Maritza Escalona

Sugarcane shoot formation in an improved temporary immersion system

A new protocol was established for sugarcane cv. C-1051-73 shoot formation in a temporary immersion system. The two-step protocol involves shoot formation in 50 ml of culture medium per explant and 1.0 mg l-1 paclobutrazol for 30 days followed by shoot elongation by exposure to 1.0 mg l-1 gibberellic acid for 15 days. The multiplication rate was doubled in comparison with the conventional micropropagation protocol (Jiménez et al., 1995) and the cost has been reduced by 46%. Three additional sugarcane varieties have been micropropagated according to this new protocol and results are comparable. Temporary immersion-derived plants have also been compared with conventionally propagated plants in sugarcane fields for more than 9 months and their agricultural indicators of performance are similar.

PHYSIOLOGY OF EFFECTS OF TEMPORARY IMMERSION BIOREACTORS ON MICROPROPAGATED PINEAPPLE PLANTLETS

SummaryTemporary immersion bioreactors are an efficient tool for plant mass propagation because they increase multiplication rate and plant quality. Little knowledge is available on the ecosystem and physiological behavior of plantlets when using this new culture technique. In order to evaluate the effects of the conditions on physiological change of pineapple plantlets, a factorial experiment was conducted, where axillary clusters were cultured under two levels of photosynthetic photon flux (PPF): 30 μmol m−2s−1 (low) and 225 μmol m−2s−1 (high), using two culture methods (conventional micropropagation in liquid medium and a temporary immersion bioreactor) during the elongation phase. CO2 concentration in the headspace volume container was measured during a whole cycle of temporary immersion (3h). At the time before the next immersion period, the levels of CO2 increased significantly to 14171 μmol mol−1 at high PPF. The maximal photosynthetic rate as well as the maximum quantum yield of photosystem II were low for plantlets cultivated in the femporary immersion bioreactor at high PPF. However, these plantlets showed large increases in sugar and nitrogen uptake and also increases in dry weight and foliar area. These results indicate that shoot growth did not totally depend on the photosynthesis process. In vitro pineapple plantlets appeared to use more nutrients in the culture medium than those from photosynthesis. In summary, temporary immersion bioreactor-derived plantlets showed remarkable nutrient uptake, indicating a higher photo-mixotrophic metabolism.

Sugarcane micropropagation and phenolic excretion

Sugarcane shoot formation was followed using a temporary immersion system. Plant fresh weight, plant dry weight, shoot number and phenolic excretion to the culture medium were recorded during shoot formation. Shoot number increased for 30 days of culture but formation of new shoots was greatly reduced from 31 to 40 days. Phenolic excretion also increased during the first 20 days of culture (gallic acid represented 82% total phenolics) and decreased during the last 10 days (31–40 days of culture). The most intensive period of phenolic excretion (11–20 days) preceded the most intensive period of shoot formation (21–30 days). The same relationship does not seem to exist between the accumulation of fresh and dry weights. Subculture onto fresh medium at the beginning of proliferation (10 days after culture initiation) was detrimental to shoot formation in the subsequent period (11–20 days). However, such a detrimental effect could be avoided if gallic acid was added to the medium. Addition of cysteine to the culture medium reduced both excretion of phenolics and shoot formation but not fresh weight. The use of temporary immersion systems, the increase of culture medium volume per initial explant and the addition of paclobutrazol promoted both phenolic excretion and sugarcane shoot formation. Results presented here indicate a relationship between phenolic excretion and shoot formation but not with accumulation of plant weight.

PHOTOSYNTHESIS AND CARBON METABOLISM IN PLANTAIN (MUSA AAB) PLANTLETS GROWING IN TEMPORARY IMMERSION BIOREACTORS AND DURING EX VITRO ACCLIMATIZATION

SummaryThe photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO2m−2s−1); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.

