Journal of Horticultural Science & Biotechnology (2006) 81 (4) 583–592
Effect of nursery irrigation regimes on vegetative growth and root development of Silene vulgaris after transplantation into semi-arid conditions By J. ARREOLA1,2, J. A. FRANCO1,3*, M. J. VICENTE1 and J. J. MARTÍNEZ-SÁNCHEZ1,3 1 Departamento de Producción Vegetal, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena, Paseo Alfonso XIII, 48, 30203 Cartagena, Spain 2 Colegio de Postgraduados, Campus Campeche, Nicaragua, 91, 3ª Planta, Col. Sta. Ana, 24050 Campeche, México 3 Unidad Asociada al CSIC de “Horticultura Sostenible en Zonas Áridas” (UPCT-CEBAS), Paseo Alfonso XIII, 52, 30203 Cartagena, Spain (e-mail:
[email protected]) (Accepted 16 February 2006) SUMMARY The influence of irrigation regimes during nursery production of seedlings on development of the aerial parts and root system after being transplanted into semi-arid conditions was investigated in Silene vulgaris (Moench.) Garcke. This Mediterranean native herb is of interest for phytoremediation of contaminated soils and for edible and medicinal uses. During the 35 d nursery period, seedlings were grown in polystyrene trays. Three irrigation treatments were used throughout the nursery period: WI, well-irrigated seedlings; MS, moderately-stressed seedlings; and HS, highly-stressed seedlings. In all treatments, seedlings were overhead irrigated on 2 d per week, and the total amounts of water applied (per tray of 176 seedlings) over the whole nursery period were: WI, 16.10 l; MS, 10.75 l; and HS, 4.50 l. After the nursery period, WI and MS plants were transplanted into transparent containers (cylindrical acrylic tubes, 8 cm in diameter and 100 cm tall) and into the field, where mini-rhizotrons were used to evaluate root system dynamics. Plants transplanted into containers were watered with two irrigation regimes: normal irrigation (NI) and deficit irrigation (DI), with 0.90 or 0.45 l plant–1 week–1, respectively. All plants transplanted into the field were watered equally, with 1.16 l plant–1 week–1. Post-transplantation growth of aerial parts and roots was studied over 120 d in the transparent containers and over 60 d in the field. MS and HS treatments during the nursery period produced seedlings that showed lower midday leaf water potential and greater root:aerial parts fresh weight (FW) ratios than the WI treatment. The MS treatment produced seedlings with the greatest length and FW of roots and with the highest quality. The HS treatment produced seedlings which were too small and over-hardened. The latter were therefore not used for posttransplantation experiments. After transplantation into transparent containers, MS seedling-derived plants showed greater root growth than WI-derived plants, especially when the water content of the substrate was low (DI treatment). Also, mini-rhizotrons allowed observation of more active root growth in MS seedling-derived plants than in WI seedling-derived plants after transplantation into the field, especially in the deepest layer of soil (50-75 cm). WIderived and MS-derived plants, under NI or DI post-transplantation treatments, showed similar FWs and dry weights (DWs) of their aerial parts, but MS-derived plants showed greater leaf:stem FW and DW ratios than WI-derived plants under DI conditions.
S
emi-arid environments tend to be sub-optimal with respect to one or more environmental parameters, such as water availability, temperature, air humidity, soil salinity or nutrient availability. Thus, transplantation from a nursery into orchards or landscape settings creates a stressful transition period that is critical to the future establishment, performance and survival of seedlings produced in the nursery. The environmental conditions and cultivation techniques used in the nursery are crucial for the establishment of seedlings and their subsequent growth. The goal of seedling pre-conditioning is to produce sturdy plants that have a high level of photosynthetic reserves, adequate morphological and physiological *Author for correspondence.
