Several experiments were conducted to evaluate the influence of explant type, sucrose level, and callus development time on sweetpotato [Ipomoea batatas (L.) Lam] in vitro culture. Shoot tip, petiole, and leaf of Selection 75-96-1 was used as explants in Murashige and Skoog (MS) media with different plant growth regulators. Calli derived from shoot tip and petiole produced 42.1% and 10.3% somatic embryos, respectively, but the leaf failed to produce somatic embryos. The effect of sucrose level was determined using shoot tip as explants. Compared with 3% sucrose in the same plant growth regulators level medium during callus initiation and callus proliferation periods, 5% sucrose level suppressed root growth and improved shoot regeneration. The callus development time was measured by using shoot tips on callus initiation medium containing 1.5 mg/L alpha-Naphthaleneacetic acid (NAA) and 0.25 mg/L Kinetin (KIN) plus 5% sucrose. When explants were cultured for less than 6 weeks during callus initiation, then transferred onto plant regeneration medium, plant regeneration via organogenesis occurred; whereas, maintaining cultures for more than 12 weeks on the same callus initiation/proliferation medium, plant regeneration was favored via embryogenesis. Explant type and other factors affecting plant regeneration noted here could be applied to protoplast culture, somatic hybridization, and transformation in sweetpotato.
L. Chen, S.O. Park, S. Dhir, and A.S. Bhagsari
S.O. Park, A. Dursun, D.P. Coyne, and G. Jung
Common bacterial blight (CBB), incited by Xanthomonas campestris pv. phaseoli (Xcp), an important disease in common bean (Phaseolus vulgaris L.) Tepary bean (P. acutifolius A. Gray) is of interest to bean breeders because of resistance to CBB. Our objective was to identify RAPD markers linked to major genes for CBB resistance using bulked segregant analysis in an F2 population from a tepary bean cross CIAT640005 (R) X Nebr#4B (S). A total of 57 RAPD primers (602 RAPD primers screened) showed polymorphisms between bulked DNA derived from R and S CBB plants. All markers showed coupling linkage with CBB resistance. A good fit to a 3:1 ratio of bands for presence and absence using 11 RAPD primers was observed in 77 F2 plants. Markers of U-15 and L-7 primers were 2.4 cM distant from the gene for resistance to Xcp strain LB-2. RAPD markers of U-10, U-20, S-12, Y-4, F-13, P-6, Q-1, and Q-ll primers were 2.4 cM distant from the gene for resistance to Xcp strain SC-4A. RAPD markers of IJ-15 and L-7 primers were 8.4 cM distant from the gene for resistance to Xcp strain EKl l. The tepary RAPD linkage group includes three molecular markers and three genes for resistance to Xcp strains EK-l l, LB-2, and SC-4A and spans a length of 19.2 cM. This data supports the presence of Xcp races.
Lianghong Chen, Ajmer S. Bhagsari, Soon O. Park, and Sarwan Dhir
This study was carried out to optimize conditions for plant regeneration of sweetpotato [Ipomoea batatas (L.) Lam] using shoot tips, petioles, and leaves of Selection 75-96-1 as explants in Murashige and Skoog (MS) with several growth regulators at different levels. Callus initiation and callus proliferation media were 9.0 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 9.0 μm 2,4-D + 1.1 μm N 6-benzyladenine (6-BA) in protocol I; 8.1 μm α-naphthaleneacetic acid (NAA) + 1.2 μm kinetin (KIN) and 5.4 μm NAA + 4.6 μm KIN in protocol II; 0.9 μm 2,4-D, and 0.9 μm 2,4-D + 1.2 μm N-isopenylamino purine (2iP) in protocol III; NAA (8.1 μm) + KIN (1.2 μm) and 2,4-D (0.9 μm) + 2ip (1.2 μm) in protocol IV, respectively. In protocol I and II, shoot tip, petiole, and leaf were used, but only petiole and leaf in protocol III and IV. In the protocol I and II, somatic embryos were obtained only from shoot tip explants; in protocol III and IV, only from petioles. The frequencies of somatic embryo development were 33.3% in protocol I, 42.1% in protocol II, 21.2% in protocol III, and 10.3% in protocol IV, respectively. The leaf explants failed to produce somatic embryos in all the experiments. In protocol I, somatic embryogenesis occurred through the well-known sequence of globular-, heart-shaped-, torpedo-, and cotyledon-type embryos. However, in protocol II, the structures resembling plumule and radicle were observed before the emergence of torpedo/cotyledon type embryo clusters. The somatic embryogenesis in protocol III and IV was similar to that in protocol I. Growth regulators influenced somatic embryo development. Further, this study showed that explant resource and growth regulators affected the frequency of plant regeneration in sweetpotato.
S.O. Park, J.M. Bokosi, and D.P. Coyne
Plant growth habit is an important trait. Our objective was to identify RAPD markers linked to major gene for indeterminate growth habit using bulked segregant analysis in an F2 population from a bean cross Chichara (indeterminate growth habit × PC-50 (determinate growth habit). A total of 132 RAPD primers (600 RAPD primer screened) showed polymorphisms between bulked DNA derived from indeterminate and determinate plants. All markers showed coupling linkage with indeterminate growth habit. RAPD markers of A-8, A-17, C-7, C-15, D-4, D-5, F-6, F-16, G-9, H-3, H-20, and I-7 were 2.2 cM distant from the gene for indeterminate growth habit. Markers of B-7, B-16, B-17, C-8, E-1, F-1, F-20 and H-l9 primers were 4.6 cM distant from the gene for indeterminate growth habit.
