Carlos Aragón

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.

New Pineapple Somaclonal Variants: P3R5 and Dwarf

The Food and Agriculture Organization has highlighted pineapple as one of the most important tropical fruits. Since classical pineapple breeding is difficult, biotechnology has emerged as an attractive instrument. We obtained two new pineapple somaclonal variants derived from in vitro culture of cv. Red Spanish Pinar: P3R5 and Dwarf. The AFLP analysis revealed an existing genetic distance. So far 44 phenotype indicators selected due to their relation to a wide range of important agricultural, morphological and physiological processes have been evaluated. P3R5 differed from the donor in 19 variables (19/44; 43.18%), while Dwarf varied in 31 indicators (31/44; 70.45%). The number of shoots was significantly different among the three plant materials. Dwarf showed two shoots per plant while P3R5 and the donor did not form any shoots. We also observed that water use efficiency, chlorophyll b concentration, total chlorophyll concentration, transpiration rate, chlorophyll a concentration, thickness of leaf photosynthetic parenchyma, fruit mass with crown, content of free phenolics and superoxide dismutase specific activity were also very different among the three plant materials. The Euclidean distances of each somaclonal variant to the donor plant material taking into consideration the genotype (AFLP) and the phenotype evaluations were also calculated. Regarding the genotype information, P3R5 is separated from cv. Red Spanish Pinar by 2.83 units of Euclidean distance, and Dwarf by 3.00 units. However, the phenotype indicators revealed higher differences: 3.74 in P3R5 and 4.71 in Dwarf. To our knowledge, this is the first report of a comprehensive analysis of pineapple somaclonal variants.

Biochemical side effects of the herbicide FINALE on bar gene-containing transgenic pineapple plantlets

Pineapple is one of the most important tropical fruits and therefore intensive genetic improvement programs are being carried out in many countries, including Cuba. Our research team has previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. Herein, we report on the biochemical side effects of the herbicide FINALE® on these transgenic plantlets during hardening. Levels of aldehydes and chlorophylls, and peroxidase activity were recorded. The transformed clone studied here, not sprayed with FINALE®, showed the following side effects because of transgenesis only. Levels of malondialdehyde, other aldehydes, chlorophyll b, and total chlorophyll pigments decreased. The most remarkable biochemical differences between transgenic and non-transgenic plantlets after application of FINALE® follow. Levels of malondialdehyde and other aldehydes in transgenic material were not decreased by FINALE®, perhaps because these levels were already low as a result of transformation. FINALE® increased peroxidase activity in transgenic plantlets but such increase was higher in non-transgenic material. The herbicide increased contents of chlorophyll pigments (a, b, total) in transformed plantlets. However, as expected, non-transgenic plantlets decreased levels of chlorophylls (a, b, total) after application of FINALE®. The genetic transformation of pineapple with the bar gene not only conferred resistance to the herbicide FINALE®, but also promoted other biochemical changes.

Phenotypic and Molecular Characterization of Phaseolus vulgaris Plants from Non-Cryopreserved and Cryopreserved Seeds

The objective of this work was to evaluate if cryostorage of Phaseolus vulgaris L. seeds induced variations in regenerated plants at the phenotypic and molecular levels. A series of agricultural traits was measured on plants grown from control, non-cryopreserved and cryopreserved seeds, and the genetic stability of plants of the second generation was analysed at selected microsatellite loci. The phenotype of the second generation plants was evaluated as well. No statistically significant phenotypic differences were observed for the parameters measured, neither in the first nor in the second generations. Averaging both treatments, about 76% of the seeds had germinated 10 days after sowing. At harvest we recorded plants with about 73 cm in height, 13 stem internodes, 25 fruits, 103 grains and 4 grains per fruit. One hundred seeds weighted about 26 g. The genetic analyses performed on the second generation plants using six nuclear Simple Sequences Repeats (SSR) markers revealed no changes in microsatellite length between control and cryopreserved samples, implying that there was no effect of seed liquid nitrogen exposure on genome integrity. The phenotypic and molecular results reported here confirm that cryostorage is an efficient and reliable technique to conserve P. vulgaris seeds and regenerate true-to-type plants.

