U
NIVERSITÀ DI
P
ISA
D
IPARTIMENTO DI
S
CIENZE
A
GRARIE,
A
LIMENTARI E
A
GRO-A
MBIENTALI
C
ORSO DI
L
AUREA
M
AGISTRALE IN
B
IOTECNOLOGIE
V
EGETALI E
M
ICROBICHE
Lepidium peruvianum: a first approach in
evaluating salinity effects on germination
RELATORE:
Prof. Lorenzo Guglielminetti
CORRELATORE:
Prof.ssa Annamaria Ranieri
CANDIDATA: Silvia Meniconi
2
INDEX
1. INTRODUCTION
1.1 Lepidium peruvianum………6
1.1.1 Botany and morphology……….. 6
1.1.2 Chemestry……… 9
1.1.3 Geographic distribution……….. 11
1.1.4 Traditional uses……….. 13
1.1.5 Investigations on maca’s medical proprieties……… 14
1.2 Plant stress: a panoramic view……… 18
1.3 Salinity stress………. 22
1.3.1 Quantifying the effects of salinty……….. 23
2. MATERIAL AND METHODS………. 28
2.1 Experimental material and growing conditions……….. 28
2.2 Biometrical analyses………... 30
2.3 Soluble sugars content analysis………... 31
2.4 Pigment analysis……….. 35
2.5 Ions……….. 35
3. RESULTS……… 36
3.1 Germination test……….. 36
3.2 Root length……….. 38
3.3 Fresh weight, dry weight and water content………... 40
3.4 Soluble sugars content (SSC)………. 45
3.5 Pigment content………. 48
3.6 Ions: Na
+content……… 49
4. DISCUSSION AND CONCLUSION………... 51
ABSTRACT
Soil salinization has often been described as a real problem that modern agriculture has
to face in order to comply with the urgency of feeding the world’s growing population.
Maca is a peruvian plant species of narrow distribution that produce an edible and
highly nourishing tuber, with pharmacological proprieties as well. Literature about this
species is scarce. In this work, the effects of salinity on seed germination, growth,
physiology, and biochemistry of yellow maca (Lepidium peruvianum) were
investigated for the first time, with the aim of establishing traits, which can provide a
basis for further investigation on this plant. The study was conducted under controlled
environment. Maca seeds and plants were subjected to five concentrations of NaCl
solutions: 0 (control), 50mM, 100mM, 200mM, 300mM. Germination percentage,
growth (root length, fresh weight and dry weight), water content, soluble sugars, ions
and pigments content were determined. At the same time, these analyses have been
carried on with same concentration of mannitol, in order to compare salinity effects
with osmotic ones. All physiological parameters assayed show to be affected
differentially depending on NaCl or mannitol treatments. In particular, 50 and 100 mM
NaCl treatment generally don’t affect severely the plant, while higher concentrations
dramatically affect all parameters. This clustered distribution appears to be different
from those obtained in mannitol treatments that instead affect physiological plant
responses in a dose-dependent way.
4
Alle mie numerose famiglie, ai miei nonni e a Lelo, alle mie amate gattine
1.INTRODUCTION
Environmental stresses represent one of the main factors which most limit agricultural productivity worldwide. These stresses not only have an impact on current crop species, but they are also significant barriers to the introduction of crop plants into areas that are not currently being used for agriculture. The beginning of 21st century is marked by global scarcity of water resources, environmental pollution and increased salinization of soil and water. It has been estimated that worldwide 20% of total cultivated and 33% of irrigated agricultural lands are afflicted by high salinity and that the salinized areas are increasing at a rate of 10% annually (B)[1].
Soil salinization has been often described as one of the main problem that modern agriculture has to face in order to comply with the urgency of feeding the world’s growing population. In view of the fact that global food production will need to increase by 38% by 2025 and by 57% by 2050 (Wild, 2003) [2] and if food supply needs to be maintained at current levels, since most of the suitable land has been cultivated and expansion into new areas to increase food production is rarely possible or desirable, the aim, therefore, should be an increase in yield per unit of land rather than in the area cultivated.
Because of this concenrs, researchers are often orientated in studing the plants response to salinity stress and in particular focusing on the germination process. Since germination is the seed’s role as reproductive unit, it is the thread of life that assures survival of all plant species and that is why, because of this role in stand establishment, seed germination remains a key to modern agriculture. Thus, especially in a world acutely aware of the delicate balance between food production and world population, a fundamental understanding of germination is essential to crop production.
In addition to this, while throught hystory thousand of plants species have been used as food supply, today only about 150 plant species are cultivated, 12 of which provide aproximately 75% of our food. This involution has increased the vulnerability of agriculture and impoverished the human diet. As a
6
result, many local crops that have traditionally being important for feeding local population are nowadays underutilized or neglected over the last 50 years.
Under such circumstances suitable biotechnology are required to improve crop productivity and thats why we’re studing plants’ responses to stresses, especially for those forgotten species that may have special features or adaptation strategies for these stressing enviroment we have been talking about.
1.1 Lepidium peruvianum
Lepidium peruvianum (Chacòn) or meyenii (Walpers) known commonly as “maca” is a cruciferous plant of the Brassicaceae family which is cultivated at an altitude of 4,000–4,500 m in the Peruvian Central Andes. This plant grows in this habitat of intense cold, extremely intense sunlight, and strong winds. Maca is traditionally used among the andean population for its nutritional and presumed medicinal properties then supported by scientific researches. Evidence from experimental studies indeed, indicates effects of maca on nutrition, fertility, memory, mood and as energizer(4)[3] Some popular plants related to maca are rapeseed, mustard, turnip, black mustard, cabbage, garden cress, and water cress (1)[4].
Over the past 20 years, interest in maca has increased in many parts of the world, and since 2005 maca is considered one of the seven Peruvian flag products (4)[3]
1.1.1 Botany and morphology
In terms of taxonomy maca, the Andean cultivated species of Lepidium, has been questioned by Chacón (1990) [5], who proposed to change its name, L. meyenii, into L. peruvianum, after morphological observations and comparative analysis of herbarium specimens in Germany and in the USA. Maca belongs to the Brassicaceae (Cruciferae) family, and it can be classified both as an annual or a biennial plant depending on the time it takes to complete its reproductive cycle that follows the
vegetative one. Normally with favourable climatic conditions it can complete its whole cycle within a year and in laboratories, can even complete its life cycle in eleven months (3)[6].
Investigations on maca’s photoperiod in Crop tests in the US have shown that maca can be successfully cultivated in winter in California and that it may also mean that photoperiod has no influence on growth and development and short days are not needed to develop the taproot and flowers, reasons why it is considered day neutral.
The edible and economic product of the plant is a radish-like tuber, formed by the taproot and the lower part of the hypocotyl. These parts of the plant swell during the vegetative phase, forming a storage organ resembling to a turnip, that reach its maximum size approximately 7 months after planting. This hypocotyl-root axis is 10-14 cm long and 3-5 cm wide on average and, due to its high water content, it reduce dramatically its size after natural drying (1)[4]. Clearly even the average weight of the tuber may vary considerably.
