Early root development of Eucalyptus pellita F. Muell. seedlings from seed and stem cutting at nursery stage

Early root development of Eucalyptus pellita F. 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Muell. seedlings from seed and stem cutting at nursery stage Affendy Hassan, Parveena Balachandran, Khairiyyah Razanah Khamis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-104802/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Eucalyptus is among the important fast-growing species, and is typically managed on short rotation to sustain the production of timber, pulpwood, charcoal, and fire-wood. Macro-propagation using cutting for larger multiplying seedlings is cheaper and efficient instead of clonal seeds for uniform plant material seedling production. However, information on root growth of Eucalyptus pellita at early development from seed and stem cutting of E. pellita seedlings is still lacking. This is probably due to the difficulty in investigation belowground, and also due to methodological problems. With such information, it is useful for forest plantation company management in enhancing the understanding on strategies to optimize yield production with the appropriate agronomic or silvicultural approach in the field planting. Therefore, the objectives of this study were; to compare the root development of two different propagation seedlings of E. pellita ; and to study the effect of various nitrogen concentration levels on two types of propagation of E. pellita seedlings. Results The study was conducted using E. pellita seedlings from two types of propagation, namely, seed and stem cuttings, along with three different nitrogen concentrations (0, 50, and 200 kg N ha -1 ). Shoot biomass, root intensity (RI), total root intensity (TRI), root biomass, root length density (RLD), and specific root length (SRL) were recorded. Dried shoot biomass, RLD and SRL of E. pellita seedlings using stem cutting were significantly higher ( P 0.05) for root biomass, TRI and RI between the propagation types of E. pellita seedlings. Conclusions: E. pellita seedlings from stem cutting was greater in terms of root distribution compared to propagation by seeds at the nursery stage, and 50 kg N ha -1 was the optimal nitrogen concentration level from the considered levels to be applied to the E. pellita seedlings. The present study therefore provides more information and understanding on E. pellita for forest plantation companies in producing plant materials using stem cutting in a cost-effective and efficient manner. This would help the forest plantation companies in planning appropriate agronomic management in the future. Forestry Root distribution root traits propagation technique stem cutting fast growing species Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Plantation forestry using Eucalyptus spp. in Sabah started in the 1970s (Harwood and Nambiar 2014 ) as part of a forest conservation effort (Zaiton et al. 2020 ). Eucalyptus is among the important fast-growing species that is typically managed on short rotation to sustain the production of timber, pulpwood, charcoal, and fire-wood (Zaiton et al. 2018 ; Zhou et al. 2018 ). Sabah Softwood Berhad (SSB) is the first private forest plantation company in Sabah that pioneered using fast-growing timber species, where E. deglupta was initially introduced during the early plantation development (Enters et al. 2002 ). However, it was unsuccessful, and was later replaced with other superior species such as Acacias , due poor growth performance (Zaiton et al. 2020 ) and foliar pathogens (Japarudin et al. 2015 ). Since nearly three decades, Acacia mangium and hybrids have been the primary species planted in Sabah, especially in some forest plantation companies such as Acacia Forest Industries Sdn Bhd (AFI), Sabah Forest Development Authority (SAFODA) and SSB. However, A. mangium and hybrids performance are affected mainly by serious fungi Ceratocystis disease (Tarigan et al. 2011 ; Japarudin et al. 2015 ), wilt (Japarudin et al. 2015 ), and Ganoderma philippii (Mohammed et al. 2014 ), which have caused death to about 10 to 20% of the Acacia trees in plantations (Wong et al. 2015 ). Therefore, E. pellita is an alternative option for the fast-growing timber production industry. Since 2008, most of forest plantation companies in Sabah and Sarawak have been involved in using Eucalyptus species in plantations (Zaiton et al. 2020 ). Eucalyptus pellita F. Muell, or red mahogany, is a medium-to-large tree that can grow up to 40 m in height and over 1 m in diameter (Harwood 1998 ). E. pellita is native to Papua New Guinea and northern Queensland, Australia (Hung et al. 2015 ; Yahya et al. 2020 ; Yew et al. 2015 ). It has good growth and a high survival rate because of its wider range of adaptability with sites and favourable stem form (Yahya 2020 ). Currently, E. pellita plays an important role in reforestation in countries such as Brazil, Cuba, Indonesia, Malaysia and the Philippines (Hung et al. 2015 ). Furthermore, E. pellita is used for a variety of products such as fine furniture (Clarke et al. 2009 ), pulp production (Eldridge et al. 1993 ; Poke and Raymond 2006 ) and high quality writing and printing paper or tissue products (Raymond 2002 ; Raymond and Schimleck 2002 ; Schimleck et al. 2006 ). In order to sustain the plant material supply with efficient and cost-effective means (Kuppusamy et al. 2019 ), macro-propagation using cutting can be used instead of clonal seeds for uniform plant material seedling production. Cutting is the most widely used technique, and is cheaper for larger multiplying seedlings of Eucalyptus , due to easier handling as compared to the micro-propagation method (Sulichantini et al. 2014 ). However, although there exist many studies on E. pellita , there is a limited amount of information on root growth of E. pellita at early development from seed and stem cutting of E. pellita seedlings. This is probably due to the difficulty in investigation belowground, and also due to methodological problems. With such information, it is useful for forest plantation company management in enhancing the understanding on strategies to optimize yield production with the appropriate agronomic or silvicultural approach. In this present study, we used two types of planting material sources from seed and stem cutting of E. pellita , and studied their root traits at three different nitrogen concentrations. We hypothesied that, both above and belowground, E. pellita seedlings from stem cutting were greater than seedlings from seed propagation. On this basis, the objectives of this study were formulated as follows: i) to compare the root development of two different propagation seedlings of E. pellita ; and ii) to study the effect of different nitrogen concentrations on two types of propagation of E. pellita seedlings. Material And Methods Experiment description This study was conducted from the 12th of April to the 30th of August 2019 at a greenhouse at the Forestry Complex of Faculty Science and Natural Resources, Universiti Malaysia Sabah (UMS), Kota Kinabalu, Sabah, Malaysia (6°02’ 08.4’’ N 116°07’ 34.4’’E). According to the Malaysia Meteorological Department 2020 ( www.met.gov.my ), the temperature was in the range of 30 to 32 °C, while rainfall distribution was in the range of 111.76 mm (April) to 304.80 mm (June), throughout the study period. A transparent plastic pot 20 cm in height × 140 mm in inner diameter, which had a total volume of 3,079 cm 3 , was used as the medium pot. The bottom of the container was created with small holes to facilitate the flow of water and air, and was covered with fine net. Each pot had four sides for grid lines, which were marked as sides A, B, C and D using a red permanent marker. The grid size was 20 × 20 mm was, and the total grid length for each side was 1.42 m. These grid lines were used to observe and count the root intensity of E. pellita . This involved repeatedly counting the number of intersections of the roots along the grid lines. During the experiment, the pot was always covered using non-transparent plastic to avoid light exposure of the soil and roots, and was opened only during the measurement process. Topsoil was taken from Tamparuli, Sabah (30 km from the Universiti Malaysia Sabah campus). The soil was open-dried for seven days in a greenhouse, sieved using a 2.0 mm soil mesh and filled into a pot. The moisture content of the soil sample before the experiment was 15.8%. After that, soil in the pot was washed with 5 L of water under low water pressure, to ensure all the nutrients in the soil were empty or low and homogenized. In this experiment, seedlings of four-week-old E. pellita propagated from seeds, and stem cutting was supplied from Acacia Forest Industries Sdn. Bhd. (AFI). Stem cutting was produced from their superior mother clonal plants. The tip was selected for cutting; the rooting duration was four weeks in a greenhouse, prior to the experiment. The seedlings were then transferred to the pot that was filled with the topsoil. The 36 E. pellita seedlings from seeds and 36 E. pellita seedlings from stem cutting, accounting for a total of 72 experimental units, including three replications (12 replicates for each fertilizer treatment), were arranged using complete randomized design (CRD). A liquid nitrogen fertilizer (AG Leader 954) was diluted and corresponded to the three different rates of 0 (control), 50 N kg ha − 1 and 200 N kg ha − 1 . No watering was done as the experiment was exposed to natural conditions. Data collection In this experiment, dried shoot biomass, root biomass, root intensity (RI), total root intensity (TRI), root length density (RLD), and specific root length (SRL) were recorded (Hassan et al. 2019 ). RI data was collected based on the method by Thorup-Kristensen ( 2001 ). RI