Establishment and optimization of a hydroponic system for root morphological and nutritional analysis of citrus

Gaofeng Zhou Qingjiang Wei Bixian Li Xiaoli Zeng Guidong Liu About the authors

ABSTRACT

The hydroponic growth system is a convenient platform for studying whole plant physiology, especially for root morphological and nutritional analysis. However, we found that most hydroponic systems described in the literature are not suitable for citrus plants. In this study, a hydroponic system for citrus was designed, comprising three principal components: power and time switch, aeration and hydroponic culture. Herein, details of the protocol were described, including equipment setup, seed pregermination and cultivation, together with preparation and transfer of nutrient solution into hydroponics. In order to demonstrate the adaptability of the trifoliate orange plant to our hydroponic system, comparative tests between soil- and hydroponically-grown plants were carried out. The results showed that the plants grew normally and there were no obvious differences between soil- and hydroponically-grown plants. In addition, nutrient deficiency and transcriptional analysis were carried out to test the efficiency, functionality and suitability of our hydroponic system for the application of physiological and molecular analysis. The results, compared with previous studies, showed that our hydroponic system delivered superior performance as regards the physiological and molecular analysis. Taken together, we established the culture system which is best suited for the growth of trifoliate oranges under hydroponic conditions. The hydroponic system described in this paper is easily constructed and controlled at a low cost. It may serve a wide gamut of experimental purposes, especially root morphological and nutritional analysis of trifoliate oranges and the system is also adaptable to other citrus plants by varying the device size.

cultivation; plant nutrition; trifoliate orange; root morphology

Introduction

Hydroponics, as a convenient method for studying plants in the laboratory and for growing commercial crops, was a term first coined by William F. Gericke in 1929 ( Hershey, 1994Hershey, D.R. 1994. Solution culture hydroponics: history and inexpensive equipment. American Biology Teacher 56: 111-118. ). It is an extremely useful technique for growing plants under controlled nutrient conditions, particularly where clean roots are needed for physiological or microscopic analysis or for RNA extraction. The detailed protocol set up in our study should make it straightforward for other laboratories to adopt the technique rapidly and successfully. For higher plants, studies relevant to nutrients (deficiency or toxicity) on root growth and development are usually carried out under hydroponic conditions because of the wide availability and easy controlability of nutrient concentration. Over the last ten years, several different types of hydroponic systems have been established and developed for Arabidopsis thaliana ( Conn et al., 2013Conn, S.J.; Hocking, B.; Dayod, M.; Xu, B.; Athman, A.; Henderson, S.; Aukett, L.; Conn, V.; Shearer, M.K.; Fuentes, S.; Tyerman, S.D. 2013. Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Methods 9: 4. ; Alatorre-Cobos et al., 2014Alatorre-Cobos, F.; Calderón-Vázquez, C.; Ibarra-Laclette, E.; Yong-Villalobos, L.; Pérez-Torres, C.A.; Oropeza-Aburto, A.; Méndez-Bravo, A.; González-Morales, S.I.; Gutiérrez-Alanís, D.; Chacón-López, A.; Peña-Ocaña, B.A.; Herrera-Estrella, L. 2014. An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions. BMC Plant Biology 14: 69. ) or model plants of other species ( Kim et al., 2005Kim, D.W.; Rakwal, R.; Agrawal, G.K.; Jung, Y.H.; Shibato, J.; Jwa, N.S.; Iwahashi, Y.; Iwahashi, H.; Kim, D.H.; Shim, I.S.; Usui, K. 2005. A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf. Electrophoresis 26: 4521-4539. ; Stefanelli et al., 2013Stefanelli, D.; Jaeger, J.; Jones, R. 2013. A new method for hydroponic tomato production. Practical Hydroponics Greenhouses 129: 25. ).

As one of the important rootstocks for the majority of the citrus species, trifoliate orange [ Poncirus trifoliata (L.) Raf.] is found and utilized in citrus-cultivated regions in China and other countries. Thus, Poncirus trifoliata could serve as a model plant in citrus (just like Arabidopsis thaliana in plant), and an excellent tool for the investigation of the molecular and physiological mechanisms of citrus. Recently, more and more studies have been reported on Poncirus trifoliata , but soil-grown Poncirus trifoliate plants take a long time to be cultivated and their growing conditions are difficult to control. Therefore, standardization of growing conditions is essential to the obtaining of experimental plant materials with high reproducibility. This may be solved by establishing a hydroponic system which is suitable for Poncirus trifoliata . However, most hydroponic systems reported are used to cultivate herbaceous rather than woody plants, such as citrus plants. The main reasons are not only that woody plant is too difficult to cultivate, but also that citrus plants depend mainly on AM (arbuscular mycorrhizas) for absorption nutrients ( Wu and Xia, 2006Wu, Q.S.; Xia, R.X. 2006. Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. Plant Physiology 163: 417-425. ), which also poses problems of cultivation under hydroponic conditions, and nutrient distribution throughout the root and enhance plant growth.

Thus, this study aimed to establish a hydroponic system for growing Poncirus trifoliata plants, which is convenient and also offers the following advantages: (i) optimization and synchronization of plant growth; (ii) easy monitoring and manipulation of mineral nutrition; and (iii) observation and sampling of roots without damage.

Materials and Methods

Plant materials

Trifoliate orange [ Poncirus trifoliata (L.) Raf.] seeds were used in this experiment.

Equipment

The following equipment were used in the present study: 15 L soil-growth pot (with 30 cm height and 25 cm internal diameter) for culturing plant seedlings, black, plastic, 8; 15 L opaque container (25 cm × 20 cm × 28 cm), measuring 2 cm from the top to board so that each container holds 14 L of solution, four in each group; plant holder, KT board for supporting the plant in hydroponic tanks (30 cm × 25 cm, 50 mm thick) with 8 wells (15 mm diameter), one for each tank; Leather punch (5 mm and 20 mm external diameter), one of each type; Black sponge strip (15 mm × 15 mm × 30 mm), 32 in each group; micro-computer time switch, one; Aquarium air pump 58 W/220 V (e.g. Risheng, ACO-003), output: 50 L min–1, pressure: 0.028 MPa, one in each group; Aquarium air stone (15 mm diameter), two in each container; aquarium air tubing (3.8 mm internal diameter and 5.0 mm external diameter), silicone flexible tube, 0.5 m × 2 in each container and 1.0 m × 4 in each group; plastic Y-shape connector, two in each container; 50 L nutrient solution stock container.

