GENETIC ENGINEERING OF SALT TOLERANCE IN ALFALFA

USING CRISPR/CAS9 TECHNOLOGY

1. Introduction
Cultivated alfalfa or lucerne (Medicago sativa spp. sativa) ) is an autotetraploid(Stanford,
1951), (2n = 4x = 32) with a genome size of 800–900 Mbp .it is the most
important perennial forage legume ) that belongs to the sub-family of Papilionoideae . found in
the world with approximately 32 million hectares cultivated mainly in the temperate regions of
both the northern and southern hemispheres. increase N2 fixation, and maximize economic
yield The primary center of origin for the genus Medicago is found in the Caucasus,
northwestern Iran, and northeastern Turkey . . It is the third most valuable crop
harvested in the U.S. behind only corn and soybeans. Alfalfa is widely
recognized as a significant component of most dairy and other animal rations
because of the multiple benefits it provides:

• Background on Alfalfa: Iran has been widely recognized as a geographical

center for alfalfa. There are two different origins of alfalfa: : one is the modern European alfalfa from
the Outer Caucasus mountain area; the other is the Central Asia. Carbonized alfalfa seeds found in
archaeological sites in Iran prove that alfalfa is an ancient crop. About 8000 years ago, humans
began to use alfalfa. Records of alfalfa from Turkey date back to 1300 BC, and alfalfa is mentioned
in the teaching books of Babylonians in 700 BC. Like some other crops, alfalfa is also transmitted
through maritime trade and military invasion. Alfalfa was the main feed of the ancient war horse.
Around 490 BC, when the Persians invaded Greece, the seeds of alfalfa were also brought to
Greece. The date of alfalfa introduction to Italy is uncertain, probably in 200 BC. At the same time
that alfalfa was introduced into Italy, Zhang Qian, the messenger sent to the western world by
Emperor Wu of the Han dynasty in 126 BC, brought alfalfa seeds to China. By 1550, alfalfa use had
expanded from Spain to France; by 1565 to Belgium and Holland; by1650 to Britain, about 1750 to
Germany and Austria, 1770 to Sweden, and in the eighteenth Century to Russia. Then in 1800
alfalfa was taken to New Zealand from Europe, and in 1806 to Australia. Alfalfa was introduced to
Peru in 1535. By the eighteenth Century, alfalfa had been introduced into Argentina, and from Peru
to Chile. Alfalfa was introduced into the United States from Mexico in 1736.

Importance of alfalfa in agriculture

high nutritional quality for livestock (protein, energy, vitamins, and minerals). high biomass production
(the record yield for one hectare of alfalfa is over 18,000 kg ha-1 of forage). ability to fix atmospheric
nitrogen (N2) ) wide adaptation to various [Link] of soil tilth when growing the
crop (excellent basis for sustainable agricultural systems). utility as a model system for genetic studies of
Alfalfa adds valuable fiber,
autotetraploid species . its ease of use with the new biotechnologies
has a faster rate of fiber digestion than grasses, is very palatable, is high in protein,
provides needed vitamins, is a good buffer in the rumen, and promotes animal
health. In addition, it provides legume nitrogen credits and has rotational yield boost
to following crops. It provides environmental benefits in soil remediation, reduced
erosion (compared to row crops), and supports varied wildlife by being a source of
cover and food from insects, spiders, mites, and earthworms

Component of alfafa
Components of alfafa meal Their percentage
crude protein 17–20%
metabolisable energy, , 1650 kcal/kg
crude cellulose 20–25%
calcium 1.50%
Total phosphorus 1.50%
Lysine 0.70%
methionine 0.25%

Another important question is can alfalfa become a viable crop in the tropics? An
ability to be used in the context of high disease and insect pest pressure (Lloyd and Lowe, 2000) with
tolerance to problem soils such as those with acidity and aluminum toxicity (Dall’Agnol et al., 2000) will
be a must for the tropics and subtropics.

