Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium

Abstract

Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby 'OM8' (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3-to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and b-glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40 days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.Abbreviations: 35S promoter -cauliflower mosaic virus 35S promoter; GUS -b-glucuronidase; hpthygromycin phosphotransferase gene; nos terminator -terminator of nopaline synthase gene; pflp -pepper ferredoxin-like protein sequence; PLB -protocorm-like body; uidA -b-glucuronidase gene

Optimization of a protocol for the production of transgenic lily plants via particle bombardment

“…유전자총 실험 조건 역시 형질전환 효율과 관련이 있는데, 입자의 종류, 크기와 양 그 리고 입자속도와 입자에 코팅되는 외래 DNA 양 및 발사횟 수 등이 주요 요인이다 (Morrish et al 1993). 또한 유전자총 발 사 전후에 삼투압조절제 등의 전처리 여부도 형질전환 효 율에 영향을 미치는데 (Watad et al 1998), 옥수수 (Vain et al 1993), 온시디움 (Li et al 2005), peanut (Deng et al 2001) 및 팔 레놉시스 (Roh et al 2014 Table 1 Effect of size of gold particle during the particle bombardment on transformation efficiency in scales of Lilium longiflorum "red flame"…”

Effects of osmoticum treatments and shooting chances on the improvement of particle gun-mediated transformation in Phalaenopsis

“…그러나 대부분 아그로박테리 움을 이용한 논문이고 10여 편 중 유전자총을 이용한 연 구는 3편에 불과하다 (Roh et al 2011). (Denchev et al 1997), 옥수수 (Vain et al 1993), peanut (Deng et al 2001)과 난과식물에서는 온시디움 (Li et al 2005 …”

Subsídios à transformação genética de plantas de Catasetum pileatum (Orchidaceae) por meio de tecidos merismáticos radiculares e caulinares

“…Oncidium (You et al, 2003;Liau et al, 2003aLiau et al, e 2003bLi et al, 2005), Brassia, Cattleya e Doritaenopsis (Knapp et al, 2000) e Calanthe (Griesbach, 1994). As publicações e seus principais atributos estão listados na tabela 1.…”

“…Entretanto, além destas estruturas serem de difícil obtenção, principalmente em orquídeas, a grande necessidade de utilização de fitohormônios e a manipulação de cultura de tecidos requerida aumentam as chances de problemas de alteração somaclonal (Phillips et al, 1994;Mishiba et al, 2005;Li et al, 2005).…”

“…A utilização de compostos fenólicos tais como a acetossiringona (AS), que mimetizam o processo dos indutores de genes de virulência, também tem se mostrado eficiente e geralmente necessário para a reprodutibilidade dos protocolos, sendo adicionada nas etapas de co-cultivo e até mesmo antes, durante o crescimento da bactéria e/ou pré-indução do material vegetal (You et al, 2003;Liau et al, 2003a;Mishiba et al, 2005b (Kuehnle & Sugii, 1992;Yu et al, 1999;Liau et al, 2004;Li et al, 2005). (Chia et al, 1994), estudos posteriores buscaram o refinamento da seleção de orquídeas por este antibiótico (Nan & Kuehnle, 1995;Yang et al, 1999;Yu et al, 2000Yu et al, e 2001 (Chia et al, 1994;Yu et al, 1999), a seleção por higromicina mostrouse mais eficiente e decisiva para a devida discriminação de tecidos transformados, sendo desta forma, utilizado na maioria dos trabalhos a partir de 1999 (Yu et al, 1999;Belarmino & Mii, 2000;Chai et al, 2002;Liau et al, 2003;Men et al, 2003;Chang et al, 2005;Chan et al, 2005;Li et al, 2005;Mishiba et al, 2005;Chin et al, 2007e Suwanaketchanatit et al (2007 …”