A method has been developed for the regeneration of the banana cultivar Dwarf Brazilian (Musa spp. AAB group). Primary somatic embryos were produced when explants of immature male flower buds were cultured on Murashige and Skoog (MS) medium plus 1 mg/l biotin, 100 mg/l malt extract, 100 mg/l glutamine, 4 mg/l 2,4-dichlorophenoxyacetic acid, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l α-naphthaleneacetic acid, 30 g/l sucrose and 2.6 g/l Phytagel, pH 5.8 (M1 medium) and then transferred to M1 medium plus 200 mg/l casein hydrolysate and 2 mg/l proline. Subsequent transfer to MS supplemented with 10% coconut water produced rapidly proliferating embryogenic callus that developed into secondary somatic embryos (SE 2 ); these were subcultured on half-strength MS supplemented with 5 mg/l 6-benzylaminopurine (BA). Differentiated embryos were transferred to MS medium supplemented with 5 mg/l BA for development, and mature SE 2 were isolated and cultured on hormone-free MS for germination and development into plantlets. Approximately 90% of the SE 2 germinated and developed into plantlets, and these were subcultured onto MS medium plus 0.1% activated charcoal, 1 mg/l BA and 1 mg/l IAA where complete plantlets developed. Morphologically normal banana plants developed from all of the regenerated plantlets, the first of which were produced within 6 months of culture initiation.

A new protocol for the production of transgenic pineapple plants was developed. Adventitious buds were induced directly from Agrobacterium-infected leaf bases and stem discs of in vitro plants, bypassing the establishment of callus cultures. Non-chimeric transgenic plants were obtained by multiple subculturing of primary transformants under increasing levels of selection. A total of 42 independent transgenic lines were produced from two cultivars with two different constructs: one containing a modified rice cystatin gene (Oc-IΔD86) and the other with the anti-sense gene to pineapple aminocyclopropane synthase (ACS). GUS histochemical staining provided the first evidence of the non-chimeric nature of the transformed plants. Their non-chimeric nature was further demonstrated by PCR analyses of the DNA extracted from individual leaves of a primary transformed plant and also from multiple plants propagated from a single transformation event. Southern hybridization confirmed random integration patterns of transgenes in the independent lines. For the Oc-IΔD86 gene, the expression at the mRNA level was detected via RT-PCR and its translation was detected by protein blot. Agronomic evaluation and bioassays of the transgenic plants will further validate the utility of this new tool for pineapple improvement.

No abstract

1997. Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers. -Physiol. Plant. 100: 341-352.Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings {Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica. The nonpathogenic mycelial extract induced expression of PR-lb and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattem of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression pattems of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattem of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattem of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.

Embryogenic cell suspensions (ECS) initiated from immature male flowers of banana cultivar 'Dwarf Brazilian' (AAB, Pome subgroup) were transformed using Agrobacterium tumefaciens containing one of four constructs derived from the replicase-associated protein (Rep) gene of the Hawaiian isolate of Banana bunchy top virus (BBTV). Each construct was engineered under control of a double CaMV 35S promoter and the AMV enchancer sequence in the binary plasmid pBI121. Constructs were transferred into A. tumefaciens strain AGL0 and used to transform banana ECS. Plantlets that survived antibiotic selection were acclimated to greenhouse conditions and challenged with viruliferous banana aphids (Pentalonia nigronervosa). Ten adult or late instar aphids were allowed to feed for 2-4 weeks on test plants. All test plants were kept in the greenhouse and monitored for symptom expression for a period of 6 months. Control plants transformed with empty vector pBI121 only were included in all tests. A total of 270 test plants and 63 control plants were screened for BBTV resistance using this approach. One of 32 test plants transformed with the M1 (mutant Rep gene) construct, 5 of 74 test plants transformed with the AS1 (antisense Rep gene) construct, 5 of 38 test plants transformed with the PR1 (partial Rep gene) construct, and 10 of 126 test plants transformed with the R/PR1 (full-length Rep gene fused to antisepses partial Rep gene) construct were found to be resistant to BBTV challenge and showed no bunchy top symptoms. All of the control plants became infected with BBTV under these experimental conditions. Plants that survived BBTV challenge were analysed by quantitative PCR (per) and Southern hybridisations to determine the number of transgenes that were present in their genomes. Results from these analyses indicated that the resistant plants contained from 2 to more than 9 copies of the NPTII (kanamycin resistance) transgene carried on the pBI121 plasmid.

Heat‐stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings (Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica. The nonpathogenic mycelial extract induced expression of PR‐1b and osmotin (PR‐5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue‐specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract‐induced and SA/MeJA‐induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat‐stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.

Osmotin gene expression is controlled by elicitor synergism. -Physiol. Plant. 95: 620-626.Arachidonic acid (AA), a fatty-acid fungal elicitor, and a celluiase preparation from Aspergillus niger, a protein-type fungal elicitor. induced osmotin gene expression. Both elicitors activated the osmotin promoter fused to a /?-gIucuronidase (GUS) reporter gene in a tissue-specific manner in tobacco seedlings [Nicotiana tabacum L. cv. Wisconsin 38). The celluiase preparation was more effective than AA at the concentrations tested and, unlike A A, also induced the accumulation of osmotin mRNA and protein. Combinations of AA and the celluiase preparation had a greater than additive effect on the activation of the osmotin promoter and the accumulation of osmotin mRNA and protein. Both AA and the celiulase preparation, when applied separately, were virtually ineffective in the induction of the osmotin promoter in cotyledon tissues. However, together they were able to induce synergisiically GUS fused to the osmotin promoter. Increases in osmotin-promoter-driven GUS activity and accumulation of osmotin mRNA induced by AA, the celluiase preparalion or their combination were reversed by norbomadiene, an ethylene action inhibitor, indicating that ethylene is involved in the induction of the osmotin gene hy these elicitors.

Arachidonic acid (AA), a fatty‐acid fungal elicitor, and a cellulase preparation from Aspergillus niger, a protein‐type fungal elicitor, induced osmotin gene expression. Both elicitors activated the osmotin promoter fused to a β‐glucuronidase (GUS) reporter gene in a tissue‐specific manner in tobacco seedlings (Nicotiana tabacum L. cv. Wisconsin 38). The cellulase preparation was more effective than AA at the concentrations tested and, unlike AA, also induced the accumulation of osmotin mRNA and protein. Combinations of AA and the cellulase preparation had a greater than additive effect on the activation of the osmotin promoter and the accumulation of osmotin mRNA and protein. Both AA and the cellulase preparation, when applied separately, were virtually ineffective in the induction of the osmotin promoter in cotyledon tissues. However, together they were able to induce synergistically GUS fused to the osmotin promoter. Increases in osmotin‐promoter‐driven GUS activity and accumulation of osmotin mRNA induced by AA, the cellulase preparation or their combination were reversed by norbornadiene, an ethylene action inhibitor, indicating that ethylene is involved in the induction of the osmotin gene by these elicitors.