`Marion' is the most important blackberry cultivar in the world, primarily due to its outstanding processing characteristics. Ideally, `Marion' could be enhanced via transformation while maintaining its fruit quality. As a successful in vitro regeneration is the prerequisite for genetic transformation, a series of experiments were conducted to optimize the conditions for in vitro regeneration for `Marion' blackberry. Parameters studied included types (three cytokinins: BA, kinetin, and zeatin; four auxins: IBA, IAA, NAA, and 2,4-D) and concentrations of plant growth regulators, explants (leaf and petiole), medium formulations (MS, WPM, and BMM), and the duration of TDZ pretreatment (3 to 6 weeks) of in vitro-grown stock plants. The highest shoot regeneration rate (65.7%) and highest number of shoots (5.1 shoots/explant) were obtained under the following conditions: stock plants were pretreated with TDZ (1 mm) for 3 weeks, followed by leaf explants dark pretreatment for 1 week on the regeneration medium (WPM with 5 mM BA and 0. 5 mm IBA). After dark treatment, regeneration plates were placed under 16-h photoperiod at light intensity of ≈50 μmol·m-2·s-1 at 23 + 2 °C for 4 weeks.
Rengong Meng, Tony H.H. Chen, and Chad E. Finn
Jing-Tian Ling and Roger J. Sauve
Leaf segments of greenhouse-grown Ulmus americana L. plants cultured on a Murashige and Skoog basal salts medium supplemented with 0.22 mg/L thidiazuron formed friable type of callus and regenerated shoots. This friable callus readily formed a cell suspension when the callus was placed in a liquid MS medium containing 2 mg/L 1-naphthaleneacetic acid and 1 mg/L 6-benzylaminopurine. Shoots were regenerated from 3-month-old suspension cell cultures after the suspension cells had been cultured on solid medium. Shoots developed roots on MS medium containing 0.1 mg/L indole-3-butyric acid. Intact plants were successfully established in soil.
Carol Gonsalves, Baodi Xue, Marcela Yepes, Marc Fuchs, Kaishu Ling, Shigetou Namba, Paula Chee, Jerry L. Slightom, and Dennis Gonsalves
A single regeneration procedure using cotyledon explants effectively regenerated five commercially grown muskmelon cultivars. This regeneration scheme was used to facilitate gene transfers using either Agrobacterium tumefaciens (using `Burpee Hybrid' and `Hales Best Jumbo') or microprojectile bombardment (using `Topmark') methods. In both cases, the transferred genes were from the T-DNA region of the binary vector plasmid pGA482GG/cp cucumber mosaic virus-white leaf strain (CMV-WL), which contains genes that encode neomycin phosphotransferase II (NPT II), β-glucuronidase (GUS), and the CMV-WL coat protein (CP). Explants treated with pGA482GG/cpCMV-WL regenerated shoots on Murashige and Skoog medium containing 4.4 μm 6-benzylaminopurine (BA), kanamycin (Km) at 150 mg·liter-1 and carbenicillin (Cb) at 500 mg·liter-1. Our comparison of A. tumefaciens- and microprojectile-mediated gene transfer procedures shows that both methods effectively produce nearly the same percentage of transgenic plants. R0 plants were first tested for GUS or NPT II expression, then the polymerase chain reaction (PCR) and other tests were used to verify the transfer of the NPT II, GUS, and CMV-WL CP genes. This analysis showed that plants transformed by A. tumefaciens contained all three genes, although co-transferring the genes into bombarded plants was not always successful. R1 plants were challenge inoculated with CMV-FNY, a destructive strain of CMV found in New York. Resistance levels varied according to the different transformed genotypes. Somaclonal variation was observed in a significant number of R0 transgenic plants. Flow cytometry analysis of leaf tissue revealed that a significant number of transgenic plants were tetraploid or mixoploid, whereas the commercial nontransformed cultivars were diploid. In a study of young, germinated cotyledons, however, a mixture of diploid, tetraploid, and octoploid cells were found at the shoot regeneration sites.
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.
Vellicce Gabriel Ricardo, Yamilet Coll, Atilio Castagnaro, and Juan Carlos Diaz Ricci
A protocol for shoot regeneration of strawberry (Fragaria ×ananassa Duch. `Pajaro') leaf disks was developed. In Murashige and Skoog basal medium with 3% of sucrose (w/v), BA (1 mg·L-1), and 2,4-D (0.1 mg·L-1), 70% of the cultivated leaf explants regenerated plants. This regeneration system was used for genetic transformation of strawberry with Agrobacterium tumefaciens strain LBA 4404 carrying the binary vector plasmid pBI121 that contains the npt II (neomycin phosphotransferase) and uidA (ß-D glucuronidase) genes. A transformation rate of 6.6%, calculated as the number of leaf disks able to regenerate kanamycin-resistant plants/total leaf disks infected, was obtained. The integration of both marker genes was evaluated in each transformed line by PCR (polymerase chain reaction) amplification of the npt II and uidA genes. High expression levels of the uidA gene were found in leaves, flower, and fruits of the transgenic lines. The protocol here reported may represent a way to conduct transformation research in strawberries. Chemical names used: benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D).
