African Journal of Biotechnology Vol. 11(11), pp. 2642-2653, 7 February, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2542 ISSN 1684–5315 © 2012 Academic Journals
Full Length Research Paper
Differential responses of Chiltepin (Capsicum annuum var. glabriusculum) and Poblano (Capsicum annuum var. annuum) hot peppers to salinity at the plantlet stage Raúl López-Aguilar, Diana Medina-Hernández, Felipe Ascencio-Valle, Enrique Troyo-Dieguez, Alejandra Nieto-Garibay, Mario Arce-Montoya, Juan A. Larrinaga-Mayoral and Gracia A. Gómez-Anduro* Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico. Accepted 31 October, 2011
Poblano (Capsicum annuum var. annuum) and Chiltepin pepper (Capsicum annuum var. glabriusculum) are considered closely related parent species. Poblano pepper is a cultivated species that has lost stress tolerance attributes, and Chiltepin is a wild species adapted to adverse environmental conditions, such as salinity stress. Differential physiological responses between the wild and cultivated species were compared in order to study the physiological and biochemical mechanisms of Chiltepin’s salinity tolerance. Salt stress treatments were applied to Chiltepin and Poblano and their growth, chlorophyll content, transpiration rate, and concentrations of anions and cations in leaves, stem and roots were measured. Dry weight and relative growth index decreased significantly with salt stress in Poblano, chlorophyll-a decreased significantly in both peppers and transpiration decreased in + – Chiltepin, with no significant changes evidenced by Poblano. Concentrations of Na and Cl increased + in stems and roots, but K declined in stems for both peppers, evidencing changes proportionally associated to the salt treatments. The accumulation of Na+ increased and the ratios K+/Na+, Ca2+/Na+, and Mg2+/Na+ decreased at all concentrations of NaCl. Sulfate, nitrate and phosphorus did not show significant differences in both species. We confirm that Chiltepin possessed salinity tolerance and also was physiologically more tolerant to salinity than Poblano. Key words: Salt stress, Capsicum, physiological traits, ion uptake, peppers.
INTRODUCTION Nearly 20% of all irrigated agricultural lands all over the world are affected by salinity (FAO 2010). Salt accumulation within the agricultural soil depth limits germination, plant growth, and completion of the life cycle of crops, as it affects several physiological and metabolic processes. High concentrations of NaCl cause ion imbalance and osmotic stress in most plant species (Maggio et al., 2000), such as increased respiration rate
*Corresponding author. E-mail:
[email protected]. Tel: +52 (612) 123-8484. Fax: +52 (612) 123-8525.
and ion toxicity, changes in plant growth, mineral distribution, membrane instability which originated from + calcium displacement by Na , and decreased efficiency of photosynthesis (Muhammad et al., 2011). Specifically, in Capsicum annum L. concentrations from 100 to 200 mM NaCl caused a reduction of vegetative growth parameters and photosynthetic pigment (Chookhampaeng, 2011). It is well known that there are differences among species in their tolerance to salinity and that these differences evolved among cultivars or varieties of a single species (Niu et al., 2010). Wild populations of sweet and hot pepper (C. annuum L.) have large genetic variation and are a valuable genetic resource that must
Lopez-Aguilar et al.
