A comparative analysis of drought and salinity tolerance in Russian alfalfa varieties according to some morphological and physiological features

Climate change is seen as a major impact to the survival of many species and ecosystems, and sustainable livestock production are main issue to end poverty and hunger in all its forms everywhere.

Sustainable livestock production globally is facing the increasing pressure of negative environmental implications, particularity because of abiotic stresses [Sejian et al., 2013]. There are several common abiotic stresses namely heat, drought, salinity and flooding. Mongolia is vulnerable to extreme climate events, and is often affected by episodes of anomalously cold winters and droughts [Morinaga Y., 2003]. In Mongolian agriculture drought and salinity are the main environmental stresses which are limiting plant growth, development, and survival rate, leading to enormous yield loss.

In Mongolia, currently the number of animals is increasing along this significant expansion herdsmen face a problem of pasture degradation and overgrazing. However, alfalfa is an essential hay product which grows in arid and semi-arid regions. Because of its depth and straight roots, this species is able to absorb even depth waters about 5 m depth and more. This advantage saves plant’s life in long term drought [Ingram J. and Bartles D. 1996].

Ghasem Ali Dianati Tilake et al., [2009], Hamidi and Safarnejad [2010], Castroluna et al., [2014], Adiyasuren [2017] are reported alfalfa seed germination under drought condition. In addition, Safarnejad [2008] established some morphological parameters of alfalfa plants and Adiyasuren [2017] presented some results of drought and salt tolerance by morphological and physiologic parameters of M.sativa, M.falcata, M. varia Marthz , M. varia Martyn.

Moreover, there is a limited comparative analysis of salt tolerance research has been done in alfalfa. Nevertheless, a comparative salinity stress tolerance research is made in Medicago falcata and Medicago truncatula by Min Liu et al., [2014] and Adiyasuren and Enkchimeg [2018] studied 4 types of alfalfa: 2 wild types and 2 varieties by physiological and morphological features.

However, the effects of drought and salt stress on alfalfa plants in laboratory conditions have been established only fewer.

The purpose of this study was to compare salt and drought stress of Russian alfalfa varieties (Vega87, Tayejnaya) by morphological and physiological parameters and to seek a possibility to plant them in Mongolian environmental condition in future to increase supply of animal feed sources.

Material and Methods

Method of Salinity Stress Tolerance

Preparation of NaCl solution

The solutions were prepared with electrical conductivity of 0 (control), 50mM (moderate level), 100mM (high level), 150mM (extreme level), 200Mm (extremely high level).

Determining salt stress tolerance by seed germination

The experiment was carried out in three replicates where 20 seeds from each cultivar were separately germinated on sheet of Whatman No.1 filter paper in Petri dishes. Priority, 10 ml from one respective test solution was poured into the plate. The plates were placed into an incubator at 25±2ºC in darkness for eight days. Germination index is the percentage of germination salinity stress tolerance index is determined as below:

Germination index: nd2 (1.00) + nd4 (0.75) + nd6 (0.50) + nd8 (0.25)

nd — day of seed germination

Seed germination index is the percentage of seed which germinate at 2nd, 4th, 6th and 8th day of observation as indicates by nd2, nd4, nd6 and nd8. Percentage of seed germination stress tolerance index is determined as below:

, . Germination index of stressed seeds

Germination index (%) —--------------------------— x 100

Germination index of control seeds

Determining salt stress tolerance by morphological parameters

The experiment was carried out in pots. Ten seeds from each cultivar were separately sown per pot at the depth pf 3 cm the experiment has three replications. The pots were placedon a growth chamber for 16 hours light photoperiod (200 µmol photons m 2, s1^, 8 hours dark and humidity 70% and a day/night temperature 20ºC, Genomics Laboratory. After 2 weeks (2–3 fully expanded leaves) watered by 0, 50mM, 100mM, 150mM, 200Mm solutions for two weeks. Phenology observation was done daily. The data for the shoot length, root length, weight of root and weight of shoot were measured and recorded as [Akbarimoghaddam et al., 2001] formulas:

