying before they reached the flowering stage. Various variations including characteristics of leaves (shape, size, hairs), stems (shape, internode length, branching), flowers (color, size, and structure), and plant stature were observed. This research demonstrates that in vitro mutation induction using gamma ray irradiation could be a useful protocol to develop new cultivars or genetic materials for further breeding of Buddleia or other related species.
Andrea Swanberg and Wenhao Dai
Two periwinkle cultivars, Pacific Coral (P1) and Sunstorm Rose (P2), were used for development of a plant regeneration system. Leaf and internodal explants collected from in vitro plants were plated onto woody plant medium (WPM) using a factorial arrangement of 6-benzyladeine (BA) and 1-naphthalene acetic acid (NAA). Shoots were successfully regenerated. Shoot production from leaf tissues was minimal for all cultivars, whereas internodal tissues showed variable rates of regeneration depending on the hormone combination. Cultivar P1 showed the maximum regeneration rate (73.3%) when internodal explants, 4 to 6 mm in length, were placed on WPM containing 5 μm BA and 5 μm NAA. Cultivar P2 showed a regeneration rate of 56.7% with a combination of 20 μm BA and 10 μm NAA. Shoot regeneration rate increased as the internodal explant size increased for P2; however, the regeneration rate decreased when the explant size was greater than 7 mm for P1. The shoot regeneration rate decreased as the period of the dark treatment of internodal explants increased in both P1 and P2. The antibiotics carbenicillin (Carb) and cefotaxime (Cef) had little effect on shoot regeneration. There was a slightly higher rate observed for P1 when Cef was added into the medium, whereas P2 showed a decrease with the addition of Cef. Carb showed no significant effect on shoot regeneration for both cultivars. Addition of both Carb and Cef to the medium slightly inhibited shoot regeneration.
Wenhao Dai, Victoria Magnusson, and Chris Johnson
Chokecherry (Prunus virginiana L.) was transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pBI121 carrying the neomycin phosphotransferase gene (nptII) and β-glucuronidase (GUS) gene (uidA). Plants were regenerated from the Agrobacterium-infected leaf tissues through organogenesis on woody plant medium (WPM) supplemented with MS (Murashige and Skoog) vitamins, 10 μm 6-benzyladenine (BA), and 250 mg·L−1 cefotaxime plus 500 mg·L−1 carbenicillin plus 15 mg·L−1kanamycin (CCK15). Transformation was verified with polymerase chain reaction (PCR) and Southern blot analysis. Four of 150 (2.67%) initial explants produced GUS- and PCR-positive shoots. Southern blot analysis confirmed that the transgenes were integrated into the chokecherry genome. Transgenic in vitro shoots were rooted in half-strength MS medium containing 10 μm naphthalene acetic acid. Rooted plants were transferred to potting mix and grown in the greenhouse. This research shows a potential for future improvement of chokecherry and other Prunus species. Chemical names used: 6-benzyladenine (BA), naphthalene acetic acid (NAA), acetosyringone (AS), 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium (X-Glu), cefotaxime, carbenicillin, kanamycin.
Wenhao Dai, Cielo Castillo, and Victoria Magnusson
In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.
Wenhao Dai, Victoria Magnusson, and Andrea Swanberg
Many woody plants, including some birch species, can be cloned using such in vitro techniques as pre-existing meristem culture, organogenesis, and embryogenesis. However, clonal fidelity of in vitro-derived plants is always a big concern because somaclonal variations may be induced during the entire in vitro process. To address this issue, we used random amplified polymorphic DNA (RAPD) markers to determine the genetic stability of in vitro-propagated plants of Betula platyphylla `Fargo'. Forty-two greenhouse-grown birch plants derived from a 10-year shoot tip culture (shoot-derived) and 42 in vitro plants regenerated from leaf tissues (regenerated) were randomly selected and evaluated for their genetic fidelity by RAPD. To date, 20 primers (C1-C20, Operon Technologies) were screened for all 84 plants. Only strong bands that are conservative were scored. Each primer generated a unique set of amplification products. Most of scoreable bands are ranged from 350 to 1800 bp. A total of 3696 fragments were amplified from 42 shoot-derived plants by all 20 primers with an average of 4.4 bands per primer, in which 6 primers produced polymorphic bands, indicating some genetic variations within shoot-derived plants. Nineteen out of 20 primers yielded 2772 clear and reproducible bands (an average of 3.47 per primer) from 42 regenerated plants with no significant variations being detected. Our preliminary results showed that in vitro regenerated plants are genetically uniform. However, a long-term tissue culture might result in a few genetic variations of birch species.
Wenhao Dai, Zong-Ming Cheng, and Wayne Sargent
A high-efficiency regeneration/transformation system was developed for three elite aspen hybrids (Populus tremuloides × P.tremula, P.tremuloides × P.davidiana, and P. × canescens × P. grandidentata). On both modified MS medium for aspen (MSA) and Woody Plant Medium (WPM) supplemented with zeatin (2.0 mg/L) and NAA (1.0 mg/L), nearly 100% leaf explants formed calli, of which 80% to 100% regenerated into shoots on both media with 2.0mg/L zeatin and 0.01 mg/L TDZ. Bacterial concentration, pH value of the co-cultivation medium, and acetosyringone were evaluated for enhancing transformation efficiency. Agrobacterial concentration at 1.0 Absorbance at OD600 was better than at 0.1, 0.5 Abs, yielding 80% and 75% of callus induction rates from agrobacterium harboring CaMV35s and Heat shock promoter constructs, respectively. The pH of co-cultivation medium, ranging 5.0 to 5.9, did not have any effect on transformation frequency. Acetosyringone was added to the co-cultivation medium and/or to the callus induction medium, the induction of kanamycin-resistant callus increased from 70% to 80% to 90% to 100%, and the size of callus also increased. Acetosyringone had no effect on shoot regeneration from kanamycin-resistant calli. Regenerated aspen shoots were screened on the kanamycin-containing medium, and confirmed by GUS histochemical assay. The GUS-positive plants were further confirmed by polymerase chain reaction, showing that the nptII, uidA, and rolB genes were integrated into the aspen genomes.
Wenhao Dai, Zong-Ming Cheng, and Wayne Sargent
Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.
Wenhao Dai, Bingcheng Sheng, and Zhen Zhang
`Xiao Fang Shi' is a rare, dwarf cultivar of persimmon (Diospyros kaki Linn cv.) recently found north of Shanghai, China. The tree starts to bear fruit at 2 years of age, while standard trees start fruiting at 5 or 6 years of age. Dwarf and standard cultivars have about equal spring shoot growth, but the dwarf cultivar has little fall growth. To determine the mechanisms of dwarfness and early fruiting, enzyme-linked immunosorbant assay (ELISA) was used to analyze the endogenous indoleacetic acid (IAA), gibberellic acid (GA1+3) and abscicic acid (ABA) contents in leaves and shoot tips of dwarf (`Xiao Fang Shi') and standard (`Da Fang Shi' and `Zhu Sha Hong') persimmon. The measurement was done during the entire growing season. The results showed that IAA, GA1+3, and ABA contents were influenced by cultivars, ages of trees, and phenophases. The dwarf cultivar `Xiao Fang Shi' has lower IAA and GA1+3 but higher ABA contents than the two standard cultivars during the growing season. These correlations are especially evident when the fruit is ripening. The correlation coefficiency between contents of IAA and GA1+3 and tree height was 0.9704, while that between ABA content and tree height is –0.9697. The low IAA and GA, and high ABA contents may be responsible for little shoot growth of the dwarf cultivar in the fall.