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Estiati, Amy
Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Agrobacterium-mediated transformation of banana Musa acuminata AA cv "“Mas Lampung” with hpt gene using sterile corm as target tissue Nena, Ade; Witjaksono, Witjaksono; Estiati, Amy
ANNALES BOGORIENSES Vol 13, No 1 (2009): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (285.979 KB) | DOI: 10.1234/27

Abstract

The protocol for  Agrobacterium-mediated transformation of local banana plants cv  “Mas Lampung” (AA) has been established. A selectable marker gene (hpt) has been used to study the transformation using  in vitro corm slices as target tissues.  Banana  in vitro corm slices were co-cultivated with the EHA105 strain of Agrobacterium tumefaciens harbouring binary vector pCAMBIA 1301 containing hygromycin resistance gene (hpt) as a selectable marker and intron-containing β-Glucuronidase (gus-intron) gene as a reporter gene driven by  CaMV 35S promoter.   Polymerase Chain Reaction (PCR) were used to examine the existence of  hpt gene in plants resulted from the transformation.  Using primer pairs specific for hpt gene, our PCR analysis on leaves showed the presence of the  hpt   transgene in banana transgenic plants at first generation (T0) of transformation.  To prove the existence of hpt gene in the fruits of transgenic banana plants, PCR analysis were also carried out.  The data showed that the  hpt  gene could be amplified  from banana fruits of tested samples.   These result demonstrates that the Agrobacterium-mediated transformation method used in this experiment has been successful to transfer gene into banana plants. Thus, the transformation method reported here could be used as a standard protocol to  transfer another useful genes into local banana plants cv. “Mas Lampung”.  Furthermore, the presence of transgene in fruits of banana transgenic plants is important achievement  especially for transgene that is expected to be expressed in the fruit including to introduce vaccine genes into banana fruits for edible vaccine.Key words:  Agrobacterium,  hpt  gene, transgenic banana Mas Lampung, Musa acuminate corm slices, PCR
Segregation Analysis of Transgenic Rice Plants CV Rojolele Harboring Cry1B Gene and Plant Selection for Potential to Yellow Stem Borer Estiati, Amy; Sulistyowati, Yuli; Zahra, Fatimah; Nurhasanah, Ade Nena; Rahmawati, Syamsidah; S-Loedin, Inez H
ANNALES BOGORIENSES Vol 16, No 1 (2012): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (698.51 KB) | DOI: 10.1234/42

Abstract

Transgenic rice plants harboring resistant gene to yellow stem borer, cry1B gene had been obtained. However, the cry1B gene in second generation of transgenic rice lines still segregating following Mendelian ratio 3:1. For further use in the breeding programs, it is important to ensure that the gene is dominant gene and the plants are homozygous for  cry1B gene. Selection  of homozygous transgenic rice lines containing  cry1B  gene at third, fourth and fifth generation had been conducted by PCR.  The presence of  cry1B  gene was determined by showing the PCR product of 1.9 kb. Segregation analysis proved that six transgenic rice  lines i.e. 3R7.8.15.1, 3R7.8.15.9, 3R7.8.15.10, 3R7.8.15.15, 3R7.8.15.21, 3R7.8.15.29 are homozygous lines for  cry1B  gene.   Moreover, bioassay studies at vegetative stage on six homozygous transgenic rice lines showed that  these transgenic rice lines are potential resistant to yellow stem borer comparing with non-transgenic plants, with the score of 0 (indicated no symptom) for six transgenic lines and score of9 (more than 60% damaged tillers ) for non-transgenic plants. However, to confirm the efficacy of  cry 1B  gene to yellow stem borer in naturalcondition, confined field trial in endemic area of yellow stem borer should be conducted. Keywords: bioassay, cry1B gene, transgenic rice, yellow stem borer
Gene flow from genetically modified rice to their weedy and wild relatives and its environmental consequences Estiati, Amy; Nugroho, Satya
ANNALES BOGORIENSES Vol 13, No 1 (2009): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/32

Abstract

Rice is one of the most important food crops in the world.  Nearly half of the world population consume rice as their staple food (FAO, 2004).  With the increasing of world population, the need to provide more food supplies including rice is obvious.  Biotechnology is expected to play major roles in the improvement of crop productivity and quality. Unlike transgenic maize, eventhough many research have been done to improve rice performance by genetic engineering, transgenic rice has not been released or commercialized.  Among those research to improve rice performance are the atempt to introduce biotic and abiotic stress tolerant traits, herbicide tolerant trait and beta-carotin biosynthetic pathway.  Currently, several genetically modified rice cultivars have been and being tested on limited field trials.Keywords : Rice, stress tolerant
Construction of Binary Vector With Wound Inducible Promoter for Hbsag Expression: Development of Plant-Based Edible Hepatitis B Vaccine from Indonesian Isolate Suhandono, Sony; Rachman, Ernawati A.Giri; Zainuddin, Ima M.; Utari, Putri Dwi; Supraba, Apsari; Estiati, Amy
ANNALES BOGORIENSES Vol 11, No 1 (2007): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/22