There are now four factors, which if capitalized upon, could make alfalfa a
viable crop in the tropics:
1 ) the availability of very nondormant, winter active germplasm (North American Alfalfa
which allows forage production on a year round basis Improvement Conference,
2 the wide availability of germplasm and the use of screening 2000)
and selection methods for the many disease, insect, and
nematode pests
3 ) the increasing interest and need in dairy production and high
quality grazing in the subtropics and tropics
4 ) the advances in research which may allow the crop to be (Bouton and Parrott, 1997).
grown in problem saline and acid, aluminum toxic soils.

An ability to provide a nondormant, acid soil tolerant, high pest resistant cultivar would be a big step
toward establishment of “tropical alfalfa” systems.

economic significance and Uses. Alfalfa is an important forage source for livestock
industries around the world because of its wide adaptability, high yield, good quality, and resistance to
frequent cuttings [3,4]. It can be used for pasture, hay, silage, dehydrated products, seed production, and
soil improvement [3,4]. Globally, alfalfa is grown on about 30 million ha [5]. In Canada, more than four
million ha of alfalfa is produced either in monoculture or in mixture with grasses [6], while approximately
seven million ha is grown in the United States [7]. Alfalfa is a moderately saline-tolerant legume [8]. A
number of alfalfa cultivars with improved salt tolerance have been developed using conventional breeding
approaches [9]. However, the genetic improvement of salt tolerance in alfalfa is challenging, mainly as the
response of alfalfa plants to salt stress is physiologically and genetically complex, because salt tolerance
 is controlled by multiple genes and involves various biochemical and physiological mechanisms [9]. Most
alfalfa use in its main growing areas is for stored feed; high quality hay, silage, and dehydrated pellets
and cubes.

salinity in alfafa
. Alfalfa is a moderately saline-tolerant legume [8]. A number of alfalfa cultivars with improved salt
tolerance have been developed using conventional breeding approaches [9]. However, the genetic
improvement of salt tolerance in alfalfa is challenging, mainly as the response of alfalfa plants to salt
stress is physiologically and genetically complex, because salt tolerance is controlled by multiple genes
and involves various biochemical and physiological mechanisms [9].

Salinity criteria
Soil salinity is one of the most influential stressors that limits agricultural production.

Criteria EC the saturation extract of more and is becoming a Shrivastava, P.;

of than 4 dS m−1 in the root zone global issue of Kumar, R. Soil
saline at 25 ◦C with 15% of land degradation, salinity: A serious
soil exchangeable sodium with more environmental issue
prevalence in arid and plant growth
and semi-arid promoting bacteria as
regions one of the tools for its
alleviation. Saudi J.
Biol. Sci. 2015, 22,
123–131. [CrossRef]
[PubMed]

Excess salts chloride; sulfate; and carbonate in the root zone, Szabolcs, I. Salt-
of sodium, calcium, magnesium, making it difficult Affected Soils; CRC
potassium for plants to Press: Boca Raton,
extract water and FL, USA, 1989
nutrients from the
soil and causing
plant injury
2. Effect of Salt Stress on Morphology, Growth, Forage
Yield and Nutritive Value
Effect of Salt Stress on Morphology, Growth, Forage Yield and Nutritive Value Salt stress
reduces plant growth by influencing turgor, photosynthesis, and the activity of specific
enzymes [21]. The influence can occur in two phases; the first phase is governed by the
osmotic effect due to high salt concentration in root zones, whereas the second phase is
governed by toxic effects due to high salt accumulation in leaf tissues [22] (Figure 2). The
presence of high root zone salt concentration causes higher osmotic pressure in soil solution
than in plant cells, reducing the ability of plants to uptake water and essential minerals like
potassium and calcium [23]. In severe salt stress, the soil solution becomes hyper-osmotic,
causing the root cells to lose water, which results in severe wilting or plant senescence [24].
Osmotic stress initially reduces leaf growth and eventually causes a reduction in shoot
development and reproductive growth [25], primarily due to the water deficit in plant tissues
[24]. Salt stress induces decreased photosynthetic rate due to osmotic stressinduced partial
stomatal closure [25]. Sodium ions absorbed by plant roots, if present at a high concentration
in cytosol, can be harmful to the plant [26]. Since sodium and potassium ions are both
monovalent cations, they compete for uptake by the plant under fully hydrated saline
conditions [27] resulting in a deficiency in potassium, an essential macronutrient required for
normal metabolic function [23,28]. Increased concentrations of sodium and chloride ions in
the cytoplasm can disrupt cellular processes, causing damage to photosynthetic apparatus as
well as the dehydration of cells [25,29]. Therefore, limiting excess amounts of sodium in
cytosol and increasing the cellular potassium to sodium ratio are crucial for salt tolerance
[30,31]. This means the maintenance of a regular photosynthetic rate and stable K+/Na+ ratio
are important traits for salt-tolerant alfalfa cultivars. The development of molecular markers
should target the specific traits mentioned above to provide informative markers.
Satges of Salinity damage Mode of damage Resistance Effect
alfafa
Seed more susceptible to Highly sensitive more resistant to KCl reduce the germination and
germination CaCl2 and NaCl salts seed vigor of alfalfa
Seedling Highly sensitive
stage
Mature Na2SO4 solution from
plant emergence to maturity
growth decreased
dramatically at 12.7
dS m−1, with no
surviving plants at 30
dS m−1 [38].

