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Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

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Abstract

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

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Abbreviations

BA:

6-Benzylaminopurine

CPW:

Cell and protoplast wash solution

2,4-D:

2,4-Dichlorophenoxyacetic acid

FDA:

Fluorescein diacetate

IBA:

Indole-3-butyric acid

ISSR:

Inter-simple sequence repeats

KM:

Kao and Michayluk medium

MS:

Murashige and Skoog medium

MSB5:

MS medium with Gamborg B5 vitamins

NAA:

α-Naphthaleneacetic acid

PCR:

Polymerase chain reaction

PGR:

Plant growth regulator

Pp:

Protoplast

Pp gfw−1 :

Protoplasts per gram fresh weight

SCoT:

Start codon targeted

TDZ:

Thidiazuron

WPM:

Woody plant medium

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Acknowledgments

The authors gratefully acknowledge Drs. Scott Merkle and Praveen K. Saxena for their constructive review and suggestions for the improvement of this manuscript. Financial support for this research was funded by the Ministry of Science, Research and Technology of Iran for the University of Kurdistan. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

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  1. Laboratory of Forest Tree Biology and Biotechnology, Department of Forestry, Faculty of Natural Resources, University of Kurdistan, Khanagah Campus, Sanandaj, 66177-1-5175, Iran

    Mohammad-Shafie Rahmani & Naghi Shabanian

  2. USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, 715 West State Street, West Lafayette, IN, 47907, USA

    Paula M. Pijut

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  1. Mohammad-Shafie Rahmani
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Correspondence to Paula M. Pijut or Naghi Shabanian.

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Rahmani, MS., Pijut, P.M. & Shabanian, N. Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin . Plant Cell Tiss Organ Cult 127, 475–488 (2016). https://doi.org/10.1007/s11240-016-1072-8

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