“Evaluation of moringa leaf extract (MLE) as a natural plant growth enhancer in wheat

under drought stress conditions”

Introduction

Wheat is one of the oldest cultivated agricultural crops serve as a source of nourishment
and industrial raw material (Mosatjeran et al., 2005). It is used as a staple food for half of the
world's population, therefore considered as a strategic crop (Emam, 2007; Bijanzadeh and
Emam, 2010). In recent years wheat yield is not according to the rising demands of rapidly
growing population especially in 3rd world countries. Nevertheless, a large number of agronomic
factors such as land preparation, sowing time, time and method of nutrients application, weed
management and irrigation scheduling significantly diversified wheat yield. However, the
potential benefits of these agronomic practices cannot be exploitated fully due to the climatic
variabilities such as drought (Bosch and Alegre, 2004), salinity, extreme temperature, low or
high solar radiation (Alexieva et al., 2001) etc.

The global warming is changing the weather patterns all over the world. These climatic
changes generally affect not only the total amount of rainfall in a particular season, but also the
frequency, duration and severity of water stress in the plants at different stages of growth (Kijne
et al., 2003). Water stress affects tillering, number of spike per m 2, number of grains per spike,
1000 grain weight and awn length etc (Plaut et al., 2004; Blum, 2005). Decrease in growth rate is
caused by reduction in radiation use efficiency when drought was imposed at various growth
stages such as tillering, booting, anthesis and grain development (Ashraf, 1998a). Water stress at
anthesis reduces pollination and thus less number of grains is formed per spike (Ashraf, 1998b).
At maturity drought stress resulted in about 10% decrease in yield (Bauder, 2001). Gupta et al.
(2001) reported that number of grains, grain yield, biological yield and harvest index decreased
to a greater extent when water stress was imposed at anthesis stage. The major implication of
drought is the generation of reactive oxygen species (ROS) i.e. superoxide radical (O -2), hydroxyl
radical (OH-) and hydrogen peroxide (H2O2) due to the oxidative stress in plants (Munne-Bosch
and Penuelas, 2003). ROS can injure cell membranes, proteins and nucleic acids (Mittler, 2002).
The response of plants to water stress depends on several factors such as developmental stage,
severity, duration of stress and cultivar genetics (Beltrano and Ronco, 2008).
 The different approaches used to alleviate drought hazards include the development of
stress tolerant plants by screening of stress resistant varieties (Ahloowalia et al., 2004),
exogenous application of plant growth regulators (PGRs), antioxidants (enzymatic and non
enzymatic), certain nutrients, organic and inorganic chemicals (Farooq et al., 2009). Among
growth regulators, cytokinins application under abiotic stress conditions can delay the leaf
senescence directly by scavenging free radicals (Shani et al., 2006). The antioxidants exist in all
plants under stress and involved in detoxification of ROS (Mano, 2002). These are enzymatic
such as SOD (super oxide dismutase), CAT (catalase), POD (peroxidase) etc, and nonenzymatic
i.e. TPC (total phenolics) and AA (ascorbic acid) (Foyer, 2002). The extract of many plants
possess significant amount of antioxidants and therefore, represent a potential source of vitamins
in addition to mineral elements K+, Ca2+ and PGRs such as seaweed extract, humic acid and
Moringa oleifera leaf extract (MLE). These are inexpensive, environmentally friendly and
feasible under natural soil and plant systems (Foidle et al., 2001; Yasmeen et al., 2012a).
Moringa oleifera commonly called as suhanjana in Pakistan. Leaves of moringa are rich in
vitamins (A, B, C), essential minerals (K, Ca, and Fe), antioxidants (ascorbate, phenolics),
proteins and zeatin (Foidle et al., 2001). It contains growth-enhancing substances and can be
used as natural source of growth promoter (Fuglie, 2000), which may reduce the adverse effects
of salinity stress by delaying the leaf senescence and scavenging the reactive oxygen species
(Yasmeen et al., 2012b).

In the view of above consideration, the present study will be conducted with following
objectives

1. to screen drought resistant and susceptible wheat varieties

2. evaluate the potential of moringa leaf extract as growth enhancer in improving
physiological and biochemical stress tolerance of screened wheat varieties under
drought stress conditions,.
 (A) . Background of the research problems to be addressed:

A recent increase in the world wheat production is not sufficient to meet the demands of a
growing population and wheat production in many regions of the world is below average because
of adverse environmental conditions. The wheat cultivation is mainly restricted to the zones with
scarcity of water (Moaveni, 2011). Major threat to reduce growth and yield of a plant is drought
stress (Souza et al., 2004). This shortage of water occurs in region of low rainfall and most of the
wheat is cultivated in such semi arid regions (Deng et al., 2004). The response of plants to water
stress depends on several factors such as developmental stage, severity and duration of stress and
cultivar genetics (Beltrano and Marta, 2008). Drought limits plant growth and field crops
production more than any other environmental stresses (Zheng et al., 2010). According to the
previous studies, there is a link between various physiological responses of crop plants to
drought and their tolerance mechanisms such as high relative water content and water potential
( Datta et al., 2011) membrane stability (Gholamin and Khayatnezhad, 2010) and pigment
content stability under stress (Ghobadi et al., 2011). Moderate to severe water stress drastically
affects various morpho-physiological traits in wheat such as chlorophyll fluorescence, water use
efficiency, dry matter yield (Ehdaie et al., 1991), reduced leaf size, stem extension, root
proliferation, reduced water use efficiency (Farooq et al., 2009), alteration in metabolic activities
(Lawlor and Cornic, 2002), inhibition of enzymatic activities (Ashraf et al., 1995), ionic
imbalance and disturbances in solute accumulation (Khan et al., 1999) or a combination of all
these factors.

Spraying the leaves of many field crops with MLE diluted with water produced some
notable effects such as a longer and more vigorous life span, heavier roots, stems, and leaves,
bigger fruits, and higher sugar levels (Foidle et al., 2001). Since M. oleifera leaves are rich in
zeatin, they can be used as a natural cytokinin source (Price, 2007; Basra et al., 2011). Moringa
leaf extract (MLE) is rich in cytokinin and its foliar application has also been reported as a high
yielding plant growth promotion agent in melon, peanut, corn, sorghum, onion and sugarcane
(Foidl et al., 2001). Being rich in zeatin, ascorbic acid, vitamin E, phenolic compounds, and
minerals (Makkar and Becker, 1996; Fuglie, 1999; Nagar et al., 2006). MLE has the potential to
promote plant growth. Calcium and potassium in moringa leaf extract play essential roles in crop
growth and development through various physiological processes (Hasegawa et al., 2000). Foliar
application has been reported to be growth and yield improving tools in various crops (Jyotsna
and Srivastava, 1998). Exogenous application of moringa leaf extract resulted in higher plant
height, number of leaves, leaf area, leaf area index, fresh and dry herb weight of basil (Prabhu et
al., 2010). According to yasmeen et al., (2012) foliar applied moringa leaf extract could improve
salinity-induced adverse effects by activation of antioxidant defense system and decrease in
accumulation of Na+ and Cl- into shoots under salinity stress conditions.
(B). Research plan: schedule / phasing:

(Screening experiment)

The various approved wheat varieties cultivated in Southern Punjab will be screened for their
tolerance or sensitivity to drought. The screening experiment will be conducted in petri dishes
under laboratory conditions. The various concentrations of polyethylene glycol-8000 (PEG) were
prepared to establish different water potentials i.e. 0, -0.5, -1.0 and -1.5 MPa (Michel and
Kaufmann, 1973). Distilled water was used as a control (0 MPa). 20 Seeds will allowed to
germinate in each petridish using Whatman No.1 filter paper. The design used will be completely
randomized design in factorial arrangement replicated three times. The number of germinated
seeds will be counted for every 12 hours and following germination variables will be estimated.

1. Mean germination time (MGT)

2. Time to 50% germination T50
3. Germination index( GI)
The experiment will be harvested on 7 th day. The data for the following parameters will be
collected.

1. Root length
2. Shoot length
3. Root fresh weight
4. Shoot fresh weight
5. Leaf chlorophyll contents
6. Leaf antioxidant status
7. Relative water content = (Fresh wt- dry wt/Turgid wt – Dry wt) * 100
8. Protein SDS-PAGE electrophoresis
On the basis of above mentioned parameters the 2 varieties from each drought resistant and
susceptible group will be selected for a pot study under glass house conditions. Three drought
stress well will be maintained i.e. well watered (100% field capacity), moderate drought stress
(75% field capacity) and severe drought stress (50% field capacity). Moisture level should be
based on soil water holding capacity. The required drought stress will be imposed after uniform
stand establishment. Exogenous application of MLE will be started after imposing drought.
 Three sprays of MLE will be done i.e at tillering, booting and heading stages. MLE will be
prepared by grinding the young frozen leaves of moringa. The extract obtained will be diluted 30
times by adding the distilled water. The design used will be completely randomized design
(CRD) with three replications. 25 seeds will be sown in each pot. Recommended dose of
fertilizer will be applied. At completion of emergence thinning of plants will be done. The data
for various growth yield and biochemical parameters will be collected.

