Diurnal rhythm in levels of haemolymph sugar and hyperglycaemic activity of eyestalk extract in the fresh water rice field crab (Oziotelphusa senex senex Fabricius)

The concentration of haemolymph sugar and the hyperglycaemic activity of eyestalk extract was measured six times (8, 12, 16, 20 and 4 h) over a 24-h period. The concentration of haemolymph sugar and hyperglycaemic activity of eyestalk extract was higher during the night (0 h through 8 h) than that noted in day time (12 h). The variations are closely related to the activity of the animal.     

Nitric oxide in fruit ripening: Trends and opportunities

Monitoring ethylene is crucial in regulating post-harvest life of fruits. The concept of nitric oxide (NO) involvement in antagonizing ethylene is new. NO mediated physiologies casted through regulation of plant hormones are widely reported during developmental and stress chemistry having no direct link with ripening. Research in NO biology and understanding its interplay with other signal molecules in ripening fruits suggest ways of achieving greater synergies with NO applications. Experiments focused at convincingly demonstrating the involvement of NO in altering ripening-related ethylene profile of fruits, would help develop new processes for shelf life extension. This issue being the central theme of this review, the putative mechanisms of NO intricacies with other primary and secondary signals are hypothesized. The advantage of eliciting NO endogenously may open up various biotechnological opportunities for its precise delivery into the target tissues.     

Micropropagation in banana using high levels of cytokinins does not involve any genetic changes as revealed by RAPD and ISSR markers

Use of high levels of growth regulators during micropropagation results in undesirable clonal variability in important commercial crops such as banana. The present study investigated the effects of high levels of cytokinins on micropropagation in banana (genotype AAB), and the genetic stability of plantlets was assessed using RAPD and ISSR markers. Cytokinins, such as BA and kinetin were added to the routine shoot multiplication medium at concentrations up to 10 mg l⁻¹. After 12 weeks of culture involving three subcultures, the maximum number of shoot buds were produced in cultures receiving either 5 mg l⁻¹ BA (80 shoot buds) or 4 mg l⁻¹ kinetin (62 shoot buds). Certain morphological abnormalities observed during proliferation of shoot buds in vitro were not observed during acclimatization ex vitro. To check the genetic stability, RAPD and ISSR profiles of micropropagated plantlets obtained from different cytokinin-treatments were compared with control microplants maintained on MS medium as well as the field-grown mother plant. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands. Thus a total of 17,400 bands were generated showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation in all the plantlets analysed. Based on these results a protocol for high rate shoot multiplication was worked out leading to uniform shoot production.     

Direct shoot and cormlet regeneration from leaf explants of ‘Silk’ banana (AAB)

Summary Direct shoot and cormlet regeneration from leaf explants were obtained in triploid dessert banana cultivar Nanjanagud Rasabale (NR) that is classified under the group ‘Silk’ and has the genotype AAB. The response for both cormlet and direct shool formation was observed only in leaf explants obtained from shoots cultured in liquid medium but not in similar explants obtained from shoots grown on gelled medium. Shoot initiation occurred after a sequential culture of leaf (sheath) explants on modified Murashige and Skoog (MS) medium supplemented with different growth regulators. In the sequence, the leaf explants were cultured first on medium with a high level (22.4 μM) of benzyladenine (BA), second on indolc-3-butyric acid (IBA) supplemented medium, and third on reduced BA medium under incubation in the dark. The highest adventitious shoot regeneration in 24% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants incubated at the first step in medium with 22.4 and 0.198 μM IBA. Further growth and complete shoot formation occurred under incubation in a 16-h photoperiod. While keeping the culture conditions constant and replacing BA with picloram (0.83–20.71 μM) in the initial step, adventious origin of cormlets occurred in 12% of the explants. However, when rhizome explants (also obtained from shoots grown in liquid medium) were cultured with various growth regulators in the first step, medium containing 2,4,5-trichlorophenoxyacctic acid (7.82 μM) produced friable callus that re-differentiated into roots only. Physical forms of the medium, ie.e. agar-gelled or liquid, imparted specific effects on the extent of multiplication of leaf-regenerated shoots with no differences in morphology and growth patterns when compared to those of meristem-derived plants.     

