The Southeast Asian Journal of Tropical Biology Vol. 31 No. 3, 2024: 391 - 401 DOI: 10.11598/btb.2024.31.3.2284 ISSN: 0215-6334 | e-ISSN: 1907-770X 391 ARTICLE HIGLIGHTS • Camellia dalatensis and Camellia capitata are critically endangered species. • Habitat loss and deforestation threaten the survival of these Camellia species. • Vegetative propagation aids conservation of critically endangered Camellia species. • Rooting success depends on cutting types, growth regulators, and substrate mix. • Research aids conservation efforts for Vietnam’s rare and vulnerable flora. Article Information Received 20 June 2024 Revised 16 August 2024 Accepted 10 September 2024 Reviewer: Eucharia Chizoba Ezigbo & anonymous *Corresponding author, e-mail: lehongen@gmail.com Research Article CONSERVATION STATUS AND PROPAGATION OF Camellia dalatensis AND Camellia capitata BY USING CUTTINGS Le Hong En1*, Do Van Duong1, Nguyen Pham Doan1, Nguyen Van Phuc1, Ngo Van Cam1, Le Thi Thuy Hoa1, Nguyen Ba Trung1, Ho Si Hung1, Ngo Giang Phi1, Nguyen Thanh Nguyen1, Hoang Thanh Truong1, Truong Quang Cuong2 1Forest Science Institute of Central Highlands and South of Central Vietnam, Da Lat City 66000, Lam Dong Province, Vietnam. 2Bidoup Nui Ba National Park, Da Lat City 66000, Lam Dong Province, Vietnam ABSTRACT Camellia dalatensis (V. D. Luong, Ninh & Hakoda) and Camellia capitata (Orel, Curry & Luu) are classified as critically endangered (CR) by the IUCN. Conservation solutions for these two species have not yet been fully implemented, while wild populations are declining. This research on vegetative propagation examined the efficacy of indole-3-butyric acid (IBA in solution or in powdered activated charcoal), cutting types (terminal shoot or stem cuttings), and substrates for the growth of cuttings (sand-coir dust mixes) on rooting efficiency. The highest rooting of C. dalatensis was observed in terminal shoot cuttings at 1,000 ppm IBA, and 25% sand and 75% coir dust substrate. Maximum values obtained were survival 95.6%, rooting percentage 88.9%, number of roots per cutting 6.9, root length 6.2 cm, and rooting index 42.9. For C. capitata, superior rooting was obtained with semi-hardwood cuttings, at 1,500 ppm IBA, and equal parts of sand and coir dust as substrate. Camellia capitata was more difficult to root, with maximum values of 65.6% survival, 52.2% rooting, 2.8 roots per cutting, 2.9 cm root length, and rooting index of 8.1. The results of this study can be used to support further propagation and conservation of these two endangered species. Keywords: Camellia capitata, Camellia dalatensis, endangered species, IBA, rooting, vegetative propagation INTRODUCTION Camellia L., the largest genus in the Theaceae family, is widely distributed from Bhutan, Northeast India, China, and Japan to Indonesia and the Philippines (Chang & Bartholomew 1984; Ming & Bartholomew 2007). Vietnam has a high diversity of Camellia and many species have been recently recorded, such as C. hiepii, C. hoaana, C. hoabinhensis, C. maianhii, C. pyriformis, and C. vanlangensis (Nguyen et al. 2023; Trinh et al. 2023; Yang et al. 2024; Trinh & Hoang 2024; Quach et al. 2024). Camellia dalatensis was described in 2012 (Tran & Luong 2012) and C. capitata in 2014 (Orel et al. 2014). These two species are classified as critically endangered (CR) by the IUCN, with low regeneration potential due to habitat changes under the impacts of climate change and human impacts, such as deforestation from shifting cultivation and overexploitation. Therefore, more quantitative information on the status and https://doi.org/10.11598/btb.2024.31.3.2284 BIOTROPIA Vol. 31 No. 3, 2024 392 conservation strategies of these threatened Camellia is needed (Rivers & Luu 2018; Orel et al. 2014). Propagation is one of the important components to provide seedlings for species conservation and development programs. Each propagation method has its advantages and