characteristics, and are capable of rapid establishment and resumption of growth following transplantation (Franco et al., 2001; 2002a, b; Bañón et al., 2002; 2003b; 2004). The use of native species as food crops, or in xerogardening, landscaping, revegetation or phytoremediation of contaminated soils, is of increasing interest because of their ability to adapt to adverse environmental conditions (Vignolio et al., 2002; Clary et al., 2004; Sánchez-Blanco et al., 2004a; Bañón et al., 2006; Franco et al., 2006). Among these, Silene vulgaris, a perennial herb and member of the Caryophyllaceae, and a native of the Mediterranean coast, is of interest for edible uses, medicinal purposes and phytoremediation of soils contaminated with heavy metals (Launert, 1981; Chaney et al., 1997; Ernst and Nelissen, 2000; Arreola
Nursery irrigation of Silene vulgaris
c
-0.1
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Midday leaf water potential (MPa)
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5
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80
C
a
70
-0.6 -0.7
a WI MS HS
-0.8 -0.9
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Root length (cm)
0.0
MATERIALS AND METHODS Plant material and study site The experiment was carried out in the south-eastern part of Murcia Province (SE Spain) on the Mediterranean coast (37º 41' N; 0º 57' W). Native S. vulgaris (Moench.) Garcke was used. Seeds of S. vulgaris used in this study were provided by the germplasm bank of the Universidad Politécnica de Cartagena, registered as accession UPCT-01-313 (collected in Cartagena, Murcia; at 37º 41' 50'' N; 1º 05' 05'' W). These seeds were collected in January 2001 and stored at 4ºC until use. The climate of the area is typically Mediterranean, with mild Winters, low rainfall and very hot, dry Summers. Rainfall is characterised by extreme interannual variations, low overall value, and a tendency to fall as storms.
Plant height (cm)
et al., 2004). For culture under semi-arid conditions, as well as to increase the efficiency of phytoremediation, it is important that a plant shows rapid and vigorous root growth after transplantation. Furthermore, root morphology determines the ability of a plant to acquire soil resources (water and nutrients) or to remove heavy metals from contaminated soils (Chaney et al., 1997; Franco et al., 2006). It is known that irrigation practices throughout the nursery period affect some morphological and anatomical aspects related to the hardening of seedlings, and to plant growth after transplantation (De Herralde et al., 1998; Franco et al., 2001; Sánchez-Blanco et al., 2004b). Among these aspects, the least studied is root development post-planting, undoubtedly due to limited accessibility for experimental observations (Franco and Abrisqueta, 1997; Franco et al., 2006). The influence of irrigation regimes, in the nursery, on root development after transplantation into containers has been studied extensively in native species (De Herralde et al., 1998; Franco et al., 2001). However, field studies of root system dynamics are particularly difficult because they require successive, non-destructive, measurements. Mini-rhizotrons allow direct, periodic observation of the root system (McMichael and Taylor, 1987; Franco and Abrisqueta, 1997; Franco and Leskovar, 2002; Franco et al., 2002a, b). The aim of this study was to ascertain the extent to which different nursery irrigation treatments influenced the development of S. vulgaris plants after transplantation into water-deficit conditions. Both the aerial parts of the plants, and the root system, were studied, the latter using transparent containers and mini-rhizotrons.
Number of leaves per plant
584
60 50
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40
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30 20
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Nursery period (weeks) FIG. 1 Dynamics of midday leaf water potential (md) of Silene vulgaris seedlings as affected by three irrigation regimes throughout the nursery period: WI (well-irrigated) seedlings, watered twice a week at 75% of the water-holding capacity (WHC); MS (moderately-stressed) seedlings, watered twice a week at 50% of the WHC; and HS (highly-stressed) seedlings, watered twice a week at 15% of the WHC. Vertical lines indicate ± SE of the means (n = 12). Mean values with a common lowercase letter, for each sampling time, are not significantly different (P < 0.05) based on the Tukey test.
10 0
WI
MS
HS
Nursery irrigation regime FIG . 2 Height (Panel A), number of leaves (Panel B) and root length (Panel C) of Silene vulgaris seedlings at the end of the nursery period (see nursery irrigation treatments in Figure 1). Vertical lines indicate SE of the means (n = 12). Mean values with a common lower-case letter are not significantly different (P < 0.05) based on the Tukey test.
