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The present study was taken up with a view to ascertain the possibility of introduction of alcohol resistant bacteria in vitro through the aseptically raised watermelon (Citrullus lanatus) seedlings in the backdrop of isolating such organisms from micropropagated watermelon stocks. Watermelon cv. Arka Manik seedlings grown in vitro from surface-sterilized seeds with the intact seed coat on MS medium appeared visibly clean largely, but upon subjecting them to tissue-indexing, the segments from the collar or root tissue showed bacterial colony growth on Nutrient Agar (NA) from 72% of such healthy seedlings and the cotyledon and hypocotyl tissue of 44% seedlings. The pooled colony growth from NA upon challenge with 90% alcohol yielded 10 distinct colony types, identified as B. pumilus (4×), B. subtilis (4×), B. cereus (1×) or B. safensis (1×) based on partial 16S rRNA sequence analysis. The shoot-tip tissue from the healthy index-negative seedlings cultured on watermelon proliferation medium partly turned index-positive within 2 - 4 sub-culture cycles while being apparently clean. On the other hand, those from the previously index-positive cultures tended to show obvious bacterial growth during subsequent in vitro culturing. The observations suggested the possibility of introduction of spore-forming Bacillus spp. through surface-sterilized seeds, their gradual emergence in vitro in visibly clean seedlings, possible transmittal of spores to the alcohol through tissue-culturing tools and the survival therein with the chances of unsuspected lateral spread. Seed coat removal followed by surface sterilization with sodium hypochlorite facilitated the raising of clean seedlings with no detectable bacterial association.

The study was taken up to assess the chances of alcohol and instrument mediated horizontal transmission of bacterial spores and to fix the sterilization needs of tools recurrently used in bacteriological or plant tissue culture work in the backdrop of encountering mixtures in purified microbial cultures and the re-emergence of contaminants in ‘bacteria-sanitized’ plant tissue cultures. The microbial inoculating needle after contaminating the loop portion with Bacillus subtilis or B. pumilus spores was flamed over a sprit-lamp or gas-burner directly for 10–40 s, or after dipping the instrument (10–12 cm) in spore-adulterated 90 % ethanol or rectified spirit (96 %). The loop and rod portions were monitored for any residual inoculum by bringing the loop region in contact with nutrient agar, or swabbing the rod portion with moistened cotton-bud and imprinting on agar medium. Direct flaming of loop region for ≥20 s eliminated the hardy spores therein, whereas alcohol-dip and flaming increased the chances of spore survival on the rod portion that were acquired from the contaminated alcohol. Similar results were observed with the set of forceps used in tissue culturing work after dip in spore-containing ethanol. Alcohol once got adulterated with the hardy spores served as a source of bacterial inoculum causing unsuspected lateral spread of contamination. Direct flaming of inoculating loop nichrome or the forceps-tips, which came in contact with the microbial inoculum, preferably over a gas flame proved satisfactory for instrument sterilization. Alternatively, the use of autoclaved micropipette tips for handling bacterial cultures in the petri-dishes or attaching the tips to a pair of sterilized forceps is suggested for handling the cultures in tubes. Exposing the forceps and scalpel in a glass bead sterilizer set at 260 °C for ≥5 min effectively sterilized the tissue culturing tools.

Transgenic lines of creeping bent grass were generated by Agrobacterium-mediated transformation with the VuNCED1 which was cloned from cow pea has a homology to 9-cis-epoxycarotenoid dioxygenase, which is supposed to be involved in abscisic acid (ABA) biosynthesis. ABA, a cleavage product of carotenoids, is involved in stress responses in plants. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. Molecular analyses of transgenic lines as performed by Southern hybridization genomic DNA-PCR revealed integration of the VuNCED1. Challenge studies performed with transgenic plants by exposure to salt stress (up to 10 dS m−1) and water stress (up to 75%) for 10 weeks, revealed that more than 50% of the transgenic plants could survive NaCl and drought stress whereas wild-type was not. ABA levels were measured under drought and normal conditions, endogenous ABA was dramatically increased by drought and NaCl stress in transgenic plants. These results indicate that it is possible to manipulate ABA levels in plants by over expressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.

