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When plants are subjected to stress conditions, they are believed to be developing defensive mechanisms. Those mechanisms could be studied by analysing and comparing the proteins from stressed and nonstressed plant materials. Photomixotrophically grown soybean suspension cultures were shocked with 150 mM, 200 mM, and 250 mM salt concentrations for 1 hr. and 3 hrs. The cells were then given 2, or 4 hr. recovery period. After treatment, proteins were quantified, using Bradford Assay, and then separated on SDS PAGE gels. In Coomasie stained gells, there were different banding patterns in shock treated samples, compared to the control. But there were no differences identified between different shocking times or recovery period treatments. The results from Silver staining and growth studies will be presented.
This study reports some factors effecting rapid regeneration of Swingle Citrumelo (Citrus paradisi × Poncirus trifoliata).
Inter-nodal stem and root sections from in vitro grown seedlings were shocked with 10, 6, 2 or 0 mg/l BAP for 48 h and then transferred to hormone-free Murashige and Tucker (1961) medium gelled with 2 g/l Gelrite. Explants were cultured horizontally or vertically to study the effect of orientation on shoot initiation.
BAP shock had a pronounced effect on shoot regeneration by root, but not by stem explants. Root explants shocked with 10 mg/l BAP had the highest regeneration frequency. Only vertically placed root and stem explants produced shoots. Shoot buds were first observed in root explants about 10 days after BAP shock. Stem cuttings were slow in producing shoot buds which were first seen after 25 days. A total of 53 shoots were regenerated from 48 root explants while the same number of stem cuttings produced only 11 shoots. When subcultured onto the same medium, more than 85% of the shoots rooted, and were recovered as plants. Explant type, explant orientation and cytokinin shock all influenced regeneration.
A suspension culture of Eucalyptus tereticornis was initiated from callus and grown for 7 months under indirect light in a Murashige and Skoog (1962) basal medium containing 3% glucose and 1 mg/l 2,4-D. Glucose was used, instead of sucrose, as it reduced production of phenolic-like compounds. The inoculum size for maximum cell yield was determined. Cells (0.1, 0.2, 0.5, 1.0, 2.5 and 5.0 g fresh weight) were cultured in basal medium for 14 days. Maximum fresh weight (mean 11.8 g) was attained from samples inoculated with at least 1.0 g of cells. Largest dry weight (mean 608 mg) occurred following, inoculation with at least 0.5 g fresh weight of cells. Inoculation with 0.5 g of cells resulted in the most rapid fresh weight doubling time (3.4 days).
After 17 months of culture, cells were grown in basal medium or m basal medium supplemented with 1 mg/l kinetin, under continuous, direct light. Growth, based on fresh and dry weight increases, was measured over the 2-week subculture period. Growth of cells was similar in both media. The cells' chlorophyll content remained low. Fresh weight doubling time averaged 3.8 days.
Viral damage is a major problem in citrus. As most citrus are asexually propagated, it is necessary to have an alternative way of regenerating virus-free plants from infected plants. Shoot apicies are the most suitable explant material for this purpose because that part of the plant is virus-free. Fifty sour orange shoot tips and 22 Swingle shoot tips, 1 mm - 1.5 mm long, were excised from in vitro germinated seedlings and cultured on semisolid Murashige and Skoog medium, without growth regulators, containing 0.2 % Gelrite. After 8-10 weeks, shoots and leaves developed in 68'% of the sour orange explants, and in 77% of the Swingle explants. Some explants produced roots, after 11-12 weeks, and could be removed from culture and established in soil medium.
Citrus is one of the major horticultural cash crops in the Southern United States. Several attempts have been made to genetically improve present citrus cultivars using biotechnology. We report transformation of citrus (Citrus sinensis (L.) Osbeck `Hamlin') suspension cultures using microprojectile bombardment.
A thin layer of seven day-old suspension cultures, grown in HH medium, were transferred to Whatman #1 filter paper at a cell density of 0.15 mg/ml fresh weight. The cells were bombarded with 1.112μM diameter, DNA-coated, tungsten particles using the Dupont PDS 1000 biolistic system. Plasmid PRT 99 GUS containing the marker genes β-Glucuronidase (GUS) and NPT II, with a 35S promoter, and NPT II terminators was used in transformation.
GUS activity was monitored on a time scale. Expression of GUS was observed after 48 hours of bombardment. Further studies are being done to enhance the transformation efficiency.