disadvantages. Propagation from seeds is easy to implement. However, these two species have disadvantages in implementing seed propagation, i.e., these two species have a small number of individuals, poor fruiting ability, and seasonal seed availability. Propagation via tissue culture has the potential to produce a large number of plants, but this technique requires high technology, high costs, and a long time to conduct. A cheaper alternative approach is to use cuttings for propagating these two Camellia species, in which this technique has the advantage of preserving the genetic characteristics of the mother trees. Propagation by using cuttings has been successful for other indigenous Camellia species in Vietnam, including C. chrysantha, C. flava, C. petelotii, and C. tamdaoensis (Nguyen et al. 2017; Nguyen et al. 2021). The aims of this study were: 1) to assess the conservation status of C. dalatensis and C. capitata in the field and 2) to determine the effects of auxin, cutting types, and substrates on root formation of the cuttings. The research results will contribute to supporting the propagation and conservation of these two threatened species of Camellia. MATERIALS AND METHODS Assessment of Conservation Status A survey of C. dalatensis and C. capitata was undertaken using the snowball sampling approach with 15 respondents (5 forest managers, 5 ethnic people living near the forest boundary, and 5 forest planters). Before completing the interview, all respondents were freely consented. The interview questionnaire had three questions: (1) Have you ever seen these two plant species?; (2) Where did you see these two plant species?; and (3) How many people know about these two species at this location? Following the interviews, we established 5 survey transects per species, each ranging in length from 2 to 3 km, to locate mature trees. The program http://geocat.kew.org/editor (Royal Botanic Garden 2024) and the IUCN Red List Criteria and Classification Guidelines (IUCN 2022) (http://www.iucnredlist.org/documents/ RedListGuidelines.pdf ) were used to calculate the Extent of Occurrence (EOO) and Area of Occupancy (AOO). Cutting Experiments Source of Mother Trees C. dalatensis was obtained from natural forest in Tram Hanh Commune, Da Lat City, Lam Dong Province, Vietnam. C. capitata was gathered from natural forests in Phuoc Cat Commune, Cat Tien District, Lam Dong Province, Vietnam. Sample Collection and Processing The branches of C. capitata and C. dalatensis were gathered in January and May of 2022, respectively. Branches with a diameter of 0.5-0.8 cm were chopped into 50-60 cm long sections, placed in foam crates, moistened with wet towels, and then transported to Da Lat City. The stems were rinsed multiple times in clean water before being cut around 1 cm from the node into 10-15 cm long portions for the research trials. Location of Propagation Experiments The propagation experiments were set up in a greenhouse at the Forest Science Institute of Central Highlands and South of Central Vietnam (FSIH), Da Lat City, Lam Dong Province, Vietnam (11o56’35” N, 108o24’23” E). The altitude of the nursery was 1,504 m, the average annual temperature was 18-25 oC, the average annual rainfall was about 2,200 mm, and the wet season happened from May to November. The misting mode in the greenhouse was set up at 20 seconds every 2 hours. Experimental Design Experiment 1 - Effect of IBA on Root Formation The experiment was undertaken with 2 types of indole-3-butyric acid (IBA) in solution and IBA powder combined with activated charcoal. Five concentrations of IBA solution were prepared (0; 500; 1,000; 1,500; and 2,000 ppm) in water. The cuttings were submerged in the IBA solution for 15 Conservation status and propagation of Camellia dalatensis and Camellia capitata - En et. al. 393 minutes. IBA-activated charcoal treatments were 0, 0.5, 1.0, 1.5, and 2.0%. The IBA was dissolved in a small