Introduction
Abiotic stress is a major global problem limiting crop productivity. Stress
factors are a serious problem limiting the yield potential of modern cultivars. Abiotic
stresses like salinity and heavy metals are the primary causes of crop loss,
particularly cereals. These stresses are increasing because of pollution, declining
availability of good quality water and land degradation. Stress results in nutritional
imbalances in the plant causing reduction in water uptake and increase in toxicity,
decreasing the product. The major abiotic stresses that limit and pose a threat to
agricultural production in many parts of the world (Altman, 2003) are salinity and
drought. Abiotic stresses are the primary cause of crop failures in India. It has been
estimated that about 8.6 million ha of land is affected by salinity in India (Pathak,
2000). Tolerance to salinity is a complex phenomenon in which primary reaction is
to overcome osmotic and ionic stresses and subsequently the secondary stresses (e.g.
oxidative stress) are combated. Glycophytes are thought to have special role in
mechanisms for salt tolerance (Levitt, 1980; Hasegawa et al., 1986; Rains, 1989; He
and Cramer, 1993; Hasegawa et al., 2000; Munns et al., 2002; Mansour et al., 2003;
Mansour and Salama, 2004).
Drought is one type of major abiotic stress, increasing day by day. Areas are
affected by water shortage regularly in different parts of the earth, often disturbing
cereal productivity. The arid and semiarid areas that are mainly affected by water
scarcity, have usually contributed only 40 % of the total yield of the entire categories
of food grains (Thakurta, 2010).
Drought Stress induces stomatal closure and this increases the oxidative
stress on the plant tissues resulting in harm to others important bio-molecules
(Sairam and Tyagi, 2004). Plants protect themselves from drought stress. Some
morphological adaptations towards drought stress leads to multiplicity of
biochemical processes and physiological processes which act as mechanism of
resistance against stress. A group of anti–oxidative enzymes like SOD, CAT, GPX,
APX etc are activated during stress to bind to the reactive oxygen species. Some
reports say that there is direct connection between enhanced antioxidant enzyme
behavior and improved tolerance to various environmental stresses (Liu et al., 1998;
Sayfzadeh and Rashidi, 2011).
1
Introduction
Salts and heavy metal effluence is one of the main problematic
environmental trouble faced by humans. Production of agricultural soils is limited
by salinity in large areas of the world. Salts and heavy metals, poses problems due to
its extensive agricultural and industrial uses. Plants are extremely sensitive to salts
and heavy metal toxicity, displaying metabolic turbulence and growth inhibition,
even at levels slightly higher than the normal. Reduction in plant height (up to
40-52%) due to salinity stress in Eleusine coracana varieties has also been observed.
Salinity reduces the capacity of plants to make use of water and causes a decline in
growth rate, as well as changes in plant metabolic processes. One of the reasons of
high salinity is higher concentration of cations such as sodium, calcium, magnesium
and anions such as chloride, phosphate, and nitrate in the soil. The effect of salinity
on plant growth is a complex characteristic that involves osmotic stress, ion toxicity,
mineral deficiencies, physiological and biochemical perturbations. Salinity also
creates certain problem of ion toxicity and high concentration of sodium also
damages the cells. The cause of reduced growth of crop in saline soils may be
imbalance in mineral nutrition, reduction in water uptake and salt toxicity to plants.
The salinity stress in crops is different at different growth stages; germination and
seedling being the most vulnerable ones (Brown and Hayward, 1956).
Toxic effects of salt also depends on the plant species and time duration.
Plants cultivated in saline soils have varied ionic composition and concentrations of
dissolved salts. Decrease in the content of total sugars also takes place due to salt
stress. At the molecular level, there are many adaptive mechanisms for salt tolerance
viz (1) ion homeostasis (2) detoxification or repair. These mechanisms control stress
damage (Zhu, 2002). Homeostasis of sodium, play a vital role for the tolerance of
salt stress.
Helicases are also affected through abiotic stress because it influences the
machinery of cellular gene expression. Molecules used in nucleic acid processing
are expected to be affected as well (Sanan et al., 2005; Puranik et al., 2011). It has
also been observed that salt stress reduces the leaf growth rate, leaf emergence rate,
and overall shoot development. Excessive toxicity of heavy metals and salts to
plants has posed severe destruction to crops and requires some strategies of
2
Introduction
protection. Eleusine coracana which mainly grows in dry condition faces the
toxicity of salts and heavy metals which may have negative effect on its yield.
