Nghiên cứu tạo dòng lan giả hạc tím Huế đột biến - Luận án TS Nguyễn Hữu Thọ

ĐẠI HỌC HUẾ VIỆN CÔNG NGHỆ SINH HỌC NGUYỄN HỮU THỌ DANH MỤC CÁC BÀI BÁO, CÔNG TRÌNH LIÊN QUAN ĐẾN LUẬN ÁN Tên luận án: Nghiên cứu tạo một số dòng lan giả hạc tím Huế (Dendrobium anosmum Lindl.) đột biến bằng phương pháp thực nghiệm. Chuyên ngành: Sinh học Mã số: 9420101 Huế, 2026 DANH MỤC CÁC BÀI BÁO, CÔNG TRÌNH LIÊN QUAN ĐẾN LUẬN ÁN 1. Nguyen Huu Tho, Nguyen Thi Oanh, Truong Thi Bich Phuong, Pham Thanh, Hoang Khanh Linh, Nguyen Thi Kim Cuc (2023). Conservation of Dendrobium anosmum ‘tim Hue’ by in vitro propagation.

Tạp chí Journal Propagation of Ornamental Plants, Tập 23, số 2, 39-48. Nguyễn Hữu Thọ, Nguyễn Thị Oanh, Trương Thị Bích Phượng, Nguyễn Thị Kim Cúc (2024). Ảnh hưởng của một số yếu tố đến nhân giống lan Giả hạc tím Huế (Dendrobium anosmum Lindl.) bằng phương pháp nuôi cấy in vitro. Hội thảo Khoa học quốc tế về “Nghiên cứu đa dạng sinh học và tiềm năng dược liệu thuộc họ Orchidaceae (Họ Lan), Theaceae (Họ trà) và Zingiberaceae (Họ Gừng) ở Việt Nam”.

Nhà xuất bản Đại học Huế, 62-68. Nguyen Huu Tho, Nguyen Thi Oanh, Truong Thi Bich Phuong, Nguyen Thi Kim Cuc (2024). Dendrobium anosmum Lindl. ‘Tim Hue’ and the method of vegetative propagation from the axillary node shoots of the stem (keikis).

Hội nghị khoa học Quốc gia lần thứ 6 về Nghiên cứu và giảng dạy sinh học ở Việt Nam, 908-915. Nguyễn Hữu Thọ, Nguyễn Thị Oanh, Lã Thị Thu Hằng, Trương Thị Bích Phượng, Nguyễn Thị Kim Cúc (2024). Nghiên cứu ảnh hưởng của Dimethyl sulfide (DMS) đến tạo biến dị lan Giả hạc tím Huế (Dendrobium anosmum Lindl). Hội nghị khoa học toàn quốc về Công nghệ sinh học, 467-473.

Nguyễn Hữu Thọ, Trương Thị Bích Phượng, Hồ Thị Xuân Tuý, Phạm Thành, Nguyễn Thị Kim Cúc (2024). Nghiên cứu phân lập vi khuẩn nội sinh từ một số giống lan rừng. Tạp chí Đại học Huế, Tập 133, số 1C. Tho Nguyen Huu, An Nguyen Hoang, Tuy Ho Thi Xuan, Cuc Nguyen Thi Kim, Phuong Truong Thi Bich (2026).

Establishment of callus-derived polyploid lines for the Purple Gia Hac orchid (Dendrobium anosmum Lindl.) via colchicine treatments. Plant Cell, Tissue and Organ Culture (PCTOC), 165(2), 36.1007/s11240-026-03456-5 Propagation of Ornamental Plants Vol. 23, № 2, 2023: 39-48 CONSERVATION OF DENDROBIUM ANOSMUM LINDL. ‘TIM HUE’ BY IN VITRO PROPAGATION Nguyen Huu Tho1,4, Nguyen Thi Oanh1, Truong Thi Bich Phuong2, Pham Thanh3, Hoang Khanh Linh3, and Nguyen Thi Kim Cuc1* 1 Institute of Biotechnology, Hue University, Road 10, Phu Thuong, Phu Vang, Thua Thien Hue, Vietnam, *E-mail: ntkcuc.vn 2 Hue University of Sciences, Hue University, 77 Nguyen Hue, Hue City, Thua Thien Hue, Vietnam 3 Hue University of Education, Hue University, 34 Le Loi, Hue City, Thua Thien Hue, Vietnam 4 College Electro-Mechanics, Construction and Agro-Forestry of Central Vietnam, KV8, Bui Thi Xuan Ward, Quy Nhon city, Binh Dinh, Vietnam Abstract Dendrobium anosmum ‘Tim Hue’ is a local orchid from the Thua Thien Hue wild forest with typical color and fragrance, needing conservation measures.

