Teknik Isolasi Dan Kultur Protoplas Tanaman Padi

Protoplastfusion or somatic hybridization technology is an alternativetechnology for production hybrids of plants that are difficultto be produced by conventional methods due to their sexualincompatibility. An experiment was conducted to developtechniques for isolation, purification, and culture of riceprotoplasts of cultivar IR64 and a wild rice species (Oryzaofficinalis). Optimization of protoplast isolation and purificationmethods from both rice genotypes were successfullydone. The highest protoplast density was obtained bydigesting embryonic callus or stems of young seedling in anenzyme solution containing of 2% cellulose, 0.1% pectolyase,0.5% macerozyme, 0.5% driselase, 5 mM ES, and 13% mannitolin CPW solution. The protoplast digestion was done forthree hours by soaking in the enzyme solution followed byshaking at 50 rpm under a room temperature. Purification ofthe protoplasts were done by separating them from plantdebris using a 25% sucrose solution. Protoplast regenerationwas not successful using although different media compositionsand conditions. Growth process from cell division tocell aggregate was only successful on IR64 protoplast cultureon a medium that contained AgNO3

Establishment of A Transient Expression Using PEG-Mediated Protoplast Transformation System in Black Rice Cempo Ireng

Indonesia black rice is a potential crop which consider to be develop as functional food because of high nutritional values. However, some agronomical traits, such as high culm and long harvesting time need to be improved for high productivity. Genetic engineering based on protoplast system is one of tools that can be used for improving black rice agronomical traits. The purpose of this study was to establish an efficient method for obtaining protoplasts, and to get information on whether the PEG-mediated transformation method can be carried out on black rice ‘Cempo Ireng’ using GFP transient expression as a marker. To get protoplast culture, we used callus and seedling as main explants. The results showed that 15th days seedling was the best explant source to get protoplast compare to callus. The combination of 1,5% macerozyme and 3% celulase was optimum to obtain the viable protoplasts. Transient expression of GFP can be done using PEG-mediated protoplast transformation in 30% concentration of PEG

A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts

BACKGROUND: Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking. RESULTS: We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7–14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60–70% transformation efficiency. In contrast, using 50 μg of a 12 kb plasmid we obtained a maximum of 25–30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes. CONCLUSION: We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown

Ligand induced cleavage and nuclear localization of the rice XA21 immune receptor

The rice XA21 receptor confers immunity to the Gram-negative bacterial pathogen, _Xanthomonas oryzae_ pv. _oryzae_ (_Xoo_) upon recognition of the conserved microbial signature AxY^S^22. Here, we demonstrate that the intracellular kinase domain of XA21 translocates to the nucleus upon AxY^S^22-mediated perception and that this translocation event is required for XA21-mediated immunity

Cisgenesis, a new tool for traditional plant breeding, should be exempted from the regulation on genetically modified organisms in a step by step approach

Modern potato breeding requires over 100,000 seedlings per new variety. Main reasons are (1) the increasing number of traits that have to be combined in this tetraploid vegetatively propagated crop, and (2) an increasing number of traits (e.g., resistance to biotic stress) originates from wild species. Pre-breeding by introgression or induced translocation is an expensive way of transferring single traits (such as R-genes, coding for resistance to biotic stress) to the cultivated plant. The most important obstacle is simultaneous transfer of undesired neighbouring alien alleles as linkage drag. Stacking several genes from different wild sources is increasing this linkage drag problem tremendously. Biotechnology has enabled transformation of alien genes into the plant. Initially, transgenes were originating mainly from microorganisms, viruses or non-crossable plant species, or they were chimeric. Moreover, selection markers coding for antibiotic resistance or herbicide resistance were needed. Transgenes are a new gene source for plant breeding and, therefore, additional regulations like the EU Directive 2001/18/EC were developed. Because of a strong opposition against genetic modification of plants in Europe, the application of this Directive is strict, very expensive, hampering the introduction of genetically modified (GM) crops and the use of this technology by small and medium-sized enterprises (SMEs). Currently, GM crops are almost the exclusive domain of multinationals. Meanwhile, not only transgenes but also natural genes from the plant species itself or from crossable plant species, called cisgenes, are available and the alien selection genes can be avoided in the end product. This opens the way for cisgenic crops without alien genes. The existing EU directive for GM organisms is not designed for this new development. The cisgenes belong to the existing breeders¿ gene pool. The use of this classical gene pool has been regulated already in agreements regarding breeders¿ rights. We are proposing a step by step approach starting with a crop and gene specific derogation and monitoring towards a general exemption of cisgenic plants from the Directive. Two examples, i.e. development of cisgenic potato for resistance to Phytophthora infestans and cisgenic apple for resistance to Venturia inaequalis are discussed shortly for illustration of the importance of cisgenesis as a new tool for traditional plant breeding. Cisgenesis is simplifying introgression and induced translocation breeding tremendously and is highly recommended for SMEs and developing countrie