1. Leaf area plant-1

2. Leaf chlorophyll contents
3. Yield traits
3.1 Number of fertile tillers plant-1

3.2 Number of spikes plant-1

3.3 Number of grains per spike

3.4 100 grain weight (g)

3.5 Biological yield plant-1

3.6 Grain yield plant-1

3.7 Harvest index (%)

5. Biochemical parameters

5.1 Leaf K+ contents

5.2 Leaf antioxidant status

5.3 Leaf total soluble protein contents

All the collected data will be analyzed using appropriate statistical package.
 REFERENCE

Ahloowalia, B.S., M. Maluszynski and K. Nitchterlein. 2004. Global impact of mutation-derived

varieties. Euphytica, 135: 187–204.

Alexieva, V., I. Sergiev, S. Mapelli, E. Karanov, 2001. The effect of drought and ultraviolet radiation on
growth and stress markers in pea and wheat. Plant Cell Environ., 24, 1337–1344

Ashraf. M. Y. I 998a. Photosynthetic efficiency of wheat under water stress conditions.

Pak. J. Scientific and Industrial Res. 41: 15 1-163.

Ashraf. M. Y. 1998b. Yield and yield components response of wheat (Triticum aestivum L.) genotypes
tinder different soil waler deficit conditions. Acta Agron. Hung. 46:45-51.

Ashraf, M. Y., A. R. Azmi, A. H. Khan, S. S. M. Naqvi and S. A. Ala. 1995. Effect of water stress on
different enzymatic activities in wheat. Acta. Physiol. Plant., 17: 615–620.

Basra, S. M. A., M. N. Irfan, I. Afzal, and M. Ashfaq. 2011. Potential of moringa leaf extract as seed
priming agent in hybrid maize. International Journal of Agriculture and Biology 13:1006-1010.

Bauder, J. 2001. Irrigating with limited water supplies. Montana State Univ. Comm. Ser. Montana Hall.
Bozeman, MT 59717, USA.

Beltrano, J. and M.G. Ronco. 2008. Improved tolerance of wheat plants (Triticum aestivum L.) to
drought stress and rewatering by the arbuscular mycorrhizal fungus Glomus claroideum: effect
on growth and cell membrane stability. Braz. J. Plant Physiol. 20(1).

Bijanzadeh, E. and Y. Emam. 2010. Effect of source-sink manipulation on yield components and
photosynthetic characteristic of Wheat Cultivars (Triticum aestivum and T. durum L.). J. Appl.
Sci. 10(7): 564-569.

Bosch, S.M. and L. Alegre. 2004. Review: Die and let live: leaf senescence contributes to plant survival
under drought stress. Functional Plant Biol. 31:203-216.
Datta, J. K., T. Mondal, A. Banerjee and N. K. Mondal. 2011. Assessment of drought tolerance of
selected wheat cultivars under laboratory condition. J. Agri.Technol. 7: 383-393.

Deng, X., L. Shan, S. Inanaga and M. Inoue. 2004. Water saving approaches for improving wheat
production. J. Sci. Food and Agri., 85 (8): 1379-1388.

Ehdaie, B., A. E. Hall, G. D Farquhar, H. T. Nguyen and J. G. Waines. 1991. Water use efficiency and
carbon isotope discrimination in wheat. Crop sci. 31: 1282-1288.

Emam, Y. 2007. Cereal production. 3rd Edn., Shiraz University Press, Iran, p. 190.

Farooq, M., A. Wahid, N. Kobayashi, D. Fujita and S.M.A. Basra, 2009. Plant drought stress, effects,
mechanisms and management. Agron. Sustain. Dev., 29: 185–212.

Foidle, N., H. P. S. Makkar and K. Becker. 2001. The potential of Moringa oleifera for agricultural and
industrial uses. In: Fuglie, L.J. (ed.), The Miracle Tree: The Multiple Attribute of Moringa, pp:
45–76.

Foyer, C.H. 2002. The contribution of photosynthetic oxygen metabolism to oxidative stress in plants. p.
33-68. In: D. Inze and M.V. Montago (eds.) Oxidative stress in plants.

Taylor and Francis, New York, USA.

Fuglie, L. J. 1999. The Miracle Tree: Moringa oleifera: Natural Nutrition for the Tropics. Church World
Service, Dakar, pp. 68; revised in 2001 and published as The Miracle Tree: The multiple
attributes of moringa, pp. 172.

Ghobadi, M., S. Khosravi, D. Kahrizi and F. Shirvani. 2011. Study of water relations, clorophyll and
their correlations with grain yield in wheat (Triticum aestivum L.) genotypes. World Academy of
Science, Engineering and Technology. 78: 582-585.

