Molecular analysis of genetic stability in long-term micropropagated shoots of banana using RAPD and ISSR markers

A large number of micropropagated plantlets of banana, Musa acuminata var. Nanjanagudu Rasabale (NR), that were developed from axillary shoot bud explants over 10 years ago were screened for genetic variation, if any, using RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers. Of the 4000 in vitro plantlets, 11 were used for screening that involved shoot cultures with distinct variation in morphological characteristics (morphotypes). Similarly, the mother maintained in the field was also subjected for genetic analysis. Out of the 50 RAPD and 25 ISSR primers screened, 30 RAPD and 5 ISSR primers produced totally 424 clear, distinct and reproducible band classes resulting in a total of 5088 bands where the banding patterns for each primer was highly uniform and comparable to the field-grown mother clone from which the cultures had been established. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and applicable for clonal propagation of banana var. NR over a long period. This is the first report on the use of genetic markers to establish genetic fidelity of long-term micropropagated banana using RAPD and ISSR.

Elicitation of peroxidase activity in genetically transformed root cultures of Beta vulgaris L

Genetically transformed roots of red beet produce copious levels of peroxidase (POD) - a multifunctional enzyme with a number of commercial applications. In an effort to elicit the POD activity, the cultures were treated with biotic elicitors such as dry cell powders of microbial cultures (0.1-0.5 percent w/v) and the respective culture filtrates (1-5 percent v/v). Similarly, abiotic elicitors, particularly metal ions (2-8 folds of that present in the nutrient medium), the plant hormone Thidiazuron (at 0.25-1 ppm) and other bio-molecules such as Glutathione (at 0.5-10 mM) and Methyl jasmonate (at 20-100 µM) were used. It was observed that dry cell powder of Candida versatilis significantly elicited the enzyme activity (3.52-fold higher than the control) followed by glutathione (3.44-fold) and Rhizophus oligosporus (3.09-fold). Among abiotic elicitors, thidiazuron, Mg and Ca salts elicited 2.49, 3.03 and 2.8 fold activities respectively. While most of the biotic elicitors were effective when added on 15th day of culture, the abiotic elicitors were effective when added on 20th day. Combination of highly effective elicitors indicated that glutathione (1 mM) and dry cell powder of R. oligosporus caused a 4-fold enhancement in enzyme activity, accounting for 10.9 x 10(6) U L-1. The present study is the first report on red beet hairy roots where a large number of elicitors have been systematically screened and their probable involvements in eliciting POD activities have been discussed.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10(6) Units L-1. Of the ten clones established using different strains of Agrobacterium rhizogenes, one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L-1) as well as POD (ranging from 8000-9000 U g-1 FW and 1.18 x 10(6) U L-1 with a specific activity of 600 U mg-1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10(6) U L-1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of Rm 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at Rm 0.06, 0.16, 0.25 and one extra band of Rm 0.575 in the spent medium where isozymes of Rm 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.