Shoot weight of stressed plants

Shoot weight stress tolerance index, gr = —-------------------—----

Shoot weight of control plants

Shoot length of stressed plants

Shoot length stress tolerance index, cm = —---------—-------—----

Shoot length of control plants

Root weight of stressed plants

Root weight stess tolerance index, gr = —------------------—----

Root weight od control plants

Root lenght of stressed plants

Root length stress tolerance index, cm---------—-------—-----

Root length of control plants

Determining salt stress tolerance by water uptake

Water uptake was recorded at 24 hours after planting. Water uptake percentage was calculated by the formula given below.

Water uptake, % = (W2-W1/W1)*100

W1— initial weight of seed

W2 — weight of seed after absorbing water

Determining drought and salinity stress by chlorophyll measurement

The Minolta SPAD-502 (Konica Minolta sensing, Japan) is a hand-held light meter used to measure the relative greenness leaves in a rapid manner. The latest model,

SPAD-502 determines the relative amount of chlorophyll present by measuring the transmittance of the leaf in two wave bands (600-700 and 400-500nm). Single fully expanded leaf samples from each species and varieties were obtained at the 14th day in stress. Triplicate reading using a SPAD-502 were taken around midpoint near the midrib of each leaf sample and averaged.

Method of determining drought stress tolerance

Preparation of osmotic solution

The solution was prepared based on Mongi Zekri methods by using polyethylene glycol (PEG-4000) with electrical conductivity of 0 (control), -0.3, -0.5, -1.0MPa osmotic potentials.

Determining drought tolerance by seed germination

All petri dishes and filter papers were disinfected in 121ºC for 25 minutes in autoclave. The experiment was carried out in 3 replicates where 20 seeds from each species and variety were separately germinated on sheet of Whatman No.1 filter paper in Petri dishes. Priority, 10ml from one respective test solution was poured into the plate. The plates were placed into an incubator at 25±2ºC in darkness for eight days. Seed germination index is the percentage of seed which geminate at 2nd, 4th, 6th and 8th day of observation. Percentage of seed germination drought stress tolerance index is determined as below:

, . Germination index of stressed seeds

Germination index (%) —--------------------------— x 100

Germination index of control seeds

Determining drought stress tolerance by water uptake

Water uptake was recorded 2 weeks of watering by PEG4000. Water uptake percentage was calculated by the formula given.

Relative saturation deficit, gr = (Saturated weight — Fresh weight) / Saturated weight

Determining drought stress tolerance by morphological parameters

Shoot weight of stressed plants

Shoot weight stress tolerance index, gr = —-------------------—----

Shoot weight of control plants

Shoot length of stressed plants

Shoot length stress tolerance index, cm = —---------—--------—-----

Shoot length of control plants

Root weight of stressed plants

Root weight stess tolerance index, gr = —------------------—----

Root weight od control plants

Root lenght of stressed plants

Root length stress tolerance index, cm —--------—-------—----

Root length of control plants

Results and Discussion

Result of salt stress tolerance in alfalfa plants

The seed germination under salt stress

Soil salinity blocks the water uptake, consequently, it negatively affects the plant growth and seed germination. Seed germination of Vega87 at 50mM was 25% and

Tayejnaya was 21.5% lower than that of control plant. When the concentration increased until 100mM, seed germination decreased in all alfalfa plants. Seed germination observed 0% at 150mM and 200mM (Table 1, Fig. 1).

Table 1

The mean of seed germination of alfalfa plants affected by different levels of NaCl, %

Fig. 1. Seed germination of alfalfa varieties affected by different levels of NaCl (Left-Vega87, Right-Tayejnaya)

Plants	0	50mM	100mM	150mM	200mM	Mean
Vega87	60	45	10	0	0	23
Tayejnaya	70	55	25	0	0	30

Means of 3 replicates (Petri dishes)

Result of relative water content

When concentration increase, water uptake ability is decreased in comparison to controls. Water uptake in Vega87 at 50mM was declined by 14.8%, at 100mM was 24.6%, at 150mM was 27.9%, at 200mM was 37.8% lower than that of control plants. Tayejnaya at 50mM was declined by 4%, at 100mM was 8%, at 150mM was 16%, at 200mM was 32% lower than control plants (Table 2).

