Phenotyping hybrids in early development stage for the future of citrus production¹

Fenotipagem de híbridos em estágio inicial de desenvolvimento para o futuro da produção dos citros

ABSTRACT

The selection of promising citrus hybrids is of great relevance for global citrus farming in the face of phytosanitary challenges and climate change that intensify extreme drought events, compromising the sustainability of citrus farming. The present study presents a method for analyzing phenotypic traits of citrus seedlings used as rootstocks with the aim of efficiently selecting hybrids with agronomic characteristics relevant to the citrus industry within a short period of time, using BRS Bravo as a control. It was later correlated with drought tolerance of plants cultivated under field conditions in the principal region of citrus industry in Brazil. The study comprised 15 elite hybrids selected from the Citrus Breeding Program of Embrapa Cassava and Fruits. The experimental design was completely randomized with five replicates. Evaluations of biometric variables (root and shoot traits) were performed after transplanting the seedlings before and after the seedlings were subjected to water deficit. Correlations were found between root system and phenotypic characteristics of seedlings and adult plants, respectively.

Key words:
Citrus spp.; Poncirus trifoliata, water deficit; rootstock

HIGHLIGHTS:

Root dry weight to aerial part dry weight ratio can be considered a crucial indicator of drought tolerance.

The significant increase in root dry biomass emerges as a key feature of interest.

Specific root length is part of the response associated with higher citrus productivity under water deficit conditions.

RESUMO

A seleção de híbridos promissores de citros é de grande relevância para a citricultura global diante dos desafios fitossanitários e das mudanças climáticas que intensificam eventos extremos de seca, comprometendo a sustentabilidade da citricultura. O presente estudo apresenta um método para análise de características fenotípicas de mudas de citros utilizadas como porta-enxertos com o objetivo de selecionar eficientemente híbridos com características agronômicas relevantes para a citricultura em um curto período de tempo, utilizando BRS Bravo como testemunha. Posteriormente, foi correlacionado com a tolerância à seca de plantas cultivadas em condições de campo na principal região da citricultura no Brasil. O estudo compreendeu 15 híbridos elites selecionados pelo Programa de Melhoramento Genético de Citros da Embrapa Mandioca e Fruticultura. O delineamento experimental foi inteiramente casualizado com cinco repetições. As avaliações das variáveis biométricas (características da raiz e da parte aérea) foram realizadas após o transplante das mudas antes e após as mudas serem submetidas ao déficit hídrico. Foram encontradas correlações entre o sistema radicular e as características fenotípicas de mudas e plantas adultas, respectivamente.

Palavras-chave:
Citrus spp.; Poncirus trifoliata, déficit hídrico; porta-enxerto

INTRODUCTION

Prolonged periods of water scarcity accompanied by elevated temperatures have caused substantial harm to citrus cultivation, leading to a decline in both fruit yield and quality (Sampaio et al., 2021). In nature, plants are exposed to multiple stresses, which simultaneously led to a selection of individuals possessing characteristics that enable their adaptation to the environment.

Studies have confirmed that certain citrus rootstocks under water deficit exhibit significant development in their roots in response to stress (Janiak et al., 2016). It is essential to have a thorough understanding of the anatomy of the root system in order to accurately estimate rootstock characteristics and maximize production (Zhang et al., 2019).

The development of citrus hybrids encounters several obstacles, among them: extended juvenile period of 5-10 years before the initiation of the first flowering makes phenotyping during this phase a daunting task (Gill et al., 2022). It is essential to acknowledge the extensive history of cultivating citrus crops as trees grafted onto specific rootstock hybrids. However, prevailing rootstocks exhibit notable limitations, and conventional breeding methodologies for rootstocks demand a minimum of 2 to 3 decades for the development and field testing of new varieties (Bowman et al., 2021). Phenotyping citrus hybrids with potential use as rootstocks during the juvenile stage is highly desirable for breeders. This leads to cost reduction, decreased field area required for selection, as well as a shorter time frame for obtaining a new variety (Salonia et al., 2020).

The present study presents a method for analyzing phenotypic traits of citrus seedlings used as rootstocks with the aim of efficiently selecting hybrids with agronomic characteristics relevant to the citrus industry within a short period of time, using BRS Bravo as a control. It was later correlated with drought tolerance of plants cultivated under field conditions in the principal region of citrus industry in Brazil (Costa et al., 2020). The traits obtained under controlled conditions were compared with those of 14 -year-old adult plants. The same rootstocks evaluated under controlled conditions had orange ‘Valencia’ [ Citrus ×sinensis (L.) Osbeck] as a scion variety in the municipality of Colômbia, Northern São Paulo state, Brazil, in an environment subjected to annual periods of intense water shortage associated with high temperatures.

MATERIAL AND METHODS

The present study was performed under controlled conditions of temperature and relative air humidity (26 °C and 70% diurnal relative air humidity and 21 °C and 80% nocturnal relative air humidity), irradiance (average 540 μmol photons m^-2 s^-1) and photoperiod of 12 hours (FITOTEC - model LT 13.76 UTFP) at the Vegetal Ecophysiology and Meteorology Laboratory of Embrapa Cassava and Fruits in the municipality of Cruz das Almas, Bahia, Brazil.