How salinity affect alfafa:

three different growth stages at which alfalfa plants may be affected
by salinity: Smith [32]
, many breeding efforts have been targeted to improve the seed germination of alfalfa under salt stress,
but the selection for salt tolerance should focus on the whole life cycle of alfalfa rather than focusing on a
particular growth stage, as there is no clear correlation between seed germination to post germination
performance [39,40].

The shoot growth of alfalfa had been found to be more adversely affected by salinity than root
growth [41]. A study on 15 alfalfa populations under salt stress imposed by a mixture of MgSO 4, Na2SO4,
CaCl2, NaCl, and KCl revealed that root mass per plant at 18.3 dS m −1 and 24.5 dS m−1 EC was reduced
by 18% and 49%, while the shoot mass was reduced by 50% and 73%, respectively [35]. The average
shoot mass of alfalfa during the first, first + second, and first + second + third cuts decreased by 50%,
44%, and 38% at 8.0 dS m −1 (chloride and sulfate salt solution) and by 80%, 73%, and 67% at 15.6 dS
m−1, respectively [16]. Salt stress in alfalfa caused a reduction in the relative growth rate [42,43]. Moderate
NaCl salt stress (9 ± 0.2 dS m−1) reduced the plant height, leaf, and stem masses of alfalfa by 32%, 34%,
and 35%, respectively [44]. Sibole et al. [45] reported that stem and petiole growth was sensitive to NaCl
salt stress in two different salt-tolerant Mediterranean Medicago species. As compared to the salt stress
imposed by NaCl, other saline solutions containing Ca2+ and K+ ions not only have less toxic effects on
plants, but also they can mitigate the negative effects on a plant under salt stress [46,47].
Though a reduction in growth rate and shoot mass were common, high genetic diversity existed
among alfalfa populations under salt stress [16,35,48], suggesting an adequate genetic variation for
further selection for improved salt tolerance. Lei et al. [49] compared the performance of seven alfalfa
cultivars under NaCl stress and found that the growth rate of salt-tolerant cultivars was less affected by a
high salt stress of ~50 dS m−1. However, in another study, no significant variation in both shoot and root
biomasses was observed between salt-tolerant and intolerant alfalfa cultivars under NaCl stress [50].
Differences among studies might be because of the genetic backgrounds of the alfalfa, as the studies
used different salt-tolerant cultivars.
The forage nutritive value was affected by the salinity and varied among the cultivars [51]. Salt
stress up to 20 dS m−1 increased the leaf-to-stem ratio of alfalfa and its crude protein content [52] and
reduced acid detergent fiber (ADF) and neutral detergent fiber (NDF) [51,53]. The reduction in stem
height may be the main factor causing changes in alfalfa nutritive value, as it increases the leaf-to-stem
ratio.

