[en] We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur-2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T0 and T1 generations to confirm the gene integration. Six events from T0 had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.
Disciplines :
Biotechnology
Author, co-author :
Lakkakula , Satish
Stanislaus, Antony Ceasar ; Université de Liège - ULiège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
Manikandan , Ramesh
Language :
English
Title :
Improved Agrobacterium-mediated transformation and direct plant regeneration in four cultivars of finger millet (Eleusine coracana (L.) Gaertn.)
Publication date :
September 2017
Journal title :
Plant Cell, Tissue and Organ Culture
ISSN :
0167-6857
eISSN :
1573-5044
Publisher :
Kluwer Academic Publishers, Netherlands
Volume :
131
Issue :
3
Pages :
547-565
Peer reviewed :
Peer reviewed
Funders :
The author L. Satish sincerely thanks the University Grants Commission, New Delhi, India for financial support in the form of UGC BSR JRF and SRF (UGC order no. F.4-1/2006 (BSR)/7-326/2011/BSR) for the Ph.D. program.
Arockiasamy, S., Ignacimuthu, S., Regeneration of transgenic plants from two indica rice (Oryza sativa L.) cultivars using shoot apex explants (2007) Plant Cell Rep, 26, pp. 1745-1753. , COI: 1:CAS:528:DC%2BD2sXhtVaksbnO, PID: 17593368
Arvinth, S., Arun, S., Selvakesavan, R.K., Srikanth, J., Mukunthan, N., Kumar, P.A., Premachandran, M.N., Subramonian, N., Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus) resistance (2010) Plant Cell Rep, 29, pp. 383-395. , COI: 1:CAS:528:DC%2BC3cXjt1CjtrY%3D, PID: 20179936
Babu, A.G., Geetha, K.N., Manjunatha, V., Shankar, A.G., An efficient high throughput plant regeneration and transformation protocol for production of transgenics tolerant to salt in finger millet (2012) Int J Forestry Crop Improv, 3, pp. 16-20
Barton, M.K., Twenty years on: the inner workings of the shoot apical meristem, a developmental dynamo (2010) Developmental Biol, 341, pp. 95-113. , COI: 1:CAS:528:DC%2BC3cXks1Wjt7o%3D
Baskaran, P., Dasgupta, I., Gene delivery using microinjection of Agrobacterium to embryonic shoot apical meristem of elite indica rice cultivars (2012) J Plant Biochem Biotechnol, 21, pp. 268-274. , COI: 1:CAS:528:DC%2BC3sXhtVSitLbP
Bayer, G.Y., Yemets, A.I., Blume, Y.B., Obtaining the transgenic lines of finger millet Eleusine coracana (L.) Gaertn. with dinitroaniline resistance (2014) Cytology Genetics, 48, pp. 139-144
Bilang, R., Zhang, S., Leduc, N., Iglesias, V.A., Gisel, A., Simmonds, J., Potrykus, I., Sautter, C., Transient gene expression in vegetative shoot apical meristems of wheat after ballistic microtargetting (1993) Plant J, 4, pp. 735-744. , COI: 1:CAS:528:DyaK2cXivFSqu7Y%3D
Ceasar, S.A., Ignacimuthu, S., Efficient somatic embryogenesis and plant regeneration from shoot apex explants of different Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn.) (2008) In Vitro Cell Dev Biol Plant, 44, pp. 427-435. , COI: 1:CAS:528:DC%2BD1cXhsVyltbbI
Ceasar, S.A., Ignacimuthu, S., Genetic engineering of millets: current status and future prospects (2009) Biotechnol Lett, 31, pp. 779-788. , COI: 1:CAS:528:DC%2BD1MXmtVKnsr8%3D, PID: 19205896
Ceasar, S.A., Ignacimuthu, S., Effects of cytokinins, carbohydrates and amino acids on induction and maturation of somatic embryos in kodo millet (Paspalum scorbiculatum Linn.) (2010) Plant Cell Tiss Organ Cult, 102, pp. 153-162. , COI: 1:CAS:528:DC%2BC3cXnvVejt7c%3D