volume of alcohol and then mixed with activated charcoal to form a slurry. The slurries were dried to evaporate the alcohol and then were ground and passed through a sieve (0.35 mm mesh size). The cuttings were briefly dipped into the powder. Experiments were carried out using washed river sand in rectangular plastic drainage baskets with dimensions of 35 cm (length) x 26 cm (width) x 10 cm (height). Rooting development was assessed on day 120. Experiment 2 - Effect of Cutting Types on Root Formation The experiment compared two treatments of cutting types, i.e., terminal shoot and semi-hard- wood stem cuttings. Sand substrate was used in combination with the optimal result of IBA in experiment 1. Rooting development was assessed on day 120. Experiment 3 - Effect of Substrates on Root Formation Four treatments of substrates were applied, i.e., 75% sand and 25% coir dust, 50% sand and 50% coir dust, 25% sand and 75% coir dust, and 100% coir dust. Rooting development was assessed on day 90. Data Analysis The experiments were conducted in a randomized block design with three replications. The data measured were survival percentage (%), rooting percentage (%), number of roots, root length (cm), and rooting index (number of roots multiplied by root length). The data were analyzed using the Duncan test in SPSS 26.0.0 software (Statistical Package for Social Sciences version 26). RESULTS AND DISCUSSION Conservation Status Camellia dalatensis has a narrow distribution in Phat Chi Village, Tram Hanh Commune, Da Lat City, Lam Dong Province. The number of mature individuals in the natural habitat was 205 plants. The Extent of Occurrence (EOO: 0.464 km2) and Area of Occupancy (AOO: 8,000 km2) for Critically Endangered (CR) C. dalatensis are illustrated in Figure 1 (upper panel). Camellia capitata occurs in Cat Tien National Park, an area managed by Lam Dong Province (Cat Tien District). There were 5 mature plants in the natural habitat. C. capitata is categorized as Critically Endangered (CR) with EOO of 0.050 km2 and AOO of 4,000 km2 (Figure 1; lower panel). Both C. dalatensis and C. capitata were shown to have a very small distribution area (Fig.2), which were threatened by logging, deforestation, planting of industrial crops, such as cashew, rubber and coffee, and the harvesting of non-timber forest products (Beech et al. 2017; Orel et al. 2014). Recently, the forests are being exploited for medicinal herbs and ornamental plants with high polyphenol contents and beautiful flowers, including these two Camellia species (Fig. 3) (Trinh 2022). Effect of IBA on Root Formation of C. dalatensis and C. capitata Asexual propagation by using cuttings is the optimal solution for preserving genetic characteristics from mature plants when propagation by using seeds is limited by low fecundity. There are many factors affecting rooting, including the environment, the types of cuttings used for propagation, and the use of plant growth regulators (PGRs) (Bhupathireddy et al. 2022; Dewi & Sabhara 2022; Eed & Burgoyne 2014; Gautam et al. 2022; Griffin et al. 1998; Ray & Ali 2017). Cutting propagation efficiency is often increased by using exogenous PGRs, including IBA treatments (Bhupathireddy et al. 2022; Gautam et al. 2022). However, the optimum concentrations and PGR forms (solution or solid form) can differ markedly between species. For example, 1,000 ppm IBA in solution gave the best results in Lycium barbarum L., but 1% IBA was optimal for Cornus mas L. (Çelik & Çetin 2021). In this study, 1,000 ppm IBA was suitable for C. dalatensis, but C. capitata required a higher concentration of 1,500 ppm IBA for obtaining the highest rooting percentage. BIOTROPIA Vol. 31 No. 3, 2024 394 Figure 1 Extent of Occurrence (EOO) and Area of Occupancy (AOO) of Camellia dalatensis (upper panel) and C. capitata (lower panel) Figure 2 Distribution map of Camellia capitata and C. dalatensis in Lam Dong Province, Vietnam