The overground part is small and flat in appearance and grows in the shape of a shrub, in close contact with the ground, so that the foliage forms a kind of mat. This could be the result of an adaptation process to prevent the impact of strong winds.
The reproductive fase growing starts from the end of the vegetative cycle, when the storage organ has reached its maximum size (7 months), growing from the base, radially and under the leaves, generative shoots, that will be reproductive branches. Whithin the following three months generative brances will produce flowering racemes bearing 50-70 flowers if they are secondary branches or close to 1000 if they are primary ones.
8
Fig. 1 – Maca’s overground part (left) and black, red and yellow maca’s tubers (source: www.themacateam.com)
The flowers of maca are inconspicuous and arranged in axillar racemes. They have four erect, concave sepals, and four small white petals. The ovary is oval and bicarpelar with a short style, which develops into a dehiscent silique of two locules, carrying one seed per locule. Only two stamens, or seldom three, with well-developed anthers are present in the flowers. Some studies suggest that maca reproduces predominantly by self-pollination. (2)[7]
Fruit will set in most of these flowers throughout this period, maturing in approximately 5 weeks, when they will start dehiscence and the seed will be released.
Approximately 85% of the fruits will bear seeds and a single plant of maca produces approximately 14 g of seeds. Seeds are small, measuring 2 mm in length, and are light tan to brown in colour [7]. Apparently seeds do not have dormancy and when once hydratated they produce a transparent mucilage sphere where the seed will be enclosed, reminding of linen seeds (fig. 2).
Fig. 2 – Maca’s seeds before (left) and after (right) hydratation (credits: Silvia Meniconi)
On the base of the tuber coloring, it is possible to differentiate four to eight “forms”. A recent study at the Bombon mesa (department of Junin, Peru) reports that six cultivars can clearly be differentiated by the color of their storage organ (yellow, purple, cream, light purple [“red”], black, and gray), and seven different shapes have been observed, from flattened circular to amorphous. [8] Furthermore it has been recently demonstrated that different types of maca (according to its color) have different biological properties [9]. (1)
1.1.2 Chemestry
We can butch chemical compound in two categories: primary metabolites, corresponing to the nutritional component of the hypocotyls, and secondary metabolites that are compound with presumed biological and medicinal properties.
Primary metabolites. Fresh hypocotyls contain 80% water and mostly we find in litterature the determination of nutrient composition conducted on dried material. It has been found that the nutritional value of the dried hypocotyl is really high, resembling to that found in cereal grains such as maize, rice and wheat. It shows a large amount of essential aminoacids and higher mineral content,
10
in particular Fe, Ca and Cu, it contains important amounts of fatty acids such as linoleic, palmitic and oleic acids and it’s also rich in sterols. Alkaloids are also present, but these have yet to be determined [10]. A more detailed composition is reported in table 1.
Secondary Metabolites. According to many studies maca contains several secondary metabolites found exclusively in this plant such as macaridine, macaene, macamides, and othrer alkaloids that are still unidentifyied [11]. We can also find a wide range of unsaturated fatty acids, such as macaenes and sterols such as beta-sitosterol, campesterol, and stigmasterol.
Different glucosinolates as the aromatic glucosinolate glucotropaeolin have been described within maca. Benzyl glucosinolate has been suggested as chemical marker for maca biological activity even if lately this suggestion has been discarded since glucosinolates may easily metabolize to isothiocyanates and other smaller metabolites [12].
It’s interestingly been observed how maca batches from different producers significantly vary in the amount of macaene, macamides, sterols, and glucosinolates fact wich it’s lately been reported in 2005 when appeared the first publication indicating that different maca color types have different properties [13]. Finally, it has been confirmed by more recent studies that maca colors can be related with variations in concentrations of distinct bioactive metabolites: these compounds individually or working in synergy may be enhancing the reported biological properties from maca. Thus, the differences in proportion of secondary metabolites between maca colors may explain different biological proprieties described for maca.
Table 1. Summary of the most prominent chemical components of maca ‘hypocotyls’[10]
1.1.3 Geographic distribution
The genus Lepidium is widely distributed throughout the world in all continents except Antarctica. The genus probably originates in the Mediterranean basin where most of the diploid species are found [14]. Little is known about the time of origin of the genus and the mechanisms responsible for its worldwide distribution. For what concern maca, it was probably domesticated in San Blas, Junin (present day: Ondores) some 1,300–2,000 years ago and nowadays it’s a crop of narrow distribution since its cultivation is restricted to the Suni and Puna ecosystems of the Departments of Junín and Cerro de Pasco of Peru at elevations above 3500 m and often reaching 4450 m in the central Andes of Peru [15]. Nowadays the largest cultivated area is found around Lake Junín at Huayre,
Maca hypocotyl primary metabolites content
Protein (% of total dry weight) 10.2
Hydrolyzable carbohydrates (% of total dry weight) 59.0
Amino acids (mg/g protein)
Glutamic acid 156.5 Serine 50.4 Aspartic acid 91.7 Glycine 68.3 Arginine 99.4 Valine 79.3
Fatty acids (% of methyl ester mix)
Palmitic acid 23.8
Linoleic acid 32.6
Saturated fatty acids 40
Unsaturated fatty acids 52.7
Sterols (% of sterol mix)
Campesteryl acetate 27.3
Sitosteryl acetate 45.5
Minerals (mg/100 g dry matter)
Fe 16.6
K 2050
12
Carhuamayo, Uco, Ondores, Junín, Ninacana and Vicco. Apparently maca occupied wider areas of cultivation in the past. In addition to Junín and Cerro de Pasco, presumably, it also was grown in Cusco and in the Lake Titicaca watershed. Some of the writers of the time mention that many natives did not have any other food but maca. It was also used as payment of taxes to the Spanish administrators [16]. Its restricted cultivation today indicates that maca may have been in danger of being phased out as a crop.
Tello et al in 1992 reported that at that time less than 50 ha are being dedicated to the production of maca in Peru and presumably in the world. Since then, things seem not to have changed much even if the popularity of this crop is steadily increasing. In fact consumption of Maca worldwide has significantly increased already during the past decade (2000-2010) as we can observe in figure 3 in which are reported data on maca export from Peru, the only country producing maca. During 2010, Peru exported maca for a value of 6,179,011.8 USD, 4.36-times higher than value exported during 2001.
Fig. 3 - Maca exported from Peru in the last 10 years. Data are in FOB values (USD). [4]
Evidence-Based Complementary and Alternative Medicine
7
Table 2: Semen variables before and 4 month after maca treatment.
Semen variable
Before maca N
=9
After maca N
=9
P value
Volume (mL)
2.23
±0.28
2.91
±0.28
<0.05
pH
7.47
±0.09
7.44
±0.07
NS
Sperm count (10
6/mL)
67.06
±18.61
90.33
±20.46
NS
Total sperm count (10
6/mL)
140.95
±31.05
259.29
±68.17
<0.05
Motile sperm count (10
6/mL)
87.72
±19.87
183.16
±47.84
<0.05
Sperm motility grade a (%)
29.00
±5.44
33.65
±3.05
NS
Sperm motility grade a + b (%)
62.11
±3.64
71.02
±2.86
<0.05
Normal sperm morphology (%)
75.50
±2.02
76.90
±1.23
NS
Data are mean±standard error of the mean. N=number of subjects, NS: not significant, source: [67].
