Introgresi Gen CsNitr1-L dari Transgenik Nipponbare ke Ciherang dan Analisis Pewarisannya pada Generasi BC3F4

CsNitr1-L gene is a gene encoding nitrites transporter and is included in the group of proton oligopeptide transporter (POT) gene family. The absorption of nitrites by plants expressing this transporter becomes efficient. The gene encoding this protein (CsNitr1-L) under the control of 35S CaMV Promoter had been introduced into rice plants (Oryza sativa L.) subspecies japonica cv. Nipponbare to transfer this gene. The japonica transgenic rice had been crossed with Ciherang variety followed by back-cross and self polination until BC3F4 generation. The aim of this study was to analyse introgression of CsNitr1-L gene in the transgenic rice BC3F4 generation. The transgenic rice plants in BC3F4 generation were selected based on the resistance to hygromicin. More than 90% population of BC3F4 are putative introgression rice lines carriying the transgene. The introgression of the transgene were indirectly confirmed by PCR analaysis using primer corresponding to hpt gene. The yield of introgression line was higher than these of original Ciherang cultivar. Four introgression lines (G3, G7, G8 and G11) that had higher yield were analysed by PCR. Result of the analysis showed that these four transgenic plants carried the introgression

Induksi Kalus Dan Regenerasi Beberapa Genotipe Gandum (Triticum Aestivum L.) Secara in Vitro

Callus Induction and In Vitro Plant Regeneration ofWheat Genotypes (Triticum aestivum L.). AtmitriSisharmini, Aniversari Apriana, and Sustiprijatno. Developmentof a reliable in vitro plant regeneration procedure forwheat is a prerequisite for its improvement by genetic transformation.The purpose of this study was to obtain methodsof callus induction and regeneration of wheat genotypes.This experiment was conducted at ICABIOGRAD. Immatureembryos from four wheat genotypes, ie Perdix, Naxos Wew,Combi and Fasan were used to induce callus formation andregeneration rate of callus. For the preparation of callusinduction medium, MS-L7 basal medium was supplementedwith combination of growth regulators 2,4 dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid(picloram). While, plant regeneration medium was preparedusing MS basal medium supplemented with combination ofthree growth regulators i.e. IAA, BAP and kinetin. The resultsshowed that genotype, in vitro culture medium and growthregulators played a dominant role in callus induction andplantlet regeneration. All the 4 genotypes responded positivelyto callus induction, however, variability was observednot only among the genotypes but also within callusinduction medium used. The best induction medium wasthe MS-L7 basal medium supplemented with combination ofphytohormon 4 mg/l 2,4-D + 2 mg/l picloram (GIK-3) whichshowed 100% callus induction frequency. Whereas, the bestregeneration medium was shown by MS basal medium withcombination of phytohormon 1.5 mg/l BAP dan 0.5 mg/lkinetin (RG3). Regarding plant regeneration, Perdix was themost responsive genotype to be regenerated with regenerationfrequency of 57.33%. The successfully acclimatizedplanlets in greenhouse were obtained from Perdix andNaxos Wew genotypes. These results will potentially facilitategenetic transformation research of wheat in Indonesia

Agrobacterium Tumefaciens-mediated In-planta Transformation of Indonesian Maize Using Pig121hm-cs Plasmid Containing Nptii and Hpt Genes

Maize (Zea mays L.) productivity in Indonesia is challenged to be increased using genetic engineering. Recent advances in Agrobacterium tumefaciens-mediated in-planta transforma-tion makes it possible to transform maize with low cost, and simple method. This study aimed to confirm pIG121Hm-Cs plasmid in A. tumefaciens, and to estimate the efficiency level of A. tumefaciens-mediated in-planta transformation of Indonesian maize by using pIG121Hm-Cs plasmid containing nptII and hpt genes. A series of studies were conducted including confirmation of gene construct of pIG121Hm-Cs plasmid in A. tumefaciens, transformation of four maize lines through A. tumefaciens-mediated in-planta technique, acclimatization of transformant plants and molecular analysis of selected plants using polymerase chain reaction (PCR). The pIG121Hm-Cs plasmid was confirmed via PCR analysis using specific primers of nptII and hpt genes and resulted 700 bp and 500 bp for fragments of nptII and hpt, respectively. After selection, acclimatization and molecular analysis steps, the efficiency levels of transformation of four maize lines were low, ranging from 3.8% to 12.8%. The level of transformation efficiency of ST-27 line was the highest accounting for 12.8% of 45 planted embryos on selection medium based on PCR analysis using specific primer for nptII gene. Overall, A. tumefaciens-mediated in planta transformation on maize floral pistil in this study proved to be successful and rapid. Therefore, this enhanced transformation method will be beneficial for Indonesian maize genetic engineering

