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

Abstract

<jats:p>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 (<jats:italic>Juglans regia</jats:italic>) 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 (GA<jats:sub>3</jats:sub>) 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 GA<jats:sub>3</jats:sub> 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<jats:sup>−1</jats:sup> GA<jats:sub>3</jats:sub> (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.</jats:p>

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

“…In the present investigation, no major differences between fluorescence peak derived from nuclei of Ruta chalepensis plantlets and ex vitro plants were found. The findings of flow cytometric analysis are consistent with those obtained from earlier research on Mentha arvensis [69], Puya berteroniana [112], Solanum lycopersicum [113], Cucumis melo [114], Bacopa monnieri [34], Curcuma zedoaria [72], and Juglans regia [115].…”

“…It has been shown that the use of RAPD and DAMD molecular markers is efficient in determining the genetic stability of regenerated plants in a variety of medicinal plant species, including Withania somnifera [108], Henckelia incana [109], Avicennia marina [110], Ruta graveolens [18], Curcuma zedoaria [72], Artemisia vulgaris [100], Anarrhinum pubescens [111], and Anthurium andraeanum [106]. In recent years, it has been shown that a flow cytometry-based approach for evaluating in vitro raised plantlets is an excellent technique for assessing the clonal integrity and ploidy status in micropropagated plants [34,71,72,112,113]. In the present investigation, no major differences between fluorescence peak derived from nuclei of Ruta chalepensis plantlets and ex vitro plants were found.…”

Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry

“…Stability in genome size has also been reported in plants regenerated by somatic embryogenesis of Coriandrum sativum [ 88 ], Hibiscus sabdariffa [ 89 ], Milkweed spp. [ 90 ], Juglans directed [ 91 ], and Rauvolfia serpentina [ 92 ] evaluated by flow cytometry.…”

Genetic characteristics and ploidy trigger the high inducibility of double haploid (DH) inducer in Brassica napus

“…Flow cytometry has been used to assess plant ploidy [ 57 ]. In this research, flow cytometry is used to identify ploidy in all parental and F 1 hybrid combinations [ 58 ].…”