Sugarcane (Saccharum sp. Hybrid) Propagated in Headspace Renovating Systems Shows Autotrophic Characteristics and Develops Improved Anti-oxidative Response

Previous results have shown that sugarcane plantlets micropropagated in Temporary Immersion Bioreactors (TIB) demonstrated a better morphology and physiological behaviour when compared to plantlets propagated in Gelled Medium (GM). The present work focuses on the onset of oxidative stress symptoms at transfer to ex vitro and during acclimatization. The specific ROS being produced were identified and tissue-located by infiltrating leaves with specific O2−.and H2O2 staining dyes, respectively NBT and DAB. TIB plantlets showed trichomes stained with NBT and DAB, their density decreasing with time. Stomata were coloured with NBT and DAB in GM and, at the end of acclimatization, plantlets from both systems presented the lowest level of staining of both stomata and trichomes. The response of the anti-oxidative system was also analysed through in vitro and in gel enzyme activities and transcription levels of genes for key response enzymes. At the end of the in vitro phase, GM plantlets showed higher activities of APX and MDHAR, while CAT, GR, GT and DHAR activities increased in TIB. At the end of acclimatization SOD and CAT increased mainly in TIB, while GM induced the increase of APX. The immunobloting of peroxiredoxins showed that Prxs were expressed at higher levels in TIB plantlets, some showing polymerization. The transcription of genes coding for key response enzymes was strongly up-regulated in GM plantlets. In conclusion and comparing with GM, TIB produced plantlets closer to autotrophy and with improved mobilization of the anti-oxidative response.

Generalized pustular psoriasis triggered by Zika virus infection

Zika virus is an emerging arbovirus, which is expanding in epidemic proportions through tropical and subtropical areas of the world. Although Zika is linked to a number of congenital and neurological complications, there is scarce knowledge on the impact of ZIKV infection in human skin. We report the case of a 68‐year old woman who presented with generalized pustular psoriasis after a preceding and otherwise uneventful episode of ZIKV infection. Based on recent experimental data on the biology of ZIKV infection in the cutaneous environment, we speculate that ZIKV may have directly triggered the development of generalized pustular psoriasis by stimulation of keratinocyte‐derived mediators of inflammation and a polyfunctional T‐cell driven immune reaction in the cutaneous milieu.

Comparison of different in vitro micropropagation methods of Stevia rebaudiana B. including temporary immersion bioreactor (BIT

Stevia rebaudiana is beneficial to treat diabetes because of its low-calorie glucoside sweeteners. Natural and vegetative propagation are inefficient. In vitro techniques are an attractive alternative but low propagation and reproducibility rates have been reported. Therefore, different ways to increase natural sweetener production in vitro, such as BIT®, are required. We compared semisolid medium, liquid medium and BIT® in terms of Stevia biomass and steviol glycosides production. At 21 days of culture, morphological quality of BIT®-derived shoots was best and coupled with shoot fresh and dry weight that were more than seven times higher in BIT® compared with micropropagation in liquid or semisolid media. In turn, the total content of steviol glycosides produced was also higher in bioreactors. The usefulness of BIT® to produce plant metabolites in vitro is again demonstrated, even if additional experiments are required to increase the economic efficiency of the process.

Salinity induces specific metabolic changes in sugarcane shoot explants in temporary immersion bioreactors

There is a great demand of salt-tolerant sugarcane planting material in Cuba. Temporary immersion bioreactors (TIB) are effective to significantly increase sugarcane in vitro shoot proliferation rate from 1:4 in conventional containers to about 1:35. Sugarcane micropropagation in TIBs under NaCl stress may help screen mutants with salinity tolerance. We developed the experiment shown here to identify a NaCl concentration able to stress shoot in TIBs. At 30 days of culture initiation with different NaCl levels (0 - 200 mM), explant multiplication rate, shoot cluster fresh mass, and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. Content of soluble phenolics in the culture medium was also evaluated. Addition of NaCl decreased shoot multiplication rate and fresh mass. Other statistically significant differences were recorded but the most important were noted in the increased contents of carotenoids, malondialdehyde, other aldehydes and soluble phenolics in the plants, and in the soluble phenolics in the culture medium. This research may be useful for future experiments of in vitro selection of new sugarcane genetic materials with NaCl tolerance. Fifty percent of multiplication rate was reduced with 89 mM NaCl which can be used to stress shoots during micropropagation in TIBs and eventually detect mutants with salt tolerance.