EFECTO DE DIFERENTES EXPLANTES IRRADIADOS EN LA REGENERACIÓN in vitro DE FRIJOL COMÚN (Phaseolus vulgaris L.) CULTIVAR "ICA Pijao" / Effect of Different Irradiated Explants in the in vitro Regeneration of Common Bean (Phaseolus vulgaris L.) Cultivar "ICA Pijao"

RESUMEN El mejoramiento genético convencional en frijol común resulta difícil debido a que presenta una base genética estrecha y muy estable. En este sentido, la combinación de la mutagénesis y el cultivo de tejidos, es una alternativa para inducir variabilidad genética en la búsqueda de tolerancia a factores bióticos y abióticos. Es por ello, que el presente trabajo tuvo como objetivo determinar el efecto de diferentes explantes irradiados en la regeneración in vitro de frijol común (Phaseolus vulgaris L.) cultivar "ICA Pijao". Se aplicaron radiaciones gamma en callos, en el nudo cotiledonal con un cotiledón (NC-1) con dosis de 0, 10, 20, 30, 40, 50 y 60 Gy y semillas con 0, 100, 200, 300 y 400 Gy. Se evaluó el porcentaje de germinación, longitud de las raíces, porcentaje de explantes que formaron callos, masa fresca (g) de los callos, número de brotes por callo y el número de brotes con raíces. La radiación gamma provocó una disminución en la masa fresca del callo y NC-1. Los callos y el NC-1 solamente formaron brotes con las dosis de 10 y 20 Gy, pero estos fueron hiperhíricos. Los resultados demostraron que la semilla irradiada fue el explante con el que se logró la regeneración in vitro de plantas con hojas definidas, por lo que se recomienda como explante inicial para el uso combinado de mutagénesis y regeneración in vitro de plantas para el cultivar P. vulgaris "ICA Pijao" a través de la organogénesis indirecta.

ABSTRACT Conventional breeding in common bean is difficult because they have a close and very stable genetic base. In this connection the combination of mutagenesis and tissue culture is an alternative to induce genetic variability in the search for tolerance to biotic and abiotic factors. For this reason, the present study aimed to determine the effect of different irradiated explants in the in vitro regeneration of common bean (Phaseolus vulgaris L.) cultivar "ICA Pijao". To do this, were applied doses gamma radiation in callus, cotiledonary node with one cotyledon (NC-1) with doses of radiation 0, 10, 20, 30, 40, 50 and 60 Gy and seeds with 0, 100, 200, 300 and 400 Gy. The length of roots in the germinated seeds, fresh mass (g) of the callus and the number of shoots per callus were determined. The gamma radiation caused a decrease in the fresh weight of callus and NC-1. The callus and NC-1, irradiated with doses of 10 and 20 Gy they formed buds but these were hyperhydric. Results demonstrated that the irradiated seed was the explant with which it was achieved regeneration of shoots with leaves defined, so it is recommended as initial explant for combined use of mutagenesis and in vitro regeneration of plants for P. vulgaris cultivar "ICA Pijao" via organogenesis indirect.

In vitro selection for Fusarium oxysporum f. sp. conglutinans resistance in brassica vegetables

Background:Fusarium wilt is an issue of concern in economically and nutritionally important Brassica vegetable cultivation. Thus, it deserves measures against the adverse production impact caused by Fusarium wilt.Methods:In this study, development of resistance to F.oxysporumf. sp. conglutinansin six white head cabbage cultivars, by in vitro chemical mutagenesis and selection, through direct and indirect organogenesis was examined. 6 day and 10 day old hypocotyl, shoot tip and calli, from 6 day old hypocotyl explants, were subjected to chemicalmutagenesis treatment (DMSO (4% v/v) + EMS (0.3% v/v) for two hours at 28±2°C) were incubated in MS shoot induction medium (MS+ NAA (0.2 mg/l), BAP (3 mg/l), GA3 (0.01 mg/L) and AgNO3 (0.5 mg/l)). Shoots developed from hypocotyl and shoot tip explants (inthe MS shoot induction medium and then in MS + NAA (0.2 mg/l), BAP (3 mg/l), GA3 (0.01 mg/l) for shoot development) and calli following mutagenesis treatment were screened for Fusarium resistance subjecting to 15% and 20% Fusariumculture filtrate for 30 and 60 day selection periodsfor each strength respectively.Results:Developed plantlets from all six cultivars tested, showed resistance toFusarium culture filtrate in the in vitro conditions with different survival frequencies ranging between 12.5% to 84.0% from hypocotyl and 0.0% to 86.7% from shoot tip explants among cultivars indicating development of resistance to Fusariumby in vitro chemical mutagenesis.Conclusions:Direct organogenesis, and 10 day old hypocotyl and 6 day old shoot tips are potential explants for successful application of in vitro chemical mutagenesis for Fusarrium resistance development in in cabbage.

