The architectural characteristics of rubber trees are increasingly important in the context of climate change. Branching, canopy shape and growth pattern have to be adapted to monocropping and intercropping systems to foster latex and wood production, wind-tolerance, light availability and microclimate for intercrops, and soil stability and fertility. This study aimed to identify key architectural traits that could be used in breeding programs as well as chromosomal regions underlying QTLs that could be targeted for marker-assisted selection. Five quantitative (height of bole, trunk girth, estimated bole volume, number of terminal branches and apical shoots) and five qualitative (tree straightness, axillary shoot score, type of crown and axillary shoot, diameter of axillary shoot) architectural traits were phenotyped in a segregating population derived from clones PB 260 and SP 217. The frequencies of categories for each qualitative architectural variable were analysed as quantitative variables. A principal component analysis performed with these traits showed that trunk girth, estimated bole volume, round crown and medium diameter of axillary branches are negatively correlated with small diameter of axillary branches and conical type of crown. Seven architectural variables have heritability greater than 0.50. Twenty quantitative trait loci and their underlying genes and functions were pinpointed using the high-density genetic map previously constructed and an improved high-quality genome of the parent clone PB 260. Of the 680 genes found in chromosomal regions under QTLs, 19 genes have a function directly involved in plant development such as transcription factors related to the regulation of shoot apical meristem (SAM) and vascular cambium activity (WUSCHEL). A literature review was also conducted to provide additional insights into tree architecture and its impacts on agricultural systems. This first genetic analysis of architectural traits in rubber revealed that seven traits (trunk girth, bole height, estimated bole volume, number of apical branches, diameter of axillary branches, number of terminal branches and type of crown) could play a major role in rubber breeding both for monoculture and agroforestry single and double row systems. Chromosomal regions harbouring developmental genes could be used to develop specific strategy of marker-assisted selection.

The challenges for the development of rubber cultivation are so agronomically significant in the face of climate change that it is crucial to introduce biotechnology into the clonal propagation of rubber trees. A protocol was developed on rubber clone PB 260. This protocol was used for two other clones, RRIM 600 and REYAN 88-13, which exhibited highly contrasting response to callus growth and embryogenic capacity. Optimization of somatic embryogenesis for these two clones led us to successful conduct genetic transformation trials. We discussed the in vitro behaviour of the calli considering the origin of the embryogenic callus and the endogenous redox status.

Rubber ( Hevea brasiliensis ) is natural rubber-producing trees that have a wide range of adaptability to climate conditions. Tapping Panel Dryness (TPD) is a physiological disorder that can affect latex production. The percentage of TPD occurrences in big plantations can reach 7-15%, while in smallholders could be greater. This study aimed to see the distribution of latex production and the occurrence of TPD in a biparental population under an intensive harvesting system. The study used a biparental population from crosses of PB 260 (TPD susceptible clone) and SP 217 (TPD resistance clone) clones planted in a small-scale clone trial plot. The plot consists of 201 genotypes and 8 control clones. Clustering based on production and dry cutting length could reveal the potential analysis of promising TPD-tolerant genotypes. This study suggests that the application of an intensive harvesting system can be considered to discard TPD-susceptible genotypes.

Tapping Panel Dryness (TPD) is a physiological syndrome affecting natural rubber production in Hevea brasiliensis that is thought to be exacerbated by climate change stress. TPD is associated with high latex viscosity and agglutination of rubber particles. Although many studies have been carried out on the physiological and molecular mechanisms associated with TPD, little has been done in the way of genetic improvement. Intensive harvesting systems with high tapping frequency and ethephon stimulation of rubber trees are known to induce early TPD occurrence. A harvesting system with daily tapping and monthly ethephon stimulation was applied for one year to a segregating population of 189 individuals obtained from a cross between the TPD-susceptible clone PB 260 and the TPD-tolerant clone SP 217. This treatment induced a dramatic increase in dry cut length after six months for 26 % of the genotypes. Heritability also peaked at 88 % four months after treatment. Of the seven quantitative trait loci identified for the dry cut length, three were uniquely detected after application of the intensive harvesting system, and two quantitative trait loci were only observed after opening of trees under a standard harvesting system. Several genes underlying quantitative trait loci revealed functions previously identified through transcriptomic analyses. These results suggest a complex genetic basis of TPD.