Soil contamination in cultivated fields by industrial effluents full with toxic
heavy metals has emerged as a new hazard to agriculture. Most of the effluents and
wastes contain heavy metals in an amount toxic to crop plants (Hutchinson and
Whitby, 1974; Temple and Bisessar, 1981; Khan et al., 2006). Extreme
accumulation of heavy metals like nickel, copper, cobalt etc occurs in the soil due to
metal mining, processing and other industrial activities (Foy et al., 1978;
Maliszewaska et al., 1985).
Heavy metals that limit growth and yield of several crop plants are important
group of soil pollutants. Heavy metals contamination causes a serious problem
because they cannot be naturally degraded, like organic pollutants and accumulate
in different parts of the food chain. Heavy metals make a major involvement to
environmental pollution as a result of human activities such as mining,
electroplating, energy smelting, sludge dumping, military operations, fuel
production, power transmission and intensive agriculture (Nedelkoska and Pauline,
2000).
They run the risk for primary consumers, secondary consumers and
ultimately humans (Zeller and Feller, 1999). During the past few years unlimited
developmental activities such as urbanization and industrialization have given rise to
serious problems of environmental contamination. Increase in the level of heavy
metals poses a pervasive risk to the natural ecosystem. Many heavy metals, in trace
amounts, are essential for various metabolic processes in organisms, but if present in
high concentration, they create physiological stress.
The abiotic stress factors discussed above decreases the growth rate of
explants in in vitro conditions also. Plant yield affected by stresses is due to less
availability of water or disorder in nutrients uptake. This may cause deficiency
symptoms or ion toxicity leading to alteration in physiological and biochemical
processes (Munns, 1993). Imbalance in phytohormone level is also one of the causes
of growth retardation in stressed plants (Pujari and Chandra, 2002).
3
Introduction
Whatever may be the mechanism of damage due to stress; development of
stress tolerant plants can only be a strategy to win over the problem of decreasing
global food production. Several such schemes have been proposed for development
of salt tolerance in wheat and other crop species (Kingsbury and Epstein, 1984;
Kelman and Qualset, 1991; Karadimova and Djambova, 1993; Pecetti and Gorham,
1997; Munns and James, 2003). Transgenesis and Classical breeding has already
been attempted to overcome stress without much success (Yoshiba et al., 1995;
Nilsen and Orcutt, 1996). Not much success is attained in this field and needs
attention as both the processes are cumbersome and time consuming. Tissue culture
techniques provide an important and easy tool in developing stress tolerant
somaclonal variants. It has been attempted to develop resistance against stress via
breeding of salt-tolerant plants (Forster et al., 2000; Yoshida, 2002; Munns, 2007;
Bhatnagar et al., 2008; Witcombe et al., 2008). Plants tolerant to salinity are
obtained through cell and tissue culture techniques by two approaches, 1) selection
of mutant cell lines (Tal, 1990, 1993, 1994; Mandal et al., 1999) and 2) screening of
germplasm of the plant for salt tolerance, in vitro (Arzani and Mirodjagh, 1999). A
good knowledge of the biology of the target plant and mechanism of stress tolerance
is prerequisite for successful application of biotechnology to the crop plants facing
salinity. However, to attain best results integration of both plant biotechnology and
classical breeding programs is must (Altman, 2003).
Production can thus be increased by developing ways to overcome stress.
Stress injuries can be reduced by reducing the nutrient and phytohormone imbalance
in the plants. Establishment of ion homeostasis is an essential requirement for plants
to survive under salt stress conditions. The study is therefore planned thinking that
alteration of nutrient level in the growing media can help us to develop stress
tolerant plants.
Some media nutrients are known to overcome the toxicity of salts and heavy
metals. At toxic concentration of salts and heavy metals, addition of these media
nutrients in media can increase the resistance of explant against the toxicity. Nutrient
levels in the medium can have profound effect on embryogenic callus induction and
regeneration. Nutrients used in plant tissue culture can be used by the plant cell as
4
Introduction
buildings blocks for the synthesis of organic molecule or as catalyzers in enzymatic
reactions. Changes in mineral salt composition and concentration can affect medium
water relations, influence the other constituents of tissue culture medium, effects the
loss and decay of plant growth regulators in the medium. Regeneration could be
improved by optimization of the nutrient of tissue culture medium.
Under in vitro conditions, variation in the tissue culture media has been
reported to vary the response of plants to stress conditions. Changing the nutrient
composition of the media altered the requirements and percentage of the explants
responding, by altering enzyme and hormonal activity (Poddar et al., 1997).
Addition of ABA to the induction medium containing 200mM NaCl is also reported
to enhance the acquired tolerance of finger millet seedling (Uma et al., 1995). Their
study also indicated that the synthesis of stress specific proteins is also co related
with the observed acquired tolerance. These results were in accordance with the
previous results of Aarti et al. (2003); Srivastava (2002). Even Jayaprakash et al.
had the same observation in 1998.