Abstract

Hepatitis B is a serious infectious disease In the third world countries including Indonesia . Vaccination is  the most effective way to prevent the spread of the disease;  therefore the demand for HBV vaccine is high.  In order to produce more vaccine at lower cost, transgenic plant can be chosen to express the vaccine with the above criteria. Several researches were successfully producing transgenic plants expressing HBsAg that formed virus-like particles and  induced  immune response  in  human.  However. HBsAg expression  in  transgenic plant needs to be  improved especially on gene  expression control system. Here, we describe the construction of HBsAg .  structural gene under the control of wound  inducible promoter, MeEFl promoter from  manihot esculenta Crantz. The HBsAg gene was amplified using PCR from HBV genome isolated from an  Indonesian patient. The gene was subsequently fused with VSPaS signal peptide, which targeted the reticulum endoplasm of plant cell . The construct was cloned into binary expression vector for Agrobacterium plant Transformation  in  near future.Keywords: HBsAg. VSPaS signal peptide and MeEFI promoter
Agrobacterium-Mediated Transformation of Javanica Rice Plants With A Cry1b Gene Under The Control of Wound-Inducible Gene Promoter Estiati, Amy; Rahmawati, Syamsidah; Astuti, Dwi; Loedin, Inez H.S
ANNALES BOGORIENSES Vol 11, No 1 (2007): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/18

Abstract

A  cry1B synthetic gene of Bacillus thuringiensis has been used  for  the transformation of the javanica rice plants  cv.  Rojolele  confer resistance  to  an  important  pest,  yellow  stem  borer  (Scirpophaga  incertulas). Embryogenic callus were co-cultivated with  the EHA 105 strain of Agrobacterium tumefaciens harbouring binary vector CAMB  IA 130 I containing cry  / B gene under  the control of wound inducible gene promoter  (mpi), hygromycin resistance gene (hpt)  as a selectable marker and intron-containing β-glucuronidase (gus - intron) gene al) a reporter gene  driven  by  CaMV35S  promoter.  Previously.  our  histochemical  assay  and  P  R  analyses.  had  proved  th integration of  cry1B gene  into  the genome of rice plants at first generation .  However,  the existence of the gene should remain  table  throughout generation.  In  this study,  the presence of the cry1B transgene in  rice  transgenic plants at  second generation was confirmed by Polymerase Chain Reaction (PCR) .  Insertion of  the cry1B gene in the genome of PCR positive plants was  verified by Southern blot analysis is and showed that integration of cry1  B  into the genomic DNA of  javanica rice plants cv. Rojolele.  An effective resistance of transgenic  plants against  stem borer was verified in  bioassays.
Insect Bioassay in Biosafety Containment to Select Transgenic Rice (Oryza sativa L.) Harboring Cry1B Gene Resistant to Yellow Stem Borer (Scrirpophaga incertulas Walk.) Estiati, Amy; Nena, Ade; Nugroho, Satya
ANNALES BOGORIENSES Vol 17, No 2 (2013): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/77

Abstract

Development of rice varieties resistant to yellow stem borer (YSB) is very crucial.  Agrobacterium-mediated transformation of Cry1B gene under wound inducible gene promoter mpi (maize proteinase inhibitor) into a local rice variety Rojolele had been conducted. PCR analysis proved that Cry1B gene had been integrated into plant genome of 3R25 and 3R5 rice lines. Segregation analysis using PCR for Cry1B gene of the two putative transgenic rice lines at third (T2), fourth (T3), fifth (T4) and sixth (T5) generations of 3R25 and 3R5 lines proved that 3R25.7.27, 3R25.7.13.8.2, 3R25.7.13.8.6, 3R25.7.13.8.8, 3R5.26.2 and 3R5.26.5 are homozygous lines for Cry1B. Insect bioassay on three randomly picked homozygous transgenic rice lines to study the efficacy of Cry1B gene toward YSB was conducted in biosafety containment by infestingYSB larvae at first instar into 3R25.7.27, 3R25.7.13.8.6 and 3R5.26.5 transgenic rice lines, using non-transgenic Rojolele, IR64 and IR74 as susceptible controls. The results showed that the precentages of deadhearts symptoms of  3R25.7.27, 3R5.26.2 and 3R25.7.13.8.6 rice lines were lower than those of the susceptible control lines with scores of 0,1 and 0, respectively. While the scores of all three susceptible control plants were 9. The results proved that lines 3R25.7.27, 3R25.7.13.8.6 and 3R5.26.2 were categorized as resistant lines while the non-transgenic Rojolele, IR64 and IR74 were categorized as susceptible lines. The results also showed that the Cry1B gene was expressed and  produced insecticidal protein CRY1B which were active against YSB to protect rice plant toward YSB infestation
Co-Authors Dwi Astuti Hery Widijanto Loedin, Inez H.S Nena, Ade Nurhasanah, Ade Nena Rachman, Ernawati A.Giri S-Loedin, Inez H Satya Nugroho SONY SUHANDONO Supraba, Apsari Syamsidah Rahmawati, Syamsidah Utari, Putri Dwi Witjaksono Witjaksono Yuli Sulistyowati Zainuddin, Ima M.