The rubber industry is facing major socio-economic and environmental constraints. Rubber-based agroforestry systems represent a more sustainable solution through the diversification of income and the provision of greater ecosystem services than monoculture plantations. Participative approaches are known for their ability to co-construct solutions with stakeholders and to promote a positive impact on smallholders. This study therefore implemented a participatory research process with stakeholders in the natural rubber sector for the purpose of improving inclusion, relevance and impact. Facilitation training sessions were first organised with academic actors to prepare participatory workshops. A working group of stakeholder representatives was set up and participated in these workshops to share a common representation of the value chain and to identify problems and solutions for the sector in Indonesia. By fostering collective intelligence and systems thinking, the process is aimed at enabling the development of adaptive technical solutions and building capacity across the sector for future government replanting programmes. The resulting adaptive technical packages were then detailed and objectified by the academic consortium and are part of a participatory plant breeding approach adapted to the natural rubber industry. On-station and on-farm experimental plans have been set up to facilitate the drafting of projects for setting up field trials based on these outcomes. Research played a dual role as both knowledge provider and facilitator, guiding a co-learning process rooted in social inclusion, equity and ecological resilience. The initiative highlighted the potential of rubber cultivation to contribute to climate change mitigation and food sovereignty, provided that it can adapt through sustainable practices like agroforestry. Continued political and financial support is essential to sustain and scale these innovations.

To prepare Hevea brasiliensis plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock-scion interaction studies. This article describes the implementation of an efficient micrografting technique from Hevea in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion.

To prepare Hevea brasiliensis plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock-scion interaction studies. This article describes the implementation of an efficient micrografting technique from Hevea in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion. Key features • This protocol requires rejuvenated in vitro plantlets. • Requires between 40 and 60 days before scion bud break. • Sterile working conditions. Graphical overview.

The Latex Diagnosis (LD) is performed through the determination of dry rubber (DRC), sucrose (Suc), inorganic phosphorus (Pi) and thiol (RSH) contents. It is carried out through the colorimetric method using a single cuvette spectrophotometer, which is time-consuming and requires a high volume of chemicals. It also needs to be executed as soon as the samples are collected so that limits the adoption of LD. The adaptation of the protocol to a multiplate reader and suitable conservation aimed to overcome the limitations. The experiments were carried out at the Indonesian Rubber Research Institute, Sembawa, Palembang, and Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta. Various combinations of solution volumes, adapted from standard protocol, were tested. The closest slope of the standard curve to control was then applied to 7 randomly selected trees. The result showed that for Suc, the composition of 10 µL samples, 30 µL of 2.5 % TCA and 210 µL anthrone reagent was suitable for determination using a multiplate reader. The combination of 40 µL of sample, 85 µL of 2.5 % TCA and 125 µL mL FeSO4 solution was suitable for Pi determination, while 100 µL samples, 5 µL DNTB and 100 µL Tris is recommended for RSH. The measurement using a multiplate reader resulted in comparable contents (average deviations were 1.83, 2.69 and 3.51 % for Suc, Pi, and RSH, respectively), lower time and chemicals consumptions, and a larger number of sample handling compared to single cuvette method. This study also tested the effect of freeze-drying for sample conservation. The result indicated that freeze-drying was able to maintain the Pi level (deviation 0.93 %). However, it increased Suc (deviation 19.43 %) and decreased ascorbate (AsA) significantly (deviation 43.60 %); therefore, freeze-drying might not be suitable for conserving serum samples in the LD method.

Circular leaf fall disease (LFD) has been spreading in South-East Asia since 2018. This disease has been considered to be the most important factor that influences natural rubber yield. After identifying Pestalotiopsis as the causal agent of circular LFD, several studies reported the potential involvement of another primary agent. The aim of the present study was to understand the genetic basis of the infection caused by Pestalotiopsis in bioassays under controlled conditions and field observations of circular disease. Individuals from a biparental population resulting from a cross between clones PB 260 and SP 217 were phenotyped. The symptoms on leaves inoculated with two isolates of Pestalotiopsis in bioassays and natural infection in the field were found to be identical. Of the two Pestalotiopsis isolates, PE-001 produced a lesion with a larger average diameter in the population compared to PE-002. The severity of the disease observed in the field increased regularly for three years. Bioassays with Pestalotiopsis isolates and field observations revealed no correlation. The classification of genotypes has made it possible to identify classes with both small lesion diameters and low disease severity. Heritability of the lesion diameter was found to be much higher in bioassays than heritability of disease severity observed in the field. The QTLs detected differed between bioassay isolates and field observations. The latter QTLs did not remain stable over time and were no longer detected when disease severity increased in March 2023. The QTLs are associated with long chromosomal regions harbouring many genes associated with the gene ontology terms "molecular function" and "biological process", the latter being associated with the response to biotic factors. The results of the bioassays confirmed Pestalotiopsis as the agent causing the symptoms of circular disease. This disease has a complex genetic basis which evolves over time.