was measured by counting the number of roots crossing the lines of 20 × 20 mm grid squares placed on the container surface view sides. RI data was recorded every week, starting from when the roots started to appear on the surface of the transparent pot, until the roots reach the bottom of the pot. Three different dates sampling procedures were carried out 4, 6 and 8 weeks after transplanting (WAT). Each sampling involved the harvesting of 12 experimental units, or four (4) replicates for each fertilizer treatment from both planting materials. Aboveground biomass was cut from the ground topsoil, washed, and placed in a labelled plastic bag. It was then kept in an oven at 70 ℃ for 48 hours, before being weighed. For root parameters, roots biomass was washed out from soil and organic matter using a sieve 2.0 mm mesh under low pressure water. It was then stored in 50% ethanol in a 50 ml eppendorf tube at 5 °C, before root image analysis. RLD (cm cm − 3 ) was determined using an EPSON® scanner and Winrhizo® software, and expressed in cm cm − 3 (Hassan et al. 2019 ). For SRL (cm g − 1 ), the length of a sub-sample was measured, was then divided by its mass (g), before being converted to actual root biomass. Statistical analysis All the mean values were subject to statistical analysis using the Statistical Package Social Science (IBM SPSS Statistics 22.0). An independent sampled T-test was used to compare the TRI, RLD, SRL, root biomass, and shoot biomass between two types of plant material E. pellita seedlings at various nitrogen concentrations for all sampling dates. Subsequently, a one-way ANOVA followed Tukey HSD’s post hoc analysis was used for RI at different nitrogen concentrations for both seed and stem cutting seedlings. In assessing the differences between the results, tests with P < 0.05 were considered statistically significant. Prior to statistical analyses, all data were tested for normality using the Shapiro-Wilk Normality test, and for homogeneity using Levene’s test. Results Dried shoot biomass for E. pellita Dried shoot biomass was harvested three times, four, six and eight weeks after transplanting (4, 6, 8 WAT), as indicated in Fig. 1 . It is clear that there was a significant difference ( P < 0.05) of dried shoot biomass of E. pellita seedlings between seed and stem cutting, especially at 6 WAT (Fig. 1 b). At 4 WAT, there was no significant difference between seed and stem cutting for both 0 and 50 kg N ha − 1 , but the shoot biomass of stem cutting was nearly double than seed under 200 kg N ha − 1 . In contrast, at 6 WAT, all the treatments showed a significant difference ( P < 0.05), where 50% of stem cutting was higher than seed. However, there was no significant difference between seed and stem cutting for all treatments at 8 WAT (Fig. 1 c). Figure 1 : Dried Shoot Biomass (g) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha − 1 ) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *( P 0.05) between the seed and stem cutting of E. pellita for all treatments in 4 WAT (Fig. 2 a). However, in 6 WAT, only for 50 kg N − 1 , the seeds of E. pellita showed a significant difference ( P < 0.05), as compared to stem cutting (Fig. 2 b). Interestingly, without fertilizer, the root biomass of E. pellita seeds witnessed a significant difference ( P < 0.05), as compared to stem cutting (Fig. 2 c). Figure 2 : Root Biomass (g) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha − 1 ) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *( P < 0.05). Error bars denote standard deviations of the mean, ( n = 4). Total Root Intensity of E. pellita from seed and stem cutting In comparison, the total root intensity (TRI) of E. pellita stem cutting was significantly higher ( P < 0.05) compared to seed cutting for all measurement dates. Despite the large variations observed in stem cutting treatment, the TRI remained to be nearly double that of seed cutting, especially at 6 and 8 WAT. Figure 3 : Total Root Intensity (Intersections m − 1 gridline) of E. pellita seedlings from seeds and stem cutting plant materials at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values tested using Independent Samples T-Test and statistically the mean values were different, (P < 0.05). Bars represent standard deviations of the mean, n = 36 (4 WAT), n = 24 (6 WAT) and n = 12 (8 WAT). Root Intensity of E. pellita at different nitrogen concentrations Figure 4 shows a comparison of root intensity (RI) of E. pellita from seed cutting (Fig. 4 a) and stem cutting (Fig. 4 b), at various measurement dates and nitrogen concentrations. According to the findings, there was no significant difference ( P > 0.05) for treatments and types of plant material for each measurement date. However, despite the large variations of RI, E. pellita stem cutting was clearly higher, and increased with the measurement dates, as compared to seed cutting (Fig. 4 b). Figure 4 : Root Intensity (intersections m − 1 gridline) of E. pellita seedlings from seeds (a) and stem cutting (b) plant materials at different nitrogen concentrations (0, 50, 200 kg N ha − 1 ) at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values were tested using ANOVA followed by Tukey HSD’s post hoc Test. The mean values were not significant different between the different N concentrations for each dates ( P > 0.05). Bars represent standard deviations of the mean, n = 12 (4 WAT), n = 8 (6 WAT) and n = 4 (8 WAT). Root Length Density of E. pellita Figure 5 showed the root length density (RLD) of E. pellita , both from seed and stem cutting, taken at three independent harvest times. Based on the results, stem cutting of E. pellita was significantly higher at 200 kg N − 1 than at the control and at 50 kg N − 1 (Fig. 5 a). At 6 WAT, all RLDs of stem cutting of E. pellita were significantly higher ( P < 0.05) compared to seed cutting, for all N concentrations (Fig. 5 b). However, the RLD of stem cutting of E. pellita was significantly higher compared to seed cutting under the control and high N concentrations, on the final measurement date (Fig. 5 c). It was also found that the RLD of E. pellita for both seed and stem cutting under fertilizer treatment decreased with the measurement dates. Figure 5 : Root Length Density (cm cm − 3 ) of E. pellita seedlings from seeds and stem cutting plant material at different nitrogen concentrations (0, 50, 200 kg N ha − 1 ) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *( P < 0.05). Error bars denote standard deviations of the mean, ( n = 4). Specific Root Length (SRL) of E. pellita The specific root length (SRL) of E. pellita was significantly higher ( P < 0.05) for all treatments and measurement dates (Fig. 6 ). At 4 WAT, SRL of stem cutting was significantly higher ( P < 0.05) compared to seed cutting, almost by three-fold, especially at high N concentrations. Similar findings were also found at 6 WAT, which was almost 50% higher ( P < 0.05) than seed cutting for all fertilizer treatments (Fig. 6 b). However, at 8 WAT, SRL was found to be significantly higher for approximately 50% of stem cutting, as compared to seed cutting, at zero and 50 kg n ha − 1 . No difference in SRL was found between stem cutting and seed cutting of E. pellita at 200 kg N ha − 1 (Fig. 6 c). Figure 6 : Specific Root Length (cm g − 1 ) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha − 1 ) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *( P < 0.05). Error bars denote standard deviations of the mean, ( n = 4). Discussion The findings demonstrate that shoot biomass of stem cutting of E. pellita seedlings was greater compared to seedlings from seed cutting. The shoot biomass is connected with root distribution in the soil, especially the fine roots from stem cuttings. The larger the fine root density, the more water and nutrients are taken up, expressed by higher shoot biomass. This is confirmed by Rostamza et al. ( 2013 ), who reported that greater root length in millet is connected to an increased shoot biomass. However, in this present study, seedlings from stem cutting were significantly higher with an increase in nitrogen levels, as compared to seedlings from seed cutting (see Fig. 1 b). However, seedlings from seed cutting were not affected under different nitrogen concentrations. At the final harvest, there was no significant difference between stem cutting and seed cutting for all fertilizer treatments (Fig. 1 c). Therefore, biomass is influenced by species, and specific silviculture such as irrigation (Toky et al. 2011 ), fertilization and water availability (Ares et al. 2009 ). In root biomass, most of the mean values did not differ for seed and stem cutting, for all treatments, except for 50 N kg ha − 1 at 6 WAT, and for zero nitrogen at 8 WAT (Fig. 2 ). However, Coleman et al. ( 2004 ) reported that root biomass tends to increase with fertilizer rate, but the proportion of root biomass tends to decrease with more fertilizer application. This argument is associated with the current findings, especially for higher nitrogen concentrations, showing that there is no increase with fertilization rate. Another explanation of the results is that seedlings from seed cutting were higher compared to stem cutting, and had no significant difference, because seed cutting produces tap root and high root mass, while stem cutting produces fibrous and fine roots. Without any statistical significance, the total RI for both propagation types of E. pellita seedlings was not different, although there was a higher distribution under stem cutting (Fig. 3 ). Looking at RI, at different nitrogen concentrations for both seed and stem cutting, the distribution of RI was higher in stem cutting compared to seed cutting, although there was no significant difference. This shows that there are more fine roots in stem cutting than in seed cutting, which and increased with the