0 1,000,000 2,000,000 3,000,000 4,000,000 5,000,000 6,000,000 7,000,000 Ma ca ex po rt ed (F O B va lu e U SD ) 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 Ano˜ y=20494x3−1E + 08x2+ 2E + 11x−2E + 14 R2=0.978
Figure 3: Maca exported from Peru in the last 10 years. Data are in
FOB values (USD).
a value of 6,179,011.8 USD, 4.36-times higher than value
exported during 2001.
Clearly, further research is required to address the
mechanisms of actions and the active principles of this
plant. However, available data suggest that maca has several
important biological properties, and scientific evidence of
these properties could be important for farmers, dealers, and
consumers. Furthermore, it is necessary to demonstrate the
biological effects of specific secondary metabolites of maca
and their actions when added as a mixture.
Maca is a plant with great potential as an adaptogen
and appears to be promising as a nutraceutical in the
prevention of several diseases. Scientific evidence showed
effects on sexual behavior, fertility, mood, memory,
osteo-porosis, metabolism, and the treatment of some tumor
entities. However, the active principles behind each effect
are still unknown. Macamides have been described as novel
compounds of maca that have not been found in any other
plant species so far [
13
]. It is suggested that this lipid
fraction of maca may be responsible for the increase in
sexual behavior [
13
,
23
]. Studies on testicular function,
spermatogenesis, fertility, mood, memory, and prostatic
hyperplasia [
16
,
35
,
42
,
75
] were performed with aqueous
extracts that contain only trace amounts of macamides [
17
].
This suggests that compounds other than macamides are
responsible for these activities.
Acknowledgments
This paper has been supported by a Grant from Fogarty
Program of the National Institutes of Health (NIH Research
Grant no. 5-D43TW005746-04 funded by the Fogarty
International Center, National Institutes on Environmental
Health Services, National Institute for Occupational Safety
and Health, and the Agency for Toxic Substances and Disease
Registry).
References
[1] C. Quiroz and R. Aliaga, “Maca (Lepidium meyenii Walp.),” in
Andean Roots and Tubers: Ahipa, Arracacha, Maca and Yacon.
Promoting the Conservation and Use of Underutilized Neglected
Crops, M. Hermann and J. Hellers, Eds., vol. 21, pp. 173–197,
International Plant Genetic Resources Institute, Rome, Italy,
1997.
[2] B. Cobo, History of the New World, Biblioteca de Autores
Espa˜noles, Madrid, Spain, 1956.
[3] G. F. Gonzales, “Biological effects of Lepidium meyenii, maca,
a plant from the highlands of Peru,” in Natural Products, VK
Singh, R Bhardwaj, JN Govil, and RK Sharma, Eds., vol. 15
of Recent Progress in Medicinal Plants, pp. 209–234, Studium
Press, Houston, Tex, USA, 2006.
[4] G. F. Gonzales, “MACA: Del alimento perdido de los Incas
al milagro de los Andes: Estudio de seguridad alimentaria
y nutricional,” Seguranc¸a Alimentar e Nutricional, Campinas,
vol. 16-17, no. 1, pp. 16–36, 2010.
[5] L. G. Valerio and G. F. Gonzales, “Toxicological aspects of
the South American herbs cat’s claw (Uncaria tomentosa) and
maca (Lepidium meyenii): a critical synopsis,” Toxicological
Reviews, vol. 24, no. 1, pp. 11–35, 2005.
[6] P. Cieza de Le ´on, Chronicle of Peru. First Part, Hakluyt Society,
London, UK, 1553.
[7] H. Ruiz, Relaci´on hist´orica del viaje a los reinos del Per´u y Chile,
1777-1778, Academia de Ciencias Exactas, F´ısicas y Naturales,
Madrid, Spain, 1952.
[8] K. Oerlemans, D. M. Barrett, C. B. Suades, R. Verkerk, and
M. Dekker, “Thermal degradation of glucosinolates in red
cabbage,” Food Chemistry, vol. 95, no. 1, pp. 19–29, 2006.
[9] N. V. Matusheski, J. A. Juvik, and E. H. Jeffery, “Heating
decreases epithiospecifier protein activity and increases
sul-foraphane formation in broccoli,” Phytochemistry, vol. 65, no.
9, pp. 1273–1281, 2004.
[10] V. Dewanto, X. Wu, K. K. Adom, and R. H. Liu, “Thermal
processing enhances the nutritional value of tomatoes by
1.1.4 Traditional uses
The first written description about maca (as a root without identification of the botanical or popular name) was published in 1553, in which Cieza de Leon, a chronicler of the Spaniard conquest of Peru, noted that in the Peruvian highlands, particularly in the province of Bombo’n (Chin- chaycocha; present day: Junin) the natives used certain roots for maintenance [17]. The roots, he was referring to were maca.
Later Father Cobo [18] was the first, in 1653, to describe this plant and its properties under the name of maca. He stated that this plant grows in the harshest and coldest areas of the province of Chinchaycocha where no other plant for man’s sustenance could be grown. Cobo also referred to the use of maca for fertility and lately in the 18th century, Ruiz referred once again to the fertility- enhancing properties and the stimulant effect of maca.
Traditionally, after being harvested maca is dried naturally and can thus be stored for many years [5]. The dried hypocotyls are hard as stone (Figure 1) so that after being naturally dried maca hypocotyls can be eating even if usually they are first boiled in water to obtain a soft product which can be consumed as juice, the most frequent form of use [3].
The boiling process seems to increase active metabolites. In fact, increased temperature affects the availability of several secondary metabolites in plants sometimes like the glucosinolates in maca. These compounds are sensitive to heating but oher metabolites, however, are increased after heating. For instance, heating decreases the activity of epithiospecifier protein and increases formation of sulforaphane, a derivative of isothiocyanates and glucosinolates, as in broccoli [19].
14
Fig. 4 - Andean people laying out maca’s tubers on the soil for natural drying (source: www.embassyofperu.org)
1.1.5 Investigations on maca’s medical proprieties
Scientific evidence showed effects of maca consumption on sexual behavior, fertility, mood, memory, osteoporosis, metabolism, and the treatment of some tumor entities. Even though many of the active principles behind each effect are still unknown, macamides, that have been described as novel compounds of maca that have not been found in any other plant species so far [4], it is suggested that this lipid fraction of maca may be responsible for the increase in sexual behavior [11]. Studies on testicular function, spermatogenesis, fertility, mood, memory, and prostatic hyperplasia [13] were performed with aqueous extracts that contain only trace amounts of macamides suggesting that other compounds besides macamides are responsible for these activities.
pharmacological effect of maca on experimental animals. The results of in vivo trial are reported in Table 2 [4].
The process of preparation of maca is important to obtain adequate biological effects. Traditionally maca is boiled or extracted in alcohol before it is consumed [20]. In experimental studies, aqueous extract of maca is only effective after boiling pulverized maca hypocotyls in water. The greatest effect on spermatogenesis was observed with the ethyl acetate fraction of the hydroalcoholic extract of black maca [42].
