Elio Jiménez

Improved production of potato microtubers using a temporary immersion system

A temporary immersion system for potato microtuber production was designed using 4-l vessels. This culture technique showed several advantages compared to solid cultures: i.e., three fold increase in shoot length, more internodes per plant and improved vigor. In the tuber induction stage, microtubers can be induced at all plant nodes, indicating that the tuberization is not restricted to specific regions. For both cultivars tested, Desiree and Atlantic, an average of 3.1 and 2.8 tubers per single node cutting was achieved after 9 weeks in culture. The size and weight of the tubers were higher than on solid media. Scale up was performed with cv. Atlantic in 10-l polycarbonate flasks and 12 units were mounted containing 150 single nodal cuttings each. An average of 2.6 tubers per inoculated cutting was obtained, with 1.3 g fresh weight per microtuber. Temporary immersion is a valuable option for potato microtuber production, as well as for shoot production during the planting season.

BIOMASS PRODUCTION OF CYMBOPOGON CITRATUS (D.C.) STAPF., A MEDICINAL PLANT, IN TEMPORARY IMMERSION SYSTEMS

SummaryIn vitro plants of lemon grass were established, starting from shoot apices derived from plants cultivated under field conditions. The effect of the immersion frequency (two, four, and six immersions per day) on the production of biomass in temporary immersion systems (TIS) of 1 liter capacity was studied. The highest multiplication coefficient (12.3) was obtained when six immersions per day were used. The maximum values of fresh weight (FW; 62.2 and 66.2 g) were obtained with a frequency of four and six immersions per day, respectively. However, the values for dry weight (DW; 6.4g) and height (8.97cm) were greater in the treatment with four immersions per day. The TIS used in this work for the production of lemon grass biomass may offer the possibility of manipulating the culture parameters, which can influence the production of biomass and the accumulation of secondary metabolites. We describe for the first time the in vitro production of Cymbopogon citratus biomass in TIS.

Effect of dissolved oxygen concentration on differentiation of somatic embryos of Coffea arabica cv. Catimor 9722

The effect of two different dissolved oxygen (DO) concentrations (50 and 80%) on differentiation of somatic embryos (SE) from cell suspensions of coffee (Coffea arabica cv. Catimor 9722) was analyzed. Two bioreactors CMF-100 (CHEMAP AG) designed for the culture of cells, with 2-l glass vessels and a maximum work volume of 1.8 l were used. Each one was equipped with a gas blending unit (air, O2, N2, CO2) for the control of DO concentration. The inoculation density of embryogenic cells was 1.0 gram of fresh weight per liter (g FW l−1). The number of somatic embryos was greater (71 072 SE l−1) with 80% DO, but the major proportion were globular and heart shaped SE (66 399 SE l−1) and only 6.6% with regard to total was torpedo shaped SE. However, the 50% DO produced the higher number in the torpedo shaped SE (7389 SE l−1) what represented 20.0% with regard to total. Thus, higher concentrations of DO induced globular and heart shaped SE differentiation, but for production of torpedo shaped SE lower concentrations DO are needed. The somatic embryos obtained in the bioreactor with 50% DO showed similar behavior to the somatic embryos obtained in the rotary shaker. After 8 weeks of culture, 49.2% germination was obtained, which allowed a total of 1725 plantlet to be transferred to conditions ex vitro. After 6 months of culture, 89.2% of conversion was achieved and 1539 plants obtained were transferred to field conditions.

Sugarcane genes differentially expressed in response to Puccinia melanocephala infection: identification and transcript profiling

