Agrobacterium-mediated transformation of the wild orchid Cattieya maxima Lindi / Transformación mediada por Agrobacterium de la orquídea silvestre Cattleya maxima Lindl / Transformação da orquídea silvestre Cattleya maxima Lindl mediada por Agrobacterium

Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.

Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.

Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.

Efficient regeneration and genetic transformation platform applicable to five Musa varieties

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.

Endogenous GUS-like activity in Capsicum chinense Jacq

The gene uidA, codes for beta-glucuronidase, which is one of the reporters more frequently utilized in transgenic plants. However, this can only be use if the selected organism does not present endogenous GUS-like activity. In tissues of C. chinense we found a GUS-like activity showing different levels of intensity. Histochemical screening showed that endogenous GUS-like activity decreased, or reduced significantly, in almost all tissues with exception of stament, when phosphate buffer was adjusted to pH 8. Subsequently, C. chinense zygotic embryo explants were transient transformed with Agrobacterium tumefaciens LBA4404 (pCAMBIA2301) and plantlets regenerated were histochemically stained in phosphate buffer pH 8. Observations of incubated tissues of C. chinense regenerants showed blue staining, suggesting expression of uidA. Incubated tissues of non-transformed regenerants did not show blue staining in phosphate buffer pH 8. The results show that for transformation experiments of C. chinense with uidA gene, pH 8 is recommended for histochemical staining.

Genotypic variation of Agrobacterium-mediated transformation of Italian ryegrass

In the present study, genotypic variation of Agrobacterium-mediated transformation of Korean Italian ryegrass has been evaluated. Mature seed-derived calli of seven cultivars were inoculated and co-cultured with Agrobacterium tumefaciens carrying the binary vector pCAMBIA1301, which contains a reporter gene (gus) and a plant selectable marker gene conferring resistance to hygromycin (hpt) in the T-DNA region. The effects of several factors such as callus type and callus age on transformation effectiveness and the expression of the GUS gene were investigated. The highest transformation effectiveness (6.7 percent) was obtained with the Hwasan 101 cultivar when 9-week-old calli (type-I) were inoculated with Agrobacterium. The overall transformation rates of the examined cultivars ranged from 0.4 percent to 6.7 percent. GUS histochemical assays, PCR, and southern analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of Italian ryegrass. Thus, evaluation of transformation effectiveness and selection of a suitable cultivar of Italian ryegrass may improve molecular breeding of this species.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus / Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.

Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens.

Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer. Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia. Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium. A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation. Transformed tissue was selected by the ability to grow on kanamycin containing medium. Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue. This transformation procedure has the potential to expand the range of genetic variation in Robinia.