Stability of T-DNA Integration in Phalaenopsis “Sogo Vivien” Transgenic Orchid Carrying 35S::Gal4::AtRKD4::GR

Endang Semiarti, Exsyupransia Mursyanti, Ahmad Suyoko, Faiza Senja Widya Perdana, Catharina Tri Widyastuti, Aditya Nur Subchan

Abstract


Orchid is an elegant ornamental plant and favoured by the society. Phalaenopsis "Sogo vivien" is a mini-sized orchid with an interesting white-striped purple petals. This study was aimed to analyze the stability of the integration of embryonic gene carrier T-DNA from Arabidobsis AtRKD4 into the P. "Sogo vivien" genome produced in 2016. The study was conducted in 3 stages: 1) Transgenic plant phenotype analysis (1 year old); 2) Examination of T-DNA integration in orchid genotypes using PCR. 3) Analysis of transgenic plant leaf explants’ ability to produce somatic embryo in vitro. In vitro cultures were performed on the base medium of New Phalaenopsis (NP), plus various concentrations of TDZ (0, 1, 2 mg.L-1) and IBA (0, 1, 2 mg.L-1) or without TDZ and IBA as controls. The transgenic Phalaenopsis ‘Sogo vivien’ were transferred to pot mediums via ex vitro with two treatments: the first leaves were cut as explants for in vitro culture, and the plants were transferred to the mixture of fern medium with shavings of bark. The integration of T-DNA in the genome was detected by DNA genome amplification from the second leaves using the AtRKD4 gene primers and the POH1 gene. The results showed that the highest number of somatic embryo (SE) propagules or protocorm like bodies (PLBs) amounted to 27 were derived from transgenic plant # 2 cultured on NP + 2 mg.L-1 TDZ +1 mg.L-1 IBA medium. The presence of AtRKD4 transgenes were detected with the amplification of 380 bp of the RKD4 gene from the genome of transgenic plant # 2 by using PCR. There were 2 out of 15 plants that positively carry the AtRKD4 gene and produce SE. Thus, the stability of the AtRKD4 carrier T-DNA integration in the genomes of transgenic plants was 13.3%.

Keywords


Somatic embryo; Phalaenopsis “Sogo vivien”; AtRKD4; stable transgenics

Full Text:

PDF

References


Bajaj, Y. P. S. 2012. Biotechnology in Agriculture and Forestry 47: Transgenic Crops II. Springer. New Delhi. p. 242.

Feng J and Cheng J. 2014. ‘A Novel In Vitro Protocol for Inducing Direct Somatic Embryogenesis in Phalaenopsis aphrodite without Taking Explants’. The Scientific World Journal. vol. 2014, Article ID 263642, 7 pages, 2014. doi:10.1155/2014/263642.

Hutami, S.2008. ‘Masalah pencoklatan pada kultur jaringan.Jurnal Agrobiogen. 4(2).83-88

Kasi, P. D. dan Semiarti E. 2008.’Pengaruh thiadizuron dan naphtalene acetic acid untuk induksi embriogenesis somatik dari daun anggrek Phalaenopsis “Sogo Vivien”. Jurnal Dinamika. 7(1):31-40.

Kim, H. J. Cho, H. S. Pak, J. H. Kim, K. J. Lee, D. H. and Chung, Y. S. 2017. Overexpression of a Chromatin Architecture-Controlling ATPG7 has Positive Effect on Yield Components in Transgenic Soybean. Plant Breed. Biotech. 5(3):237-242.

Kumar A, Neumann K and Sopory S K. 2008. Recent Advances in Plant Biotechnology and its Applications.I.K. International. New Delhi. p. 718.

Koi, S. Hisanaga, T. Sato, K. Shimamura, M. Yamato, K. T. Ishizaki, K. Kohchi, Y. and Nakajima, K. 2016. An evolutionary conserved plant RKD factor controls germ cell differentiation. Curr. Biol. 26(13): 1775-1781.

McKinley, M. 2005. Complete Guide To Orchids: Ortho Books. American Orchid Society. USA. p.137.

Mursyanti, E., Aziz-Purwantoro, S. Moeljopawiro and E. Semiarti. 2015. Induction of Somatic Embryogenesis through Overexpression of AtRKD4 Genes in Phalaenopsis ‘Sogo Vivien’. Indonesian Journal of Biotechnology. 20(1): 26-37.

Mursyanti, E., Aziz-Purwantoro, S. Moeljopawiro and E. Semiarti. 2016. Micropropagation of Mini Orchid Hybrid Phalaenopsis ‘Sogo Vivien’, Journal of Tropical Biodiversity and Biotechnology. 1(1): 45-53.

Ravanfar, S. A. Aziz, M. A. Shabanimofrad, M. and Samarfard, S. 2013. Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers. Electronic Journal of Biotechnology. 16(5): 1-4.

Semiarti E., Indrianto A., Purwantoro A., Isminingsih S., Suseno, N., Ishikawa T., Yoshioka Y., Machida Y.,and Machida C. 2007. Agrobacterium-mediated transformation of the wild orchid species Phlaenopsis amabilis. Plant Biotechnology 24: 265-272.

Sivanesan I, Hwang SJ and Jeong B R. 2008. ‘Influence of plant growth regulators on axillary shoot multiplication and iron source on growth of Scrophularia takesimensis Nakai- a rare enemic medicinal plant’.African Journal of Biotechnology. 7(24):4484-4490.

Sukumar E, Essa M M and Manickavasagan A. 2012. Dates: Production, Processing, Food and Medicinal Values. CRC Press. New York. P. 361.

Vendrame, W. A. Maguire, I. and Carvalho, V. S. 2007. In vitro Propagation and Plantlet Regeneration from Doritaenopsis Purple Gem ‘Ching Hua’ Flower Explants. Hort. Science. 42:1256-1258.

Waki, T., T. Hiki, R. Watanabe, T. Hashimoto & K. Nakajima. 2011. The Arabidopsis RWP-RK protein

RKD4 triggers gene expression and pattern formation in early embryogenesis. Current Biology. 21(15): 1277-1281.




DOI: https://doi.org/10.14421/biomedich.2018.71.5-13

Refbacks

  • There are currently no refbacks.




Copyright (c) 2018 Endang Semiarti, Exsyupransia Mursyanti, Ahmad Suyoko, Faiza Senja Widya Perdana, Catharina Tri Widyastuti, Aditya Nur Subchan



Biology, Medicine, & Natural Product Chemistry

ISSN 2089-6514 (paper) - ISSN 2540-9328 (online)
Sunan Kalijaga State Islamic University &
Society for Indonesian Biodiversity

Creative Commons License
This work is licensed under a CC BY-NC