A protocol for in vitro multiplication of Morella sp: a protected species in the Tambillo Community Protected Area - Ecuador

doi:10.21203/rs.3.rs-3214781/v1

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Research Article

A protocol for in vitro multiplication of Morella sp: a protected species in the Tambillo Community Protected Area - Ecuador

https://doi.org/10.21203/rs.3.rs-3214781/v1

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published 13 Dec, 2023

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The objective of this research was to develop a protocol for the in vitro establishment and multiplication of Morella sp. (Myricaceae). Tender apical shoots of Morella sp. were used as explants, which were disinfected under aseptic conditions with a 2% NaOCl solution for 4 minutes. Ninety percent of the apical shoots were free from contaminants. In the in vitro multiplication stage, the increase in the number of leaves was observed, being directly proportional to the concentration of the basal medium with WPM salts and vitamins + 0.25 mg.L^-1 gibberellic acid (GA₃) and different doses of 6-benzylaminopurine (BAP). The explants cultured in the basal medium with WPM salts and vitamins at 100% supplemented with 0.25 mg.L^-1 GA₃ + 1.5 mg.L^-1 BAP, generating an average of 6.4 leaves per explant. Regarding in vitro rooting, no statistically significant differences (p > 0.05) were found in the number of roots per explant among the evaluated treatments of indole-3-butryic acid (IBA) and indole-3-acetic acid (IAA). However, the number and length of roots per explant were higher in the treatments supplemented with IBA, these showed the highest number of roots per explant (2.4 roots) at a dose of 0.75 mg.L^-1 and a higher average length of 3.7 cm at a dose of 1 mg.L^-1 IBA. The results obtained in this research lay the basis for further optimization of the micropropagation protocol for mass production of Morella sp. plants and to achieve the goal of reforesting Tambillo, the first community protected area in Ecuador.

Laurel de cera

in vitro

6-benzylaminopurine (BAP)

indole-3-butryic acid (IBA)

indole-3-acetic acid (IAA)

Ecuador is the fifth most mega-diverse country in the world due to its high species diversity. This diversity is influenced by the country's location on the equator, the convergence of ocean currents, the presence of the Andes Mountain range, and the Amazon forests (Mittermeier & Mittermeier, 1997). Unfortunately, the current situation of Ecuador's native forests is alarming due to the decline in forest cover. For example, the coastal rainforests have decreased to only 3% of their original coverage (Izko & Burneo, 2003). In the highlands, natural vegetation has decreased by 570,552.2 hectares from 1999 to 2003, representing an 8.6% decline (Villamarín-Cortez et al. 2009). Despite this, certain areas of protected areas have been identified where different types of forests are conserved, such as tropical rainforests, cloud forests, and montane forests (Yánez et al. 2016).

The Tambillo Community Protected Area was established as the first community protected area in Ecuador and is located in San Miguel de Cuyes de Gualaquiza (Morona Santiago) (MADS, 2018). Within the forest of the Tambillo Community Protected Area, which covers approximately 2,000 hectares and has an altitude range between 2,500 and 3,200 meters above sea level, two dominant vegetation types can be found: forests and grasslands, with small strips of shrub vegetation in transitional areas. In addition to the aforementioned vegetation, various trees and shrubs are found, such as Prumnopitys montana (Mollón), Clusia sp. (Duco), Weinmannia ovata (Sarar), Hedyosmum sprucei (Toronjil de cerro), Drimys granadensis (Halo), Morella sp. (Laurel de cera) (Alba, 2012).

Morella sp. (Myricaceae) is a forest species that grows spontaneously in pastures, roadside slopes, and near rivers and streams (Paz & Paz, 2012). This species is cultivated in countries such as Bolivia, Ecuador, Peru, and Colombia, and is native to the northern part of the American continent (Quijano & Pino, 2007). In Ecuador, the species is recorded in the provinces of Azuay, Morona Santiago, Cañar, Carchi, Chimborazo, Cotopaxi, El Oro, Pichincha, and Tungurahua (Inga, 2017). Due to its multipurpose nature, several studies highlight its importance within agroforestry systems (Cabrera, 2011) and its industrial uses, such as wax extraction for panela production, candle making, soap production, floor wax, and bronze casting processes (Paz & Paz, 2012).

