Somatic Embryogenesis and Genetic Transformation in Pinus radiata

Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

“…Embryo development medium (EDM) (Walter et al 1998a) with a combination of 4.5 lM 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma-Aldrich Co., USA) and 2.7 lM 6-benzylaminopurine (BAP, Sigma) was used for initiation. Before sterilization, the pH of the medium was adjusted to 5.7 and then 3 g L -1 gellan gum (GelriteÒ, Duchefa Biochemie B.V., The Netherlands) were added.…”

Detection of asymptomatic fungal microorganisms in Pinus radiata tissue culture material

“…Ten different genotypes were used for the embryogenic tissue and another 10 for the mature organogenesis shoots so 30 different genotypes were tested in total. Embryogenic tissue was initiated using techniques described by Walter and Grace (2000) and Walter et al (2005). The adventitious-axillary organogenesis shoots were prepared from excised cotyledons that were cryopreserved at − 196°C and then had shoots initiated adventitiously.…”

Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants

“…The copy number assessment is particularly important with the use of direct DNA delivery transformation methods such as biolistic technologies which result in very high copy number of the introduced gene and also in fragmented copies integrated into the genome (Walter et al 2005). Transgenic plants with a single transgene copy are usually preferred because multiple copies often bring on undesirable effects such as gene silencing (Matzke and Matzke 1995;Kumpatla et al 1997).…”

“…Transgenic plants with a single transgene copy are usually preferred because multiple copies often bring on undesirable effects such as gene silencing (Matzke and Matzke 1995;Kumpatla et al 1997). The issue of gene silencing and gene arrangement based on multiple and fragmented copies appears of great importance to genetic engineering in trees, since in these organisms, transgene is harbored by only one of the two allelic chromosomes and is expected to be expressed correctly and to remain unmodified over a period of 20 years or more (Walter et al 2005). This condition becomes permanent, given that no further manipulation such as crossing or selffertilisation are viable approaches for obtaining homozygous single-copy clones (the genotypes usually selected for commercialisation) due to long juvenile period, high level of heterozygosity leading to inbreeding depression and to self-incompatibility and sterility phenomena.…”