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A fully in vitro protocol towards large scale production of recombinant inbred lines in wheat (Triticum aestivum L.)

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Abstract

A bottleneck for genetic research and breeding of crop plants is the time taken to producing large pure line segregating populations so called recombinant inbred lines (RILs). One way to overcome this problem is through use of the single-seed-decent (SSD) process under in vitro conditions. A number of factors that may affect in vitro SSD approach of wheat including temperature, light duration and intensity, salt strength and carbohydrate concentration were investigated in this study. Under the in vitro conditions, 45 days per generation was achieved for an early flowering wheat genotype Emu Rock, allowing eight generations per annum; 58 days per generation was achieved for mid flowering genotypes, allowing six generations per annum. The results showed that a variation of growth environment before and after three-leaf stage allowed in vitro seed-set with a relatively short generation time. Specifically, the plantlets were first grown under 22 °C with a light intensity of 145 μmol m−2 s −1 (16 h d−1) for 20 days (around three-leaf stage), and then moved to an environment of 28 °C and 500 μmol m−2 s−1 (20 h d−1) light. The culture medium was 1/2 strength Murashige and Skoog (MS) with modification of adding ten times of extra KH2PO4 and 4% sucrose. The fully in vitro protocol resulted in 100% flowering rate and average seed set rate of 91.5% in Emu Rock and Zippy. It can be further fine-tuned to suit different genotypes and it has a potential for factory scale mass-production of RILs for genetic studies and practical breeding programs.

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Acknowledgements

Financial supports from the Australian Council of Grain Grower Organisations (COGGO) Research Fund 2014–2015, National Natural Science Foundation of China (41373118), and Guangzhou Education Bureau Innovation Team Project (No.13C02) are appreciated. Y. Yao would like to thank China Scholarship Council (CSC) for her visiting scholarship at The University of Western Australia.

Author contributions

YY, HL, ZL, GY conducted experimental design, data analysis and manuscript writing. PZ was involved in strategic experimental input. YY and HL conducted the in vitro experiments.

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Authors and Affiliations

  1. School of Life Science, Key Laboratory for Functional Study on Plant Stress-Resistant Genes, Guangzhou University, Guangzhou, 510006, China

    Yan Yao & Ping Zhang

  2. Academy of Agriculture and Animal Husbandry Sciences in Inner Mongolia, 22 Zhaojun Road, Yuquan District, Huhehot, 010031, China

    Zhanyuan Lu

  3. School of Plant Biology, Faculty of Science and The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA, 6009, Australia

    Hui Liu & Guijun Yan

Authors
  1. Yan Yao
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  2. Ping Zhang
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  3. Hui Liu
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  4. Zhanyuan Lu
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  5. Guijun Yan
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Correspondence to Zhanyuan Lu or Guijun Yan.

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Yao, Y., Zhang, P., Liu, H. et al. A fully in vitro protocol towards large scale production of recombinant inbred lines in wheat (Triticum aestivum L.). Plant Cell Tiss Organ Cult 128, 655–661 (2017). https://doi.org/10.1007/s11240-016-1145-8

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  • DOI: https://doi.org/10.1007/s11240-016-1145-8

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