J. ARREOLA, J. A. FRANCO, M. J. VICENTE and J. J. MARTÍNEZ-SÁNCHEZ Nursery conditions S. vulgaris seeds were sown manually on 18 January 2004 and, during the 35 d nursery period, the seedlings were grown in polystyrene trays (60 cm 41 cm 5.3 cm) with 176 cells of 26.4 cm3 of capacity (one seedling per cell). Three irrigation treatments were evaluated throughout the nursery period: WI (well-irrigated) seedling were watered twice a week at 75% of the waterholding capacity (WHC); MS (moderately-stressed) seedling were watered twice a week at 50% of the WHC; and HS (highly-stressed) seedling were watered twice a week at 15% of the WHC. In all three treatments, seedlings were overhead irrigated by hand from a watering can, and the total amounts of water applied (per tray of 176 seedlings) throughout the whole nursery period were: WI, 16.10 l; MS, 10.75 l; and HS, 4.50 l. The substrate used was a mixture of black peat and sand [1:1 (v/v)], and its main physical characteristics were as follows: bulk density, 850 kg m–3; total pore space, 66% (v/v); volume of air after irrigation, 18% (v/v); WHC, 4.5 g g–1 DW; and easily available water, 23% (v/v). Seedlings were grown in a metal-framed structure (2.8 m in height) covered with a plastic black shade screen (50% transmissivity). The following conditions
0.7 b
0.5 0.4 0.3 c
0.2
0.000
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FW ratio of roots:aerial parts
Aerial parts fresh weight (g)
Evaluation during the nursery period Measurements of midday leaf water potential (md) were made every week on leaves from the top third of the seedlings using a portable pressure chamber (Scholander et al., 1965). At the end of the nursery period, seedling height, the number of leaves per seedling, the fresh weights (FW) of both aerial parts and roots, and the FW ratio for roots:aerial parts were measured. Total root length was measured using a digital image analysis system WinRhizo LA 1600 (Regent Instruments Inc., Quebec, Canada). HS seedlings were not used in the following experiments (i.e., transplantation into transparent containers or into the field). Although they had good roots:aerial parts ratios, these plants were too small and had over-hardened. Transplantation into transparent containers After the nursery period, the WI and MS seedlings were transplanted into transparent containers (cylindrical acrylic tubes 8 cm in diameter, 100 cm tall, 5 l capacity) on 24 February 2004. The substrate used was a mixture of black peat and sand [1:1 (v/v)] with the same physical characteristics mentioned above in the section on nursery conditions. The aerial environment was the same as the field. Plants transplanted into containers were watered with two irrigation regimes: normal irrigation (NI) and deficit irrigation (DI), with 0.90 and 0.45 l plant–1 week–1, respectively.
0.1
Root fresh weight (g)
prevailed during the nursery phase: minimum temperature, 6° – 9ºC; maximum temperature, 13° – 17ºC; minimum relative humidity (RH), 48 – 64%; and maximum RH, 74 – 90%. Three replications of each irrigation treatment were planted with four trays per replication. The design was totally random. Measurements were made on 20 randomly selected plants per tray. A fresh sample was used on each measurement date.
A
a 0.8
0.6
585
a
0.07 a
0.06 0.05 0.04
b
0.03 0.02 0.01
a
WI
MS
HS
Nursery irrigation regime FIG . 3 Fresh weights (in g) of aerial parts (Panel A) and roots (Panel B) of Silene vulgaris seedlings at the end of the nursery period (see nursery irrigation treatments in Figure 1). Vertical lines indicate SE of the means (n = 12). Mean values with a common lower-case letter are not significantly different (P < 0.05) based on the Tukey test.
0.00
WI MS HS Nursery irrigation regime FIG . 4 Fresh weight (FW) ratios of roots:aerial parts of Silene vulgaris seedlings at the end of the nursery period (see nursery irrigation treatments in Figure 1). Vertical lines indicate SE of the means (n = 12). Mean values with a common lower-case letter are not significantly different (P < 0.05) based on the Tukey test.
Nursery irrigation of Silene vulgaris
586
Deficit irrigation
Normal irrigation
Plant height (cm)
25
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10 a 5
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Root length (m)
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16
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16
Weeks after transplantation
FIG . 5 Post-transplantation development of plant height and root length of Silene vulgaris plants grown in transparent containers under two irrigation treatments [normal irrigation, NI (Panels A and C) and deficit irrigation, DI (Panels B and D)] as influenced by irrigation regimes throughout the nursery period: WI (well-irrigated) seedlings, watered twice a week at 75% of the water-holding capacity (WHC); and MS (moderately-stressed) seedlings, watered twice a week at 50% of the WHC. Vertical lines indicate ± SE of the means (n = 9). Mean values with a common lower-case letter, within each panel and for each measurement time, are not significantly different (P < 0.05) based on the Tukey test.
