Friable embryogenic callus of American ginseng (Panax quinquefolium L.) was induced from root pith on Murashige and Skoog medium supplemented with 2 mg 2,4-D and 1 mg KIN/liter. Optimal callus growth occurred on medium containing 1.5 mg dicamba/liter. Plants were regenerated on MS medium supplemented with various concentrations of plant growth regulators (PGRs); the best PGR combination was 0.5 mg IBA and 0.1 mg NAA/liter. Chemical names used: (2,4-dichlorophenoxy) acetic acid (2,4-D); 3,6-dichloro-o-anisenic acid (dicamba); 6-benzylaminopurine (BA); gibberellic acid (GA); indole-3-butyric acid (IBA); kinetin (KIN); and naphthaleneacetic acid (NAA).
Andrew S. Wang
Larry J. Shoemake, Michael A. Arnold and Fred T. Davies Jr.
A series of six experiments was conducted over eight years to investigate impacts of provenance on transplant establishment in landscapes and the role of adventitious root regeneration in differential genotypic responses during establishment of Platanus occidentalis L. Fall, spring, and summer transplants of container-grown half-sib families (HSF = seedlings derived from a single mother tree with unknown male parentage), including two selections native to Brazos County, Texas (Brazos-C, Brazos-D), one native to Cookeville, Tenn. (Cookeville), two Kentucky/Tennessee HSF from the Westvaco Corp. (WV-10, WV-14), and two Texas HSF from the Texas Forest Service tree improvement program (TFS-09, TFS-24), were established to determine field/landscape growth responses. Subsequent studies were conducted to investigate differential leaf gas exchange responses of TFS-09 and Cookeville during moderate water deficits and to determine root regeneration potential (RRP) responses of TFS-09, Brazos-C, WV-14, and Cookeville HSF following fall, spring, and summer transplant. To investigate consistency of within-family genotypic responses and to determine relationships among adventitious root initiation from shoot cuttings, RRP, and landscape establishment, five seedlings of TFS-09 and five from Cookeville HSF were clonally propagated and ramets tested under field and RRP conditions similar to those with seedling-derived plants. Regionally native HSF consistently grew taller, had larger trunk diameters, and often had greater survival during the first 3 years in the landscape than HSF not native to the region in which the studies were conducted. Rapidity of root regeneration among HFS at the time of transplant was the best root growth related predictor of successful landscape establishment. Some growth advantages were found using genetically improved HSF, but not as consistent an improvement as with the use of seedlings from regional provenances. Within-family variation in landscape performance was greater with nonregional Cookeville clones than with regional TFS-09 clones, however there was overlap among the more vigorous Cookeville clones and the least vigorous TFS-09 clones. Increased rapidity of root regeneration and drought adaptations related to leaf morphology and gas exchange characteristics may be involved in enhanced growth responses of Texas regional genotypes. No consistent relationships were found among adventitious rooting responses from shoot cuttings and subsequent RRP of the same genotypes from root tissues or their growth during the first 3 years in landscapes.
Hristina H. Stamenova-Berberova and Paul E. Read
Native plants are often ignored in horticulture because they may lack major ornamental traits and many of them are difficult to propagate. Creamy indigo (Baptisia bracteata Mnhl.) is a North American legume with considerable potential as a container-grown or ornamental plant for managed landscapes. Nodal explants from aseptically germinated seedlings were evaluated for axilary shoot and leaf development. The explants were cultured on Murashige and Skoog medium (MS) containing adenine sulfate at 80 mg•L-1, 30% sucrose, and different levels of N-6-benzyladenine (BA) (0.5,1.0,2.0 mg•L-1) supplemented with indole-3-acetic acid (IAA) (0.05, 0.1 or 0.5 mg•L-1) or with IAA omitted. Shoot regeneration occurred within 2 to 3 weeks. The best medium for shoot regeneration was MS supplemented with BA at 1.0 mg and IAA at 0.1 mg•L-1. Shoots were transferred onto rooting medium consisting of Ω MS supplemented with 1.0 mg alpha-naphthaleneacetic acid (NAA) and 1.0 mg indole-3-butyric acid (IBA)/L and 20% sucrose. Rooting took place within 3 to 5 weeks. Plantlets were then planted in soil mix, placed under a polyethylene tent for 2 weeks, and transferred into the greenhouse for further growth.
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.
Mahmoud A. Hassan and Harry Jan Swartz
An efficient protocol for plantlet regeneration using petioles and internodes of two blackberry cultivars has been developed for use in genetic transformation. Maximum shoot regeneration was induced on MS medium supplemented with 5 μM Thidiazuron (TDZ) and 0.5 μM IBA. Preconditioning the source shoots on 0.5 μM TDZ containing MS medium did increase the number of regenerated shoots/explant, but did not affect the regeneration percentage. The antibiotic, kanamycin, significantly reduced the growth and regeneration of explants at 10 mg/L or higher. In contrast, cefatoxime at 100-500 mg/L increased explant growth and percentage regeneration.
