Generation of Variability for Salt Tolerance in Rice Using Tissue Culture Techniques

The study deals with the generation of variability for salt tolerance in rice using tissue culture techniques. Rice is the staple food of more than half of the world’s population. The management of drought, salinity and acidity in soils are all energy intensive agricultural practices. The Genetic variability is the basis of crop improvement. Somaclonal and androclonal variation can be effectively used for this purpose. In the present study, eight isozymes were studied and esterase and isocitric dehydrogenase was found to have varietal specific, developmental stage specific and stress specific banding pattern in rice. Under salt stress thickness of bands and enzyme activity showed changes. Pokkali, a moderately salt tolerant variety, had a specific band 7, which was present only in this variety and showed slight changes under stress. This band was faint in tillering and flowering stage .Based on the results obtained in the present study it is suggested that esterase could possibly be used as an isozyme marker for salt tolerance in rice. Varietal differences and stage specific variations could be detected using esterase and isocitric dehydrogenase . Moreover somaclonal and androclonal variation could be effectively detected using isozyme markers.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

Use of ovary culture techniques in reproductive toxicology

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved. Acknowledgements The author's studies in this field are supported by MRC grants G1002118 (NS and RAA) and G110357 (RAA), MR/L010011/1 (PAF), the European Community's Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 212885 (PAF) and the Wellcome Trust (080388 to PAF). AS was funded by a BBSRC CASE Studentship co-funded by AstraZeneca.

Comparison of human alveolar osteoblasts cultured on polymer-ceramic composite scaffolds and tissue culture plates

The effects of medical grade polycaprolactone–tricalcium phosphate (mPCL–TCP) (80:20) scaffolds on primary human alveolar osteoblasts (AOs) were compared with standard tissue-culture plates. Of the seeded AOs, 70% adhered to and proliferated on the scaffold surface and within open and interconnected pores; they formed multi-layered sheets and collagen fibers with uniform distribution within 28 days. Elevation of alkaline phosphatase activity occurred in scaffold–cell constructs independent of osteogenic induction. AO proliferation rate increased and significant decrease in calcium concentration of the medium for both scaffolds and plates under induction conditions were seen. mPCL–TCP scaffolds significantly influenced the AO expression pattern of osterix and osteocalcin (OCN). Osteogenic induction down-regulated OCN at both RNA and protein level on scaffolds (3D) by day 7, and up-regulated OCN in cell-culture plates (2D) by day 14, but OCN levels on scaffolds were higher than on cell-culture plates. Immunocytochemical signals for type I collagen, osteopontin and osteocalcin were detected at the outer parts of scaffold–cell constructs. More mineral nodules were found in induced than in non-induced constructs. Only induced 2D cultures showed nodule formation. mPCL–TCP scaffolds appear to stimulate osteogenesis in vitro by activating a cellular response in AO's to form mineralized tissue. There is a fundamental difference between culturing AOs on 2D and 3D environments that should be considered when studying osteogenesis in vitro.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.

Strategic banana tissue culture industry development and biosecurity act

Application and development of activities based on in vitro technologies delivering research, industry development and biosecurity activities to sustain and improve the Australian banana industry.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Seed production and culture techniques of Genetically Improved Farmed Tilapia (GIFT) in brackishwater environment

Effects of different levels of salinity on survival, growth and gonadal development of Genetically Improved Farmed Tilapia (GIFT) were studied under laboratory conditions in glass aquarium, for a period of ten weeks. The initial individual size of the GIFT was 20.23±4.45 and the salinity levels tested were 0, 5, 10, 15 and 20 ppt. The highest survival of 87.5% was found in 0 ppt and the lowest 60.5% in 20 ppt. Though the survival decreased progressively with increased salinity, there were no significant differences (P>0.05) among 0, 5, and 10 ppt. Similar to what has been observed in survival, the specific growth rate (SGR %/day) also decreased as of 1.30, 1.24, 1.08, 0.90 and 0.71, respectively, with the increased salinity of 0, 5, 10, 15 and 20 ppt. The gonadal development was highest in 0 ppt with a GSI value of 3.75 and lowest of 2.01 in 20 ppt. In the second experiment, gonadal development and seed production performance of GIFT in brackishwater condition were investigated for a period of three months. Each of the three fine meshed hapas of 20 square meters made from nylon net was placed in a freshwater (0 ppt) and in a brackish water (10-15 ppt) pond of the Brackishwater Station (BS). GIFT of 65 g average weight from a single cohort were stocked into three hapas at a rate of 2 per m. The male vs female ratio was 1:3. The development of gonad was faster with the higher gonadosomatic index (GSI %) of 3.85 % in freshwater condition than that of 2.73 % in brackish water. Within three months of the study period, a total of 70,510 and 44,250 GIFT fry were produced respectively, in freshwater and brackishwater conditions. Finally under third experiment, a participatory on-farm trial was carried out to evaluate the production performance of GIFT in monoculture and in polyculture with silver barb in coastal freshwater pond conditions. Nine ponds were selected for three treatment combinations of GIFT monoculture (T1), GIFT and silver barb polyculture (T2), and silver barb monoculture (T3). The ponds have been stocked in April, 05 at a density of 25,000 fry per ha. Fishes were fed with rice bran at the rate of 6% bw per day. In one month culture period, GIFT attained an average weight of 16.27 g in monoculture and 17.23 g in polyculture, against an average stocking weight of 0.37 g. Silver barb reached an average weight of 16.62 g in polyculture with GIFT and 10.01 g in monoculture, against an average stocking weight of 3.79 g.