1. A series of technical breakthrough of pepper transformation Pepper is known as a difficult plant to transform and has resisted the efforts of many laboratories for many years. To obtain a successful transformation system for pepper plants, we developed a selection method that eliminated shoots gr...
1. A series of technical breakthrough of pepper transformation Pepper is known as a difficult plant to transform and has resisted the efforts of many laboratories for many years. To obtain a successful transformation system for pepper plants, we developed a selection method that eliminated shoots grown directly from explant and saved shoots grown indirectly from calli (callus-mediated shoot formation; CMSF). These indirect shoots grown from the callus showed a high probability of being transformed. Several genes such as TMV-CP, CMV-CP, CaPPI1 and CaWRKY were used to transform commercially important chill pepper inbred lines by Agrobacterium co-culture. Transformation was confirmed by Southern and Northern blot analyses. Although the transformation rate was low (0.07-0.2 %), this is the best rate ever obtained and this transformation proved reproducible. Another advanced method, we have developed, was a transformation system by inducing callus and shoot before and after co-culturing with Agrobacterium, respectively.? It is called as callus induced transformation (CIT) and the transformation rate was ranged from 0.5-1.8 % depending on the genotype. This method is a stable and reliable and has been worked for many different inbred lines including chilli and paprika.? On top of this method, we have also developed a multi-gene co-transformation technique to enhance the number of transformed pepper in the same experiment period. Pepper explant was transformed by the same Agrobacterium strain (EHA105) independently containing different vectors in which different genes were inserted.? By doing this way, even in a same working period, more and different kinds of pepper transformants were obtained by multi-gene co-transformation comparing to the general transformation method with a? gene in a single vector.? 2. Green fluorescent protein expression in pepper as a monitoring system of genetic transformation 1) Callus induction In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacterium-mediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of T0, T1 and T2 peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.46-1.83% depending on the genotype, which was higher than that obtained by CIT without GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for maintaining the tissue culture process. A co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues was monitored to select the right transformant. 2) Embryo induction from microspore The aim of this research is to establish the conditions for Agrobacterium-mediated genetic transformation using microspore. The embryo induction from microspore was examined under several kanamycin concentration in media, and the induction rate decreased about 4, 8, 10 times when the kanamycin concentration increased 10, 50, 100 mg/L, respectively. This indicates the transformation rate would be much lower if the selection pressure is high by kanamycin. In order to apply the GFP gene as a reporter gene for Agrobacterium-mediated genetic transformation, GFP expression from the microspore-mediated embryos was observed using GFP filter under microscope. The GFP expression occurred when the microspore cultured toward embryo development for 12, 24 and 48 days. The microspore formed a cluster by microspore division from 12 days culture and continuously become bigger mass. We obtained a total of 8 GFP-expressing embryos suggesting that the transformation of microspore occurred. However, those young embryos did not fully developed for some unknown reason. Further study pertinent to culture conditions is required to fulfill the Agrobacterium-mediated genetic transformation using microspore 3. Development of GM peppers tolerant to Pepper mottle virus (PepMoV) Virus infection to chili pepper causes a heavy loss in productivity. In Korea, CMV (cucumber mosaic virus) is the most frequently occurring virus with a single infection rate of 45%. However, a total occurrence of CMV by co-infection, either couple or multiple, with BBWV (broad bean wilt virus), PepMoV (pepper mottle virus) and PMMoV (pepper mild mottle virus) covers over 90% in the field cultivation. The PepMoV is transmitted by several aphid species, and it has been considered the most frequently detected potyvirus when it co-infects with CMV or PMMoV. Since it is hard to develop a variety that is resistant to PepMoV using the classical breeding practice, we have developed transgenic peppers using Agrobacterium-mediated transformation with a HC-Pro gene of the PepMoV. A large number of T1 peppers were tested for resistance to the PepMoV, and T1 peppers tolerant of PepMoV were selected. After consequent self-crossing, the T2 and T3 peppers highly tolerant of PepMoV were selected. The selected T3 peppers are under the safety assessment study. Presently, GM pepper lines (BC1F1) were selected by back-crossing with 3 elite lines.