Hevea brasiliensis, a natural rubber producing species, is widely cultivated due to its high rubber yield potential. Natural rubber is synthesised in the rubber particles of laticifers. Latex diagnosis (LD) was established to characterise the physiological state of the laticiferous system by measuring its physiological parameters, i.e., sucrose, inorganic phosphorous (Pi), thiols and total solid content (TSC). Rubber clones are often classified in three groups i.e., quick starters, medium starters and slow starters. To better understand the genetic bases of latex yield, a biparental population was generated from a cross between the quick-starter clone PB 260 and the medium-starter clone SP 217. LD was performed during the peak latex production season and used to calculate sucrose loading. The agronomic and physiological parameters associated with latex yield led to the classification of genotypes according to the rubber clonal typology and to the identification of quantitative trait loci (QTL) using a high-density map. Inorganic phosphorous content was positively associated with yield during the first year of production thus enabling identification of quick-starter clones. In addition, the LD-based clonal typology led to determine the long-term yield potential and the use of appropriate ethephon stimulation. QTL analysis successfully identified several QTLs related to yield, sucrose, Pi and TSC. One QTL related to sucrose loading was identified in the same position as the QTL for sucrose on linkage group 1. To our knowledge, this is the first study to report QTL analysis for this trait. The use of a high-density map enables the identification of genes underlying QTLs. Several putative genes underlying QTLs related to yield, sucrose and TSC were identified.

Agroforestry is often seen as a sustainable land-use system for agricultural production providing ecosystem services. Intercropping with food crops leads to equal or higher productivity than monoculture and results in food production for industry and subsistence. Low rubber price and low labor productivity in smallholdings have led to a dramatic conversion of rubber plantations to more profitable crops. The literature analysis performed in this paper aimed at better understanding the ins and outs that could make rubber-based agroforestry more attractive for farmers. A comprehensive search of references was conducted in March 2023 using several international databases and search engines. A Zotero library was set up consisting of 415 scientific references. Each reference was carefully read and tagged in several categories: cropping system, country, main tree species, intercrop type, intercrop product, level of product use, discipline of the study, research topic, and intercrop species. Of the 232 journal articles, 141 studies were carried out on rubber agroforestry. Since 2011, the number of studies per year has increased. Studies on rubber-based agroforestry systems are performed in most rubber-producing countries, in particular in Indonesia, Thailand, China, and Brazil. These studies focus more or less equally on perennials (forest species and fruit trees), annual intercrops, and mixed plantations. Of the 47 annual crops associated with rubber in the literature, 20 studies dealt with rice, maize, banana, and cassava. Agronomy is the main discipline in the literature followed by socio-economy and then ecology. Only four papers are devoted to plant physiology and breeding. The Discussion Section has attempted to analyze the evolution of rubber agroforestry research, progress in the selection of food crop varieties adapted to agroforestry systems, and to draw some recommendations for rubber-based agroforestry systems associated with food crops.

Tapping Panel Dryness (TPD) is a physiological disorder affecting natural rubber production in Hevea brasiliensis. TPD is associated with clonal susceptibility and overexploitation of rubber trees. Most studies are based on a binary point view of the absence or presence of TPD. This study sets out to characterize the dynamic of the TPD onset through the monthly monitoring of the dry cut length. This reveals the presence of dry spots on the tapped panel of any trees. The frequency of these dry spots increases dramatically in trees developing high level of TPD. Brown bast is an irreversible form of TPD. Brown bast is correlated to a high level of dry cut length. Application of an intensive harvesting system induces early TPD occurrence, which facilitates the study of TPD. Among latex diagnosis parameters, only sucrose content is significantly associated with TPD. Other parameters are more prone to environmental effects and are not reliable as physiological markers. These findings explain the contradictory conclusions of some papers. This study suggests to use intensive harvesting system and monitor the dry cut length for genetic analysis of TPD.