measurement date (Fig. 4 ). The findings of this study are in agreement with prior work that has proven that more root distribution, especially for fine roots, is closely associated to soil water and nutrients (Zhang et al. 1995 ; Wang 1990 ; Zhao et al. 1990 ). This explains the presence of more fine roots after stem cutting on the soil. In RLD, stem cutting was also significantly higher at high nitrogen concentrations, compared to seed cutting. In the subsequent measurement date, all of the nitrogen levels under stem cutting increased significantly compared to seed cutting; this also applies on the final measurement date, except under 50 kg N ha − 1 (Fig. 5 ). However, the values decreased with the measurement dates. The RLD of seedlings from seed cutting were neither affected by different nitrogen levels, nor by measurement dates. Similarly, in SRL, stem cutting was also significantly higher compared to seed cutting, as found in RLD. Despite stem cutting being higher compared to seed cutting, especially in shoot biomass, in SRL and RLD, these growth parameters were not affected by different nitrogen levels. Especially at high nitrogen concentrations (Fig. 6 c), the SRL did not differ between the propagation types. Previous work reported various responses of fertilizer rates against Eucalyptus in Brazil (Goncalves et al. 2004 ; Goncalves et al. 2008 ; Stape et al. 2010 ), in Australia (Smethurst et al. 2004 ; Mendham et al. 2008 ) and in South Africa (du Toit et al. 2010 ). Nevertheless, fertilizer responses varied, depending on the species and sites considered (Halomoan et al. 2015). Eucalyptus in Brazil and South Africa responded to fertilization when water was available (Stape et al. 2010 ; du Toit et al. 2010 ). Stape et al. ( 2010 ) reported that the application of very high rates and excessive nitrogen levels in Brazil did not show any significant effects on Eucalyptus productivity (Halomoan et al. 2015). Fertilizer rates from 50 to 100 kg N ha − 1 increased biomass, but then biomass decreased at a rate of 200 kg N ha − 1 (Halomoan et al. 2015); this was also reported in the present study. Graciano et al. ( 2006 ) reported that P applications affected E. grandis biomass more than N applications in Argentina. However, we cannot validate this argument, since this present study tested E. pellita seedlings at a nursery scale. In this case, a rate of 50 kg N ha − 1 could be cheaper and more efficient to absorb by E. pellita , as opposed to 200 kg N ha − 1 . Chen et al. ( 2011 ) reported that moderate nitrogen fertilizer increased the root intensity in soil layers. As explained above, the root distribution from propagation seeds is less compared to stem cutting. With the short period for the experiment under a small pot, we cannot observe the difference between the nitrogen concentrations. This needs to be done in a larger field. In the comparison between seed and stem cutting, as the above findings, propagation by stem cutting of E. pellita was proved to be viable and productive in terms of root performance at the nursery stage. Although there are works in the related literature that have proved that seed cutting is still the better propagation method (Kiragu et al. 2015 ), producing plant material using stem cutting is not only more efficient and faster, but would also be able to reduce the production costs and time spent for upkeep and maintenance in the nursery. Partelli et al. ( 2014 ) also supported the fact that the cuttingpropagated method for coffee is more productive than the seedpropagated method. Furthermore, Naidu and Jones ( 2015 ) also suggested a superior initial survival and growth of E. dunnii minicuttings compared to seedlings based on early indications. This finding has also proven that secondary branches as semi-hard wood cuttings could be the most effective propagation material of Jatropha curcas (Santoso and Parwata, 2014 ). Notwithstanding this, rooting ability of cuttings from woody or perennial plants declined with an increase in the age of the mother plants (Santoso and Parwata 2014 ). Root ability of cutting formation becomes more difficult with a farther position from the apical shoot (Hartmann et al. 2002 : Wilson 1993 ), due to differences in the type and number of carbohydrates and other stored materials (Hartmann et al. 2002 ; Leakey 1999 ). Therefore, root system characteristics are known to differ according to species, genotype, plant age, physiological status of mother plant (Henning 2003 ), season, climate, plant density, root diameter, biotic stresses, and soil texture and structure (Lynch 1995 ). Also, the growth rate of stem cutting depends on age variation, position in stem, and diameter of stem (Kraiem et al. 2010 ). The present study therefore provides more information and understanding on E. pellita for forest plantation companies in producing plant materials using stem cutting in a cost-effective and efficient manner. Further research is required on the root aspect, especially in real field conditions, as the soil is more heterogenous and exhibits different environmental conditions. Such findings will help these companies take agronomic measures and a silvicultural approach in the future. Conclusions To conclude, E. pellita seedlings from stem cutting were greater in terms of root distribution compared to propagation by seed cutting, at the nursery stage. In addition, aboveground biomass of stem cutting was also higher in E. pellita seedlings than of seed cutting. The 50 kg N ha − 1 was the optimal nitrogen concentration to be applied to the E. pellita seedlings. This is because excessive fertilizer application not only increases fertilizer costs, but may also not necessarily result in an increased volume yield or shoot biomass. Moreover, it is harmful to the soil. Research on the root distribution of these two types of propagation in real field soil merits further investigation, as different environmental factors may affect the growth performance of E. pellita . Thus, this would help the forest plantation companies in planning appropriate agronomic management in the future. Abbreviations N Nitrogen; RI:Root intensity; TRI:Total root intensity; RB:Root biomass; RLD:Root length density; SRL:specific root length; WAT:Weeks after transplanting. Declarations Acknowledgement The authors acknowledge the staff of Acacia Forest Industries Sdn Bhd (AFI) and technical staffs of Forestry Complex, Faculty of Science and Natural Resources, UMS during the study in technical support, as well as the anonymous reviewers, for their thoughtful comments and suggestions on the manuscript. Authors' contributions AH designed the study, supervised data collection and contributed to and edited manuscripts. PB and KRK collected literatures, prepared field experiments, data collection, laboratory analysis, and prepared all figures. All authors read and approved the final manuscript. Funding The authors thank to Acacia Forest Industries Sdn Bhd (AFI) for its kind support during the study, especially in sponsoring the seedlings and technical support. Availability of data and material Not applicable Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests Author details Forest Plantation and Agroforestry Programme, Forestry Complex, Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia. References Ares A, Burner DM, Brauer DK (2009) Soil phosphorus and water effects on growth, nutrient and carbohydrate concentrations, d 13C, and nodulation of mimosa ( Albizia julibrissin Durz.) on a highly weathered soil. Agroforest Syst 76:317-325. doi: 10.1007/s10457-009-9213-2. Chen SX, Arnold R, Li ZH, Li TH, Zhou GF, Wu ZH, Zhou QY (2011) Tree and stand growth for clonal E. urophylla × grandi s across a range of initial stockings in southern China. 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Hortic. 125:146-150. doi:10.1016/j.scienta.2010.03.008 Kuppusamy S, Ramanathan S, Sengodagounder S, Seniappan C, Brindhadevi K, Kaliannan T (2019) Minicutting - A powerful tool for the clonal propagation of the selected species of the Eucalyptus hybrid clones based on their pulpwood studies. Biocatalysis Agric. Biotech. 22:1-4. doi:10.1016/j.bcab.2019.101357. Leakey RRB (1999). Nauclea diderrichii : rooting of stem cuttings, clonal variation in shoot dominance, and branch plagiotropism. Trees, 4:164-169. doi:10.1007/BF00225781 Lynch L (1995) Root architecture and plant productivity. Plant Phys. 109:7‑13. doi:10.1104/pp.109.1.7 Mendham DS, Grove TS, O’Connell AM, Rance SJ (2008) Impacts of inter-rotation site management on soil nutrients and plantation productivity in Eucalyptus globulus plantations in South-Western Australia. In: Nambiar EKS (ed) Site Management and Productivity in Tropical Plantation Forests. Center for International Forestry Research. Bogor. Mohammed CL, Rimbawanto A, Page DE (2014) Management of basidiomycetes root- and stem-rot diseases in oil palm, rubber, and tropical hardwood plantation crops. For Path. 44:428–446. doi:10.1111/efp.12140 Naidu D, Jones N (2015) The Journey to Successful Commercial Propagation and Deployment of Eucalyptus dunnii Cuttings. In: IUFRO Eucalypt Conference 2015. 21-24 October, 2015, Zhanjiang, Guangdong, China. Partelli FL, Covre AM, Oliveira MG, Alexandre RS, da Vitória EL, da Silva, MB (2014) Root system distribution and yield of 'Conilon' coffee propagated by seeds or cuttings. Pesq. agropec. bras. Brasília. 