According to the IUCN Red List of Threatened Species, species of the genus Morella are classified as "critically endangered" (IUCN, 2022), likely due to various anthropogenic activities, such as agriculture, illegal logging, periodic burning and forest fires, illegal mining, resulting in deforestation, soil cover fragmentation, biodiversity loss, and changes in microclimates in affected regions (Orozco et al. 2011). Laurel de cera has both economic and environmental significance. On one hand, it can be implemented in agroforestry systems and temporary crops, and its essential oil can be extracted. On the other hand, Laurel de cera contributes significantly to environmental protection due to its soil and watershed protection properties, thanks to its abundant and deep roots (Cabrera, 2011; Palacios, 2016). It is considered a valuable component for the recovery of degraded areas, as it naturally regenerates through seed dispersal (Salazar, 2022).

This forest species propagates sexually, with birds being the natural dispersal medium, forming groups of trees of varying sizes. However, the germination of Laurel de cera seeds using conventional methods is inefficient due to their low germination capacity (10–20%) (Cabrera, 2011). Therefore, it is necessary to investigate pre-germination methods for sexual reproduction of the species and to explore asexual reproduction using biotechnological tools such as in vitro plant tissue culture (Sharry et al. 2015).

In vitro plant tissue culture is a valuable tool that allows the production of healthy plants in a short period and in large quantities, and it is particularly useful for species that have difficulties with conventional propagation methods (Bonilla-Morales & Hernández-Castañeda, 2012). Therefore, the purpose of this research was to evaluate the response of Morella sp. (Myricaceae) to in vitro propagation. It is important to note that the plants generated in this research will be used for the reforestation program in the Multipurpose Project in the Tambillo Community Protected Area.

Location and plant material

This research was conducted in the Biotechnology Laboratory of the Faculty of Agronomic Engineering at the Technical University of Manabí, located in the Lodana parish of Santa Ana canton at an altitude of 50 meters above sea level (01°12'25''S 80°22'15''W). Seeds of mature Morella sp. (Myricaceae) trees were collected from within the Tambillo Community Protected Area, located in the San Miguel de los Cuyes parish of Morona Santiago province (latitude 03°17'20.0" South and longitude 78°52'42.4" West). To establish a mother plant bank, the seeds were washed, and the wax covering them was manually removed. Additionally, they underwent pre-germination treatments, including soaking the seeds in water and in a GA₃ solution at concentrations of 250 ppm and 500 ppm for 24 and 48 hours, respectively. The treated seeds were then sown in germination trays with 50 cells (54.5 cm in length and 29 cm in width), using peat moss (Floria peat) as the substrate, and placed in a shade house.

In vitro establishment of Morella sp.

The explants were disinfected according to the procedures described by Cevallos and Loor (2023) in the Biotechnology Laboratory of the Faculty of Agronomic Engineering at the Technical University of Manabí (unpublished data). The explants were washed with water and liquid soap for 15 minutes with agitation, followed by several rinses with running water. In a laminar flow hood, the explants were immersed in 70% ethanol for 1 minute and then rinsed with sterile distilled water. One hundred explants were cut into approximately 1 cm in length and soaked in a 200 ppm cysteine solution until planting. They were then planted in 150 x 25 mm test tubes with 10 ml of WPM basal medium (Woody Plant Medium) at 100% strength, supplemented with sucrose as a carbohydrate source at 3%, agar at 0.7%, and cysteine at 100 ppm. The pH of the media was adjusted to 5.8, and they were sterilized for 20 minutes at 121°C and 1.5 atm. The culture conditions were maintained at a temperature of 24 ± 2°C and a photoperiod of 16/8 hours light/dark. In this phase, the production of new leaves and contamination of explants by fungi and bacteria were evaluated.