Equipment setup

As shown in Figure 1A , the KT board (30 cm × 25 cm, 50 mm thick) was drilled with 8 wells (20 mm diameter) with a leather punch (20 mm external diameter) to make the plant holder. Additionally, 2 wells (5 mm diameter) for aquarium air tubing (3.8 mm internal diameter and 5.0 mm external diameter) were also drilled with the leather punch (5 mm external diameter).

Figure 1
–The component dimensions and assembly of hydroponic system. A) The dimensions of plant holder. The numbers indicate the dimensions of holes and edges. B) The assembly and dimensions of aeration system.

Aeration of each hydroponics tank was provided via a single aquarium air tube (3.8 mm internal diameter and 5.0 mm external diameter) from a 4-outlet aquarium air pump (58 W, 50 L min–1 maximum), with a plastic Y-type connector fitted inline to permit the use of 2 aquarium air stones (15 mm diameter) in each tank. A micro-computer time switch (HHQ 4) was used to control the aeration time ( Figure 1B ).

Protocol

The general workflow for the trifoliate orange hydroponic system is shown in Figure 2 . Step by step instructions for setting up our hydroponic system are described in the following sections. Critical points and important notes are also annotated where appropriate.

Figure 2
– Simplified trifoliate orange hydroponics growth method. Flow chart outlining the timeline and the key steps in the process. Timing (in bold) on right of arrows indicate time between steps (d = days). Images on right-hand panel show setup of seed germination and representative images of seedling plants.

Seed germination

1) The seeds of trifoliate orange were selected and then rinsed repeatedly in distilled water to remove sediment and floating seeds. Then the seeds were soaked in distilled water for 30 min.

NOTE (for future application): If the storage time of seeds are too long, the seeds have to be soaked in 35-40 °C warm distilled water for 1 h, then soaked in cold distilled water overnight.

2) Selected trifoliate orange seeds were surface-sterilized in a 5 % sodium hypochlorite solution for 15 min and 70 % ethanol for 1.5 min, followed by thorough washing in distilled water.

3) The seeds were placed on a porcelain tray with moistened gauze and transferred to an incubator at 30 °C in the dark with 75 % relative humidity. Then they were moistened every day with distilled water till seed germination.

NOTE (for future application): The incubator should be cleaned up and surface-sterilized with 70 % ethanol before seed pregermination. The porcelain tray and gauze should also be sterilized. Before moistening the seeds, distilled water must be preheated in an incubator for at least 1 h.

Seedling culture

1) After germination, the seeds were transferred into 14 L plastic soil-growth pots (20 plants per pot) filled with vermiculite.

NOTE (CRITICAL POINT): Often, trifoliate orange seeds will germinate in one week, but most seeds will germinate 10-15 days after pre-germination. Therefore, in order to obtain more uniform seedlings, those seeds which fail to germinate within 10-15 days after pre-germination should be abandoned.

NOTE: Vermiculite should be sterilized in 121 °C for 30 min before being used to germinate the seeds. Vermiculite was selected as the seedling media because it is easy to clean up. Thus, there was no contamination of the nutrient solution when the seedlings were transferred to a hydroponics container.

NOTE: The plastic pot must be deep since the taproot of trifoliate oranges is long.

In order to preserve the humidity and temperature of the pot, all the pots were covered with plastic mulch. The plastic mulch applied in this step required a fine light transmission property, and were removed once the seedlings had two leaves.

3) The plants were transferred to a growth chamber and submitted to the following conditions (28 °C day and 22 °C night with 75 % relative humidity, and light intensity of 800 µmol m–2 s–1 of photosynthetically active radiation) and 14/10 h photoperiod. They were then irrigated twice a week, until the plants had 4 leaves (approximately 3 weeks later).

Preparing nutrient solution

1) The modified Hoagland’s N°.2 nutrient solution was used in this protocol, which contained 6 mM KNO3, 4 mM Ca(NO3)2, 1 mM NH4H2PO4, 2 mM MgSO4, 9 µM MnCl2, 0.8 µM ZnSO4, 0.3 µM CuSO4, 0.01 µM H2MoO4 15 µM H3BO3 and 50 µM Fe-EDTA ( Hoagland and Arnon, 1950Hoagland, D.R.; Arnon, D.I. 1950. The water-culture method for growing plants without soil. California Agricultural Experiment Station, Davis, CA, USA. (Circular. California Agricultural Experiment Station, 347). ). The detailed nutrient solution formulae are shown in Table 1 .

Table 1
– The modified Hoagland’s N°. 2 nutrient solution formula.

2) The modified Hoagland’s N°. 2 nutrient solution used in this protocol was divided into two stock solutions, one with six macronutrients and the other with seven micronutrients.

CRITICAL POINT: Na2Fe-EDTA solution is difficult to prepare, thus the 1000X Na2Fe-EDTA stock solution should be prepared according to the following procedure: 1) 0.1 mol EDTA-Na2 solution: 37.7 g EDTA-Na2 + 600 mL H2O should be heated to dissolve and set the volume to 1 L; 2) 0.1 mol FeSO4 solution: 27.8 g FeSO4 7H20 + 600 mL H2O constant volume to 1 L; 3) 0.05 mol Na2Fe-EDTA solution: mixed in equal volumes of the two solutions.

NOTE: All the reagents used to make the nutrient solutions should be guaranteed reagent (GR) with a green label.

NOTE: Nutrient solution level should be 2 cm below the container rim.

NOTE: All the prepared stock nutrient solutions and the working nutrient solutions must be kept in opaque containers to exclude light to prevent moss breeding.

Transferring to hydroponics

1) When the plants had 4 leaves (about 3 weeks), 32 seedlings from each group were selected by uniform size and transferred into hydroponics. The seedlings were fixed on a black plant holder and then transferred to a growth chamber under the following conditions (28 °C day and 22 °C night with 75 % relative humidity, and light intensity of 800 µmol m–2 s–1 of photosynthetically active radiation) and 14/10 h photoperiod.

2) Plants were pre-cultured with 1/2 Hoagland’s N°.2 nutrient solution for 2-3 weeks until the new white root appeared, and then the solution was replaced for experimentation. The solution was ventilated for 20 min every 2 h and changed twice a week until analysis. The pH of the nutrient solution was adjusted to 6.0 with 0.1 M NaOH.

CRITICAL POINT: Citrus seedlings grown in nutrient solutions consume a lot of water through transpiration. Thus, the nutrient solution level decreases and a large number of roots are exposed to the air. Moreover, concentration of the nutrient solutions also increase as a result of plant transpiration, and plant growth and development will be influenced by the high nutrient solution concentration. Therefore, the nutrient solution level should be checked every day, and the hydroponic container filled with water, not with nutrient solution.

