Location

The study was carried out under greenhouse conditions in the drought resistance area of the Postgraduate College in Montecillo, Texcoco, State of Mexico (19° 29’ north latitude, 98° 53’ west longitude and altitude of 2 250 m).

Vegetal material

The experiment included 10 varieties of lucerne obtained from different commercial houses. Four groups of 100 seeds in each variety were chosen at random and germinated in Petri dishes with Whatman^® brand filter paper, under laboratory conditions (temperature of 20 °C). The germination percentage ranged from 95 to 98% for all varieties.

Experimental design and soil moisture treatments

A randomized complete block experimental design with four replications and two soil moisture treatments (irrigation and drought) was used; the blocks were rotated inside the greenhouse every week to reduce the effect of the microenvironment on the plants. The experimental unit was an individual plant grown in a 1m high and 4” diameter PVC tube, with a 1.1 m high cylindrical poly bag and the same diameter as the PVC tube.

The sowing was carried out in boxes for storage, on March 15, 2017, by placing five seeds of the same weight in each cell; when the seedlings were 20 days after sowing (dds), the most vigorous seedling in each cell was chosen to be transplanted into the PVC tubes.

The PVC tubes were filled with soil dried in the sun for two weeks; the soil used is of a sandy loam texture with an apparent density of 1.12 T m^-3, field capacity (CC) of 41.6% and percentage of permanent wilting (PMP) of 28.2% (Central University Laboratory, Autonomous University Chapingo, Texcco, Mexico, 2016). Fertilization was done 44 days after transplantation (ddt) with the formula 60-140-00, using urea and triple calcium superphosphate as sources of nitrogen and phosphorus; a second and third fertilization were done with the same formula at 240 and 420 ddt.

The soil moisture treatments were: irrigation, where the soil water content remained close to CC from the transplant date (20 dds) to 406 ddt (I₁) and from 406 ddt to the conclusion of the experiment (688 ddt) (I₂) and drought, where the application of water was suspended for a period of 61 days [345 to 406 ddt, March to May 2018; (D₁)] and a second 68-day period [620-688 ddt; November 2018 to February 2019; (D₂)].

Measured and calculated variables

The foliar water potential (ψ), osmotic (π) and turgidity (P), the photosynthesis rate (A), transpiration rate (E) and stomatal conductance (g), were measured at 345, 360, 377, 391 and 406 ddt in all plants in irrigation (I₁) and drought (D₁), and at 620, 635, 658, 673 and 688 ddt in I₂ and S₂.

ψ (MPa) was measured on a complete stem with the upper leaflets fully expanded and exposed to solar radiation, between 11:00 and 13:00 h, using the pressure pump or Scholander pump (^{Barrios-Gómez et al., 2011}).

The π (MPa) was measured in the samples of the leaflets used to measure the ψ; the leaflets were placed in 5 ml plastic microtubes, which were placed in a thermos with liquid nitrogen at a temperature of -195 °C for 5 minutes. The π was determined in a 100 µl aliquot of each sample, using a Typ 6M 10/25 µl model osmometer, Löser brand (Germany) in the laboratory. The P (MPa) was calculated as the difference between the potentials ψ and π (p= ψ - π), for each sample analyzed (^{Begg and Turner, 1976}). The osmotic adjustment (OA, MPa) of the plants was calculated as the difference between π determined in irrigation (π_I) and π measured in drought (π_D), for each variety (OA = π_I - π_D) ^{Morgan (1995)}.

The net photosynthesis rate (A, µmol CO₂ m^-2 s^-1), the transpiration rate (E, mmol H₂O m^-2 s^-1) and the stomatal conductance (g_i, mol H₂O m^-2 s^-1) were measured by placing a fully expanded sheet inside the assimilation chamber of the infrared gas analyzer (IRGA, model LI-6400, LI-COR Inc; Lincoln, Nebraska, USA), configured for an open system, where measurements they are based on the differences in the concentration of CO₂ and H₂O in the leaf and the concentration of CO₂ and H₂O in a stream of air flowing through the leaf. The transpiratory efficiency or efficiency in the use of the instantaneous water of the entire plant, measured by the gas exchange (W, mol CO₂ mol H₂O^-1) was calculated by dividing A by E (W= A/E) (^{Farquhar, 1991}).