be conserved and used in breeding programs (Hernández-Verdugo et al., 2001). Many studies of comparative responses to salt stress of wild and domesticated species have been done, mainly in grasses and grains (Colmer et al., 2006), whereas only a few have been conducted on vegetables. Tomato is one of the most studied plants; its relative wild species (Solanum lycopersicum, S. esculentum, S. pimpinellifolium, S. cheesmanii, S. chilense, S. peruvianum), among others, have been studied as genetic resources that could be used to increase salt tolerance in domesticated tomato varieties (Cruz et al., 1990; Hajer et al., 2006). Peppers possess a high agricultural, economic and commercial importance for African, Asian and LatinAmerican countries. Poblano peppers (C. annuum var. annuum L.) are a cultivated species moderately sensitive to salt stress (Rhoades et al., 1992); their growth is noticeably retarded by accumulation of Na+ and Cl– at 60 mM NaCl (Silva et al., 2008), and photosynthetic pigments are decreased by 100 to 200 mM NaCl (Chookhampaeng, 2011). Chiltepin pepper (C. annuum var. glabriusculum) (Pickersgill, 1984) is considered a closely related parent species of Poblano pepper. It is well known by farmers of several countries that Chiltepin varieties or ecotypes are adapted to stressed ecosystems (Votava et al., 2002) and they have high genetic variability (Hernández-Verdugo et al., 2001). In this study, we compared the responses of the wild Chiltepin and the cultivated Poblano varieties that noticeably differ in several physiological and biochemical features when cultivated under saline and non-saline conditions and investigated the physiological mechanisms of Chiltepin salinity tolerance. MATERIALS AND METHODS
2643
appearance of each variety were chosen for the saline treatments and separated in four replicates for each treatment. After one week, different concentrations of NaCl were added (0, 50, 100, 200, and 300 mM NaCl using Hoagland’s solution) for 30 days in order to know both pepper species responses to several NaCl treatments. Each pot was irrigated daily using 100 ml of solution. A total of twenty plants (four plants by treatment) were used to assess morphometric variables and transpiration and chlorophyll. After measurements, plants were dried to obtain dry weight and mineral analysis.
Measuring growth Plants were harvested after 30 days of treatment; they were washed for 3 min with distilled water to remove all peat moss particles and were dissected into leaves, stems and roots. The number of leaves, length of stems and roots of each plant were recorded. Leaf area was measured with a foliar area meter (LI3000, LI-COR Biosciences). However, leaf area index (LAI) was defined as leaf area per unit area of land (LAI=LA/P), where P was used to represent the land area, and LA was used to represent the total leaf area above the land area (Breuer et al., 2003). It was measure as the average of the mayor and minor diameter of the projected shadow over the soil surface on which plant grows (fallen leaves not included). Leaves, stems and roots of each plant were weighed using an analytical balance to obtain fresh weight of vegetative parts per each individual plant. Subsequently, vegetative parts were separately placed in paper bags and dried at 80°C for 72 h in a forced hot air flow oven, or until reaching a constant weight, in order to obtain dry weight. The dried material was pulverized for a mineral analysis later.
Measuring chlorophyll Chlorophyll concentration was measured in four plants from each treatment. Samples were taken from fully expanded leaves (leaf number 4 from the top) and extraction was performed by 10 min with 80% (v/v) acetone. The extract was filtered and the absorbance was measured with a spectrophotometer at 645 and 665 nm (U-1100, Hitachi). Concentrations of chlorophyll-a and chlorophyll-b were estimated according to Arnon (1949).
Plant material Measuring transpiration Two Capsicum species widely grown in semi-arid and subtropical zones of Mexico and other Latin American countries were chosen for this study; Poblano (C. annuum var. annuum L.) and Chiltepin hot peppers (C. annuum var. glabriusculum). Poblano seeds were from Japan Sakata Seed Company, and Chiltepin seeds were local varieties, gotten from Etchojoa, Sonora, México.
Growth conditions and salt treatments Seeds of both varieties were disinfected with a 5% sodium hypochlorite solution for 5 min and soaked 3 h in deionized water. 25 seeds of each variety were placed in Petri dishes with cotton saturated with deionized water as a substrate for germination. Seeded Petri dishes were placed in a germination chamber at 25°C at 70% RH, with a 12-h photoperiod. After three weeks of germination, Poblano and Chiltepin were transferred into plastic pots (26 cm high, 14.5 cm bottom diameter, 17.5 cm top diameter) filled with peat moss (Sun Gro Horticulture). Plants were irrigated daily for five weeks with 100% Hoagland’s solution (Hoagland and Arnon, 1938), and 20 plants of uniform size and homogeneous
Two measurements were performed using plants of 9 weeks after germination. The first was done after 23 days of initial salt stress and the second was done in day 30. The internal variation is considered in the statistical analysis of Figure 3. Transpiration was measured in µg cm2 s1 with a steady state porometer (LI-1600, LICOR Biosciences Li-Cor, Inc., Lincoln, Nebraska, USA). Measurements were performed between 10:00 and 11:00 h on the third fully expanded leaf of each plant (counting from the plant’s growth point); although the average leaf temperature was 29.8°C, with an instant simultaneous radiation of 400 µE s -1m-1.
