Abstract
Maize is an important crop for billions of people globally. The existing immature embryo-based regeneration protocol of maize has major limitations due to the non-availability of explants throughout the year, limited durability for culturing, and its laborious nature. Mature embryos, especially in tropical maize, are considered recalcitrant towards tissue culture. Therefore, standardization of a robust regeneration and transformation protocol in tropical maize using mature embryos or seeds as starting material is long envisaged. Considering this, in this study, 28 diverse tropical maize genotypes were evaluated for their embryogenic callus induction potential using two different explants (nodal explants and split embryo region) under two different callusing media. Out of 28 genotypes, better callus induction was achieved in four genotypes (BML 6, DHM 117, DMRH 1301, and DMRH 1308) from nodal explants. Further, in vitro regeneration was standardized using 22 different combinations of various auxins and cytokinins. Out of 28 genotypes, two recently commercialized and high-yielding cultivars (DMRH 1301 and DMRH 1308) demonstrated the best callusing and regeneration capability with an average regeneration percentage of 60.4% and 53.6%, respectively. Using the nodal explants-derived embryogenic calli, the genetic transformation was successfully carried out using the ‘Biolistic’ approach, and up to ~ 5% transformation efficiency was achieved. This efficient regeneration and transformation protocol can overcome the major limitations associated with the existing immature embryo-based protocol in tropical maize as mature seeds can be obtained easily in ample quantity round the year. Such a generalized and reproducible protocol has the potential to be a major tool for maize improvement using transgenic and genome-edited techniques.
Key message
The standardized protocol not only overcomes the major limitations associated with the existing and predominately used immature embryo-based protocol but it is easier, reproducible, and has either higher or comparable callusing, regeneration, and transformation efficiency.
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Data availability
The datasets supporting the conclusions are included in the article.
References
Abhishek A, Karjagi CG, Nath R, Bhardwaj M, Ramteke PW, Kumar P, Dass S, Kumar RS (2014) Differential effect of immature embryo’s age and genotypes on embryogenic type II callus production and whole plant regeneration in tropical maize inbred lines (Zea mays L.). Indian J Genet Plant Breed 74(3):317–324. https://doi.org/10.5958/0975-6906.2014.00849.9
Agarwal A, Yadava P, Kumar K, Singh I, Kaul T, Pattanayak A, Agrawal PA (2018) Insights into maize genome editing via CRISPR/Cas9. Physiol Mol Biol Plants 24:175–183. https://doi.org/10.1007/s12298-017-0502-3
Aguado-Santacruz GA, Garcia-Moya E, Aguilar-Acuna JL, Moreno-Gomez B, Preciado-Ortiz ER, Jimenez-Bremont JF et al (2007) In vitro plant regeneration from quality protein maize. In Vitro Cell Dev Biol Plant 43:215–224. https://doi.org/10.1007/s11627-007-9042-9
Ahmadabadi M, Ruf S, Bock R (2007) A leaf based regeneration and transformation system for maize(Zea mays L.). Transgenic Res 16:437–448. https://doi.org/10.1007/s11248-006-9046-y
Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split seed: a new tool for maize researchers. Planta 223:1355–1360. https://doi.org/10.1007/s00425-006-0237-9
Anami SE, Mgutu AJ, Taracha C, Coussens G, Karimi M, Hilson P, Lijsebettens MV, Machuka J (2010) Somatic embryogenesis and plant regeneration of tropical maize genotypes. Plant Cell Tissue Org Cult 102:285–295. https://doi.org/10.1007/s11240-010-9731-7