Water uptake by alfalfa plants affecting by salt stress, g

Table 2

Plants	0	50mM	100mM	150mM	200mM
Vega87	3.0 + 50.01	2.6 + 0.02	2.3 + 0.01	2.2 + 0.01	1.9 + 0.02
Tayejnaya	2.5 + 0.02	2.4 + 0.01	2.3 + 0.03	2.1 + 0.01	1.7 + 0.01

Means of 3 replicates±SE.

Result of chlorophyll content

The chlorophyll content was declined because of induction of chlorophyllase enzyme. Although, accumulation of minerals in chloroplast is one of the reasons of decreasing chlorophyll under salt stress condition. Based on chlorophyll content of alfalfa plants can be arranged in the following order: 1) Tayejnaya, 2) Vega87 (Fig. 2).

VEGA87                  TAYEJNAYA

■0 ■ 50mM ■ 100mM ■ 150mM  200mM

Fig. 2. The effect of different NaCl concentration on chlorophyll content after 14 days of salt stress. Means of 3 replicates + SE.

Result of morphological parameters by measuring root, shoot length and weight

Shoot, root length and weight were measured after 14 days of irrigating with different concentration of NaCl.

Table 3

The shoot length of alfalfa plants affected by different level of NaCl, cm

Plants	0	50mM	100mM	150mM	200mM
Vega87	— 15.1 + 3.5	— 13 + 0.2	— 10.9 + 0.5	— 9.8 + 1.1	1.08 + 0.06
Tayejnaya	15.7 + 4	11.5 + 0.5	11.2 + 0.3	10.5 + 2.1	5.2 + 0.05

The values are the mean of 3 replications.

The final shoot length is measured at 14th day of salt stress.

Mean of shoot length varies between 5.2-1.08 cm. The longest shoot length observed in Tayejnaya. Shoot length in Tayejnaya was 26% longer than Vega87 (Table 3, Fig. 3).

Fig. 3. Alfalfa plant’s growth affected by salt after 14days. A-Vega87, B-Tayejnaya (Left side 0, 50, 100, 10, 200mM).

Table 4

The shoot weight of alfalfa plants affected by different level of NaCl, g

Plants	0	50mM	100mM	150mM	200mM
Vega87	0.6 + 0.01	0.27 + 0.03	0.14 + 0.01	0.10 + 0.02	0.01 + 0.01
Tayejnaya	0.5 + 0.02	0.15 + 0.01	0.13 + 0.02	0.11 + 0.03	0.05 + 0.01

The values are the mean of 3 replications.

The shoot weight is illustrated at 14th day.

As a result of comparing by shoot weight Vega87 was 9.5% lower than Tayejnaya.

The significantly lowest shoot weight observed in Vega87 (Table 4).

Table 5

The root length of alfalfa plants affected by different level of NaCl, cm

Plants	0	50mM	100mM	150mM	200mM
Vega87	9 + 3	7.5 + 0.3	5.5 + 0.2	5.1 + 0.9	1.1 + 1
Tayejnaya	7 + 2.6	6.5 + 0.2	6.3 + 0.3	6.2 + 0.6	3.4 + 0.6

The values are the mean of 3 replications.

The percentage is illustrated at 14th day.

While increasing NaCl concentration root length of alfalfa is declined. Root length of Vega87 was decreased by 16.6% at 50mM, at 100mM — 38.8%, 150mM — 43.3% in comparison to control plant. Tayejnaya showed the declined root length by 7.1 at 50mM, 10% at 100mM, 11.4% at 150mM than control. Among species Tayejnaya had longer root length than Vega87 (Table 5).

