Chokecherry (Prunus viginiana L.) is an important shrubby species for agroforestry planting in the northern Great Plains states. The X-disease is a serious limiting factor for its utilization. The objective of this research was to produce clonal materials for studying the host and X-disease phytoplasma interactions and for screening X-disease resistant chokecherry germplasms. Shoot tips of 1–2 cm in length were isolated from 1-year old seedling plants, sterilized, and initiated on three basal media supplemented with 5 μm BA and 5 μm IBA. After five weeks, an average of 4.8, 2.2 and 0.3 new shoots were produced on Murashige and Skoog (MS) medium, woody plant medium (WPM) and Knop's medium, respectively. The newly formed shoots were subcultured on MS medium with 5 m BA and 5 m IBA. MS and DKW media gave significantly higher proliferation rates (12–13 shoots after 4 weeks) than WPM (5.5 shoots). Microshoots rooted in half-strength MS medium supplemented with 5 and 10 μm of either IBA or NAA. The shoots were either placed on the medium for 19 days, or for 5 days then transferred to a hormone free medium for 14 days. On the media with IBA, 80% to 90% of the microshoots rooted with an average of 2.4 roots per shoot and there were no differences in rooting percentage and root number. When shoots were exposed to NAA for 5 days, 66.7% of shoots on medium with 5 μm NAA, and 83.3% on the medium with 10 m NAA formed an average of 2.2 roots per shoot; but when the shoots were exposed to NAA for 19 days, 36.4% of shoots on the medium with 5 m NAA and 30% on the medium with 10 μm of NAA formed an average of 0.53 roots per shoot. These rooted shoots are under acclimation to the ambient environment.
Zhen Zhang and Zong-Ming Cheng
Zhen Zhang and Zong-Ming Cheng
Jasmonates are a group of native plant bioregulators that occur widely in the plant kingdom and exert various physiological activities when applied exogenously to plants. We investigated the effect of free jasmonic acid (JA) on stem and root growth and tuberization of potato in vitro nodal culture. Nodal cuttings of three potato cultivars, Norchip, Red Pontiac, and Russet Burbank, were cultured in 2.5 × 15 cm test tubes containing either nodal culture (MS with 2% sucrose) or tuber-inducing (MS with 8% sucrose and 11.5 μm kinetin) medium. The media were supplemented with JA at 0, 0.1, 0.5 1.0, 5.0, 10.0 and 50 m. The cultures were maintained under a 16-hour photoperiod at 24°C for 6 weeks. Potato cultivars showed different sensitivities to JA in stem growth. Norchip is the most and Red Pontiac the least sensitive cultivar. On the nodal culture medium, stem length of Norchip was promoted at 0.1–5 μm, and inhibited at 10–50 μm of JA, but that of Red Pontiac was promoted by JA at all concentrations tested. The number of nodes increased significantly on media with JA than that on medium without JA. The number of adventitious roots did not, but the lateral roots increased significantly when JA was added to the medium. On tuber-inducing media, stem length and node number did not appear to be affected by addition of JA to the medium. The number of axillary shoots increased significantly on the media with low concentrations of JA (0.1–5 μm). No microtubers formed on both media from all three cultivars in 6 weeks.
Nian-Oing Shi and Zong-Ming Cheng
Some antibiotics mimic plant hormones on cell growth and plant regeneration. Cefotaxime and carbenicillin were tested in American elm for induction of embryogenesis from cotyledonary explants, which normally show organogenesis. Cotyledons from 1-week-old in vitro germinated seedlings were placed on a shoot regeneration medium (a modified MS medium containing 15 μ M BA, B5 vitamins and 0.3% gelrite) with various levels of cefotaxime and carbenicillin. One hundred percent of explants showed embryogenesis in the medium supplemented with 125 μg/ml cefotaxime; 75% explants regenerated somatic embryos in medium with 500 μg/mg carbenicillin; and only 50% explants produced somatic embryos in the medium with both of these antibiotics. In control medium without antibiotics, 100% explants regenerated shoots, instead of somatic embryos. Further studies are necessary to determine the nature of these antibiotics on shifting developmental pathways and their stimulatory effect on embryogenesis from American elm cotyledons.
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.
James A. Kapaun and Zong-Ming Cheng
Plants were regenerated from leaf tissue of greenhouse-grown seedlings of Siberian elm (Ulmus pumila L.). Shoot regeneration was induced on Murashige and Skoog (MS) medium containing 5 to 10 μm of BA. Up to 55% of the leaf explants formed shoots with an average of 2.4 shoots per explant. Addition of 2.5 or 5 μm of IBA failed to enhance regeneration. Thidiazuron at 0.5 or 1.0 μm also induced shoot regeneration, but the shoots failed to elongate as well as shoots regenerated from media containing BA. Incubation in darkness for 7, 14, or 21 d had little effect in promoting shoot regeneration, except that incubation for 21 d increased shoot regeneration on the medium with 5.0 μm BA. Genotypes differed in shoot regeneration potential, with regeneration frequencies ranging from 13% to 55%. Regenerated shoots were micropropagated on Driver and Kuniyuki Walnut medium. Ninety percent of microcuttings rooted directly in potting soil. This regeneration system will be valuable for genetic transformation and cell selection of Siberian elm. Chemical names used: 6-benzylaminopurine (BA); indole-3-butyric acid (IBA); N-phenyl-N′ -1,2,3-thiadiazol-5-ylurea (thidiazuron, TDZ).