Conservation status and propagation of Camellia dalatensis and Camellia capitata - En et. al. 395 Figure 3 Appearance of Camellia capitata and C. dalatensis Notes: a. Seedlings of C. dalatensis in their natural habitat; b. Flower of C. dalatensis; c. Flower of C. capitata; d. Seedlings of C. capitata in their natural habitat. Differences in PGR requirements for the rooting of cuttings not only vary between plant species, but also depend on the position in the plant where the cuttings are sourced. Cuttings taken from mature trees often have reduced endogenous auxin content compared to cuttings taken from juvenile plants. Hence, it is necessary to undertake experiments on cuttings responses toward PGR dosages to ensure that optimal PGR dosages are being used for the correct cutting type. Also, at very high concentrations, the rooting efficiency can be significantly reduced, such as in Prunus laurocerasus L. at 2 g/L IBA (Sulusoglu & Cavusoglu 2010). Thus, depending on the type of tree and the age of the tree, the rooting auxin treatment needs to be adjusted to the appropriate concentration, and it is necessary to have specific studies for each tree species. The effectiveness of using IBA in propagation of threatened Camellia species are shown in Tables 1 and 2. There were significant (P < 0.05) effects of IBA rates on survival percentage, rooting percentage, number of roots, root length, and rooting index. For C. dalatensis, the survival percentage ranged from 65.56% to 80%; the rooting percentage ranged from 14.45% to 45.56%; the number of roots ranged from 0.57 to 3.80; the root length ranged from 1.67 cm to 4.23 cm; and the rooting index ranged from 1.12 to 16.32. The most effective treatment was 1,000 ppm IBA. For C. capitata, the survival percentage ranged from 28.89% to 40.00%; the rooting percentage ranged from 7.78% to 31.11%; the number of roots ranged from 0.30 to 1.90; the root length ranged from 0.40 cm to 2.13 cm; and the rooting index ranged from 0.15 to 4.09. The most effective treatment was 1,500 ppm IBA. BIOTROPIA Vol. 31 No. 3, 2024 396 Table 1 Effect of IBA concentration on Camellia dalatensis root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index 500 ppm 74.45±2.22ab 32.22±2.94bc 1.73±0.35bcd 2.43±0.15b 4.29±1.11c 1,000 ppm 77.78±1.11ab 45.56±2.94a 3.80±0.32a 4.23±0.38a 16.32±2.76a 1,500 ppm 76.67±1.93ab 37.78±4.01ab 3.73±0.90a 2.90±0.75b 11.72±4.83ab 2,000 ppm 76.67±1.93ab 31.11±2.94bc 2.50±0.61ab 2.80±0.45b 6.85±2.06bc 0.5% 72.22±2.94b 22.22±2.94de 1.33±0.12bcd 2.87±0.15b 3.85±0.50c 1.0% 80.00±1.92a 35.56±1.11b 2.10±0.35bc 2.83±0.38b 5.73±0.65bc 1.5% 77.78±1.11ab 25.55±2.22cd 2.07±0.19bc 2.57±0.46b 5.29±1.10bc 2.0% 65.56±1.11c 20.00±1.92de 0.63±0.29cd 1.70±0.32b 1.12±0.67c Not using PGRs 74.44±1.11ab 14.45±2.22e 0.57±0.42d 1.67±0.22b 1.12±0.91c Mean 75.06±0.93 29.38±1.95 2.05±0.25 2.67±0.18 6.25±1.10c P-value 0.001 0.000 0.000 0.012 0.001 Notes: *Mean values in columns followed by different letters are statistically significantly different with P ≤ 0.05 using Duncan’s multiple range test. Table 2 Effect of IBA concentration on Camellia capitata root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index 500 ppm 38.89±1.11ab 16.67±1.93c 0.83±0.90bcd 1.67±0.12c 0.99±0.21bc 1,000 ppm 40.00±1.92a 25.55±2.22b 1.37±0.12ab 1.53±0.12bc 2.10±0.25b 1,500 ppm 40.00±1.92a 31.11±1.11a 1.90±0.15a 2.13±0.24a 4.09±0.70a 2,000 ppm 31.11±1.11bcd 25.57±1.11b 0.87±0.09bcd 1.23±0.27c 1.05±0.23bc 0.5% 36.67±3.85abc 13.33±1.93c 0.57±0.09cd 1.23±0.12c 0.68±0.06bc 1.0% 32.22±2.94abcd 14.44±1.11c 1.00±0.47bc 1.80±0.15ab 1.94±1.05b 1.5% 32.22±4.01abcd 14.44±2.94c 1.40±0.21ab 1.23±0.90c 1.71±0.23b 2.0% 26.67±1.93d 11.11±1.11cd 0.97±0.15d 1.00±0.21c 1.02±0.35bc Not using PGRs 28.89±2.22cd 7.78±1.11d 0.30±0.26 0.40±0.12d 0.15±0.11c Mean 34.07±1.16 17.78±1.52 1.02±0.54 1.30±0.10 1.53±0.25 P-value 0.009 0.000 0.001 0.000 0.001 Notes: *Mean values in columns followed by different