WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Misra et al. World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 6.647 Volume 6, Issue 10, 294-304 Review Article ISSN 2278 – 4357

SODIUM AZIDE INDUCED MUTAGENESIS IN WHEAT

Shailja Dubey1, Renu Bist1 and Shrilekha Misra*2

1Dept. of Bioscience and Biotechnology, Banasthali University, Rajasthan, India. 2Department of Internal Medicine, Ohio State University, Columbus, OH, USA.

ABSTRACT Article Received on 04 August 2017, Sodium azide, a chemical mutagen has become important tool to

Revised on 25 August 2017, enhance agronomic traits of crop . It is being used to produce Accepted on 15 Sept. 2017,

DOI: 10.20959/wjpps201710-10199 resistance in various susceptible crops to improve their yield and quality traits against harmful pathogens. There are several mutagens available for crop improvement and each mutagen has its important *Corresponding Author Shrilekha Misra role as positive or negative effects on crops. Sodium azide creates Department of Internal point mutation in the genome of plants through metabolite and thus Medicine, Ohio State produced protein in mutant plants has different function compared to University, Columbus, OH, the normal plants. The mutant plants produced by the treatment of USA. sodium azide are capable to survive under various adverse conditions and have improved yields, increased stress tolerance, longer shelf life and reduced agronomic input in comparison to normal plants.

KEYWORDS: Sodium azide, crop improvement, wheat, breeding, mutagenesis.

INTRODUCTION Wheat (Triticum spp.) is the second-most produced crop on Earth and provides a large fraction of the dietary protein and total food supply. Wheat is one of the major cereals in the world and is one of the main sources of calories and protein. Approximately 85% and 82% of the global population depends on wheat for basic calories and protein, respectively.[1] It is grown all throughout the world in a wide variety of climates. It is a staple food for more than 35% of the world population. Moreover, this cereal is used in the production of a variety of wheat products, such as leavened bread, flat and steamed breads, cakes, pasta, biscuits, noodles, couscous and beer.[2] Beyond its use for human consumption, wheat is also used for the development of non-food products such as fuel. Wheat is grown on more land area than any other commercial crop and is the most important staple food for humans. World trade in

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wheat is greater than for all other crops combined.[3] Globally, wheat is the leading source of vegetable protein in human food, having higher protein content than either maize (corn) or rice, the other major cereals.

Taxonomic Hierarchy Kingdom : Plantae (Unranked) : Angiosperms (Unranked) : Monocots (Unranked) : Order : Family : Subfamily : Pooideae Tribe : Triticeaes Genus : Triticum L.

Description of plant Wheat is a widely grown winter crop. A wide range of varieties are available including dwarf, semi-dwarf and bearded varieties. Wheat has erect seedling. Leaves are 10-20 cm long and up to 15 mm wide. Seedlings begin to tiller soon after emergence, with multiple tillers forming from the plant base. Leaves are largely flat, up to 40 cm long and 20 mm wide. The ligule is membranous, 0.5-2 mm long and broadly rounded. The auricles are membranous. Form on the end of the stems. Heads are 5-10 cm in length, up to 2 cm in width and flattened with 2 rows of alternating seeds. They can be bearded, with stiff terminal awns up to 6 cm in length, or may be awn less. Seeds are typically around 4-5 mm in length.

Cultivation Wheat is cultivated in tropical and sub-tropical regions and under both rainfed and irrigated cultivation. However, crop production is severely affected by adverse environmental stresses.[4]

Land Preparation The wheat crop requires a well-pulverized but compact seed bed for good and uniform germination. Three or four ploughing in the summer, repeated harrowing in the rainy season, followed by three or four cultivations and planking immediately before sowing produce a good, firm seed bed for the dry crop on alluvial soils. For the irrigated crop, the land is given www.wjpps.com Vol 6, Issue 10, 2017. 295 Misra et al. World Journal of Pharmacy and Pharmaceutical Sciences

a pre-sowing irrigation (palewa or raund) and the number of ploughings is reduced. Where white ants or other pests are a problem, Aldrin 5% or BHC 10% dust at the rate of 25 kg/ha should be applied to the soil after the last ploughing or before planking.

Worldwide production of wheat Currently India is second largest producer of Wheat in the world after China with about 12% share in total world wheat production. In 2016 world production of wheat was 851 million tons, making it, the third most-produced cereal after maize (844 million tons) and rice (672 million tons). In 2017, world production of wheat was 982 million tons, making it the second most-produced cereal after maize (817 million tons) and with rice as close third (679 million tons). In terms of total production tonnages used for food, it is currently second to rice as the main human food crop and ahead of maize, after allowing for maize's more extensive use in animal feeds.

