Agrobacterium Tumefaciens EHA105 Research Articles

Optimization of Agrobacterium tumefaciens-Mediated Transformation Systems in Tea Plant (Camellia sinensis)

In this study, an efficient plant regeneration protocol in vitro and transformation by Agrobacterium-mediated method of Camellia sinensis was achieved, which would lay the foundation for genetic improvement of tea plant by genetic engineering technology. The cotyledon callus of C. sinensis were used as the receptors for transformation by Agrobacterium tumefaciens EHA105 containing PS1aG-3. Some factors which affected the result of Agrobacterium-mediated transformation of C. sinensis were studied on the basis of GUS transient expression system. The optimum system of Agrobacterium-mediated transformation was that the cotyledon callus were pre-cultured for 3 d, and then infected by EHA105 for 15 min followed by 3 d co-culture in the dark on the YEB medium containing 150 µmol⋅L−1 acetosyringone (AS). The transient expression rate of GUS gene was 62.6%. After being delayed selective culture for 3 d, infected callus were transferred into the differentiation medium and the root induction medium both of which were supplemented with 100 mg⋅L−1 spectinomycin, and then resistant seedlings of C. sinensis were obtained. The conversion rate was 3.6%.

벼에서 밀 고분자 글루테닌 단백질(TaGlu-Ax1) 발현을 통하여 쌀가루 가공적성 증진을 위한 마커프리(marker-free) 형질전환 벼의 개발

밀의 고분자 글루테닌 서브유닛[high molecular-weight glutenin subunit (HMW-GS)]은 밀가루의 성질을 결정하는데 가장 중요한 요소이며 가공적성을 나타내는데 중요한 역할을 수행한다. 우리는 Agrobacterium 동시 형질전환법을 이용하여 한국 밀 품종인 ‘조경’으로부터 밀 HMW-GS을 암호화하는 TaGlu-Ax1 유전자를 가지는 marker-free 형질전환 벼를 생산하였다. TaGlu-Ax1 유전자의 종자 특이적 발현을 위하여 밀에서 존재하는 TaGlu-Bx7 유전자의 자체 프로모터를 벡터 내에 삽입하였다. 동시 접종을 위해서 오직 TaGlu-Ax1 유전자와 hygromycin phosphotransferase II (HPTII) 저항성 유전자만으로 구성된 두 종류의 발현 카세트를 독립적으로 Agrobacterium EHA105에 도입하였고, TaGlu-Ax1와 HPTII가 도입된 각각의 EHA105 Agrobacterium을 3:1 비율로 혼합하여 벼 캘러스에 접종하였다. 210개의 HPTII 저항성 형질전환체 중에서 벼 게놈에 TaGlu-Ax1과 HPTII가 모두 삽입된 20개의 형질전환 라인을 획득하였다. TaGlu-Ax1와 HPTII가 벼 게놈에 도입된 것을 Southern blot을 통해서 다시 확인하였다. 형질전환 벼 T₁ 세대의 종자에서 밀 TaGlu-Ax1 유전자가 전사와 번역되어 오직 TaGlu-Ax1만을 가지는 marker-free 식물체를 T₁세대에서 성공적으로 선발할 수 있었다. TaGlu-Ax1 유전자가 발현되는 marker-free 형질전환 식물체는 야생형(wild type)과의 표현형 차이는 없었다. 형질전환 벼의 쌀가루의 제빵적성을 비교하였을 때 TaGlu-Ax1 유전자만이 발현되어서는 제빵적성이 더 나아지지 않았다. 그러므로 더 많은 밀 고분자 및 저분자 글루테닌, 글리아딘의 유전자의 집적과 조합이 쌀가루 가공적성을 증진시키는데 필요하다. 결론적으로 TaGlu-Ax1 marker-free 형질전환 벼는 쌀가루 가공적성을 증진시키는데 좋은 재료로 사용될 것이다. High-molecular-weight glutenin subunits (HMW-GSs) are extremely important determinants of the functional properties of wheat dough. Transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding a HMG-GS were produced from the Korean wheat cultivar ‘Jokyeong’ and used to enhance the bread-making quality of rice dough using the Agrobacterium-mediated co-transformation method. Two expression cassettes with separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately into the Agrobacterium tumefaciens EHA105 strain for co-infection. Rice calli were infected with each EHA105 strain harboring TaGlu-Ax1 or HPTII at a 3:1 ratio of TaGlu-Ax1 and HPTII. Among 210 hygromycin-resistant T₀ plants, 20 transgenic lines harboring both the TaGlu-Ax1 and HPTII genes in the rice genome were obtained. The integration of the TaGlu-Ax1 gene into the rice genome was reconfirmed by Southern blot analysis. The transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in rice T₁ seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened in the T₁ generation. There were no morphological differences between the wild-type and marker-free transgenic plants. The quality of only one HMW-GS (TaGlu-Ax1) was unsuitable for bread making using transgenic rice dough. Greater numbers and combinations of HMW and LMW-GSs and gliadins of wheat are required to further improve the processing qualities of rice dough. TaGlu-Ax1 marker-free transgenic plants could provide good materials to make transgenic rice with improved bread-making qualities.