49(5):349-355. doi:10.1590/S0100-204X2014000500004 Poke FS, Raymond CA (2006) Predicting extractives, lignin, and cellulose contents using near infrared spectroscopy on solid wood in Eucalyptus globulu s. J of Wood Chem Tech. 26:187–199. doi:10.1080/02773810600732708 Raymond CA (2002) Genetics of Eucalyptus wood properties. Ann of For Sci. 59:525–531. doi:10.1051/forest:2002037 Raymond CA, Schimleck LR (2002) Development of near infrared reflectance analysis calibrations for estimating genetic parameters for cellulose content in Eucalyptus globulus . Can. J of For Res. 32:170–176. doi:10.1139/x01-174 Rostamza M, Richards RA, Watt M (2013) Response of millet and sorghum to a varying water supply around the primary and nodal roots. Ann of Bot. 112:439-446. doi:10.1093/aob/mct099 Santoso BB, Parwata GA (2014) Seedling Growth from Stem Cutting with Different Physiological Ages of Jatropha curcas L. of West Nusa Tenggara Genotypes. Int J of App Sci and Tech. 4(6):5-10. Schimleck LR, Kube PD, Raymond CA, Michell AJ, French J (2006) Extending near infrared reflectance (NIR) pulp yield calibrations to new sites and species. J of Wood Chem Tech. 26:299–311. doi:10.1080/02773810601076683 Smethurst P, Holz G, Moroni M, Bailie C (2004) Nitrogen management in Eucalyptus nitens plantations. For Ecol Manage 193: 63-80. doi:10.1016/j.foreco.2004.01.023 Stape JL, Binkley D, Ryan MG, Fonseca S, Loos RA, Takahashi EN, Silva CR, Hakamada RE, Ferereira JMA, Lima AMN, Gava JL, Leite FP, Andrade HB, Alves JM, Silva GGC, Azevedo MR (2010) The Brazil Eucalyptus Potential Productivity Project: Influence of water, nutrients, and stand uniformity on wood production. For Ecol Manage 259: 1684-1694. doi:10.1016/j.foreco.2010.01.012 Sulichantini ED, Sutisna M, Sukartiningsih, Rusdiansyah (2014) Clonal Propagation of Two Clones Eucalyptus pellita F. Muell By Mini-Cutting. Int J of Sci Eng. 6(2):112-116. doi:10.12777/ijse.6.2.117-121 Tarigan M, Roux J, van Week M, Tjahjono B, Wingfield MJ (2011) A new wilt and die-back disease of A. mangium associated with Ceratocystis manginecans and C. acaciicvora sp. nov. in Indonesia. South Afr J Bot.77:292–304. doi:10.1016/J.SAJB.2010.08.006 Thorup-Kristensen K (2001) Are differences in root growth of nitrogen catch crops important for their ability to reduce soil nitrate-N content, and how can this be measured? Plant Soil. 230(2):185–195. doi:10.1023/A:1010306425468 Toky OP, Riddle-Black D, Harris PJC, Vasudevan P, Davies PA (2011) Biomass production in short rotation effluent-irrigated plantations in North-West India. J Sci Ind Res. 70: 601-609. Wong SK, Ahmad Zuhaidi Y, Charles GDC, Peter KCS (2015) Recommending Eucalyptus Species for Soft Loan Financing. Working paper presented at the 1st Technical Meeting on Forest Plantation Programme, Malaysian Timber Industry Board (MTIB), Kuala Lumpur. Malaysia. Yahya AZ (2020) Planting of Eucalyptus in Malaysia. Acta Sci Agric. 4(2):139-140. doi: 10.31080/ASAG.2020.04.0785 Yahya AZ, Hassan NH, Loon NT, Heng LH, Zorkarnain FA (2020). Comparing the early growth performance of plantation–grown Eucalyptus hybrid and Eucalyptus pellita , south Johore, Peninsular Malaysia. World J of Adv Res Rev. 6(2), 234-238. doi:10.30574/wjarr Yew HS, Su HK, Lung NM, Ak Penguang SJ, Meder R (2015) Assessment of Plantation-Grown Eucalyptus pellit a in Borneo, Malaysia for Solid Wood Utilisation. In: IUFRO Eucalypt Conference 2015. 21-24 October, 2015, Zhanjiang, Guangdong, China. Wang J (1990) The root system development of Cicer milkvetchin - the first year of growth. Pratac Sci. 7(1): 53-60. Wilson PJ (1993) Propagation characteristics of Eucalytus globules Labill. spp. globules stem cutting in relation to their original position in the parent shoot. J of Hort Sci. 68(5):715-724. doi:10.1080/00221589.1993.11516404 Zaiton S, Paridah MT, Hazandy AH, Azim RARA (2018) Potential of Eucalyptus Plantation in Malaysia. The Malay For. 81(1):64-72. Zaiton S, Sheriza MR, Ainishifaa R, Alfred K, Norfaryanti K (2020) Eucalyptus in Malaysia: Review on Environmental Impacts. J of Lands Ecol. 13(2):79-94. doi:10.2478/jlecol-2020-0011 Zhang D, Jiang X, Zhao S (1995) Further thoughts on growth redundancy. Acta Pratac Sci. 4(3): 17-22. Zhao M, Tan C, He D (1990) A study of root system of Artemisia dalailamen . Pratac. Sci. 7(3): 55-57. Zhou X, Zhu H, Wen Y, Goodale UM, Li X You Y, Liang H (2018) Effects of understory management on trade-offs and synergies between biomass carbon stock, plant diversity and timber production in eucalyptus plantations. For Ecol Manage. 410:164–173. doi:10.1016/j.foreco.2017.11.015 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-104802","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":4479471,"identity":"45c63b94-a4bb-4a28-b79d-3186675771de","order_by":1,"name":"Affendy Hassan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYDAC5oMPGBjYGOQgPDZitLAlG4BUGpOuJbGBaC38bcyMjwvKDqdvuHb8AcOHssMMujMS8GuROMbMbDzj3OHcDbdzDBiBDAazGwS0MNzvPybN2wbWwsAMZBDWIn+Mmf03UGW6we30B8x/idFicIyZDWR4gsHtBANmRmK0GAL9Is1zLt1wJtAvB3vOpfOYnXmAX4vcMWbGzzxl1vJ8t9MfPvhRZi1ndpyALVDQDCYPADEPgwBxWuqQ2PwHiNIyCkbBKBgFIwcAANpMRjCLj2evAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8713-4973","institution":"Universiti Malaysia Sabah","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Affendy","middleName":"","lastName":"Hassan","suffix":""},{"id":4479472,"identity":"fdcd1a99-2cb8-4ea2-b40f-aa9fdc5fb25d","order_by":2,"name":"Parveena Balachandran","email":"","orcid":"","institution":"Universiti Malaysia Sabah Fakulti Sains dan Sumber Alam","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Parveena","middleName":"","lastName":"Balachandran","suffix":""},{"id":4479473,"identity":"457194e7-b6e4-4df7-9b6e-2ddacd807a8c","order_by":3,"name":"Khairiyyah Razanah Khamis","email":"","orcid":"","institution":"Universiti Malaysia Sabah Fakulti Sains dan Sumber Alam","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Khairiyyah","middleName":"Razanah","lastName":"Khamis","suffix":""}],"badges":[],"createdAt":"2020-11-08 16:48:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-104802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-104802/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3574004,"identity":"ecb80e62-f2b8-4ab3-ab8b-de5e94867b8e","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39285,"visible":true,"origin":"","legend":"Dried Shoot Biomass (g) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha-1) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(P\u003c 0.05). Error bars denote standard deviations of the mean, (n= 4).","description":"","filename":"Figure1.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/c667bd74f4191ec12ebdd838.JPG"},{"id":3574005,"identity":"b0d0bf77-35bc-48d4-83c9-6d04c0319485","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41619,"visible":true,"origin":"","legend":"Root Biomass (g) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha-1) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(P\u003c 0.05). Error bars denote standard deviations of the mean, (n= 4).","description":"","filename":"Figure2.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/9def4dc82f7a09062181d185.JPG"},{"id":3574006,"identity":"6bedd2b7-e97d-4018-be12-89ed288cfc84","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34761,"visible":true,"origin":"","legend":"Total Root Intensity (Intersections m-1 gridline) of E. pellita seedlings from seeds and stem cutting plant materials at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values tested using Independent Samples T-Test and statistically the mean values were different, (P\u003c0.05). Bars represent standard deviations of the mean, n=36 (4 WAT), n=24 (6 WAT) and n= 12 (8 WAT).","description":"","filename":"Figure3.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/8e67cc669a89b77006e7c186.JPG"},{"id":3574007,"identity":"e347f85d-c056-41cc-acbf-fc33172f0058","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39859,"visible":true,"origin":"","legend":"Root Intensity (intersections m-1 gridline) of E. pellita seedlings from seeds (a) and stem cutting (b) plant materials at different nitrogen concentrations (0, 50, 200 kg N ha-1) at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values were tested using ANOVA followed by Tukey HSD’s post hoc Test. The mean values were not significant different between the different N concentrations for each dates (P\u003e0.05). Bars represent standard deviations of the mean, n=12 (4 WAT), n=8 (6 WAT) and n= 4 (8 WAT).","description":"","filename":"Figure4.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/3b3e384fb6baec90cc2e0849.JPG"},{"id":3574008,"identity":"081695a3-e0ec-4801-a594-dd94da348c9f","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36411,"visible":true,"origin":"","legend":"Root Length Density (cm cm-3) of E. pellita seedlings from seeds and stem cutting plant material at different nitrogen concentrations (0, 50, 200 kg N ha-1) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(P\u003c 0.05). Error bars denote standard deviations of the mean, (n= 4).","description":"","filename":"Figure5.