1. A series of technical breakthrough of pepper transformation Pepper is known as a difficult plant to transform and has resisted the efforts of many laboratories for many years. To obtain a successful transformation system for pepper plants, we developed a selection method that eliminated shoots grown directly from explant and saved shoots grown indirectly from calli (callus-mediated shoot formation; CMSF). These indirect shoots grown from the callus showed a high probability of being transformed. Several genes such as TMV-CP, CMV-CP, CaPPI1 and CaWRKY were used to transform commercially important chill pepper inbred lines by Agrobacterium co-culture. Transformation was confirmed by Southern and Northern blot analyses. Although the transformation rate was low (0.07-0.2 %), this is the best rate ever obtained and this transformation proved reproducible. Another advanced method, we have developed, was a transformation system by inducing callus and shoot before and after co-culturing with Agrobacterium, respectively.? It is called as callus induced transformation (CIT) and the transformation rate was ranged from 0.5-1.8 % depending on the genotype. This method is a stable and reliable and has been worked for many different inbred lines including chilli and paprika.? On top of this method, we have also developed a multi-gene co-transformation technique to enhance the number of transformed pepper in the same experiment period. Pepper explant was transformed by the same Agrobacterium strain (EHA105) independently containing different vectors in which different genes were inserted.? By doing this way, even in a same working period, more and different kinds of pepper transformants were obtained by multi-gene co-transformation comparing to the general transformation method with a? gene in a single vector.? 2. Green fluorescent protein expression in pepper as a monitoring system of genetic transformation 1) Callus induction In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacterium-mediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of T0, T1 and T2 peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.46-1.83% depending on the genotype, which was higher than that obtained by CIT without GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for maintaining the tissue culture process. A co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues was monitored to select the right transformant. 2) Embryo induction from microspore The aim of this research is to establish the conditions for Agrobacterium-mediated genetic transformation using microspore. The embryo induction from microspore was examined under several kanamycin concentration in media, and the induction rate decreased about 4, 8, 10 times when the kanamycin concentration increased 10, 50, 100 mg/L, respectively. This indicates the transformation rate would be much lower if the selection pressure is high by kanamycin. In order to apply the GFP gene as a reporter gene for Agrobacterium-mediated genetic transformation, GFP expression from the microspore-mediated embryos was observed using GFP filter under microscope. The GFP expression occurred when the microspore cultured toward embryo development for 12, 24 and 48 days. The microspore formed a cluster by microspore division from 12 days culture and continuously become bigger mass. We obtained a total of 8 GFP-expressing embryos suggesting that the transformation of microspore occurred. However, those young embryos did not fully developed for some unknown reason. Further study pertinent to culture conditions is required to fulfill the Agrobacterium-mediated genetic transformation using microspore 3. Development of GM peppers tolerant to Pepper mottle virus (PepMoV) Virus infection to chili pepper causes a heavy loss in productivity. In Korea, CMV (cucumber mosaic virus) is the most frequently occurring virus with a single infection rate of 45%. However, a total occurrence of CMV by co-infection, either couple or multiple, with BBWV (broad bean wilt virus), PepMoV (pepper mottle virus) and PMMoV (pepper mild mottle virus) covers over 90% in the field cultivation. The PepMoV is transmitted by several aphid species, and it has been considered the most frequently detected potyvirus when it co-infects with CMV or PMMoV. Since it is hard to develop a variety that is resistant to PepMoV using the classical breeding practice, we have developed transgenic peppers using Agrobacterium-mediated transformation with a HC-Pro gene of the PepMoV. A large number of T1 peppers were tested for resistance to the PepMoV, and T1 peppers tolerant of PepMoV were selected. After consequent self-crossing, the T2 and T3 peppers highly tolerant of PepMoV were selected. The selected T3 peppers are under the safety assessment study. Presently, GM pepper lines (BC1F1) were selected by back-crossing with 3 elite lines.
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