This study focuses on in vitro propagation of ‘Tim Hue’. Nodal segments used as initial explants were disinfected in 2% NaClO and 0.1% HgCl2 for 10 min, resulting in 66. Axillary shoot induction was best (88.9%) in Murashige and Skoog (MS) medium contain- ing 1.5 mg l-1 BAP and shoot multiplication was optimal in the same medium supplemented with 1.5 mg l-1 BAP and 0.5 mg l-1 IAA, with an estimated average of 6.0 cm in length and 4.7 leaves after eight weeks of culture. Also, MS medium supplemented with 1 g l-1 activated charcoal, and 0.7 mg l-1 NAA or 0.5 mg l-1 IAA was found the most efficient medium for the rooting of shoots.

‘Tim Hue’ plantlets were well acclimatized to the nursery conditions with a survival rate of 97.8% on coconut coir and rice husk coal (1 : 1, w : w) or 95.6% on fern board with sphagnum moss. Key words: conservation, Dendrobium anosmum Lindl. ‘Tim Hue’, in vitro propagation INTRODUCTION Among floral varieties, orchids are famous orna- The overall decline in biodiversity has dramatically mentals with a global marketing industry, but they increased in recent years, therefore, it is imperative are recently at risk of extinction in nature (Zhao et al. to implement conservation strategies to conserve and 2021).

Previously, growing orchids in the wild mainly propagate plant genetic resources from each geographi- depended on vegetable regeneration because their seeds cal area (Deplazes-Zemp 2018). Previously, valuable lack endosperm and have a very low germination rate clonal plants, that have unique characteristics were (Zhao et al. To overcome that fact, the micro- conserved by traditional vegetative propagation instead propagation technique has been successfully applied of sexual reproduction (Rajasekharan and Sahijram to generate orchid plantlets from seeds and somatic 2015), however these techniques are difficult to mass tissues (Chugh et al. 2009) that are important to the production of seedlings (Högberg 2003).

To overcome market and maintain the orchid gene pool (Reddy et the disadvantage of the traditional method, in vitro al. So far, many orchid species have been suc- tissue culture has been applied to generate mass plant- cessful cultured by seed germination in aseptic condi- lets from a few initial explants (Hussain et al. tions as Laelia anceps Lindl. (Ramírez-Mosqueda et Commonly, apical meristem or axillary buds, which al.

2019), Epidendrum nocturnum (De Stefano et al. retain good developmental capacity, were preferred 2022), Vanda pumila Hook. (Maharjan et al. 2019), as initial materials to differentiate tissues in in vitro Dendrobium secundum (Ramasoot et al.

Sofar bium anosmum Lindl. (Nguyen et al. 2022b), however, different crops, flowers, and fruitswere successfully it is valuable to note that the in vitro propagation of micropropagation using artificial media with suitable orchid seeds may generate heterozygous plantlets with growth hormone regulators (Hasnain et al. Therefore, some group, recently, tried to Received: April 13, 2023 Accepted: May 1, 2023 39 Nguyen Huu Tho et al.

Conservation of Dendrobium anosmum ‘Tim Hue’ by in vitro propagation use somatic tissues as initial material to micropropaga- To evaluate the effect of different factors on disinfec- tion of valuable orchid, such as Dendrobium palpebrae tion, the percentage of infected explants was counted Lindl. trankimianum after a week. Yukawa (Bing et al. 2018), Phalaenopsis amabilis cv.

Detroit (Balilashaki et al. Axillary shoot induction Vietnam has various native orchids with unique The disinfected nodal segments were transferred to ornamental characteristics, and some of them were modified MS medium, and supplemented with 0.5, propagationand conservation by somatic in vitro culture 1.0 mg l-1 of 6-benzylaminopurine (BAP) (Bing et al. 2018, Nguyen et al. 2018, Vu et al.

for axillary shoot induction. Three replications, each However, it needs to pay more attention to saving containing 10 explants were used per treatment. The natural orchids. Dendrobium anosmum ‘Tim Hue’, percentage of regenerated shoots from buds, the number originated from Thua Thien Hue wild forest, is an at- and the average length of shoots were monitored after tractive orchid species with violet flowers and a sweet, eight weeks.