As regards effects on humans, a recent study was designed to investigate on health status in a population from the Peruvian central Andes (Carhuamayo, 4100 m) which traditionally consumes maca and compared it with a population from the same place which does not consume maca. The study, based on a survey, assessed maca consumption, sociodemographic aspects, health status, and fractures in men and women aged 35–75 years old. In a subsample were assessed the hepatic and kidney functions and hemoglobin values. From the sample studied, 80% of the population consumed maca and 85% of them consume maca for a nutritional purpose.
Maca is used since childhood and mainly after hypocotyls it is naturally dried. The consumption is mainly as juices, and the variety that they consume is a mixture of different colors of the hypocotyls. Maca consumption is associated with higher score in health status (Fig.4), lower rate of fractures, and lower scores of signs and symptoms of chronic mountain sickness. In addition, maca consumption is associated with low body mass index and low systolic blood pressure.
Hepatic and kidney function, lipidic profile, and glycemia were normal in the population consuming maca.
16
Evidence-Based Complementary and Alternative Medicine
5
fruits like oranges and grapefruit and fruit juices [
54
], which
are frequently used because of their favorable properties on
health. MTCA has been described on the fermented garlic
extract [
55
,
56
], and its concentration increases with time,
in turn increasing its antioxidant activity. Moreover, MTCA
is detected in several foods, and in some, in concentrations
relatively high (greater than the ones found by Piacente in
maca) suggesting that claims are overestimated.
In a recent paper, several arguments indicate that MTCA
in maca is safe [
57
]. In addition, maca is not mutagenic
but it contains several beneficial compounds, some of which
have anticarcinogenic properties [
5
,
58
]. The consumption
of maca must not generate concern, taking in account that, as
mentioned in the French alert [
53
], it has not been reported
any toxicity in the case of maca traditional consumption that
requires a boiling process. MTCA is a natural constituent of
several plants and on consumption of such plants there is no
toxicity found. This suggests that as a multicomponent it may
lose its adversity as drug action.
Furthermore, a recent study was designed to investigate
health status in a population from the Peruvian central
Andes (Carhuamayo, 4100 m) which traditionally consumes
maca and compared it with a population from the same
place which does not consume maca. The study, based on
a survey, assessed maca consumption, sociodemographic
aspects, health status, and fractures in men and women aged
35–75 years old. In a subsample were assessed the hepatic and
kidney functions and hemoglobin values. From the sample
studied, 80% of the population consumed maca. 85% of
them consume maca for a nutritional purpose.
Maca is used since childhood and mainly after hypocotyls
it is naturally dried. The consumption is mainly as juices,
and the variety that they consume is a mixture of different
colors of the hypocotyls. Maca consumption is associated
with higher score in health status (
Figure 2
), lower rate
of fractures, and lower scores of signs and symptoms of
chronic mountain sickness. In addition, maca consumption
is associated with low body mass index and low systolic blood
pressure.
Hepatic and kidney function, lipidic profile, and
glyce-mia were normal in the population consuming maca. In
summary, this study demonstrated in a population
tradition-ally using maca that consumption of this food is safe [
4
].
9. Maca and Sexual Function
Sexual dysfunctions are highly prevalent in our society
worldwide, and the occurrence of sexual dysfunctions
in-creases directly with age for both men and women [
59
]. They
occur in 20–30% of men and 40–45% of women according
to 18 descriptive epidemiological studies from around the
world [
60
].
Most sexual problems relate to sexual desire (interest in
sex) in both females and males and male erectile dysfunction
(ED) [
60
]. Interest in medicinal plants to treat sexual
dysfunctions has increased in the last 20 years [
61
].
Maca has been described to improve sexual behavior
in experimental animals [
13
,
22
,
23
], although conflictive
30 35 40 45 50 40 45 50 55 60 65 70 75 Sc or e of h ea lt h st at u s Age (years) R2=0.0042 y=113.98x−0.269 R2=0.1241 y= −0.0405x + 47.43 Carhuamayo (4100 m)
Figure 2: Score of health status from men and women residents of Carhuamayo, Junin at 4100 m in the Peruvian Central Andes. Upper line: population consuming extracts of maca. Bottom line:
population not consuming maca; Source: [4].
results may be observed [
24
]. Traditionally maca has been
referred to as a plant to improve fertility [
2
] and as an
energizer [
3
]. In a randomized study we were unable to
demonstrate effect of maca on penile erection in apparently
healthy adult men after 12 weeks of treatment with
gela-tinized maca compared with results using placebo [Gonzales,
unpublished data].
Recently, a systematic review has been performed on
effect of maca on sexual function in humans [
62
]. In this
review, according to the authors only four randomized
clinical trials (RCT) met all the inclusion criteria [
49
,
63
–
65
].
According to the review, two RCTs suggested a significant
positive effect of maca on sexual dysfunction or sexual
desire in healthy menopausal women [
49
] or healthy adult
men [
63
], respectively, while the other RCT according to
the reviewers failed to show any effects in healthy cyclists.
However, analyzing results from such study, authors showed
that maca extract significantly improved the self-rated sexual
desire score compared to the baseline test (P
=0.01), and
compared to the placebo trial after supplementation (P
=0.03) [
64
]. The effect in this study was as early as 14 days
of treatment which is significantly shorter that that showed
with gelatinized maca in which effects were observed after 8
weeks of treatment.
A further RCT assessed the effects of maca in patients
with mild erectile dysfunction using the International Index
of Erectile Dysfunction-5 and showed significant effects on
subjective perception of general and sexual well-being [
65
].
A study was not included in the systematic review
because no placebo effect was assessed [
66
]. In such
study, maca was administered in two doses (1.5 g/day and
3–0 g/day) to patients with selective-serotonin reuptake
inhibitor-(SSRI-)induced sexual dysfunction. The Arizona
Sexual Experience Scale (ASEX) and the Massachusetts
General Hospital Sexual Function Questionnaire
(MGH-SFQ) were used to measure sexual dysfunction.
Subjects on 3.0 g/day maca had a significant
improve-ment in ASEX (from 22.8
±3.8 to 16.9
±6.2; z
= −2.20,
Table 2 - Properties of maca after in vivo administration in different animals species (left) and Properties of maca observed in clinical trials on humans (right) [4]
Fig. 5 - Score of health status from men and women residents of Carhuamayo, Junin at 4100 m in the Peruvian Central Andes. Upper line: population consuming extracts of maca. Bottom line: population not
consuming maca [4].
Forsch Komplementmed 2009;16:373–380
Lepidium meyenii (Maca): Biological Properties 375
Experimental Studies
A summary of biological properties is presented in table 2.
Nutritional Properties
The nutritional properties of maca as described traditionally [2] have been proven scientifically in studies on rats [15] and fish [16, 17]. The nutritional components of maca have been described early [18].
Male Reproduction
The effects of maca on fertility were described as early as 1653 [2]. At an international level, the first description that maca improves sexual behavior in rodents, was published in 2000 [19]. In 2001, maca was reported to increase sperm count in men [20].
