Elimination of pathogen from noble canes, saccharum officinarum, by tissue culture methods for conservation and molecular analysis to detect genetic changes.
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MRR-18-00080
Title
Elimination of pathogen from noble canes, saccharum officinarum, by tissue culture methods for conservation and molecular analysis to detect genetic changes.
New sugarcane varieties are interspecific hybrids derived mainly from crosses between the noble canes (Saccharum ofjicinarum) and the wild type species of Saccharum spontaneum. Through a number of selection programmes, varieties with enhanced disease resistance, high sucrose content and increased water/drought tolerance are obtained. Owing to the importance of noble canes in breeding programmes, this project was carried out in order to eliminate the various pathogens infecting them and to introduce a long term in vitro system for their conservation. A survey was carried out among an existing collection of noble canes to determine the level of infection with the following pathogens; sugarcane bacilliform virus (SCBV), sugarcane yellow leaf virus (Sc YL V), Xanthomonas campestris pv . vasculorum (XCV) and Xnnthomonas albilineans (Xalb). All varieties tested for SCBV by PCR using primers SCBVF5 and SCBVR5 were found infected by the virus. ScYLV was detected in the noble canes by tissue blot immunoassay (TBIA) using leaf midribs and stems. The pathogen was found in 39 (out of 65) varieties with an incidence as high as 90% observed in several varieties. However, presence of the virus was not always associated with the typical symptoms of yellowing of the leaves. The stalk imprint method on Wilbrink medium and the diffusion test were used for the detection of XCV and Xalb. XCV was present in eight varieties of noble canes whereas Xalb was not detected in any plant. A more effective tissue culture medium (TCM) was devised consisting of phytagel (1.8 g/1) instead of type A agar (7 g/1) and a 2:1 (13.7: 6.3 g/1) sucrose I glucose carbon source instead of 20 g/1 sucrose as sole carbon source. Using this modified medium, high level of regeneration was observed with apical meristem and axillary buds. Similarly, embryogenic callus could be obtained from 95% of the varieties initiated. Out of nine varieties tested for ScYLV by RT-PCR using primer pairs YLS 111 and YLS 462, plantlets from seven varieties were found freed from the virus. These were regenerated from apical meristem, axillary buds and from callus. Sc YLV was eliminated from all plants derived from callus culture. Inability of the virus to colonise the somatic embryos or apical meristem may explain their absence from plantlets regenerated from such explants. Plantlets (variety M168/33) regenerated from the three types of explants and presumably freed from the virus were potted and transferred to the glasshouse. After nine months, the virus remained undetected in these plants. However, all plantlets remained infected with SCBV following tissue culture. The ability of an antiviral agent, ribavirin, to eliminate SCBV and Sc YLV was investigated. This compound was found to be phytotoxic to in vitro plantlets at concentration above 30 mg/1 in tissue culture medium. Treatment at 10-30 mg/1 failed to eliminate either virus after 7-10 subcultures. The evaluation of antibiotics for the elimination of XCV from noble canes was also carried out. Cefotaxime, tetracycline and rifampicin were found to have bacteriocidal activity against XCV in TCM. The minimal bacteriocidal concentration (MBC) of the antibiotics in TCM were as follows: tetracycline; 50 mg/1, rifampicin; 15 mg/1 and cefotaxime; 90 mg/1. However, exposure to strong light led to the inactivation of the antibiotics, their activity falling from 2MBC to MBC within 4 days for tetracycline, 6 days fer nfampicin and 7 days for cefotaxime. Under low light intensity, their activity remained above that of MBC for at least 19 days. The phytotoxicity of the antibiotics on sugarcane plantlets was also assayed. Exposure for two months with tetracycline at 2MBC led to the bleaching of newly formed leaves and eventual death of the plants. No phytotoxic effect could be associated with cefotaxime and rifampicin. The use of heat on the elimination ofXCV was further studied. The thermal death point (TDP) ofXCV was found to be 60°C for 30 min. Likewise axillary buds from some sugarcane varieties were found to be able to resist such temperature. These results suggest that heat could potentially be use for the elimination of XCV by treatment of axillary buds. Finally, the effect of encapsulation/dehydration on the regeneration of somatic embryos and micromeristems (variety M 168/33) was studied. Three treatments were carried out: 1) direct regeneration, 2) regeneration following encapsulation in 3% alginate beads and 3) regeneration following encapsulation and dehydration. Encapsulation seemed to have minor effects on the regeneration of somatic embryos or meristems. However, no regeneration was observed following dessication of both encapsulated explants.