In vitro multiplication of Morella sp.

In a laminar flow hood, explants from six-week-old seedlings obtained in the in vitro establishment phase were sectioned. The explants were subcultured in 150 x 25 mm test tubes with 10 ml of WPM basal medium at 50% and 100% strength, supplemented with 0.25 mg.L^− 1 GA₃ and three doses of BAP (1-1.5 and 2 mg.L^− 1). The explants were maintained under the same environmental conditions as the establishment phase for 8 weeks, following the methodology described by Hernández-Jerez et al. (2013) with modifications. In this phase, the number of leaves and shoots were evaluated using a completely randomized design (CRD) 1 x 2 + 2 (1 growth regulator x 2 concentrations of the culture medium + 2 control treatments), with 5 replicates per treatment.

In vitro rooting of Morella sp.

Shoots from explants obtained in the in vitro multiplication phase, which had at least 2 cm in length, were selected. They were then subcultured in 150 x 25 mm test tubes with 10 ml of WPM basal medium at 100% strength (which showed the best responses in the multiplication phase), supplemented with different concentrations of auxins: Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA) at three doses (0.5–0.75 and 1 mg.L^− 1). The explants were maintained under the same temperature and photoperiod conditions as the previous phases for 8 weeks. In this phase, the number and length of roots (cm) were evaluated. The treatments were distributed in a completely randomized design (CRD) 2 x 3 + 1 (2 growth regulators x 3 concentrations + 1 control) with 7 replicates per treatment.

Statistical analysis

The assumptions of normality (Shapiro-Wilk test) and homogeneity of variances (Bartlett's test) were analyzed. When these assumptions were not met, the data were transformed to √y obs + 0.5. Analysis of variance (ANOVA) was performed, followed by the Scott-Knott multiple comparison test (p ≤ 0.05) and the Dunnett test (p ≤ 0.05) to compare treatment means with controls. The chi-square test was used to analyze variables in the establishment phase. Polynomial and linear regression adjustments were used to estimate the parameters of the different equation models. All calculations and statistical analyses were performed using the "ExpDes.pt" and "Treatments.ad" packages available in R software version 4.3.0 (R Core Team, 2023).

In the in vitro establishment phase, using a previous disinfection process with sodium hypochlorite (2%), 2–4 leaves per explant of Morella sp. were obtained, indicating a limited likelihood (p = 0.3331) of obtaining a different number of leaves than the expected values (Table 1). On the other hand, with the NaOCl (2%) disinfection protocol for 4 minutes, the percentage of contamination by fungi and bacteria was only 10%, indicating a low likelihood (p = 0.1653) of obtaining contamination levels above the expected values. Similarly, Ramírez et al. (2014) mention that contamination can be reduced by adding NaOCl at 2% and applying it for 5 minutes. Additionally, Núñez et al. (2017) report that disinfection with 3% sodium hypochlorite for 10 minutes yielded the best response in Caesalpinia spinosa (Mol.) O. Kuntz. On the other hand, Méndez & Abdeknour (2014) indicate that in the in vitro establishment of Terminalia amazonia, treatments with 3% NaOCl resulted in a higher number of aseptic explants. Additionally, the physiological age of the plant material used to initiate in vitro cultures is an important factor that significantly affects the disinfection process (López-Gómez et al.2010; Pedroza et al.2007).