NOTE: If not all the 32 plant wells (each group) are filled with plants, or plants are sampled during the experiment, unused holes must be covered with black sponge strip (15 mm × 15 mm × 30 mm) to exclude light from the growth solution.

NOTE: The plant holder provides a useful handling tool for transferring the seedlings to experimental chambers or different nutrient solutions.

Nutrient deficiency treatments and sampling

The 2-month-old trifoliate orange seedlings of uniform size were selected and transferred into our hydroponic culture system as described above. These plants were precultured for 2-3 weeks until the new white root appeared, and then transferred into new hydroponic containers (easily transferred from one container to another new one) with 0 µM Fe-EDTA for Fe-deficiency treatment and 0 µM H3BO3 for B-deficiency treatment, respectively. For investigating the physiological and root morphological changes of trifoliate orange seedlings in response to Fe- and B-deficiency, the samples were harvested randomly after ten weeks of treatment. For the microarray analysis of B-deficient experiment, the root tissue samples were taken at 3 h, 6 h and 12 h after treatment and frozen immediately at each prescribed point in time.

Plant-growth parameters measurement

After ten weeks of treatment, 32 plants per group were harvested randomly and the pant-growth parameters including leaf area, dry weights and seedling height were determined using the method previously reported ( Zhou et al., 2014Zhou, G.F.; Peng, S.A.; Liu, Y.Z.; Wei, Q.J.; Han, J.; Islam, M.Z. 2014. The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency. Trees 28: 295-307. ).

Root morphology analysis

32 seedlings (8 plants in each hydroponic container or soil pot, 4 replicates) were randomly sampled in each group, and the total root length, root surface area, root volume and root number were then analyzed as described by Zhou et al. (2014)Zhou, G.F.; Peng, S.A.; Liu, Y.Z.; Wei, Q.J.; Han, J.; Islam, M.Z. 2014. The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency. Trees 28: 295-307. . Length of primary root (cm) was measured using a scaled ruler, and then the root density (number/cm) was calculated.

Determination of mineral nutrients

The mineral concentration of P, K, Ca, Mg, Fe, Mn, Zn, B, Cu, Na, Al, Ni, Cr and Co in the different plant tissues were determined using the method described by Storey and Treeby (Storey and Treeby, 2000). Briefly, 0.50 g of each sample was dry-ashed in a muffle furnace at 500 °C for 6 h, followed by dissolution in 0.1 N HCl, and then the mineral nutrients concentration were determined using ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

Microarray analysis

The gene expression profiles in root tissue were investigated using microarray analysis after 3, 6 and 12 h B-deficient stress and the corresponding non-stress controls were investigated by microarray analysis. Fluorescent dye-labeled cDNA and hybridization were prepared according to a previous published protocol ( Guo et al., 2005Guo, Y.; Guo, H.Y.; Zhang, L.; Xie, H.Y.; Zhao, X.; Wang, F.X. 2005. Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. Journal of Virology 79: 14392-14403. ). Microarray data and EST sequence analysis had been carried out during our previously published study ( Zhou et al., 2015Zhou, G.F.; Liu, Y.Z.; Sheng, O.; Wei, Q.J.; Yang, C.Q.; Peng, S.A. 2015. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray. Frontiers in Plant Science 5: 795. ).

Results

Comparison of soil-grown and solution-grown plants

In order to compare the growth and development of citrus between soil-grown and hydroponic, a number of growth indicators were identified. Under both soil and hydroponic conditions, the plants can grow normally, with no obvious difference in biomass and plant height ( Table 2 ). However, the leaf area and number of leaves in hydroponic cultured plants was much higher than in soil cultured plants ( Table 2 ). These results indicated that the growth and development of trifoliate orange seedlings were not influenced by nutrients in our hydroponics system.

Table 2
– Comparative growth indicators of soil-grown and hydroponically-grown plants.

The effects of hydroponics on the root-morphological traits of trifoliate orange seedlings were also determined. As shown in Table 3 , the length of primary root and root total length (cm) were higher in hydroponics than in soil, but there was no remarkable difference in root surface area and root volume. In contrast, the number of lateral root and root density were lower in hydroponics compared to that in soil.

Table 3
– Root morphology of trifoliate orange seedlings grown in both soil and hydroponics.

We tested a number of plant growth solutions and found that a modified 1/2 Hoagland’s solution was a simple, defined and affordable media, which supported fine plant growth as described above. To further investigate the effects of our hydroponic solution on plant nutrient status, the plant ionome of trifoliate orange seedlings were determined and compared to plants grown in soil. The ionomic analysis results after ten weeks growth are shown in Table 4 . A ratio was drawn up to compare the ionome of soil-grown plants to hydroponics plants which showed lines are similar in nutrient content for most essential nutrients, except for Mn and Cu in root. As for non-essential nutrients, there were no significant differences between Na, Al and Co, since they are not essential nutrients required for plant growth and thus they were not added to the hydroponic solutions.

Table 4
– Comparative ionomics of soil-grown and hydroponically-grown plants in the leaf and root.

Applications of our hydroponic system

1) Plant nutrient deficiency experiments

In order to demonstrate the efficiency and functionality of our hydroponics system, micro-mineral nutrient deficiency (Fe- and B-deficiency) experiments were performed on trifoliate orange seedlings, respectively. After ten weeks of nutrient-deficient treatments, typical symptoms in leaf and root architecture of trifoliate orange seedlings ( Figure 3A -C and 4A-F) began to appear. As shown in Figure 3C , the serious vein swelling and cracking of the leaves were observed under B-deficient conditions. In the case of Fe-deficiency, there was leaf etiolation in the young leaves at the top of the plant ( Figure 3B ). In contrast, there was no change in the control plants ( Figure 3A ).

Figure 3
– Hydroponically cultured trifoliate orange seedlings and morphological symptoms caused by Fe and B-deficiency treatment. All the trifoliate orange seedlings were grown under hydroponic conditions and treated for 10 weeks. A) Control; B) Fe-deficiency; C) B-deficiency.

Root morphological traits under these nutrient deficiencies were also examined in this experiment ( Figure 4A -F). Results showed that the primary root length, root total length, root surface area, root volume and root number were decreased significantly by B-, and Fe-deficiency treatments ( Figure 4A , B, C, D and E). In contrast, the root density increased markedly in all three nutrient deficiency treatments compared with control plants ( Figure 4F ). In the case of B-deficiency treatment, the root morphological traits were inhibited or improved more seriously than those under Fe-deficiency conditions.