Three replications of each treatment were planted with three plants per replication and one plant per container. The design was totally random. At the same time, additional containers with WI seedlings received the above-mentioned irrigation treatments in order to measure, periodically, the water content of the substrate. Evaluation of root development in transparent containers To study the dynamics of root growth, transparent containers were used. These were placed in opaque PVC containers to prevent light influencing root development and were removed only when measurements were to be taken through the transparent sides of the containers. Root length was measured at bi-weekly intervals over 120 d. Counting started on 11 March 2004 and finished on 10 July 2004. Evaluation of aerial parts in transparent containers Plant height and numbers of leaves per plant were measured every 2 weeks over 120 d. At the end of the experiment (10 July 2004), the FWs and dry weights (DWs) of the aerial parts, and leaf:stem FW and DW ratios were measured.
Transplantation into the field In a parallel experiment, WI and MS seedlings were transplanted into the field in order to study the effect on root development of the previous nursery irrigation treatments. The soil had a silty loam texture, and showed no significant variation throughout the depth studied (0 – 100 cm). Average analytical data for the total soil profile were: 24.4% clay, 35.9% silt and 34.7% sand. Water retention properties within the plant root zone were relatively uniform with depth. All plants transplanted into the field were irrigated identically, receiving the same volume of water at each irrigation (twice a week). The amount of water applied weekly was 1.16 l plant–1. Four replications of each nursery seedling treatment (WI and MS) were planted with a row of 15 plants per replication. The design was totally random. Evaluation of root development in the field with mini-rhizotrons During the growth of S. vulgaris in the field, root system development was studied using the minirhizotron technique (Franco and Abrisqueta, 1997).
Aerial parts FW (g)
50
Aerial parts DW (g)
7 6 5 4 3 2 1
Leaf:stem FW ratio
3.0
Leaf:stem DW ratio
J. ARREOLA, J. A. FRANCO, M. J. VICENTE and J. J. MARTÍNEZ-SÁNCHEZ
3.0
a
A
a
40 b
30
b
20 10
2.5
a
B a a a
C a
ab b
where RLD is the root length density (cm cm–3 soil), N is the number of roots observed in each tube section, A is the area of the tube outer wall in the given section (cm2), and d is the tube external diameter (cm). The tube diameter is retained in the equation for dimensional consistency.
1.5 1.0 0.5
2.5
a
D
b b
2.0
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1.5
tube-soil interface. The total length of buried tube was 87.5 cm, reaching a maximum depth of 75 cm. The mouth of the tube was closed with a rubber stopper. The part of the tube protruding from the soil surface was covered with a black plastic sheet, an insulating material and an opaque PVC cap to prevent light from entering the tube and to prevent it from becoming hot, both of which would have favoured the absence of roots around the tube near the soil surface (McMichael and Taylor, 1987; Franco and Abrisqueta, 1997). The tubes were installed on 20 February 2004, plants were transplanted on 24 February 2004, and root counting started on 10 March 2004. Roots were observed by means of a borescope (Model R-160-143-090-351; Olympus KeyMed Group, Southend-on-Sea, Essex, UK) equipped with a halogen light source (Model ILK-6A; Olympus Optical Co., Hamburg, Germany). Counting was carried out between 10 March and 25 April 2004 at bi-weekly intervals. Roots were counted in each 5 cm tube section. Data were then grouped for each of five sections, corresponding approximately to three layers in the soil: 0 – 25 cm, 25 – 50 cm and 50 – 75 cm. The length of root per unit of soil volume was obtained by applying the formula used by Upchurch and Ritchie (1983): RLD = N d A–1 d–1
ab
2.0
587
Evaluation of aerial parts in the field During growth of S. vulgaris, plant height and the numbers of leaves per plant were measured every 2 weeks. Measurements were taken of six randomly selected plants per replication. At the end of the experiment, the FWs and DWs of the aerial parts were also determined.
1.0 0.5 WI
MS
WI
MS
Normal irrigation Deficit irrigation FIG . 6 Fresh weights (FW) of aerial parts (Panel A), dry weights (DW) of aerial parts (Panel B), leaf:stem FW ratios (Panel C) and leaf:stem DW ratios (Panel D) of Silene vulgaris plants 16 weeks after transplantation into transparent containers under two irrigation treatments (normal irrigation and deficit irrigation) as influenced by irrigation regime throughout the previous nursery period (see nursery irrigation treatments in Figure 5). Vertical lines indicate SE of the means (n = 9). Mean values with a common lower-case letter are not significantly different (P < 0.05) based on the Tukey test.