Latex diagnosis is widely adopted in natural rubber-producing countries to optimize the natural rubber production through a physiological-based latex-harvesting system management. This study is the first bibliographical searching and meta-analysis on the variation of latex physiological parameters i.e. sucrose, inorganic phosphorus, thiols, and total solid content. The study used information extracted from 158 scientific papers. Descriptive statistics, agglomerative hierarchical clustering, and principal component analysis were performed to characterize applications of latex diagnosis, how often parameters are used and interpreted as well as the variation of its parameter values. From 158 papers, latex diagnosis parameters were used in 114 agronomy, 22 in physiology, and 22 in breeding papers. The agglomerative hierarchical clustering analysis indicated that sucrose and inorganic phosphorus contents were clustered together and total solid and thiols contents were located in another cluster. The average values of the total solid content, sucrose, inorganic phosphorus and thiols were 43.9%, 9.4 mM, 16.5 mM, and 0.52 mM, respectively. The percentage of interpretation is 63.3% for sucrose, 54.4% for inorganic phosphorus, 47.5% for thiols, and 41.1% for the total solid content. The low interpretation of thiols and total solid contents question their relevance in latex diagnosis. The low adoption of latex diagnosis in breeding could hinder the selection of activities leads to a limitation of selection for long-term high yielding and stress-adapted clones.

Latex diagnosis (LD) is applied to optimize the natural rubber production and prevent tapping panel dryness (TPD), a physiological syndrome affecting latex production in Hevea brasiliensis. The reduced thiol content (RSH) is one of the biochemical parameters associated with the risk of TPD. However, RSH is difficult to interpret because of the influence of the environment. In order to better understand the regulation of antioxidants and to better interpret RSH, a key parameter of LD, this study analysed in latex both oxidised and reduced forms of ascorbic acid (AsA) and glutathione, and their cofactors as well as other latex diagnosis parameters in response to harvesting stress (tapping and ethephon stimulation) and TPD occurrence. The content of antioxidants in latex had a high variability among five rubber clones. The concentration in AsA was about ten times higher than GSH in laticifer, GSH accounting for about 50% of RSH. For short-term harvesting stress, RSH increased with tapping frequency and ethephon stimulation. TPD is associated with high latex viscosity and bursting of lysosomal particles called lutoids, as well as for several rubber clones with lower RSH and GSH contents. These results suggest that a high level of RSH shows the capacity of laticifer metabolism to cope with harvesting stress, while a drop in RSH is the sign of long stress related to lower metabolic activity and TPD occurrence. RSH remains an essential physiological parameter to prevent TPD when associated with reference data under low and high harvesting stress. This study paves the way to understand the role of AsA and GSH, and carry out genetic studies of antioxidants.

It is predicted that drought will be more frequent and sustained in the future, which may affect the decline of rubber tree production. Therefore, it is critical to research some of the variables related to the drought-resistance mechanism of the rubber tree. As a result, it can be used to guide the selection of new rubber drought-resistance clones. The goal of this study was to identify drought-resistance mechanisms in rubber clones from the high drought factor index (DFI) group using ecophysiological and biochemical variables. The treatments consist of two factors, namely water deficit and contrasting clones based on the DFI variable. The first factor consisted of three levels, namely normal (fraction of transpirable soil water (FTSW) > 0.75), severe water deficit (0.1 < FTSW < 0.20), and recovery condition (FTSW > 0.75 after rewatering). The second factor consisted of seven clones, namely clones G239, GT1 (low DFI), G127, SP 217, PB 260 (moderate DFI), as well as G206 and RRIM 600 (high DFI). RRIM 600 had the highest DFI among the other clones as a drought-tolerance mechanism characteristic. Furthermore, clones RRIM 600, GT1, and G127 had lower stomatal conductance and transpiration rate than drought-sensitive clone PB 260. As a result, as drought avoidance mechanisms, clones RRIM 600, GT1, and G127 consume less water than clone PB 260. These findings indicated that clone RRIM 600 was a drought-resistant clone with drought tolerance and avoidance mechanisms.