Nawab Ali, Robert Skirvin, Walter E. Splittstoesser and William L. George
Cucumber (Cucumis sativus L.) seeds of `Marketer', `Marketmore', `Wisconsin SMR-18', `Tablegreen', `Spotfree', and `China' were stored at 3C and 38% relative humidity for up to 26 years. Seed older than 13 years did not germinate. Cultivars stored 10 years gave 80% germination, except Wisconsin SMR-18' (40%). Ten-year-old seeds were separated from their seedcoats, and cotyledons were excised into six segments. Explants were placed on Murashige and Skoog medium with all combinations of BAP (0, 1,2, and 3 mg·liter-1) and NAA (0, 0.1,0.2, and 0.3 mg·liter-1). Plants were obtained from culture for all cultivars grown on medium containing NAA and 1 mg BAP/liter. No plants were regenerated when BAP or NAA was lacking. Chemical names used: benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA).
Partially expanded male catkins of swamp white oak (Quercus bicolor Willd.) and red oak (Quercus rubra L.) were cultured on Murashige and Skoog (MS) medium supplemented with BA or 2,4-D. Explants on 2,4-D produced a yellow embryogenic callus originating from the junction of the pedicel and peduncle. Subsequent transfers to MS with BA and then MS without growth regulators resulted in callus proliferation. After 10 to 14 weeks in culture, white embryoids developed from the callus of Q. bicolor. Separated and individually cultured embryoids underwent direct, repetitive embryogenesis. Upon transfer to l/2-strength MS, embryoid germination and plant regeneration occurred. Callus of Q. rubra degenerated after 5 months in culture, failing to yield embryogenic structures. Chemical names used: dichlorophenoxyacetic acid (2,4-D); benzyladenine (BA).
Yaseen Mohamed-Yaseen, Raymond J. Schnell, Robert J. Knight and T.L. Davenport
A procedure was developed to regenerate plants via tissue culture from embryonic axes of mature avocado seeds. Explants were cultured in Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and naphthalene-acetic acid (NAA) or thidiazuron (TDZ) and NAA. Culture were kept in the dark for 7-10 days to reduce browning resulting from phenolic oxidation. Multiple shoots (5-8) were formed after transfer to light. Further multiplication were achieved using different combination of BA and NAA or TDZ and NAA. Shoots were cultured in MS supplemented with 2mg/l indolebutyric acid (IBA) for 2 weeks then transferred to MS supplemented with lg/l activated charcoal for root induction. Complete plants were obtained in vitro.
Immature leaf laminae and petioles of `Regale' and `Fry' muscadine grapes (Vitis rotundifolia Michx.) were cultured on Nitsch and Nitsch (NN) medium supplemented with 9.0 μm 2,4-D and 4.4 μm BA, and gelled with agar. Callus and original explant tissues were transferred to NN medium containing 10.7 μm NAA and 0.9 μm BA to proliferate embryogenic callus, which, when transferred to NN medium without growth regulators, yielded globular embryos. The embryos matured and germinated after being subcultured to fresh medium without growth regulators. Somatic embryogenesis incidence was greater from petioles than laminae: 90% of `Regale' and 50% of `Fry' petioles formed embryos, compared with 14% and 2% of laminae, respectively. Culturing germinated somatic embryos on NN medium with 1 μm BA enhanced shoot growth. Regenerated plants flowered and appeared morphologically normal. Chemical names used: N-(phenylmethyl)- 1H -purin-6-amine (BA); 2,4-dichlorophenoxyacetic acid (2,4-D); α- naphthaleneacetic acid (NAA).
Lunique Estime, Marie O'Shea, Michael Borst, Jennifer Gerrity and Shih-Long Liao
Typha latifolia L. (broadleaf cattail) callus was initiated from leaf sections, as well as from pistillate and staminate spikes. Two basal media in combination with three growth regulator regimes were tested for their capacity to induce callus from the explants. Pistillate spikes maintained in the dark on B5 medium supplemented with 5 mg·L-1 dicamba and 1 mg·L-1 BA produced the fastest growing cell line compared to other explants and media combinations. A growth curve in suspension culture was generated for this cell line on B5 medium. The mass of the callus increased by 150% by the end of the growth curve. Upon transfer of the callus to MS medium without growth regulators but with 3% sucrose and 3% phytagel, plants could be regenerated from 22% of the cultures. Chemical names used: 3,6-dichloro-2-methoxybenzoic acid (dicamba); N 6-benzyladenine (BA).