The mini-rhizotron tubes were placed just under the plant rows. Cylindrical acrylic tubes, 1 m long, 80 mm in external diameter and 74 mm in internal diameter, were permanently installed at an angle of 66º to the soil surface to prevent abnormal growth of the roots at the
Measurement of water content In the transparent containers, changes in substrate water content with depth were determined by the gravimetric method three times per replication: after initial irrigation, 8 weeks after transplantation and 16 weeks after transplantation. Three replications were used in each determination. In the field, changes in soil water content with depth were determined by time domain reflectometry (Moisture Point, Model MP-917; Environmental Sensors Inc., Victoria, Canada). Soil water content was monitored every 25 cm, from 0 – 75 cm depth. Three replications were used. Statistical analysis All sets of data were subjected to ANOVA, and a Tukey test was used to check significance by using the SPSS Program, version 12.0. Figures were made with the help of SIGMA Plot 2000 and the standard errors of the means (SE) are represented.
588
Nursery irrigation of Silene vulgaris
Normal irrigation
Root length (cm)
2800
Deficit irrigation D 0-33 cm deep
A 0-33 cm deep
2400 WI MS
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FIG . 7 Root development in three layers of the soil profile [0-33 cm deep (Panels A and D), 33-66 cm deep (Panels B and E) and 66-100 cm deep (Panels C and F)] for Silene vulgaris plants grown in transparent containers under two irrigation treatments (normal irrigation and deficit irrigation) as influenced by irrigation regime throughout the previous nursery period (see nursery irrigation treatments in Figure 5). Vertical lines indicate ± SE of the means (n = 9). Mean values with a common lower-case letter, within each panel and for each measurement time, are not significantly different (P < 0.05) based on the Tukey test.
J. ARREOLA, J. A. FRANCO, M. J. VICENTE and J. J. MARTÍNEZ-SÁNCHEZ RESULTS Nursery period Irrigation regimes during nursery production had a considerable influence on md throughout the nursery period (Figure 1). Seedlings that underwent the WI treatment showed the highest md values, while seedlings that underwent the HS treatment showed the lowest values. There was a progressive decrease in md in all treatments over time. At the end of the nursery period, the height (Figure 2A) and number of leaves per seedling (Figure 2B) showed significant differences due to the effects of the irrigation regimes. Seedlings that underwent HS had a notable reduction in height and number of leaves. Seedlings produced under MS showed greater root lengths than the WI and HS seedlings (Figure 2C). The WI seedlings had the highest FW values for their aerial parts (Figure 3A), but root FWs were highest in the MS seedlings (Figure 3B). The HS seedlings showed notably lower values for both parameters than WI and MS seedlings (Figure 3). Root:aerial part FW ratios were greater in plants under water stress treatments (MS and HS), with no significant differences (Figure 4). Transparent containers Post-transplantation, the development of plant height was similar for the WI and MS seedling-derived plants, under both normal irrigation (NI; Figure 5A) as well as under deficit irrigation (DI; Figure 5B). During the first weeks, MS plants grew faster than WI plants. Four weeks
589
after transplantation under NI treatment, and 6 weeks under DI treatment, the heights of the seedling-derived MS plants equalled those of WI seedling-derived plants, which had been taller when transplanted (Figure 2A). After 16 weeks in the transparent containers, there were no longer any significant differences in the FWs (Figure 6A) or DWs (Figure 6B) of the aerial parts between treatments. However, post-transplantation, the NI treatment produced plants with more FW than the DI treatment (Figure 6A). Nursery irrigation regimes did not affect the leaf:stem FW (Figure 6C) and DW (Figure 6D) ratios of plants undergoing NI treatment. However these ratios were greater in MS plants than in WI plants undergoing DI treatment. Seedlings which underwent MS treatment throughout nursery production showed greater root growth after transplantation into transparent containers than those which underwent WI treatment (Figure 5C, D). There were larger differences under deficit irrigation conditions (DI treatment). The MS plants showed greater root elongation under DI treatment (Figure 5D) than under NI treatment (Figure 5C). The greatest root growth occurred in the intermediate soil layer (33 – 66 cm deep; Figure 7B, E), which is the layer that maintained greater humidity throughout the experiment (Figure 8). The greatest differences between MS and WI seedling-derived plants occurred in the deepest layer of the substrate (66 – 100 cm) when DI treatment was used (Figure 7F). In this layer, low substrate water content was observed throughout the experiment (Figure 8).