Assem SK, Hussein EHA, El-Akkad TA (2008) Genetic transformation of Egyptian maize lines using the late embryogenesis abundant protein gene, HVA1, from barley. Arab J Biotechnol 11(1):47–58. https://vlibrary.emro.who.int/imemr/genetic-transformation-of-egyptian-maize-lines-using-the-late-embryogenesis-abundant-protein-gene-hva1-from-barley-2/
Barloy D, Beckert M (1993) Improvement of regeneration ability of androgenetic embryos by early anther transfer in maize. Plant Cell Tissue Org Cult 33:45–50. https://doi.org/10.1007/BF01997597
Bohorova NE, Luna B, Brito RM, Huerta LD, Hoisington DA (1995) Regeneration potential of tropical, sub tropical, mid altitude and highland maize inbreds. Maydica 40:275–281. https://agris.fao.org/agris-search/search.do?recordID=IT9661482
Bohorova N, Zhang W, Julstrum P, McLean S, Luna B, Brito RM et al (1999) Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos. Theor Appl Genet 99:437–444. https://doi.org/10.1007/s001220051255
Brettschneider R, Becker D, Lörz H (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94:737–748. https://doi.org/10.1007/s001220050473
Carvalho CHS, Bohorova N, Bordallo PN, Abreu LL, Valicentle FH, Bressan W, Paiva E (1997) Type II callus production and plant regeneration in tropical maize genotypes. Plant Cell Rep 17:73–76. https://doi.org/10.1007/s002990050355
Duncan DR, Wiliams ME, Zehr BE, Widholm JM (1985) The production of callus capable of plant regeneration from immature embryos of numerous Zea mays (L.) genotypes. Planta 165:322–332. https://doi.org/10.1007/BF00392228
El-itriby HA, Assem SK, Hussein EHA, AbdeL-Galil FM, Madkour MA (2003) Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system. In Vitro Cell Dev Biol Plant 39:524–531. https://doi.org/10.1079/IVP2003439
FAO (2018) World food and agriculture—statistical pocketbook. FAO, Rome. http://www.fao.org/faostat. Accessed 15 Mar 2021
Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE et al (2000) Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol Plant 36:21–29. https://doi.org/10.1007/s11627-000-0007-5
Frame B, Main M, Schick R, Wang K (2011) Genetic transformation using maize immature zygotic embryos. In: Thorpe TA, Yeung EC (eds) Plant embryo culture. Methods in molecular biology (methods and protocols), vol 710. Humana Press, New York, pp 327–341. https://doi.org/10.1007/978-1-61737-988-8_22
Geetha S, Joshi JB, Kumar KK, Arul L, Kokiladevi E, Balasubramanian P, Sudhakar D (2019) Genetic transformation of tropical maize (Zea mays L.) inbred line with a phytase gene from Aspergillus niger. 3 Biotech 9(6):208. https://doi.org/10.1007/s13205-019-1731-7
Huang XQ, Wei ZM (2004) High frequency plant regeneration through callus initiation from mature embryos of maize (Zea Mays L). Plant Cell Rep 22:793–800. https://doi.org/10.1007/s00299-003-0748-9
Ishida Y, Saito H, Ohta SH, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750. https://doi.org/10.1038/nbt0696-745
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Bio Rep 5:387–405. https://doi.org/10.1007/BF02667740
Joshi JB, Yatish KR, Joel AJ, Kumar KK, Kokiladevi K, Arul L, Gnanam R, Balasubramanian P, Sudhakar D (2014) A high-throughput regeneration protocol for recalcitrant tropical Indian maize (Zea mays L) inbreds. Maydica 59(3):211–216. https://journals-crea.4science.it/index.php/maydica/article/view/1001
Kaul J, Nara U, Kumar R, Kumar RS, Kumar V (2012) A compendium of hybrids and composites of maize (1993-2012). Technical Bulletin No. 2012/5; pp. 172 + iv. Directorate of Maize Research, New Delhi. https://iimr.icar.gov.in/wp-content/uploads/2020/03/A-Compendium-of-Hybrids-and-Composites-of-Maize.pdf, https://issuu.com/kisanadmin/docs/a_compendium_of_hybrids_and_composites_of_maize/149
Kumar KK, Maruthasalam S, Loganathan M, Sudhakar D, Balasubramaniam P (2005) An improved Agrobacterium mediated transformation protocol for recalcitrant elite indica rice cultivars. Plant Mol Biol Rep 23:67–73. https://doi.org/10.1007/BF02772648
