A protocol for efficient transformation and regeneration of Carica papaya L.

Abstract

A reproducible and effective biolistic method for transforming papaya (Carica papaya L.) was developed with a transformation-regeneration system that targeted a thin layer of embryogenic tissue. The key factors in this protocol included: 1) spreading of young somatic embryo tissue that arose directly from excised immature zygotic embryos, followed by another spreading of the actively growing embryogenic tissue 3 d before biolistic transformation; 2) removal of kanamycin selection from all subsequent steps after kanamycin-resistant clusters were first isolated from induction media containing kanamycin; 3) transfer of embryos with finger-like extensions to maturation medium; and 4) transferring explants from germination to the root development medium only after the explants had elongating root initials, had at least two green true leaves, and were about 0.5 to 1.0 cm tall. A total of 83 transgenic papaya lines expressing the nontranslatable coat protein gene of papaya ringspot virus (PRSV) were obtained from somatic embryo clusters that originated from 63 immature zygotic embryos. The transformation efficiency was very high: 100% of the bombarded plates produced transgenic plants. This also represents an average of 55 transgenic lines per gram fresh weight, or 1.3 transgenic lines per embryo cluster that was spread. We validated this procedure in our laboratory by visiting researchers who did four independent projects to transform seven papaya cuhivars with coat protein gene constructs of PRSV strains from four different countries. The method is described in detail and should be useful for the routine transformation and regeneration of papaya.

Transgenic plants of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. ex Steud., from microprojectile bombardment of highly chlorophyllous embryogenic cells

“…In our case, however, we found kanamycin to be a very efficient selectable agent at moderately high levels for B. gracilis. This concentration of kanamycin is comparable to that utilized previously in some dicots such as papaya (Cai et al 1999) or tobacco (Holmström et al 2000). Studies with other morphogenic callus lines of B. gracilis (Aguado-Santacruz et al 2001a) have also shown the efficiency of the aminoglycoside antibiotic, paramomycin, as a selective agent, at similar concentrations to those reported in this work for kanamycin (150 mg/l).…”

Etiologia e estratégias de controle de viroses do mamoeiro no brasil

“…Esta estratégia de controle abriu uma nova possibilidade para solução do problema da mancha anelar, de forma mais eficiente e possivelmente mais duradoura (Fitch et al, 1992;Tennant, 1996;Cai et al, 1999;Souza Jr., 1999). No entanto, quando desafiadas por isolados de outras regiões geográficas, inclusive do Brasil, as plantas da Linha 55-1 apresentaram-se susceptíveis à infecção por PRSV-P (Tennant et al, 1994).…”

Recent Advances in Fruit Species Transformation

“…The PRSV resistant papaya has been commercialized, reached to end user and improved papaya is now under production in Hawaii (Tripathi et al, 2008). This study was followed by other reports mainly on improvement of PRSV tolerance in papaya via microprojectile bombardment-based transformation (Tennant et al, 1994;Cai et al, 1999;Guzman-Gonzalez et al, 2006). The deployment of transgenic papayas has showed that virus CP protein supplies durable and stable resistance to homologous strains of PRSV (Fermin et al, 2010).…”