Volumetric water content (cm3 cm-3) 0.05 0.10 0.15 0.20
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Depth (cm)
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FIG . 8 Soil volumetric water content between 10–100 cm depth in transparent containers at three different dates during the experimental period: Panel A, after initial irrigation; Panel B, 8 weeks after transplantation; and Panel C, 16 weeks after transplantation. Two irrigation regimes were used: normal irrigation (NI) and deficit irrigation (DI).
Nursery irrigation of Silene vulgaris
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a 14 a
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RLD (cm cm-3)
Plant height (cm)
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Weeks after transplantation FIG . 9 Post-transplantation development of plant height in Silene vulgaris plants grown in the field as influenced by irrigation regime throughout the nursery period (see nursery irrigation treatments in Figure 5). Vertical lines indicate ± SE of the means (n = 4). For each time point, mean values with a common lower-case letter are not significantly different (P < 0.05) based on the Tukey test.
Field period Soil water content after transplantation was kept between 0.06 – 0.17 cm3 cm–3, values similar to those which were maintained during the NI treatment in the experiment in transparent containers. Furthermore, the lowest humidity values occurred in the top 25 cm, and in the deepest layer (50 – 75 cm) of the soil profile. Both WI and MS seedling-derived plants rapidly reached the same height following transplantation into the field (Figure 9). Both also showed similar numbers of leaves per plant and FWs and DWs of aerial parts throughout the experiment (data not shown). MS plants had more root growth following transplantation into the field than WI plants (Figure 10). Eight weeks after transplantation, MS plants had approx. double the RLD in the lower soil layers (Figure 10B, C) than WI plants.
DISCUSSION During nursery production of S. vulgaris, irrigation treatment notably affected seedling characteristics. The MS treatment produced the most robust seedlings, with the greatest root length (Figure 2C) and root FW (Figure 3B). These seedlings had roots:aerial parts FW ratios (Figure 4) almost double those of seedlings obtained with the WI treatment. Several authors have determined that deficit irrigation during nursery production influenced the growth of the aerial parts and roots (Hipps et al., 1996; Bañón et al., 2002; 2003a; 2004; 2006; Sánchez-Blanco et al., 2002; 2004a; Cameron et al., 2004). Different studies have also demonstrated that an increment in the root:shoot ratio (caused either by reducing the shoot growth rate, or by increasing root growth relative to shoot growth) as a result of the hardening and acclimation processes during the nursery period (i.e., pre-conditioning), increases plant growth and reduces the mortality rate after transplantation under semi-arid conditions (Fernández et al., 2004; 2006; Franco et al., 2006).
RLD (cm cm-3)
4
RLD (cm cm-3)
2
A 0-25 cm deep
WI MS
0.25 0.20 0.15 0.10 0.05
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B 25-50 cm deep
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Weeks after transplantation FIG . 10 Post-transplantation development of root length density (RLD) in three layers of the soil profile [0-25 cm deep (Panel A), 25-50 cm deep (Panel B) and 50-75 cm deep (Panel C)] of Silene vulgaris plants grown in the field as influenced by irrigation regime throughout the previous nursery period (see nursery irrigation treatments in Figure 5). Vertical lines indicate ± SE of the means (n = 4). Means with a common lower-case letter, within each panel and for each time of measurement, are not significantly different (P < 0.05) based on the Tukey test.