The experiment was conducted using a fully randomized design, with 15 hybrids represented by nucellar seedlings and five replicates subjected to a soil water regime, using BRS Bravo as a control.

The following hybrids used in the study were obtained from the Citrus Breeding Program of Embrapa Cassava and Fruits - PMG Citrus hybrids, which are elite materials used as rootstock (Table 1).

Table 1
Citrus rootstock^* used in the study

Variety	Parents
BRS O S Passos	Pera Sweet orange [Citrus ×sinensis (L.) Osbeck] × Yuma citrange [C. ×sinensis × Poncirus trifoliata (L.) Raf.]
BRS Victoria	Rangpur lime (C. ×limonia Osbeck) × Barnes trifoliate orange (P. trifoliata)
BRS Bandeirante	Sunki mandarin (C. sunki (Hayata) hort. ex Tanaka) × Coleman citrange (C. ×sinensis × P. trifoliata)
BRS Hleão	Sunki mandarin × Benecke trifoliate orange
BRS O Sempionato	Sunki mandarin × Swingle citrumelo (C. × paradisi Macfad. x P. trifoliata) (L.) Raf.] ‘Swingle’
BRS Pompeu	Sunki mandarin × Swingle citrumelo
BRS Cunha Sobrinho	Sunki mandarin × Swingle citrumelo
BRS Stuchi	Sunki mandarin × Rangpur lime (C. × limonia Osbeck); × trifoliata (P. trifoliata)
BRS Donadio	Sunki mandarin × Flying Dragon trifoliate orange (P. trifoliata var. monstrosa (T. Itô) Swingle)
BRS Matta	Sunki mandarin × Flying Dragon trifoliate orange
BRS N Gimenes Fernandes	Florida Rough lemon (C. × jambhiri Lush.) × Rangpur lime (C. × limonia Osbeck); × trifoliata (P. trifoliata)
BRS L Navarro	Sunki mandarin× citrangequat ‘Thomasville’ [Fortunella margarita (Lour.) Swingle x (C. ×sinensis x P. trifoliata)];
LCR × TR - 001	Rangpur lime × P. trifoliate
BRS S Moreira	Rangpur lime × Swingle citrumelo
TSK × TRBK- CO1	Sunki mandarin × Benecke trifoliate orange - Colômbia

• ^*
  Rootstocks obtained by the Citrus Breeding Program of Embrapa.
• ¹
  Hybrid obtained by USDA (United States Department of Agriculture)

The traits obtained under controlled conditions were compared with those of 14 -year-old adult plants. The same rootstocks evaluated under controlled conditions had orange ‘Valencia’ [Citrus × sinensis (L.) Osbeck] as a scion variety in the municipality of Colômbia, Northern São Paulo state, Brazil, in an environment subjected to annual periods of intense water shortage associated with high temperatures. BRS Bravo (Sunki mandarin × Rangpur lime) was used as a control in this study because it showed good agronomic performance, including drought tolerance, in the different regions in which it was evaluated by Ramos et al. (2015) and Carvalho et al. (2016).

The seeds from the 15 hybrids included in the present study were germinated in 100 mL tubes and placed in trays with coconut fiber as the substrate. Once the seeds had sprouted and the seedlings had developed four or more distinct leaves, ir was identified the individuals of nucellar and hybrid origin by examining the characteristics of their leaves. Individuals resulting from sexual reproduction were excluded. Ninety days after identifying the seedlings that came from nucellar origin and were genetically identical to the selected hybrids for this study, they were transplanted into PVC tubes. These tubes had a height of 0.3 m, a diameter of 0.075 m and capacity of 1.200 mL. The tubes were filled with washed and sterilized sand, and were filled to their maximum capacity for water retention, with a moisture level of 0.15 cm³ cm ^-3. The transplanting was performed 90 days after the seeds were planted. A graphic representation of the study is shown in Figure 1.

Figure 1
Graphic representation of the study, transplanting performed 90 days after sowing 80 seedlings (5 per hybrid), with 30 days of acclimatization and 40 days of water deficit

In order to maintain the substrate close to the field capacity, seedlings were irrigated with 'Forth Nutri ^®' nutrient solution at a rate of 0.72 mg of the solution per liter of water using the same volume (100 mL) for all hybrids. To estimate the soil water content at field capacity in the washed sand, samples were taken from five tubes containing the same amount of washed sand. The weight of each tube, including the dried sand, was obtained. The tubes were then placed in a container with a 0.50 m water column to promote capillaritybased sand column saturation. After the tubes containing sterilized sand were re moved, the excess water was allowed to drain naturally over the course of 24 hours, at which point the mass of the tubes plus moist sand was recorded (M1). During the acclimatization period, irrigation was performed at least twice a week to maintain the water content close to field capacity. The water consumed (water extraction plus evaporation) was calculated using the mass balance (M1) - (tube + sand moisture) before each irrigation.