The root weight of alfalfa plants affected by different level of NaCl, g

Table 6

Plants	0	50mM	100mM	150mM	200mM
Vega87	0.55 + 0.08	0.18 + 0.01	0.15 + 0.02	0.14 + 0.02	0
Tayejnaya	0.49 + 0.1	0.28 + 0.02	0.22 + 0.01	0.21 + 0.01	0.06 + 0.01

The values are the mean of 3 replications.

The final seed germination percentage is illustrated at 14th day.

Based on root weight, Tayejnaya showed highest than that of Vega87 by 24.4% (Table 6).

Result of drought stress

Result of seed germination under drought condition

The data indicated the significant variation among alfalfa varieties (Table 7, Figure 4). PEG4000 with -1.0MPa osmotic solution inhibited all alfalfa plants seed germination. As shown in Table 7, seed germination decreased with escalating PEG 4000 concentration.

Table 7

The seed germination of alfalfa plants in different osmotic solution level, %

Plants	0	-0.3МПа	-0.5МПа	-1МПа	Mean
Vega87	70	15	0	0	21.25
Tayejnaya	85	70	10	0	41.25

Means of 3 replicates.

The final seed germination percentage is illustrated at 8th day.

Fig. 4. Seed germination of alfalfa plants affected by different levels of osmotic solution (left side-Vega87, right side-Tayejnaya)

Result of water uptake

For species Vega87, water uptake was decreased by 31.2% at -0.3MPa, 37.7% at -0.5MPa, 60.7% at -1.0MPa compare to control (Table 8). Whereas, the lowest water uptake was observed in Tayejnaya variety by 24% at -0.3MPa, 32% at -0.5MPa and 60% at -1.0MPa (Table 8).

Table 8

Effects of PEG 4000 concentration on water uptake by alfalfa plants, g

Plants	0	-0.3MPa	-0.5MPa	-1MPa
Vega87	3.05 + 0.1	2.1 + 0.2	1.7 + 0.3	1.2 + 0.2
Tayejnaya	2.5 + 0.2	1.9 + 0.1	1.6 + 0.2	1 + 0.03
The values are the mean of 3 replicates + SE.

Chlorophyll content

Drought stress imposed at the vegetative, flowering stages and total chlorophyll content. Chlorophyll content varied among the accessions under well-watered (control)

— conditions. Mild water stress (-0.3MPa) had little effect on chlorophyll content of the alfalfa plants. All alfalfa plants showed the sharp decrease in chlorophyll content at 1.0MPa solution (Figure 5, 6). The chlorophyll was measured after 14 days after the seedlings of the seeds were planted in the soil. After the second leaf, it has been stressed for 14 days without irritation. The highest chlorophyll content observed in Tayejnaya with 3.07 % difference.

S T RESS WITHOUT

STRESS

AFFECTED PERIOD

RECOVERY PERIOD

Fig. 5. Chlorophyll content in alfalfa plants under drought stress after 14^th days, SPAD value. The columns indicate the mean of 3 replications

Fig. 6. Seedling affected by PEG4000 for 14 days (A-Vega87, B-Tayejnaya)

Result of morphological parameters by measuring root, shoot length and weight

After 2 weeks of drought treatment shoot, root length and weight were recorded. The longest shoot length was recorded in Tayejnaya. Tayejnaya had the longest root length than Vega87 varienties (Table 9).

Table 9

Alfalfa plants	Shoot length, cm	Shoot weight, g	Root length, cm	Root weight, g
Control Vega87	18.4±2.5	0.15±0.01	10.3±0.4	0.13±0.01
Control Tayejnaya	19.4±0.7	0.16±0.03	11.4±0.7	0.13±0.01
Vega87	10.1±1.0	0.07±0.01	7.2±0.4	0.07±0.06
Tayejnaya	12.2±3.9	0.07±0.06	9.1±1.0	0.11±0.01

