Initiation and Cytological Aspects of Somatic Embryogenesis in Dendrobium candidum Wall ex Lindl.

in HortScience

To find the characteristics of somatic embryogenesis of orchids and elucidate the mechanism, we had previously established an efficient plant regeneration system via somatic embryogenesis in Dendrobium candidum Wall ex Lindl. In this study, a detailed cytological investigation was carried out on the initiation and developmental process of somatic embryogenesis. Based on our observations, the somatic embryogenesis in D. candidum originated from the transition of an embryonic callus cell to the initial somatic embryo cell, and the somatic embryos initiated from those cells. During the transition process, condensation and devacuolation successively occurred in the cytoplasm of the embryonic callus cells, giving rise to the formation of a typical initial somatic embryo cell with dense cytoplasm and a clear nucleus. One of the two pathways in somatic embryogenesis is the single-cell-derived somatic embryo which is generated from an inner initial somatic embryo cell in embryonic callus and develops into a globular somatic embryo in a way similar to zygotic embryogenesis and then keeps developing into a protocorm-like body (PLB). The other is a multiple-cell-derived somatic embryo which is generated from peripheral grouped initial somatic cells in embryonic calli and directly forms globular embryo or multicellular somatic proembryo, lacking the typical early stages of embryogenesis. Both pathways were observed in the somatic embryogenesis system, indicating that the culture system in D. candidum can be a useful tool for investigating the mechanisms underlying orchid embryogenesis.

Contributor Notes

This work was supported by the Grant (No. 31371232) from the National Natural Science Foundation of the People’s Republic of China.

Co-first authors.

Corresponding author. E-mail: wanjunwang@home.swjtu.edu.cn.

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Article Figures

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    Cytological characters of embryonic callus cell and initial somatic embryo cell. (A) Embryonic callus cells with loosened cytoplasmic materials. (B and C) Cytoplasmic materials were getting condensed, and a clear denser cytoplasm was formed in some cells (arrow). (D) Initial somatic embryo cells with a denser cytoplasm, clear nuclear and small vacuole (arrow). Bar = 50 μm.

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    Development of somatic embryos derived from epidermal layer of embryonic callus. (A) Some of the initial somatic embryo cells on the surface of embryonic callus grouped together (bar = 50 μm). (B) The initial somatic embryo cells located in the sublayer of embryonic callus gathered together (bar = 100 μm). (C) A young globular somatic embryo on the surface of callus (bar = 50 μm). (D) A multicellular somatic proembryo formed from the gathered multiple initial somatic embryo cells in the sublayer of embryonic callus (bar = 50 μm). (E) A bigger somatic proembryo developed from the multiple initial somatic embryo cells in the sublayer of embryonic callus is getting ready to stick out (bar = 50 μm). (F) A globular somatic embryo originated from the multiple initial somatic embryo cells in the sublayer of embryonic callus (bar = 200 μm). (GI) Scanning electron microscopic observation on somatic embryo initiation derived from epidermal layer of embryonic callus. (G) The surface of embryonic callus before somatic embryo formation (bar = 100 µm). (H) Somatic embryos (arrow) on the surface of callus (bar = 100 µm). (I) A globular somatic embryo (arrow) on the surface of the callus (bar = 200 µm).

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    Cell division pattern of the inner initial somatic embryo cell at the earliest stage of somatic embryogenesis. (A) An initial somatic embryo cell inside the embryonic callus (bar = 30 μm). (B) The first cell division of the inner initial somatic embryo cell results in the formation of a smaller “apical cell” and a larger “basal cell” (bar = 30 μm). (C) The “basal cell” divided earlier than “apical cell” (bar = 50 μm). (D) A three-celled somatic embryo (bar = 50 μm). (E) A four-celled somatic embryo (bar = 50 μm). (F) A five-celled somatic embryo (bar = 50 μm).

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    Somatic embryo differentiation. (A) A cone-shaped somatic proembryo with a suspensor-like structure within callus (bar = 100 μm). (B) Pear-shaped somatic embryo generated inside the embryonic callus would develop into a globular somatic embryo and stick out of the callus before the differentiation stage (bar = 200 μm). (C) A cup- or heart-shaped somatic embryo differentiated from a globular somatic embryo (bar = 250 μm). (D) A landmine-shaped somatic embryo with promeristem (arrow) and a rhizoid (star) (bar = 500 μm).

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