Gholamin, R., and M. Khayatnezhad. 2010. Study of some physiological responses of drought stress in
hexaploid and tetraploid wheat genotypes in Iran. J. Sci. Res. 6: 246-250.

Gupta, N. K., S. Gupta and A. Kumar. 2001. Effect of water stress on physiological attributes and their
relationship with growth and yield of wheat cultivars at different stages. J. Agron. Crop Sci. 186:
55-62.
Hasegawa, P. M., R. A. Bressan, J. K. Zhu and H. J. Bohnert. 2000. Plant cellular and molecular
responses to high salinity. Annu. Rev. Plant Physiol. 51: 463-499.

Jyotsna, V. and A. K. Srivastava. 1998. Physiological basis of salt stress resistance in pigeon pea
(Cajanus cajan L.). II. Pre-sowing seed soaking treatment in regulating early seedling
metabolism during seed germination. Plant Physiol. Bioc. 25: 89-94.

Kijne, J. W., R. Barker and D. Molden. 2003. Water Productivity in Agriculture: Limits and
Opportunities for Improvement. CABI Publishing, CAB International, Wallingford, Oxon, U.K.

Khan, A. H., S. M. Mujtaba and B. Khanzada, 1999. Response of growth, water relation and solute
accumulation in wheat genotypes under water deficit. Pakistan J. Bot., 31: 461–468.

Lawlor, D. W. and G. Cornic. 2002. Photosynthetic carbon assimilation and associated metabolism in
relation to water deficits in higher plants. Plant Cell Environ., 25: 275–294.

Makkar, H. P. S. and K. Becker. 1996. Nutritional value and antinutritional components of whole and
ethanol extracted Moringa oleifera leaves. Anim. Feed Sci. Technol. 63(1): 211-228.

Mano, J. 2002. Early events in environmental stresses in plants – Induction mechanism of oxidative
stress in plants. p. 217- 245. In: D. Inze and M.V. Montagu (eds.). Taylor & Francis, London.

Moaveni, P. 2011. Effect of water deficit stress on some physiological traits of wheat (Triticum
aestivum). Agricul Sci Res J. 1: 64 - 68.

Mostajeran, A., R. Amooaghaei and G. Emtiazi. 2005. The effect of Azospirillum brasilense and pH of
irrigation water on yield, protein content and sedimentation rate of protein in different wheat
cultivars. J. Iranian Biol. 3: 248-260.

Munné-Bosch S., Alegre L. 2004. Die and let live: leaf senescence contributes to plant survival under
drought stress. Functional Plant Biology 31, 203-216.

Nagar, P. K., R. I. Iyer and P. K. Sircar. 2006. Cytokinins in developing fruits of Moringa
pterigosperma Gaertn. Physiol Plant. 55(1): 45-50.

Plaut, Z., B. J. Butow, C. S. Blumenthal and C. W. Wrigley. 2004. Transport of dry matter into
developing wheat kernels. Field Crops Res. 86:185-198.
Prabhu, M., A. R. Kumar and K. Rajamani. 2010. Influence of different organic substances on growth
and herb yield of sacred basil (Ocimum sanctum L.). Indian J. Agric. Res., 44 (1): 48-52.

Price, L. M. 2007. The moringa tree. ECHO Technical Note. 19 p. Educational Concerns For Hunger
Organization ECHO, North Fort Myers, Florida, USA.

Shani, E., O. Yanai, and N. Ori. 2006. The role of hormones in shoot apical meristem function. Plant
Biology 5(9):488-489.

Souza, R. P., E. C. Machado, J. A. B. Silva, A. M. M. A. Lagoa and J. A. G. Silveira. 2004.

Photosynthetic gas exchange, chlorophyll fluorescence and some associated metabolic changes
in cowpea (Vigna unguiculata) during water stress and recovery. Environ. Exp. Bot. 51:45–56.

Yasmeen, A., S.M.A. Basra, R. Ahmad, and A. Wahid. 2012a. Performance of late sown wheat in
response to foliar application of Moringa oleifera lam. leaf extract. Chil. J. Agric. Res. 72: 92-
97.

Yasmeen, A., S. M. A. Basra, M. Farooq, H. Rehman, N. Hussain and H. R. Athar. 2012b. Exogenous
application of moringa leaf extract modulates the antioxidant enzyme system to improve wheat
performance under saline conditions. Plant Growth Regul, doi. 10.1007/s10725-012-9764-5.

Zheng, J., Fu, J., Gou, M., Huai, J., Liu, Y., Jian, M., Huang, Q., Guo, X., Dong, Z., Wang, H., and
Wang, G. 2010. Genome-wide transcriptome analysis of two maize inbred lines under drought
stress. Plant Mol. Biol. 72: 407-423.