Sperm Count: Scientific studies have shown that maca may
increase daily sperm production (DSP), epididymal sperm count or vas deferens sperm count in both healthy rats [20, 21] or when spermatogenesis was disturbed by different ex-perimentally-induced abnormal conditions such as exposure to high altitude [22], injection of malathion [23], or adminis-tration of lead acetate [24].
One of the effects of maca administered for 7 days of treat-ment was that it increased the lengths of stages VIII (spermia-tion) in a dose-response fashion [22]. This allows an increased number of sperm in the epididymis. Also, an increase in the frequency of stages IX–XI of mitosis was observed after 7, 14, or 21 days of treatment with maca [20, 25].
More recently, it has been described that black maca has a better effect on sperm count than yellow maca, whereas red maca was without any effect. Effects on increased epididy-mal sperm motility were observed only with black maca after 42 days of treatment. As compared to the control group, red Maca neither affected testicular and epididymal weight nor epididymal sperm motility and sperm count [12].
In studies with varying treatment periods between 1–84 days, the first action of black maca could be observed at the epididymal level in terms of an increased sperm count after 1 day of treatment; whereas an increase in sperm count in the vas deferens was only observed at day 3 of the treatment. Fi-nally, an increase in DSP was observed after 7 days of treat-ment. Testicular testosterone was not affected even after 7 days of treatment with black maca [26]. Treatment for 84 days with yellow or black maca increased epididymal sperm count with-out affecting DSP. Maca seems to modulate sperm count at the reproductive tract level [27].
Aqueous extract of maca is only effective after boiling pul-verized maca hypocotyls in water. The greatest effect on sper-matogenesis was observed with the ethyl acetate fraction of the hydroalcoholic extract of black maca [28].
Prostatic Hyperplasia: Prostate growth is mainly regulated
by androgens, especially by dihydrotestosterone (DHT) [29]. However, estrogens also play an important role in this proc-ess, as 3-β-diol is responsible for tissue apoptosis through its union with estrogen receptors [30]. An alteration to the bal-ance of testosterone and estrogen might contribute to the for-mation of benign prostatic hyperplasia (BPH) [31].
Prostate pathologies in adult men occur frequently, caused by an abnormal prostate growth which can be malignant. BPH is associated with a series of symptoms of the lower urinary tract [32]. The incidence of BPH increases proportionally with age, starting at about 50 years of age [33].
The treatment of choice for prostatic hyperplasia is an inhibitor of 5-alpha-reductase II (5AR-II), an enzyme that specifically inhibits the conversion from testosterone to DHT in the reproductive tract. Red maca has been shown to ef-fectively reduce prostate size in rats and mice in which hy-perplasia had been induced with testosterone enanthate [11, 34]. When red maca treatment was compared to the 5AR-II inhibitor, it was observed that red maca reversed the effect of testosterone enanthate even more efficiently than the con-ventional treatment [27]. Moreover, only maca reversed the effect of testosterone enanthate on the intraprostatic concen-tration of zinc [personal data]. Histological analyses revealed that BPH increased the acinar and stromal areas [34]. Both treatments reduced the acinar area, but only maca also re-duced the stromal area [34]. The 5AR-II inhibitor acts on the androgenic pathway, regulating the prostatic growth, but this organ is regulated by androgens and estrogens. These results combined with the fact that red maca does not exert its action on another androgen-dependent organ, such as the seminal vesicle, suggests that maca acts on the estrogen pathway.
Table 2.Properties of maca after in vivo administration in different species
Property Reference
Rats
Increase sperm count Gonzales et al., 2004 Chung et al., 2005 Increase male sexual behavior Zheng et al., 2000
Cicero et al., 2001, 2002 Nutritional Canales et al., 2000 Anti-stress Tapia et al., 2000
Lopez-Fando et al., 2004 Prevent testosterone-induced
prostatic hyperplasia Gonzales et al., 2005 Against osteoporosis Zhang et al., 2006 Learning and memory Rubio et al., 2006
Mice
Increase male sexual behavior Zheng et al., 2000 Increase embryo survival Ruiz-Luna et al., 2005
Guinea pigs
Increase number of offsprings Alvarez, 1993
Fish
Nutritional Lee et al., 2004, 2005 Increase embryo survival Lee et al., 2004
378 Forsch Komplementmed 2009;16:373–380 Gonzales/Gonzales/Gonzales-Castañeda
Clinical Studies
Several clinical trials have assessed the efficacy and safety of maca consumption. Two of them included healthy men [44, 66, 67], and four trials included participants with pathological con-ditions [68–72]. The majority of these studies focused on the effects of maca on sexual behavior and sperm count (table 3).
In one clinical trial performed in healthy men, using a dou-ble-blind placebo-controlled, randomized, parallel-group de-sign, treatment with three different schedules of gelatinized maca (45 men in total; 30 men were administered 1,500 mg/ day maca, 15 men were administered 3,000 mg/day maca) was compared with placebo (15 men). Treatment with maca as compared to placebo increased sexual desire [44, 67] after 8 weeks of treatment; improved mood and anxiety, and in-creased activity [73]. No difference was observed between groups taking 1,500 or 3,000 mg/day of maca. A second study in 9 men who had received maca for 4 months showed an in-crease in sperm count and sperm motility [66]. Serum hor-mone levels were not affected by treatment with maca [66].
In three other studies on patients with sexual dysfunction, treatment with maca improved libido and sexual well-being. In fact, maca at a dose of 2,400 mg improved the perception of general and sexual being in men with mild erectile dysfunction after 12 weeks of treatment [72]. Similarly, a double-blind, randomized, parallel-group study was performed in women with sexual dysfunction due selective serotonin reuptake in-hibitors (SSRIs), in order to determine the effect of maca on sexual dysfunction. Patients were administered either 1.5 or 3.0 g/day of maca. Improvements in sexual dysfunctions were observed at a dose of 3 g/day [70]. In postmenopausal women, treatment with 3.5 g/day of maca for 6 weeks reduced psycho-logical symptoms including anxiety and depression and re-duced measures of sexual dysfunction [69].
As maca may increase sexual behavior in rodents [19, 37, 38] and men [67, 69, 70, 72], it is assumed that these effects are due to an increase in testosterone concentrations or to a testosterone-like action. However, only one study has demon-strated an increase in serum testosterone levels in mice [42]; in other studies, serum testosterone levels were not affected by administration of maca to rats [26] or men [44, 66, 67]. In recent studies, maca powder and maca extract were unable to activate androgen receptor-mediated transcription in prostate cancer cell lines [39] or in a yeast-based hormone-dependent reporter assay [69].
Maca has been shown to reduce scores in depression and anxiety inventories, act as an energizer, and increase sexual de-sire and sperm count [66, 67, 69], leaving unaffected serum lev-els of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, testosterone, and estradiol [44, 66, 69, 73].
Finally, in a randomized double-blind study on 95 patients with osteoarthritis, a combination of Uncaria gianensis (300 mg) and maca (1,500 mg) was administered twice a day for 8 weeks and compared with a treatment with glucosamine sulfate.
Both treatments substantially improved pain, stiffness, and functioning in the patients [68]. However, as the study did not include a placebo control group, glucosamine effects remain unclear.
A summary of the significant results yielded in clinical tri-als is presented in table 3.
