A simple method of plant sectioning using the agarose embedding technique for screening intracellular green fluorescent protein

It is difficult to observe plant tissue sections transformed using the agroinfiltration method under a fluorescent microscope. This is due to the softness of the post‐transformation plant. This research was conducted to optimize the sectioning of tobacco stems transformed through the agarose embedding technique. Optimization was conducted at various agarose concentrations: 2%, 4%, and 6%, followed by five minutes of incubation at various temperatures: –80 °C, 4 °C, and 25 °C. The stems were then cut using a scalpel and examined under a fluorescence microscope. The results showed that the embedding method using 6% agarose was more effective at producing a tobacco stem section than 2% or 4% agarose. Meanwhile, incubation at 25 °C was better suited to the transformed tobacco stems than at 4 °C or –80 °C. Green Fluorescent Protein (GFP) could be determined under a fluorescent microscope when using the optimum method. Thus, the optimum method for creating sections of transformed tobacco stems by embedding was to use 6% agarose followed by incubation at 25 °C for 5 min. The optimum result can be applied to obtain a slight section of tobacco stem in order to observe a recombinant protein or other anatomical structures

Isolation and Molecular Identification of Endophytic Bacteria From Rambutan Fruits (Nephelium lappaceum L.) Cultivar Binjai

Interactions between plants and endophytic bacteria are mutualistic. Plant provides nutrient for bacteria, and bacteria will protect the plant from pathogen, help the phytohormone synthesis and nitrogen fixation, and also increase absorption of minerals. These bacteria called plant growth-promoting bacteria. The aim for this study is to identify endophytic bacteria on rambutan (Nephelium lappaceum L.) cultivar Binjai with 16S rRNA. Sequencing results showed that the bacteria is derived from genus Corynebacterium, Bacillus, Chryseobacterium, Staphylococcus and Curtobacterium,which suspected play a role as plant growth-promoting bacteria

Measuring Leaf Chlorophyll Concentration from Its Color: A Way in Monitoring Environment Change to Plantations

Leaf colors of a plant can be used to identify stress level due to its adaptation to environmental change. For most leaves green-related colors are sourced from chlorophyll a and b. Chlorophyll concentration is normally measured using a spectrophotometer in laboratory. In some remote observation places, it is impossible to collect the leaves, preserve them, and bring them to laboratory to measure their chlorophyll content. Based on this need, measurement of chlorophyll content is observed through its color. Using CIE chromaticity diagram leaf color information in RGB is transformed into wavelength (in nm). Paddy seed with variety name IR-64 is used in observation during its vegetation stage t (age of 0-10 days). Light exposure time {\tau} is chosen as environmental change, which normally should be about 12 hours/day, is varied (0-12 hours/day). Each day sample from different exposure time is taken, its color is recorded using HP Deskjet 1050 scanner with 1200 dpi, and its chlorophyll content is obtained from absorption spectrum measured using Campspec M501 Single Beam UV/Vis Spectrophotometer after it is rinsed in 85 % acetone solution and the information from the spectrum is calculated using Arnon method. It has been observed that average wavelength of leaf color {\lambda}avg is decreased from 570.55 nm to 566.01 nm as is measured for t = 1 - 10 days with {\tau} = 9 hours/day, but chlorophyll concentration C is increased from 0.015 g/l to 3.250 g/l and from 0.000 g/l to 0.774 g/l for chlorophyll a and b, respectively. Other value of {\tau} gives similar results. Based on these results an empirical relation between concentration of chlorophyll a Cc-a and its wavelength {\lambda}avg can be formulated.Comment: 8 pages, 5 figures, conferene paper to be presented in Padjadjaran International Physics Symposium 2013 (PIPS 2013), 7-8 May 2013, Jatinangor, Indonesi

Isolation of MA-ACS Gene Family and Expression Study of MA-ACS1 Gene in Musa acuminata Cultivar Pisang Ambon Lumut