Unfortunately, this orchid is subject to indiscriminate exploitation and is at risk of extinction Multiplication of shoots from wild habitats. Moreover, the ability to propagate The isolated axillary shoots were cultured in modi- ‘Tim Hue’ in the wild is rare due to the low frequency fied MS medium containing mixed treatments of dif- of seed germination and the infrequent bud develop- ferent concentrations (0.7 mg l-1) ment from stocks. Therefore the aim of our study was to of indole-3-acetic acid (IAA) and BAP. The shoot study the effect of different plant growth regulators and proliferation coefficient, average shoot length, number their concentrations for in vitro propagation by axillary of leaves, and shoot morphology were monitored after shoots and acclimatization of the produced plantlets.

8 weeks of culture. Shoot proliferation coefficient (H) was calculated as: MATERIALS AND METHODS number of shoots after 8 weeks H= Plant material and surface disinfection of the explants initial number of nodal segments D. anosmum ‘Tim Hue’ from the wild forest in Thua Thien Hue province, Vietnam was collected and Rooting of adventitious shoots grown in the net house (Institute of Biotechnology, Adventitious shoots (3-4 leaves and 3-4 cm in Hue University). length) were excised and cultured to modified MS Healthy, disease-free nodal segments about 15-20 cm medium supplemented with α-naphthaleneacetic acid long of D.

anosmum ‘Tim Hue’ were collected in June/ (NAA) (0.0 mg l-1) or IAA July 2021. They were washed under running water, and (0.0 mg l-1) and 1 g l-1 activated gently shaken with a diluted commercial soap for 5 min, charcoal (Nguyen et al. The rooting ability is then rinsed several times with tap water to completely determined by the percentage of rooted shoots, the remove the residue of soap. The isolated nodal segments average length, and number of roots after eight weeks.

6-7 cm in length, were placed in a 500 ml Erlenmeyer Each treatment had six replications, and five shoots flask and washed again every 2 min by shaking them were used in each replication. in distilled water 2-3 times. Disinfection was carried out using 2% NaClO for 10 min (David et al. 2022), Acclimatization of the plantlets 3% H2O2 for 10-20 min (De Stefano et al.

2022), and The plantlets with 4-5 cm in length, 5-6 roots, and 0.1% HgCl2 for 5-10 min (Bing et al. 2018) and a dif- 5-6 true leaves were acclimated in a net house covered ferent combination of 2% NaClO, 3% H2O2, and 0.1% with a plastic roof and sun mesh shade to maintain HgCl2 (Table 1). Finally, the explants were rinsed with PPDF between 18.5-37 µmol s-1m-2, earthen floor with sterile distilled water 3-4 times before embedding in tarps, and equipped with an irrigation system to produce modified plant growth regulators (PGRs)-free MS me- mish which helps to maintain the temperature in the dium (Murashige and Skoog 1962) supplemented with range 25-32°C. All plants in aseptic culture bags were 100 ml l-1 coconut water; 7 g l-1 agar, 30 g l-1 sucrose, hung on cloth ropes in the net house for two weeks and 60 g l-1 potatoes paste).

The nutrient medium was before taking out and planting in different substrates adjusted to pH 5.8 before being autoclaved and then (Teixeira da Silva et al. After acclimatization of poured into a square-bottomed polypropylene (PP) bag plantlets in the aseptic condition in the net house for with approximately 70 ml of medium/bag (Nguyen et two weeks, the agar-containing medium was removed al. 2022c) and the in vitro culture conditions were set from the roots by washing in tap water, and the plantlets at 25 ± 2°C, and photosynthetic photon flux density were planted in mixed coconut coir and rice husk char- (PPDF) was between 13.5-27 µmol s-1m-2 for 8 h/day coal (1 : 1, w : w; CT1) (Nguyen et al. 2021); coconut if not further note.

coir and sphagnum moss (1 : 1, w : w; CT2); coconut 40 Propagation of Ornamental Plants Vol. 23, № 2, 2023: 39-48 coir and pine bark (1 : 1, w : w; CT3); sphagnum moss that the 2% NaClO and 0.1% HgCl2 combination was (CT4); Coconut coir (CT5); fern board (20 × 15 cm) better for disinfection than 2% NaClO and 3% H2O2, with sphagnum moss (Kang et al. 2020) (CT6) or Star but the long-term exposure to HgCl2 could be toxicity apple wood board (20 × 15 cm) with sphagnum moss or induced tissue degeneration leading to an increased (Pradhan et al. After 12 weeks, the sur- number of dead explants (Table 1).

Our results were vival rate, stem length and diameter, number of leaves, consistent with the observation of Hashim et al. (2021) leaf size, shape, and color of plantlets were monitored. who performed the similar experiment in Clinacanthus Three replications, each containing 10 plantlets were nutans (Burm. used per treatment.