Evaluations of biometric variables (root and shoot traits) were performed after transplanting the seedlings before and after the seedlings were subjected to water deficit. At the end of the experiment (160 days after sowing, 30 days after the imposition of acclimatization, 40 days after the imposition of water deficit with suppression of irrigation), plants were segmented into aerial part (stems and leaves) and roots. Roots were washed, conditioned in 30% ethanol and refrigerated until they were digitalized in scanner. The process was completed using the software WinRizho version 2013d at a resolution of 400 dpi to obtain root diameter (RD, mm); total root length (TRL, cm) for different classes of root diameter; root length density (RLD, cm cm ^-3); and specific root length (SRL, cm g ⁻¹). Then the final dry weight of the aerial part (DWAP) and the ratio between the dry weights of root and the aerial part (DWR/DWAP) were determined in both cases using a forced air oven at 65 ^ºC for a period of 72 hours.

The results underwent analysis of variance, and multivariate analysis. The Dunnett test was employed at a p ≤ 0.05 significance to compare each hybrid with the control hybrid BRS Bravo. Correlations were calculated between the vari ables, and a matrix was constructed to display the correlation values. The analyses were conducted using the R program (R Core Team, 2021).

The results from measuring biometric variables on 15 hybrids that were being studied using BRS Bravo as a control were compared to those from Costa et al. (2020), who used the same hybrids grafted on a 'Valencia' orange scion during the fruit production stage in an experiment in the municipality of Colômbia, Northern region of Sã o Paulo state, which had a strong water deficit and was very hot.

RESULTS AND DISCUSSION

The examination of the dry weight variables of the roots and aerial part of the seedlings is shown in Figure 2. Regarding these variables, it is noted that BRS Bravo exhibited greater values compared to BRS HLeão, and there were no significant differences identified among the other hybrids.

Figure 2
Significant mean values for dry weight of the roots (DWR) (A) and dry weight of aerial part (DWAP) (B), and total dry weight (TDW) (C) of nucellar seedlings (rooted cuttings or plants originated from seeds) of 15 citrus hybrids under water deficit

No significant differences were identified in the DWR/ DWAP ratio between the hybrids analyzed in comparison with BRS Bravo. It is assumed that plants with a higher DWR/ DWAP ratio have a greater adaptability to efficiently exploit the water content available in the soil. This characteristic can be considered a crucial indicator of drought tolerance, highlighting the relevance of the DWR/DWAP ratio as a potential marker to evaluate plant performance under water scarcity conditions. The increase in the proportion between the mass of roots and shoots was identified by Silva et al. (2019) in citrus rootstocks subjected to water deficit conditions. This significant increase in root dry biomass emerges as a key feature of interest. This characteristic can play a crucial role in mitigating the effects of water scarcity, minimizing transpira tion losses, and improving efficiency in water and nutrient capture (Diaz-Espejo et al., 2012).

Among the hybrids analyzed, it was observed that the root systems had greater thickness, expressed in terms of root diameter (RD), compared to the BRS Bravo hybrid. The following hybrids stand out in this regard: BRS H Montenegro, BRS L Navarro, BRS Cunha Sobrinho, BRS O S Passos, BRS Victoria, BRS S Moreira, BRS Pompeu, and BRS HLeã o (Figure 3A). With regard to the variable specific root length (SRL), the BRS Bravo hybrid showed superiority in relation to the majority of hybrids evaluated, with the exception of Citrandarin 1697, BRS N Gimenes Fernandes, BRS L Navarro, BRS Donadio, and BRS Bandeirante (Figure 3B).

Figure 3
Significant mean values for root diameter (RD) (A), specific root length (SRL) (B), root length density (RLD) (C), and total root length (TRL) (D) of nucellar seedlings (rooted cuttings or plants originated from seeds) of 15 citrus hybrids under water deficit

The total root length was categorized into different classes considering the diameter of roots: LC1 (TRL from 0 to 0.5 cm), LC2 (TRL from 0.51 to 1.0 cm), LC3 (TRL from 1.01 to1.5 cm), LC4 (TRL from 1.51 to 2.0 cm), and LC5 (TRL from 2.01 to 2.50). BRS Bravo outperformed most hybrids, with the exception of BRS H Montenegro and BRS N Gimenes Fernandes, in the first class (LC1), which comprises fine roots (Figure 3C). This outcome is consistent with the low RD and SRL values observed for this particular hybrid. Most hybrids did not differ significantly in the root diameter range of 0.5 to 1.0 cm (class LC2). Nevertheless, BRS Bravo

showed superiority compared to BRS S Moreira, BRS Stuchi, and BRS HLeão. This differentiation shows that BRS Bravo is more effective than the previously listed hybrids in this specific range of root diameter.

Concerning the third class (LC3), characterized by root diameters of 1.01 to 1.5 cm, it was observed that the performance of BRS Bravo was superior to that of the hybrids BRS O Sempionato, BRS Stuchi, BRS HLeão, and BRS Bandeirante. In the fourth class (LC4), where the root diameter was between 1.51 and 2.0 cm, BRS Moreira was different from BRS Bravo, but no other hybrids were statistically different. In the fifth class (LC5), which includes diameters between 2.01 and 2.5 cm, there were no significant differences between the hybrids tested compared to the performance of BRS Bravo.