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/ef5df1b8fcf2610e5bc2444c.JPG"},{"id":3574009,"identity":"a7ea0df1-e1dd-4cba-982b-c0988c21b241","added_by":"auto","created_at":"2020-11-13 18:32:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":40981,"visible":true,"origin":"","legend":"Specific Root Length (cm g-1) of E. pellita seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200 kg N ha-1) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(P\u003c0.05). Error bars denote standard deviations of the mean, (n= 4).","description":"","filename":"Figure6.JPG","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/6479f57a0aa13a7d6f40f58a.JPG"},{"id":13614785,"identity":"6b019240-26db-4366-ba44-54016cb3fa8f","added_by":"auto","created_at":"2021-09-17 06:42:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":476599,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-104802/v1/1f5da877-f372-4e20-92f3-03976f630c11.pdf"}],"financialInterests":"","formattedTitle":"Early root development of Eucalyptus pellita F. Muell. seedlings from seed and stem cutting at nursery stage","fulltext":[{"header":"Background","content":" \u003cp\u003ePlantation forestry using \u003cem\u003eEucalyptus\u003c/em\u003e spp. in Sabah started in the 1970s (Harwood and Nambiar \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) as part of a forest conservation effort (Zaiton et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eEucalyptus\u003c/em\u003e is among the important fast-growing species that is typically managed on short rotation to sustain the production of timber, pulpwood, charcoal, and fire-wood (Zaiton et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Sabah Softwood Berhad (SSB) is the first private forest plantation company in Sabah that pioneered using fast-growing timber species, where \u003cem\u003eE. deglupta\u003c/em\u003e was initially introduced during the early plantation development (Enters et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, it was unsuccessful, and was later replaced with other superior species such as \u003cem\u003eAcacias\u003c/em\u003e, due poor growth performance (Zaiton et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and foliar pathogens (Japarudin et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince nearly three decades, \u003cem\u003eAcacia mangium\u003c/em\u003e and hybrids have been the primary species planted in Sabah, especially in some forest plantation companies such as Acacia Forest Industries Sdn Bhd (AFI), Sabah Forest Development Authority (SAFODA) and SSB. However, \u003cem\u003eA. mangium\u003c/em\u003e and hybrids performance are affected mainly by serious fungi \u003cem\u003eCeratocystis\u003c/em\u003e disease (Tarigan et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Japarudin et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), wilt (Japarudin et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eGanoderma philippii\u003c/em\u003e (Mohammed et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which have caused death to about 10 to 20% of the \u003cem\u003eAcacia\u003c/em\u003e trees in plantations (Wong et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, \u003cem\u003eE. pellita\u003c/em\u003e is an alternative option for the fast-growing timber production industry. Since 2008, most of forest plantation companies in Sabah and Sarawak have been involved in using \u003cem\u003eEucalyptus\u003c/em\u003e species in plantations (Zaiton et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEucalyptus pellita\u003c/em\u003e F. Muell, or red mahogany, is a medium-to-large tree that can grow up to 40\u0026nbsp;m in height and over 1\u0026nbsp;m in diameter (Harwood \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). \u003cem\u003eE. pellita\u003c/em\u003e is native to Papua New Guinea and northern Queensland, Australia (Hung et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yahya et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yew et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). It has good growth and a high survival rate because of its wider range of adaptability with sites and favourable stem form (Yahya \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Currently, \u003cem\u003eE. pellita\u003c/em\u003e plays an important role in reforestation in countries such as Brazil, Cuba, Indonesia, Malaysia and the Philippines (Hung et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, \u003cem\u003eE. pellita\u003c/em\u003e is used for a variety of products such as fine furniture (Clarke et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), pulp production (Eldridge et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Poke and Raymond \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and high quality writing and printing paper or tissue products (Raymond \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Raymond and Schimleck \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Schimleck et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn order to sustain the plant material supply with efficient and cost-effective means (Kuppusamy et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), macro-propagation using cutting can be used instead of clonal seeds for uniform plant material seedling production. Cutting is the most widely used technique, and is cheaper for larger multiplying seedlings of \u003cem\u003eEucalyptus\u003c/em\u003e, due to easier handling as compared to the micro-propagation method (Sulichantini et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, although there exist many studies on \u003cem\u003eE. pellita\u003c/em\u003e, there is a limited amount of information on root growth of \u003cem\u003eE. pellita\u003c/em\u003e at early development from seed and stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e seedlings. This is probably due to the difficulty in investigation belowground, and also due to methodological problems. With such information, it is useful for forest plantation company management in enhancing the understanding on strategies to optimize yield production with the appropriate agronomic or silvicultural approach. In this present study, we used two types of planting material sources from seed and stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e, and studied their root traits at three different nitrogen concentrations. We hypothesied that, both above and belowground, \u003cem\u003eE. pellita\u003c/em\u003e seedlings from stem cutting were greater than seedlings from seed propagation. On this basis, the objectives of this study were formulated as follows: i) to compare the root development of two different propagation seedlings of \u003cem\u003eE. pellita\u003c/em\u003e; and ii) to study the effect of different nitrogen concentrations on two types of propagation of \u003cem\u003eE. pellita\u003c/em\u003e seedlings.\u003c/p\u003e "},{"header":"Material And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperiment description\u003c/h2\u003e \u003cp\u003eThis study was conducted from the 12th of April to the 30th of August 2019\u0026nbsp;at a greenhouse at the Forestry Complex of Faculty Science and Natural Resources, Universiti Malaysia Sabah (UMS), Kota Kinabalu, Sabah, Malaysia (6\u0026deg;02\u0026rsquo; 08.4\u0026rsquo;\u0026rsquo; N 116\u0026deg;07\u0026rsquo; 34.4\u0026rsquo;\u0026rsquo;E). According to the Malaysia Meteorological Department 2020 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.met.gov.my\" target=\"_blank\"\u003ewww.met.gov.my\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e), the temperature was in the range of 30 to 32\u0026nbsp;\u0026deg;C, while rainfall distribution was in the range of 111.76\u0026nbsp;mm (April) to 304.80\u0026nbsp;mm (June), throughout the study period. A transparent plastic pot 20\u0026nbsp;cm in height\u0026thinsp;\u0026times;\u0026thinsp;140\u0026nbsp;mm in inner diameter, which had a total volume of 3,079\u0026nbsp;cm\u003csup\u003e3\u003c/sup\u003e, was used as the medium pot. The bottom of the container was created with small holes to facilitate the flow of water and air, and was covered with fine net. Each pot had four sides for grid lines, which were marked as sides A, B, C and D using a red permanent marker. The grid size was 20\u0026thinsp;\u0026times;\u0026thinsp;20\u0026nbsp;mm was, and the total grid length for each side was 1.42\u0026nbsp;m. These grid lines were used to observe and count the root intensity of \u003cem\u003eE. pellita\u003c/em\u003e. This involved repeatedly counting the number of intersections of the roots along the grid lines. During the experiment, the pot was always covered using non-transparent plastic to avoid light exposure of the soil and roots, and was opened only during the measurement process. Topsoil was taken from Tamparuli, Sabah (30\u0026nbsp;km from the Universiti Malaysia Sabah campus). The soil was open-dried for seven days in a greenhouse, sieved using a 2.0\u0026nbsp;mm soil mesh and filled into a pot. The moisture content of the soil sample before the experiment was 15.8%. After that, soil in the pot was washed with 5\u0026nbsp;L of water under low water pressure, to ensure all the nutrients in the soil were empty or low and homogenized.\u003c/p\u003e \u003cp\u003eIn this experiment, seedlings of four-week-old \u003cem\u003eE. pellita\u003c/em\u003e propagated from seeds, and stem cutting was supplied from Acacia Forest Industries Sdn. Bhd. (AFI). Stem cutting was produced from their superior mother clonal plants. The tip was selected for cutting; the rooting duration was four weeks in a greenhouse, prior to the experiment. The seedlings were then transferred to the pot that was filled with the topsoil. The 36 \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and 36 \u003cem\u003eE. pellita\u003c/em\u003e seedlings from stem cutting, accounting for a total of 72 experimental units, including three replications (12 replicates for each fertilizer treatment), were arranged using complete randomized design (CRD). A liquid nitrogen fertilizer (AG Leader 954) was diluted and corresponded to the three different rates of 0 (control), 50\u0026nbsp;N kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 200\u0026nbsp;N kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. No watering was done as the experiment was exposed to natural conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData collection\u003c/h2\u003e \u003cp\u003eIn this experiment, dried shoot biomass, root biomass, root intensity (RI), total root intensity (TRI), root length density (RLD), and specific root length (SRL) were recorded (Hassan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). RI data was collected based on the method by Thorup-Kristensen (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). RI was measured by counting the number of roots crossing the lines of 20\u0026thinsp;\u0026times;\u0026thinsp;20\u0026nbsp;mm grid squares placed on the container surface view sides. RI data was recorded every week, starting from when the roots started to appear on the surface of the transparent pot, until the roots reach the bottom of the pot.