Impact of soil salinity on alfalfa growth and productivity. Salt

stress reduces plant growth by influencing turgor, photosynthesis, and the activity of specific enzymes
[21]. The influence can occur in two phases; the first phase is governed by the osmotic effect due to high
salt concentration in root zones, whereas the second phase is governed by toxic effects due to high salt
accumulation in leaf tissues [22] (Figure 2). The presence of high root zone salt concentration causes
higher osmotic pressure in soil solution than in plant cells, reducing the ability of plants to uptake water
and essential minerals like potassium and calcium [23]. In severe salt stress, the soil solution becomes
hyper-osmotic, causing the root cells to lose water, which results in severe wilting or plant senescence
[24]. Osmotic stress initially reduces leaf growth and eventually causes a reduction in shoot development
and reproductive growth [25], primarily due to the water deficit in plant tissues [24]. Salt stress induces
decreased photosynthetic rate due to osmotic stress-induced partial stomatal closure [25]. Sodium ions
absorbed by plant roots, if present at a high concentration in cytosol, can be harmful to the plant [26].
Since sodium and potassium ions are both monovalent cations, they compete for uptake by the plant
under fully hydrated saline conditions [27] resulting in a deficiency in potassium, an essential
macronutrient required for normal metabolic function [23,28]. Increased concentrations of sodium and
chloride ions in the cytoplasm can disrupt cellular processes, causing damage to photosynthetic
apparatus as well as the dehydration of cells [25,29]. Therefore, limiting excess amounts of sodium in
cytosol and increasing the cellular potassium to sodium ratio are crucial for salt tolerance [30,31]. This
means the maintenance of a regular photosynthetic rate and stable K+/Na+ ratio are important traits for
salt-tolerant alfalfa cultivars. The development of molecular markers should target the specific traits
mentioned above to provide informative markers.

4. Effect of Salt Stress on Oxidative Stress and Anti-Oxidative Activities

Salinity induces oxidative stress in plants at the sub-cellular level [60]. Salt stress increases the
accumulation of superoxide radicals (O2−) and hydrogen peroxide (H2O2) in cell compartments including
chloroplast and mitochondria [60]. Therefore, lipid peroxidation and protein oxidation occur in the
apoplastic space. An increase in peroxidase (POX) reduces H 2O2 to water using various substrates as
electron donors in salt-tolerant alfalfa [61,62], suggesting that the analysis of antioxidant enzymes could
be useful in understanding the salt tolerance of alfalfa. During seed germination and seedling growth,
increased salt stress also increases the activities of superoxide dismutase (SOD), catalase (CAT), and
ascorbate peroxidase activity (APX)—and the increase is higher in salt-tolerant alfalfa cultivars [62,63].
Under salt stress, the unsaturated fatty acids of plant membranes are decomposed to malondialdehyde
(MDA), and the rate of lipid peroxidation in terms of MDA can be used as a biochemical indicator to
evaluate salt-tolerant cultivars [30,64]. The salt tolerance of alfalfa is also improved by melatonin
application, which acts as an antioxidant in scavenging H2O2 and enhances antioxidant enzymes’ activities
[65]. Under salt stress, tolerant alfalfa shows less severe cell membrane damage and lower accumulation
of reactive oxygen species (ROS) than salt sensitive types [66].