English

Jatropha curcas L. oil has been shown to be suitable for the production of biodiesel. However, this species has not been domesticated yet. Genetic breeding through conventional methods is time consuming and costly, hence, genetic transformation could contribute positively to the improvement of interesting traits. Although in vitro regeneration and stable genetic transformation has been pursued for several years, variation in transformation efficiency remains strongly genotype-dependent and indicates that protocols optimization is still needed. Thus, this study was carried out to introduce a chitinase gene from the Trichoderma viride fungus into the genome of a J. curcas superior genotype by inoculating leaf explants with Agrobacterium tumefaciens EHA 105 strain in axenic conditions. Some key parameters such as pre-culture period and antibiotic doses were optimized with 500 mg.L-1 cefotaxime and 100 mg.L-1 kanamycin concentrations being suitable for A. tumefaciens inhibition and explant selection, respectively. The best transformation efficiency (50%) was obtained when leaf explants were incubated on a culture medium promoting shoot regeneration at 15 days before the induction of the transformation process. Plants where chitinase gene amplicons could be detected were assessed for transgene copy number and expression levels by quantitative real-time polymerase chain reaction (PCR). One, two and three copies of the introduced gene were confirmed in nine transgenic lines with two of them that were assessed for gene expression and showed quantitative variation for this variable. These results bring valuable information for further gene insertions in breeding programs of J. curcas for fungal disease resistance.

Agrobacterium-mediated transformation of thin cell layer explants of Scutellaria ocmulgee small: a rare plant with anti-tumor properties

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shoot-derived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6- benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 µM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the β glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Plant tissue culture independent Agrobacterium tumefaciens mediated In-planta transformation strategy for upland cotton (Gossypium hirsutum).

A new method of transgenic development called “In-planta” transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.

Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80%, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination.

Efficient Agrobacterium-mediated transformation of Artemisia annua L. using young inflorescence

Artemisia annua L. is currently the only practical source of artemisinin, an effective antimalarial agent. Development of an efficient gene delivery system is crucial for genetic manipulation of this medicinal plant to enhance artemisinin production. Here, we report a protocol for efficient Agrobacterium-mediated transformation and plant regeneration of A. annua using young inflorescence as explants. We evaluated the influence of explant types and Agrobacterium strains on transformation and regeneration efficiency. Young inflorescence and leaf explants were co-cultivated with Agrobacterium tumefaciens strains, EHA105 or LBA4404, carrying the pCAMBIA2301 binary vector containing a s-glucuronidase reporter. Shoot regeneration was observed when explants were incubated on modified Murashige and Skoog (MS) medium supplemented with 0.5 mg L−1 6-benzylaminopurine and 0.3 mg L−1 α-naphthalene acetic acid. Shoot bud development from young inflorescence explants was observed within 7 wk of culture whereas those from the leaf explants regenerated after 9 wk. PCR and Southern blot analyses showed that 95% of the putative transgenic lines were positive for the GUS reporter gene. The maximum transformation frequency of 17% was achieved by co-cultivation of A. tumefaciens strain EHA105 with young inflorescence, representing a twofold improvement compared to using leaf explants. Molecular and genetic analysis of T1 progeny further confirmed the inheritance of the introduced gene. The transformation protocol using young inflorescence of A. annua reduces the transformation time and increases the efficiency; hence, this represents a practical platform for metabolic engineering of this important medicinal plant.