Temperature data

The maximum and minimum daily temperature (°C) of the air inside the greenhouse was measured with a maximum and mine mercury column thermometer, Taylor brand (England). The maximum and minimum mean temperatures recorded in the greenhouse during the entire experiment were 31.9 and 8.4 °C, respectively.

Moisture content in the soil

The soil water content was determined by the gravimetric method every third day with a Tor-Rey brand electronic scale, PCR Series model, adding the amount of evapotranspirated water in each experimental unit, to keep the soil moisture content close to CC in all the plants, except during the drought treatments (D₁ and D₂), in which the application of water was suspended and only the weight of each tube was recorded, to estimate the evapotranspiration occurred in the plants subject to water deficit.

Statistical analysis

The data obtained was analyzed with the GLM procedure (SAS Inst. Inc., version 9.12), version for Windows 10, under a split plot design with two main factors; the greatest effect was the soil moisture treatment and the least treatment was the genotype (^{Hinkelmann and Kempthorne, 2008}).

The means of the soil moisture treatments and genotypes were compared using the minimum significant difference (MSD, p< 0.05), under the following statistical model: Y_ij= µ + T_i + G_j + T*G_jj + E_ij. Where: Y_ij= value of the response variable in soil moisture treatment (T) of level i, genotype (G) of level j; µ= general mean, T_i = effect of soil moisture treatments at level i = irrigation and drought, and G_j = effect of the genotype of the different varieties of lucerne at level j= San Miguel, Oaxaca, Genex, Júpiter, Milenia, San Isidro, Aragón, Cuf 101, Victoria and Atlixco, and E_ij= experimental error.

Soil water content

In irrigation (I₁ and I₂) the water content of the soil remained close to CC during the experiment, while in drought (D₁ and D₂), the availability of soil moisture decreased below the PMP (percentage of soil moisture, where the availability of humidity for the plant is reduced to zero) between 361 to 407 ddt in D₁ and 634 to 687 ddt in D₂, and it remained in this condition for longer in D₂ (53 d) than in D₁ (46 d).

A severe decrease in soil moisture content below the root absorption limit (PMP), can greatly affect lucerne morphology, physiology, and yield (^{Pembleton et al., 2009}). However, dehydration of the plant can be delayed by a longer absorption period or reduced transpiration; when root growth and absorption stop in a drying soil, lucerne can maintain water deficit by reducing it (Erice et al., 2010) and then stop its vegetative growth (^{Sheaffer et al., 1988}).

Water relations

The drought was more severe and lasted longer in D₂ than in D₁; ψ, π and p in irrigation (I₁ and I₂) were consistently higher (p≤ 0.01) than in drought (D₂ and D₂) on average of all genotypes. In I₁ the ψ, π and p remained at an average of -0.6, -1.6 and 1 MPa during the period of water deficit (Figures 1a, b and c), while in D₁the ψ, π and p decreased (p≤ 0.01) from - 0.6, -1.6 and 1 MPa, at the beginning of the water deficit period (345 ddt) at -3.1, -3.5 and 0.4 MPa at the end of the drought period (406 ddt) (Figures 1d, e and f).

Figure 1 Water, osmotic and turgor potential in irrigation (I₁) (a, b and c), and drought (D₁) (d, e and f), for 10 varieties of lucerne in greenhouse conditions. Montecillo, Texcoco, State of Mexico. 

In D₁ the Júpiter variety had high (p≤ 0.05) ψ at 392 and 407 ddt (Figure 1d), and π at 361, 378 and 407 ddt; the Atlixco variety had high (p≤ 0.05) π at 361 and 407 ddt and the Victoria variety showed high (p≤ 0.05) π at 361 ddt (Figure 1e).

