Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 yearsin vitro
Micropropagated grape (Vitis vinifera L.) cv. Arka Neelamani cultures showed a decline in root and shoot growth performance after 6-7 yr of continuous in vitro culture. Indexing the culture medium using nutrient agar or 523 bacteriological medium (Viss et al., 1991) revealed covert bacteria in 75-100% cultures. Testing the tissue from different parts of in vitro plantlets on nutrient agar showed bacteria comprising of six or more morphotypes in 100% of root and collar tissue samples but less frequently in stem segments. The shoot tips had the lowest incidence of bacterial association. The whole shoots treated with NaOCl (4% chlorine) or HgCl2 (0.1%) showed endophytic bacterial survival. Culturing the HgCl2-treated (5 min) shoot tips on antibiotic overlaid medium (1 ml of 50 mg l−1 gentamycin and/or cefazolin) in culture tubes (150×25 mm) for 1 mo. facilitated the cleansing of cultures with 75% recovery of contaminant-free shoots as monitored through indexing for the next 2 yr. Repeated indexing of medium and tissue from various plant parts during the first two to four subculture cycles following antibiotic treatment was instrumental in reliably identifying clean cultures and preventing bacterial re-emergence. Antibiotic incorporation in the medium was detrimental to grape microcuttings. Bacteria-freed cultures showed 80-100% rooting and a high number of plantlets that could be acclimatized. The plants put in the field after 8 yr of active micropropagation showed some juvenile characteristics initially, which disappeared in 6-8 mo., and the pruned shoots showed flowering and bunch development within 1 yr of field planting. This indicated the feasibility of keeping grape plants in vitro for long periods if covert microbes were eliminated.