\u003c/p\u003e \u003cp\u003eThree different dates sampling procedures were carried out 4, 6 and 8 weeks after transplanting (WAT). Each sampling involved the harvesting of 12 experimental units, or four (4) replicates for each fertilizer treatment from both planting materials. Aboveground biomass was cut from the ground topsoil, washed, and placed in a labelled plastic bag. It was then kept in an oven at 70 ℃ for 48 hours, before being weighed. For root parameters, roots biomass was washed out from soil and organic matter using a sieve 2.0\u0026nbsp;mm mesh under low pressure water. It was then stored in 50% ethanol in a 50\u0026nbsp;ml eppendorf tube at 5\u0026nbsp;\u0026deg;C, before root image analysis. RLD (cm cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) was determined using an EPSON\u0026reg; scanner and Winrhizo\u0026reg; software, and expressed in cm cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (Hassan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For SRL (cm g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the length of a sub-sample was measured, was then divided by its mass (g), before being converted to actual root biomass.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the mean values were subject to statistical analysis using the Statistical Package Social Science (IBM SPSS Statistics 22.0). An independent sampled T-test was used to compare the TRI, RLD, SRL, root biomass, and shoot biomass between two types of plant material \u003cem\u003eE. pellita\u003c/em\u003e seedlings at various nitrogen concentrations for all sampling dates. Subsequently, a one-way ANOVA followed Tukey HSD\u0026rsquo;s post hoc analysis was used for RI at different nitrogen concentrations for both seed and stem cutting seedlings. In assessing the differences between the results, tests with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Prior to statistical analyses, all data were tested for normality using the Shapiro-Wilk Normality test, and for homogeneity using Levene\u0026rsquo;s test.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cp\u003e \u003cb\u003eDried shoot biomass for\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e\u003c/p\u003e \u003cp\u003eDried shoot biomass was harvested three times, four, six and eight weeks after transplanting (4, 6, 8 WAT), as indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It is clear that there was a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) of dried shoot biomass of \u003cem\u003eE. pellita\u003c/em\u003e seedlings between seed and stem cutting, especially at 6 WAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). At 4 WAT, there was no significant difference between seed and stem cutting for both 0 and 50\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, but the shoot biomass of stem cutting was nearly double than seed under 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. In contrast, at 6 WAT, all the treatments showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), where 50% of stem cutting was higher than seed. However, there was no significant difference between seed and stem cutting for all treatments at 8 WAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: Dried Shoot Biomass (g) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Error bars denote standard deviations of the mean, (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot Biomass of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThere was no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the seed and stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e for all treatments in 4 WAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). However, in 6 WAT, only for 50\u0026nbsp;kg N\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the seeds of \u003cem\u003eE. pellita\u003c/em\u003e showed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as compared to stem cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Interestingly, without fertilizer, the root biomass of \u003cem\u003eE. pellita\u003c/em\u003e seeds witnessed a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as compared to stem cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: Root Biomass (g) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Error bars denote standard deviations of the mean, (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal Root Intensity of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e \u003cb\u003efrom seed and stem cutting\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn comparison, the total root intensity (TRI) of \u003cem\u003eE. pellita\u003c/em\u003e stem cutting was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to seed cutting for all measurement dates. Despite the large variations observed in stem cutting treatment, the TRI remained to be nearly double that of seed cutting, especially at 6 and 8 WAT.\u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e: Total Root Intensity (Intersections m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gridline) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and stem cutting plant materials at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values tested using Independent Samples T-Test and statistically the mean values were different, (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Bars represent standard deviations of the mean, \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;36 (4 WAT), \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;24 (6 WAT) and \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;12 (8 WAT).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot Intensity of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e \u003cb\u003eat different nitrogen concentrations\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows a comparison of root intensity (RI) of \u003cem\u003eE. pellita\u003c/em\u003e from seed cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) and stem cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), at various measurement dates and nitrogen concentrations. According to the findings, there was no significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for treatments and types of plant material for each measurement date. However, despite the large variations of RI, \u003cem\u003eE. pellita\u003c/em\u003e stem cutting was clearly higher, and increased with the measurement dates, as compared to seed cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e: Root Intensity (intersections m\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e gridline) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds (a) and stem cutting (b) plant materials at different nitrogen concentrations (0, 50, 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at three selected dates of root measurement (4, 6 and 8 weeks after tranplanting, WAT). The mean values were tested using ANOVA followed by Tukey HSD\u0026rsquo;s post hoc Test. The mean values were not significant different between the different N concentrations for each dates (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05). Bars represent standard deviations of the mean, \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;12 (4 WAT), \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8 (6 WAT) and \u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;4 (8 WAT).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot Length Density of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e showed the root length density (RLD) of \u003cem\u003eE. pellita\u003c/em\u003e, both from seed and stem cutting, taken at three independent harvest times. Based on the results, stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e was significantly higher at 200\u0026nbsp;kg N\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e than at the control and at 50\u0026nbsp;kg N\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). At 6 WAT, all RLDs of stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to seed cutting, for all N concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). However, the RLD of stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e was significantly higher compared to seed cutting under the control and high N concentrations, on the final measurement date (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). It was also found that the RLD of \u003cem\u003eE. pellita\u003c/em\u003e for both seed and stem cutting under fertilizer treatment decreased with the measurement dates.