The greater root-length of the MS seedlings can have a considerable effect in minimising localised water depletion around roots, thus minimising resistance to water transport to the roots (Franco et al., 2006). On the other hand, the greater proportion of roots in nurserystressed seedlings, compared to the well-irrigated seedlings, would favour an accumulation of solutes for osmotic adjustment in the roots to enable them to maintain the gradient necessary to absorb water, even when in short supply in the soil (Leskovar and Stoffella, 1995; Franco et al., 2001; 2002a, b; Bañón et al., 2006). With HS treatment, seedlings were obtained with the same roots:aerial parts FW ratios as obtained using the MS treatment. However, these seedlings were too poor
J. ARREOLA, J. A. FRANCO, M. J. VICENTE and J. J. MARTÍNEZ-SÁNCHEZ in quality to be transplanted. They were too small (Figure 3A) and had under-developed root systems (Figure 3B) due to over-hardening caused by the water stress being too severe, as their md values showed throughout the nursery period (Figure 1); values much lower than those of WI and MS seedlings. The severity of any water restriction during nursery production is critical. In general, seedlings growing under very high or very low moisture conditions are affected adversely, while those growing under moderate moisture conditions exhibit optimum growth. A desirable level of deficit irrigation results in stocky, stress-resistant seedlings (Franco et al., 2006); but, if the water restriction is too severe, seedlings die or are over-hardened, slowing new shoot and root growth (Liptay et al., 1998). In the present study, after transplantation into transparent containers (Figure 5) or into the field (Figure 9), MS seedling-derived plants, that had been smaller than WI plants at the time of transplantation (Figure 2A), showed more rapid vegetative growth and equalled the WI plants in height within a few weeks. Similarly, Hipps et al. (1996) and Franco et al. (2002b) found that, after transplantation, shoot growth of seedlings that had previously received a high rate of irrigation was less than in those that had received a low rate. When DI treatment was employed after transplantation, leaf:stem FW (Figure 6C) and DW (Figure 6D) ratios were greater for MS plants than for WI plants. This fact is notable as leaves are the edible part of the S. vulgaris plant. MS seedling-derived plants had greater posttransplanting root growth (Figure 5; Figure 10) than WI plants, especially when the soil moisture level was low. At the end of the experiments there were important differences in the deepest soil layer, both in the transparent containers under DI (Figure 7F) and in the field (Figure 10 C). Franco et al. (1999; 2001; 2002a, b) have observed that water-stressed plants in the nursery showed greater and more rapid root growth than wellirrigated plants following transplantation, particularly when the soil water content was low. It is known that root activity depends strongly on soil water content. Soil strength increases with decreasing soil moisture. As soil strength increases, the root elongation rate decreases due to increasing mechanical impedance. This effect is greater than the direct effect of the low matric potential (Clark et al., 2003). However, the present study found that root growth of pre-conditioned plants can be
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maintained, or even increased, under low soil water contents. Pritchard et al. (2000) and Neumann (2003) observed similar responses promoted by water stress. It should be pointed out that RLD alone cannot be considered a reliable morphological marker for drought tolerance (Vamerali et al., 2003) as its relationship with water uptake can become very tenuous at very low water potentials. Nevertheless, higher RLD does allow plants to take up water at a lower matric potential which may become an advantage for MS seedling-derived plants. In addition, higher RLD in deep soil layers must be useful to ensure water capture when water in the upper soil layers is exhausted. RLD values measured with the mini-rhizotrons in the top layer of soil (Figure 10A) were very low. Low topsoil root activity, induced by lack of moisture, should result in a lower uptake of topsoil nutrients, or heavy metals during phytoremediation of soils. In addition, roots in the upper soil layers may have an important role in capturing water efficiently after dry periods (Franco et al., 2002b). Nevertheless, the very low RLD values may have been due to the fact that mini-rhizotrons frequently tend to underestimate root measurements near to the surface of the soil (Franco and Abrisqueta, 1997; Machado and Oliveira, 2003). In conclusion, nursery irrigation regimes have a significant effect on the characteristics of seedlings. The application of a pre-conditioning water-stress treatment throughout nursery production can reduce the shock of transplantation, which is a key obstacle to plantation success under semi-arid conditions. Preconditioning affects post-transplanting vegetative growth and produces greater root growth, especially when soil moisture content is low. Greater and faster root growth is important for S. vulgaris during cultivation to allow greater nutrient and water uptake, and for phytoremediation when roots explore a greater volume of contaminated soil to remove heavy metals. This research was supported by the SENECA Foundation (PI-75/00819/FS/01), by CICYT-FEDER (AGL2000-0521) and by Consejería de Industria y Medio Ambiente de la Región de Murcia (2I04SU011). The authors gratefully acknowledge financial assistance to Jesús Arreola provided by the Pablo García Foundation (Government of Campeche State) and by the Colegio de Posgraduados, México.
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