Based on the data from Costa et al. (2020), correlations were established between the field response of the evaluated rootstock varieties and the variables of this study, carried out under controlled conditions (Table 2). A notable and positive correlation was identified between fruit production (GP, kg per plant) and drought tolerance (DT), plant height (PH, m), stem diameter (SD, m), in addition to crown volume (CV - m³), while it showed a negative correlation with root length density (RLD). There was also a positive correlation between drought tolerance (DT) and plant height (PH), stem diameter (SD) and crown volume (CV). A strong negative correlation was observed between production efficiency (PEF) and plant height (PH), stem diameter (SD) and crown volume (CV). Additionally, plant height (PH) showed strong and positive correlations with stem diameter (SD) and crown volume (CV).

Table 2
Correlation matrix from variables obtained through biometric evaluations in the field* and laboratory in nucellar seedlings (rooted cuttings or plants originated from seeds) for 15 hybrids of citrus under water deficit

Correlation matrix
Variables	GP	DT PEF	PH	SD	VOL	DWR	DWAP	TDW	DWR/ DWAP	SRL	RLD	RD	LC1	LC2	LC3	LC4	LC5
GP	0.52	-0.40	0.64	0.74	0.73	-0.24	-0.25	-0.24	0.08	-0.22	-0.29	0.15	-0.25	-0.35	0.03	0.19	0.42
DT		-0.21	0.54	0.47	0.47	-0.12	0.01	-0.04	-0.11	-0.28	-0.32	0.43	-0.33	-0.25	0.19	0.33	0.38
PEF			-0.7	-0.7	-0.7	0.02	-0.02	-0.01	0.06	-0.06	-0.06	-0.1	-0.1	0.01	-0.1	-0.24	-0.17
PH				0.84	0.89	-0.11	-0.04	-0.07	-0.06	-0.18	-0.2	0.29	-0.2	-0.2	0.14	0.32	0.32
SD					0.98	-0.14	-0.07	-0.09	-0.06	-0.08	-0.11	0.16	-0.1	-0.2	0.08	0.29	0.29
VOL						-0.14	-0.06	-0.09	-0.06	-0.10	-0.13	0.19	-0.1	-0.2	0.09	0.31	0.29
DWR							0.61	0.82	0.33	-0.17	0.55	0.01	0.43	0.59	0.44	0.22	0.17
DWAP								0.94	-0.47	0.09	0.55	-0.04	0.44	0.6	0.38	0.1	0.09
TDW									-0.16	-0.03	0.61	-0.02	0.48	0.67	0.46	0.16	0.16
DWR/DWAP										-0.34	-0.05	0.10	-0.06	-0	0.08	0.12	0.2
SRL											0.68	-0.81	0.75	0.43	-0.15	-0.45	-0.38
RLD												-0.65	0.96	0.82	0.25	-0.21	-0.12
RD													-0.75	-0.34	0.33	0.49	0.41
LC1														0.6 4	0.13	-0.26	-0.2
LC2															0.33	-0.12	-0.1
LC3																0.18	0.28
LC4																	0.17
LC5																	1

• ^*
  Costa et al. (2020); GP - Gathered production; DT - Drought tolerance; PEF - Production efficiency; PH - Plant height; SD - Stem diameter; VOL - Crown volume; DWR - Final dry weight of roots; DWAP - Dry weight of the aerial part; TDW - Total dry weight, DWR/DWAP - Ratio of dry weight of roots and aerial part; SRL - Specific root length; RLD - Root length density; RD - Root diameter; LC1 - Diameter class 0-0.5 mm; LC2 - 0.5-1.0 mm; LC3 - 1.0-1.5 mm; LC4 - 1.5-2.0 mm and LC5 - 2.0-2.5 mm. Values in blue indicate p ≤ 0.01, in brown p ≤ 0.05, and in black p > 0.05

Regarding plant traits obtained under controlled conditions, it was observed that the dry weight of roots (DWR) and aerial part (DWAP) showed a significant positive correlation with total dry weight (TDW). Furthermore, the relationship between the dry weights of the roots and the aerial part (DWR/DWAP) showed a high positive correlation with the roots and an inverse correlation with the aerial part, as shown in Table 2. A positive correlation was also identified between dry weight (TDW, DWR, DWAP) and the variables RLD, TRL, LC1, LC2, and LC3, highlighting the relevance of the total length of fine roots in promoting plant growth. Specific root length (SRL) revealed a high positive correlation with both total root length (TRL) and root length density (RLD), in addition to showing positive correlations with the LC1 and LC2 root classes. On the other hand, there was a negative correlation between SRL and the root classes LC4 and LC5, as well as with the average root diameter (RD). As expected, RD exhibited a high negative correlation with LC2 and a positive correlation with LC3, LC4, and LC5 root classes, aligning with established scientific predictions (Tables 3 and 4).