Modes of Action
According to the results obtained in different studies, maca may act as an antioxidant [74] or as an immunomodulator [16, 75] de-pending on its biological properties. The maca constituent with antioxidant capacity has high polarity and can be extracted by methanol [17]. However, it has been shown that neither aque-ous nor methanolic extracts of maca prevent oxidative damage in hepatocytes intoxicated by t-butyl hydroperoxide [43].
It has also been suggested that some effects of maca act through paracrine control affecting activity of IGF-1 [75]. In fact, maca enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7. Interleukine-1 beta (IL-1 beta) has sev-eral deleterious effects on chondrocytes. Maca may prevent most of these effects. The authors suggest that maca may act on fertility and fetal development also by activating IGF-1 production in target tissues [75].
Toxicity
Maca has been used for centuries in the Central Andes of Peru and no toxic effects have been reported if it was con-sumed after boiling [3]. Previous review data on in vivo and in vitro studies with maca indicate that its use is safe [3]. Further evidence shows that aqueous and methanolic extracts of maca do not display in vitro hepatotoxicity [43]. Moreover, freeze-dried aqueous extract of maca (1 g/kg BW) in mice did not reveal any toxic effect on the normal development of pre-implanted mouse embryos [76].
Table 3. Properties of maca observed in clinical trials on humans Property Reference
Increase sperm count and
sperm motility Gonzales et al., 2001 Increase sexual desire Gonzales et al., 2002;
Zenico et al., 2009; Dording et al., 2008; Brooks et al., 2008
Anti-stress Gonzales et al. (unpublished data) Decrease score for anxiety
and depression Gonzales, 2006 Energizer Gonzales, 2006 Improves pain, stiffness and
However, the most important commercial aspect about maca’s employement in medical field is its aphrodiasiac power. Sexual dysfunctions are highly prevalent in our society worldwide, and the occurrence of sexual dysfunctions increases directly with age for both men and women [12]. Most sexual problems relate to sexual desire (interest in sex) in both females and males and male erectile dysfunction (ED). Interest in medicinal plants to treat sexual dysfunctions has increased in the last 20 years.
Maca has been described to improve sexual behavior in experimental animals [11], although conflictive results may be observed in human [14]. Traditionally maca has been referred as an energizer that may be the reason why maca its considered sexual stimulant and an healt enancher as well. In a randomized studywe were unable to demonstrate effect of maca on penile erection in apparently healthy adult men after 12 weeks of treatment with gelatinized maca compared with results using placebo [Gonzales, unpublished data]. According to a recent review, two randomized clinical trials (RTC) suggested a significant positive effect of maca on sexual dysfunction or sexual desire in healthy menopausal women or healthy adult men , respectively, while the other RCT according to the reviewers failed to show any effects in healthy cyclists. However, analyzing results from such studies, authors showed that maca extract significantly improved the self-rated sexual desire score compared to the baseline test (P = 0.01), and compared to the placebo trial after supplementation (P = 0.03) [4]. Lots of other clinical trials have been conducted to test other maca’s effects on healt. These are reported in table 2.
Mode of action. According to the results obtained in different studies, maca may act as an antioxidant or as an immunomodulator [4] depending on its biological properties. The maca constituent with antioxidant capacity has high polarity and can be extracted by methanol. However, it has been shown that neither aqueous nor methanolic extracts of maca prevent oxidative damage in hepatocytes intoxicated by t-butyl hydroperoxide.
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In fact, maca enhanced basal IGF-1 mRNA levels in human chondrocytes by 2.7. Interleukine-1 beta (IL-1 beta) has several deleterious effects on chondrocytes. Maca may prevent most of these effects. The authors suggest that maca may act on fertility and fetal development also by activating IGF-1 production in target tissues. [4]
Fig. 6 - Some maca commercial products (source: www.terrasoul.com)
In summary maca is a plant with great potential as an adaptogen and appears to be promising as a nutraceutical in the prevention of several diseases.
1.2 Plant stress: a panoramic view
Though the term “stress” has been defined exactly in mechanics, in the biology field it has been given several different meanings. Probably due to an extension of the physical meaning to living organisms, many of these definitions converge in attributing “stress” to any environmental “unfavourable” factor for the subject under consideration. In this way, we can try to give a generic definition of stress as an altered physiological condition caused by factors that tend to alter a “normal” equilibrium triggering
Commentato [SM1]: Adaptogens or adaptogenic su bstances, compounds, herbs[1] or practices refer to the pharmacological concept whereby administration results in stabilization of physiological processes and promotion of homeostasis, for example, decreased cellular sensitivity to stress.[2]
different responses.
Talking about plants, since they are sessile organisms, they are used to adapt their metabolism and physiologic processes to environmental changes which fluctuate regularly and predictably over daily and seasonal cycles. Thus, every deviation of a factor (biotic or abiotic one) from its optimum does not necessarily result in stress. Consequently, we can arbitrarily say that a stress, in the sense of a stress inducer, is an unusual or a usual factor, of the biotic and abiotic environment, modified in such a way (excess or deficit), that it has the capability of causing metabolism disfunctions or aberrant physiology. This mostly results in a strain for the plant to adaptate to the new condition. As we already said, plants are confined to the place where they grow and so they have a limited capacity to avoid unpredictable unfavourable changes in their environment like extremes of temperature, water shortage, unbalanced light or mineral nutrients, attack by pathogenic bacteria, fungi, viruses and viroids (Fig. 7). This could be the reason why they have developped ingenious molecular strategies to defend themselves against such biotic and abiotic stresses, coming most often combined with an alteration of growth and developmental patterns. This explains why the concept of stress is intimately associated with the external conditions that adversely affect growth, development or productivity [21]. That’s the reason why environmental stresses represent one of the main factors which most limit agricultural productivity worldwide.
Fig. 7 – Biotic and abiotic stresses that affect plant growth, development and yield (source: biologydiscussion.com)
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However, duration, severity, and rate at which a stress is imposed, togheter with the plant’s features (including organ or tissue identity, development age, and genotype), influence how a plant responds. Depending on how the plant responds to a stress we can identify three possibilities: in one case, tolerance, plants have mechanisms that maintain high metabolic activity (similar to that in the absence of stress) under mild stress and reduced activity under severe stress. In contrast, mechanisms of avoidance, involve a reduction of metabolic activity upon exposure to extreme stress. Commonly, a plant species may have several tolerance or avoidance mechanisms, or a combination of both. In the case of susceptibility, the stress esposure brings the plant to death.
We can group the first two case into a more general concept, that concern those mechanisms that permit the plant survival and that allow it to tolerate or avoid a stress termed resistance (fig.8). Furthermore, another important distinction in plant stress physiology must be made between plant responses, specifically between adaptation and acclimation. Adaptation occurs by various mechanisms at the genetic level in populations over many generations, like a kind of micro-evolutionary processes that change gene frequencies of a population over time.
Fig.8 - Plant responses to environmental stress in correspondence with stress and plant characteristics. [21] state, upon exposure to extreme stress (Osmond et al.