The varieties Júpiter, Atlixco and Milenia showed ψ similar to Genex at 361 ddt, Júpiter showed ψ similar to 361 ddt, Genex showed ψ similar to Jupiter at 392 and 407 ddt (Figure 1e).

The Genex, Milenia and Aragón varieties had π similar to Jupiter, Atlixco and Victoria at 361 ddt, the Atlixco and Victoria varieties had π similar to Jupiter at 378 ddt; the Júpiter, Atlixco and Milenia varieties showed π similar to Genex at 392 ddt and the Milenia, Victoria and Aragón varieties were π similar to Genex, Júpiter and Atlixco (Figure 1e). The Milenia variety showed p similar to Genex at 378 ddt (Figure 1f).

In I₂ the ψ, π and p were -0.6, -1.6 and 1 MPa on average during the period of soil water deficiencies (Figures 2a, b and c) while in D₂, the ψ, π and P decreased (p≤ 0.01) from - 0.6, -1.6 and 1 MPa, at the beginning of the drought (620 ddt) to -3.5, -3.7 and 0.2 MPa at the end of the water deficit period (688 ddt) (Figures 2d, e and f).

Figure 2 Water, osmotic and turgor potential in irrigation (I₂) (a, b and c) and drought (D₂) (d, e and f), for 10 varieties of lucerne under greenhouse conditions. Montecillo, Texcoco, State of Mexico. 

In D₂ the Genex variety showed high (p≤ 0.05) ψ at 634, 672 and 687 ddt (Figure 2d) and π at 657, 672 and 687 ddt (Figure 2e). The Atlixco variety had high (p≤ 0.05) ψ at 634 ddt (Figure 2d), π at 634 ddt (Figure 2e) and p at 634 ddt (Figure 2f). The Milenia variety presented high (p ≤ 0.05) ψ at 634, 657 and 687 ddt (Figure 2d), π at 672 ddt (Figure 2e) and p at 657 ddt (Figure 2f).

The Genex variety showed ψ similar to Milenia at 657 ddt; Atlixco and Milenia varieties had ψ similar to Genex at 672 ddt and Atlixco showed ψ similar to Genex and Milenia at 687 ddt (Figure 2d). The Milenia variety had π similar to Atlixco at 634 ddt; the Atlixco and Milenia varieties had π similar to Genex at 657 ddt, the Atlixco variety had π similar to Genex and Milenia at 672 ddt and the Atlixco and Milenia varieties were similar in π to Genex at 687 ddt (Figure 2e). The Genex and San Isidro varieties showed p similar to Atlixco at 634 ddt (Figure 2f).

A water deficit in the soil (≈ 60% of the extractable moisture) in adult plants of the varieties Cuf 101, Moapa 69 and WL 318 of lucerne, linearly reduced the ψ of all genotypes until the availability of humidity was very low to a depth of 2.4 m (≈ 0.14 m³ m^-3), with a differential response between varieties (^{Grimes et al., 1992}). The reduction in ψ of -0.5, -0.5 and -0.35 MPa with respect to the well-irrigated lucerne control, decreased dry matter production by 29, 23 and 20% (^{Godoy-Ávila et al., 2003}). However, it has been determined that resistance to drought in lucerne germplasm is associated with a high capacity of tolerance to wilting of the plant, maintenance of the green leaf area and greater number of stems per plant under stress conditions, in addition to a wide genetic variability in physiological and morphological characters of the plant (^{Anicchiarico et al., 2013})

Osmotic adjustment

The OA increased (p≤ 0.05) as the drought worsened with values from 0.39 to 1.7 MPa in D₁ (361 to 407 ddt) and from 0.46 to 1.96 MPa in D₂ (634 to 687 ddt), on average of all genotypes; the longest drought period in D₂ was reflected in a greater capacity for osmotic adjustment (Figure 3). The OA is one of the adaptive cellular mechanisms to drought, salinity and freezing in plants, the dehydration of the cell enters a state of osmotic tension due to the increase in the cellular concentration of solutes (^{Blum, 2015}).