Breeding Breeding is method generally used for crop improvement and genetic variations. Genetic variation is necessary to adapt the changing environmental conditions and promote the survival of species. The common method for improvement of economically important crop is mutation. Mostly conventional mutation methods were used for improvement of characters like disease and pest control, yield and quality in crop plants. The mutant varieties of some agronomically important crops like wheat, cotton and barley covers the majority of crop lands.[5]

The utilization of induced mutations in crop improvement is called Mutation “Breeding”. Mutation is a sudden heritable change in an organism. The mutation produced by changes in the base sequence of gene, is called point mutation or gene mutation. Mutations are known to enhance the genetic variability of crop plants and result in large number of high yielding varieties of several crops.[6,7] Generally there are two types of mutations are occurred: Spontaneous and Induced mutations.

Spontaneous mutations Mutation occur in nature are called spontaneous mutation. This type of mutation occurs in organism without any treatment at low rate in the nature. Different genes and organism shows different type of mutation rate.

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Induced mutations Mutation induced in an organism or plant by treatment with physical and chemical mutagens are called induced mutations. The agents which are used to induce mutation are called mutagens. Mutagens used for the induced mutations may be broadly classified into two groups.

Physical mutagens Physical mutagens include various types of radiations, viz. gamma rays, x-rays, alpha particles, beta particles, ultra violet rays, fast and thermal neutrons. Change in pH value (acidity) or temperature shocks are also included in physical mutagens (Table 1).

Table 1: Properties of various physical mutagens. Types of Radiations Properties X-Rays Penetrating and non particulate, sparsely ionizing. Gamma Rays Very penetrating and non particulate, sparsely ionizing. Alpha Particle Less penetrating, Particulate, positively charged, densely ionising. Beta Particle More penetrating than alpha particles, particulate, negatively charged and sparsely ionizing. Fast and thermal neutrons Highly penetrating, Particulate, neutral particles, densely ionising. UV Rays Low penetrating, non ionizing

Chemical mutagens The first report of mutagenic action of a chemical was given by Charlotte Auerbach (1942), who showed that nitrogen mustard could cause mutation in cells. Chemical mutagen causes single base-pair (bp) mutation or single-nucleotide polymorphisms (SNPs) because they are more commonly called to, rather than translocations and deletions. In contrast to MNU and EMS (both are liquid), azide is a solid in its ground state and the step of the first dissolving and the acutely toxic and volatile substance. The target of mutation breeding is to create maximum genetic changes with a less decrease in viability. The chemical mutagens can be divided into four groups (Table 2).

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Table 2: Properties of various chemical mutagens. Mutagen group Chemicals Mode of action Ethyl Methane Sulphonate (EMS), Sodium Azide Alkalyting (Nan3), Methyl Methane Sulphonate (MMS), AT↔ GC Transition agents Diethyl Sulphate (DES), Dimethyl Sulphate (DMS) AT↔GC Transition, 5- Bromouracil, 2- Aminopurine, 6- Ami Base analogs deletion, addition, Nopurine frameshift Acridine dyes Acriflavin, Proflavin AT↔GC Transition Others Mustard gas, Caffeine, Azide, Phenol etc. Transition

Sodium Azide Sodium azide (NaN3, Mol.Wt 65.02) is a colorless, odorless and crystalline solid.[8] It is relatively safe to handle, inexpensive, non-carcinogenic and is also the least dangerous and the most efficient chemical mutagen. Sodium azide is a common laboratory chemical and is widely used in industry, agriculture, medical practice, and organic synthesis research. It is a common bactericide, pesticide, and industrial nitrogen gas generator, and known to be highly mutagenic in several organisms, including plants and animals.[9,10,11]

The mutagenic effect of sodium azide is due to presence of a metabolite of azide.[12] Azide also creates point mutation in genome. In barley and bacteria many studies have been performed to understand its mutagenic mechanism.[13,14] It affects plant growth, their different parts and developmental phenomena by disturbing the metabolic activity. Sodium Azide causes cytotoxicity in several animal and plant test systems, by inhibiting the protein synthesis and replicative DNA synthesis at low dosages[15] and induces chromosomal aberrations such as break, gap, isochromatid break and exchange the cells.[16]

The joint report of FAO/IAEA division as in Vienna records depicts that about 2965 crop cultivars, with one or more applicable traits produced from these mutations, were released worldwide during the last 40 years.[17] Some examples of several varieties of wheat (e.g., durum wheat), barley, cotton, rice, grapefruit and sunflower, gives an enormous positive economic impact.[18,19]

Structure of sodium azide Sodium azide is an ionic solid. Two crystalline forms are known, rhombohedral and hexagonal. Both adopt layered structures. The azide anion is very similar in each form, being centrosymmetric with N–N distances of 1.18 Å. The Na+ion has octahederal geometry. Each azide is linked to six Na+ centers, with three Na-N bonds to each terminal nitrogen center. www.wjpps.com Vol 6, Issue 10, 2017. 298 Misra et al. World Journal of Pharmacy and Pharmaceutical Sciences

Mutagenic effects of sodium azide on crop plants It is well known that sodium azide induces chromosomal aberrations only at a very low rate compared to other mutagenic treatments. Is has been shown that L-azido alanine does not interact with DNA directly under in vitro conditions. Mutagenic activity of this compound was attenuated by a deficiency in the excision of UV-like DNA damage in both plants and bacteria; therefore it seems that a lesion recognizable by the excision-repair mechanisms must be formed to evoke the effect. Mutagenesis proceeds from this by „direct mispairing‟.[20]