Ceasar, S.A., Ignacimuthu, S., Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants (2011) Plant Cell Rep, 30, pp. 1759-1770. , PID: 21584677
Ceasar, S.A., Ignacimuthu, S., Finger millet [Eleusine coracana (L.) Gaertn.] (2015) Part of the Methods in Molecular Biology book series (MIMB, vol 1223), 1, pp. 135-142. , In:, Wang, K, (ed), Agrobacterium Protocols, vol, Springer, New York
Chandrashekar, A., Finger millet: Eleusine coracana Chap. 6 (2010) Adv Food Nutri Res, 59, pp. 215-262. , COI: 1:CAS:528:DC%2BC3cXhtFSnurbN
Cho, M.J., Choi, H.W., Okamoto, D., Zhang, S., Lemaux, P.G., Expression of green fluorescent protein and its inheritance in transgenic oat plants generated from shoot meristematic cultures (2003) Plant Cell Rep, 21, pp. 467-474. , COI: 1:CAS:528:DC%2BD3sXlsFGktQ%3D%3D, PID: 12789450
Cho, M., Wu, E., Kwan, J., Yu, M., Banh, J., Linn, W., Anand, A., Zhao, Z., Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line (2014) Plant Cell Rep, 33, pp. 1767-1777. , COI: 1:CAS:528:DC%2BC2cXht1antb%2FE, PID: 25063322
Devi, P., Sticklen, M., Culturing shoot-tip clumps of pearl millet [Pennisetum glaucum (L.) R. Br.] and optimal microprojectile bombardment parameters for transient expression (2002) Euphytica, 125, pp. 45-50. , COI: 1:CAS:528:DC%2BD38XksFeiu7s%3D
Devi, P.B., Vijayabharathi, R., Sathyabama, S., Malleshi, N.G., Priyadarisini, V.B., Health benefits of finger millet (Eleusine coracana L.) polyphenols and dietary fiber: a review (2014) J Food Sci Technol, 51, pp. 1021-1040. , COI: 1:CAS:528:DC%2BC2cXptVCnu7w%3D, PID: 24876635
Dey, M., Bakshi, S., Galiba, G., Sahoo, L., Panda, S.K., Development of a genotype independent and transformation amenable regeneration system from shoot apex in rice (Oryza sativa spp. Indica) using TDZ. 3 (2012) Biotech, 2, pp. 233-240
Do, P.T., Lee, H., Mookkan, M., Folk, W.R., Zhang, Z.J., Rapid and efficient Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) employing standard binary vectors and bar gene as a selectable marker (2016) Plant Cell Rep, 35, pp. 2065-2076. , COI: 1:CAS:528:DC%2BC28XhtVOnsrbM, PID: 27350252
Dosad, S., Chawla, H.S., In vitro plant regeneration and transformation studies in millets: current status and future prospects (2016) Indian J Plant Physiol, 21, pp. 239-254. , COI: 1:CAS:528:DC%2BC28XhsFSgtbjF
Doyle, J.J., Doyle, J.L., Isolation of plant DNA from fresh tissue (1990) Focus, 12, pp. 13-15
Gupta, P., Raghuvanshi, S., Tyagi, A.K., Assessment of the efficiency of various gene promoters via biolistics in leaf and regenerating seed callus of millets, Eleusine coracana and Echinochloa crusgalli (2001) Plant Biotechnol, 18, pp. 275-282. , COI: 1:CAS:528:DC%2BD38XjsVyksQ%3D%3D
Hema, R., Vemanna, R.S., Sreeramulu, S., Reddy, C.P., Senthil-Kumar, M., Udayakumar, M., Stable expression of mtlD gene imparts multiple stress tolerance in finger millet (2014) PLoS ONE, 9 (6). , PID: 24922513
Ignacimuthu, S., Ceasar, S.A., Development of transgenic finger millet (Eleusine coracana (L.) Gaertn.) resistant to leaf blast disease (2012) J Biosci, 37, pp. 1-13
Jagga-Chugh, S.J., Kachhwaha, S., Sharma, M., Kothari-Chajer, A., Kothari, S.L., Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn (2012) Plant Cell Tiss Organ Cult, 109, pp. 401-441. , COI: 1:CAS:528:DC%2BC38XmvVKhsL8%3D