Regenerasi Empat Genotipe Gandum (Triticum Aestivum L.) Pasca Transformasi Melalui Agrobacterium Tumefaciens

Genetic transformation ofwheat mediated by Agrobacterium tumefaciens has notbeen established yet. This research aimed to obtain themost responsive wheat genotype to be transformed, toselect the most effective combination of growth regulator forregenerating transforman calli, and to determine thetransformation efficiency. Transformation mediated by A.tumefaciens was performed on four genotypes of wheat,namely Combi, Fasan, Perdix, and Naxos-Wew.Transformed calli with green spots in selection media weretransferred to regeneration media containing 25 mg/lhygromycin, i.e. R1H25 and R2H25. The obtained plantletswere analyzed by PCR using specific primers for hygromycinphosphotransferase gene. The results showed that Fasanwas the most responsive genotype in callus formation(95.47%) and regeneration both in R1H25 (27%) and R2H25(28.6%) media. Fourteen plantlets were succesfullyacclimatized and PCR analysis showed that there were fourpositive transformants containing the hpt gene. The resultsare expected to provide information on the development oftransgenic wheat in Indonesia, specifically in the success ofcallus formation and regeneration of wheat aftertransformation using A. tumefaciens

Introduksi Konstruk Gen CsNitr1-L dengan Promotor Ubiquitin melalui Agrobacterium Tumefaciens dan Deteksi Molekulernya pada Padi Kultivar Nipponbare

Nitrogen based fertilizers such as urea and NPK are primary needs for rice farmers. To get significant improvement of crop yield, the more quantity of fertilizers are applied. It make negative impact for surrounding environment. Based on that, the efforts should be done to suppress the demand of fertilizers such as by developing Nitrogen Use Efficiency crops. CsNitr1-L is one of gene that related to Nitrogen Use Efficiency trait in plant. The objectives of this research are to develop the construction of CsNitr1-L gene candidate in pCAMBIA1300-Ubi1 promoter and to obtain the transformants of rice cultivar Nipponbare which contain the construction of CsNitr1-L gene candidate. The construction of pCAMBIA1300::Ubi1::CsNitr1-L has successfully assembled and was transformed to immature embryo of rice cultivar Nipponbare using Agrobacterium tumefaciens strain LBA4404. It was obtained 146 lines of T0 Nipponbare. PCR analysis of T0 Nipponbare lines showed that 66 of them was identified as positive T0 lines contained hptII and CsNitr1-L genes. Transformation efficiency obtained was 11,9%. The result of analysis copy number using Southern Hybridization in positive PCR of T0 lines randomly indicated that 4 lines have a single copy of transgene. Based on these results, it can be concluded that CsNitr1-L gene construct was successfully introduced into the genome of the rice plant cultivar Nipponbare and the positive PCR of T0 lines containing the gene of hptII and CsNitr1-L, also a single copy of the transgene was obtained

Induksi Dan Regenerasi Kalus Jagung Yang Ditransformasi Dengan Gen CsNitr1-L Melalui Penembakan Partikel

The success in development of transgenic plants is influenced by the regeneration system. The objective of the study was toassess the response of maize genotypes to regeneration system of organogenesis and embryogenesis, after transformed withCsNitr1-L gene through particle bombardment. Induction and callus regeneration of maize immature embryos of inbred linesUlt:cm.1#, ARC 178-123-112-XB3, and AZ2 were conducted through organogenesis, whereas those inbred lines AZ1, AZ2,P4G19(S)C2.59.3.3.1.3 and P4S3.29.4.4.1 were conducted through embryogenesis somatic. Transformation of CsNitr1-L gene wasdone with the distance of bombardment of 7 cm and 9 cm and calli were then selected using 10 mg/l hygromycin. All explants(100%) of inbred lines Ult:cm.1# formed organogenic callus, while callus formation of ARC 178-123-112-XB3 was 94.3% and AZ2was 60.5%. Ult:cm.1# was the most responsive line to the regeneration of organogenesis and produced 24 green shoots,compared with ARC 178-123-112-XB3 which produced one green shoot and AZ2 that did not produce green shoots. The highestpercentage of embryogenic calli formed through somatic embryogenesis was obtained on inbred lines AZ1 (85.4%) and thelowest was on P4S3.29.4.4.1 (18.9%). Inbred lines AZ1 had the highest percentage of regeneration (50.7%) and produced 62plants, followed by P4G19(S)C2.59.3.3.1.3 that produced 17 plants (2.8%) and P4S3.29.4.4.1 which produced two plants.Preliminary identification on 31 putative transgenic plants through PCR analysis produced 22 plants (70.96%) that contained nptIIgene