Directional breeding of high oil content peanut variety Yuhua 9 by in vitro mutagenesis and screening / 生物工程学报

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.

Breeding of peanut variety Yuhua 4 by in vitro mutagenesis / 生物工程学报

The embryonic leaflets of peanut (Arachis hypogaea) variety Huayu 20 were used as explants and pingyangmycin as a mutagen to induce somatic embryos. Four weeks after the inoculation, the survived explants were transferred to somatic embryo germination medium containing screening reagent hydroxyproline, and finally 15 regenerated plants were obtained. Pedigree breeding method was used during the following selection breeding, and three lines with significantly increased yield and 23 lines with high oil content were obtained from these mutant offsprings. The line with both high yield and high oil content has passed peanut variety multi-location in Anhui province and was named "Yuhua 4". Its yield was 16.63% higher than that of the control variety Baisha 1016, ranking the first in all the testing varieties. Yuhua 4 showed the characteristics of early maturity, small pod and high oil content. The oil content of kernels was 56.10%, higher than that of original parent Huayu 20 with 49.50% oil content, tested by the Ministry of Agriculture of Oil and Products Quality Supervision, Inspection and Test Center (Wuhan), and the yield was 15% higher than that of Huayu 20. It was concluded that in vitro mutagenesis and target screening was an effective way on creating new germplasm and breeding new variety in peanut.

Nuclear protein binding patterns in the 5'-upstream regulatory elements of HLA class I genes

The expression of MHC class I genes has been thought to be regulated by two major cis-acting regulatory elements. The first region, enhancer A (Enh A) spanning from positions -210 to -165 contains perfect palindrome (PP), TGGGGATTCCCCA. The PP is well-conserved both in mouse and human MHC class I genes, even though the PP is disrupted by 2 bp substitutions (TGAGGATTCTCCA) in HLA-C genes. Three proteins binding to the Enh A of HLA-A and -B locus genes, but very weakly or nearly not to the Enh A of HLA-C locus gene have been identified. To determine functional importance of the PP for binding of trans-acting protein, mutant DNA probes were made by site-directed in vitro mutagenesis and then electrophoretic mobility shift assay was performed. HLA-A mutant DNA probe, in which the PP is disrupted, shows the same nuclear protein binding pattern as that of the HLA-C gene, and HLA-C mutant DNA probe, in which the PP is introduced, shows the same nuclear protein binding pattern as that of the wild type HLA-A gene. These data suggest that the perfect palindrome and its cognate DNA binding nuclear protein play an important role in the HLA class I gene regulation, and thus the lower expression of HLA-C antigen may be ascribed to no or very weak factor binding to the nonpalindromic sequences of HLA-C upstream DNA.

T4 RNA ligase: New structural studies on an unusual but useful enzyme.

Gene 63 from bacteriophage T4 encodes a single polypeptide with two independent enzyme activities, RNA ligase and tail fibre attachment. The DNA sequence of gene 63 has been determined and the gene cloned in an expression plasmid (pDR540) that contains the inducible tac promoter. Escherichia coli cells containing the plasmid (KR54) produce about 5–10 % of their soluble protein as RNA ligase. A convenient isolation procedure for the enzyme is described from KR54 cells and the isolated product is indistinguishable from that obtainable from T4-infected Escherichia coli. The enzyme reacts reversibly with ATP in the presence of Mg2+ to give a covalent AMP-enzyme adduct. It is shown by FAB mass spectrometric analysis of chymotryptic fragments of the adenylylated enzyme that the AMP is bound covalently to lysine residue 99. Methods of in vitro mutagenesis are described for gene 63 cloned in a bacteriophage M13 vector. By deletion mutagenesis it was shown that the C-terminal 20 % of the protein is not crucial for the RLi activity but the TFA activity, as measured by a complementation assay, is reduced. A method is described for the introduction of point mutations in gene 63 by use of AMV reverse transcriptase for error-directed repair polymerisation in gapped DNA heteroduplexes. In addition, a synthetic oligonucleotide mismatched at the 3’ end was used as a primer for reverse transcriptase catalysed repair polymerisation to force a single base change in the codon for Lys-99 to give the codon for Asn. The mutant protein has no detectable RNA ligase activity but retains tail fibre attachment activity.