EFFECT OF TEMPERATURE ON ROOTS AND SHOOTS FORMATION IN THREE VANILLA SPECIES (ORCHIDACEAE) UNDER CONTROLLED CONDITIONS †

°C (20.64 ± 0.26 cm) and the lowest value at 35 °C (1.22 ± 0.49 cm), contrary to V. insignis , where the increase in temperature at 35 °C favored root elongation (4.76 ± 0.07 cm), while the lowest growth was observed at 25 °C (1.74 ± 0.13 cm). V. pompona did not present significant differences in root growth at the three temperatures evaluated. Similarly, the increase in temperature showed a distinct influence on the promotion and growth of the shoot. V. planifolia only produced shoots at 32 °C (8.4 ± 0.4 cm) and V. pompona generated them at 32 and 35 °C, being those of greater length at 35 °C (3.3±0.13 cm) and did not produce at 25 °C; while V. insignis showed the highest shoot growth at 25 °C (7.22 ± 0.2 cm) and the lowest at 32 °C (6.56 ± 0.12). At 38 °C, 100% mortality was observed in the three species. Implications. The growth of roots and shoots of V. planifolia are affected by an increase in temperature above 32 °C, therefore, conservation and genetic improvement programs are needed that could start from the qualities that V. pompona and V. insignis showed. Conclusion. Temperature ranges between 25 and 35 °C have a differential effect on the promotion and growth of roots and shoots for the species V. planifolia , V. insignis and V. pompona .


INTRODUCTION
Temperature is one of the most important factors for the growth and development of plants because they require specific values to function optimally (Gray andBrady, 2016, Hatfield andPrueger, 2015). Alterations in global temperature are expected due to climate change, but a greater impact on temperature is expected in the tropics (Armenta-Montero et al., 2022). An increase in temperature between 3 and 5 °C is expected (Hatfield andPrueger, 2015, Luo et al., 2020), but some forecasts anticipate an increase of up to 11 °C (Faget et al., 2013, Reddy et al., 2017. This may cause severe thermal and water stress that could affect plant organ production and strengthening (Hatfield and Prueger, 2015).
Plants respond intraspecifically to increments in temperature. An increment between 1-6 °C above the optimal range can reduce root production and length, as observed in Solanum lycopersicum, L., Pisum sativum L., and Oryza sativa L. (Faget et al., 2013, Fahad et al., 2019; while the same interval favors Lactuca sativa L., Zea mays L., Brassica napus L. and Helianthus annuus L. (Faget et al., 2013, Gray andBrady, 2016). Similarly, the stem may be favored by an increase in temperature, as in O. sativa and Triticum aestivum L. (Fahad et al., 2019), or it may reduce its size, as occurs in Gossypium hirsutum L. (Reddy et al., 2017). Regarding orchids, temperatures upper than the optimum range may have a negative effect on growth and development (Zhang et al., 2018). A little increase in temperature might favor plant growth, as it has been seen in Phalaenopsis spp. (Zhang et al., 2018); however, higher temperatures (5 °C above the optimal range) reduce leaf development, and photosynthetic activity, as well to cause a high percentage of the inflorescences abort, as reported in Cymbidium sazanami, Cypripedium flavum, Phalaenopsis spp., or Odontioda spp. (Zhang et al., 2005(Zhang et al., , 2018; whereas an increase in temperature may cause an increase in root length, as in C. tracyanum, or a decrease, as in C. sinense . Vanilla (Vanilla planifolia Andrews) is a species of global commercial interest due to the use of its fruit in the food and aromatic industries (Ranadive, 2018). Despite its economic importance, in Mexico V. planifolia is in the category "subject to special protection" within NOM-059 (SEMARNAT, 2010). It has been exclusively propagated asexually, which has generated genetic erosion (Menchaca-García, 2018), which has made vanilla susceptible to several factors including higher temperatures (Hernández-Hernández, 2018). Its optimal cultivation temperature ranges between 21 and 27 °C (Hernández-Ruíz et al., 2016;Hernández-Ruíz et al., 2020). Values outside of this range can cause alterations in physiology such as difficulty rooting, reduction in stem growth and decreased fruit production (Hernández-Hernández, 2018).
Wild species within the genus Vanilla may be more resistant to possible increments in temperature. This is the case of V. pompona Schiede and V. insignis Ames (Cameron, 2018). V. pompona is the second most utilized species of vanilla because its fruit can be mainly used for perfume (Maruenda et al., 2013). On the other hand, V. insignis has not been used in the industry due to its low concentration of vanillin, but it has other metabolites that confer it an aromatic potential (Cameron, 2018, de Oliveira et al., 2022. Both species are able to grow in arid environments with periods of prolonged drought and temperatures between 30.9 and 34.5 °C (Flores-Jiménez et al., 2016, Soto-Arenas andDressler, 2009). Thus, they may be able to tolerate thermal increments. Therefore, the objective of this study was to determine the effect of temperature on the development of roots and shoots of V. planifolia, V. pompona and V. insignis under controlled conditions.