Plant’s morphological parameters

Discussion

According to Castroluna (2014), Bolormaa (2016) and Adiyasuren et al., (2017) research of salt stress tolerance in alfalfa showed declining result in seed germination and vegetative stage. Soil salinity blocks the water uptake, consequently, it negatively affects the plant growth and seed germination. Seed germination of Vega87 at 50mM was lower than control by 25% and seed germination of Tayejnaya at 50mM was 21.5% lower than control. When the concentration increased until 100mM seed germination decreased in all alfalfa plants. Seed germination observed 0% at 150mM, 200mM whereas Mongolian M. varia Marthz and M.varia Martyn seed germination showed 0% at 200mM. Adiyasuren et al., (2017) concluded that the NaCl is one the reason for inhibiting the Medicago L. plants seed germination. Similarly, Bolormaa B (2016) reported that drought stress caused by PEG6000 decreased seed germination percentage in two Mongolian (var. Burgaltai and Nutag Belcheer -2) and two Taiwanese (Known Your alfalfa, Middle east) varieties especially at -1.8MPa and osmotic potential solution obtained 0% of alfalfa.

Ghasem Ali Dianati Tilake et al., (2009) and Adiyasuren et al., (2016) used PEG6000 and 4000 to make drought in laboratory. The drought stress caused by PEG showed a negative effect on seed germination. It can be clearly seen that due to rise of PEG concentration seed germination rate went down gradually. Our research revealed that Russian Vega87 stopped germinating at -0.5MPa while Tayejnaya’s germination stopped at – 1.0 MPa. Likewise, Ghasem Alit Dianati Tilakte et al., (2009) and Adiyasuren et al., (2016) showed that Medicago L. seed germination stopped at -0.8 MPa and – 1.0 MPa respectively.

Plant growth is a function of complex interplay between sources and sink limitations of the two main organs of a plant, the root system and the shoot, establishing functional equilibrium. The permanent or temporary water deficit severely hampers the plant growth and development more than any other environmental factor (Anjum et al., 2011). Salinity effects are the results of complex interactions among morphological, physiological, and biochemical processes including seed germination, plant growth, and water and nutrient uptake (Akbarimoghaddam et al.,2011; Singh and Chatrah, 2011). Our research data gives a declining result in both morphological and physiological features, particularly Vega87 was more sensitive than Tayejnaya to salt stress. Noticeably, shoot and root weight presented the lowest result compare to other parameters.

According to Chazen et al., (1995) established that polyethylene glycol (PEG) is a marker under laboratory conditions for inducing drought stress because of its reputation as an osmotic agent resulting in increasing solute potential and blockage of absorption of water by the root system. Safarmajed A (2008) and Bolormaa (2016) reported that shoot and root length and weight were reduced with an increase in PEG concentration. Adiyasuren et al., (2016) concluded that M.sativa (Russian wild type plant) was more tolerant to drought stress compare to other wild type – M.falcata while our result shows that Tayejnaya is more tolerance to drought than Vega87 respectively.

In addition, both water deficit and salt stresses have a detrimental effect on plant growth that might be related to a reduced leaf expansion rate, lesser final leaf size, diminished leaf production rate and accelerated senescence (Greenway and Munns, 1980; Munns, 2002; Munné-Bosch and Alegre, 2004; Parida and Das, 2005). Chlorophyll content in both Russian alfalfas showed a slight decrease depending on the concentration of salt and PEG solution. After drought treatment on alfalfa it recovered quickly based on chlorophyll content. The highest chlorophyll observed in Tayejnaya compared to Vega87 due to low relative water content which causes inhibition of photosynthesis.

Conclusion

Tayejnaya is more salt and drought tolerant than Vega87 by morphological and physiological measurements. Chlorophyll content declined in both alfalfas due to the concentration of NaCl and PEG solution. However, after drought treatment Tayejnaya recover was higher than Vega87. Thus, Russian Tayejnaya is convenient to plant in Mongolian salty and dry environmental conditions. Meanwhile, it protects soil from degradation and improves vegetation cover.

Acknowledgements

This study is supported by Winter feeding management of growing beef cattle project funded by KOPIA MONGOLIA CENTER in Ulaanbaatar, Mongolia.