\u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e: Root Length Density (cm cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and stem cutting plant material at different nitrogen concentrations (0, 50, 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Error bars denote standard deviations of the mean, (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpecific Root Length (SRL) of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eE. pellita\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe specific root length (SRL) of \u003cem\u003eE. pellita\u003c/em\u003e was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for all treatments and measurement dates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e). At 4 WAT, SRL of stem cutting was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to seed cutting, almost by three-fold, especially at high N concentrations. Similar findings were also found at 6 WAT, which was almost 50% higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than seed cutting for all fertilizer treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, at 8 WAT, SRL was found to be significantly higher for approximately 50% of stem cutting, as compared to seed cutting, at zero and 50\u0026nbsp;kg n ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. No difference in SRL was found between stem cutting and seed cutting of \u003cem\u003eE. pellita\u003c/em\u003e at 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003e: Specific Root Length (cm g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of \u003cem\u003eE. pellita\u003c/em\u003e seedlings from seeds and stem cutting plant materials at different nitrogen concentrations (0, 50, 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at three selected dates of root measurement; 4 weeks after transplanting, WAT (a), 6 WAT (b), and 8 WAT (c). The mean values were tested using Independent Samples T-Test. All mean values were significant different *(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Error bars denote standard deviations of the mean, (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eThe findings demonstrate that shoot biomass of stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e seedlings was greater compared to seedlings from seed cutting. The shoot biomass is connected with root distribution in the soil, especially the fine roots from stem cuttings. The larger the fine root density, the more water and nutrients are taken up, expressed by higher shoot biomass. This is confirmed by Rostamza et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), who reported that greater root length in millet is connected to an increased shoot biomass. However, in this present study, seedlings from stem cutting were significantly higher with an increase in nitrogen levels, as compared to seedlings from seed cutting (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, seedlings from seed cutting were not affected under different nitrogen concentrations. At the final harvest, there was no significant difference between stem cutting and seed cutting for all fertilizer treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Therefore, biomass is influenced by species, and specific silviculture such as irrigation (Toky et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), fertilization and water availability (Ares et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn root biomass, most of the mean values did not differ for seed and stem cutting, for all treatments, except for 50\u0026nbsp;N kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 6 WAT, and for zero nitrogen at 8 WAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, Coleman et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) reported that root biomass tends to increase with fertilizer rate, but the proportion of root biomass tends to decrease with more fertilizer application. This argument is associated with the current findings, especially for higher nitrogen concentrations, showing that there is no increase with fertilization rate. Another explanation of the results is that seedlings from seed cutting were higher compared to stem cutting, and had no significant difference, because seed cutting produces tap root and high root mass, while stem cutting produces fibrous and fine roots.\u003c/p\u003e \u003cp\u003eWithout any statistical significance, the total RI for both propagation types of \u003cem\u003eE. pellita\u003c/em\u003e seedlings was not different, although there was a higher distribution under stem cutting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Looking at RI, at different nitrogen concentrations for both seed and stem cutting, the distribution of RI was higher in stem cutting compared to seed cutting, although there was no significant difference. This shows that there are more fine roots in stem cutting than in seed cutting, which and increased with the measurement date (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The findings of this study are in agreement with prior work that has proven that more root distribution, especially for fine roots, is closely associated to soil water and nutrients (Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Wang \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). This explains the presence of more fine roots after stem cutting on the soil.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn RLD, stem cutting was also significantly higher at high nitrogen concentrations, compared to seed cutting. In the subsequent measurement date, all of the nitrogen levels under stem cutting increased significantly compared to seed cutting; this also applies on the final measurement date, except under 50\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the values decreased with the measurement dates. The RLD of seedlings from seed cutting were neither affected by different nitrogen levels, nor by measurement dates. Similarly, in SRL, stem cutting was also significantly higher compared to seed cutting, as found in RLD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite stem cutting being higher compared to seed cutting, especially in shoot biomass, in SRL and RLD, these growth parameters were not affected by different nitrogen levels. Especially at high nitrogen concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), the SRL did not differ between the propagation types. Previous work reported various responses of fertilizer rates against \u003cem\u003eEucalyptus\u003c/em\u003e in Brazil (Goncalves et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Goncalves et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Stape et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), in Australia (Smethurst et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mendham et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and in South Africa (du Toit et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Nevertheless, fertilizer responses varied, depending on the species and sites considered (Halomoan et al. 2015). \u003cem\u003eEucalyptus\u003c/em\u003e in Brazil and South Africa responded to fertilization when water was available (Stape et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; du Toit et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Stape et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) reported that the application of very high rates and excessive nitrogen levels in Brazil did not show any significant effects on \u003cem\u003eEucalyptus\u003c/em\u003e productivity (Halomoan et al. 2015). Fertilizer rates from 50 to 100\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e increased biomass, but then biomass decreased at a rate of 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Halomoan et al. 2015); this was also reported in the present study. Graciano et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reported that P applications affected \u003cem\u003eE. grandis\u003c/em\u003e biomass more than N applications in Argentina. However, we cannot validate this argument, since this present study tested \u003cem\u003eE. pellita\u003c/em\u003e seedlings at a nursery scale.\u003c/p\u003e \u003cp\u003eIn this case, a rate of 50\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be cheaper and more efficient to absorb by \u003cem\u003eE. pellita\u003c/em\u003e, as opposed to 200\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Chen et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) reported that moderate nitrogen fertilizer increased the root intensity in soil layers. As explained above, the root distribution from propagation seeds is less compared to stem cutting. With the short period for the experiment under a small pot, we cannot observe the difference between the nitrogen concentrations. This needs to be done in a larger field.\u003c/p\u003e \u003cp\u003eIn the comparison between seed and stem cutting, as the above findings, propagation by stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e was proved to be viable and productive in terms of root performance at the nursery stage. Although there are works in the related literature that have proved that seed cutting is still the better propagation method (Kiragu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), producing plant material using stem cutting is not only more efficient and faster, but would also be able to reduce the production costs and time spent for upkeep and maintenance in the nursery. Partelli et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) also supported the fact that the cuttingpropagated method for coffee is more productive than the seedpropagated method. Furthermore, Naidu and Jones (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) also suggested a superior initial survival and growth of \u003cem\u003eE. dunnii\u003c/em\u003e minicuttings compared to seedlings based on early indications. This finding has also proven that secondary branches as semi-hard wood cuttings could be the most effective propagation material of \u003cem\u003eJatropha curcas\u003c/em\u003e (Santoso and Parwata, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotwithstanding this, rooting ability of cuttings from woody or perennial plants declined with an increase in the age of the mother plants (Santoso and Parwata \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Root ability of cutting formation becomes more difficult with a farther position from the apical shoot (Hartmann et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e: Wilson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), due to differences in the type and number of carbohydrates and other stored materials (Hartmann et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Leakey \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Therefore, root system characteristics are known to differ according to species, genotype, plant age, physiological status of mother plant (Henning \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), season, climate, plant density, root diameter, biotic stresses, and soil texture and structure (Lynch \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Also, the growth rate of stem cutting depends on age variation, position in stem, and diameter of stem (Kraiem et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study therefore provides more information and understanding on \u003cem\u003eE. pellita\u003c/em\u003e for forest plantation companies in producing plant materials using stem cutting in a cost-effective and efficient manner. Further research is required on the root aspect, especially in real field conditions, as the soil is more heterogenous and exhibits different environmental conditions. Such findings will help these companies take agronomic measures and a silvicultural approach in the future.