Table 1

Production of new leaves and percentage of contamination of *Morella* sp. explants after disinfection with 2% sodium hypochlorite.
Especie	Production of new leaves			Contaminación de explantes
Especie	Number of leaves per explant	Observed frequency (%)	Expected frequency (%)	Type of contamination	Observed frequency (%)	Expected frequency (%)
Morella sp.	2	29	33,3	Fungi	2	5
	3	41	33,3	Bacteria	8	5
	4	30	33,3	No contamination	90	90
X²	2,19			3,60
p-value	0,3331^ns			0,1653^ns
Chi-square values for the adherence test; ns = non-significant values

The number of leaves per explant in Morella sp. was significantly impacted by the interaction between the culture medium and BAP concentrations, with statistical differences (p ≤ 0.05) among the treatments (Fig. 1A-C). Explants established in WPM basal medium with 100% salts and vitamins, supplemented with 0.25 mg.L^− 1 GA₃ + 1.5 mg.L^− 1 BAP, showed the highest average number of leaves per explant (6.4) (Table 2; Fig. 1D-F). The use of higher doses may not result in further leaf development. Explants exposed to the interaction between WPM 50% supplemented with 0.25 mg.L^− 1 GA₃ and different concentrations of BAP recorded the lowest number of leaves per explant, without showing statistical differences (p > 0.05) compared to the control treatments.

Table 2

Effect of woody plant medium (WPM + 0.25 GA₃) and concentrations of 6-benzylaminopurine on the number of leaves and shoots of *Morella* sp. under *in vitro* conditions
	Number of leaves			Number of shoots
Treatments	6-Benzylaminopurine (mg.L^− 1)			6-Benzylaminopurine (mg.L^− 1)
	1^ns	1,5	2^ns	1	1,5	2^ns
WPM 50% + 0.25 GA₃	3.6 ± 0.8	4.2 ± 0.4 b	3.8 ± 0.2	1.8 ± 0.6 b*⁺	1.6 ± 0.6 b*⁺	2.2 ± 0.4 *⁺
WPM 100% + 0.25 GA₃	4.4 ± 0.6	6.4 ± 0.4 a*⁺	3.0 ± 0.3	3.4 ± 0.5 a*⁺	4.0 ± 0.3 a*⁺	3.4 ± 0.2 *⁺
Control 1	4.6 ± 0.4			0.0
Control 2	3.8 ± 0.6			0.0
Means followed by the same letter in the column do not differ significantly according to the Scott-Knott test (p ≤ 0.05). ns = no significant differences. The means of the common treatments followed by "*" and "+" differ from control 1 (WPM 50% + 0.25 GA3) and control 2 (WPM 100% + 0.25 GA3), respectively, according to the Dunnett test (p ≤ 0.05).

The findings of this research indicate that carefully choosing the culture medium and its concentration is essential to achieve a positive response in the in vitro propagation of Morella sp. In this regard, it can be determined that the WPM culture medium plays a crucial role in the response efficiency of the explants and is widely employed in in vitro culture of woody species. Multiple studies have described WPM as one of the most commonly used media for woody and shrub species, as it is specifically designed and adapted for their requirements (Naranjo, 2022; Poothong & Reed, 2014).

The use of WPM at 100% concentration has shown a positive effect on the response of explants in the in vitro propagation of apple tree (Malus domestica) (Cabral et al.2022). Similarly, Jiménez et al. (2020) reported that in the in vitro propagation of Buddleja incana Ruíz & Pav, the WPM culture medium at 100% concentration yielded the best response among the treatments under study. Conversely, in other research on forest species (Bursera laxiflora S. Watson; Bertholletia excelsa Bonpl.), it was highlighted that using WPM at 50% concentration resulted in the best response in in vitro propagation (Mc-Caughey et al.2020; Robles et al.2023). Variable responses to the concentration of the WPM basal medium with salts and vitamins may be influenced by the specific nutritional requirements and characteristics of each species (García et al. 2001; Rueda et al. 2013).

A commonly adopted practice in in vitro propagation is the addition of growth regulators to the culture medium, which has proven to be effective in enhancing the in vitro multiplication phase (Bhojwani & Dantu, 2013). The combination of cytokinins and auxins in the medium is beneficial for plant propagation as these phytohormones work together to stimulate the growth and development of explants (Alcantara et al.2019). Furthermore, Arteaga (2022) revealed that the inclusion of growth regulators (BAP 0.3 mg.L^− 1 and 0.5 mg.L^− 1 GA₃) resulted in positive effects on the multiplication of Actinidia chinensis. Silva et al. (2020) determined that the best average results in terms of leaf number were obtained with the addition of 2 mg.L^− 1 BAP and 5 mg.L^− 1 GA₃ in Salix babylonica L.

