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

Roh, Hee Sun; Kim, Jong Bo

doi:10.5010/jpb.2014.41.4.216

Cited by 2 publications

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“…To use particle bombardment method for the production of transgenic Alstroemeria plants, size and volumes of gold particle and the number of shooting are important factors (Morrish et al 1993). Furthermore, pre-treatment of osmoticum can be influenced on transformation efficiency (Watad et al 1998) and effects of osmoticum treatments were tested in Corn (Vain et al 1993), Lily (Kim 2017), Oncidium (Li et al 2005) and Phalaenopsis (Roh and Kim 2014). Therefore, factors influence on transformation efficiency in particle bombardment should be optimized to develop elite transgenic Alstroemeria plants which can be used for further breeding program.…”

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confidence: 99%

An efficient protocol for the production of transgenic Alstroemeria plants via particle bombardment

Kim

2020

J Plant Biotechnol

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Alstroemeria plants were transformed by using an improved particle-gun-mediated transformation system. Friable embryogenic callus (FEC) induced from the leaves with axil tissues of Alstroemeria plant was used as the target tissue. Also, FEC was transformed with the bar gene was used as a selectable marker. In the case of plasmid pAHC25, 7.5% of the twice-bombarded FEC clumps showed blue foci, whereas the clumps with single bombardment showed only 2.3%. Additionally, a 90° rotation with double bombardment led to a higher frequency (6 times) of luciferase gene expression in PBL9780 than the control treatment. After 8 weeks of bombardment, more than 60 independent transgenic lines were obtained for pAHC25 and nearly 150 independent transgenic lines were obtained for PBL9780, all of which were resistant to PPT and demonstrated either GUS or luciferase activity. Regarding effect of osmotic treatment (0.2 M mannitol) with 7 different periods, the highest transient gene expression was obtained in 8 h before and 16 h after transformation in both pAHC25 and PBL9780. Compared with the control, at least three times more GUS foci and photons were observed in this treatment. With respect to different combinations of mannitol and sorbitol with 8 h before and 16 h after transformation, high numbers of transient and stable transgene expressions were observed in both 0.2 M mannitol and 0.2 M sorbitol used in the osmotic pre-culture. This combination showed the highest transformation efficiency in both pAHC25 (8.5%) and PBL9780 (14.5%). In the control treatment, only 10% of the FEC clumps produced somatic embryos. However, by using 0.2 M mannitol and 0.2 M sorbitol, the frequency of somatic embryos increased to 36.5% (pAHC25) and 22.9% (PBL9780). Of the somatic embryos produced, at least 60% germinated. Approximately 100 somatic embryos from these 210 independent transgenic lines from 2 plasmids developed into shoots, which were then transferred to the greenhouse. PCR analysis confirmed the presence of the bar gene. This is the report on the production of transgenic Alstroemeria plants by using particle bombardment with a high efficiency, thereby providing a new alternative for the transferring of gene of interests in Alstroemeria in the breeding program in the future.

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Section: Introductionmentioning

confidence: 99%

An efficient protocol for the production of transgenic Alstroemeria plants via particle bombardment

Kim

2020

J Plant Biotechnol

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“…한편 유전자총을 이용한 형질전환 나리식물체 개발에 관한 연구에서는 1998년 최초로 Watad et al (1998) (Hoshino et al 2008;Kim et al 2007;Lin et al 2000), 히야신스 (Popowich et al 2007), 심비디움 (Roh et al 2011 and2013), 팔레놉시스 (Roh et al 2014)나 튤립 (Wilmink et al 1992) 등과 비교하면 형질전환연 구가 비교적 많이 수행되었음을 알 수 있다. 나리 형질전환 연구에 많이 사용된 아그로박테리움 방법은 비교적 안정성 과 재현성이 높고, 형질전환 시 유전자 copy수가 낮게 들어 가는 장점이 있지만 (Hiei et al 1997), 숙주식물에 제한이 있 고 박테리아 배양 및 공동배양에 시일이 오래 걸리는 단점 도 있다 (Hiei et al 1994).…”

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“…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"…”

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

Kim

2017

J Plant Biotechnol

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This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Abstract Transgenic lily plants have been obtained after particle bombardment, using PDS-1000/He system and scale explants of lilies, followed by PPT (D-L-phosphinothricin) selection. In this study, scales of the lily plants cv. 'red flame' were bombarded with a plasmid containing the bar gene as a selectable marker, and the AtSIZ gene as a gene of interest, showing salt tolerance and drought tolerance respectively, and both being driven by the CaMV 35S promoter. For optimization of a protocol, factors which optimized and showed a high transformation efficiency under following conditions, were considered: a bombardment pressure of 1100 psi, a target distance of 6 cm and 1.0 µm of gold particle, and 24-h pre-culture and post-culture on MS medium containing 0.2 M sorbitol and 0.2 M mannitol as osmoticum agents. After bombardment, all the bombarded scales of lily were transferred to MS medium without selective agents, for a week. Subsequently, these bombarded scales were transferred to a selection MS medium containing 10 mg/l PPT, and incubated for a month for further selection, after which they were cultured for another 4-8 weeks with a 4-week subculture regime on the same selection medium. After transferring into hormone-free MS medium, the PPT-resistant scales with shoots were successfully rooted and regenerated into plantlets. PCR analysis revealed that the surviving putatively transformed plantlets indicated the presence of both the bar gene and the AtSIZ gene. In conclusion, when 100 scales of lily cv. Red flame are bombarded, this study produced approximately 17-18 transgenic plantlets with an optimized bombardment protocol. The protocol described here can contribute to the breeding program of lilies.

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

Cited by 2 publications

References 12 publications

An efficient protocol for the production of transgenic Alstroemeria plants via particle bombardment

An efficient protocol for the production of transgenic Alstroemeria plants via particle bombardment

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

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