Table 3
Mean values for dry weight of roots (DWR) and the aerial part (DWAP), ratio of dry weight of roots/dry weight of the aerial part (DWR/DWAP), total dry weight (TDW), root diameter (RD), and specific root length (SRL) of nucellar seedlings (rooted cuttings or plants originated from seeds) of 15 citrus hybrids under water deficit

Hybrids	DWR (g)	DWAP (g)	DWR/DWAP	TDW (g)	RD (mm)	SRL (cm g-1)
Citrandarin 1697	0.320ns	0.512ns	0.602ns	0.814ns	0.562ns	1.409.500ns
BRS 0 Sempionato	0.270ns	0.370ns	0.728ns	0.640ns	0.542ns	1.902.920**
BRS H Montenegro	0.282ns	0.400ns	0.716ns	0.682ns	0.642**	740.740**
BRS N Gimenes Fernandes	0.358ns	0.504ns	0.722ns	0.862ns	0.530ns	1.473.970ns
BRS L Navarro	0.186ns	0.308ns	0.596ns	0.494ns	0.572*	1.297.006ns
BRS Cunha Sobrinho	0.286ns	0.312ns	0.906ns	0.598ns	0.658**	803.59**
BRS 0 S Passos	0.298ns	0.492ns	0.606ns	0.790ns	0.566*	1.163.186*
BRS Donadío	0.256ns	0.512ns	0.512ns	0.768ns	0.558ns	1.363.934ns
BRS Matta	0.308ns	0.400ns	0.800ns	0.708ns	0.562ns	1.083.634**
BRS Victoria	0.276ns	0.318ns	0.874ns	0.594ns	0.662**	758.804**
BRS S Moreira	0.272ns	0.424ns	0.678ns	0.696ns	0.762**	514.878**
BRS Stuchi	0.256ns	0.352ns	0.736ns	0.608ns	0.544ns	1.072.528**
BRS Pompeu	0.242ns	0.432ns	0.557ns	0.674ns	0.607**	1.081.475**
BRS HLeáo	0.184*	0.234*	0.786ns	0.418*	0.576**	1.085.186**
BRS Bandeirante	0.222ns	0.314ns	0.738ns	0.526ns	0.506ns	1.549.084ns
Average	0.292	0.388	0.758	1.146	0.51	1.586.740

• ^*
  - Significant at p ≤ 0.05 by Dunnett's t-test;
• ^**
  - Significant at p ≤ 0.01 by Dunnett's t-test; ns - Not significant at p > 0.05 by Dunnett's t-test

Table 4
Mean values of root length density (RLD) and total root length (TRL), and diameter classes of 0.0-0.5 cm (LC1), 0.5-1.0 cm (LC2), 1.0-1.5 cm (LC3), 1.5-2.0 cm (LC4), and 2.0-2.5 cm (LC5) of nucellar seedlings (rooted cuttings or plants originated from seeds) of 15 citrus hybrids under water deficit

Hybrids	Mean values
	RLD (cm cm-3)	TRL (cm)	LC1 (cm)	LC2 (cm)	LC3 (cm)	LC4 (cm)	LC5 (cm)
Citrandarin 1697	0.320ns	424.380ns	245.140*	157.778ns	10.800ns	3.930ns	6.692ns
BRS 0 Sempionato	0.210**	282.538**	183.294**	83.146ns	6.984**	3.924ns	5.188ns
BRS H Montenegro	0.160**	209.170**	94.226ns	94.482ns	10.390ns	4.494ns	5.567ns
BRS N Gimenes Fernandes	0.380ns	509.724ns	342.790ns	140.698ns	15.046ns	4.624ns	6.550ns
BRS L Navarro	0.170**	228.332**	126.718**	85.798ns	9.684ns	3.602ns	2.528ns
BRS Cunha Sobrinho	0.170**	227.364**	101.334**	105.388ns	10.168ns	4.098ns	6.360ns
BRS 0 S Passos	0.250*	337.650*	207.616**	109.582ns	11.448ns	3.772ns	5.224ns
BRS Donadío	0.260*	343.784*	204.744**	121.506ns	9.864ns	2.740ns	4.906ns
BRS Matta	0.250*	335.460*	197.482**	118.702ns	11.048ns	3.136ns	5.050ns
BRS Victoria	0.160**	205.588**	97.696**	85.646ns	12.616ns	3.540ns	6.088ns
BRS S Moreira	0.110**	140.282**	63.478**	49.316**	14.812ns	6.092**	6.580ns
BRS Stuchi	0.200**	267.494**	184.230**	67.530*	5.686**	3.618ns	6.396ns
BRS Pompeu	0.200**	261.112**	139.205**	102.857ns	10.022ns	4.237ns	4.740ns
BRS HLeáo	0.150**	197.392**	123.214**	59.124*	7.400**	3.168ns	4.452ns
BRS Bandeirante	0.290*	343.828*	235.352*	99.134ns	4.740**	2.044ns	2.492ns
Average	0.34	451.028	317.232	112.894	13.418	2.596	4.840

• ^*
  - Significant at p ≤ 0.05 by Dunnett's t-test;
• ^**
  - Significant at p ≤ 0.01 by Dunnett's t-test; ns - Not significant at p > 0.05 by Dunnett's t-test

Significant correlations were identified between the traits observed in the field by Costa et al. (2020) and those observed in plants cultivated under controlled conditions in this study. Various variables, including fruit production (GP, kg per plant), drought tolerance (DT), plant height (PH, m), stem diameter (SD, m), crown volume (CV, m ³), and production efficiency (PEF), were analyzed and documented in Table 2. Field-based fruit production (GP) showed significant positive correlations with TRL of roots with a diameter greater than 2.0 cm (LC5) and negative correlations with root length density (RLD), total root length (TRL), and TRL of LC2 root class. Additionally, a positive correlation was observed between drought tolerance (DT) and root diameter (RD) in classes LC4 and LC5. Plant height (PH) exhibited positive correlations with RD and LC4, while crown volume (CV) and stem diameter (SD) showed robust positive correlations with LC4 and LC5 root classes. Regarding production efficiency (PEF), a negative correlation was observed with coarse roots (LC4).