letters are statistically significantly different with P ≤ 0.05 using Duncan’s multiple range test. For C. dalatensis and C. capitata, the liquid growth regulator was more effective compared to the charcoal powder in promoting rooting. Results on survival percentage and rooting percentage are compared in Figure 4. the most effective treatment, whether the activated charcoal-contained powder or liquid plant growth regulator. Effect of Cutting Types on Root Formation Cutting types are divided into softwood (non- lignified) cuttings, semi-hardwood (partly lignified) cuttings, and hardwood (lignified portion) cuttings (Griffin et al. 1998; Islam et al. 2010). Most propagators of woody plants use semi-hardwood cuttings for higher rooting efficiency compared to softwood and hardwood cuttings (Alkaç et al. 2022). In each cutting type, the cutting position, whether terminal shoot or stem cuttings, also has a great impact on rooting efficiency. In most species, Liquid IBA solutions are easy to prepare, while the phloem and xylem of cuttings have better access to exogenous hormones than hormones combined with powders. However, powder containing activated charcoal is an effective adsorbent for many pollutants (organic, inorganic, microbial, and biological) (Mohammad-Khah & Ansari 2009). Therefore, when the activated charcoal- contained powder is dry-mixed with a plant growth regulator, the dry mix can reduce infection. Therefore, in this research, we sought to identify Conservation status and propagation of Camellia dalatensis and Camellia capitata - En et. al. 397 Figure 4 Comparisons on survival and rooting percentages for cuttings dipped in liquid IBA or IBA in charcoal for Camellia dalatensis (a) and C. capitata (b) Notes: DD = IBA solution, DR = IBA powder, DC = no IBA. cuttings from terminal shoot have higher rooting efficiency. However, the use of appropriate plant growth regulators can affect the rooting efficiency of stem cuttings and ensure propagation efficiency with the number of rooted cuttings higher than that of the terminal shoot cuttings (Solikin 2019). Choosing the right cuttings for propagation Table 3 Effect of cutting types on Camellia dalatensis root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index TC 90.00±1.92 58.89±1.11 5.10±0.59 4.50±0.12 23.08±3.20 SC 67.78±2.94 31.11±2.94 1.97±0.15 2.33±0.20 4.62±0.62 Mean 78.88±5.21 45.00±6.39 3.53±0.75 3.42±0.50 13.85±4.38 P-value 0.003 0.001 0.007 0.001 0.005 Notes: *Mean values in columns followed by different letters are statistically significantly different with P ≤ 0.05 using Duncan’s multiple range test; TC = Terminal shoot cuttings; SC = Stem cuttings. Table 4 Effect of cutting type on Camellia capitata root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index TC 45.56±1.11 25.56±1.11 1.80±0.06 1.87±0.19 3.36±0.37 SC 37.78±2.22 34.45±2.22 1.50±0.06 2.00±0.10 3.00±0.19 Mean 41.67±2.06 30.00±2.28 1.65±0.08 1.93±0.10 3.18±0.20 P-value 0.035 0.023 0.021 0.056 0.436 Notes: *Mean values in columns followed by different letters are statistically significantly different with P≤0.05 using Duncan’s multiple range test; TC = Terminal shoot cuttings; SC = Stem cuttings. and increasing the efficiency of propagation are necessary solutions. For some Camellia spp., it is common to choose semi-hardwood cuttings because this cutting type provides the best results. In this study, semi- hardwood cutting was used and divided into terminal shoot cuttings and stem cuttings (Tables 3 and 4). BIOTROPIA Vol. 31 No. 3, 2024 398 Based on Experiment 1, the IBA concentrations used were 1,000 ppm for C. dalatensis and 1,500 ppm for C. capitata. The parameters of survival percentage, rooting percentage, number of roots, root length, and rooting index were significantly different for C. dalatensis (P < 0.05). Terminal shoot cuttings provided better results compared to stem cuttings with the survival percentage of 90.00% compared