Figure 4
– Effects of Fe and B-deficiency on root morphology of trifoliate orange seedlings under hydroponic conditions. A) primary root length; B) root total length; C) root surface area; D) root volume; E) root number; F) root density. All the trifoliate orange seedlings were grown under hydroponic conditions and treated for 10 weeks. Data are presented as means ± SE of 8 replicates (n = 8, one plant for each replicate). Significance of ANOVA: * p < 0.05; ** p < 0.01; *** p < 0.001. BD = B-deficiency; FeD = Fe-deficiency; CK = Control.

2) Transcriptional responses to B-deficiency

In order to demonstrate the application of our hydroponic system on the molecular level, transcription analysis was performed on the root of trifoliate orange under B-deficiency conditions. In this study, two-month-old trifoliate orange seedlings were grown in our hydroponic system with 0 µM H3BO3 and the root tissue samples were harvested at three prescribed points in time (3 h, 6 h and 12 h). After microarray analysis, a total of 63 differentially expressed genes (FDR < 0.01 and fold change > = 2) from trifoliate orange were identified under B-deficiency conditions. As shown in Figure 5A , the expression patterns of trifoliate orange showed that the largest number of up-regulated genes appeared at 6 h, but, in contrast, the down-regulated genes appeared at 6 h after B-deficient treatment. Out of these genes, there were 6, 43 and 20 genes up-regulated and 13, 3, and 7 down-regulated by B-deficient stress at 3 h, 6 h and 12 h, respectively ( Figure 5B ). However, only one gene out of the up-regulated genes and no genes from amongst the down-regulated genes were common to all three prescribed points in time.

Figure 5
– The transcriptome change in the root of trifoliate orange seedling under B-deficiency hydroponic conditions. A) Number of differentially expressed genes significantly up- or down-regulated in trifoliate orange root in response to boron deficiency stress at various time points. B) Venn diagram illustrates the number of common or distinct regulated genes up- or down-regulated by B-deficiency stress over the sample time points. C) Distribution of differentially expressed genes of trifoliate orange root under B-deficiency conditions based on MIPS functional categories.

Distribution of differentially expressed genes of trifoliate orange root by B-deficiency stress were shown in Figure 5C , a total of 63 unique genes were grouped into ten functional categories based on the Munich Information Center for Protein Sequences’ (MIPS) functional categories. The majority of these differentially expressed genes in trifoliate orange are involved in metabolism (11 %), subcellular localization (10 %), cell transport, transport facilitation and transport routes (11 %), and cell rescue, defense and virulence (13 %). Interestingly, there is a large number of genes regulated by B-deficiency in this study encoding unclassified (10 %) and unknown (22 %) proteins, and these genes may be responsible for the responsive mechanism of trifoliate orange to B-deficiency.

In this study, we focused on genes involved in plant metabolism, subcellular localization, and cellular transport related genes ( Table 5 ). In the metabolism category, there were four gene encoding key enzymes in the lignin biosynthesis metabolism which were significantly up-regulated under B-deficiency, including phenylalanine ammonia-lyase ( PAL ; JK817683), 4-coumarate: CoA ligase (4CL; JK817661), cinnamoyl-CoA reductase 4 ( CCR4 ; KJ817664) and peroxidase ( POD ; JK817712). There are another six genes involved in the cell wall metabolism (belonging to the subcellular localization category) which were significantly affected under B-deficiency. These six genes are xyloglucan endotransglycosylase/hydrolase 9 genes ( XTH9 ; JK817615), proline-rich cell wall protein 2 ( PRP2 ; JK817604), glucan endo-1.3-beta-glucosidase (JK817631), polygalacturonases ( PG ; JK817590), expansion ( EXP ; JK817639), and pectin methylesterase ( PME ; JK817660). As for the cellular transport category, genes involved in transmembrance transport were also identified, amongst which there were three aquaporin genes [ PIP1 ; 3 (JK817607); TIP2 ; 2 (JK817649) and NIP5 ; 1 (JK81752)] that were highly regulated under B-deficiency stress.

Table 5
– A list of some important differentially expressed genes in the root of trifoliate orange under hydroponic boron deficiency stress. Focused on genes involved in plant metabolism, subcellular localization and cellular transport, transport facilitation and transport route related genes.

In addition, the differentially expressed genes in trifoliate orange roots were also clustered using the hierarchical correlation and average linkage clustering in the TreeView 3.0 software program ( Figure 6 ). The clustering analysis of expression patterns showed that most of these genes were up-regulated at various prescribed points in time, especially at the 6 h point.

Figure 6
– TreeView representation of the differentially expressed genes of trifoliate orange root by B-deficient stress. Hierarchical clustering of 63 differentially expressed genes that showed a fold change of at least ± 2 and FDR-corrected P values < 0.01 at any time point. The signals are shown on a red-green color scale, where red represents higher expression and green represents lower expression.

Discussion

The establishment and advantages of our hydroponic system for trifoliate orange seedlings

In this study, we established a culture system which is best suited to the growth of trifoliate orange seedling in hydroponics, based on a hydroponic device which can be quickly and cheaply constructed, and is easy to control ( Figure 2 ). This hydroponic system consists of three principal sections: power and time switch section, aeration section and plant hydroponic culture section. We described step by step a detailed protocol for setting up this hydroponic system, including equipment setup, seed pregermination and culture, nutrient solution preparing and transferring into hydroponics. As we know, hydroponics was a term first coined by William F. Gericke in 1929 ( Hershey, 1994Hershey, D.R. 1994. Solution culture hydroponics: history and inexpensive equipment. American Biology Teacher 56: 111-118. ). Increasingly, hydroponic culture systems were established and optimized for Arabidopsis ( Conn et al., 2013Conn, S.J.; Hocking, B.; Dayod, M.; Xu, B.; Athman, A.; Henderson, S.; Aukett, L.; Conn, V.; Shearer, M.K.; Fuentes, S.; Tyerman, S.D. 2013. Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Methods 9: 4. ; Alatorre-Cobos et al., 2014Alatorre-Cobos, F.; Calderón-Vázquez, C.; Ibarra-Laclette, E.; Yong-Villalobos, L.; Pérez-Torres, C.A.; Oropeza-Aburto, A.; Méndez-Bravo, A.; González-Morales, S.I.; Gutiérrez-Alanís, D.; Chacón-López, A.; Peña-Ocaña, B.A.; Herrera-Estrella, L. 2014. An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions. BMC Plant Biology 14: 69. ), rice ( Kim et al., 2005Kim, D.W.; Rakwal, R.; Agrawal, G.K.; Jung, Y.H.; Shibato, J.; Jwa, N.S.; Iwahashi, Y.; Iwahashi, H.; Kim, D.H.; Shim, I.S.; Usui, K. 2005. A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf. Electrophoresis 26: 4521-4539. ), barley ( Battke et al., 2003Battke, F.; Schramel, P.; Ernst, D. 2003. A novel method for in vitro culture of plants: cultivation of barley in a floating hydroponic system. Plant Molecular Biology Reporter 21: 405-409. ), lettuce ( Kratky, 1993Kratky, B.A. 1993. A capillary, noncirculating hydroponic method for leaf and semi-head lettuce. HortTechnology 3: 206-207. ), tomato ( Stefanelli et al., 2013Stefanelli, D.; Jaeger, J.; Jones, R. 2013. A new method for hydroponic tomato production. Practical Hydroponics Greenhouses 129: 25. ) amongst other crops. However, all these hydroponic culture systems were for herbaceous plants rather than for woody plants, (not to mention citrus plants). Thus, our culture system represents the first hydroponic culture system for woody plants, especially for citrus plants.