The dendrogram shown was generated using biometric variables that were collected in the laboratory and in the field. These variables were also utilized in the formulation of the correlation matrix, as detailed in Table 2. Analysis of the distance matrix using single linkage revealed the formation of three distinct groups, with the associated cophenetic correlation coefficient being significant (r = 0.7405976***), indicating an agglutination of the hybrids into three clusters, as evidenced in the patterns of similarity illustrated in Figure 4. The smallest distances, indicative of greater similarities, were observed between the hybrids BRS O Sempionato and BRS Stuchi (group G2) and between BRS H Montenegro and BRS Victoria (group G1). The first group (G1) comprises the hybrids BRS S Moreira, BRS Cunha Sobrinho, BRS H Montenegro, and BRS Victoria, as shown in Figure 4.

Figure 4
Dendrogram for the distance matrix by the single linkage method with the formation of three groups: G1 - Group 1; G2 - Group 2 and G3 - Group 3

The second group (G2) comprises the following hybrids: BRS L Navarro, BRS HLeão, BRS Matta, BRS O S Passos, BRS Pompeu, BRS O Sempionato, and BRS Stuchi, exhibiting high tolerance to drought in the field as rootstocks for orange ‘Valencia’ and as nucellar seedlings in juvenile stage with satisfactory performance concerning the specific root growth. However, BRS Matta was the only hybrid in this group that did not do well in drought when grown with ‘Valencia’ oranges, as described by Costa et al. (2020). In the present study, these same hybrids, as nucellar seedlings in the juvenile phase, exhibit the characteristics SRL, TRL, LC1, and LC2 giving them a good performance with regard to specific root growth.

The third group (G3) is constituted by the hybrids BRS N Gimenes Fernandes, BRS Bravo, BRS Bandeirante, Citrandarin 1697, and BRS Donadio. It comprehends hybrids showing higher means for SRL and lower means for RD. All individuals in G3 except BRS Bandeirante showed high values for DWR, setting this group as second statistically when considering the magnitude of the variables mentioned in Table 3.

It was found that the mean values for the variable DWR/ DWAP were high. The mean values for RLD and TRL were also high in this group, except for BRS Donadio and BRS Bandeirante, which had median values for TRL.

When evaluating elite citrus rootstock materials under field conditions, strong negative correlations were observed between production efficiency (PEF) and plant height (PH), stem diameter (SD), and crown volume (CV). This evidence suggests that smaller p lants may exhibit high production efficiency in the field, as demonstrated by Costa et al. (2020). Plant height (PH) exhibited strong positive correlations with stem diameter (SD) and crown volume (CV). However, these relationships with elite materials are not in accordance with certain rootstocks in the breeding program that exhibit high production efficiency (PEF), showing for example a large crown volume; it is merely a matter of different sources of variability.

In relation to TRL, RLD, and SRL, BRS Bravo was superior to the majority of hybrids evaluated, except Citrandarin 1697 and BRS N Gimenes Fernandes. These are important traits of root architecture that prove to be significant in influencing plant anchorage and productivity under drought conditions (Comas et al., 2013), especially when the purpose is to select hybrids adapted to access water and nutrients in deeper layers of soil by increasing the total root length (TRL) and root length density (RLD) (Lynch, 2013). In the case of citrus plants growing in cohesive soil, plant productivity is highly correlated with soil water in the shallow soil profile (Sampaio et al., 2021), which is associated with a greater total root length density in this profile influencing hybrid vigor (Meneses et al., 2020). Silva et al. (2019), while evaluating some hybrid rootstocks, verified BRS Bravo exhibiting higher TRL when compared to all other individuals evaluated.

The findings of the current study are intriguing, revealing negative correlations of TRL (total root length), LC1 (0-0.5 cm) and LC3 (0.5-1.0 cm) with both productivity and drought tolerance. This outcome could be attributed to the elite nature of the evaluated hybrids, potentially indicating robust plant vigor. Although not explicitly clarified by the correlations with PH, CV, and SD, this connection becomes apparent when analyzing the correlations between productivity and vigor in the field (Table 2). SRL, strongly correlated with TRL, particularly with fine roots (LC1 and LC2), is one of the morphological variables associated with the root system capacity for water and nutrient uptake (Syvertsen & Graham, 1985; Comas et al., 2013). There is evidence suggesting that species with high SRL have higher plasticity of root growth and higher physiological capacity for water and nutrient uptake, but with less root longevity and dependence on mycorrhizae when compared with species with low SRL (Eissenstat, 1992). Mycorrhizal activity may cause positive alterations in SRL, as demonstrated by Fitter (2004). This may be part of the response associated with the higher productivity of fruits in some combinations of scion/rootstock in citrus under water deficit conditions.