to 67.78%, the rooting percentage reaching 58.89% compared to 31.11%, 5.10 roots per cutting compared to 1.97 roots per cutting, the root length reached 4.50 cm compared to 2.33 cm, the rooting index reached 23.08 compared to 4.62. Similar results have been reported by Wazir (2014) in propagating C. japonica by using terminal cuttings with 1,000 ppm IBA where the highest percentage of rooting was 84.96%, and the longest root length was 18.3 cm. For C. capitata, the parameters of survival percentage, rooting percentage, and number of roots per cutting were significantly different (P < 0.05). However, root length and rooting index were not statistically significantly different (P > 0.05). The survival percentage of terminal shoot cuttings compared to stem cuttings was 45.56% compared to 37.78%. However, the rooting index for terminal shoot cuttings was about 9% lower than that of stem cuttings (25.56% versus 34.45%), and the number of roots per cutting was 1.80 compared to 1.50. The root length ranged from 1.87 cm to 2.00 cm (average 1.93 cm) and the rooting index range was 3.00 to 3.36 (average 3.18) for both cutting types. Comparing the rooting parameters, the effective cutting type for C. capitata propagation was semi- hardwood stem cuttings. At the time of gathering cuttings in the field, the C. capitata had begun to sprout. Therefore, the terminal shoot cuttings were very immature and weak, so they were easy to be infected by fungi. Besides, we collected the stem cuttings at the same time that the cuttings were partially lignifying, to have better auxin synthesis; thereby, the rooting percentage of stem cuttings was higher than that of terminal shoot cuttings in C. capitata. Effect of Substrates on Root Formation The growing substrate environment should have good porosity to support smooth water draining and provide a suitable temperature and humidity regimes for root development. The substrate media can be combined with other factors, such as plant growth regulators and cutting types, to bring the highest efficiency to the propagation process (Johnson et al. 2005; Silva et al. 2012). The selected growing substrate for propagation in this study was a mixture of sand and coir dust. Sand ensures good drainage, and coir dust improves water retention in the rooting environment. In this experiment, 1,000 ppm IBA was used for semi- hardwood cuttings of C. dalatensis, while 1,500 ppm IBA was used for semi-hardwood cuttings of C. capitata. Rooting results are shown in Table 5 (C. dalatensis), Table 6 (C. capitata), and Figure 5. Table 5 Effect of substrate type on Camellia dalatensis root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index A1 94.44±1.11 75.56±1.11c 4.93±0.30c 4.60±0.06c 22.73±1.65c A2 95.56±2.94 83.33±1.93b 5.13±0.18bc 5.33±0.15b 27.41±1.47bc A3 95.56±1.11 88.89±2.22a 6.87±0.27a 6.23±0.15a 42.88±2.71a A4 95.56±2.94 81.11±1.11b 6.10±0.50ab 5.60±0.20b 34.26±3.59b Mean 95.28±0.10 82.22±1.61 5.76±0.27 5.44±0.19 31.82±2.53 P-value 0.978 0.003 0.011 0.000 0.002 Notes: *Mean values in columns followed by different letters are statistically significantly different with P ≤ 0.05 using Duncan’s multiple range test; A1 = 75% sand and 25% coir dust; A2 = 50% sand and 50% coir dust; A3 = 25% sand and 75% coir dust; A4 = 100% coir dust. Conservation status and propagation of Camellia dalatensis and Camellia capitata - En et. al. 399 Table 6 Effect of substrate type on Camellia capitata root formation Treatment Survival percentage (%) Rooting percentage (%) Number of roots per cutting Root length (cm) Rooting index A1 64.45±2.22 47.78±2.22 2.03±0.09b 2.33±0.12b 4.72±0.05b A2 65.55±2.22 52.22±2.94 2.83±0.20a 2.87±0.17a 8.12±0.69a A3 65.56±2.94 53.33±1.93 2.57±1.19a 1.83±0.07c 4.73±0.50b A4 66.66±3.33 52.22±2.22 2.57±0.33a 1.77±0.09c 4.54±0.28b Mean 65.56±1.84 51.39±1.19 2.50±0.11 2.20±0.14 5.53±0.49 P-value 0.952 0.406 0.026 0.001 0.001 Notes: *Mean values in columns followed