1987). Commonly, a plant species may have several tolerance or avoidance mechanisms, or a combination of both. For instance, drought stress may induce drought tolerance that can be followed by desiccation tolerance: in the later “dormant” state, the organism can survive the dry state for long periods, i.e. years. Notice that the ability to rehydrate without damage can be considered as a part of the desiccation toler-ance. The other issue is immediate or delayed DAM-AGES, through somaclonal variation, mutation, neo-plastic progression, ultimate death via necrosis and/or apoptosis (Figure 3).
The intensity of stress (pressure to change exerted by a stressor) is not easily quantified. Stress could occur at a low level, creating conditions that are mar-ginally non-optimal, with little effect expected. How-ever, if this mild stress continues for a long time, becoming chronic stress, the physiology of plants is likely to be altered. In contrast, conditions could be-come difficult quickly, resulting in acute condition. This shock pattern of stress is likely to induce signif-icant changes in a short time frame. Toxicologists, particularly those in the area of pollution studies, have developed the concept of dose. Dose is defined to be the magnitude of perturbation times the length of time the stress is applied. It thus accounts for the influence of both intensity and duration on
physiolog-ical performance. Stress can be dramatic when it is applied for a short duration and high intensity, or when it is applied for a long duration at low intensity. Plant responses to chronic stress and acute stress may be very different even though the dose is the same.
In plant stress physiology an important distinction must be made between ultimate (ADAPTATION) and proximal (ACCLIMATION) plant responses. Adapta-tion occurs by various mechanisms at the genetic level in populations over many generations. Micro-evolutionary processes change gene frequencies of a population over time. In a stressful environment, it is logical to assume that specific genotypes with appro-priate gene combinations (those that confer the abil-ity to survive and reproduce) are dominant in the pop-ulation. Those particularly favourable gene combinations in plants that inhabit stressful environ-ments are called adaptations.
Populations that have adapted through evolution-ary processes acting at the genetic level to a particu-lar climatic regime are by no means static systems. On the contrary, plants have an incredible ability to adjust physiological and structural attributes on the scale of seconds or seasons within a single genotype: this is acclimation. In other words, during acclima-tion, an organism alters its homeostasis, its steady-state physiology, to accommodate (further) shifts in its external environment. For instance, prolonged
ex-Figure 3.Plant responses to environmental stress in correspondence with stress and plant characteristics.
As a matter of fact in a stressful environment, it is logical to assume that specific genotypes with appropriate gene combinations (those that enhance the enviromental fitness) became dominant in the population. Those particularly favourable gene combinations in plants, that inhabit stressful environments, are called adaptations. This are definable kind of ultimate responses. On the contrary, plants have an incredible ability to adjust physiological and structural attributes on the scale of seconds or seasons within a single genotype and this is the mechanism on wich acclimation occurs. In other words, during acclimation, an organism alters its homeostasis, its steady state physiology, throught an adjustment of plant growth and metabolism, to accommodate shifts in its external environment. On a long-term scale, acclimation is enhanced in plants because of the modular nature of metabolism and growth. Plant parts can be abscised and regrown in a new morphology or anatomy, and specific organs can be enhanced by increasing their numbers or size. On the other side, a short-term basis (such seconds or minutes), the proteosome can be modified, like metabolism regulators can be released or activated, or transcription and translation can be up or down regulated.
On the base of this facts, contrary to the general opinion, stresses must not be automatically associated with adverse detrimental effects.
1.3 Salinity stress
In this work we focus on salinity stress, that is so much strongly related to osmotic stress, that it’s often hard to distinguish one from another. We can identify two main effects of an excess of salt in the plant’s growth medium: a short term and a long term response. The first one is the one much more related with osmotic stress, and as a matter of fact the earliest response of a non-tollerant plant exposed to salinity is a reduction of germination and growth rate, synthoms that are strongly related with a difficulty in water up-take due to external osmotic pressure. In shoots it would appear at first
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sight that leaves grow more slowly after exposure to salinity because of a water deficit: the response is very rapid, is usually proportional to the osmotic potential of the external solution, and is easly reversible [22]. When plants are exposed to salinity in laboratory experiments, there is a rapid and temporary drop in growth rate followed by a gradual recovery to a new reduced rate of growth. The temporary effects are clearly due to rapid and often transient changes in plant water relations. The subsequent changes in growth rate, and the molecular or metabolic events occurring, are not so easily ascribed to water stress or to salt-specific effects.
Litterature shows more than once that during a short time in salinity, there will be a significant decrease in growth rate, but the decrease may be the same for species that have quite different reputations for salt tolerance. For example, durum wheat has the reputation of being more salt-sensitive than bread wheat, but over short periods of time in salinity, no differences have been found between durum and bread wheat cultivars, nor between barley and triticale cultivars [23]: what else could act so quickly but a fall in leaf water potential due to a decrease in root water potential caused by the external salt?
Also in roots, there are rapid and transient reductions in growth rates after sudden increases in NaCl, and similar changes occur with KCl and mannitol, strenghtening the hypotesis that, as well as what occurs in leaves, these effects are entirely due to sudden changes in cell water relations. [24] Long term effects of salinity stress are salt-specific ones. These effects become visible after several days and under high salinity, and if the plant has a poor ability to exclude NaCl, marked injury in older leaves might occur within days, as found for white lupin once the salinity increased above 100 mM NaCl [23]. In salt-sensitive species salt injury is due to Na+ or Cl– (or both) accumulating in transpiring leaves to excessive levels, exceeding the ability of the cells to compartmentalize these ions in the vacuole, or for the unability of the plant to keep the ions out. When vacuoles becomes ‘full’, Na+ or Cl– must build up in the cytoplasm where they will inhibit enzyme activity, or they can build up in the cell walls and dehydrate the cell. [24]
Focusing on germination, salt and osmotic stresses could reduce it either by limiting water absortion by the seeds, by affecting the mobilisation of stored reserves or by directly affecting the structural organisation or synthesis of proteins in germinating embryos. In particular it occurs by the inhabit of enzymes involved in the solubilization of proteins and by reduction of α-amilase activity that would induce the conversion of starch into sugars. Furthermore we can frequently detect an inhibition of gliossisome malate syntase and isocitrate syntase catalysis that are those enzymes involved in the glucose syntesis starting from free fat acids.
These parameters could be affected by both the ionic and the osmotic components of salt stress although the relative importance of each component is a matter of debate and could differ among species and even among cultivars of a given species [25]. However, we find as a rule in litterature that under salt stress germination is reduced in most non-halophyte plants.
1.3.1 Quantifying the effects of salinity
When studing the effects of salinity on a species it is possible to determinate how much the stress affect the normal functions of the plant by using several aproaches. In this work, we focus on germination that can be defined, in a agricultural prospective, as the emergence and development from the seed embryo of those essential structures wich are indicative of the ability to produce a adult plant under favorable conditions.
This definition focuses on the reproductive ability of the seed, an essential objective in agriculture, even if mainly physiologists confine germination in being the emergence of the radicle through the seed coat without considering the other essential structures such as the epicotyl or hypocotyls that become the above ground parts of a successful seedling.
Studing the effects of a stress on plant germination would mean evaluate it starting from the seed’s imbibition to the developement of roots and cotyledons and it is possible to quantify the effects mainly by monitoring germination rate, plant growth, photosyntesis, water relations, ion relation, and soluble
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sugars content.