[This corrects the article DOI: 10.1016/j.heliyon.2022.e09840.].

Climate change has an impact. Droughts will become more common in the future and as a result is the drop of rubber production. Drought tolerant rubber genotypes, may reduce drought risk, could be discovered by selection of germplasm and crossing progenies. As perennial plant, conventional rubber selection of rubber needs long time. Molecular detection such as QTL (precise and fast detection) for screening to get drought tolerant rubber genotypes is very important to be conducted. A variable widely used to screen stress tolerance genotypes is Performance Index (PI). Measurement of PI in normal, moderate, and severe drought stress enables the calculation of the Drought Factor Index (DFI). DFI as an integrative parameter for drought tolerance trait provides simple and robust method to quantify the tolerance ability of plants to withstand drought condition. This research is the first application of DFI to select drought-tolerant rubber genotypes and to identify QTLs related to DFI. This research aimed to select candidate drought-tolerant genotypes from the biparental population PB 260 x SP 217 as well as to identify QTLs related to DFI variable. One hundred and thirty-two genotypes and four control commercial clones, namely PB 260, SP 217, GT1, and RRIM 600 have been successfully screened using DFI variable. Normal, moderate, and severe drought stresses were calibrated for each plant by monitoring the fraction of transpirable soil water (FTSW). Due to the skewed distribution of DFI data that was generated in the phenotyping step, the Kruskal-Wallis non-parametric test was used to test the significance of each QTLs using MapQTL version 6 software. From this research, it can be concluded that DFI can be used as a parameter for selection of drought tolerant rubber clones. Based on DFI calculation, the top ten high DFI genotypes/clones were genotype number 34, 206, 056, 007, 205, 225, 066, 235, 109, and clone RRIM 600. Furthermore, the most significant detection of QTL for DFI variables was found in locus g5TA2155, located at 0.553 cM in LG5. It indicated that this locus contained genes controlling DFI trait.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.

1 CIRAD UMR Innovation, France, Montpellier, eric.penot@cirad.fr 2 UGM, Yogyakarta, Indonesia ariniwu@ugm.ac.id 3 UGM, Yogyakarta, Indonesia yekti@ugm.ac.id 4 IRRI/Bogor, Indonesia gedewibawa@yahoo.co.id 5 UPLB Philippines, Crop Institute 6 PSU, Thailande 7 MRB production division, Malaysia. 8 CIRAD Montpellier France , UMR Eco & sols 9 UGM Faculty of Forestry, Indonesia. 10 IRRI/Bogor, Indonesia 11 IRRI/Seambawa, Indonesia 12 CIRAD Montpellier France , UR ABCYSS 13 Cifor. Rome/Italy 14 UGM, Plant pathology , Yogyakarta, Indonesia 15 CIRAD UMR AGAP 16 Wageningen University 17 Yunnan University

Natural rubber is an important industrial raw material and is commercially produced by rubber trees (Hevea brasiliensis). The sucrose transporter HbSUT3 plays an essential role in rubber production. Its expression in latex (cytoplasm of rubber-producing laticifers) is induced by bark treatment with Ethrel, an ethylene releaser, and the inducing effect correlates well with Ethrel-stimulated rubber yield increase. However, the mechanisms of ethylene induction on HbSUT3 expression are not known. Here, five Ethylene Response Factor (ERF) genes were identified from the cDNA library of Hevea latex by yeast one-hybrid screening with the promoter of HbSUT3 gene as bait. As revealed in a tobacco (Nicotiana tabacum) protoplast transient expression system, these HbERFs were mainly localized in the nucleus and four of them exhibited apparent transactivation activity. Of the five HbERF genes, HbERF-IXc4 was the most frequently screened in yeast one-hybrid, accounting for 65% of the ERF clones obtained. Moreover, among the five HbERFs, HbERF-IXc4 showed the strongest transactivation capacity when expressed in tobacco protoplast, the highest transcript abundance in latex and a close expressional correlation with its target gene, HbSUT3, in response to the Ethrel treatment. Taken together, our results indicate that ERFs, especially HbERF-IXc4, are critically involved in the activation of HbSUT3 expression in latex after Ethrel treatment on Hevea bark, and thus the stimulated latex yield.