Cisgenesis: an important sub-invention for traditional plant breeding companies

Modern plant breeding is highly dependent on new technologies to master future problems. More traits have to be combined, frequently originating from wild species. Traditional breeding is connected with linkage drag problems. The crop plant itself and its crossable species represent the traditional breeders gene pool. GM-breeding is a new way of improving existing varieties. Transgenes originate from non-crossable species and are representing a new gene pool. For release of GM-plants into the environment and onto the market in Europe Directive 2001/18/EC has been developed, primarily based on GM-technology and not on gene source. In society, opposition against GM crops is complicating the implementation of GM crops. In this paper, it is shown that not only transgenes, representing a new gene pool but also cisgenes and intragenes are available, representing the breeders gene pool. Cisgenes are natural genes and intragenes are composed of functional parts of natural genes from the crop plant itself or from crossable species. Cisgenesis is the combined use of only cisgenes with marker-free transformation, mimicking linkage drag free introgression breeding in one step. Therefore, cisgenesis is a new sub-invention in the traditional breeding field and indicates the need for reconsideration of GM Directives. Inventions are frequently containing not only hardware elements, but also software and orgware elements. For cisgenesis it is foreseen that the technical (hardware) and bioinformatic (software) elements will develop smoothly, but that implementation in society is highly dependent on acceptance and regulations (orgware). It could be made in a step by step approach by specific crop-gene derogations from the Directive, followed by adding cisgenesis to annex 1b of Directive 2001/18/EC for exemption. At present GM crops can only be introduced by large companies. An open innovation approach for cisgenesis by public private partnership including traditional SMEs has been discussed. Cisgenesis has been exemplified for resistance breeding of potato to Phytophthtora infestans

Genome-Wide Profiling of Circular RNAs in the Rapidly Growing Shoots of Moso Bamboo (Phyllostachys edulis).

Circular RNAs, including circular exonic RNAs (circRNA), circular intronic RNAs (ciRNA) and exon-intron circRNAs (EIciRNAs), are a new type of noncoding RNAs. Growing shoots of moso bamboo (Phyllostachys edulis) represent an excellent model of fast growth and their circular RNAs have not been studied yet. To understand the potential regulation of circular RNAs, we systematically characterized circular RNAs from eight different developmental stages of rapidly growing shoots. Here, we identified 895 circular RNAs including a subset of mutually inclusive circRNA. These circular RNAs were generated from 759 corresponding parental coding genes involved in cellulose, hemicellulose and lignin biosynthetic process. Gene co-expression analysis revealed that hub genes, such as DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1), MAINTENANCE OF METHYLATION (MOM), dicer-like 3 (DCL3) and ARGONAUTE 1 (AGO1), were significantly enriched giving rise to circular RNAs. The expression level of these circular RNAs presented correlation with its linear counterpart according to transcriptome sequencing. Further protoplast transformation experiments indicated that overexpressing circ-bHLH93 generating from transcription factor decreased its linear transcript. Finally, the expression profiles suggested that circular RNAs may have interplay with miRNAs to regulate their cognate linear mRNAs, which was further supported by overexpressing miRNA156 decreasing the transcript of circ-TRF-1 and linear transcripts of TRF-1. Taken together, the overall profile of circular RNAs provided new insight into an unexplored category of long noncoding RNA regulation in moso bamboo