Musa acuminata cultivar pisang ambon lumut is a native climacteric fruit from Indonesia. Climacteric fruit ripening process is triggered by the gaseous plant hormone ethylene. The rate limiting enzyme involved in ethylene biosynthesis is ACC synthase (ACS) which is encoded by ACS gene family. The objective of this study is to identify MA-ACS gene family in M. acuminata cultivar pisang ambon lumut and to study the MA-ACS1 gene expression. The result showed that there were nine M. acuminata ACS gene family members called MA-ACS1–9. Two of them (MA-ACS1 and MA-ACS2) were assessed using reverse transcriptase PCR (RT-PCR) for gene expression study and it was only MA-ACS1 correlated with fruit ripening. The MA-ACS1 gene fragment has been successfully isolated and characterized and it has three introns, four exons, and one stop codon. It also shows highest homology with MACS1 gene from M. acuminata cultivar Hsian Jien Chiao (GenBank accession number AF056164). Expression analysis of MA-ACS1 using quantitative PCR (qPCR) showed that MA-ACS1 gene expression increased significantly in the third day, reached maximum at the fifth day, and then decreased in the seventh day after harvesting. The qPCR expression analysis result correlated with the result of physical analysis during fruit ripening

Analysis of MeEf1A6 Gene Promoter Activity with In-vitro and In-vivo using Transient and Stable Expression Techniques in Tobacco Plant (Nicotiana tabacum)

The promoter is a part of the gene that functions in carrying out the gene expression, and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculenta Elongation Factor 1 Alfa - 6) is a promoter derived from cassava plants (Manihot esculenta). In previous studies, the MeEF1A6 promoter was successfully isolated, introduced, and characterized into the pBI121 plasmid, replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in-vivo and in-vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was introduced into Agrobacterium tumefaciens strain AGL1 and LBA4404. The promoter's work was then analyzed by the result of introducing it into the tobacco plant using the transient and stable transformation. The whole part of explants was used for transient study and tested in a minimum of two biological replicates. Sixty sheets of explant leaves that have been cut were used for stable transformation. The promoter work analysis was carried out with the GUS gene expression that integrated with the promoter with histochemical GUS assay. The transient produced a blue color in the roots, stems, and leaves on the whole repetition. The transverse incision in the stem shows the blue color on the epidermis and procambium tissue. Stable transformation using AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 plantlets that were successfully grown. Some plantlets are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants, indicating that the MeEF1A6 promoter has been successfully introduced. The results indicate that the MeEF1A6 promoter could work on plant tissue in roots, stems, leaves, and tissues that connect meristems such as procambium in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter performs work constitutionally as a constitutive promoter. 

TRANSFORMASI MENGGUNAKAN Agrobacterium tumefaciens PADA TUNAS DAUN Kalanchoe mortagei DAN Kalanchoe daigremontiana 1 DAN 2

Cocor bebek adalah tumbuhan sukulen yang mampu memproduksi tunas adventif (reproduksi vegetatif) pada tepian daunnya. Kemampuan reproduksi vegetatif ini menghasilkan tanaman yang sama dalam waktu yang singkat, sehingga memungkinkan untuk dijadikan sebagai bioreaktor protein rekombinan. Transformasi dilakukan menggunakan Agrobacterium tumefaciens pada tunas daun cocor bebek dari spesies Kalanchoe mortagei dan Kalanchoe daigremontiana. Optimasi dilakukan mencakup: galur A. tumefaciens, kerapatan optis dari kultur A. Tumefaciens, konsentrasi acetosyringone, teknik ko-kultivasi, pH medium dan komposisi medium ko-kultivasi. Hasil optimasi transformasi secara transien menunjukan bahwa perbedaan galur A. tumefaciens, kerapatan optis, konsentrasi acetosyringone menghasilkan ekspresi transien yang relatif sama secara kualitatif. Berdasarkan uji GUS teknik ko-kultivasi dengan infiltrasi vakum dan pH medium 5,5 menghasilkan ekspresi transien lebih baik dibandingkan dengan perendaman dan pH medium 7,0. Medium ko-kultivasi M9 menghasilkan ekspresi transien yang lebih baik dibandingkan dengan medium ½MS0. Tunas daun K. daigremontiana 2 menunjukan ekspresi transien yang lebih baik dibandingkan K. mortagei dan K. daigremontiana 1

Effect of Terbinafin and DMSO on The Gene Expression Level of Squalene Synthase (Sqs) and Amorpha-4,11-Diene Synthase (Ads) in Artemisia Annua L

