In vitro response of vanilla (Vanilla planifolia Jacks. ex Andrews) to PEG-induced osmotic stress

Drought-induced water stress affects the productivity of the Vanilla planifolia Jacks. ex Andrews crop. In vitro culture technique is an effective tool for the study of water stress tolerance mechanisms. This study aimed to evaluate the morphological, physiological and biochemical response of V. planifolia under in vitro water stress conditions induced with polyethylene glycol (PEG). In vitro regenerated shoots of 2 cm in length were subjected to different concentrations of PEG 6000 (0, 1, 2 and 3% w/v) using Murashige and Skoog semi-solid culture medium. At 60 days of culture, different growth variables, dry matter (DM) content, chlorophyll (Chl), soluble proteins (SP), proline (Pro), glycine betaine (GB), stomatal index (SI) and open stomata (%) were evaluated. Results showed a reduction in growth, Chl content, SP, SI and open stomata (%) with increasing PEG concentration, whereas DM, Pro and GB contents rose with increasing PEG concentration. In conclusion, PEG-induced osmotic stress allowed describing physiological and biochemical mechanisms of response to water stress. Furthermore, the determination of compatible Pro and GB osmolytes can be used as biochemical markers in future breeding programs for the early selection of water stress tolerant genotypes.

Features of cultivation of the deadhead hawk moth Acherontia atropos (Linnaeus, 1758) on artificial nutrient media

The results of testing of the method of cultivation of the deadhead hawk moth Acherontia atropos (Linnaeus, 1758) using solid culture medium are given in this article. Optimal cultivation conditions for all caterpillar stages of this species are discussed. Information about bacterial infection of caterpillars which is caused by Gram-negative rod-shaped bacterial flora, is also noted in the results. Methods of treatment of this bacterial infection were selected empirically and also given use of bactericidal antibiotics (ceftriaxone) and selenopiran. We also trialled the use of a similar method of cultivation on some specific media for rare species of Lepidoptera like Proserpinus proserpina, Eudia pavonia, Saturnia pyri and some other species.

Purification of Pectin Lyase Enzyme from Bacillus pumilus Bacteria by Three-Phase Partitioning Method (TPP), Nanoflower Preparation and Investigation of Fruit Juice Clarification

In this study, firstly, Bacillus pumilus bacteria were isolated from tomato vegetables and identified. Then, the new pectin lyase enzyme was purified from B. pumilus, characterized, and hybrid nanoflower pectin lyase (hNF-PL) was synthesized the first time in this study. For this purpose, PL enzyme was produced in a solid culture medium using B. pumilus bacterium, and PL was purified in 191.8 folds with a yield of 78.2% using the three-phase partitioning (TPP) technique. Using SDS-PAGE, PL enzyme was determined to have a single subunit, and molecular weight was defined as 32.88 kDa with gel chromatography technique. This is the very first study to easily immobilize purified PL enzyme onto nanoflower chitosan/calcium pyrophosphate hybrid NPs. The synthesized nanoflower hNF-PL structure was characterized by SEM, FT-IR, XRD, and TEM chromatographic methods. In the final phase of the study, the effects of the pure PL and hNF-PL enzymes on the clarification and cleavage rate of fruit juices obtained from black grape, pomegranate, peach, red apple, and plum were investigated. Under the light shed by this study determined that the hNF-PL enzyme clarified the fruit juices more effectively than the pure PL enzyme.

Comparison of Different Semi-Automated Bioreactors for In Vitro Propagation of Taro (Colocasia esculenta L. Schott)

Taro is important for its nutritional content, medicinal use, and bioethanol production. The aim of the present study was to compare different semi-automated bioreactors (SABs) during in vitro multiplication of C. esculenta. The SABs used were temporary immersion bioreactors (TIBs), SETIS™ bioreactors and ebb-and-flow bioreactors; semi-solid culture medium was used as a control treatment. At 30 d of culture, different developmental variables, determination of chlorophyll, stomatal content, and survival percentage during acclimatization were evaluated. SABs increased the shoot multiplication rate relative to the semi-solid medium; however, the SETIS™ bioreactor showed the highest shoot production, with 36 shoots per explant, and the highest chlorophyll content. The stomatal index was higher in the semi-solid medium compared to the SABs, while the percentage of closed stomata was higher in the SABs than in the semi-solid culture medium. The survival rate during acclimatization showed no differences among the culture systems assessed, obtaining survival rates higher than 99%. In conclusion, the SETIS™ bioreactor showed the highest multiplication rate; however, other bioreactor alternatives are available for semi-automation and cost reduction for micropropagation of C. esculenta.