Figure 3 Osmotic adjustment capacity of the plant in D₁ (a) and D₂ (b) and their respective irrigation periods (I₁ and I₂), for 10 varieties of lucerne under greenhouse conditions. Montecillo, Texcoco, State of Mexico. 

In D₁ the Genex, Jupiter and Atlixco varieties had high OA values ( p≤ 0.05) at 361, 378, 392 and 407 ddt, the Victoria variety showed higher OA (p≤ 0.05) at 361 and 378 ddt (p≤ 0.05) and the variety Aragon had a higher OA (p≤ 0.05) at 407 ddt (Figure 3a).

In D₂, the Genex, Milenia and Atlixco varieties showed high OA (p≤ 0.05) at 634, 657 and 672 ddt, the Oaxaca variety showed high OA (p≤ 0.05) at 672 and 687 ddt, the Cuf 101 variety showed high OA (p≤ 0.05) at 634 ddt, and the Jupiter variety had high OA (p≤ 0.05) at 657 ddt (Figure 3b). The Genex, Milenia and Atlixco varieties showed an OA similar to Oaxaca at 687 ddt (Figure 3b).

Drought significantly reduced leaf dry weight in three lucerne populations (Chemini, Ganouch, and Tebelbou), with a significant increase in the content of proline, total soluble sugars, amino acids, and potassium in leaves and roots; the Chemini population showed the highest content of solutes, while Tebelbou exhibited the lowest content of proline and the other osmolytes (^{Abid et al., 2016}).

The maintenance of the physiological processes and the hydric status of the leaf suggests that lucerne has some important mechanisms to maintain cellular function when the plant is subject to hydric deficit, in such a way that the maintenance of cellular function under hydric deficit can be related to the processes of antioxidation and OA (^{He et al., 2012}).

An examination of the individual behavior of the lucerne genotypes used in the present study under water deficit conditions, indicated that the Genex, Atlixco and Milenia varieties in D₁ and the Genex, Jupiter, Atlixco and Milenia varieties in D₂ maintained higher values for the potential ψ, π and p. The high OA capacity observed in the two drought periods (D₁ and D₂), possibly was one of the mechanisms that contributed to maintaining the survival of the plant, the turgidity of plant organs and tissues, and consequently, the growth and accumulation of dry matter.

Gaseous exchange relationships

The rate of A, E and g in irrigation (I₁ and I₂) were higher (p≤ 0.05) than in drought (D₁ and D₂), while W in irrigation (Figure 4) was lower (p≤ 0.05) than in drought (Figure 5) on average of all genotypes. In D₁ (345 to 406 ddt) the A, E and g decreased (p≤ 0.05) to 4.4 µmol CO₂ m^-2 s^-1, 0.022 mol H₂O m^-2 s^-1 and 0.7 mmol H₂O m^-2 s^-1 (p≤ 0.05) with respect to the values observed at the beginning of the drought treatment (A=15.3 µmol CO₂ m^-2 s^-1, E= 3.6 mmol H₂O m^-2 s^-1 and g= 0.133 mol H₂O m^-2 s^-1) (Figures 4a, b and c) and the W increased (p≤ 0.05) from 0.0043 to 0.0058 mmol CO₂ mmol H₂O^-1 (Figure 4d).

Figure 4 Net assimilation rate (a), transpiration rate (b), stomatal conductance (c) and efficiency in the use of water (d) in irrigation (I₁) and drought (D₁) (e, f, g and h), for 10 varieties of lucerne in greenhouse conditions. Montecillo, Texcoco, State of Mexico. 

Figure 5 Net assimilation rate (a), transpiration rate (b), stomatal conductance (c) and efficiency in the use of water (d) in irrigation (I₂) and drought (D₂) (e, f, g and h), for 10 varieties of lucerne in greenhouse conditions. Montecillo, Texcoco, State of Mexico. 