A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization.

Different types of ventilation of the culture vessel with and without the ethylene inhibitor KMnO 4 in the culture media were investigated in terms of their effect on hyperhydricity of in vitro cultured potato shoots. The shoots grown in a completely sealed vessel (CSV) were severely vitrified, but the shoots cultured in a gas permeable vessel (GPV) showed normal shoot proliferation. Dry weight percentage and chlorophyll content were lower in the vitrified shoots than in the normal shoots. The levels of ethylene and CO 2 were significantly higher in the CSV, but little ethylene was detected in the GPV system. When an ethylene scrubber (perlite treated with KMnO 4 ) was applied to the CSV, the shoots exhibited normal growth without any sign of vitrification. In addition, no ethylene was found in CSV with the ethylene scrubber (CSVE). These results indicate that hyperhydricity is related to the ethylene accumulated in the culture vessel during the in vitro culture of potato shoots. It is suggested to use CSVE to reduce the ethylene level and overcome hyperhydricity.

Potato microtubers produced in vitro of the cultivar ‘Superior’ were assessed to decide up to what size it can be used for the seed potato with respect to storability, dormancy period, and sprouting vigor. The larger microtubers lost moisture content more slowly and retained firmness longer when stored at 4 °C. In the sprouting test, the larger ones had less period of dormancy and showed more vigorous sprouting ability. The starch increased with increasing size of the microtuber and showed the distribution of about 70–80% of dry matter content. The amounts of sugars were positively co-related with the size of the potato. The internal factors such as dry matter and carbohydrate content reveal that potato microtuber follows the field-grown potatoes in all aspects. The results suggest that the size of microtubers can be used as an index for grading their quality as seed potatoes, and the size of the microtuber should be at least 0.5 g to be used as seed potato.

Plant differentiation, including morphogenesis, growth, development and plant senescence result from a complex interaction between different cells, tissues and organs. Analysis at the molecular level of the mechanism of differentiation is still difficult due to the lack of knowledge on i) the nature of the morphogenic signal, ii) the perception and transduction mechanisms and iii) the localization of target and responsive cells. In order to answer the question of how plant cells sense and process signals for differentiation, our strategy is to select for signals that induce a specific response in defined target cells that are also the responsive cells. In thin cell layer (TCL) systems, the responsive cells are well defined and are not scattered as observed in heterogenous organisms, organs or callus. Chemical signals such as kinetin, zeatin and oligosaccharides induce specific differentiation patterns, which is referred to as the specific program of differentiation.

Fresh pollen of 16 promising genotypes of gladiolus was tested for their pollen viability. Modified cellophane method was employed to assess the pollen viability. Pollen germination media consisted of 15% sucrose supplemented with 300 ppm calcium nitrate, 200 ppm magnesium sulphate, 100 ppm potassium nitrate and 100 ppm boric acid. Highly significant variations were observed for percentage of pollen germination, non-germinated pollen and sterile pollen. The highest pollen germination (76.41%) was in genotype Hybrid selection 88-10-22, and did not differ significantly from Gladiolus callianthus (75.41%), Sapna (75.10 %), Hybrid selection 86-32-11, (73.28 %), Kum Kum (69.41%), Poonam (69.22 %), Hybrid selection 87-22-1 (67.87 %), Hybrid selection 87-1-1 (67.61 %), Psittacinus hybrid (64.64%) and Darshan (63.97%). The lowest non- germinated pollen (10.47%) was in genotype Gladiolus callianthus, and insignificantly followed by Hybrid selection 88-10-22 with 18.77 % and Hybrid selection 87-22-1 with 18.95 %. The lowest percentage of sterile pollen was noticed in genotype Sapna (2.82%) followed by Poonam with 4.00 % Hybrid selection 88-10-22 with 4.82% and Hybrid selection 82-11-27 with 5.22%. Key words: Gladiolus; germination; pollen DOI: 10.3126/njst.v11i0.4089Nepal Journal of Science and Technology 11 (2010) 47-50