Jha, P., Shashi, Rustagi, A., Agnihotri, P.K., Kulkarni, V.M., Bhat, V., Efficient Agrobacterium-mediated transformation of Pennisetum glaucum (L.) R. Br. using shoot apices as explant source (2011) Plant Cell Tiss Organ Cult, 107, pp. 501-512. , COI: 1:CAS:528:DC%2BC3MXhsVyrtrjF
Karthikeyan, A., Pandian, S.K., Ramesh, M., Transgenic indica rice cv. ADT 43 expressing a D1-pyrroline-5-carboxylate synthetase (P5CS) gene from Vigna aconitifolia demonstrates salt tolerance (2011) Plant Cell Tiss Organ Cult, 107, pp. 383-395. , COI: 1:CAS:528:DC%2BC3MXhsVyrtrjN
Karthikeyan, A., Pandian, S.K., Ramesh, M., Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43) (2011) Plant Sci, 181, pp. 258-268. , COI: 1:CAS:528:DC%2BC3MXovF2ms7g%3D, PID: 21763536
Karthikeyan, A., Shilpha, J., Pandian, S.K., Ramesh, M., Agrobacterium-mediated transformation of indica rice cv. ADT 43 (2012) Plant Cell Tiss Organ Cult, 109, pp. 153-165. , COI: 1:CAS:528:DC%2BC38XktVGrsb8%3D
Latha, A.M., Rao, K.V., Reddy, V.D., Production of transgenic plants resistant to leaf blast disease in finger millet (Eleusine coracana (L.) Gaertn.) (2005) Plant Sci, 169, pp. 657-667. , COI: 1:CAS:528:DC%2BD2MXntVCrsr0%3D
Latha, M.A., Rao, K.V., Reddy, T.P., Reddy, V.D., Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew (2006) Plant Cell Rep, 25, pp. 927-935. , COI: 1:CAS:528:DC%2BD28XotFChtLc%3D, PID: 16607531
Ling, H.Q., Kriseleit, D., Ganal, M.W., Effect of ticarcillin potassium clavulanate on callus growth and shoot regeneration in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.) (1998) Plant Cell Rep, 17, pp. 843-847. , COI: 1:CAS:528:DyaK1cXltFKls7o%3D
Mayavan, S., Subramanyam, K., Jaganath, B., Sathish, D., Manickavasagam, M., Ganapathi, A., Agrobacterium-mediated in planta genetic transformation of sugarcane setts (2015) Plant Cell Rep, 34, pp. 1835-1848. , COI: 1:CAS:528:DC%2BC2MXhtFChsbnI, PID: 26152769
Mbithi-Mwikya, S., Ooghe, W., Camp, J.V., Ngundi, D., Huyghebaert, A., Amino acid profiles after sprouting, autoclaving, and lactic acid fermentation of finger millet (Eleusine coracana) and kidney beans (Phaseolus vulgaris L.) (2000) J Agric Food Chem, 48, pp. 3081-3085. , COI: 1:CAS:528:DC%2BD3cXks1Grur0%3D, PID: 10956072
Mousa, W.K., Schwan, A., Davidson, J., Strange, P., Liu, H., Zhou, T., Auzanneau, F., Raizada, M.N., An endophytic fungus isolated from finger millet (Eleusine coracana) produces anti-fungal natural products (2015) Front Microbiol, 6, p. 1157. , PID: 26539183
Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue cultures (1962) Physiol Plant, 15, pp. 473-497. , COI: 1:CAS:528:DyaF3sXksFKm
Patel, M., Dewey, R.E., Qu, R., Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection (2013) Plant Cell Tiss Organ Cult, 114, pp. 19-29. , COI: 1:CAS:528:DC%2BC3sXpvFelsLo%3D
Perales, M., Reddy, G.V., Stem cell maintenance in shoot apical meristems (2012) Curr Opin Plant Biol, 15, pp. 10-16. , COI: 1:CAS:528:DC%2BC38XhsFCrsrc%3D, PID: 22079787
Rahman, H., Jagadeeshselvam, N., Valarmathi, R., Sachin, B., Sasikala, R., Senthil, N., Sudhakar, D., Muthurajan, R., Transcriptome analysis of salinity responsiveness in contrasting genotypes of finger millet (Eleusine coracana L.) through RNA-sequencing (2014) Plant Mol Biol, 85, pp. 485-503. , COI: 1:CAS:528:DC%2BC2cXotFynsb0%3D, PID: 24838653
Satish, L., Ceasar, S.A., Shilpha, J., Rency, A.S., Rathinapriya, P., Ramesh, M., Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine coracana (L.) Gaertn.) (2015) In Vitro Cell Dev Biol Plant, 52, pp. 192-200
