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Agrobacterium-mediated transformation of banana Musa acuminata AA cv "“Mas Lampung” with hpt gene using sterile corm as target tissue Witjaksono, Witjaksono; Nena, Ade; 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
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