A GENOTYPE-INDEPENDENT AGROBACTERIUM MEDIATED TRANSFORMATION OF GERMINATED EMBRYO OF COTTON (GOSSYPIUM HIRSUTUM L.) (original) (raw)
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Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton 'Lashata' were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agarsolidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.
International Journal of Current Microbiology and Applied Sciences, 2017
The purpose of this study was to develop an efficient protocol for genotype independent gene transformation in cotton (Gossypium hirusutum) a worldwide commercially important fibre crop, to reduce the adverse impact of harmful chemicals used to control biotic stress. Most cotton varieties remain recalcitrant and amenable to genetic manipulation to protocols so far developed. The commercially significant Indian cotton cultivars NH-615 and NH-635 were successfully transformed using shoot apex as explants. Shoot apices were aseptically isolated from 6 day old seedlings and co cultivated with Agrobacterium tumifaciens strain EHA 105 harbouring the recombinant vector pCAMBIA containing Cry1Ac gene under control of CaMV 35S promoter; neomycin phosphotransferase (nptII) gene as selectable marker. Inoculated explants were placed for two days on co cultivation medium. Transformed shoots were selected on MS (Murashige and Skoog 1962.) basal medium supplemented with 75mg/l kanamycin and 200mg/l cefotaxime. Multiple shoots subsequently regenerated on MS + 0.5mg/l BAP resulted in high kanamycin resistant multiple shoot induction (16.5 and 13 plants of NH-615 and NH- 635 respectively by applying RBD statistical analysis). A total 40 explants were cultured under each treatment in 4 replications. At the same time a tissue culture independent Agrobacterium mediated in planta transformation protocol was followed to overcome recalcitrance in cotton regeneration. Germinating seedlings of NH-615 with just emerging plumules were inoculated with a separate strain of Agrobacterium LBA4404 carrying gene construct PBI121 that carries GUS (β- glucoronidase) and selectable marker gene nptII to confirm the transformability of the cultivar. Maximum of the germinated plants were positive for GUS showing either tissue specific expression or blue spots in at least one plant part. Callus derived from cotyledonary nodes of NH-615 also showed transformation efficiency by blue colour formation in GUS histochemical analysis. This research is the foremost and successful transformation protocol for the genetic improvement of university developed cotton cultivar NH-615 and NH-635 and this protocol will be useful to research students as well as cotton breeders to develop biotic stress resistant cotton which is one of the important perspectives of AICRP under Cotton Research Station Nanded, VNMKV Parbhani.
Journal of Genetic Engineering and Biotechnology, 2016
A new method of transgenic development called ''In-planta" transformation method, where Agrobacterium is used to infect the plantlets but the steps of in vitro regeneration of plants is totally avoided. In this study, we have reported a simple In-planta method for efficient transformation of diploid cotton Gossypium hirsutum cv LRK-516 Anjali using Agrobacterium tumefaciens EHA-105 harbouring recombinant binary vector plasmid pBinAR with Arabidopsis At-NPR1 gene. Four day old plantlets were used for transformation. A vertical cut was made at the junction of cotyledonary leaves, moderately bisecting the shoot tip and exposing meristem cells at apical meristem. This site was infected with Agrobacterium inoculum. The transgenic events obtained were tested positive for the presence of At-NPR1 gene with promoter nptII gene. They are also tested negative for vector backbone integration and Agrobacterium contamination in T0 events. With this method a transformation frequency of 6.89% was reported for the cv LRK-516.
Genetic Transformation of Cotton (Gossypium hirsutum) Using Marker Genes
Transformation of novel candidate gene for cotton fiber improvement is using a unique Agrobacterium Mediated Transformation approach. The detection of NPTII (kanamycin resistance) as a transgenic marker gene and 35SCaMV as promoter in cotton has been carried out. Transgenic cotton plants were also confirmed by using VirG gene of Agrobacterium. Genomic DNA from transgenic cotton samples were isolated using Fermentas DNA extraction kit. The purity of DNA samples was estimated using UV-Vis spectrophotometry at 260 nm and 280 nm. They gave an absorbance ratio (A260/A280) of 1.74-1.79 which indicated its purities. In order to identify the transgenic cotton, a 151bp fragment of the NPTII gene (kanamycin resistant gene), 210bp fragment of 35SCaMV promoter and 410bp of VirG gene of Agrobacterium were amplified using polymerase chain reaction (PCR). The results showed that both NPTII gene and 35SCaMV promoter are present in putative transgenic cotton plants but no amplification was found in untransformed plant.
Stable genetic transformation in cotton (Gossypium hirsutum L.) using marker genes
2014
Article history: Received 24 October 2013 Revised form 29 October 2013 Accepted 3 November 2013 Available online 25 January 2014 Transformation of novel candidate gene for cotton fiber improvement is using a unique Agrobacterium Mediated Transformation approach. The detection of NPTII (kanamycin resistance) as a transgenic marker gene and 35SCaMV as promoter in cotton has been carried out. Transgenic cotton plants were also confirmed by using VirG gene of Agrobacterium. Genomic DNA from transgenic cotton samples were isolated using Fermentas DNA extraction kit. The purity of DNA samples was estimated using UV-Vis spectrophotometry at 260 nm and 280 nm. They gave an absorbance ratio (A260/A280) of 1.74-1.79 which indicated its purities. In order to identify the transgenic cotton, a 151bp fragment of the NPTII gene (kanamycin resistant gene), 210bp fragment of 35SCaMV promoter and 410bp of VirG gene of Agrobacterium were amplified using polymerase chain reaction (PCR). The results sho...