• CRISPR/Cas9 Technology: Overview of CRISPR/Cas9, its

mechanism, and applications in plant biotechnology.
2. Literature Review
• Previous Studies on Salt Tolerance in plant : Summarize
key research on enhancing salt tolerance in various crops.
• Signalling pathway of salinity in Alfalfa
• Genetic Basis of Salt Tolerance: Key genes and pathways
involved in salt tolerance.
• CRISPR/Cas9 in Plant Science: Review of CRISPR/Cas9
applications in crop improvement, with a focus on salt
tolerance.
3. Conventional breeding to improve salt tolerance in alfafa
The classical strategy to obtain crops more tolerant to drought and salinity
is through breeding. In this approach the genetic variability underlying
stress resilience is identified by screening germplasm collections. Beneficial
traits are subsequently introduced into cultivars/lines through different
mating designs .A key obstacle hindering the generation and
commercialisation of stress tolerant crops is the direct transfer of studies
performed under laboratory conditions to the field. While survival (or
recovery) is the major trait from a purely physiological perspective, it is crop
yield the likely determinant of successful stress tolerant crops from an
agronomical point of view.
4. Effect of Salt Stress on Physiological Responses in Alfalfa
The mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress are complex
processes involving photosynthesis, detoxifying, antioxidant, secondary metabolism, and ion
transport [54]. Salinity alters photosynthetic pigments (i.e., chlorophyll content), therefore it
reduces the maximum photochemical efficiency of alfalfa genotypes [55,56] and increases the
dark respiration rate in alfalfa [43]. Salinity reduces membrane stability, relative water content,
and growth rates and increases lipid peroxidation, proline, and hydrogen peroxide (H 2O2) contents
in the leaf tissue of alfalfa [57]. In fact, studies have shown that an increased level of proline
content is associated with improved salt tolerance [41,58]. In addition, effective osmoregulation in
salt-tolerant alfalfa cultivars is also associated with increased levels of sucrose and pinitol in
leaves and a high accumulation of starch in roots [50]. The pinitol accumulation is a characteristic
of halophytic plants growing in a saline environment. Anower et al. [59] characterized the
physiological traits of two alfalfa half-sib (HS) families, HS-A and HS-B, selected for improved salt
tolerance. Under salt treatment, HS-B showed a greater leaf number (72%) and stem length
(44%) and HS-A displayed better leaf production (84%) compared to the unselected initial
population. This improved growth was associated with a 208% and 78% greater accumulation of
chlorophyll content in HS-B and HS-A, respectively. An increase in protein concentration in salt-
tolerant alfalfa cultivars had been reported in previous studies [51,53]; it may be associated with
an increase in chlorophyll and enzyme activities in the leaves. It is common for plant breeders to
measure the plant vigor and shoot mass to determine the performance of alfalfa under salt stress.
However, this analysis could be enhanced by the determination of leaf-to-stem ratio and certain
physiological traits such as chlorophyll content and protein concentration.
5.
4. Mutation breeding to improve salt tolerance
5. Transgenic approach to develop salt tolerance variety of
alfafa its genetic improvement for salt tolerance is challenging, as alfalfa’s response to salt stress is
Generating salt tolerance forage plant is essential for
genetically and physiologically complex.
use of the land affected by high salinity. A salt tolerance gene rstB was used as a
selectable marker gene in Agrobacterium-mediated transformation of tobacco under a
selective regime of 170 mM NaCl. The transgenic plants showed clear improvement in
salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was
introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal
phenotype was observed among the transgenic plants when compared with wild type
(wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on
the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed
improved germination rate and seedling growth under salt-stress condition. Hindered
Na+ accumulation, but enhanced Ca2+ accumulation was observed on the rstB T1 plants
when subjected to salt-stresses. Enhanced calcium accumulation in transgenic
plants was also verified by cytohistochemical localization of calcium. Under salt-stress
of 50 mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt
plants had no flower formation. The results demonstrated that the expression
of rstB gene improved salt tolerance in transgenic alfalfa. It was estimated that about
20% of the world's irrigated land were severely affected by high soil salinity, which has
been a major constraint to agricultural productivity [1], [2], [3]. With the population
increasing globally, the fertile lands are mainly used for food grain planting. There is a
need to exploit the marginal lands, such as the saline soil, for forage production. Now,
forage has been part of the most critical components in sustainable agriculture and
global food security by providing part of the feed requirement of ruminants for meat and
milk production [4], [5]. To meet the requests, forage cultivars with high salt tolerance
are needed.
The cultivated alfalfa is tetraploid (2n = 4x = 32) with a genome size of 800–900 Mbp
[12]. Alfalfa is generally self-incompatible with inbreeding depression, and insect-
assisted pollination. Its cultivars are thus heterogeneous and synthetic. All these
characters are therefore difficult to perform genomic studies or to carry out salt
tolerance breeding directly [13]. Compared with traditional breeding methods, direct
genetic transformation has been used widely and regarded as a powerful tool to
improve single traits of plants. Since the success of first Agrobacterium-mediated
genetic transformation of alfalfa [14], transgenic approaches have been employed to
improve alfalfa in the following aspects: insects resistance [15], down-regulation of lignin
content for better digestibility [16], [17], [18], [19], nutritional quality [20], drought
tolerance [21], [22], herbicide resistance [7] and salt tolerance [23], [24], [25]. Many
genes that have been found to involve in different salt tolerance mechanisms were
introduced into various plant genomes with positive results, such
as MSPRP2 [26], Alfin1 [20], SOS1 [27], osDREB [28], SsNHX1 and AVP1 [29], OsbZI
P23 [30], GmDREB1 [31], TaNHX2 [24], GsZFP1 [25], etc. RstB gene [Rhizobium salt
tolerance B, GI:2865454, open reading frame (ORF) 2] was cloned from Sinorhizobium
fredii strain RT 19 isolated from root nodules of soybean growing in high-saline soil.
Expression of rstB gene had restored salt-tolerance of a salt-sensitive mutation strain
RC-3 of S. fredii in a complementary experiment [32]. Ectopic expression of rstB gene in
tobacco conferred salt-tolerance in transgenic plants and protected the root tip cells
from necrosis under stress of 100 mM NaCl [33]. In this research, rstB was introduced
into alfalfa genome to improve salt tolerance of alfalfa. Growth and physiological
responses were examined in both transgenic and non-transgenic alfalfa plants under
salt stress and non-stress conditions. The results indicate that the rstB transgene
enhanced salt tolerance of alfalfa. Many genes that have been found to involve
in different salt tolerance mechanisms were introduced into various plant
genomes with positive results, such
as MSPRP2 [26], Alfin1 [20], SOS1 [27], osDREB [28], SsNHX1 and AVP1 [2
9], OsbZIP23 [30], GmDREB1 [31], TaNHX2 [24], GsZFP1 [25], etc.
RstB gene [Rhizobium salt tolerance B, GI:2865454, open reading frame
(ORF) 2] was cloned from Sinorhizobium fredii strain RT 19 isolated from
root nodules of soybean growing in high-saline soil. Expression of rstB gene
had restored salt-tolerance of a salt-sensitive mutation strain RC-3 of S.
fredii in a complementary experiment [32]. Ectopic expression of rstB gene
in tobacco conferred salt-tolerance in transgenic plants and protected the
root tip cells from necrosis under stress of 100 mM NaCl [33]. In this
research, rstB was introduced into alfalfa genome to improve salt tolerance
of alfalfa. Growth and physiological responses were examined in both
transgenic and non-transgenic alfalfa plants under salt stress and non-stress
conditions. The results indicate that the rstB transgene enhanced salt
tolerance of alfalfa.

