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Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.)

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Abstract

Somatic embryogenesis was obtained from immature cotyledon explants that were cultured on half-strength Murashige and Skoog (MS) salts and vitamins with 5.4 μM naphthaleneacetic acid (NAA) and 0.2 μM thidiazuron (TDZ) plus a 4 × 4 factorial combination of 0, 9.8, 24.6, or 49.2 μM indole-3-butyric acid (IBA) and 0, 8.9, 22.2, or 44.4 μM 6-benzyladenine (BA). The addition of 44.4 μM BA improved the percentage of cotyledon explants that produced somatic embryos to >20%, if 9.8 or 24.6 μM IBA was also present. Somatic embryogenesis was >30% when seeds were harvested on 31 July or 15 August. The addition of 50 or 70 g l−1 sucrose enhanced embryogenesis. Histological examination showed that somatic embryos originated from epidermis cells of zygotic embryos. A peak germination rate (69%) was attained when somatic embryos were desiccated for 10 min before they produced green cotyledons and elongating shoot tips. Of the germinated embryos from this desiccation treatment, 65.6% also grew roots and therefore converted into plants. Chilling somatic embryos at 4°C for 15 days resulted in the highest germination rate (69.4%), which was significantly higher than those without chilling treatment (27.6%). However <10% of the chilled germinated embryos formed roots and grew into plants. Plantlets from somatic embryos were transplanted into a 2 vermiculite: 1 sphagnum peat medium, where they had a survival rate of 80.8%, and had no morphological abnormalities.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

6-Benzyladenine

IBA:

Indole-3-butyric acid

MS:

Murashige and Skoog medium

NAA:

Naphthaleneacetic acid

PGR:

Plant growth regulator

TDZ:

Thidiazuron, N-phenyl-N′-1,2,3-thiadiazol-5-yl urea

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Acknowledgments

Funding was provided by four grants from National Natural Science Foundation of China (Project No. 30872008, 30040041, 30170772, 30371155). We thank Dr. David South, professor at Auburn University in USA for his advice and comments. We also thank Dr. Peng Zhang, Ms. Gun-Jun Sun, Ms. Nan Li, Ms Li-Jie Zhang, Ms. Dan–Dan Feng, Ms. Yu Zhang, Ms. Yan-Shuang Tan, Mr. Jian Huang, Ms. Ai-Zhi Wang, Ms. Li-Wei Zhang, Ms. Hong-Mei Wang and Mr. Li-Jiang Hu, Dr. Ling Yang, Ms. Chun-Ping Liu, and Dr. Yu-Hua Li from the School of Forestry, Northeast Forestry University for assistance with the experiments.

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Authors and Affiliations

  1. College of Life Science, Shanxi University, No. 92, Wucheng Road, Taiyuan, 030006, China

    Dong-Mei Kong

  2. Department of Plant, Soil and Agricultural Systems, Southern Illinois University, MC 4415, Carbondale, IL, 62901, USA

    John E. Preece

  3. State Key Laboratory of Forest Genetics and Tree Breeding, Northeast Forestry University, Hexing-Lu 26, Harbin, 150040, China

    Hai-Long Shen

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  1. Dong-Mei Kong
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  2. John E. Preece
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Correspondence to Dong-Mei Kong.

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Kong, DM., Preece, J.E. & Shen, HL. Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.). Plant Cell Tiss Organ Cult 108, 485–492 (2012). https://doi.org/10.1007/s11240-011-0062-0

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  • DOI: https://doi.org/10.1007/s11240-011-0062-0

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