As a model plant in citrus (just like Arabidopsis thaliana in plant), Poncirus trifoliata is an excellent tool for the investigation of the molecular and physiological mechanisms of citrus. In this study, we compared plant growth and development between soil-grown and hydroponics, and a number of growth indicators were determined. Under both soil and hydroponic conditions, the plants can grow normally, and no obvious difference was found in biomass nor plant height ( Table 2 ). These results indicated that our our hydroponic culture system is best suited to the cultivation of trifoliate orange seedling.

In addition to the general advantages of the hydroponic system, such as control of mineral nutrition and access to the root system, there are the many other advantages to our hydroponic system described above. First, our hydroponic system is a simple, quickly and cheaply constructed system. It consists of three principal sections: power and time switch section, aeration section and plant hydroponic culture section. Details of the protocol of the system are presented below, including equipment setup, seed pregermination and culture, nutrient solution preparing and transferring into hydroponics ( Figure 1A and B; Figure 2 ). Second, our hydroponic system is convenient and quick for transferring the plants from one hydroponic solution to another in new nutrient solutions. In this study, nutrient deficiency (Fe- and B-deficiency) experiments were performed on trifoliate orange seedlings ( Figure 3B and C; Figure 4A -F). At the beginning of these experiments, the plant materials were grown in normal nutrient hydroponic solution for 2-3 weeks, and were then transferred to Fe- and B-deficient nutrient solutions without changing the plant holder for ten weeks, respectively. Results revealed that our hydroponic system is convenient and quick in plant material treatment. Third, the tissue samples cultured in our hydroponic system can be harvested quickly and without damage. The tissue samples are clean for RNA isolation, especially for root.

In addition, our hydroponic system can be easily adapted for other citrus plants, such as for Carrizo citrange ( C . sinensis L. × P . trifoliata L.), Red tangerine ( C . Reticulata B.), Cleopatra mandarin ( C . reshni Hort.), Fragrant citrus ( C . junos Sieb.), Sour orange ( C . aurantium L.) and others by changing the hole size and other conditions ( Mei et al., 2011Mei, L.; Sheng, O.; Peng, S.A.; Zhou, G.F.; Wei, Q.J.; Li, Q.H. 2011. Growth, root morphology and boron uptake by citrus rootstock seedlings differing in boron-deficiency responses. Scientia Horticulturae 129: 426-432. ; Zhou et al., 2014Zhou, G.F.; Peng, S.A.; Liu, Y.Z.; Wei, Q.J.; Han, J.; Islam, M.Z. 2014. The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency. Trees 28: 295-307. ). Furthermore, we also adapted our system to investigate the salt stress on trifoliate orange on the addition of excessive sodium (Na+) and (or) chloride (Cl) to the standard nutrient solution ( Wei et al., 2013Wei, Q.J.; Liu, Y.Z.; Sheng, O.; An, J.C.; Zhou, G.F.; Peng, S.A. 2013. Overexpression of CsCLCc , a chloride channel gene from Poncirus trifoliata , enhances salt tolerance in Arabidopsis . Plant Molecular Biology Reporter 31: 1548-1557. ). This is an additional advantage of our hydroponic system.

Performance of our hydroponic system on root morphological and nutritional analysis

The way roots develop in soil can have a critical effect on plant growth and impact crop yield ( de Dorlodot et al., 2007de Dorlodot, S.; Forster, B.; Pagès, L.; Price, A.; Tuberosa, R.; Draye, X. 2007. Root system architecture: opportunities and constraints for genetic improvement of crops. Trends in Plant Science 12: 474-481. ; Postma and Lynch, 2011Postma, J.A.; Lynch, J.P. 2011. Root cortical aerenchyma enhances the growth of maize on soils with suboptimal availability of nitrogen, phosphorus, and potassium. Plant Physiology 156: 1190-1201. ). Root washing is the most common method used to study the root system of plants grown in their natural soil environment ( Gregory, 2006Gregory, P.J. 2006. Plant, Root and the Soil. Blackwell, Oxford, UK. ; Smit et al., 2000Smit, A.L.; Bengough, A.G.; Engels, C.; van Noordwijk, M.; Pellerin, S.; van de Geijn, S.C. 2000. Root Methods: A Handbook. Springer, Berlin, Germany. ). However, this method often leads to the underestimation of fine root through breakage during the washing process. Recently, a new method has been developed that can recover the complete structure of the plant root system from soil via X-ray μ-Computed Tomography, but this method process is too complex and the equipment too expensive ( Mairhofer et al., 2013Mairhofer, S.; Zappala, S.; Tracy, S.; Sturrock, C.; Bennett, M.J.; Mooney, S.J.; Pridmore, T.P. 2013. Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography. Plant Methods 9: 8. ). In contrast, using our hydroponic system to investigate the plant root system architecture is easy and cheap. Moreover, our hydroponic system can be used to investigate the dynamic of plant root system development without root breakage and damage. In our method, as shown in Table 3 , the length of primary root and root total length were higher in hydroponics than in soil, but there was no noticeable difference in root surface area and root volume. However, the number of lateral root and root density were lower in hydroponics compared to that in soil. These results are similar to a previous study on the root of honey locust, whose results showed the root systems grown in solution had longer primary roots, fewer lateral roots and root hairs, and a greater distance between the tip of the primary root and the junction of the youngest secondary root and the primary root than root systems grown in sand ( Graves, 1992Graves, W.R. 1992. Influence of hydroponic culture method on morphology and hydraulic conductivity of root of honey locust. Tree Physiology 11: 205-211. ).