BAP is a growth regulator that promotes plant growth and is widely used in the in vitro propagation of various species. Due to its ability to stimulate the photomorphogenic development of plants, it plays a crucial role in leaf generation and increased shoot production at the vegetative level (Moncada et al.2004; Pedroza-Manrique & Bejarano-Tibocha, 2008). In this regard, Ventura (2016) describes that a concentration of 1 mg.L^− 1 BAP stimulates a higher number of leaves per explant (3.9) in Physalis peruviana L. Similarly, Ruiz et al. (2020) state that the addition of 1 mg.L^− 1 BAP to the WPM culture medium during the multiplication phase of Swietenia macrophylla King resulted in the best average values of leaf number per explant (4.80).

The number of leaves was not affected by the change in BAP concentrations in WPM 50% + 0.25 mg.L^-1 GA₃ (Fig. 2A). However, when WPM 100% + 0.25 mg.L^-1 GA₃ (Fig. 2B) was supplemented with different BAP concentrations, changes in the number of leaves per explant were observed, indicating that increasing the BAP doses up to 1.5 mg.L^-1 resulted in an increase in the number of leaves in Morella sp. explants. However, higher doses may limit their response. This is consistent with Montes-Salazar et al. (2016), who reported that the presence of high doses of BAP (3 and 5 mg.L^-1) in WPM medium does not significantly affect the in vitro propagation of Cedrus atlantica.

Regarding the number of shoots, the Morella sp. explants did not show statistical significance (p > 0.05) in the interaction between the medium concentration and BAP dosage (Table 3), nor did the number of shoots increase as the concentrations of the aforementioned phytohormone were increased. In a study conducted by Núñez et al. (2017), the effect of various BAP doses (0.25–0.50 and 1 mgL^-1) on the multiplication of Caesalpinia spinosa (Mol.) O. Kuntz was evaluated. The results indicate that BAP did not have a significant effect on the number of shoots (2.8). Conversely, Abbasi et al. (2013) highlight that BAP is effective in shoot production due to the ability of plant tissues to metabolize it more efficiently than other regulators, or its capacity to stimulate zeatin production. Similarly, Kharel et al. (2022) describe that the addition of 6 mg.L^-1 BAP resulted in the highest number of shoots in Vaccinium corymbosum L.

Table 3

Effect of indole-3-butyric acid and indole-3-acetic acid on the number of roots and root length (cm) under *in vitro* conditions.
	Number of roots (cm)			Root length (cm)
Tratamientos	Dose (mg.L^− 1)			Dose (mg.L^− 1)
	0,5^ns	0,75	1^ns	0,5^ns	0,75^ns	1
Indole-3-butyric acid	1,7 ± 0,2	2,4 ± 0,2 a+^*	3,0 ± 0,2 +^*	1,1 ± 0,5	2,0 ± 0,4	3,7 ± 0,3 a+^*
Indole-3-acetic acid	1,6 ± 0,4	1,4 ± 0,2 b	2,1 ± 0,4	1,6 ± 0,2	1,9 ± 0,3	2,0 ± 0,0 b
Control	1,1 ± 0,1			1,3 ± 0,2
Means followed by a different letter in the column differ from each other according to the Scott-Knott test (p ≤ 0.05). ns = no significant differences. Means of common treatments followed by * differ from the control treatment (WPM 100%) according to the Dunnett test (p ≤ 0.05).