Kumar R, Mamrutha HM, Kaur A, Venkatesh K, Grewal A, Kumar R, Tiwari V (2017) Development of an efficient and reproducible regeneration system in wheat (Triticum aestivum L.). Physiol Mol Biol Plants 23(4):945–954. https://doi.org/10.1007/s12298-017-0463-6
Kumar K, Aggarwal C, Sapna B, Singh I, Yadava P (2018) Microbial genes in crop improvement. In: Prasad R, Gill SS, Tuteja N (eds) Crop improvement through microbial biotechnology. Elsevier, Amsterdam, pp 39–56. https://doi.org/10.1016/B978-0-444-63987-5.00003-7
Kumar B, Singh SB, Yadav OP et al (2019) Newly released high yielding single cross maize hybrids for different ecologies of India. Indian J Agric Sci 89(2):371–375
Kumar K, Gambhir G, Dass A et al (2020) Genetically modified crops: current status and future prospects. Planta 251:91. https://doi.org/10.1007/s00425-020-03372-8
Malini N, Ananadakumar CR, Ramakrishnan SH (2015) Regeneration of Indian maize genotypes (Zea mays L.) from immature embryo culture through callus induction. J Appl Nat Sci 7(1):131–137. https://doi.org/10.31018/jans.v7i1.576
Manivannan A, Kaul J, Singode A, Dass S (2010) Callus induction and regeneration of elite Indian maize Inbreds. Afr J Biotechnol 9(44):7446–7452. https://doi.org/10.5897/AJB10.500
Mórocz S, Donn G, Nérneth J, Dudits D (1990) An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture. Theor Appl Genet 80:721–726. https://doi.org/10.1007/BF00224183
Muppala S, Gudlavalleti PK, Pagidoju S, Malireddy KR, Puligandla SK, Dasari P (2020) Distinctive response of maize (Zea mays L.) genotypes in vitro with the acceleration of phytohormones. J Plant Biotechnol 47:26–39. https://doi.org/10.5010/JPB.2020.47.1.026
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Pareddy DR, Petolino JF (1990) Somatic embryogenesis and plant regeneration from immature inflorescences of several elite inbreds of maize. Plant Sci 67(2):211–219. https://doi.org/10.1016/0168-9452(90)90245-J
Petrillo CP, Carneiro NP, Purcino AAC, Carvalho CHS, Alves JD, Carneiro AA (2008) Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines. Pesq Agropecu Bras 43(3):371–378. https://doi.org/10.1590/S0100-204X2008000300012
Rakshit S, Rashid Z, Sekhar JC, Fatma T, Dass S (2010) Callus induction and whole plant regeneration in elite Indian maize (Zea mays L.) inbreds. Plant Cell Tissue Organ Cult 100:31–37. https://doi.org/10.1007/s11240-009-9613-z
Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. PNAS 81(24):8014–8018. https://doi.org/10.1073/pnas.81.24.8014
Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Sigla-Pareek SL (2011) An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7(1):49. https://doi.org/10.1186/1746-4811-7-49
Sairam RV, Parani M, Franklin G, Lifeng Z, Smith B, MacDougall J et al (2003) Shoot meristem an ideal explant for Zea mays L. transformation. Genome 46:323–329. https://doi.org/10.1139/g02-120
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Shah TR, Prasad K, Kumar P (2016) Maize—a potential source of human nutrition and health: a review. Cogent Food Agric 2:11166995. https://doi.org/10.1080/23311932.2016.1166995
Shiva PN, Bhojaraja R, Shivbachan SK, Priya GGH, Nagraj TK, Prasad V et al (2009) Marker-free transgenic corn plant production through co-bombardment. Plant Cell Rep 28:1655–1668. https://doi.org/10.1007/s00299-009-0765-4
Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling derived maize callus. Plant Cell Rep 25(4):320–328. https://doi.org/10.1007/s00299-005-0058-5
Tiwari S, Agrawal PK, Pande V, Gupta HS (2015) Callus induction and whole plant regeneration in sub-tropical maize (Zea mays L.) using mature embryos as explants. Indian J Genet 75(3):330–335. https://doi.org/10.5958/0975-6906.2015.00052.8