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eTo conclude, \u003cem\u003eE. pellita\u003c/em\u003e seedlings from stem cutting were greater in terms of root distribution compared to propagation by seed cutting, at the nursery stage. In addition, aboveground biomass of stem cutting was also higher in \u003cem\u003eE. pellita\u003c/em\u003e seedlings than of seed cutting. The 50\u0026nbsp;kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was the optimal nitrogen concentration to be applied to the \u003cem\u003eE. pellita\u003c/em\u003e seedlings. This is because excessive fertilizer application not only increases fertilizer costs, but may also not necessarily result in an increased volume yield or shoot biomass. Moreover, it is harmful to the soil. Research on the root distribution of these two types of propagation in real field soil merits further investigation, as different environmental factors may affect the growth performance of \u003cem\u003eE. pellita\u003c/em\u003e. Thus, this would help the forest plantation companies in planning appropriate agronomic management in the future.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNitrogen; RI:Root intensity; TRI:Total root intensity; RB:Root biomass; RLD:Root length density; SRL:specific root length; WAT:Weeks after transplanting.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the staff of Acacia Forest Industries Sdn Bhd (AFI) and technical staffs of Forestry Complex, Faculty of Science and Natural Resources, UMS during the study in technical support, as well as the anonymous reviewers, for their thoughtful comments and suggestions on the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAH designed the study, supervised data collection and contributed to and edited manuscripts. PB and KRK collected literatures, prepared field experiments, data collection, laboratory analysis, and prepared all figures. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank to Acacia Forest Industries Sdn Bhd (AFI) for its kind support during the study, especially in sponsoring the seedlings and technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForest Plantation and Agroforestry Programme, Forestry Complex, Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAres A, Burner DM, Brauer DK (2009) Soil phosphorus and water effects on growth, nutrient and carbohydrate concentrations, d 13C, and nodulation of mimosa (\u003cem\u003eAlbizia julibrissin\u003c/em\u003e Durz.) on a highly weathered soil. 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J Sci Ind Res. 70: 601-609.\u003c/p\u003e\n\u003cp\u003eWong SK, Ahmad Zuhaidi Y, Charles GDC, Peter KCS (2015) Recommending \u003cem\u003eEucalyptus\u003c/em\u003e Species for Soft Loan Financing. Working paper presented at the 1st Technical Meeting on Forest Plantation Programme, Malaysian Timber Industry Board (MTIB), Kuala Lumpur. Malaysia.\u003c/p\u003e\n\u003cp\u003eYahya AZ (2020) Planting of \u003cem\u003eEucalyptus\u003c/em\u003e in Malaysia. Acta Sci Agric. 4(2):139-140. doi: 10.31080/ASAG.2020.04.0785\u003c/p\u003e\n\u003cp\u003eYahya AZ, Hassan NH, Loon NT, Heng LH, Zorkarnain FA (2020). Comparing the early growth performance of plantation\u0026ndash;grown \u003cem\u003eEucalyptus\u003c/em\u003e hybrid and \u003cem\u003eEucalyptus pellita\u003c/em\u003e, south Johore, Peninsular Malaysia. World J of Adv Res Rev. 6(2), 234-238. doi:10.30574/wjarr\u003c/p\u003e\n\u003cp\u003eYew HS, Su HK, Lung NM, Ak Penguang SJ, Meder R (2015) Assessment of Plantation-Grown \u003cem\u003eEucalyptus pellit\u003c/em\u003ea in Borneo, Malaysia for Solid Wood Utilisation. In: IUFRO Eucalypt Conference 2015. 21-24 October, 2015, Zhanjiang, Guangdong, China.\u003c/p\u003e\n\u003cp\u003eWang J (1990) The root system development of Cicer milkvetchin - the first year of growth. Pratac Sci. 7(1): 53-60.\u003c/p\u003e\n\u003cp\u003eWilson PJ (1993) Propagation characteristics of \u003cem\u003eEucalytus globules\u003c/em\u003e Labill. spp. globules stem cutting in relation to their original position in the parent shoot. J of Hort Sci. 68(5):715-724. doi:10.1080/00221589.1993.11516404\u003c/p\u003e\n\u003cp\u003eZaiton S, Paridah MT, Hazandy AH, Azim RARA (2018) Potential of \u003cem\u003eEucalyptus\u003c/em\u003e Plantation in Malaysia. The Malay For. 81(1):64-72.\u003c/p\u003e\n\u003cp\u003eZaiton S, Sheriza MR, Ainishifaa R, Alfred K, Norfaryanti K (2020) Eucalyptus in Malaysia: Review on Environmental Impacts. J of Lands Ecol. 13(2):79-94. doi:10.2478/jlecol-2020-0011\u003c/p\u003e\n\u003cp\u003eZhang D, Jiang X, Zhao S (1995) Further thoughts on growth redundancy. Acta Pratac Sci. 4(3): 17-22.\u003c/p\u003e\n\u003cp\u003eZhao M, Tan C, He D (1990) A study of root system of \u003cem\u003eArtemisia dalailamen\u003c/em\u003e. Pratac. Sci. 7(3): 55-57.\u003c/p\u003e\n\u003cp\u003eZhou X, Zhu H, Wen Y, Goodale UM, Li X You Y, Liang H (2018) Effects of understory management on trade-offs and synergies between biomass carbon stock, plant diversity and timber production in eucalyptus plantations. For Ecol Manage. 410:164\u0026ndash;173. doi:10.1016/j.foreco.2017.11.015\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Root distribution, root traits, propagation technique, stem cutting, fast growing species","lastPublishedDoi":"10.21203/rs.3.rs-104802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-104802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\u003cp\u003e\u003cem\u003eEucalyptus\u003c/em\u003e is among the important fast-growing species, and is typically managed on short rotation to sustain the production of timber, pulpwood, charcoal, and fire-wood. Macro-propagation using cutting for larger multiplying seedlings is cheaper and efficient instead of clonal seeds for uniform plant material seedling production. However, information on root growth of \u003cem\u003eEucalyptus pellita\u003c/em\u003e at early development from seed and stem cutting of \u003cem\u003eE. pellita\u003c/em\u003e seedlings is still lacking. This is probably due to the difficulty in investigation belowground, and also due to methodological problems. With such information, it is useful for forest plantation company management in enhancing the understanding on strategies to optimize yield production with the appropriate agronomic or silvicultural approach in the field planting. Therefore, the objectives of this study were; to compare the root development of two different propagation seedlings of \u003cem\u003eE. pellita\u003c/em\u003e; and to study the effect of various nitrogen concentration levels on two types of propagation of \u003cem\u003eE. pellita\u003c/em\u003e seedlings. \u003c/p\u003e\u003cp\u003eResults\u003c/p\u003e\u003cp\u003eThe study was conducted using \u003cem\u003eE. pellita\u003c/em\u003e seedlings from two types of propagation, namely, seed and stem cuttings, along with three different nitrogen concentrations (0, 50, and 200 kg N ha\u003csup\u003e-1\u003c/sup\u003e). Shoot biomass, root intensity (RI), total root intensity (TRI), root biomass, root length density (RLD), and specific root length (SRL) were recorded. Dried shoot biomass, RLD and SRL of \u003cem\u003eE. pellita\u003c/em\u003e seedlings using stem cutting were significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) compared to seed. Whereas, there were no significant differences\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05) for root biomass, TRI and RI between the propagation types of \u003cem\u003eE. pellita\u003c/em\u003e seedlings. \u003c/p\u003e\u003cp\u003eConclusions:\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. pellita\u003c/em\u003e seedlings from stem cutting was greater in terms of root distribution compared to propagation by seeds at the nursery stage, and 50 kg N ha\u003csup\u003e-1\u003c/sup\u003e was the optimal nitrogen concentration level from the considered levels to be applied to the \u003cem\u003eE. pellita\u003c/em\u003e seedlings. The present study therefore provides more information and understanding on \u003cem\u003eE. pellita\u003c/em\u003e for forest plantation companies in producing plant materials using stem cutting in a cost-effective and efficient manner. This would help the forest plantation companies in planning appropriate agronomic management in the future.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Early root development of Eucalyptus pellita F. Muell. seedlings from seed and stem cutting at nursery stage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-13 18:32:06","doi":"10.21203/rs.3.rs-104802/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"430db76a-99c6-40e4-9a88-e15b2ecc0635","owner":[],"postedDate":"November 13th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1069995,"name":"Forestry"}],"tags":[],"updatedAt":"2020-11-14T20:52:00+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-13 18:32:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-104802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-104802","identity":"rs-104802","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}