Effects of Preharvest Methyl Jasmonate Elicitation and Electrical Stimulation on Camptothecin Production by In Vitro Plants of Ophiorrhiza ridleyana Craib

To enhance plant camptothecin (CPT) production in vitro, 5-month-old Ophiorrhiza ridleyana Craib plant cultures were treated with solutions of methyl jasmonate (MeJA) dissolved in ethanol, which were applied to the surface of the solid culture medium. It was demonstrated that the maximum CPT content in the tissue-cultured plants was achieved after 12 h elicitation with 50 µM MeJA. The mean CPT contents in roots and stems were 50.8 and 67.0 µg/g DW, respectively, which were approximately 1.8- and 2.6-fold higher, respectively, than those of the control. However, MeJA elicitation showed no significant effect on CPT accumulation in O. ridleyana leaves. Moreover, it was found that direct electric current (DC) stimulation also significantly increased CPT accumulation in O. ridleyana. The treatment with DC at 20 mA for 3 min of stimulation enhanced 3-fold the CPT content in roots, stems, and leaves to 41.9, 36.0 and 19.6 µg/g DW, respectively, which were approximately 1.5-, 1.7- and 1.4-fold higher, respectively, as compared to those of the control. The results demonstrate that preharvest treatment by MeJA elicitation and electrical stimulation can be beneficial for secondary metabolite production of CPT in tissue-culture plants of O. ridleyana.

In Vitro Screening of Sugarcane Cultivars (Saccharum spp. Hybrids) for Tolerance to Polyethylene Glycol-Induced Water Stress

Water stress caused by drought affects the productivity of the sugarcane crop. A breeding alternative is the selection of drought-tolerant sugarcane cultivars. The objective of this study was the in vitro screening of cultivars tolerant to water stress using polyethylene glycol (PEG) as a stressing agent. Cultivars (cv) Mex 69-290, CP 72-2086, Mex 79-431 and MOTZMex 92-207 were subjected to different concentrations of PEG 6000 (0, 3, 6 and 9% w/v) using Murashige and Skoog semi-solid culture medium. At 30 days of culture, different developmental variables and dry matter (DM), total protein (TP), proline (Pr) and glycine-betaine (GB) contents were evaluated. The results showed reduced development in cv CP 72-2086, Mex 79-431 and MOTZMex 92-207 with increasing PEG concentration. The cv Mex 69-290 showed tolerance to osmotic stress of −0.45 MPa using 3% PEG. Overall, TP content decreased with increasing PEG concentration, while DM, Pr and GB contents rose with increasing PEG concentration in all evaluated cultivars. Our results suggest that cv Mex 69-290 has a slight tolerance to water stress and could be used for rainfed cultivation with low rainfall or reduced irrigation for better water use efficiency. In conclusion, the in vitro screening technique of cultivars tolerant to PEG-induced water stress is an alternative for early determination of drought stress in sugarcane.

Potential absorption of mercury-contaminated substrate by Trichoderma sp isolated from Brazil Nuts and Amazon Soil

Mercury is an inorganic contaminant with serious harmful consequences to the environment. There has been a continuous rise in its level due to industrialization and other anthropogenic activities, such as the burning of coal and petroleum products, use of mercurial fungicides in agriculture and mercury catalyst in industries, and production of waste by paper industries. Five strains of Trichoderma sp., a filamentous fungi, were used in this study to evaluate their resistance to high concentrations of mercury for the purpose of using them for bioremediation. The solid culture medium used was prepared with malt agar 2% with pH 7.0 in which the strains of Trichoderma sp. were inoculated. The minimum inhibitory concentration (MIC) of the selected Trichoderma sp. isolates was calculated considering the time for growth and concentration of the mercury salt (Hg(NO3)2). At a mercury concentration of 50 mg/mL, maximum growth was first observed in TCH 1 (89.42 ± 0.63 mm) followed by TCH 2 isolate (87.33 ± 0.58 mm). At this concentration, all isolates reached the maximum mycelia growth. When the concentration of 200 mg/L Hg(NO3)2 was used, complete growth inhibition of the isolates was observed. Scanning electron microscopy suggested that differences in sporulation between the control and mercury treatment groups. In conclusion, it can be stated that Trichoderma isolates have great potential for bioremediation of sources contaminated with mercury.