Ion analysis of tissues Cations were extracted using acid solution of H2SO4, HNO3 and HClO4 at a ratio of 1:10:4 (v/v). Ca, Mg, K, and Na concentrations in the extract were determined by atomic absorption spectroscopy (AA-660, Shimadzu), and phosphorus was quantified with the molybdenum blue method. Anions of Cl-, NO3, and SO4 were extracted with boiling water and their concentrations
2644
Afr. J. Biotechnol.
Table 1. Effects of increasing NaCl concentration on different growth parameters of two plant parts. The values are means ± SD of 4 replicates.
Species
NaCl (mM)
Length (cm plant-1)
Poblano
0 50 100 200 300
Root 35 ± 3.2c 29 ± 0.5b b 29 ± 1.1 30 ± 0.6b a 15 ± 0.8
Stem 45 ± 2.6b 44 ± 4.0b b 42 ± 5.5 36 ± 4.4a a 36 ± 3.6
a
Chiltepin
0 50 100 200 300
34 ± 1.4 41 ± 2.7b b 42 ± 3.7 ab 39 ± 4.0 c 47 ± 2.3
21 ± 6.8 23 ± 4.7a a 22 ± 2.3 22 ± 4.1a a 22 ± 1.5
a
Leaves number
Total leaf area 2 -1 (cm plant )
Leaf area index
54 ± 8c 47 ± 11bc b 33 ± 8 ab 33 ± 13 a 26 ± 12
226 ± 149a 336 ± 102a a 347 ± 78 293 ± 162a a 219 ± 131
1.67 ± 0.51b 1.73 ± 0.39b b 1.46 ± 0.81 ab 1.09 ± 0.36 a 0.54 ± 0.45
a
185 ± 69 240 ± 118ab bc 292 ± 89 ab 207 ± 158 ab 226 ± 149
a
0.92 ± 0.34 1.19 ± 0.59a a 1.45 ± 0.44 1.03 ± 0.78a a 1.12 ± 0.74
135 ± 32 114 ± 19a a 124 ± 40 a 89 ± 12 a 108 ± 30
a
Different letters in each column indicate significant differences at P < 0.05, according to Tukey’s test.
Table 2. Effect of salinity induced by NaCl on relative growth index (RGI) of dry weight of plant parts. Values are the average of 4 replicates.
Species
0 Poblano
Chiltepin
RGI
NaCl (mM) Root
Stem
c
b
100
100
ab
b
b
100
50 100 200 300
80 73ab 42a 35a
0
100
50 100 200 300
128a 140a 94a a 122
a
Leaf b
91 75ab 48a 37a
75 87b 70b 26a
100
a
100
118a 197a 133a a 161
132a 196a 228a a 178
a
Different letters in each column indicate significant differences at P < 0.05, according to Tukey’s test. RGI= Weight in saline treatment *100/weight in control (0 mM NaCl).
were determined by ion chromatography (HIC-6A, Shimadzu).
Experimental design and statistical analyses The experimental design was completely random with four independent replicates per treatment. For this purpose, one replicate was considered a pot containing one plant. The data obtained for each variable was analyzed through one-way ANOVA, considering NaCl-salinity as the main source of variance. Tukey’s test was used to compare means when the F-test was significant at P < 0.05. Regression and correlation analyses were performed in order to find the numerical tendency of the dry matter as affected by salinity (NaCl, mM), as the independent variable. Statistical procedures were carried out using the computer software Statistica 6.0 (StatSoft).
RESULTS Growth responses Salt stress effects on growth-related parameters of both Capsicum sp. are shown in Table 1. For Poblano pepper, the values of the evaluated parameters decreased significantly (P < 0.05) with salt stress, starting mainly from 200 mM NaCl, with the exception of total leaf area. On the contrary, for Chiltepin pepper treated with NaCl, no significant changes were observed in stem length, leaf number, leaf area index (Table 1), and relative growth index (Table 2) although it recorded higher values than Poblano pepper in root length and leaf area.
Lopez-Aguilar et al.
2645
Figure 1. Variations in dry weight of leaves, stem and root of Poblano and Chiltepin peppers exposed to salt stress induced by NaCl for 30 days. Values are the average of 4 replicates ± S.D. Different letters indicate significant differences from each other (p