S.O. Park, A. Dursun, and D.P. Coyne
Common bacterial blight (CBB), incited by Xanthomonas campestris pv. phaseoli (Xcp), is an important disease of common bean (Phaseolus vulgaris L.). Tepary bean (P. acutifolius A. Gray) is of interest to bean breeders because of resistance to CBB. The objective was to identify RAPD markers linked to major dominant genes for CBB resistance and purple flower color using bulked segregant analysis in an F2 population from a tepary bean cross Nebr#19 [resistant (R) to CBB and white flower color] × Nebr#4B [susceptible (S) to CBB and purple flower color]. Ten RAPD primers (600 RAPD primers screened) showed polymorphisms between bulked DNA derived from R and S plants. All markers showed coupling linkage with CBB resistance. The RAPD marker of G-14 primer was 5.2 cM distant from the gene for resistance to Xcp strain LB-2. The RAPD marker of L-18 primer was 6.8 cM distant from the gene for resistance to Xcp strain SC-4A. The RAPD marker of G-14 primer was 26.2 cM distant from the gene for resistance to Xcp strain EK-11. Seven RAPD primers showed polymorphisms between bulked DNA derived from purple and white flower plants. All markers showed coupling linkage with the gene for purple flower color. The RAPD marker of Y-6 primer was 3.6 cM distant from the gene for purple flower color.
S.O. Park, L.H. Chen, S.K. Dhir, and A.S. Bhagsari
The objective was to determine optimum conditions for embryogenic callus, embryo, organogenesis, and embryogenesis developed from leaf, petiole, stem, and tip tissues of the sweetpotato `Jewel' cultivar and from subcultured callus. Embryogenic callus was developed from stem and tip tissues on MS medium containing combinations of BA and NAA only under light conditions. Plant regeneration via organogenesis was developed from stem and tip tissues on medium including 1, 3 and 4 mg/L BA under dark and light conditions, while no plant regeneration via organogenesis was developed from leaf and petiole tissues. Frequencies for plant regeneration via organogenesis from the tissues were very low. No plant regeneration via embryogenesis was developed from the four tissues on medium having any combinations of BA+NAA and of kinetin+NAA. Embryogenic callus was observed in the subculture of callus developed from petiole and tip tissues on medium containing 0.2 and 2 mg/L 2,4-D only under dark conditions. Embryo was found in the subculture of callus from the tissues on medium containing 0.2 mg/L 2,4-D only under both conditions. Plant regeneration via embryogenesis was obtained in the subculture of callus from the tissues. Plant hormones and other factors affecting plant regeneration from the four tissues of the `Jewel' cultivar and other elite cultivars are currently being investigated at our lab for its application in transformation.
J.D. Chung, Y.K. Park, S.O. Jee, H.Y. Kim, and M.S. Cho
This experiment was conducted to identify the effect of various growth retardants on the growth of Aerides japonicum in vitro. Paclobutrazol was found the most effective retardant for reducing the leaf growth of seedling. Ancymidol and uniconazole also showed retarding effects on leaf growth of one, whereas Daminozide didn't. When growth retardants were added to culture medium, leaf length of seedlings was gradually shortened and leaf width became wider than that of control. However, root length was shorter and number of roots and root diameter were greatly increased. On the contrary, at 0.05 and 0.1 ppm uniconazole, growth of leaf and root were enhanced. It was showed that the possibility of using as an additive for good growth of Aerides japonicum seedling in vitro. The activity of GA-like substances was higher in the portion in which growth of seedlings were promoted. It was identified by anatomical observations that the number of stomata and thickness of cell layer in leaf were increased by treatment of retardants.
N. Mutlu, D.P. Coyne, S.O. Park, and J.R. Steadman
Common bacterial blight (CBB) in common bean (Phaseolus vulgaris L.), caused by Xanthomonas campestris pv. phaseoli (Xcp), reduces bean yields and quality throughout the world. Pinto `Chase' is a high-yielding variety with moderate resistance to Xcp derived from great northern Nebraska #1 selection 27, whose resistance is derived from an unknown tepary (P. acutifolius) bean source. XAN-159 is a black mottled small seeded breeding line with different genes for high resistance to Xcp derived from a different tepary source (PI 319443). Our objective was to pyramid different genes for Xcp resistance from the donor parent XAN-159 into the rust-resistant recurrent parent Pinto `Chase' using the classical back-cross breeding method with confirmation of resistance using RAPD molecular markers. Resistance was confirmed in some BC2F2 generation plants. Seven RAPD markers and the V locus (flower color) previously identified were confirmed in the BC1 and BC2 populations. Smaller seed size, purple flower color, and black mottled seed coat color were coinherited with resistance to Xcp. However, a recombinant plant with enhanced CBB resistance and moderate-sized pinto seed was identified. Backcross breeding is being continued.