by different letters are statistically significantly different with P ≤ 0.05 using Duncan’s multiple range test; A1 = 75% sand and 25% coir dust; A2 = 50% sand and 50% coir dust; A3 = 25% sand and 75% coir dust; A4 = 100% coir dust. The effects of media types and composition on rooting efficiency have been reported in many plants (Eed & Burgoyne 2014). Air content and oxygen diffusion rate in media are important for rooting (Ercisli et al. 2002). The characteristics of the species also influence rooting success. In particular, Camellia impressinervis grows well in high moisture and well drained soil (Tran 2018), in which their cuttings had the best rooting results in a growth medium of 100% sand with high air content and oxygen diffusion capacity (Tran et al. 2020). Figure 5 Rootings of Camellia cuttings Notes: a. C. dalatensis terminal shoot cuttings with 1,000 ppm IBA in a substrate of 25% sand mixed with 75% coir dust; b. C. capitata semi-hardwood stem cuttings with 1,500 ppm IBA in a substrate of 50% sand mixed with 50% coir dust. For C. dalatensis, there was no statistically significant differences in survival percentage (P > 0.05) between treatments, with a mean value of 95.28%. However, rooting percentage, number of roots, root length, and rooting index were significantly different (P < 0.05). Rooting percentage ranged from 75.56% to 88.89% (average 82.22), number of roots per cutting ranged from 4.93 to 6.78 (average 5.76), root length ranged from 4.60 cm to 6.23 cm (average 5.44 cm), and rooting index ranged from 22.73 to 42.88 (average 31.82). The treatment with the best results for C. dalatensis was 25% sand:75% coir dust, which may be related to the wet environment where this species occurs. C. dalatensis was distributed in Dalat City which has high humidity and high annual rainfall. For C. capitata, there was no significant difference between the survival and rooting percentages (P > 0.05), the average survival percentage was 65.56% and the average rooting percentage was 51.39%. The number of roots per cutting ranged from 2.03 to 2.83 (average 2.50), the root length ranged from 1.77 cm to 2.87 cm (average 5.44 cm) and the rooting index ranged from 4.54 to 8.12 (mean 5.53). Treatment with the best results for C. capitata was the treatment with equal parts of sand and coir dust. C. capitata occurs in Cat Tien District which has lower humidity and rainfall compared to that in Dalat City. Therefore, this species was able to grow better in the medium which had similar conditions to their natural habitat. BIOTROPIA Vol. 31 No. 3, 2024 400 CONCLUSION Camellia capitata and C. dalatensis have very limited distributions area. The declining natural populations face the risk of extinction due to fragmentation, deforestation, illegal logging, and clearing for agriculture. Vegetative propagation protocols are essential to help domesticate these species and conserve genetic diversity. For propagation by using cuttings, root formation was effective using liquid IBA at 1,000 ppm for C. dalatensis and at 1,500 ppm for C. capitata. Rooting formation was the highest for terminal shoot cuttings of C. dalatensis and semi-hardwood stem cuttings of C. capitata. Sand and coir dust substrates with a ratio of 25:75% for C. dalatensis and 50:50% for C. capitata were the best substrates for root formation of cuttings. ACKNOWLEDGMENTS The authors would like to thank BCGI (Botanic Gardens Conservation International) for their financial support and the Southern Institute of Ecology, Forest Science Institute of Central Highlands and South of Central Vietnam, Cat Tien National Park, and Lam Vien Special-use Forest Management Board created the most favorable conditions for us to complete this study. REFERENCES Alkaç OS, Öcalan ON, Güneş M. 2022. Effect of silver nanoparticles treatments on some characteristics of “Santander” lily cultivar. 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