In vitro Regeneration and Microscopic Studies of Castanea sativa Mill.

Siti Aishah, Zakeri (2024) In vitro Regeneration and Microscopic Studies of Castanea sativa Mill. Masters thesis, Universiti Malaysia Sarawak.

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Abstract

Castanea sativa Mill. (European chestnut) belongs to the Fagaceae family and is known among multipurpose plants. It is a significant industrial crop that is appreciated and processed globally to produce goods in various industries including food, medicine, furniture, and timber industry. At present, it is considered a staple food throughout southern Europe, Turkey, southwestern Asia and eastern Asia, which infers the essential for rapid multiplication and higher yield production of the species. Yet, there is still a lack of research on the propagation of C. sativa Mill. especially in Malaysia. Thus, the present study focuses on investigating the optimum medium for the in vitro propagation of C. sativa to produce plantlets on a large scale besides establishing the sterilization protocol for seed germination. Initially, the selected seeds were surface sterilized and cultured on MS basal medium. After 2 months, the nodal segments of aseptic seedlings were sectioned into smaller pieces and cultured on various treatments for shoot induction. Then, the cultures were maintained in the culture room. Among the parameters recorded were the percentage of shoot production, mean number of shoots produced, mean shoot height, mean number of leaves, and mean leave length. Furthermore, the 6-month-old plantlets regenerated in vitro were subjected to morphological and anatomical studies. The present study has discovered the sterilization protocol with 80% seed germination. Besides, the stage of seedling growth and development have been examined starting with the seed germination on the first week of culture, followed by seedling emergence and the appearance of true leaves and hairy roots after one month of culture. Additionally, direct in vitro propagation of C. sativa is influenced by either internal or external factors. Among the main factors include plant growth regulators and plant additives. Current findings clarified the treatments tested mostly promoted 100% shoot production whereby the explants could produce approximately 1-2 shoots per explant (≈1 shoot per explant) with 1.0-2.0 cm shoot height (≈2.0 cm) besides producing 1-4 leaves (2 leaves) and the leaves length within the range of 0.3-2.0 cm (1.0 cm) after 2 months of culture. Analysis of the results disclosed the most recommended medium for regeneration of the plant species in vitro was MS supplemented with CW + AC + 3.0mg/L BAP + 1.0mg/L NAA whereby 100% explants producing shoot within 2 months, with an average of 2 shoots per explant, 2.0 cm shoot height and 4 leaves with 1.0 cm length. Moreover, the morphological study has revealed the stomata dispersed widely on the abaxial leaf with the size of stomata opening, 12.45 μm length and 2.7 μm width. Meanwhile, the anatomical study disclosed unique internal structures of the leaf blade and midrib including the vascular bundles (phloem and xylem) and mesophyll cells (palisade and spongy). Hence, the present findings provide some basic information and understanding on the propagation of plant species in vitro. Nonetheless, further research should be performed to improve the protocol for seed sterilization and shoot induction of C. sativa Mill. in vitro. In fact, more parameters should be considered for data analysis as well as the other factors that could influence the growth and performance of C. sativa Mill. Keywords: European chestnut, micropropagation, seed germination, shoot induction, plant growth regulator.

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