It was reported that addition of Ca+2 reduced the adverse effects of salinity
on plants (La Haye and Epstein, 1969; Cramer et al., 1990). Stress-induced changes
in the cytosolic concentration of Ca were also reported by Knight (2008). Exogenous
Ca+2 reduced the perception of stress by the cytoplasm. It has been suggested that
Ca+2 displaces Na+2 from plasmalemma of salt stressed root cells thus decreasing the
influx of ions into the cytoplasm (Cramer et al.,1985; Lynch et al., 1987). Extra Ca+2
added to the medium possibly have the same role in maintaining membrane integrity
and contribute to the ability of plants to resist salt stress.
Even Zeid (2009) observed that addition of arginine and urea reduces salinity
stress. Proline is also a well known and characterized osmoprotectant that occurs
commonly in higher plants. It accumulates in large amounts than other amino acids
in salt stressed plants to overcome stress conditions (Eyidogan and Oz, 2007). It has
been reported that in transgenic plants there are a number of genes which are
activated only under drought/salt stress. These genes might play an important role in
stress management and it is thought that addition of nutrients in the medium
mediate gene activation.
5
Introduction
Agriculture is strongly affected by abiotic stresses, influencing plant
productivity globally. Biotic and abiotic stresses like low and high temperature,
drought, water, salinity, heavy metals, and pesticides etc affects yield greatly,
specially of cereals. These stresses cause osmotic and nutritional imbalance in the
millets.
Millets represents the small-seeded group of the family Poaceae. The
specialty of millets is that they can be grown under extreme environmental
conditions and therefore, especially suited to areas with inadequate moisture or
short-growing cycle and poor soil fertility (Baker, 2003). Although millets are a lot
in number, the most widely cultivated ones are pearl millet- Pennisetum glaucum
(L.)R. Br., finger millet -Eleusine coracana (L.) Gaertn, tef - Eragrostis tef (Zucc.),
foxtail millet - Setaria italica (L.) P. Beauvois, proso millet -Panicum miliaceum
(L.), barnyard millet - Echinochloa crusgall (L.)P. Beauvois, and kodo millet –
Paspalum scrobiculatum (L.). Millets play key role in the maintenance of food
security in the developing world since they are the major food and feed sources.
Belton and Taylor (2004) reviewed that together with sorghum, millets account for
about half of the total cereal production in Africa. In addition to its nutritional value,
millets are also considered as healthy food. In developing countries millets are a rich
a source of human and livestock nutrition (NAS, 1996). Millets contain high amount
of vitamin, calcium, iron, potassium, magnesium, and zinc. The grains of most
millet do not contain gluten, a substance that causes celiac disease or other forms of
allergies (Leder, 2004).
Six millet species namely kodo, finger, proso, foxtail, little and pearlmillet
have an anti-proliferative property and potential in the prevention of cancer
initiation, due to the presence and amount of phenolic extracts (Chandrasekara and
Shahidi, 2011). Maize, Sorghum and Millets follow the C4 photosynthesis system.
Thus they avoid photorespiration and maximally utilize the limited moisture in the
semi-arid regions (Warner and Edwards, 1988). New approaches and technologies
for generating new varieties are necessary to meet the strong increase in cereal
demand worldwide. Creation of transgenic plants with desirable traits is one in these
methods. Initially it was difficult to transform cereal crops due to their recalcitrance
6
Introduction
to in vitro regeneration and their resistance to Agrobacterium-mediated infection.
Later, efficient transformation methods have been established for the major cereals
including rice and maize. The optimization of regeneration method is prerequisite to
increase the efficiency of transformation. Optimization of various factors including
the right type of explants and the proper composition of the medium establishes
efficient regeneration system.
Once optimum regeneration methods are established, valuable agronomic
and nutritional traits like resistance to biotic and abiotic stresses, biofortification of
useful nutritional elements, and altered architecture of the plant, could be routinely
transferred in millets.
Cereals constitute a major source of food for the human population of the
world. Important cereals in day to day use are rice, wheat and millets. Of the all the
millets Eleusine contains more percentage of different nutrients as compared to rice
and wheat. It is a rich source of calcium, iron, fiber, magnesium and potassium.
Eleusine is nutritionally very rich cereal and is a staple food for poor and invalids
(Panda, 1999). Due its nutritional superiority and requirement by poor people
production needs to be improved. Moreover previous work has proved Eleusine to
be a favorable material for tissue culture studies. Eleusine coracana belonging to
family Poaceae was therefore used as a model plant in the present study.