Satish, L., Rathinapriya, P., Ceasar, S.A., Rency, A.S., Pandian, S., Rameshkumar, R., Subramanian, A., Ramesh, M., Effects of cefotaxime, amino acids and carbon source on somatic embryogenesis and plant regeneration in four Indian genotypes of foxtail millet (Setaria italica L.) (2016) In Vitro Cell Dev Biol Plant, 52, pp. 140-153. , COI: 1:CAS:528:DC%2BC2MXhslWrsr%2FN
Satish, L., Rathinapriya, P., Rency, A.S., Ceasar, S.A., Pandian, S., Rameshkumar, R., Ramesh, M., Somatic embryogenesis and regeneration using Gracilaria edulis and Padina boergesenii seaweed liquid extracts and genetic fidelity in finger millet (Eleusine coracana) (2016) J Appl Phycol, 28, pp. 2083-2098
Satish, L., Rency, S.A., Rathinapriya, P., Ceasar, S.A., Pandian, S., Rameshkumar, R., Rao, T.B., Ramesh, M., Influence of plant growth regulators and spermidine on somatic embryogenesis and plant regeneration in four Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn.) (2016) Plant Cell Tiss Organ Cult, 124, pp. 15-31. , COI: 1:CAS:528:DC%2BC2MXhsFKgtbrK
Sharma, M., Kothari-Chajer, A., Jagga-Chugh, S., Kothari, S.L., Factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Eleusine coracana (L.) Gaertn (2011) Plant Cell Tiss Organ Cult, 105, pp. 93-104. , COI: 1:CAS:528:DC%2BC3MXjsVWmu78%3D
Sood, S., Kumar, A., Babu, B.K., Gaur, V.S., Pandey, D., Kant, L., Pattnayak, A., Gene discovery and advances in finger millet [Eleusine coracana (L.) Gaertn.] genomics—An important nutri-cereal of future (2016) Front Plant Sci, 7, p. 1634. , PID: 27881984
Sticklen, M.B., Oraby, H.F., Shoot apical meristem: a sustainable explant for genetic transformation of cereal crops (2005) In Vitro Cell Dev Biol Plant, 41, pp. 187-200. , COI: 1:CAS:528:DC%2BD2MXos1alt7c%3D
Tang, H., Ren, Z., Karzal, G., An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effect on the proliferation of somatic embryos and regeneration of transgenic plants (2000) Plant Cell Rep, 19, pp. 881-887. , COI: 1:CAS:528:DC%2BD3cXmt12isrg%3D
Tran, T.N., Sanan-Mishra, N., Effect of antibiotics on callus regeneration during transformation of IR 64 rice (2015) Biotechnol Rep, 7, pp. 143-149
Travella, S., Ross, S.M., Harden, J., Everett, C., Snape, J.W., Harwood, W.A., A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques (2005) Plant Cell Rep, 23, pp. 780-789. , COI: 1:CAS:528:DC%2BD2MXitFCgt74%3D, PID: 15761662
Visarada, K.B.R.S., Kishore, N.S., Advances in genetic transformation (2015) Sorghum Molecular Breeding, pp. 199-215. , Madhusudhana R, Rajendrakumar P, Patil JV, (eds), Springer, New York
Wilmink, A., Dons, J.J.M., Selective agents and marker genes for use in transformation of monocotyledonous plants (1993) Plant Mol Biol Rep, 11, pp. 165-185. , COI: 1:CAS:528:DyaK3sXlsFKitLw%3D
Yookongkaew, N., Srivatanakul, M., Narangajavana, J., Development of genotype-independent regeneration system for transformation of rice (Oryza sativa ssp. indica) (2007) J Plant Res, 120, pp. 237-245. , COI: 1:CAS:528:DC%2BD2sXivVSht74%3D, PID: 17139419
Zhang, S., Cho, M.-J., Kopret, T., Yun, R., Bregitzer, P., Lemaux, P.G., Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings (1999) Plant Cell Rep, 18, pp. 959-966. , COI: 1:CAS:528:DyaK1MXmtF2ntbg%3D
Zhong, H., Sun, B., Warkentin, D., Zhang, S., Wu, R., Wu, T., Sticklen, M.B., The competence of maize shoot meristems for integrative transformation and inherited expression of transgenes (1996) Plant Physiol, 110, pp. 1097-1107. , COI: 1:CAS:528:DyaK28XitlWjtr4%3D, PID: 12226244