6. application of crispr In salinity stress

7. Limitation of crispr system
8. References
Bouton, J. H., "Alfalfa" (2021). IGC Proceedings (1993-2023). 22.
[Link]

SUN Qi-zhong, LIU Qian, LI Feng, TAO Ya, XU Li-jun. A brief review of the origin and dissemination
of Alfalfa[J]. Acta Prataculturae Sinica, 2019, 28(6): 204-212.

1.[Link]
alfalfa-heichel-martin
2.[Link]
scroll=top
2. (Stanford, 1951).
3. [Link]
[Link], B.P.; Baenziger, H.; Bailey, L.D.; Gross, A.T.H.; Hanna, M.R.; Michaud, R.; Richards, K.W.;
Waddington, J. Agriculture Canada: Growing and Managing Alfalfa in Canada; Publication 1705/E;
Agriculture Canada: Ottawa, ON, Canada, 1982.

4. Coburn, F.D. The Book of Alfalfa: History, Cultivation and Merits. Its Uses as a Forage and Fertilizer;
Orange Judd Co: New York, NY, USA, 1907

5. Yuegao, H.; Cash, D. Global status and development trends of alfalfa. In Alfalfa Management. Guide
for Ningxia; Cash, D., Ed.; United Nations Food and Agriculture Organization: Beijing, China, 2009; pp. 1–
14.