In this study, in order to demonstrate the performance of our hydroponic system on root morphologic analysis, we also examined critically the root morphology of trifoliate orange under Fe- and B-deficiency stress. Fe and B were selected in this verification experiment, and Fe- and B-deficiency are frequently observed in the citrus growing regions of China and other countries ( Forner-Giner et al., 2010Forner-Giner, M.A.; Llosá, M.J.; Carrasco, J.L.; Perez-Amador, M.A.; Navarro, L.; Ancillo, G. 2010. Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock poncirus trifoliata (L.) Raf. Journal of Experimental Botany 61: 483-490. ; Peng et al., 2010Peng, L.Z.; Zhang, G.Y.; Chun, C.P. 2010. Study on the relationship between leaf yellowing with vein swelling or cracking and Mg, B element concentrations in Newhall Navel Orange leaves. South China Fruit 39: 1-5. ; Chen et al., 2012Chen, L.S.; Han, S.; Qi, Y.P.; Yang, L.T. 2012. Boron stresses and tolerance in citrus. African Journal of Biotechnology 11: 5961-5969. ; Liu et al., 2012Liu, G.D.; Wang, R.D.; Wu, L.S.; Peng, S.A.; Wang, Y.H.; Jiang, C.C. 2012. Boron distribution and mobility in navel orange grafted on citrange and trifoliate orange. Plant and Soil 360: 123-133. ). After ten weeks of Fe- and B-deficiency stress, typical changes in root morphology in trifoliate orange. As shown in Figure 4A -F, the root morphology of trifoliate orange was influenced significantly, in comparison with the control plants, and all the results are in agreement with previous studies ( Mei et al., 2011Mei, L.; Sheng, O.; Peng, S.A.; Zhou, G.F.; Wei, Q.J.; Li, Q.H. 2011. Growth, root morphology and boron uptake by citrus rootstock seedlings differing in boron-deficiency responses. Scientia Horticulturae 129: 426-432. ; Han et al., 2012Han, J.; Zhou, G.F.; Li, Q.H.; Liu, Y.Z.; Peng, S.A. 2012. Effects of magnesium, iron, boron deficiency on the growth and nutrition absorption of four major citrus rootstocks. Acta Horticulturae Sinica 39: 2105-2112. ; Cao et al., 2013Cao, X.; Chen, C.L.; Zhang, D.J.; Shu, B.; Xiao, J.; Xia, R.X. 2013. Influence of nutrient deficiency on root architecture and root hair morphology of trifoliate orange ( Poncirus trifoliata L. Raf.) seedlings under sand culture. Scientia Horticulturae 162: 100-105. ; Zhou et al., 2014Zhou, G.F.; Peng, S.A.; Liu, Y.Z.; Wei, Q.J.; Han, J.; Islam, M.Z. 2014. The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency. Trees 28: 295-307. ). In sum, all the results indicated that the root growth and development of trifoliate orange seedlings were not significantly influenced by the nutrient solution in our system and the typical changes in root morphology can be found under nutrient deficiency conditions using our hydroponic system.

The efficiency and functionality of our proposed hydroponic system is demonstrated by the nutrient deficiency experiments. It is known that B is essential for the growth and development of higher plants ( Warington, 1923Warington, K. 1923. The effect of boric acid and borax on the broad bean and certain other plants. Annals of Botany os-37: 629-672. ). Although citrus plants are not classified as the species the most sensitive species to B-deficiency, the occurrence of B-deficiency has been reported in major citrus producing countries of the world, such as Spain, the United States, Brazil and China ( Shorrocks, 1997Shorrocks, V.M. 1997. The occurrence and correction of boron deficiency. Plant Soil 193: 121-148. ; Han et al., 2008Han, S.; Chen, L.S.; Jiang, H.X.; Smith, B.R.; Yang, L.T.; Xie, C.Y. 2008. Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings. Journal of Plant Physiology 165: 1331-1341. ; Chen et al., 2012Chen, L.S.; Han, S.; Qi, Y.P.; Yang, L.T. 2012. Boron stresses and tolerance in citrus. African Journal of Biotechnology 11: 5961-5969. ; Liu et al., 2013Liu, Y.Z.; Li, S.; Yang, C.Q.; Peng, S.A. 2013. Effects of boron-deficiency on anatomical structures in the leaf main vein and fruit mesocarp of pummelo [ Citrus grandis (L.) Osbeck]. Journal of Horticultural Science and Biotechnology 88: 693-700. ). Moreover, it is hard to create B-deficiency growth conditions for the culturing of plant materials because B is a micro-nutrient for plants. In this study, a complete set of B-deficiency growth conditions was established using our hydroponic system. To demonstrate the application of our hydroponic system under B-deficiency conditions, physiological and molecular experiments were carried out on trifoliate orange seedlings. As shown in Figures 3A-C and 4A-F, after ten weeks of B-deficient treatment the typical symptoms of B-deficiency were observed in the leaves and root of trifoliate orange.

Performance of our hydroponic system on molecular level

To demonstrate the application of our hydroponic system on the molecular level, microarray analysis was carried out on trifoliate orange root under B-deficiency conditions. According to the results of our transcription analysis, a total of 63 unique genes significantly changed by B-deficiency stress in the root of trifoliate orange ( Figure 5A and B). Among these genes, there are several gene encoded key enzymes involved in the cell wall metabolism, such as XTH9 , PRP2 , glucan endo-1.3-beta-glucosidase, PG , EXP , and PME ( Figure 5C and Table 5 ). Except for PG and glucan endo-1.3-beta-glucosidase gene, which were up-regulated under B-deficiency, the other four genes were down-regulated markedly. It is known that the plant cell wall plays a very important role in plant growth and development, and the cell wall mediates the responses of plants to environment and pathogen-induced stress ( Farrokhi et al., 2006Farrokhi, N.; Burton, R.A.; Brownfield, L.; Hrmova, M.; Wilson, S.M.; Bacic, A. 2006. Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes. Plant Biotechnology Journal 4: 145-167. ). On the other hand, B has been established as an essential element for the structure and functions of the cell wall ( O’Neill et al., 2004O’Neill, M.A.; Ishii, T.; Albersheim, P.; Darvill, A.G. 2004. Rhamnogalacturonan. II. structure and function of a borate cross-linked cell wall pectic polysaccharide. Annual Review of Plant Biology 55: 109-139. ). Taken together, our results demonstrate that the expression of several enzyme genes involved in cell wall metabolism were significantly changed in trifoliate orange roots under B-deficiency conditions. Similar results were also reported in Arabidopsis ( Camacho-Cristóbal et al., 2008Camacho-Cristóbal, J.J.; Herrera-Rodríguez, M.B.; Beato, V.M.; Rexach, J.; Navarro-Gochicoa, M.T.; Maldonado, J.M. 2008. The expression of several cell wall-related genes in Arabidopsis roots is down-regulated under boron deficiency. Environmental and Experimental Botany 63: 351-358. ).

