Marceline Egnin and C.S. Prakash
This study aimed to optimize factors for the efficient delivery of foreign genes into sweetpotato using Agrobacterium tumefaciens and develop transgenic plants. Disarmed Agrobacterium C58 carrying a binary vector pBI 121C2H with gusA, nptll, and the nutritional protein asp-l genes was used to cocultivate (4 days) petiole explants of the sweetpotato genotype P1318846-3. Pre-incubation of petioles for 3 days on MS medium with 2,4-D (0.2 mg·liter–1) before infection resulted in higher transformation. Putative transgenic shoots were obtained by transfer of petioles to MS medium with TDZ (0.2 mg·liter–1) and kanamycin (80 to 140 mg·liter–1). The PCR amplification of gusA, nptll, and asp-1 genes in the 37 putative transgenic shoots showed that six plants contained the three genes. However, none of these plants showed histochemical expression of the gusA gene. The introduced gene may have been methylated resulting in the lack of its expression. DNA blot hybridization studies are underway to verify the presence and integration of the transgenes.
G. S. Varadarajan and C. S. Prakash
The sweet potato (Ipomoea batatas) and its relatives (the batatas complex) appear to have evolved in the New World and radiated over several geographic centers in the tropics and subtropics. Traditional studies on taxonomy, cytogenetics, and reproductive biology of the batatas complex have enabled us to investigate certain evolutionary aspects. We conclude that this complex is a monophytetic, “polyploid pillar”, evolved by chromosome doubling (euploidy) and interspecific hybridization. We apply molecular genetic techniques to detect variation [restriction fragment length polymorphism (RFLP) and DNA fingerprinting analyses] to reexamine some of the evolutionary issues that could not be satisfactorily addressed by the conventional approaches, e. g., phylogenetic history of the batatas group, the diploid ancestors of the polyploid members, homology/diversity of genome(s) within the entire group. We find DNA variation in the hypervariable or multiple copy regions of the genome in Ipomoea species. In addition, we are investigating polymorphism in unique/low copy regions using a battery of DNA sequences from homologous as well as heterologous sources. The success of this study will hopefully shed a new light on the subject of evolutionary biology and may also have potential applications in the sweet potato breeding.
C.S. Prakash, G. He and R. Jarret
The PCR-based DNA amplification fingerprinting (DAF) approach was used to investigate the evolutionary relationships among 30 U.S. sweetpotato cultivars. Phenogram and pairwise similarity matrix based on Jaccard's coefficients showed relationships among U.S. cultivars and their progenitors to be consistent with the pedigree history. The genetic variability of U.S. cultivars was relatively low (compared to a sample of global collection). Many older U.S. cultivars formed a cluster in the principal coordinate analysis, suggesting their narrow genetic base, but new cultivars, such as `Regal' and `Excel', showed greater divergence. Somatic mutants showed close genetic similarity with their wild types and yet distinct in fingerprint profiles (e.g., `Resisto' and `Copper Resisto'; `Redmar' and `Goldmar'). All cultivars showed unique DAF profiles, and thus, the DAF approach enabled cultivar identification. `Centennial' showed high similarity to major U.S. cultivars such as `Jewel' and `Rojo Blanco'. `Regal' and its open-pollinated offspring `Excel' showed high similarity with each other. `Jewel', the most leading sweetpotato cultivar in the United States, clustered closely to its parent `Nugget' (83%). Carver, a selection from a cross `Centennial' × `Jewel', showed 75% similarity with `Jewel' and 63% similarity to `Centennial'. `Scarlet', a mutant of `Jewel', appeared in the same cluster as `Jewel' but showed only 68% similarity. Our results show that DAF may be an useful approach in elucidating evolutionary relationships among sweetpotato cultivars.
C.S. Prakash, O. Zheng and A. Porobodessai
Stable, transgenic, sweetpotato plants have been developed using an improved somatic embryogenesis consisting of l) stage I—explants incubated in darkness for 14 days on MS medium with 2,4D (2.5 mg·liter–1) and 6-BAP (0.25 mg·liter–1) and 2) stage II—culture in light for 14 to 28 days on MS medium with ABA (2.5 mg·liter–1). Petiole or leaf explants of the genotype PI318846-3 were co-cultivated with Agrobacterium tumefaciens EHA 101 containing gusA::nptII fusion gene. Transgenic somatic embryos were selected on a kanamycin medium (100 mg·liter–1). The PCR analysis of the transgenic sweetpotato plants showed the presence of foreign genes in the sweetpotato genome. About 100 transgenic plants are being maintained under laboratory and greenhouse conditions. All the transgenic plants showed a strong expression of gusA gene in the histochemical GUS assay but showed quantitative differences in the chemiluminescent assay. The CaMV35S promoter shows a differential expression because there was some degree of tissue- and organ-specificity in the gusA expression. All transgenic plants appear normal with no phenotypic aberrations and are being tested for productivity traits.