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.     

Food-grade chemical and biological agents permeabilize red beet hairy roots,assisting the release of betalaines

Hairy root cultures of red beet, Beta vulgaris L., were permeabilized under the functions of food-grade chemical and biological agents cetyl trimethylammonium bromide (CTAB), Triton X-100, Tween-80, Lactobacillus helveticus, Saccharomyces cereviseae, and Candida utilis, as well as cell fractions of L. helveticus, for the recovery of betalaines with or without oxygen stress. Tween-80 (0.15%), Triton X-100 (0.2%), and CTAB (0.05%), in combination with oxygen stress, released 45%, 70%, and 90% pigment into the medium, respectively, with significantly lesser levels in agitated cultures receiving similar treatments. The release was rapid (1 h) in CTAB treatment with a much slower release in Tween-80. CTAB (0.002%) was found to be also useful in effluxing betalaines (80%) from hairy roots grown in a bubble column reactor. Viability of permeabilized hairy roots, tested on agar medium, was not affected by any level of CTAB treatment and was significantly retarded at higher levels of Triton X-100 and Tween-80. An altogether new approach of pigment release using biological agents such as live cells of food-grade microbes was used where C. utilis, L. helveticus, and S. cereviseae released 60%, 85%, and 54% betalaines, respectively, in 24 h, though lower level treatments also released similar levels of pigment by 48 h. Dried whole cell powder of L. helveticus, its total insoluble carbohydrate, and free lipid fractions released 10%, 0%, and 85% pigment, respectively. An extended study with a bubble column reactor using the free lipid fraction of L. helveticus showed 50% and 84% pigment release in 8 and 12 h, respectively, exhibiting good viability when plated on agar medium. Even in the bioreactor, replenishment of medium 8 h after treatment with free lipid of L. helveticus allowed regrowth of hairy roots. The high level of pigment release recorded here, using CTAB or lipid of L. helveticus, appears useful for developing processes for in situ recovery of betalaines. The live microbes, applicable only for batch cultures, are expected to impart improved sensory/nutraceutical effects to the recovered pigment and hence may add value to the product receiving the red beet pigment thus produced.     

Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers

A cultivar of dessert banana, namely, Nanjanagudu Rasabale (NR), classified under group “silk” (of genotype AAB), is seriously under the threat of extinction due to its susceptibility to bacterial wilt and bunchy-top virus disease. A regeneration protocol using tissue culture method was developed (Venkatachalam et al. 2006), where a large number of plantlets were regenerated from leaf base explants. Simultaneously, a micropropagation protocol was also developed where high levels of up to 53.28 μM of benzylamino purine (BAP) and 55.80 μM of kinetin (Kn) were used. The progressive increase of cytokinins levels resulted in concomitant increase in shoot number, with a maximum of 80 shoot buds per segment in BAP (31.08 μM). The plantlets were analyzed for their genetic stability using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation among the plantlets analyzed. The present study has established for the first time that the regeneration and rapid micropropagation protocol developed through the present study will be of great use in conserving the endangered cultivar – NR – without risk of genetic instability.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Factors influencing direct shoot regeneration from ovary explants of saffron

Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant. The best response towards caulogenesis (28%) with highest shoot numbers per ovary was observed when full strength Murashige and Skoog (1962) medium was supplemented with naphthaleneacetic acid and benzyladenine. Incubation in the dark at 20 °C was beneficial for induction of shoot buds. Ovaries of different growth stages having stigmas of pale yellow, pale orange and bright orange regenerated a maximum mean number (3.8 – 4.2) of shoots per ovary. Further development of ovary-derived shoots was influenced by the composition of basal salts in the culture medium where full strength Murashige and Skoog salts gave the best response of those tested. Regenerated shoots produced normal photosynthetic leaves and corms. This revised version was published online in June 2006 with corrections to the Cover Date.