There are three aquaporin genes [ PIP1 ; 3 (JK817607); TIP2 ; 2 (JK817649) and NIP5 ; 1 (JK81752)] were highly up-regulated under B-deficiency stress ( Table 5 ). As we know, aquaporins are water channel proteins of intercellular and plasma membranes which are involved in many functions of plants, such as nutrient acquisition, carbon fixation, cell signaling and stress responses ( Maurel, 2007Maurel, C. 2007. Plant aquaporins: novel functions and regulation properties. FEBS Letters 581: 2227-2236. ). To date, it has been reported that three subgroups (NIPs, PIPs and TIPs) of the major intrinsic protein (MIP) family were involved in B transmembrane transport ( Takano et al., 2006Takano, J.; Wada, M.; Ludewig, U.; Schaaf, G.; Von Wirén, N.; Fujiwara, T. 2006. The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. Plant Cell 18: 1498-1509. ; Tanaka et al., 2008Tanaka, M.; Wallace, I.S.; Takano, J.; Roberts, D.M.; Fujiwara, T. 2008. NIP6;1 is a boric acid channel for preferential transport of boron to growing shoot tissues in Arabidopsis . Plant Cell 20: 2860-2875. ; Fitzpatrick and Reid, 2009Fitzpatrick, K.L.; Reid, R.J. 2009. The involvement of aquaglyceroporins in transport of boron in barley roots. Plant Cell Environment 32: 1357-1365. ; Pang et al., 2010Pang, Y.Q.; Li, L.J.; Ren, F.; Lu, P.L.; Wei, P.C.; Cai, J.H. 2010. Overexpression of the tonoplast aquaporin AtTIP5 ; 1 conferred tolerance to boron toxicity in Arabidopsis . Journal of Genetics Genomics 37: 389-397. ). In this study, it was also found that the expression levels of three genes matched previous reports, including the higher expression of TIP2 ; 2 , NIP5 ; 1 and PIP1 ; 3 in the trifoliate orange root.

Taken together, all these results described above demonstrated that our hydroponic system delivered a more than satisfactory performance for studying plant responses to B-deficiency (or other nutrient deficiency or toxicity) at the molecular level.

Conclusions

In this study we have described, step by step, a protocol for setting up a simple, quickly and cheaply constructed hydroponic system which has standardized growth conditions for growing trifoliate orange and other citrus plants. Quality and versatility of our hydroponic system are demonstrated by profiling and comparing with soil-grown trifoliate orange seedlings, including biomass, ionomics, and root morphology. The results showed that our hydroponic system is best suited to the growth of trifoliate orange seedling. In order to test the performance of our hydroponic system on mineral nutrient deficiency, Fe- and B-deficiency treatment experiments were carried out on trifoliate orange seedlings, respectively. Moreover, to further demonstrate the application of our hydroponic system on the molecular level, transcriptional analysis was also carried out on the root of trifoliate orange under B-deficiency conditions. According to our results and previous reports, our hydroponic system performed better in terms of a physiological and molecular analysis of trifoliate orange. In conclusion, the system should be suitable for many experimental purposes, but especially for root morphologic and nutritional analysis of trifoliate orange, and it can also be adapted to other citrus plants by varying the device number and (or) size.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (N°.31501717 and 31701871), and Key Research and Development Project of Jiangxi Provincial (N°. 20161BBF60069).