Manoj Singlachar, Robert R. Tripepi, and Mary W. George
Attempts to regenerate plants from leaf explants of Rosa xhybrida L. on Murashige and Skoog medium have met with limited success. We report improved regeneration of somatic embryos and adventitious shoots from leaves of `Golden Emblem' on N6 medium. Leaf explants were obtained from microshoots that had been in culture for 4 years. Leaves were placed on N6 medium containing various combinations of 0, 0.4, or 1.0 μM 2,4-D and kinetin for 20 days with an initial dark treatment of 12 days, then transferred to a medium without plant growth regulator. Adventitious shoots and somatic embryos were observed 3 weeks after transferring to medium without plant growth regulators. Leaf explants placed on media without 2,4-D failed to form embryos or shoots. The best combinations of 2,4-D and kinetin (0.4 and 0.4 μM, 0.4 and 1.0 μM, or 1.0 and 1.0 μM) induced regeneration percentages ranging from 21% to 39%. N6 appears to improve regeneration of somatic embryos and adventitious shoots from `Golden Emblem' leaf explants, but the interaction between media formulation and duration of exposure to 2,4-D and kinetin needs to be examined.
Azza Abdel-Aziz Tawfik and P. E. Read
Regeneration from callus of rosemary has not been reported. Leaf segment, meristem-tip and shoot-tip explants of Rosmarinus officinalis were cultured on a Murashige and Skoog (MS) medium supplemented with five concentrations of the cytokinin thidiazuron (TDZ) alone or in combination with 3-indoleacetic acid (IAA). Callus was formed on the base and leaves of the shoottips after 6 weeks when cultured under cool white fluorescent light (26 u mol·S-1 m-2) on MS containing 0.5, 1.0, 1.5 or 2.0 mg/l TDZ. Calti were transferred to fresh MS medium supplemented with 0.2, 0.4, 0.6, 0.8 or 1.0 mg/l TDZ or 2.0, 4.0, 6.0 or 8.0 mg/l benzyladenine (BA) where shoot formation occurred. Essentiality of IAA was not clear from these experiments and further research is underway to refine regeneration protocol
Chiwon W. Lee, Joel T. Nichols, Lijuan Wang, and Shanqiang Ke
Excised leaf sections of lance coreopsis cultured on Murashige Skoog (MS) medium produced adventitious shoots in response to BA. When the combinations of 0, 0.5, 1, or 2 μm NAA with 0, 5, 10, 20, or 40 μm BA were tested, shoots were induced by any of the four BA concentrations used in the medium, regardless of the presence of NAA. The average number of shoots formed per leaf section ranged from 1.4 to 4.3 seven weeks after culture initiation. Roots were induced at the base of individual shoots on the same regeneration medium when cultures were kept longer than 7 weeks. The rooted plants were transferred successfully into soil. The regenerated plants had the same growth and flowering characteristics as the seed-grown plants. Chemical names used: benzyladenine (BA); naphthaleneacetic acid (NAA).
Karen E. Hokanson and Margaret R. Pooler
Callus formation and adventitious shoot regeneration in vitro from mature stored seed were evaluated in eight ornamental cherry (Prunus) taxa: P. campanulata Maxim., P. maackii Rupr., P. sargentii Rehd., P. serrula Franch., P. serrulata Lindl., P. subhirtella Miq., P. virginiana L., and P. yedoensis Matsum. Several portions of the embryo (cotyledons and hypocotyl sections) and nine combinations of growth regulators (BA, 2,4-D, IBA, NAA, and TDZ) were compared. Effects of embryo portions and growth regulator treatments were generally small within taxa, but shoot formation differed among taxa. About 20% to 50% of the embryos from P. virginiana and P. serrula and ≈5% to 30% of those from P. maackii produced shoots. The other taxa generally did not produce shoots. Regeneration from mature stored seed in the responsive taxa represents a potential system for genetic transformation. Chemical names used: 6-benzyladenine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); indole-3-butyric acid (IBA); α-naphthaleneacetic acid (NAA); thidiazuron (TDZ).
Jing-Tian Ling, Henock Zerit, and Roger J. Sauve
Seeds of Chinese elm cultivar King's Choice were collected from field-grown plants and germinated aseptically. Hypocotyl segments were excised from 2-week-old seedlings and cultured on Murashige and Skoog (MS) medium supplemented with TDZ alone or in combination with 0.05 mg/L NAA. At least 50% of explants produced shoots 4 weeks after culture initiation. At thidiazuron (TDZ) from 0.05 to 5.0 mg/L, the number of shoots/explant increased as concentration increased. Addition of 0.05 mg/L NAA stimulated shoot regeneration when TDZ concentration was 0.5 mg/L or less, but suppressed it if TDZ concentration was higher than 0.5 mg/L. Regenerated shoots elongated quickly on MS medium supplemented with 1 mg/L gibberellic acid and initiated rooting on MS medium containing 0.1 mg/L indole-3-butyric acid.