SYSTEMATIC POSITION
Kingdom- Plantae
Order - Poales
Family- Poaceae
Genus- Eleusine
Species-coracana
7
Introduction
Figure:1 Eleusine coracana -Plant
Figure:2 Eleusine coracana -Seeds
E.coracana originated in hot moderate area of the Earth from Africa to Japan
and as well in Australia. It is there in archaeological report of
early African
agriculture in Ethiopia back 5000 years, that it may be originated somewhere in the
region of Uganda country (National Research Council, 1996). It is a significant
staple crop in various area of Africa and has been cultured in Eastern and Southern
Africa from the time of the iron era. Earlier then maize, Eleusine was the essential
crop of the Southern Africa. It was introduced some 3000 years ago in India.
Eleusine is a tufted yearly grass that grows up to a height of 211-621 mm
tall. The leaves and culms are normally green in color. The leaf blades are strongly
keeled, shiny, and hard to break. Length of leaf was 221-500 mm approximately and
7-10 mm width (Fig-1). It has tough root system (Van Wyk & Van Oudtshoorn,
1999). The height of spikelets is 6-8 mm long and 3-5 mm broad, spikelets do not
disarticulate at ripeness. E.coracana ligule is a fringed membrane and the culms,
leaf sheaths are highly flattened. The seeds of Eleusine can be easily detached from
the seed coat (Van Wyk & Gericke, 2000). The seeds are globose in shape (GibbsRussell et al., 1989) (Fig-2).
Eleusine can be developed in any type of soil type and shows good
production, when rainfall is higher than 800 mm per year (Van Wyk & Gericke,
2000). On lower rainfall E. coracana are mostly produced on reddish-brown lateritic
soils with better drainage and sufficient water holding capability (National Research
8
Introduction
Council, 1996). Eleusine has a better capacity to use rock posphate than other
cereals (Flach et al., 1987).
Eleusine has high nutritive value; protein contents vary from 6-14%, fat 114%, food energy 323-350 Kcal and iron 5 mg per 100g. Of the essential amino
acids, the most remarkable is methionine which is reported to be 3% in Eleusine, an
incomparable figure for a cereal grain (National Research Council, 1996). It is
considered to be more nutritious as compared to other cereals and millets.
Table 1: Nutrient composition of Eleusine as compared to rice and wheat
(per 100 gm).
Protein(g) Fat(g)
Crude fibre
(g)
Mineral
matter(g)
1.3
3.6
2.7
6.8
0.5
0.2
0.6
11.8
1.5
1.2
1.5
Crop
Carbohydrate (g)
Eleusine
72
7.3
Rice
78.2
Wheat
71.2
Source: Panda (1999)
Table 2: Mineral content of Eleusine as compared to rice and wheat
(Milligram per 100 gms. of edible protein).
Crop
P
S
Ca
Mg
K
Na
Fe
Cu
Cl
Eleusine
17.4
160
344
401
408
11.0
0.5
0.6
44
Rice
4.0
79
9
38
117
10.0
1.4
0.4
13
Wheat
4.9
128
41
139
284
17.1
4.9
0.5
47
Source: Panda (1999)
Decreasing production due to abiotic stress like salinity and heavy metal is a
serious problem in Eleusine and requires immediate attention by the researchers.
Although traditional and modern breeding approaches are common methods of
overcoming stress, genetic transformation for protecting crop plants from major
stresses is also gaining impetus (Sreenivasulu et al., 2007). Breeding and
development of transgenic for stress tolerance are under progress but are very
complex processes (Babu et al., 2003) and are time consuming and require space
9
Introduction
and labor. But looking to the economics and urgent need of the solution, it is now
time to shift to some easy approach. Keeping this in mind the study was performed
under in vitro conditions. This would help to find an easier, faster, and approachable
solution in limited time and space. Moreover Eleusine is highly responsive in in
vitro studies and gives quick results making the chances of success even brighter. It
is therefore taken as a model plant to study the effect of abiotic stress factors on
plant regeneration and growth. If growing conditions can be redesigned to better
cope with detrimental effects of stresses, agricultural produce can be increased to a
great level.
By the help of the present research work, we have tried to develop stress
tolerance in Eleusine through variations in the tissue culture media. Extrapolating
the same results under field conditions might be helpful in proposing simpler
solutions for the farmers. The main goal of the present study is to develop conditions
that can support and produce economic yields even under moderately saline
conditions. The study was therefore performed with the following objectives:
Objectives:
•
To develop an efficient protocol for plant regeneration system (in vitro)
using callus derived from explants – seeds.
•
To study the influence of various stress inducers on Eleusine coracana tissue
culture and establishment of the detrimental level of each.
•
To optimize the culture condition by adding nutrients with an objective for
minimizing stress injuries and maximizing the yield.
10