EFECTO DEL DÉFICIT HÍDRICO SOBRE CAMBIOS MORFO-FISIOLÓGICOS Y BIOQUÍMICOS EN PLANTAS MICROPROPAGADAS DE PIÑA ‘MD-2’ EN LA ETAPA FINAL DE ACLIMATIZACIÓN

The molecular techniques need of protocols that allow determine levels of genetic change inside the populations in different environmental conditions. So much the optimization of the isolation of the DNA, as that of the working conditions of the amplifications, they are fundamental to reach the success of the molecular analyses, therefore the present research has like objective: optimize protocols of extraction of DNA and of the molecular marker type RAPD (Random Amplified Polymorphic DNA) in Anonaceas. For the DNA extraction were used the Kit Nucleon PHYTOpure and DNeasy® of QIAGEN. The working conditions of the protocol of amplification were fittedand changed the concentrations of DNA and of the used chokers. Followed by this, a tes was realized with 10 fatteners of the series OPH and five of the series OPA, to select more polimorphiss. The first results obtained with the Kit Nucleon PHYTOpure showed an DNA of low quality, due to the high fenolization of the vegetable material, not like that with the Kit DNeasy® of QIAGEN, who allowed obtain an DNA of quality, purity and homogeneity to an approximate concentration of 30 ng μ L-1. The biggest amplification products were obtained touse 3 ng μ L-1 of choke and 2 ng μ L-1 of DNA. Four chokers that presented major polymorphism were OPA-16, OPH-03, OPH-13 and OPH-18. The results of this research allowed optimize the working conditions of the technical RAPD for the characterization of the collection ex-situ of Anonaceas under our environmental conditions.Theobroma cacao L. is known in folk medicine as an antiseptic, diuretic and antiparasitic. Foods derived from this plant are rich in natural products of high added value, including phenolic compounds. As in vitro cultivation handle is an alternative source for the production of these metabolites. The present study was conducted to obtain phenolic compounds from callus culture with embryogenic structures. Culture conditions (agitation, light and glucose) were established to increase the concentration of phenols in calluses and elicitors to achieve the increase in callus and excretion into the culture area. The accumulation of phenolic compounds was favored with the additional supplement of glucose, growth in agitation and darkness. The addition of random hydroxylated cyclodextrins allowed the increase in the specific yield of phenols and biomass.The genus Azotobacter and Bacillus have a potential to atmospheric nitrogen fixation, mineral elements solubilization and producing a group of stimulator of vegetal growth substances. In addition, Bacillus is recognizing by its antagonistic activity. The reasons justify its selection with actives principles of biofertilizer products. The presence of fungus diseases in vegetable cultures is a problem for Cuban agriculture. The objective of the present work was to evaluate the antagonistic activity of Azotobacter and Bacillus strains against fungus that caused diseases in vegetables cultures. For those were employed Fusarium chlamydosporum, Corynespora casiicola and Cladosporium oxysporum. All strains belong at the INIFAT collections. For the development of work, “Dual Culture Bring” Method was used with permitting the selections those to possess previous activity and descript, in turn, principle affectations caused to fungi structures. Results emit that inside of two genus exist strains that inhibit the micelial growth. Among strains of Azotobacter, five result promissory against Cladosporium oxysporum, two respond to Fusarium chlamydosporum and only one was effective against Corynespora cassiicola. The activity shows for Bacillus genus is major. In this case, two strains show activity against Corynespora cassiicola, six against Cladosporium oxysporum and eight against Fusarium chlamydosporum. Highlight the finished that exists strains of Azotobactersp capable of inhibit more than one fungi species, which results very new for being quoted few times the genus activity against vegetable pathogens.