Ari Indrianto

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

Abstract. The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo.

High-frequency genetic transformation of Phalaenopsis amabilis orchid using tomato extract-enriched medium for the pre-culture of protocorms

Summary The development of an efficient methodology for the genetic transformation of orchids is needed in order to support thegenetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process.Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.

Agrobacterium-Mediated Transformation of Indonesian Orchids for Micropropagation

Endang Semiarti*1, Ari Indrianto1, Aziz Purwantoro2, Yasunori Machida4 and Chiyoko Machida3 1Faculty of Biology, Universitas Gadjah Mada, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 2Faculty of Agriculture, Universitas Gadjah Mada, Jl. Flora, Bulaksumur, Yogyakarta 3College of Biotechnology and Bioscience, Chubu University, Kasugai, 4Division of Biological Sciences, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 1,2Indonesia 3,4Japan

Flavonoid Production, Growth and Differentiation of Stelechocarpus burahol (Bl.) Hook. F. and Th. Cell Suspension Culture

BACKGROUND AND OBJECTIVE Stelechocarpus burahol is a plant containing flavonoid compounds that have the potential for use as an antihyperuricemic for gout medication. This study was performed to assess flavonoid production, growth and cell differentiation of S. burahol in cell suspension culture. METHODOLOGY Mesocarp was planted in Murashige and Skoog (MS) medium supplemented with 7.5 mg L-1 picloram for the induction of callus. Non-embryonic callus obtained was used in the formation of cell suspension cultures. Growth of cells was determined by fresh and dry weights. During the culturing, the fresh weight, dry weight and flavonoid content were determined as a result of culture status. RESULTS The growth of the S. burahol cell suspension was slow, the stationary phase occurred at 30 days. The production of flavonoids was not in line with the growth of cells and the maximum production occurred on the 15th day of the log phase. The globular-shaped cells dominated the cell suspension culture at all ages. Fluorescein diacetate (FDA) staining of cells derived from cell cultures aged for 36 days showed that some cells were still viable. CONCLUSION The results show that flavonoid production, growth and cell differentiation of a S. burahol cell suspension culture differed according to the culture age.

Intergenus Protoplast Fusion between Pichia manshurica and Rhodosporidium paludigenum to Increase the Production of Inulinase

The purpose s of this study was to identify the optimum concentration of the lytic enzyme Glucanex for protoplast isolation and to conduct fusion for the purpose of increasing inulinase production . The study performs the protoplast fusion technique using Pichia manshurica and Rh odosporidium paludigenum . Protoplast fusion consists of a series of stages: protoplast isolation , protoplast fusion , protoplast regeneration , and analysis of hybrid fusion results . Protoplast isolation and fusion success rate are determined by various factors, including age of the culture , media type, and type of lytic enzymes used . H ybrid results were analyzed using a fungicide as a marker and measuring specific growth rate ( μ) of the hybrid compared with parental growth rates. Results demonstrated that a concentration of 4 mg/ mL of Glucanex produces the greatest number of protoplasts, 7.2 x 10 10 ( c ell/mL) for P. manshurica and 8.8 x 10 10 ( c ell/mL) for Rh . paludigenum . The results of analysis of hybrid fusions indicate that the study has identified a new fusant , called fusant F4 . Fusant F4 is capable of producing the highest inulinase, 0.6892 IU, compared with parentals P. manshurica, 0557 IU, and Rh . paludigenum, 0.3263 IU. Fusant F4 has specific growth rate ( μ) of 0.3360/h and generation time ( g) of 2.0629 h .