Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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The effects of cytokinin and plating density on protoplast culture of sunflower

  • Chitpan Kativat (School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology) ;
  • Witsarut Chueakhunthod (School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology) ;
  • Piyada Alisha Tantasawat (School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology)
  • Received : 2022.06.23
  • Accepted : 2022.10.18
  • Published : 2022.12.31
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Abstract

Sunflower (Helianthus annuus L.) protoplasts were isolated from seven-day-old etiolated hypocotyls of 10 A line and four-week-old fully expanded young leaves of PI 441983 line in vitro seedlings using an enzymatic method. Purified protoplasts were collected by filtration and floatation in sucrose solution. Semi-solid protoplast culture was performed using the L4 regeneration protocol with various culture media and plating densities to achieve the highest efficiencies for protoplast culture of hypocotyl and mesophyll protoplasts of 10 A and PI 441983 lines, respectively. The concentrations in liquid L'4M medium and different plating densities were evaluated in two types of cytokinins, the adenine-type 6-benzyladenine (BA) and the phenylurea-type thidiazuron (TDZ). The highest colony formation was achieved in both sunflower lines when 0.5 mgL-1 BA and 0.5 mgL-1 TDZ were applied with a high plating density (3 × 105 protoplasts mL-1). These conditions led to 38.45% and 39.40% colony formation for hypocotyl protoplasts of the 10 A line and mesophyll protoplasts of the PI 441983 line, respectively. Moreover, many hypocotyl protoplast-derived colonies developed into micro-calli. In addition, superior development of both sunflower protoplasts was observed with all plating densities when BA was used in combination with TDZ. This finding will be applicable to future sunflower hybrid production via somatic hybridization.

Keywords

Acknowledgement

We gratefully acknowledge Dr. Paisan Laosuwan and Dr. Thitiporn Machikowa for providing the sunflower seeds for the current study. We are also very grateful to Peter C. Bint for proofreading our manuscript. This work was supported by Suranaree University of Technology and by Thailand Science Research and Innovation (TSRI). The authors would also like to acknowledge the Center of Excellence on Agricultural Biotechnology (AG-BIO), S&T Postgraduate Education and Research Development Office (PERDO) Commission on Higher Education (CHE), Ministry of Education and grants from National Research Council of Thailand (NRCT).

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