C.S. Prakash, U. Varadarajan and A. S. Kumar
Development of a gene transfer system will enable rapid introduction of agronomically useful genes into elite cultivars of sweet potato. We compared microprojectile bombardment and Agrobacterium cocultivation approaches to introduce foreign genes into the genome of two sweet potato cultivars. Chimeric marker genes (gusA and kan) were successfully introduced into cvs. Jewel and TIS-70357 using both approaches. However, transgenic plants were generated in vitro using only the Agrobacterium approach. Callus and root isolates with stable expression of gusA gene were obtained using the microprojectile method. Expression of the screenable marker gusA gene was detected by histochemical assays. Integration of the introduced gene into the genome of sweet potato was confirmed by polymerase chain reaction (PCR) amplification of the kan gene and Southern blot analyses. Transgenic sweet potato plants from two cultivars are being raised and studied for quantitative expression and localization of the introduced genes. These results show that foreign genes can be successfully introduced and expressed in sweet potato. Current efforts are directed at optimizing several variables to increase the transformation efficiencies and to generate transgenic cultivars with foreign genes of agricultural importance.
C.S. Prakash, Guohao He and Robert L. Jarret
The polymerase chain reaction (PCR)-based DNA amplification fingerprinting (DAF) approach was used to investigate genetic relationships among 30 U.S. sweetpotato (Ipomoea batatas L. Lam.) genotypes including heirloom cultivars and recent releases. Phenogram, pairwise similarity matrix, and principal coordinate plots were developed based on Jaccard's coefficients using band-sharing data generated by seven octamer primers. All cultivars showed unique fingerprint patterns indicating the utility of DAF in cultivar identification. Many heirloom cultivars such as `Creole' and `Porto Rico' were readily differentiated from recently developed cultivars. Modern cultivars such as `Jewel', `Carver', `Nugget', and `Scarlet' exhibited a high degree of similarity reflecting ancestral relatedness. `Regal' and `Excel', recently developed using a population-based breeding approach, showed greater divergence from all other cultivars. Those cultivars, developed as a result of somatic mutations, exhibited high levels of genetic similarity to their normal-type parents and yet had distinct fingerprint profiles. With few exceptions, genetic relationships derived from DAF data appear to be consistent with available pedigree information.
Ramana M. Gosukonda, C.S. Prakash and Ananta Porobo Dessai
Studies were conducted to improve adventitious shoot regeneration in sweetpotato [Ipomoea batatas (L.) Lam.], specifically to extend the protocol to many genotypes and to elicit production of multiple shoots per explant. The use of a two-stage procedure where excised petioles were incubated on Murashige and Skoog (MS) (1962) medium with 2,4-D (0.2 mg·liter–1) for 3 days and transferred to a second medium containing MS salts with thidiazuron and 2iP (0.05 mg·liter–1 each) resulted in shoot regeneration from eight of 13 genotypes tested, including elite sweetpotato cultivars such as `Jewel' and `Rojoblanco'. PI 318846-3 was the most regenerable genotype, with up to 77% of explants producing one to three shoots per explant. The orientation of the petiole on the nutrient medium was critical; those placed vertically inverted developed multiple shoots. Wounding explants through epidermal peeling with normal horizontal orientation of the explants during incubation also resulted in multiple shoot production (about three shoots per explant). Interference with auxin transport due to explant inversion or wounding may have stimulated increased shoot induction. Chemical names used: 2,4 dichlorophenoxyacetic acid (2,4-D); N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron); N 6-(2-isopentenyl) adenine (2iP).
Essie T. Blay, Anant Porobo-Dessai and C. S. Prakash
Explants of sweet potato (Ipomoea batatas) and garden egg plant (Solanum integrifolium) were cocultivated with disarmed strains of Agrobacterium tumefaciens containing binary vectors with gusA, gusA-nptll fusion or gusA-intron genes. We examined whether the addition of vir gene inducers during cocultivation would improve the transformation in both crops. Acetosyringone and galacturonic acid were tested individually and in combination. A very high GUS expression was detected histochemically in both plant species. The frequency and extent of transformation varied with the type of explant, petioles being the most responsive. The presence of the vir inducing substances in the medium influenced the percent explant area transformed but did not appreciably affect the frequency of transformation. The selective proliferation of the transformed tissue and organogenesis was achieved by the culture of explants on MS medium supplemented with antibiotics.
C. S. Prakash, R. Gosukonda, A. Porobo Dessai, E. Blay and K. Dumenvo
Lack of suitable methods to develop adventitious plantlets in vitro is a limiting factor in producing transgenic sweetpotato plants through gene transfer. Studies were conducted to develop an in vitro high frequency regeneration protocol for sweetpotato that is rapid and consistent. When 27 genotypes of sweetpotato were screened, five were identified as highly regenerative (318846-3, PI 531143, Hi Dry, Rojoblanco and Beauregard). High frequency regeneration of shoots (in 60 to 80% explants) was observed within 30 days when leaf explants with intact petioles from the apical portions of the in vitro shoots were cultured on a MS medium with 2,4-D (0.2 mg/l) for three days and then transferred to a medium with zeatin riboside (ZR) (0.2 mg/l). However, thidiazuron (0.2 mg/l) had to be substituted for ZR to achieve regeneration of shoots from petiole (0.5 to 1 cm) explants (the most responsive organ for transformation by Agrobacterium). Petiole explants developed shoots efficiently (80-90%) and rapidly (10 to 21 d), but were specific to the genotype 318846-3. The resulting plantlets were vigorous and normal, and were transferred to the green house with little or no mortality.