The concentration of the growth medium was the only factor that positively affected the increase in the number of shoots per explant, with the highest response observed in the treatments with WPM 100% (Fig. 3). Similarly, Delgado & Hoyos (2016) describe that in the in vitro multiplication of Aniba perutilis Hemsl, the best response in terms of the number of shoots (0.6) was obtained in WPM 100% medium. Likewise, Ruiz et al. (2020) indicate that in the in vitro multiplication of Swietenia macrophylla (Mahogany), the WPM 100% growth medium produced the highest average values in the number of shoots (1.30).

No statistical differences (p > 0.05) were observed in the number of roots per explant in the interaction between the growth regulators (IBA and IAA) and their doses (0.5, 0.75 and 1 mg.L^− 1). The number of roots per explant was mainly influenced by the type of growth regulator used, with IBA reporting the highest number of roots per explant, with a mean of 2.3 roots, which were higher than the control treatment (Table 4). Eras et al. (2019) evaluated the effect of IBA in Cinchona officinalis L., highlighting an increase in the number of roots per explant (0.8224). Similarly, in the rooting of Bertholletia excelsa Bonpl., IBA was found to generate the best response in the number of roots per explant (4) (Ancasi et al.2023).

This response could be attributed to the fact that auxins play an important role in various stages of plant development, including growth and root formation (Alcantara et al.2019). Additionally, it has been reported that IBA, as a growth regulator, can stimulate plant development by promoting root formation. Similarly, Báez-Pérez et al. (2015) describe that the use of IBA can have a positive impact on plant growth by promoting nutrient absorption, improving root development, and optimizing metabolic functions.

The length of roots was higher in treatments supplemented with IBA. In the treatment with the addition of 1 mg.L^− 1 IBA, Morella sp. explants showed an average root length of 3.7 cm, higher than the treatments with IAA and the control treatment (Table 3). Patiño et al. (2014) mentioned that IBA at concentrations of 0.5 and 1.0 mg.L^− 1 resulted in the best average root length values (3.1 and 2.7 cm). It is important to note that these findings indicate that IBA is the most suitable growth regulator for this species at the evaluated doses. Similarly, Cancino-Escalante et al. (2015) reported that the addition of 2 mg.L^− 1 IBA resulted in the longest root length (4.9 cm).

The number of roots/explants increased as the concentrations of IBA were increased (Fig. 4A), as demonstrated by the regression analysis, where the linear coefficient of the equation was highly significant (p ≤ 0.01). However, the different concentrations of IAA did not increase the number of roots (Fig. 4B), and no relationship was found through the tested linear regression analysis. It is worth noting that an increase in the concentration of IAA in the culture media may result in a decrease in root length. Patiño et al. (2014) reported that the number of roots/explants increased by 55% with a higher dose of IBA (1 mg.L^− 1), resulting in an average of 15.3 roots. Many studies on in vitro rooting have shown that the inclusion of auxins such as IBA or IAA in the culture medium is necessary to obtain a better response in root formation (Suárez et al.2006; Valverde-Cerdas et al.2008). This is because they are directly involved in physiological processes: 1) cell division and elongation, 2) tissue differentiation (Kravchenko et al.2004).

On the other hand, root length showed a linear growth in response to increasing concentrations of IBA (Fig. 5A), with high statistical significance (p ≤ 0.01) for the regression coefficients, which is a good indicator of the quality of our linear model. Conversely, the linear regression fit demonstrated that the increase in IAA concentrations did not significantly induce the growth of root length in Morella sp. explants under in vitro conditions (Fig. 5B). Domínguez et al. (2019) determined that the addition of 1 mg.L^− 1 of IBA resulted in an 85% increase in the response of explants to in vitro rooting of Paulownia elongata x Fortunei. Additionally, Bohórquez-Quintero et al. (2016) concluded that treatment with the highest dose of IBA (5 mg.L^− 1) resulted in the longest average root length (13 cm) in Espeletia paipana S.