Germination rate. Water is a basic requirement for germination. It is essential for enzyme activation, breakdown, translocation, and use of reserve storage material. This is the starting point for developing the other seedling’s structures and then for plant growth. When water uptake is prevented by the high osmotic potential in the soil, due to the presence of high concentration of solutes, that will result in a delay or in a defect or lack of germination. In seeds water relations are mainly related to their imbibition. Some seeds possess mucilage, as in the case of maca, which is extruded from the epidermal cells of the seed as it imbibes water. The mucilage serves to increase the contact of the seed with the soil by increasing the number of pathways through which water may be absorbed by the seed that usually leads to increasing germination. Mostly, imposition of all stresses resulted in significant decreases in germination rate, but for example in the case of sorghum the maximum decrease occurred due to NaCl and PEG treatments [26] and similar declines in seed germination have been reported in the literature. The decrease in germination rate, particularly under saline and osmotic stresses, may be due to the fact that seeds seemingly develop an osmotically enforced “dormancy” under water stress conditions. This may be an adaptive strategy, to prevent germination under stressful environment thus ensuring proper establishment of the seedlings [27]. Studing the effects of salt on germination, will requires in the first place the monitoring of germination percentage in different conditions of salt concentration referred to a control, durig a time lapse, to evaluate effects on delay or inability of seed germination.
Plant growth. In an experimental setting, one of the first observable responses after salinity imposition is a reduction in shoot growth [28] ascribable in the first place and in an early response to water deficit. To describe this reduction in plant growth, two distinct approaches can be used: a destructive harvest, or a non – destructive one. The first one involves separation of plants into parts, such as shoot from root in order to record data of fresh and dry mass, that can also be combined with
other measurements, such as root length, plant height and leaf area. Those operations can be easily made. The non - destructing one, for exemple digital imaging, otherwise requires more specialized and expensive equipment. In any case, in order to determinate an hypotetic relative decrease in plant biomass, wich will represent a reduction in growth, we need to compare the total biomass of stressed and control plants during and, especially, at the end of the experiment since as already said salinity stress also affects cell expansion in young leaves, generally causing a decrease in leaf area maybe just due to a reduction of water-uptake.
In particular, a recognizable indication of salinity stress is a reduction in shoot growth, which, in turn, can change the allocation of biomass between roots and shoots that generally results in decrease of root mass upon salt stress to a greater extent than under control conditions. In addition to this, relative leaf dry weight (RLDW), that is the ratio leaf’s dry mass of the treatment compared to the control, provides a measure of the effect of salinity on what is effectively leaf thickness: reduction in RLDW under salinity stress may be adaptive since it may be related to the leaf’s thicker cell walls to reduce water loss and so have a greater diluition of solutes [28].
Plant water relation. When possibly the seedling has estabilished, one of the main components of a plant’s water relations is water content in tissues, that is the output of water potential and hydraulic conductivity, namely the maintenance of water levels in tissue and that is the primary determinant of cellular growth and function. The water fraction of a tissue can be easly assessed by subtracting dry weight to fresh weight. Plants under stress often lose some water from their tissues, which can have rapid and large effects on cell expansion, cell division, stomatal opening, abscisic acid (ABA) accumulation, etc. [29]. Of course, a plant with a higher relative water fraction (RWF, the WF under stress conditions relative to control conditions) is better able to maintain its water content in the shoot upon salt stress.
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conditions, as the accumulation of toxic ions such as sodium can perturb the plant’s ability to control accumulation of other ions. In most species, Na+ appears to accumulate to toxic levels before Cl- does; thus, we focus here on Na+, because reducing Na+ in the shoot, while maintaining K+ homeostasis, is a key component of salinity tolerance in many cereals and other crops.. In litterature it is often found recommend calculating Na+and K+ concentrations (mM), rather than Na+and K+ content (umol g-1 dry mass) because the latter does not account for differences in the water status of, and thus differences in concentration in the aqueous phase, which is the primary factor directly affecting transport and biochemical processes, might be missed. It’s also important to distinguish ions concentration between leaves and roots.
In litterature we find that measurements of ion contents in plants under salt stress revealed that halophytes accumulate salts whereas glycophytes tend to exclude the salts [30].
This is becouse glycophytic plants leaves cannot retain high levels of salt without injury, and by excluding salt from absorption they are often not challenged by toxic salt concentrations in their natural habitats. By comparison, halophytes preferentially accumulate salt in the leaves, adopting different strategies and sometimes these is used to balance the osmotic potential of the salts outside the plant [30]. This difference between halophytes and glycophytes in their contrasting adaptive strategies is significant.
Photosintesis. Upon salinity stress, a substantial decrease in a plant’s stomatal aperture can be observed, but the rates of photosynthesis per unit leaf area sometimes remain unchanged (Munns and Tester, 2008). Stomatal closure is usually a consequence of water retention related problem and this usually results in internal reduction of CO2 that brings to a decrease in the activity of several enzymes including RuBisCO (Chaves et al., 2009), limiting carboxylation and reducing the net photosynthetic rate. [28] Chlorophyll is the principal agent responsible for photosynthesis and, under adverse conditions, chlorophyll level represents a good indicator of photosynthetic activity [31]. For such
reason, we find in many works the determination of photosynthetic pigments content in order to infer the effect of salt concentrations on the photosynthetic activity, for example quantifying the amount of chlorophyll in the leaf (expressed, for instance, as µg Chl g-1 tissue o µg Chl cm-2 tissue) [28]. For this purpouse several aproaches are used like non- invasive methods that capture photosynthetic responses include measurements by infrared gas analysers (IRGAs) and pulse amplitude-modulated (PAM) chlorophyll fluorometers, or invasive ones like a spectrophotometric analysis of leaves extract.
Sugars content. Carbohydrates, especially starch, represent the major reserve substance in most seeds. In normal condition during early germination, mobilisation of storage carbohydrates occurs, especially after radicle emergence and once the high molecular weight carbohydrates are mobilised, they are converted into soluble forms as sucrose, glucose and fructose, that are readily transportable to sites where they are required for growth. Furthermore, these sugars belong to those compounds called compatible solutes or osmolytes which do not interfere with normal biochemical reactions and they acccumulates in the cytoplasm to achieve ion balance in the vacuoles, thus involved in a mechanism known as osmotic adjustment. Yancey (2005) describes compatible solutes as neutral molecules, non- toxic, that stabilize proteins and membranes and prevent denaturation at high salt concentrations and, even at low concentrations, compatible solutes avoid water loss, ion imbalance, reducing intracellular concentration of salts (. In addition to glucose, fructose and sucrose, compatible solutes accumulated in the cytoplasm of plant cells under salt stress include proline, valine, isoleucine, aspartic acid, pinitol, betaine, mannitol and inositol [31].
It is well known that salt tolerant plant since they collect salt from the cytoplasm into the vacuole creates a strong osmotic gradient across the vacuolar membrane. This gradient is balanced by an increase in the synthesis of solute molecules in the cytoplasm.
Osmotic adjustment is regarded as an important adaption of plants to salinity because it helps to maintain turgor and cell volume. [30]
