• 한국초지조사료학회지
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• 제22권2호
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• pp.101-106
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• 2002

환경 스트레스에 의해 야기되는 활성 산소종에 의한 피해에 내성을 가지는 식물의 개발을 위하여 딸기 유래의 cytosolic ascorbate peroxidase 유전자(ApxSC7)를 Agrobacterium tume-faciens LBA4404를 매개로 형질전환 시켰다. Hygromycin으로 선발된 캘러스로부터 재분화 된 식물체는 야생형과 비교하여 형태적으로 차이를 나타내지 않았다. PCR 및 Southern blot 분석을 통하여 형질전환 식물체의 염색체 내에 ApxSC7 유전자가 integration 되었음을 확인하였다. 담배 잎으로부터 total RNA를 분리하여 Northern blot 분석을 실시한 결과, 도입된 유전자가 형질전환 식물체 내에서 지속적으로 발현된다는 것을 확인하였다.

• 한국초지조사료학회지
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• 제20권2호
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• pp.103-108
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• 2000

To produce of transgenic orchardgrass, the seed-derived calli of orchardgrass (Dactylis glomerata L.) co-cultivated with Agrobacterium turnefaciens EHAlOl harboring binary vector pIG121-Hm were selected with hygromycin and then transferred onto N6 regeneration medium containing 1 rngl l of NAA, 5 rngl l of kinetin, 250 rngl l of carbenicillin and 50 mg/ l of hygromycin. The efficiency of transformation was differed on cultivars, that is, 'Potomac' appeared 12% of transformation efficiency while 'Amba' did 5.5%. The addition of acetosyringone during co-cultivation was a key to successhl transformation of orchardgrass. Transgene fragments were identified by PCR analysis and the constitutive expression of GUS gene was confirmed by Northern blot analysis. (Key words : Acetosyringone, Agrobacterium tumefaciens, Orchardgrass (Dactylis glomerata L.), Transformation)

• Journal of Plant Biotechnology
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• 제36권4호
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• pp.336-343
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• 2009

Tall fescue is an open-pollinated, perennial, cool season grass species widely used for forage and turf. Tremendous progress has been made in genetic transformation of tall fescue in the past decade. Methods for generating transgenic tall fescue plants have been developed based on biolistic transformation and Agrobacterium-mediated transformation. Potentially useful agronomic genes have been tested to environmental stress tolerance, herbicide tolerance and improve forage quality in tall fescue plants. We review progress in biotechnological improvement of tall fescue and discuss future molecular breeding of this species.

• 한국초지조사료학회지
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• 제43권1호
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• pp.42-49
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• 2023

The plant-specific NAC transcription factors control various biological processes, including plant development and stress responses. We have isolated an ANAC032 gene, one of the NAC transcription factor family, which was highly activated by multi-abiotic stresses, including high salt and drought in Arabidopsis. Here, we generated transgenic plants constitutively expressing ANAC032 and its knockout to identify the functional roles of ANAC032 in Arabidopsis under abiotic stress responses. The ANAC032-overexpressing plants showed enhanced tolerance to salinity and drought stresses. The anac032 knockout mutants were observed no significant changes under the high salt and drought conditions. We also monitored the expression of high salt and drought stress-responsive genes in the ANAC032 transgenic plants and anac032 mutant. The ANAC032 overexpression upregulated the expression of stress-responsive genes, RD29A and ERD10, under the stresses. Thus, our data identify that transcription factor ANAC032 plays as an enhancer for salinity and drought tolerance through the upregulation of stress-responsive genes and provides useful genetic traits for generating multi-abiotic stress-tolerant forage crops.

• 한국초지조사료학회지
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• 제29권4호
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• pp.299-306
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• 2009

저온 스트레스에 대한 내성을 지닌 신품종 톨 페스큐를 개발할 목적으로 CaMV35S 프로모터 하류에 NDP kinase 2 유전자가 항상적으로 발현하도록 제작한 벡터를 Agrobacterium법을 이용하여 톨 페스큐에 도입하였다. Hygromycin이 첨가된 선발배지에서 내성을 나타내며 재분화된 형질전환 식물체를 pot로 이식하여 기내순화 시킨 후, Southern blot 분석을 실시하여 본 결과, NDP kinase 2 유전자가 형질전환 식물체의 genome에 정상적으로 도입되었음을 확인하였다. 프로테옴 분석을 통하여 도입유전자의 조절을 받는 항산화관련 유전자들의 발현이 유도되었음을 확인 할 수 있었다. 형질전환 식물체 잎 절편을 산화스트레스 중의 하나인 저온 스트레스를 처리하여 세포의 손상 정도를 조사한 결과, 비형질전환체에 비해 형질전환체는 강한 내성을 나타내었다. 또한 유식물체 수준에서 저온 스트레스를 처리하여 내성을 비교한 결과, 비형질전환체에 비해 형질전환체는 높은 내성을 나타내었다. 이 형질전환 톨페스큐는 산화스트레스 내성 품종개발을 위한 소재로 활용 될 수 있을 것이다.

