Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

Abstract

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p < 0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD 600 concentration of 0.6 for 30 min, and co-cultivated with acetosyringone 100 µM for 5 days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.

Optimizing the potential utilization of bioreactors for the mass propagation of Indonesian Dendrobium varieties

“…Banana extract (BE) noted had optimal effect on regeneration and preparation of well rooted shoots (Nurfadilah et al, 2018;Sidhu and Zafar, 2018;Novita et al, 2022) with 50 g L -1 for Cymbidium pendulum (Kaur and Bhutani, 2012), 100 ml L -1 for Dendrobium sp. (Islam et al, 2016), 150 g L -1 for D. lasianthera (Utami et al, 2016), and 100 g L -1 for D. lineale (Mustika and Semiarti, 2021). These results confirmed that AC, AA, PVP, Glutamine, and BE had high potential applied in establishing in vitro protocol for D. Syifa Agrihorti and D. Dian Agrihorti dealing with browning and EC regeneration.…”

Application of CRISPR/Cas9 genome editing system for molecular breeding of orchids

“…In this result the transformation efficiency was low (Table 2), it might be due to the low efficiency in T DNA integration into the plant genome. Our previous study in Dendrobium phalaenopsis, efficiency transfor mation reached until 12% (Setiari et al 2018) and Utami et al (2018) got high transformation efficiency in D. lasianthera that the transformation efficiency up to 70%. This might be caused by specific character of each species related to their own genetic make up (Men et al 2003).…”

“…Transformation efficiency in this study were lower com pared to the previous study. In Dendrobium phalaenopsis, transformation efficiency reached until 12% (Setiari et al 2018) and D. lasianthera up to 70% (Utami et al 2018). This might be caused by each species has their own ge netic make up (Men et al 2003).…”

“…This further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process in account for the susceptibility of plant cells to Agrobacterium infec tion as well as the identification of plant genes involved in the transformation process (Tie et al 2012). Utami et al (2018) used the five different infection period of Agrobac terium for another purpose. According to our result, futher research is necessary to increase mutation frequency for mutant propagation.…”

Establishment and validation of a callus tissue transformation system for German chamomile (Matricaria chamomilla L.)

“…When the co-culture time is too long, overgrowth of A. tumefaciens may occur, which greatly reduces plant viability and transformation efficiency. Utami et al found that the highest infestation efficiency was achieved when the explants were co-cultured with the strain for 5 days [ 30 ]; the lowest infestation efficiency was achieved when the explants were co-cultured with the strain for 1 day. In this study, the optimum co-culture time of German chamomile was 3 days, and the transformation effect was the best and transformation rate was the highest.…”