Production of herbicide-resistant medicinal plant Salvia miltiorrhiza transformed with the bar gene.

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections.

Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

Cloning and Genetic Transformation of CnCHS Gene from Camellia nitidissima

Using total cDNA from the petals of Camellia nitidissima as template,we amplified the full length chalcone synthase( CHS) cDNA of C. nitidissima successfully by the specific primers designed according to the CnCHS sequence in genbank. The amplified fragment of Cn CHS was inserted into PMD18-T vector and then transformed into E. coli DH5α. After the compounded plasmid was identified by enzyme digestion and sequencing,it was digested and connected to the expression vector pCAMBIA1300. The compounded plasmid pCAM-CnCHS was identified and the sense expression vector of CnCHS was constructed successfully. Then,the pCAM-CnCHS was transformed into Agrobacterium tumefaciens EHA105 for plant infections,and 18 transgenic tobaccos containing CnCHS were obtained. The transgenic plants were then identified by PCR and Southern blotting,and the transgenic tobaccos which contain single copy of CnCHS gene were obtained with the positive rate of 67%. The genetic transformation system of CnCHS to tobaccos was constructed successfully,which would lay the foundation for further function research and regulation effects on flower color of CnCHS.

Micropropagation and DNA delivery studies in onion cultivars of Bellary, CO3

Onion is one of the most important vegetables grown all over the world. In the present study, rapidly growing and maintainable callus was accomplished using the basal meristem plate and twin scale leaves as explants in Bellary and CO3 varieties for callus induction and in vitro bulb formation. The two cultivars were transformed with Agrobacterium tumefaciens EHA 105 harboring the binary vector pCAMBIA 1301 containing the GUS gene with different concentrations of acetosyringone. The outcome of this study revealed that the frequency of callus induction was maximum on Murashige and Skoog medium (MS) fortified with B5 vitamins supplemented with 0.5 and 1 mg L−1 picloram. Regeneration of plantlets from the callus was observed on MS supplemented with 0.5 mg L−1 each of BAP and KIN and 0.1 mg L−1 NAA. In vitro bulb response was observed on MS with B5 vitamins supplemented with 2.0 mg L−1 BAP. GUS gene integration in the transgenic plants was confirmed by PCR. The maximum GUS gene expression was observed at a concentration of 150 µM acetosyringone in the transformed plants by histochemical assay. This protocol allows effective genetic transformation and regeneration system of onion via in vitro bulb development.

In vitro propagation and preliminary results of Agrobacterium-mediated genetic transformation of Cordyline fruticosa