Sodium azide was used for the first time as a mutagen by Nilan et al.[21] in barley, when an increase in the frequency of chlorophyll mutations was observed in a dose-dependent manner, at the concentration range 1-4 mM at pH 3, with highest frequency, 17.3% recorded for 4 mM. A high frequency of chlorophyll mutations was also observed in barley variety Aramir after combined treatment with 1 mM NaN3 (3 hours, pH=3) followed by 0.7 mM MNU with a 6-hour inter-incubation (germination) period between treatments. Most of work has been done to induce mutation by chemical mutagens in crop plants. For herbicide tolerance in soyabeen,[22] early flowering in the spring rape,[23] and effect on seed germination[24] in wheat and cucumber,[25] improvement in chlorophyll content and genetic variability in chili.[26,27] Sodium azide has been used in a number of crops for several biotic and abiotic stresses such as zea mays resistant against pathogen Striga.[28] Triticum aestivum (durum wheat) for salt tolerance, Oryza sativa for reduced amylase content,[29] for enhanced yield.[30] The mutational effects of this mutagen has been observed on tomato and it was very effective in inducing mutations with respect to germination percentage, root length, seedling height, seedling survival, number of branches per plant, and yield per plant respectively.[31] Effect of sodium azide on seed germination and plant survival was reported in Stevia rebaudiana seeds by[32] and in Nigella, Plantago and Trigonella by.[33]

Mutagenic effects of sodium azide on wheat The mutagenesis work done on wheat, reported by.[34,35,36] Being a polyploidy Triticum aestivum offers different type of opportunity to explosion of mutation, recombination an increasing genetic variability in quantitavely inherited characters.[37] Sodium azide is a strong mutagen, and growth of plant parts are strongly inhibited with increasing its concentration and treatment duration. The effect of different concentration of sodium azide treatment on root length was clearly observed in different crops and wheat. The effect of different percentage of sodium azide was observed in the seed germination, root and shoot growth on

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bread wheat cultivar “Gerek-79”. Thus the most potential doses were applied for successful mutagenic studies such as Tilling.[38] In wheat, sodium azide reduced the root depth and shoot height.[39]

The greater sensitivity at higher mutagenic level has been attributed to various factors such as changes in the metabolic activity of the cells, inhibitory effects of mutagens and to disturbance of balance between promoter and inhibitors of growth regulators.[40]

Applications of Sodium Azide Azide ion plays an important role in causing of mutation by interacting with enzymes and DNA in the cell. These azide anions are strong inhibitors of cytochrome oxidase which in turn inhibits oxidative phosphorylation process. It is used in agriculture for pest control of soil-borne pathogens such as Meloidogyne incognita or Helicotylenchus dihystera. It is also used as a mutagen for crop selection of plants such as rice, barley and wheat.

The application of sodium azide on crop is easy and inexpensive for improvement of agronomic traits. The mutagenic effects of sodium azide appear soon after sowing the seeds and can be observed by naked eyes. However, sodium azide has been being used in various crops to improve their yield and quality traits and create resistance to them against biotic and abiotic stresses.

Future aspects Development of biotic and abiotic stress resistant crops is necessary to reduce the esnvironmental pollution caused by hazardous and non-hazardous materials. The conventional breeding method takes several years to develop new cultivars from wild species. Chemical mutagens are potential tools and being highly used in wheat and other crops to improve their quality and yield traits. These mutagens are easy to apply on crops and inexpensive to develop resistant varieties. The mutant plant species can be easily selected from wild plants by PCR and non PCR based techniques. The mutant plants produced by sodium azide are capable to tolerate various biotic and abiotic stress conditions, and avoid application of hazardous pesticides against harmful pathogens. Therefore, it should be apply on various crops, which are susceptible to harmful pathogens and create resistance to them against these pathogens.

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CONCLUSION The application of sodium azide on wheat is easy and inexpensive for improvement of agronomic traits. The mutagenic effects of sodium azide appear soon after sowing the seeds and can be observed by naked eyes. However, sodium azide has been being used in various crops to improve their yield and quality traits and create resistance to them against biotic and abiotic stresses. There are quite a few techniques capable of recognizing the presence of single base changes caused by sodium azide in plant genes. The point mutation caused by sodium azide in any plant species can be detected by these PCR and non PCR based techniques. Therefore, sodium azide (NaN3) should be used to create mutation in many crops like wheat, which are highly susceptible for harmful pathogens and made them economically inexpensive and beneficial for farmers.

Conflict of interest There is no conflict of interest among the authors.

ACKNOWLEDGEMENTS The authors sincerely express their gratitude to Department of Bioscience and Biotechnology, Banasthali University, Rajasthan and Department of Science and Technology (DST), India, for giving the facilities for completion of the research.

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