Germination of Persian Walnut Somatic Embryos and Evaluation of their Genetic Stability by ISSR Fingerprinting and Flow Cytometry

in HortScience

Somatic embryos (SEs) can play important roles in genetic manipulation and breeding. They can be used as targets for induced mutagenesis, as material for cryopreservation and germplasm conservation, and for transformation or gene editing in support of plant improvement and proof of gene function. However, germination rates of walnut (Juglans regia) SEs are low, and the genetic stability of plantlets regenerated from them has not been explored. Here, we studied first the effects of gibberellic acid (GA3) and low temperature storage (LTS) on germination of walnut somatic embryos. Second, we assessed the genetic fidelity of plantlets regenerated from these SEs by comparing them to each other and to their cultivar of origin. Results showed that GA3 and LTS increased the walnut SE germination rate. The best rate was observed when SEs were subjected to LTS for 60 d followed by culture on a medium with either 1 or 3 mg·L−1 GA3 (56.6% and 46.6% germination respectively). Genetic stability was evaluated, using flow cytometry and 15 sets of ISSR primers. Flow cytometry indicated that all samples (i.e., regenerated and parental counterpart) showed the same peak. Amplified fragments of inter simple sequence repeats (ISSR) primers ranged in size from ≈200 to 1800 bp. All ISSR profiles of regenerants were monomorphic. Results did not show any genetic differences among plantlets regenerated from SEs or from their parental counterpart. Due to this apparent genetic stability, walnut SEs can be useful for genetic transformation and germplasm conservation.

Contributor Notes

We would like to thank the University of Tehran, Center of Excellence for Walnut Improvement and Technology, Iran National Foundation for Science and Technology (INSF), and the Biotechnology Development Council of the Islamic Republic of Iran (grant number 960805) for their support.

Corresponding author. E-mail: kvahdati@ut.ac.ir.

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Article Figures

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    Somatic embryos and plant regeneration of J. regia. (A) Somatic embryos 12 d after subculture. (B) Globular embryos formed after 2 weeks. (C) and (D) mature and torpedo somatic embryos. (E) Somatic embryo before transferring to germination medium (bar = 3 mm). (F) Necrotic somatic embryos on the germination medium. (G) SE with only shoot formation. (H) SE with only root formation. (I) SE with shoot and root meristem. (J) Growing regenerated shoots developed from a somatic embryo on DKW medium under in vitro conditions (bar = 6 mm).

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    Flow cytometry analysis of J. regia. (A) Typical flow cytometry profile of plants regenerated by SEs (all samples showed 2 at channel 100). (B) Flow cytometry of the parental counterpart, the cultivar Chandler.

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    ISSR fingerprint profile obtained with primer (AGTG)4 (A), (GA)8-TC (B), and CAC-(TGT)5 (C), respectively. M = mother plant (the cultivar Chandler); 1–7 regenerants from ‘Chandler’ somatic embryos.

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