Wayne A. Sargent and Zong-Ming Cheng
Hybrid aspen is a major source of fiber in the north-central United States. One major problem for utilizing genetically improved aspen, and a variety of other woody species, is the difficulty in rooting hardwood cuttings. The objective of this project was to construct and confirm the function of genetic transformation vectors with two rooting genes (iaaM from Agrobacterium tumefaciens and rolB from A. rhizogenes) using three promoters (CaMV35S, soybean heat shock-inducible and poplar wound-inducible promoters). The gene constructs can be used to transform aspen for improvement of rooting hardwood cuttings and to elucidate rooting mechanisms. Each of these six gene constructs was inserted contiguously upstream from the promoter-less-glucuronidase (GUS) gene in the plasmid pBI 101. The engineered plasmids were transformed into A. tumefaciens strain LBA 4404 by electroporation. The functions of the genes were confirmed by transforming Nicotiana tabacum with these constructs. In plants transformed with iaaM and rolB under the CaMV35S promoter, the GUS gene expressed constitutively. These plants were also morphologically abnormal, with callus (and sometimes root) formation on stem tissue. The plants transformed with heat shock and Win6 constructs had no root formation and little or no GUS expression, without induction. After induction with heat shock (42°C for 1 hr per day for 7 days) or wounding (wounding the plants daily for 7 days), root formation occurred and GUS expression increased significantly with each gene, respectively. No roots developed in the non-transformed control shoots. The transformed plants with the regulatory promoters appeared morphologically normal. These constructs are currently being tested in hybrid aspen.
James A. Kapaun and Zong-Ming Cheng
Four aminoglycoside antibiotics were evaluated for their effects on shoot regeneration from leaf explants of Siberian elm (Ulmus pumila L.) seedlings and their potential use as selective agents in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene. Kanamycin at 100 mg·L–1 or higher concentration reduced shoot regeneration, with complete inhibition at 225 mg·L–1, and was considered a suitable selective agent. Neomycin completely inhibited shoot regeneration at 450 mg·L–1, but all explants remained green; therefore, it may also be used as a selective agent. Geneticin significantly inhibited shoot formation at 1 mg·L–1 and completely killed the explants at 4 mg·L–1 after 1 week. Geneticin was too toxic for direct selection, but may be useful in a delayed selection scheme or for confirmation of transformation. Paromomycin was least effective in inhibiting shoot formation; 13% of explants still regenerated shoots on the medium with the highest concentration tested (400 mg·L–1). Both neomycin and paromomycin precipitated in media containing Phytagel as a gelling agent if antibiotic stock solutions were added to the medium without adjusting their pH. Precipitation was prevented by adjusting the pH of the stock solutions from 6.2 (neomycin) or 6.9 (paromomycin) to above 9, or by using agar as a gelling agent. The precipitation was not affected by the concentrations of salts in the media.
Chao Dong, Xue Li, Yue Xi and Zong-Ming Cheng
Pyracantha coccinea is a thorny evergreen shrub native to southeast Europe to southeast Asia. It is a popular ornamental plant because of its showy bright red fruits and small white flowers. However, in vitro vegetative propagation of P. coccinea has not been studied. Nodal segments with one or two axillary buds (1 to 1.5 cm in length) were cut and disinfected in a solution of 0.1% (v/v) mercuric chloride (HgCl2) for 5 minutes, and proliferated on Murashige and Skoog (MS) basal medium supplemented with various concentrations 6-benzylaminopurine (6-BA). After 4 weeks, newly formed shoots were transferred to proliferation and rooting media containing various concentrations of indole-3-butyric acid (IBA). Establishment of axillary buds was significantly better with an establishing rate of 67% on basal MS medium augmented with 6.6 µm 6-BA. The best medium for proliferation of shoots was three-fourth basal MS supplemented with 1.5 µm IBA, with a proliferation rate of 3.4 axillary bud. The optimum rooting medium was one-fourth MS basal medium containing 93 µm IBA. Rooting of shoots was as much as 77%. Rooted plantlets were transferred to pots containing vermiculite:perlite:peat (6:1:2) and acclimatized to ambient greenhouse conditions with a 95% survival rate. This protocol can be used for in vitro propagation of P. coccinea.
Shanqiang Ke, Chiwon W. Lee and Zong-Ming Cheng
Coleoptile tissues excised from young seedlings of `Touchdown' Kentucky bluegrass (Poa pratensis L.) were bombarded with the disarmed Agrobacterium tumefaciens strain EHA 101 carrying rolC (from A. rhizogenes), NPT II and GUS genes. These tissues were then cultured on Murashige and Skoog (MS) medium containing 0.2 mg·L–1 picloram, 0.01 mg·L–1 naphthaleneacetic acid (NAA), 150 mg·L–1 kanamycin, and 50 m acetosyringone. Calli formed on this medium within 2 weeks. The regenerated plants from these calli were analyzed for the presence of the GUS and rolC genes by histochemical GUS assay, PCR, and Southern hybridization. Only 3.7% of the regenerants were transformed when determined by the GUS assay. A similar frequency of transformation in the regenerated plants was obtained after bombarding the coleoptile tissues with the DNA isolated from the pGA-GUSGF-rolC plasmid. Most of the putative transformants were either albinos or variegated plants that are composed of both albino and green tissues.