A method for in vitro propagation through axillary shoot proliferation and induction of adventitious shoots as well as Agrobacterium-mediated transformation of Cordyline fruticosa is presented. In vitro Cordyline shoots were cultured on MS medium supplemented 0–4mgl−1 thidiazuron (TDZ) for their multiplication. MS medium supplemented with 0.1mgl−1 TDZ proved to be the most effective in which 14 shoots per explants were obtained. Leaf segments were excised from in vitro axillary shoots and cultured on MS medium supplemented with TDZ (0–5mgl−1) for callus induction. Following induction, the non-embryogenic calluses were proliferated on MS medium without plant growth regulators. A rapid and efficient method for producing transgenic plants has been developed for Cordyline via Agrobacterium-mediated transformation. Leaf segments from in vitro shoots were co-cultivated with Agrobacterium tumefaciens EHA105, which harbored the binary vector carrying the bar and β-glucuronidase (GUS) genes in the T-DNA region. The results showed that the number of plants expressing GUS gene first increased with inoculation time and bacterial density (OD600), and then dramatically decreased with the increment of both factors. The highest percentage of transformation efficiency (85%) was obtained when the leaf segment explants were inoculated with Agrobacterium for up to 40min at OD600 of 1.0 followed by 80 and 75% at OD600 of 0.8 and 1.2, respectively. The transgenes were confirmed in transgenic plants using GUS primers and gave the expected band size of 750bp. The results suggested that infection time and Agrobacterium density could have some effects on the transformation efficiency. Southern blot analysis was used for definitive confirmation of tDNA integration in Cordyline and confirmed the results of histochemical GUS assay and PCR amplifications. Regenerated plantlets were acclimatized in greenhouse.

Root and shoot parts of strawberry: factories for production of functional human pro-insulin.

Diabetes, a disease caused by excessive blood sugar, is caused by the lack of insulin. For commercial production, insulin is made in bacteria or yeast by protein recombinant technology. The focus of this research is evaluating another resource and producing of recombinant insulin protein in as strawberry as this plant has high potential in production of pharmaceutical proteins. Strawberry is a suitable bioreactor for production of recombinant proteins especially edible vaccines. In this research, human pro-insulin gene was cloned in pCAMBIA1304 vector under CaMV35S promoter and NOS terminator. Agrobacterium tumefaciens LBA4404, AGL1, EHA105, EHA101, C58, C58 (pGV2260) and C58 (pGV3101) strains were used for transformation of pro-insulin gene into strawberry cv. Camarosa, Selva, Sarian Hybrid, Pajaro, Paros, Gaviota, Alpine. Additionally, Agrobacterium rhizogenes K599, R1000, A4 and MSU440 strains were utilized for gene transformation into hairy roots. PCR analysis indicated the presence of transformed human pro-insulin gene in the strawberry and hairy roots. Also, its transcription was confirmed using RT-PCR. Furthermore, the analysis of plants, fruits and hairy roots at the level of proteins using dot blot, ELISA, SDS-PAGE and ECL tests re-confirmed the expression of this protein in the transgenic plants as well as hairy roots. Protein purification of human pro-insulin from transgenic tissues was performed using affinity chromatography. Finally, the bioassay of recombinant pro-insulin was performed. The analysis of second generations of transgenic plants (T1) at DNA and protein levels was also performed as a complementary experiment. This study opens a new avenue in molecular farming of human pro-insulin through its mass production in roots and shoots of strawberry.

Transient expression of siRNA targeted against the TYLCV AV1, AC1 and AC3 genes for high resistance in tomato

Tomato yellow leaf curl virus (TYLCV) is a globally devastating disease that affects the production of many crops, particularly tomato, leading to considerable economic losses. RNA interference, which allows sequence-specific gene silencing at the post-transcriptional level, is an effective method to obtain virus-resistant lines of crops. Agrobacterium-mediated transient expression assay has emerged as a rapid and useful approach to evaluate gene functions in plants without the need to produce transgenic lines. In this study, we transformed the plant expression vector pBIN438-AV1–AC1–AC3(i/r), which consists of the inverted repeat of ΔAV1, ΔAC1, and ΔAC3 fusion fragments into Agrobacterium tumefaciens EHA105 to generate tomato lines with high viral resistance. The viral resistance of the fusion gene was evaluated by transient expression. The test plants did not show disease symptoms at 35 d postinoculation with TYLCV. This study provided an important method for plant antiviral breeding and safe tomato production.

Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Transient expression of &#946-glucuronidase in recalcitrant Ugandan sweetpotato and putative transformation with two cry genes

Sweetpotato ( Ipomoea batatas Lam.) has high potential to contain hunger, malnutrition and poverty in sub-Saharan Africa (SSA), since it gives early yield with few inputs. However, productivity of the crop in Africa is very low due to various challenges, such as severe viral diseases and increasing attacks by sweetpotato weevils, Cylas puncticollis and C. brunneus . Effective resistance to weevils has not been identified in the sweetpotato gene pool. On the other hand, the weevil-resistance genes, cry7Aa1 and cry3Ca1 were assembled into a plasmid vector for use in genetic transformation of African sweetpotato cultivars. The parameters for efficient transfer of these genes and the conditions for de novo regeneration optimised in preliminary studies were used in the genetic transformation of Ugandan landrace ‘Kyebandula’ with Agrobacterium tumefaciens EHA 105 harbouring the plasmid pCIP84, which contains cry7Aa1 , cry3Ca 1 and nptII in its T-DNA. Fifty-four percent of the explants formed adventitious buds. With a mean of 7 buds formed per explant, 6.0% explants formed shoots with a mean of one shoot per explant for those explants that formed shoots on medium containing 50 mg L -1 kanamycin as a selection agent. PCR analysis using primers for cry7Aa1 showed that the transformation efficiency could be as high as 2%. These data highlight the potential of genetic transformation in transferring resistance genes and pave way for enhancement of food security through production of adapted sweetpotato weevil resistant cultivars. Key Words : Agrobacterium tumefaciens, b-glucuronidase, Ipomoea batatas

빵과 면의 가공적성 증진을 위한 밀 저장단백질 Glu-1Dy10을 발현하는 마커프리 형질전환 벼 개발

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, making it less suitable than wheat for many food products such as bread and noodles. The high-molecular weight glutenin subunits (HMW-GS) of wheat play a crucial role in determining the processing properties of the wheat grain. This paper describes the development of marker-free transgenic rice plants expressing a wheat Glu-Dy10 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation. Two expression cassettes, consisting of separate DNA fragments containing Glu-1Dy10 and hygromycin phosphotransferase II (HPTII) resistance genes, were introduced separately into Agrobacterium tumefaciens EHA105 for co-infection. Each EHA105 strain harboring Glu-1Dy10 or HPTII was infected into rice calli at a 3: 1 ratio of Glu-1Bx7 and HPTII. Among 290 hygromycin-resistant T0 plants, we obtained 29 transgenic lines with both the Glu-1Dy10 and HPTII genes inserted into the rice genome. We reconfirmed the integration of the Glu-1Dy10 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the Glu-1Dy10 in transgenic rice seeds were examined by semi-quantitative RT-PCR and Western blot analysis. The marker-free plants containing only the Glu-1Dy10 gene were successfully screened in the T1 generation.

A simple and reliable multi-gene transformation method for switchgrass

A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Protocol for Direct Transformation through Gene Gun in Capsicum annuum L.Cv. Mathania

protocol for direct transformation through biolistic gun has been established for chilli (Capsicum annuum L. cv. Mathania). A disarmed strain of Agrobacterium tumefaciens EHA 105 carrying a binary vector plasmid p35SGUSINT has been used for transformation. This vector contains neomycin phosphotransferase gene (npt II), whose expression confirms Kanamycin resistance in transformants. In addition to npt II, plasmid encodes -glucuronidase, reporter enzyme used for studying the expression of foreign genes in plants. We report for the first time, the use of Nitrogen gas for the biolistic transformation of chilli. We found physical parameter like 6 cm target distance with 900 psi rupture disk for partical gun experiment the most efficient for transient GUS expression. In case of nitrogen gas the frequency of transient GUS expression was better both in leaves (67.74%) and hypocotyl (69.18%) and frequency of conversion of transient to stable transformation was 3.4% in leaves and 4.0% in hypotocyls as against 63.91 % transient GUS expression showed by leaves and 63.77% showed by hypocotyls when helium gas was used in biolistic transformation and stable transformation frequency was 3.0% in leaves and 4.0% in hypocotyls. The transgenic nature of the regenerated plants was confirmed by the histochemical staining of GUS, and polymerase chain reaction (PCR) analysis of npt II gene.