AbstractArtemisinin, a secondary metabolite from Artemisia annua L. is a sesquiterpene lactone that has antimalaria activity but produced at low quantities by the plant. Low levels of artemisinin in the plant is related to the biosynthetic pathways influenced by specific enzymes that play role in the formation of artemisinin. Farnesyl diphosphate (FDP), which is the main precursor of artemisinin, also known as the precursor for the formation of sterols. Compared with the other compounds, sterol biosynthetic pathway is the biggest competitor of the artemisinin production since sterols are needed by plants to regulate membrane fluidity and permeability. This research aimed to study the effect of terbinafin and DMSO as sterol synthesis inhibitors on the regulation of the artemisinin biosynthetic pathway by analyzing the expression level of two genes linked, squalene synthase (SQS) and amorpha-4,11-diene synthase (ADS) using quantitative PCR (qPCR) and the amount of artemisinin is determined using high performance liquid chromatography (HPLC). The results showed that at mRNA levels, terbinafin 30μM had no significant effect on SQS and ADS expression levels, but it increased the amount of artemisinin at 50hours incubation up to 1.36 times higher than control. DMSO increased the expression level of ADS up to 3-4 times and increased the content of artemisinin up to 2.42 times higher than control at 50hours incubation tim

Transient transformation of artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) gene into Artemisia annua L.

Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13)  double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens-mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303-dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of  A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA-dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed  A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA-dbr2 construct and transformation into  A. annua have been successfully performed

Expression profiling of the CHS8, CHI1A, IFS2, and CHR genes in black soybean seed [Glycine max (L). Merr.] of F4 generation

Black soybean [Glycine max (L.) Merr.] produces isoflavones as secondary metabolites, which have many benefits for human health and plant defense system. Expression profiling can guide potential work in functional genomics of the isoflavone biosynthesis pathway. Previous studies showed the vital role of the CHS8, CHI1A, and IFS2 genes in isoflavone biosynthesis. However, expression profiling of these genes in the local black soybean varieties is still limited. This study investigated the gene expression levels of the CHS8, CHI1A, IFS2, and CHR genes in local varieties, namely, UP106 (high isoflavone) and UP122 (low isoflavone) and its progenies, i.e., UP106xUP122 and UP122xUP106. Relative gene expression profiling was conducted on the basis of Reverse Transcriptase Polymerase Chain Reaction (RT‐PCR) with ACT2/7 as a housekeeping gene. As a result, the expression level of CHS8 in UP122 is lower than that in UP106. No significant difference in the expression level of CHI1A was observed in all samples. The expression levels of CHS8 and CHI1A in both progenies were higher than that in the parental line, whereas the expression levels of IFS2 in both progenies were lower than that in the parental line. CHS8 and IFS2 expression from UP106xUP122 was higher than that from UP122xUP106, whereas CHI1A expression from UP122xUP106 was higher than that from UP106xUP122. CHR showed a high expression in the reciprocal cross; however, this expression did not exceed from UP106. In conclusion, the crossing between parental lines did not affect the gene expression level in the isoflavone biosynthesis pathway

Konstruksi vektor biner untuk ekspresi gen dip22 yang diisolasi dari tebu varietas M 442-51 pada tanaman

Sugarcane is the principle plant for producing sugar in Indonesia. Water supply is one key element in the agronomy of sugarcane. Sugarcane is a high biomass crop which requires large amounts of water. Low yields of sugar observed in water stressed plants indicate that sugarcane is very sensititive to drought. A number of genes that respond to drought, salt, and cold stress at the trasnscriptional level have been reported. dip22 (drought inducible protein) protein isolated from drought resistance variety M 442-51 was predicted to be a protein regulator to water stress in sugarcane. Increasing of tolerance to water stress by over expression of dip22 genes in high yield sugarcane variety hopefully will maintain sugar production. The goal of this research was to construct a binary vector for dip22 gene expression in plant. dip22 gene from mutated PCR was cloned to pGEM ® €“T Easy and transformed to Escherichia coli strain DH5a. And then, these gene was isolated again from pGEM ® €“T Easy-dip22 (pGdip) plasmid using restriction enzymes NcoI and PmlI. pCAMBIA 1303 plasmid is an expression vector which has the constitutive promoter CaMV35S. Recombinant plasmid was transformed to Escherichia coli strain DH5a for plasmid propagation through DNA replication. Recombinant plasmid was isolated, and digested with NcoI and PmlI to examine the presence of dip22 gene in the pCAMBIA 1303 plasmid. The recombinant plasmid was transformed to A. tumefaciens strain LBA 4404. Plasmid isolated from A. tumefaciens was digested with Bst XI and Bst EII to examine the similarity between pCAMBIA 1303-dip22 (pCdip) from Escherichia coli and A. tumefaciens. The result by electrophoresis showed that both plasmids had the same size after digested. It was concluded that the transformed A. tumefaciens strain LBA 4404 bacteria has pCAMBIA 1303-dip22 (pCdip) plasmid indeed. Therefore, this construct of dip22 gene in binary vector can be used for improving drought tolerance in plant