Plant Cell Reports, 2003
In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L. cv. Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation. We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol. Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends. A majority of these embryos (more than 75%) were b-glucuronidase (GUS)-positive. Embryos with an abnormal appearance showed a very poor conversion to plantlets. However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end. These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds. Regenerated plants tested positive for the transgene by Southern analysis. An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented.
A simple and rapid protocol was developed for Agrobacterium-mediated genetic transformation of pCAMBIA1304 vector harbouring uidA and hptII genes into the Embryonic Shoot Apical Meristem (ESAM) of germinated cotton seeds through microinjection method. Various parameters influencing transformation were standardized for getting high frequency transformation. ESAM of the cotton seeds was microinjected with bacterial inoculum for direct gene delivery to get high frequency of transformation. After co-cultivation, seeds were transferred to selection medium containing Cefotaxime and Hygromycin-B. Resistant plants were grown inside plant growth chamber for acclimatization. GUS analysis was performed to check the expression of uidA gene in resistant cotton plants. The method was so simple and rapid by which more transgenic plants were developed within a short period of time with 20% transformation frequency.
Plant Cell, Tissue and Organ Culture (PCTOC), 2010
A highly efficient and reproducible regeneration system based on somatic embryogenesis in Gossypium hirsutum cv. Narasimha (NM), which has superior fiber qualities and is also used as a female parent in several hybrid cottons, has been developed. Embryogenic callus was obtained form both hypocotyls and cotyledonary leaves on Murashige and Skoog (MS) medium containing kinetin and 2,4-dichlorophenoxyacetic acid. Somatic embryogenesis was observed on hormone-free MS medium, but embryos did not grow well beyond globular stage. However, somatic embryos germinated well on MS medium containing B5 vitamins; addition of zeatin was found to be beneficial for their normal development. Most importantly, the media and culture conditions developed for NM were also found to be suitable for high-frequency somatic embryogenesis in Coker 310. In addition, the newly developed regeneration protocol has been successfully tested for genetic transformation through co-cultivation with Agrobacterium using embryogenic calli as explants. Molecular analysis confirmed the stable integration and expression of marker gene, green fluorescent protein (GFP). These results show that it is now possible to introduce foreign gene(s) directly into elite cultivar Narasimha with similar efficiency to in traditionally used Coker lines in a relatively short period of time. Development of efficient regeneration and transformation systems as demonstrated here should augment the introduction of new traits directly into cultivated varieties/ hybrids, reducing the time required for back-crossing and the costs for seed production, besides aiding genomic research in cotton.
2016
Cotton (Gossypium hirsutum L.; Family: Malvaceae) is an important industrial crop worldwide. Because of the available non-effective regeneration protocols, transformation efficiency of cotton cultivars has remained poor. In the present study, a highly efficient embryo apex (EA) based Agrobacterium-mediated cotton transformation and regeneration protocol was developed. The EA of cotton cv. HS6, selected as explants, were cultured on Murashige and Skoog (MS) medium containing combinations of various plant growth regulators [6-benzylaminopurine (BAP), kinetin (Kin) & naphthalene acetic acid (NAA) ] to induce shoot regeneration. The regeneration percentage was nearly 2-fold higher when MS medium was supplemented with BAP (2.0 mg/L), Kin (1.5 mg/L), proline (250 mg/L) and charcoal (200 mg/L). The age of explants, bacterial inoculum, incubation time of explants in bacterial suspension and co-culture conditions were optimized. A total number of 70 explants were inoculated with Agrobacteriu...
An insight into cotton genetic engineering (Gossypium hirsutum L.): current endeavors and prospects
Cotton (Gossypium hirsutum L.) is the most significant cash crop and backbone of global textile industry. The importance of cotton can hardly be over emphasized in the economy of cotton-growing countries as cotton and cotton products contribute significantly to the foreign exchange earnings. Cotton breeders have continuously sought to improve cotton's quality through conventional breeding in the past centuries; however, due to limited availability of germplasm with resistant to particular insects, pests and diseases, further advancements in cotton breeding have been challenging. The progress in transformation systems in cotton paved the way for the genetic improvement by enabling the researchers to transfer specific genes among the species and to incorporate them in cotton genome. With the development of first genetically engineered cotton plant in 1987, several characteristics such as biotic (insects, viruses, bacteria and fungi) resistance, abiotic (drought, chilling, heat, salt), herbicide tolerance, manipulation of oil and fiber traits have been reported to date. Genetic engineering has emerged as a necessary tool in cotton breeding programs, strengthening classical strategies to improve yield and yield contributing factors. The current review highlights the advances and endeavors in cotton genetic engineering achieved by researchers worldwide utilizing modern biotechnological approaches. Future prospects of the transgenic cotton are also discussed.