According Selle et al. (2010), the most active part of the root system for water and nutrient absorption essential for plant growth and development is associated to thin roots (< 2 cm), which account for the greatest part of the superficial root area and length of the root system in citrus plants (Rewald et al., 2011). In general, they are more important than thick roots in the process of water and nutrient absorption (Pierret et al., 2005). Santos et al. (2005) verified that citrus roots with diameters ranging from 0.5 to 2.0 cm extracted higher quantities of water from the soil and therefore were more efficient when supplying water and nutrients to the plant.

In the present work, high correlations were found between productivity and drought tolerance and the TRL of thicker roots, as well as negative correlations with fine roots (Table 2). The correlations between drought tolerance (DT) in the field and seedling traits, especially RD, LC4, and LC5, highlight the importance of thicker roots, which are responsible for the structural plant foundation that supports the formation of absorption roots. In this context, Comas et al. (2013) stated that thick woody roots serve as perennial structures, anchoring or sustaining the plant and contributing to the storage of carbohydrates and nutrients during the water transport process. Although these roots do not directly extract water and nutrients from the soil, they play a crucial role in obtaining such elements from deeper layers of the soil, potentially collaborating with water absorption by superficial roots through hydraulic elevation.

On the other hand, the negative correlation between thicker roots and production efficiency (PEF) is a clear indication that a large number of coarse roots in citrus plants is associated with high vigor and crown volume. This association leads to increased overall plant production but lower production efficiency per plant. These results suggest that citrus plants with fewer coarse roots and a higher density of fine roots are better adapted to the future citrus industry due to their nature as less vigorous rootstocks, such as dwarf and semidwarf rootstocks. These rootstocks, particularly when used in scenarios with higher plant density (Girardi et al., 2021; Hayat et al., 2022), play an important role in the management of HLB (Moreira et al., 2019). Depending on the scion and rootstock, they could enhance plant production, production efficiency (kg m ^-3) and land use efficiency (Costa et al., 2021; Girardi et al., 2021).

Guo et al. (2008) reinforce this viewpoint by explaining that, despite the limited capacity of woody roots to absorb water and nutrients, their anatomical characteristics confer an elevated tolerance to environmental stresses, including water deficits. This partly accounts for the notable longevity and reduced mortality rates observed in plants with substantial presence of woody roots in their root systems. Moreover, Janiak et al. (2016) state that drought-tolerant plants exhibit a proclivity for the development of deeper root systems, enhancing access to available soil water. Bai et al. (2019) in their study involving wheat (Triticum spp.) confirmed that during drier years deeper root systems were linked to increased yield. Furthermore, they observed a strong correlation between root diameter and yield, the underlying reasons for which remain yet to be fully elucidated. The correlations between the TRL (total root length) of thicker root classes and RD with plant vigor clearly suggest the importance of anchoring the plant enhancing drought tolerance and maximizing total plant production. This is particularly significant due to the large size of these plants (Table 2).

Conversely, it is noteworthy that G3 (Figure 4 and Table 5) comprises hybrids exhibiting higher means for SRL and lower means for RD, thus corroborating the findings of Henry (2012). These studies assert that woody plants adapted to drought conditions tend to have thinner roots with a smaller diameter and higher SRL. Field observations (Costa et al., 2020) showed that these hybrids, when used as rootstocks for orange 'Valencia' exhibit adequate production efficiency and high tolerance to drought. However, an exception is noted for BRS N Gimenes Fernandes, which showed medium tolerance, and for BRS Bandeirante, which displayed the lowest tolerance to drought among the evaluated rootstocks, based on the leaf curling trait.

Table 5
Values of variables that contributed to the formation of groups in the dendrogram

Hybrids			Variables
	SRL	TRL		LC1	LC5
Group 1
BRS S Moreira	514.9	140.28		63.48	6.58
BRS Cunha Sobrinho	803.6	227.36		101.34	6.36
BRS H Montenegro	740	209.17		94.23	5.58
BRS Victoria	758.8	205.59		97.7	6.09
BRS L Navarro	1297	228.33		126.72	2.53
Average	823	202.1		96.7	5.4
Group 2
BRS HLeão	1085.2	197.39		123.21	4.45
BRS Matta	1083.6	335.46		197.48	5.05
BRS O S Passos	1163.2	337.65		207.62	5.22
BRS Pompeu	1081.5	261.11		139.2	4.74
BRS O Sempionato	1092.9	282.54		183.29	5.19
BRTS Stuchi	1072.5	267.49		184.23	6.4
Average	1096.5	280.3		172.5	5.2
Group 3
BRS N Gimenes Fernandes	1474	509.72		342.79	6.55
BRS Bravo	1586.7	451.03		317.23	4.84
BRS Bandeirante	1549.1	343.83		23535	2.49
Citrandarin 1697	1409.5	424.38		245.14	6.69
BRS Donadio	136390	34378		204.75	4.91
Average	1476.6	414.5		269.1	5.1

Silva et al. (2019) observed notable root length densities, especially in the diameter class of 0-0.5 cm, among a diverse set of citrus hybrids with the potential for utilization as rootstocks, highlighted by the hybrid BRS Bravo. Arunyanark et al. (2009) observed drought directly linked to root length and density in addition to the presence of thinner roots important for water and nutrient absorption. These results also demonstrate high variability in response among elite hybrids with respect to the total root length of thinner root classes, LC1 (0-0.5 cm), followed by LC3 (0.5-1.0 cm) and LC2 (1-1.5 cm). The total root length up to 0.5 cm explains the outcomes for the total root length (Figure 5), highlighting its significance as an important trait for utilization in phenotyping studies.