References

  • Alatorre-Cobos, F.; Calderón-Vázquez, C.; Ibarra-Laclette, E.; Yong-Villalobos, L.; Pérez-Torres, C.A.; Oropeza-Aburto, A.; Méndez-Bravo, A.; González-Morales, S.I.; Gutiérrez-Alanís, D.; Chacón-López, A.; Peña-Ocaña, B.A.; Herrera-Estrella, L. 2014. An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions. BMC Plant Biology 14: 69.
  • Battke, F.; Schramel, P.; Ernst, D. 2003. A novel method for in vitro culture of plants: cultivation of barley in a floating hydroponic system. Plant Molecular Biology Reporter 21: 405-409.
  • Camacho-Cristóbal, J.J.; Herrera-Rodríguez, M.B.; Beato, V.M.; Rexach, J.; Navarro-Gochicoa, M.T.; Maldonado, J.M. 2008. The expression of several cell wall-related genes in Arabidopsis roots is down-regulated under boron deficiency. Environmental and Experimental Botany 63: 351-358.
  • Cao, X.; Chen, C.L.; Zhang, D.J.; Shu, B.; Xiao, J.; Xia, R.X. 2013. Influence of nutrient deficiency on root architecture and root hair morphology of trifoliate orange ( Poncirus trifoliata L. Raf.) seedlings under sand culture. Scientia Horticulturae 162: 100-105.
  • Chen, L.S.; Han, S.; Qi, Y.P.; Yang, L.T. 2012. Boron stresses and tolerance in citrus. African Journal of Biotechnology 11: 5961-5969.
  • Conn, S.J.; Hocking, B.; Dayod, M.; Xu, B.; Athman, A.; Henderson, S.; Aukett, L.; Conn, V.; Shearer, M.K.; Fuentes, S.; Tyerman, S.D. 2013. Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Methods 9: 4.
  • de Dorlodot, S.; Forster, B.; Pagès, L.; Price, A.; Tuberosa, R.; Draye, X. 2007. Root system architecture: opportunities and constraints for genetic improvement of crops. Trends in Plant Science 12: 474-481.
  • Farrokhi, N.; Burton, R.A.; Brownfield, L.; Hrmova, M.; Wilson, S.M.; Bacic, A. 2006. Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes. Plant Biotechnology Journal 4: 145-167.
  • Fitzpatrick, K.L.; Reid, R.J. 2009. The involvement of aquaglyceroporins in transport of boron in barley roots. Plant Cell Environment 32: 1357-1365.
  • Forner-Giner, M.A.; Llosá, M.J.; Carrasco, J.L.; Perez-Amador, M.A.; Navarro, L.; Ancillo, G. 2010. Differential gene expression analysis provides new insights into the molecular basis of iron deficiency stress response in the citrus rootstock poncirus trifoliata (L.) Raf. Journal of Experimental Botany 61: 483-490.
  • Graves, W.R. 1992. Influence of hydroponic culture method on morphology and hydraulic conductivity of root of honey locust. Tree Physiology 11: 205-211.
  • Gregory, P.J. 2006. Plant, Root and the Soil. Blackwell, Oxford, UK.
  • Guo, Y.; Guo, H.Y.; Zhang, L.; Xie, H.Y.; Zhao, X.; Wang, F.X. 2005. Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. Journal of Virology 79: 14392-14403.
  • Han, J.; Zhou, G.F.; Li, Q.H.; Liu, Y.Z.; Peng, S.A. 2012. Effects of magnesium, iron, boron deficiency on the growth and nutrition absorption of four major citrus rootstocks. Acta Horticulturae Sinica 39: 2105-2112.
  • Han, S.; Chen, L.S.; Jiang, H.X.; Smith, B.R.; Yang, L.T.; Xie, C.Y. 2008. Boron deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings. Journal of Plant Physiology 165: 1331-1341.
  • Hershey, D.R. 1994. Solution culture hydroponics: history and inexpensive equipment. American Biology Teacher 56: 111-118.
  • Hoagland, D.R.; Arnon, D.I. 1950. The water-culture method for growing plants without soil. California Agricultural Experiment Station, Davis, CA, USA. (Circular. California Agricultural Experiment Station, 347).
  • Kim, D.W.; Rakwal, R.; Agrawal, G.K.; Jung, Y.H.; Shibato, J.; Jwa, N.S.; Iwahashi, Y.; Iwahashi, H.; Kim, D.H.; Shim, I.S.; Usui, K. 2005. A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf. Electrophoresis 26: 4521-4539.
  • Kratky, B.A. 1993. A capillary, noncirculating hydroponic method for leaf and semi-head lettuce. HortTechnology 3: 206-207.
  • Liu, G.D.; Wang, R.D.; Wu, L.S.; Peng, S.A.; Wang, Y.H.; Jiang, C.C. 2012. Boron distribution and mobility in navel orange grafted on citrange and trifoliate orange. Plant and Soil 360: 123-133.
  • Liu, Y.Z.; Li, S.; Yang, C.Q.; Peng, S.A. 2013. Effects of boron-deficiency on anatomical structures in the leaf main vein and fruit mesocarp of pummelo [ Citrus grandis (L.) Osbeck]. Journal of Horticultural Science and Biotechnology 88: 693-700.
  • Mairhofer, S.; Zappala, S.; Tracy, S.; Sturrock, C.; Bennett, M.J.; Mooney, S.J.; Pridmore, T.P. 2013. Recovering complete plant root system architectures from soil via X-ray μ-Computed Tomography. Plant Methods 9: 8.
  • Maurel, C. 2007. Plant aquaporins: novel functions and regulation properties. FEBS Letters 581: 2227-2236.
  • Mei, L.; Sheng, O.; Peng, S.A.; Zhou, G.F.; Wei, Q.J.; Li, Q.H. 2011. Growth, root morphology and boron uptake by citrus rootstock seedlings differing in boron-deficiency responses. Scientia Horticulturae 129: 426-432.
  • O’Neill, M.A.; Ishii, T.; Albersheim, P.; Darvill, A.G. 2004. Rhamnogalacturonan. II. structure and function of a borate cross-linked cell wall pectic polysaccharide. Annual Review of Plant Biology 55: 109-139.
  • Pang, Y.Q.; Li, L.J.; Ren, F.; Lu, P.L.; Wei, P.C.; Cai, J.H. 2010. Overexpression of the tonoplast aquaporin AtTIP5 ; 1 conferred tolerance to boron toxicity in Arabidopsis . Journal of Genetics Genomics 37: 389-397.
  • Peng, L.Z.; Zhang, G.Y.; Chun, C.P. 2010. Study on the relationship between leaf yellowing with vein swelling or cracking and Mg, B element concentrations in Newhall Navel Orange leaves. South China Fruit 39: 1-5.
  • Postma, J.A.; Lynch, J.P. 2011. Root cortical aerenchyma enhances the growth of maize on soils with suboptimal availability of nitrogen, phosphorus, and potassium. Plant Physiology 156: 1190-1201.
  • Shorrocks, V.M. 1997. The occurrence and correction of boron deficiency. Plant Soil 193: 121-148.
  • Smit, A.L.; Bengough, A.G.; Engels, C.; van Noordwijk, M.; Pellerin, S.; van de Geijn, S.C. 2000. Root Methods: A Handbook. Springer, Berlin, Germany.
  • Stefanelli, D.; Jaeger, J.; Jones, R. 2013. A new method for hydroponic tomato production. Practical Hydroponics Greenhouses 129: 25.
  • Storey, R.; Treeby, M.T. 2000. Nutrient uptake into navel orange during fruit development. Journal of Horticultural Science Biotechnology 77: 91-99.
  • Takano, J.; Wada, M.; Ludewig, U.; Schaaf, G.; Von Wirén, N.; Fujiwara, T. 2006. The Arabidopsis major intrinsic protein NIP5;1 is essential for efficient boron uptake and plant development under boron limitation. Plant Cell 18: 1498-1509.
  • Tanaka, M.; Wallace, I.S.; Takano, J.; Roberts, D.M.; Fujiwara, T. 2008. NIP6;1 is a boric acid channel for preferential transport of boron to growing shoot tissues in Arabidopsis . Plant Cell 20: 2860-2875.
  • Warington, K. 1923. The effect of boric acid and borax on the broad bean and certain other plants. Annals of Botany os-37: 629-672.
  • Wei, Q.J.; Liu, Y.Z.; Sheng, O.; An, J.C.; Zhou, G.F.; Peng, S.A. 2013. Overexpression of CsCLCc , a chloride channel gene from Poncirus trifoliata , enhances salt tolerance in Arabidopsis . Plant Molecular Biology Reporter 31: 1548-1557.
  • Wu, Q.S.; Xia, R.X. 2006. Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. Plant Physiology 163: 417-425.
  • Zhou, G.F.; Peng, S.A.; Liu, Y.Z.; Wei, Q.J.; Han, J.; Islam, M.Z. 2014. The physiological and nutritional responses of seven different citrus rootstock seedlings to boron deficiency. Trees 28: 295-307.
  • Zhou, G.F.; Liu, Y.Z.; Sheng, O.; Wei, Q.J.; Yang, C.Q.; Peng, S.A. 2015. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray. Frontiers in Plant Science 5: 795.

  • Edited by: Mohammad Bagher Hassanpouraghdam

Publication Dates

  • Publication in this collection
    04 Nov 2019
  • Date of issue
    2020

History

  • Received
    26 Sept 2018
  • Accepted
    20 Dec 2018
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