ABSTRACT
The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.
A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.
ABSTRACT
The transformation of coffee plantlets with the cry1ac gene of Bacillus thuringiensis was achieved by biolistic using either the whole pUBC plasmid or only the ubi-cry1ac-nos genetic cassette. The cry1ac gene was inserted into coffee plants in order to confer resistance to the leaf miner Leucoptera coffeella, an insect responsible for considerable losses in coffee crops. Bearing in mind that the genetic cassettes used for this study lack reporter genes and/or selection marker genes, the parameters for the transformation procedure by biolistic were previously standardised with a plasmid carrying the gus reporter gene. The presence of the cry1ac gene in young plantlet tissues was determined by PCR, Southern blot and reverse transcription-PCR. Our results show that the obtainment of viable coffee plantlets, transformed by bombardment with the cry1ac gene and without selection markers nor reporter genes, is feasible.
A transformação das plântulas de café com o gene cry1ac de Bacillus thuringiensis foi realizada por biobalística, utilizando todo o pUBC plasmídeo ou só o cassete genético UBI-cry1ac-nos. O gene cry1ac foi inserido no cafeeiro a fim de conferir resistência à folha mineiro Leucoptera coffeella, um inseto responsável por perdas consideráveis nas culturas de café. Tendo em conta que ao plasmídeo e ao cassete genético utilizados para este estudo faltam genes repórteres e/ou de seleção, os parâmetros para o processo de transformação por biobalística foram previamente padronizados com um plasmídeo transportando o gene repórter gus. A presença do gene cry1ac em tecidos de jovens plântulas foi determinada por PCR, Southern blot e transcrição reversa-PCR. Nossos resultados mostram que a obtenção de plântulas de café, transformado por bombardeamento com o gene cry1ac sem genes de seleção genética nem repórteres é viável.
Subject(s)
Animals , Bacterial Proteins/genetics , Coffea/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Plants, Genetically Modified/genetics , Transformation, Genetic/genetics , Blotting, Western , Biolistics/methods , Lepidoptera , Polymerase Chain Reaction , Plant Diseases/parasitology , Plant Diseases/prevention & controlABSTRACT
The aim of this work was to optimize the biolistic delivery parameters that affect the DNA delivery and stable expression of marker genes into coffee tissues (Coffea arabica. L. cvs. Caturra and Catuaí). The effect of osmotic preculture length, osmotic concentration of medium, Helium pressure and target distance on transient expression of the uidA gene in coffee leaves and somatic embryos were tested. The highest transient uidA expression was obtained when Caturra (18.3±2.8) and Catuaí (6.8±2.0) leaves and Catuaí embryos (80.0±7.4) were cultured for 5h on Yasuda medium complemented with 0.5M Mannitol +0.5M Sorbitol. The combination of 1100psi and a target distance of 9cm resulted in the highest number of blue spots per Caturra leaf segment (23.6±3.9), whereas for the Catuaí variety the combination of 1100psi and a target distance of six (10.2±1.9) and nine (8.2±1.9) cm gave the highest number of blue spots per leaf segment. The optimized protocol was tested with pCAMBIA 1 301 (uidA gene and the hpt gene), pCAMBIA 1 305.2 (uidA version GUSPlus and the hpt gene) and pCAMBIA 1 301-BAR (uidA gene and the bar gene). The highest number of blue spots was obtained when Caturra (54.6±5.7) and Catuaí (28.9±4.3) leaves were bombarded with pCAMBIA 1 305.2. Selection of bombarded coffee tissues with 100mg/l hygromicyn caused the oxidation of tissues. Rev. Biol. Trop. 57 (Suppl. 1): 151-160. Epub 2009 November 30.
La presente investigación tuvo como objetivo optimizar los parámetros que afectan la incorporación y expresión de genes marcadores mediante biobalística en segmentos de hoja y embriones somáticos de café (Coffea arabica. L. cvs. Caturra y Catuaí). La mayor expresión transitoria del gen uidA en segmentos de hoja de Caturra (18.3±2.8) y Catuaí (6.8±2.0) y embriones somáticos de Catuaí (80.0±7.4) se obtuvo al cultivar los explantes por cinco horas previo al bombardeo en el medio Yasuda complementado con 0.5M mannitol+0.5M sorbitol. Asimismo, se obtuvo una mayor expresión transitoria del gen uidA al bombardear los segmentos de hoja de Caturra y Catuaí y embriones somáticos de Catuaí con una presión de helio de 1 100psi y una distancia de bombardeo de 6 o 9 cm.
Subject(s)
Osmotic Pressure , Coffee/classification , Helium , Coffee Industry , Costa RicaABSTRACT
As the major commercial source of natural rubber,Hevea brasiliensis attracts much attention.However,the heterozygous nature,long breeding cycle are strong limitations for conventional breeding.While genetic engineering,which can be used to widen the germplasm base and produce desirable agronomic traits quickly and efficiently,offers a viable alternative approach to complement traditional breeding.Comprehensive analysis indicates that in the past two decades,with calli derived from immature anther or integumental tissues of immature fruit as receptors,both biolistic and Agrobacterium-mediated transformation methods were employed for developping rubber genotypes with improved latex yield,tolerance to tapping panel dryness syndrome,producing high-value recombinant proteins,etc.Being recalcitrant to tissue culture,the transformation efficiency of Hevea is comparatively low,and the procedures are still needed to optimize.Finally,breeding objectives and strategies to improve transformation efficiency were also proposed in the review.
ABSTRACT
Embryonic callus of Kentucky bluegrass were used as material for genetic transformation with the DREB1A gene by particle bombardment. Parameters of biolistic bombardment were studied. The optimal methods showed as follows: plasmid DNA are coated by Ca(NO_3)_2 and PEG4000, 1?m golds are the vectors of plasmid DNA, 6 cm bombardment height distance, 1 time and without osmotic treatment are favorable to transgenic efficiency. The concentration hygromycin (Hy) , which is the selection mark for cultivar‘Baron’ is 100mg/L.