Figure 5
Mean values of total root length within classes of root diameter, specifically, 0 - 0.5 cm (LC1) (A), 0.5 - 1.0 cm (LC2) (B), 1.0 - 1.5 cm (LC3) (C), and 1.5-2.0 cm (LC4) (D), in nucellar seedlings (rooted cuttings or plants originated form seeds) across 15 citrus hybrids under water deficit conditions

These findings suggest the potential use of evaluated traits for selecting new hybrids since Ramos et al. (2015) and Carvalho et al. (2016), while studying rootstocks combined with ‘Valência’ and ‘Pera’ (C. × sinensis) oranges, respectively, verified that rootstock BRS Bravo induced high tolerance to drought and high production of fruits. Those results were confirmed by Costa et al. (2020) in environment under strong water deficit associated with high temperatures while simultaneously analyzing a set of variables. In that study which continued the work of Ramos et al. (2015), it was verified the rootstock BRS Bravo when compared to the Rangpur lime ‘Santa Cruz’ (C. × limonia Osbeck) showed higher production efficiency and fruit production, with higher contents of total soluble solids, in addition to high tolerance to drought and reduction of crown size, a fundamental characteristic for high density plantations, which are used in the contemporary citrus production.

Ramos et al. (2015), Carvalho et al. (2016), and Costa et al. (2020) evaluated the hybrid BRS Bravo in various regions as a rootstock for scion varieties 'Pera' and 'Valencia,' confirming its high tolerance to drought and its ability to induce high fruit production. Regardless of the climatic conditions and the scion onto which the hybrid BRS Bravo was grafted, its production potential and drought tolerance remained unaffected, making it suitable for adaptation in different cultivation regions. This establishes it as a potential option for diversifying rootstocks in Brazilian orchards, alongside Rangpur.

Despite group G1 being associated with lower root length, the hybrids included in this group exhibit a higher mean for DWR compared to DWR/DWAP, as a result of low SRL and a large percentage of coarse roots (Table 5), suggesting greater vigor in the plant and potentially better drought tolerance as a result of the complex relationship between root structure, root function, and the adaptation of plants to the environment (Freschet et al., 2021) and hybrid (Sampaio et al., 2021), as observed in field evaluations by Costa et al. (2020). The exception was the hybrid BRS Victoria, which exhibited moderate tolerance, as indicated by leaf curling in the crown during periods of severe water deficit (Costa et al., 2020), low vigor of sweet orange plants, and a significant decrease in productivity in drought years (Sampaio et al., 2021). Such results corroborate the high positive correlations between variables of seedlings with low presence of thin roots and large root diameter (RD) with plant production (GP) and drought tolerance (DT) (Table 2). In this case, associated with high plant vigor under field conditions, with exceptions such as the case of BRS Victoria, exhibiting high production efficiency (PEF, kg fruit m ^-3 crown) and good performance under drought conditions, especially for high -density orchards, as pondered by Sampaio et al. (2021).

It was possible to find correlations between the traits of the root system of seedlings and the phenotypic characteristics of adult plants under field conditions, but the analyses are still limited since the present work was performed using elite hybrids. The uncertainty and variability observed in certain results suggest the necessity of incorporating new non -elite hybrids in future evaluations, aiming to improve correlations with plant characteristics related to performance in the field, such as yield, productivity, efficiency, and vigor, among others, and the reliability of the phenotypic pro cess. It is worth emphasizing that these results should not be considered entirely conclusive and absolute, since, as discussed, drought tolerance mechanisms are diverse. However, the results do indicate a direction for orienting a phenotyping program, focusing on the most suitable traits to express the desired behavior of plants adapted to specific soil types and climate conditions.

Conclusions

• 1.Correlation was found between root system and phenotypic characteristics of seedlings and adult plants, respectively.

• 2. There is necessity of incorporating new non -elite hybrids in future evaluations.

• 1
  Research developed at Brazilian Agricultural Research Corporation - EMBRAPA. Cruz das Almas - BA- Brazil
• Ref.288331
• Financing statement:
  Research funding: CNPq (research grant 311318/2022-3; coordinated by MACF), Embrapa (research project: 20.22.03.033.00.00/ 03 - 2022 coordinated by MACF), and FAPED (Research Project: FAPED/CNPTIA/ BCB_ZARC/CNPMF -23800.22/0109-7; coordinated by MACF).

Acknowledgments:

The authors would like to thank Conselho Nacional de Desenvolvimento Cientí fico e Tecnológico (CNPq), Embrapa, and Foundation for Research and Development Support for the research grants awarded to execute this project.

Data Availability Statement:

There is no data underlying the article texts.

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Edited by

• Editors:
  Lauriane Almeida dos Anjos Soares & Carlos Alberto Vieira de Azevedo

Publication Dates

• Publication in this collection
  21 Aug 2026
• Date of issue
  2026

History

• Received
  09 July 2024
• Accepted
  26 Apr 2026
• Published
  31 July 2026