Brown rust caused by the fungus Puccinia melanocephala is a major disease of sugarcane (Saccharum spp.). A sugarcane mutant, obtained by chemical mutagenesis of the susceptible variety B4362, showed a post-haustorial hypersensitive response (HR)-mediated resistance to the pathogen and was used to identify genes differentially expressed in response to P. melanocephala via suppression subtractive hybridization (SSH). Tester cDNA was derived from the brown rust-resistant mutant after inoculation with P. melanocephala, while driver cDNAs were obtained from the non-inoculated resistant mutant and the inoculated susceptible donor variety B4362. Database comparisons of the sequences of the SSH recombinant clones revealed that, of a subset of 89 non-redundant sequences, 88% had similarity to known functional genes, while 12% were of unknown function. Thirteen genes were selected for transcript profiling in the resistant mutant and the susceptible donor variety. Genes involved in glycolysis and C4 carbon fixation were up-regulated in both interactions probably due to disturbance of sugarcane carbon metabolism by the pathogen. Genes related with the nascent polypeptide associated complex, post-translational proteome modulation and autophagy were transcribed at higher levels in the compatible interaction. Up-regulation of a putative l-isoaspartyl O-methyltransferase S-adenosylmethionine gene in the compatible interaction may point to fungal manipulation of the cytoplasmatic methionine cycle. Genes coding for a putative no apical meristem protein, S-adenosylmethionine decarboxylase, non-specific lipid transfer protein, and GDP-l-galactose phosphorylase involved in ascorbic acid biosynthesis were up-regulated in the incompatible interaction at the onset of haustorium formation, and may contribute to the HR-mediated defense response in the rust-resistant mutant.

Selection and characterisation of sugarcane mutants with improved resistance to brown rust obtained by induced mutation

The brown rust susceptible sugarcane genotype B4362 was subjected to in vitro tissue culture and physical and chemical mutation induction procedures. Five brown rust resistant mutants with hypersensitive response to Puccinia melanocephala were selected out of a total population of 11 167 regenerated plants. High selection frequency was obtained with both mutagenic treatments, although chemical mutagenesis (NaN3) resulted in higher selection frequencies for brown rust resistance than gamma irradiation (60Co). The brown rust resistant mutants showed variations in molecular, morphological, and agronomic traits. Traits such as internode shape, bud shape, leaf sheath hairiness, outer auricule shape, intensity of flowering, stool growth habit, number of stalks per stool, and smut susceptibility were modified in brown rust resistant mutants. In addition, sugar yield was improved in two mutants with increments in stalk length, stalk number, and stalk diameter. Mutation induction proved to be suitable for the generation of new sources of brown rust resistance in sugarcane.

A green fluorescent protein-transformed Mycosphaerella fijiensis strain shows increased aggressiveness on banana

The fungal pathogen Mycosphaerella fijiensis, causal agent of black leaf streak disease of bananas and plantains, was transformed with a green fluorescent protein-carrying construct by using a restriction enzyme-mediated integration methodology. A quantitative polymerase chain reaction was adapted to estimate transgene copy number and pathogenicity assays with three banana genotypes with dissimilar reactions to M. fijiensis infection were performed to characterize the transformants. Transgene insertion varied from one to five copies per genome among four random selected transformants. All M. fijiensis strains produced typical symptoms of the black leaf streak disease on the three banana genotypes assayed. Interestingly, the GFP-18 transformant showed increased aggressiveness on susceptible ‘Grande naine’ and resistant ‘Yangambi km5’ plants demonstrating that mutation events in M. fijiensis can increase virulence.

Early regulation of primary metabolism, antioxidant, methyl cycle and phenylpropanoid pathways during the Mycosphaerella fijiensis-Musa spp. interaction

Black leaf streak disease (BLSD) caused by Mycosphaerella fijiensis is considered the most destructive and costly foliar disease that affects bananas and plantains. In spite of some recent progress regarding the study of M. fijiensis-Musa spp. interaction, there is still limited information for this pathosystem. To gain insights into the molecular mechanisms behind the M. fijiensis-Musa spp. interaction, gene expression and biochemical analysis related with primary metabolism, methyl cycle, antioxidant and phenylpropanoid pathways were conducted. Early screening of the resistant cultivar (cv.) ‘Calcutta 4’ (Musa AA) and the susceptible cv. ‘Grande naine’ (Musa AAA) plants infected with M. fijiensis was effective at looking for defense-related genes. Quantitative PCR experiments showed up-regulation of Photosystem I reaction center subunit N chloroplastic-like (primary metabolism) and S-adenosyl-L-methionine synthetaste (methyl cycle) genes in the incompatible interaction, as well as down-regulation of the phenylpropanoid pathway genes in the susceptible cv. ‘Grande naine’ as main finding of this study. Improved knowledge concerning the M. fijiensis-Musa spp. interaction could help to establish innovative approaches for plant breeding programs against BLSD.