6. Statistics Canada. Census of Agriculture, Hay and Field Crops. Table 32-10-0416-01. 2016. Available
online: [Link] (accessed on 5 February 2020).

7. USDA–NASS. Crop Production 2018 Summary. USDA–NASS. 2018. Available online:

[Link] gov (accessed on 9 February 2020).

8. Maas, E.V.; Hoffman, G.J. Crop salt tolerance-current assessment. J. Irrig. Drain. Div. 1977, 103, 115–
134.

9. Flowers, T.J. Improving crop salt tolerance. J. Exp. Bot. 2004, 55, 307–319. [CrossRef] [PubMed]

11. Shrivastava, P.; Kumar, R. Soil salinity: A serious environmental issue and plant growth promoting
bacteria as one of the tools for its alleviation. Saudi J. Biol. Sci. 2015, 22, 123–131. [CrossRef] [PubMed]
12. Kassas, M. Seven paths to desertification. Desert. Control Bull. 1987, 15, 24–26.

13. Thomas, D.S.G.; Middleton, N.J. Salinization: New perspectives on a major desertification issue. J.
Arid Environ. 1993, 24, 95–105. [CrossRef]

14. Tanji, K.K. Nature and extent of agricultural salinity. In Agricultural Salinity Assessment and
Management; Tanji, K.K., Ed.; American Society Civil Engineers: New York, NY, USA, 1990; pp. 1–13.

15. FAO. Global Network on Integrated Soil Management for Sustainable Use of Salt-Affected Soils; FAO
Land and Plant Nutrition Management Service: Rome, Italy, 2005; Available online:
[Link] (accessed on 11 February 2020).

16. Steppuhn, H.; Acharya, S.N.; Iwaasa, A.D.; Gruber, M.; Miller, D.R. Inherent responses to root-zone
salinity in nine alfalfa populations. Can. J. Plant Sci. 2012, 92, 235–248. [CrossRef]

17. Steppuhn, H. What is soil salinity? In Proceedings Soil Salinity Assessment Workshop; Alberta
Agriculture: Lethbridge, AB, Canada, 1996; pp. 1–5.

18. Wiebe, B.H.; Eilers, R.G.; Eilers, W.D.; Brierley, J.A. Application of a risk indicator for assessing trends
in dryland salinization risk on the Canadian Prairies. Can. J. Soil Sci. 2007, 87, 213–224. [CrossRef]

19. Martinez, J.; Manzur, C.L. Overview of salinity problems in the world and FAO strategies to address
the problem. In Proceedings of the International Salinity Forum, Riverside, CA, USA, 25–27 April 2005;
pp. 311–313.

20. FAO; IIASA; ISRIC; ISS-CSA; JRC. Harmonized World Soil Database (Version 1.2); FAO: Rome, Italy;
IIASA: Laxenburg, Austria, 2008.
 B.S. Dien et al.

Enhancing alfalfa conversion efficiencies for production of sugars

and ethanol by altering lignin composition
Bioresour. Technol.
(2011)
 L. Tang et al.

Overexpression of GsZFP1 enhances salt and drought tolerance in

transgenic alfalfa (Medicago sativa L.)
Plant Physiol. Biochem.
(2013)
 J.L. Dong et al.

Oral immunization with pBSVP6-transgenic alfalfa protects mice

against rotavirus infection
Maas, E.V.; Hoffman, G.J. Crop salt tolerance-current assessment. J. Irrig. Drain. Div. 1977, 103, 115–134.
9. Flowers, T.J. Improving crop salt tolerance. J. Exp. Bot. 2004, 55, 307–319. [CrossRef] [PubMed]

10. Szabolcs, I. Salt-Affected Soils; CRC Press: Boca Raton, FL, USA, 1989.

11. Shrivastava, P.; Kumar, R. Soil salinity: A serious environmental issue and plant growth promoting
bacteria as one of the tools for its alleviation. Saudi J. Biol. Sci. 2015, 22, 123–131. [CrossRef] [PubMed]