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.
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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