Development of a method to rapidly assess resistance/susceptibility of Micro-Tom tomatoes to Tomato yellow leaf curl virus via agroinoculation of cotyledons

Objective Tomato yellow leaf curl virus (TYLCV) is one of the pathogens severely damaging tomato crops. Therefore, methods to treat or prevent TYLCV infection need to be developed. For this purpose, a method to conveniently and quickly assess infection of tomatoes by TYLCV is desired. In the present study, we established a quick method to evaluate TYLCV infection using cotyledons of Micro-Tom, a miniature tomato cultivar. Results First, we constructed a binary plasmid harboring 1.5 copies of the TYLCV genome and transformed Agrobacterium with the plasmid. By injecting agroinoculum from the resulting transformant into the branches of Micro-Tom, we confirmed the susceptibility of Micro-Tom to TYLCV. To shorten the evaluation process of TYLCV infection further, we agroinoculated cotyledons of Micro-Tom 10 days after sowing seeds. We consistently observed typical symptoms of TYLCV infection on true leaves 10 days after agroinoculation. Molecular analysis detected TYLCV progeny DNA in all leaves demonstrating symptoms 6 days after agroinoculation. Therefore, our new protocol enabled assessment of TYLCV infection within 20 days after sowing seeds. Thus, agroinoculation of Micro-Tom cotyledons will accelerate the process of screening TYLCV-resistant Micro-Toms and enable screening of larger numbers of plants more quickly, contributing to the development of TYLCV-resistant tomatoes.

Agrobacterium?mediated Genetic Transformation of Two Varieties of Brassica juncea (L.) Using Marker Genes

Protocol for Agrobacterium?mediated genetic transformation using hypocotyl and cotyledonary leaf with petiole from two local varieties of Brassica juncea was established by optimizing various factors influencing transformation. GUS histochemical assay revealed that the cotyledonary leaf with petiole and hypocotyl explants had positive interaction with the Agrobacterium strain LBA4404 containing the binary plasmid pBI121 which has marker genes like, GUS and nptII. Maximum transformation was obtained with bacterial suspension having an optical density of 0.8 at 600 nm, 30 min of incubation and 72 hours of co?cultivation. The transient and stable integration of the marker genes were confirmed through histochemical GUS assay, as well as PCR analysis.Plant Tissue Cult. & Biotech. 26(1): 55-65, 2016 (June)

Penyisipan Gen Inhibitor α-amilase pada Plasmid Biner pCambia 1301

<p class="p1">The experiment was conducted at the Molecular Biology Laboratory of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor. The objective was to construct <em>-ai </em>gene on a binary plasmid <em>p</em>Cambia 1301. This experiment was carried out using construction method by ligation process between fragments of <em>α-ai </em>gene from <em>p</em>TA<span class="s1">3 </span>plasmid and <em>p</em>Cambia 1301 on <em>Hind</em>III site. The result of ligant transformation into <em>E. coli </em>DH5<em>α </em>was 182 surviving colonies on YEP medium containing kanamycin. DNA samples were obtained from 60 randomly selected colonies. The restriction pattern was tested by digesting each DNA sample using <em>Hind</em>III showed colonies containing two fragments expected of sizes wich are 11.837 and 4.887 kb. Two colonies are predicted containing of <em>α-ai </em>gene on its the binary plasmid. Advanced tests using restriction enzymes <em>Bam</em>HI and <em>Xba</em>I showed two directions (right and left) of <em>α-ai </em>gene. The right direction was shown by <em>p</em>Cambia-<em>α-ai</em>1 from colony number 43. This plasmid showed expected fragments of sizes 13.485 and 3.219 kb when digested with <em>Bam</em>HI and two fragments of sizes 15.421 and 1.303 kb when digested with <em>Xba</em>I. The left direction was shown <em>p</em>Cambia-<em>α-ai</em>2 from colony number 58. This plasmid also demon-strated expected fragments of sizes 15.026 and 1.698 kb when digested with <em>Bam</em>HI and two fragments of sizes 13.082 and 3.642 kb when digested with <em>Xba</em>I. Both <em>p</em>Cambia-<em>α-ai</em>1 and <em>p</em>Cambia-<em>α-ai</em>2 were transformed into <em>A. tumefaciens </em>LBA4404.</p>

In vitro Regeneration and Agrobacterium-mediated Transformation of Potato (Solanum tuberosum L.) Cultivars Grown in Mexico

Prior to Agrobacterium-mediated genetic transformation in vitro regeneration protocol was established for three potato cultivars (Alfa, Cambray Rosa Morelos and Atlantic) grown in Mexico using leaf, node and internodal explants. Regeneration protocol was developed with or without the intervention of callus. Two potato cultivars, namely, Cambray Rosa Morelos and Alpha were transformed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing the GUS and nptII genes. GUS histochemical assay and PCR analysis were conducted on rooted shoots grown in media without hormones but supplemented with antibiotics. Transformed shoots tested positive through GUS histochemical assay and integration of nptII gene was confirmed by PCR analysis DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14193 Plant Tissue Cult. & Biotech. 22(2): 93-105, 2012 (December)

Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation withAgrobacterium tumefaciensLBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker andβ-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated withAgrobacterium tumefacienson regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls ofCarica papayacv. Shahi showed the highest positive GUS activities compared toCarica papayacv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls ofC. papayacv. Shahi andC. papayacv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformedC. papayacv. Shahi also showed the maximum number of plant regeneration compared to that ofC. papayacv. Ranchi.

Agrobacterium-mediated Genetic Transformation of Local Cultivars of Chickpea (Cicer arietinum L.)

Agrobacterium-mediated genetic transformation protocol was established for two chickpea (Cicer arietinum L.) varieties, namely Barichhola-4 (Bch-4) and Barichhola-5 (Bch-5). Transformation ability of different explants such as decapitated embryo with single cotyledon disc (DEC), decapitated embryo (DE) and slice embryo decapitated at shoot end with single cotyledon disc (SEC) were tested using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing the GUS and nptII genes. Maximum transformation ability was exhibited by explants of decapitated embryo (DE) from Barichhola-5 (Bch-5). The optimum regeneration from the transformed tissue was achieved on MS supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn and 0.2 mg/l NAA along with double the amount of CaCl2 and KNO3. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin to 150 mg/l. Stable expression of the GUS gene was detected in various parts of the transformed shoots through GUS histochemical assay. Stable integration of nptII gene within the genomic DNA from these transformed shoots was confirmed through PCR analysis.DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11258 Plant Tissue Cult. & Biotech. 22(1): 41-50, 2012 (June)

A Second T-Region of the Soybean-Supervirulent Chrysopine-Type Ti Plasmid pTiChry5, and Construction of a Fully Disarmed vir Helper Plasmid

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadori opines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 coinherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.