In D₁ the Milenia variety showed the highest values (p≤ 0.05) of At 361, 392 and 407 ddt (Figure 4a); E at 361, 378, 392 and 407 ddt (Figure 4b) and g at 361, 378, 392 and 407 ddt (Figure 4c). The Genex variety had high values (p≤ 0.05) of A, E and g at 378 ddt (Figure 4a, b and c). The Oaxaca variety showed high value (p≤ 0.05) for W at 361 and 407 ddt, the Aragón variety had high value (p≤ 0.05) for W at 378 ddt and the Cuf 101 variety showed high value (p≤ 0.05) for W at 392 ddt (Figure 4d).

The Jupiter variety had A similar to Milenia at 361 ddt, the Milenia variety had A similar Genex at 378 ddt, the Genex and Jupiter varieties had A similar to Milenia at 392 ddt and 407 (Figure 4a), the Genex and Júpiter were similar to Milenia in E at 361, 392 and 407 ddt and the Júpiter variety was similar to Genex and Milenia in E at 378 ddt (Figure 4b). The Genex and Jupiter varieties were similar to Milenia in g at 361, 392 and 407 ddt, the Jupiter variety was similar in g to Genex and Milenia at 378 ddt (Figure 4c). The Cuf 101 variety was similar to Oaxaca in W at 361 ddt, the San Miguel variety had a W similar to Aragon at 378 ddt, the Aragon variety was similar in W to Cuf 101 at 392 ddt and the Victoria variety had a W similar to Oaxaca at 407 ddt (Figure 4d).

In D₂ (620-688 ddt), the rate of A, E and g decreased (p≤ 0.05) to 4 µmol CO₂ m^-2 s^-1, 0.019 mol H₂O m^-2 s^-1 and 0.7 mmol H₂O m^-2 s^-1 with respect to the values observed at the beginning of the drought treatment (A= 12.5 µmol CO₂ m^-2 s^-1, E= 2.8 mmol H₂O m^-2 s^-1 and g= 0.100 mol H₂O m^-2 s^-1) (Figures 5a, b and c), and the W increased from 0.0046 to 0.0061 mmol CO₂ mmol^-1 H₂O^-1 (Figure 5d).

The Milenia variety showed the highest values (p≤ 0.05) for A, E and g at 634, 657, 672 and 687 ddt; the Genex variety had high values (p≤ 0.05) for A, E and g at 672 and 687 ddt (Figures 5a, b and c). The San Isidro variety showed the highest value (p≤ 0.05) for W at 657 ddt, the Cuf 101 variety had a high value (p≤ 0.05) of W at 672 ddt and the San Miguel variety showed high value (p≤ 0.05) for W at 687 ddt (Figure 5d).

The Genex variety showed values of A, E and g similar to Milenia at 634 (Figures 5a, b and c), Jupiter had A similar to Milenia at 657 ddt, the varieties Jupiter, Atlixco and Oaxaca showed A similar to Genex and Milenia at 672 ddt and the varieties Júpiter and Oaxaca had A similar to Genex and Milenia at 687 ddt (Figure 5a). The Júpiter, Genex, Atlixco and Oaxaca varieties had an E similar to Milenia at 657 ddt (Figure 5b). The varieties Jupiter and Genex had Milenia had a g similar at 657 ddt (Figure 5c) and the Atlixco variety showed E and g similar to Genex and Milenia at 672 and 687 ddt (Figures 5b and c). The Victoria variety had a W similar to Cuf 101 at 672 ddt and San Miguel at 687 ddt (Figure 5d).

In another study, it was observed that the soil water deficit severely reduced the net assimilation rate, stomatal conductance, water use efficiency, relative water content, leaf area and chlorophyll content a and b in various lucerne populations, with an increase in the efficiency in the use of water and internal concentration of CO₂; additionally, it was determined that the most drought tolerant population reduced their leaf area as much as possible and maintained the highest values for water use efficiency (^{Abid et al., 2016a}).

Lucerne is generally considered a highly drought-sensitive species, especially in the vegetative and reproductive stages, and cultivated in dry areas is moderately drought-tolerant, but under conditions of severe water stress suffers considerable production losses. The main effects of water stress are manifested in a reduction in germination and growth, severe damage to the photosynthetic apparatus, and a decrease in net photosynthesis and nutrient absorption (^{Nadeem et al., 2019}).