• 한국초지조사료학회지
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• 제21권3호
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• pp.151-156
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• 2001

내열성 유전자인 BcHSP17.6를 갖도록 제작한 발현벡터 pBKH4를 Agrobacterium tumefaciens LBA 4404에 도입후, Agrobacterium과 알팔파 캘러스의 공배양을 통해 감염시킨 캘러스를 $100{\mu}g/m{\ell}$의 kanamycin과 $500{\mu}g/m{\ell}$의 cefotaxim을 첨가한 SH-kc배지에서 배양하며 형질전환된 캘러스를 선발하였다. 식물체 재분화는 SH- nk-c, SH-sp-c, SH-11b-c, SH-1BA 배지에서 약 4개월간 배양하여 재분화를 완성하였으며, 재분화된 알팔파의 genomic DNA를 분리한 후, PCR 분석 및 Southern blot 분석을 실시하여 알팔파의 형질전환을 확인하였다.

• 한국초지조사료학회지
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• 제20권1호
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)

• 한국초지조사료학회지
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• 제18권1호
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• pp.69-76
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• 1998

This study was conducted to construct of the transgenic plants wliich are resistant to oxidative stresses including ozone with B. mpestris cytosolic glutathione reductase cDNA using the binary vector system of Agrobacterium tumefaciens. The 1.8kb B. campestris cytosolic GR cDNA was subcloned into the unique Sma I site of the plant transformation vector pBKSI- I, downstream of the constitutive CaMV 35s promoter and upstream of the nos termination sequence, in place of the uidA (GUS) reporter gene. The resulting plant transformation vector, pBKS-GRI, was introduced into A. tumefaciens LBA4404 by two cycles of tkeze-thaw method. The B. nqus cotyledonary petioles were transformed by the Agrubaferium harboring pBKS-GRI. Transformed shoots were induced and selected on regeneration medium supplemented with kanarnycin. The shoot formation was increased remarkably by addition of Ag$NO_3$, in MS media. The transgenic plants were analyzed for the presence of the B. campestris GR gene by Southern blot analysis and it was confirmed that a foregin gene was stably integrated into the genomes of B. nqus plants.

• 한국초지조사료학회지
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• 제27권2호
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• pp.109-116
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• 2007

환경스트레스에 강한 내성을 지닌 신품종 톨페스큐를 개발할 목적으로 산화스트레스에 의해 강하게 유도되는 SWPA2 promoter 하류에 CuZnSOD와 APX 유전자가 엽록체에 동시에 발현하도록 제작한 벡터를 Agrobacterium법을 이용하여 톨 페스큐에 도입하였다. Hygromycin이 첨가된 선발배지에서 내성을 가지며 재분화된 형질전환 식물체를 pot로 이식하여 기내 순화시킨 후, Southern 분석을 실시하여 본 결과, 발현벡터의 T-DNA 영역이 형질전환 식물체의 genome에 성공적으로 도입되었음을 확인하였다. 형질전환 식물체 잎 절편을 산화스트레스와 중금속을 포함하고 있는 용액에 처리하여 엽록체의 손상정도를 조사한 결과, 비형질 전환체에 비해 형질전환체는 강한 내성을 나타내었다. 또한 유식물체 수준에서 MV를 처리하여 내성을 비교한 결과, 비형질전환체에 비해 형질전환체는 손상을 덜 받았다. 이와 같은 연구결과는 CuZnSOD와 APX 유전자를 엽록체에 동시발현시키는 기술이 다양한 환경스트레스에 대해 복합재해내성을 가지는 다양한 작물을 개발하는데 유용하게 이용될 수 있음을 나타낸 결과이다.

• Asian-Australasian Journal of Animal Sciences
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• 제17권10호
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• pp.1390-1394
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• 2004

We have achieved efficient transformation system for forage-type tall fescue plants by Agrobacterium tumefaciens. Mature seed-derived embryogenic calli were infected and co-cultivated with each of three A. tumefaciens strains, all of which harbored a standard binary vector pIG121Hm encoding the neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) and intron-containing $\beta$-glucuronidase (intron-GUS) genes in the T-DNA region. Transformation efficiency was influenced by the A. tumefaciens strain, addition of the phenolic compound acetosyringone and duration of vacuum treatment. Of the three A. tumefaciens strains tested, EHA101/pIG121Hm was found to be most effective followed by GV3101/pIG121Hm and LBA4404/pIG121Hm for transient GUS expression after 3 days co-cultivation. Inclusion of 100 $\mu$M acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression observed in targeted calli. Vacuum treatment during infection of calli with A. tumefaciens strains increased transformation efficiency. The highest stable transformation efficiency of transgenic plants was obtained when mature seed-derived calli infected with A. tumefaciens EHA101/pIG121Hm in the presence of 100 $\mu$M acetosyringone and vacuum treatment for 30 min. Southern blot analysis indicated integration of the transgene into the genome of tall fescue. The transformation system developed in this study would be useful for Agrobacterium-mediated genetic transformation of tall fescue plants with genes of agronomic importance.