The results obtained in this research suggest that Morella sp. explants respond better to a concentration of 1 mg.L^− 1 of IBA, as it generated the best average values for the studied variables in the rooting phase. In addition to this, Phillips & Garda (2019) describe that IAA is a natural auxin, sensitive to light, and easily degrades, while synthetic auxins like IBA are chemically more stable and can be 10 to 1000 times more potent. Therefore, the quantities used are relative to potency, which varies among plant species.

The evaluated rooting variables (number of roots and root length) showed a significant degree of association (p < 0.01), meaning that as the number of roots increased, their length improved considerably (Fig. 6). Some authors suggest that the correlation between the number and length of roots is an important factor for achieving higher survival rates during the ex vitro establishment process. Having a greater number and length of roots enhances their anchoring capacity (Flores-Escobar et al.2008; González, 2006).

On the other hand, in the multiplication phase, the studied variables (number of leaves and number of shoots) showed a low degree of relationship. The observed response in Morella sp. explants could be attributed to the interaction between phytohormones present in the 100% WPM growth medium and their effect on plant growth. Some authors, such as Lolaei et al. (2013), mention that GA₃ has a significant influence on plant growth and development and can increase the number of leaves when used in low concentrations. Similarly, Xu et al. (2016) indicate that treatments with added GA₃ were crucial for leaf elongation and internode elongation. Alcantara et al. (2019) concluded that GA₃ promotes consistent tissue growth and enhances the elongation of roots and young leaves.

Tissue culture, specifically micropropagation, emerges as an applied tool to address issues in nature, particularly in protected forests. If natural restoration of affected areas within an ecosystem is not occurring, anthropogenic intervention is necessary to restore certain characteristics, particularly in the soil, allowing for the harmonious reestablishment of existing flora and fauna in the long term (CAF, 2005; Tedesco et al.2023).

Near protected areas, there are communities that coexist with nature and view these areas as an opportunity for growth and economic development through sustainable and responsible management of natural resources. Therefore, the intervention needs to be a collaborative effort between the local community and the scientific community, always focusing on solving real problems with practical solutions (Ortega, 2020).

The Morella sp. seedlings obtained through in vitro propagation will be reintroduced in the middle zone of the Tambillo Community Protected Area, with the expectation that, after their adaptation, they will contribute to soil restoration. In the medium term, their biomass can be utilized for the generation of value-added products, contributing to the economic growth of the Tambillo Community Protected Area.

The addition of 2% sodium hypochlorite for 4 minutes to Morella sp. explants prevented contamination in 90% of the explants. The best response for the number of leaves variable in the in vitro multiplication phase was observed in explants cultured in 100% WPM basal medium supplemented with 0.25 mg.L^− 1 GA₃ + 1.5 mg.L^− 1 BAP. The addition of 1 mg.L^− 1 IBA to the 100% WPM basal medium resulted in the highest average number and length of roots during in vitro rooting, highlighting the importance of selecting the appropriate concentration and type of growth regulator, as well as the culture medium, for the micropropagation of Morella sp. shoot tips.

BAP 6-benzylaminopurine

IBA indole-3-butryic acid

IAA indole-3-acetic acid

GA₃ gibberellic acid

WPM Woody Plant Medium

Acknowledgements

We are thankful to the Universidad Técnica de Manabí and the Cooperative for Community Development of Jima Ltda and the Tambillo Forest Initiative for the support provided.

Data availability statement

Data supporting this study are not publicly available. Please contact [email protected]

Funding

This research was financed by Rufford Foundation through project 35230-1.

Competing interests

The authors declare that there are no conflicts of interest.

Author Contributions

Kevin Brayan Murillo Borrero: Conceptualization, Methodology. Liliana Corozo-Quiñónez: Investigation; Resources; Writing - original draft. Mateo León Durán: Writing - original draft. Fátima Macías Ponce: Writing - review & editing. Miryan Pinoargote: Writing - review & editing. Luis Alberto Saltos-Rezabala: Data Curation, Formal analysis, Visualization.

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A protocol for in vitro multiplication of Morella sp: a protected species in the Tambillo Community Protected Area - Ecuador

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