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- Author or Editor: Stephen L. Sinden x
Cultured leaf explants obtained from 36 accessions of the wild tomato species, Lycopersicon hirsutum Humb. and Bonpl., were evaluated for morphogenic capacity in response to three cytokinins (zeatin, BA, and kinetin) in combination with IAA. Media containing 0.1 μm IAA plus 4.6 or 9.2 μm zeatin were optimal for shoot induction. Cotyledon explants were superior to true leaf explants for obtaining shoot formation. Morphogenic responses of L. hirsutum f. typicum and L. hirsutum f. glabratum were clearly accession-dependent and ranged from exceptional with numerous shoots produced to recalcitrant with no shoots produced. The high morphogenetic capacity of leaf explants from L. hirsutum f. typicum accession 128644 was also evident in protoplast-derived calli that readily regenerated shoots. Chemical names used (E)-2-methyl-4-(1H-purin-6-ylamino)-2-buten-1-ol (zeatin), N-(phenylmethyl) -1H-purin-6-amine (BA), N- (2-furanylmethyl) -1H- purin-6-amine (kinetin), 1H- indole-3-acetic acid (IAA).
Abstract
Certain plant species have been propagated clonally over long periods of time using classical techniques. Variants occasionally appear, but usually in relatively low frequencies (31). Thus, “carbon copies” of the parent plant might have been expected when tissue explants and single cells were first isolated and regenerated into plants by cell-culture techniques. Most regenerated plants are, in fact, apparent exact copies of the parent plant (40, 41). However, some culture systems now available for commercial propagation and research purposes appear to produce cell cultures and regenerated plants with considerable phenotypic variability.
Leptine glycoalkaloids found in certain genotypes of Solanum chacoense, a wild potato relative, are resistance factors against the Colorado potato beetle (CPB). To efficiently introgress CPB resistance through leptine production into the cultivated potato, an understanding of leptine inheritance is important. Analysis of sibs within PI lines revealed a wide segregation for level of leptines. Leptine levels ranged from not detectable to 120 mg/100 g fresh weight among six sibs in a PI line, suggesting leptine production may be controlled by a few major genes. TLC analysis of F2 and backcross progeny, however, indicated that several genes probably control leptine level. This apparent multigene control of leptine level may make it difficult to incorporate leptine synthesis into the cultivated potato. Therefore, we are presently identifying microsatellite and RAPD markers associated with leptine synthesis to enable marker-assisted selection and facilitate the incorporation of leptine synthesis into the cultivated potato.
Somatic fusion hybrids created between tomato and Solanum ochranthum, a wild nontuber-bearing diploid species that is genetically isolated from tomato, were evaluated in an effort to introgress traits from S. ochranthum into tomato. Pollen stainability and pollen tube growth examination demonstrated that little or no viable pollen was present in tetraploid and hexaploid fusion hybrids. Aneuploidy was noted in a small percentage of these hybrids. Use of tetraploid and hexaploid fusion hybrids as female parents in backcrosses to diploid and tetraploid tomato was studied. Chemical treatments that induce either chromosomal recombination or reduction may be advantageous for overcoming difficulties in introgression of these wide hybrids into tomato.
Incorporation of genes from wild species has been a major contributor to tomato improvement in recent years. Solanum ochranthum, a woody non-tuber bearing species, is a potential source of resistance against tomato diseases and insect pests but is genetically isolated from tomato. Somatic hybridization methods were developed to facilitate the use of S. ochranthum for tomato germplasm improvement. Leaf mesophyll protoplasts of S. ochranthum and a Lycopersicon esculentum hybrid were chemically fused with polyethylene glycol. The protoplasts were initially cultured in Shepard's CL, a MS based medium, containing 1 mg·1-1 NAA, 0.5 mg·1-1 BAP and 0.5 mg·1-1 2,4-D. Hybrid regenerants and regenerants of the L. esculentum parent were recovered; S. ochranthum did not regenerate. Hybridity was established by morphological characters, peroxidase isozyme and RAPD markers. Use of these somatic hybrids for tomato improvement was evaluated.
Leaf callus of Ipomoea cordatotriloba was initiated by culturing explants on Linsmaier and Skoog medium containing 3 g yeast extract/liter, 18.9 μm ABA, 2.3 μm 2,4-D, and 0.15 m sucrose. Calluses were transferred to Murashige and Skoog media containing 17.8 μm BA and 0, 1, 10, or 100 μm PCIB. The number of shoots from calluses grown on medium containing 10 μm PCIB increased significantly, and the percentage of calluses exhibiting shoot regeneration almost doubled compared to calluses grown on regeneration medium without PCIB. Protoplasts isolated from stem and petiole tissues of in vitro-grown plants were cultured in Kao and Michayluk 8p medium to the callus stage. Calluses (4-6 mm) were transferred to the callus induction and regeneration media used to regenerate leaf-explant callus. Of the protoplast-derived calluses cultured on media containing 10 or 100 μm PCIB, ≈13% and 18%, respectively, regenerated shoots after 2 months; none regenerated on the medium without PCIB. Chemical names used: abscisic acid (ABA); 2,4-dichlorophenoxyacetic acid (2,4-D); N6-benzyladenine (BA); α -p-chlorophenoxyisobutyric acid (PCIB).
The use of wild Ipomoea species in sweetpotato improvement may be facilitated by the use of in vitro techniques such as somatic hybridization. Plant regeneration from callus cultures is essential to the successful application of these in vitro techniques. This is the first report of plant regeneration of I. cordatotriloba from protoplast derived calli. Protoplasts isolated from petiole and stem tissues of in vitro grown I. cordatotriloba were initially cultured on KM8p media. All calli cultured regenerated roots after 1 month on regeneration media. Approximately 13% and 19%, respectively, of the calli cultured regenerated shoots after 2 months on media containing 10 and 100 uM parachlorophenoxy isobutyric acid (PCIB). Regenerated shoots developed into whole plants when transferred to MS media without hormones. The regenerated plants closely resembled the parent's morphology.
Use of wild species for in vitro sweetpotato improvement has been limited, in part, by the lack of suitable regeneration systems for these species. Shoot regeneration in 4 closely related species, I. batatas, I. cordatotriloba, I. trifida and I. triloba, were evaluated. Callus was initiated using methods described by Otani and Shimada (1988). Calli were transferred to regeneration media containing 17.75 uM BAP and 0, 1, 10 and 100 uM PCIB. Organogenesis was enhanced by the presence of PCIB. With I. cordatotriloba calli grown on media with 10 uM PCIB, a 2-fold increase in the percentage of calli exhibiting shoot regeneration was observed as compared to calli grown on media with BAP alone. A significant increase in the average number of shoots per callus was also observed. The other species examined appeared to be less sensitive than I. cordatotriloba to the PCIB treatments.