One cm segments from adventitious roots of sweet potato (Ipomoea batatas (L.) Lam.) will regenerate shoots when cultured on Murashige and Skoog salts and vitamins plus either sucrose (1-3%) or fructose (1-6%). The best source for adventitious roots is sweet potato shoot cultures maintained in Magenta vessels. A low concentration of cytokinin (0.02 mg/liter) promotes shoot formation. Higher levels of cytokinin (0.1-0.5 mg/liter) encourage callus growth. The maximum average number of shoots formed per root segment attained thus far is 0.5. Attempts are being made to increase the frequency of shoot formation. Regeneration of shoots from roots also may be a useful method for obtaining plants from protoplasts of sweet potato. Protoplasts can be isolated from mesophyll tissue and petioles of in vitro grown plants. Plating efficiency of up to 12% routinely can be obtained. Shoot formation directly from callus is sporadic; root formation is more frequent.
Peggy Ozias-Akins and Srini Perera
Peggy Ozias-Akins and Robert L. Jarret
The nuclear DNA content of 53 accessions from 24 Ipomoea (Convolvulaceae) species, including four sweetpotato cultivars, was determined by flow cytometry of DAPI-stained nuclei. Ploidy level and DNA content were significantly correlated within the genus, but more highly so within species that contained multiple cytotypes. DNA content of cultivated Z. batatas (L.) Lam. (4.8 to 5.3 pg/2C nucleus) and feral tetraploid I. batatas (3.0 to 3.5 pg/2C nucleus) was estimated from the known DNA content of chicken erythrocytes (2.33 pg), which were used as an internal standard. Tetraploid forms of Z. cordato-triloba Dennstedt also were identified. Ploidy analysis using flow cytometry is rapid and suitable for large-scale experiments such as studying the genetic structure of populations of Z. batatas and related species. Chemical name used: 4′,6-diamidino-2-phenylindole (DAPI).
Srini C. Perera and Peggy Ozias-Akins
Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).
Jason J. Goldman, Wayne W. Hanna and Peggy Ozias-Akins
`TifEagle' (2n = 3x = 27) hybrid bermudagrass [Cynodon dactylon (L.) Pers. (2n = 4x = 36) × Cynodon transvaalensis Burtt-Davy (2n = 2x = 18)] is an ultradwarf cultivar for greens, and `TifSport' (2n = 3x = 27) is a more versatile hybrid used on fairways, athletic fields, and lawns. To develope a transformation system and determine if somaclonal variation was present in regenerated plants, both cultivars were tested for their ability to produce embryogenic callus from which plants could be regenerated. Sliced nodes of both cultivars and immature inflorescences from `TifSport' were used as the explant sources. Cultures were initiated on Murashige and Skoog medium supplemented with 6.79 μm 2,4-D and 0.044 μm BA (`TifSport' and `TifEagle') or 6.79 μm 2,4-D plus 200 mg.L-1 casein hydrolysate (`TifSport'). In total, 51 plants were regenerated from callus of a single node of `TifEagle'. Nodes from `TifSport' did not produce embryogenic callus. In total, 29 plants were regenerated from callus of `TifSport' produced from immature inflorescences. These plants were grown in the field for at least one season, and 5-cm-diameter plugs were harvested, repotted in a greenhouse, and allowed to reestablish. Data on canopy height, leaf width, leaf length, and number of stolons were collected. Seven `TifEagle'-derived entries (14%) were not significantly different (α = 0.05) from `TifEagle' harvested from the breeder plot in Tifton, Ga., for all measured traits, and 41%, 24%, and 22% differed by one, two, or three measurements, respectively. Flow cytometry indicated that 33% (13 plants) of the `TifEagle' regenerants were hexaploid (2n = 6x = 54) and the rest remained triploid. One `TifSport' regenerant was significantly different (α = 0.05) for plant height.
Peggy Ozias-Akins, Edward L. Lubbers and Wayne W. Hannna
Apomixis is asexual reproduction through seed. Apomixis in the genus Pennisetum is of the gametophytic (aposporous) type. Genes for apomixis have been transferred from a wild apomictic species (P. squamulatum) to pearl millet (P. glaucum) by conventional breeding to produce an obligately apomictic backcross 3 (BC3) plant (Dujardin and Hanna, 1989, J. Genet. Breed. 43:145). Molecular markers based on restriction fragment length polymorphisms and random amplified polymorphic DNAs were identified in BC3 that were shared only with the apomictic parent. Segregation of these informative markers in a BC4 population indicated that three linkage groups from P. squamulatum were present in BC3 and that minimal recombination between these alien chromosomes and those of the recurrent parent occurred. Transmission of only one of the linkage groups was required for transfer of apomixis. Recombination is essential for genetic mapping, thus we are beginning to map the informative molecular markers in an F, interspecific cross between pearl millet and P. squamulatum, a population that segregates for apomictic and sexual reproduction.
Myneni Aruna, Max E. Austin and Peggy Ozias-Akins
Cultivars of the economically important rabbiteye blueberry (Vaccinium ashei Reade) were differentiated at the DNA level using the technique of randomly amplified polymorphic DNA. Single decanucleotide primers of arbitrary sequence were used to amplify genomic DNA by the polymerase chain reaction. All cultivars tested exhibited a unique set of collective amplified fragments of distinct molecular weight. A blind fingerprinting experiment resulted in identification of unknown samples without ambiguity. We also clarified the genetic identity of two wild selections of rabbiteye blueberry, `Ethel' and `Satilla', which have been maintained as two different selections, hut are considered by some blueberry breeders to be of the same genetic constitution. The technique also verified the probable identity of two cultivars in a commercial blueberry field by comparing their amplified DNA patterns with those of standard cultivars. No variation was observed between the amplification profiles of `Brightwell' and its presumed sport. A cultivar key based on 11 markers amplified by four primers is presented.