MATERIALS AND METHODS
We used cuttings of V. planifolia (PL), V. pompona (PO) and V. insignis (IN), the cuttings of the three species came from the environmental management unit "vainillales Tlali Nantli" (SEMARNAT-UMA-IN-VIV-0281-VER/19), located in Mesa de Guadalupe, Veracruz. The climate is warm sub-humid, with a temperature range of 14-26 °C, and a precipitation range of 1100-1600 mm. The biological material was donated to the germplasm bank of the Universidad Veracruzana orchidarium (SEMARNAT-UMA-IN-VIV-0129-VER/11), where this research was carried out. The three species were identified using the dichotomous key for the genus Vanilla proposed by Soto-Arenas and Dressler (2009). Cuttings were washed with water and detergent for 10 minutes; they were then immersed in household bleach solution (0.5 mL/L) for five minutes and were rinsed twice with plenty of sterilized distilled water. We used the method proposed by Adame-García et al. (2015), where short cuttings of approximately 20 cm in length with two knots and at least one leaf are placed on Styrofoam trays with absorbent paper moistened with 20 mL of sterilized distilled water, inside transparent plastic bags. These wet chambers were hydrated weekly with 20 mL of sterilized distilled water, to maintain a relative humidity of 100%. To evaluate the effect of temperature on rooting and shoot production, cuttings were placed in incubation stoves under controlled conditions at 25, 32, 35 and 38 °C, in darkness, for six weeks. Five cuttings of each species were used for each of the four temperatures evaluated. We used 20 cuttings in total per species (n = 60). In order to homogenize the biological material, in each species the cuttings were obtained from a single mother plant (approximately five meters in length). At the end of the exposure period, the survival percentage of the cuttings was assessed, as well as the number of roots produced, the length and diameter of the primary root, the number of shoots produces and the length and diameter of the shoots. The growth rate of the primary root and the vegetative shoot was evaluated using the value of weekly measurements with the formula: Where 1 corresponds to the value of the initial measurement and 2 corresponds to the value of the following measurement. Positive values of this variable indicate growth, while values close to zero indicate a decrease in size (Paine et al., 2012). For each variable, we determined the differences between groups using a two-way analysis of variance (ANOVA) and a Tukey post hoc test (p ≤ 0.05) in R (R Core Team, 2020). We additionally counted the number of cuttings that lost their leaf throughout the duration of the experiment.