Wang K, Frame B (2009) Biolistic gun-mediated maize genetic transformation. In: Scott MP (ed) Transgenic maize. Methods in molecular biology, vol 526. Humana Press, Totowa. https://doi.org/10.1007/978-1-59745-494-0_3
Wang D, Zhao Q, Zhu D, Ao G, Yu J (2006) Particle-bombardment mediated co transformation of maize with a lysine rich protein gene (sb401) from potato. Euphytica 150:75–85. https://doi.org/10.1007/s10681-006-9095-6
Yadava P, Abhishek A, Singh R, Singh I, Kaul T, Pattanayak A, Agrawal PK (2017) Advances in maize transformation technologies and development of transgenic maize. Front Plant Sci 7:1949. https://doi.org/10.3389/fpls.2016.01949
Acknowledgements
The research work was carried out under the IIMR institutional in-house project (Project Code: AR:DMR:16:02) funded by the Indian Council of Agricultural Research (ICAR). The research was also supported in part by funds from the National Agricultural Science Fund (NASF/GTR-5004/2015-16/204) for which authors express their sincere thanks to NASF. The authors thank Dr. S. R. Bhat (Ex-Emeritus Scientist, ICAR-NIPB) for giving his valuable inputs and guidance during executing experiments. We are also thankful to Dr. R. C. Bhattacharya for his kind inputs in regeneration experiments. We are thankful to Dr. Sadhu Leelavathi, ICGEB, New Delhi for their help in molecular confirmation work. The authors also acknowledge the maize partners for sharing germplasm. AKJ acknowledges ICAR-IIMR support in the form of YP fellowship. AA, GG and CA, and AT acknowledge NASF support in the form of RA, SRF, and LA fellowships, respectively.
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SR and KK conceived the idea. AKJ, KK, AA, GG, CA, AT, and PS performed the experiments. AKJ, KK, and BK analyzed the data. PP carried out Southern and northern blot experiments. KK wrote the primary draft, which was further augmented, edited, and improved by SR and BK. BK and CGK provided the seeds of genotypes used in the study. All the authors read and approved this article for publication.
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Communicated by Francisco de Assis Alves Mourão Filho.
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11240_2021_2207_MOESM2_ESM.tif
Supplementary material 2 (TIF 4280.4 kb)—Supplementary Fig. 1. A pictorial representation for the response of embryogenic calli towards combinations of auxins (IAA, NAA) and cytokinins (BAP, Kinetin) in regeneration media.
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Supplementary material 3 (TIF 1956.1 kb)—Supplementary Fig. 2. Callusing and regeneration in BML 6 inbred line from nodal-explants derived embryogenic calli.
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Supplementary material 4 (TIF 12204.1 kb)—Supplementary Fig. 3. Molecular characterization of putative transformants through Southern (a) and northern (b) hybridization using gusA gene probe. Lane M represents molecular marker, WT corresponds to the negative control (Wild type, untransformed maize plant) (a) while different independent lines of DMRH 1301 and DMRH 1308 are represented by letter L followed by a number (a and b). Lines showing stable integration of the transgene (a) and expression of the transgene (b) are marked with asterisk * symbol. The lower panel in (b) corresponds to RNA gel representing equivalent loading pattern of total RNA used for northern blot.
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Kumar, K., Jha, A.K., Kumar, B. et al. Development of an efficient and reproducible in vitro regeneration and transformation protocol for tropical maize (Zea mays L.) using mature seed-derived nodal explants. Plant Cell Tiss Organ Cult 148, 557–571 (2022). https://doi.org/10.1007/s11240-021-02207-y
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DOI: https://doi.org/10.1007/s11240-021-02207-y