RESULTS
We obtained a 100% survival rate of cuttings for the three vanilla species subjected to 25, 32 and 35 °C, throughout the duration of the experiment. In contrast, mortality was 100% at 38 °C after one week of thermal exposure. Increased temperature had an effect on the time it took for roots to appear in the different treatments (

DISCUSSION
In other crops, studies have found that roots can be affected at the cellular level by exceeding their optimum temperature (Faget et al., 2013, Zhao et al., 2021. The optimum temperature of PL is between 21-27 °C (Hernández-Ruíz et al., 2016). Thus, the increase in temperature in PL interferes with the development of roots. In contrast, in PO and IN the appearance of new roots was favored by the increase in temperature. Both species showed the highest production at 32 °C. Additionally, IN showed the greatest root length at 35 °C, and PO the largest diameter at the same temperature, because they are species with a greater tolerance to high temperatures (Cameron, 2018). Moreover, plants with larger roots and rapid expansion achieve greater adaptation to areas with warmer climates in the face of higher temperatures (until 5 °C above the optimum) (Luo et al., 2020).
Similarly to root production, new vegetative shoots presented an intraspecific response to the increase in temperature. In the case of PL, the highest shoot production at 32 °C indicates that the optimal point for superior development in the species is at this temperature. (Hatfield and Prueger, 2015). However, higher temperatures can inhibit shoot formation (Hasanuzzaman et al., 2013, Zhao et al., 2021. Furthermore, the optimal temperature for the root is usually lower (Gaveliené et al., 2022), as observed in this species. Shoot production in PO was favored by the increment in temperature. In contrast, species in which their roots were favored by the increase in temperature usually exhibit the reverse behavior in stem development (Fahad et al. , 2019;Hatfield and Prueger, 2015) and this was the case of IN. The decrease in stem growth and development is associated with effects on photosynthesis: higher than optimal temperatures affect enzymatic activity within chloroplasts as well as denaturation of thylakoidal membranes, reducing the photosynthetic rate (Zhao et al., 2021), in addition to causing dehydration and stomatal closure (Hasanuzzaman et al., 2013). At 32 and 35 °C the cuttings of the three species lost their original leaves (those from the vine from which they were obtained). This happened because the leaves of these species are water and nutrient reserves, due to they are CAM plants, and therefore are able to use these resources to survive until they produce new roots and shoots (Edwards, 2019).
In general, the maximum temperature for the development of both roots and shoots, under the conditions of this experiment, was 32 °C. This is 5 °C higher than the optimal temperature previously reported for PL, and within the optimal range for both PO and IN (Flores-Jiménez et al., 2016;Hernández-Ruíz et al., 2016;Hernández-Ruíz et al., 2020). However, temperature higher than 32 °C would be detrimental to both PL and IN. PO would continue to show optimal responses, as long as prolonged periods at 38 °C do not occur, since all of the species in this study died at a temperature beyond what was tolerated. In future climate change scenarios, PL could be negatively affected if average temperatures exceed 32 °C, so it is necessary to implement conservation plans to avoid crop losses. Such measures may include the use of 50 to 80% shade meshes with a sprinkler irrigation system which may help mitigate insolation, but may also increase costs (Hernández-Hernández, 2018).Vanilla is currently grown at higher altitudes that can exceed 1000 meters above sea level. Displacement of this crop given the current climate change trend has been observed (Menchaca-García et al., 2019), so it is necessary to implement studies that determine the geographical areas in which vanilla plants can develop with adequate growth, flowering and fruiting based on environmental tolerance ranges. PO was able to develop in higher temperature ranges compared to the other species in this study. Thus, we proposed that, to promote its cultivation, vanilla fields exclusive to this species or in association with PL must be establish. In turn, cultivating PO jointly with PL could contribute to the development of genetic improvement programs through species hybridization (Barreda-Castillo et al., 2022) where tolerant plants to these and other scenarios of interest may be obtained.

CONCLUSIONS
The root and shoot production of the three species studied were affected by temperature. The increase in temperature reduced root growth in V. planifolia and promoted it in V. insignis. Prolonged exposure to 25 °C only stimulated the production of shoots in V. insignis. Prolonged exposure to 32 °C was optimal for all three species, as both shoots and roots were produced. V. pompona had greater tolerance to increments in temperature by managing to maintain root and shoot production even at 35 °C. Prolonged exposure to average temperatures greater than or equal to 38 °C was harmful to all three vanilla species.