Introduction

Man discovered agriculture almost 10 000 years ago. Agriculture is domestication of wild plants for personal use of humans. Food is the necessity for sustainability of human life on this planet. Various crops have been harvested since thousands of years. However, it was not possible to cope with the demand of food by using conventional methods of cultivation. There was need to develop new approaches to improve the quality and quantity of yield. As the global world, population is rapidly and alarmingly increasing, new methods have been introduced for better production, improved nutrient content and disease resistant crops. Since start, man has been trying to manipulate different ideas and techniques to save the crops from different diseases by using conventional methods. Unfortunately, conventional methods are no more serviceable towards the current needs. Although, from past five decades global food grain production is growing as with increasing population but still 1 billion persons of the world are malnourished because of food insecurity (Hazell and Wood, 2008). It has been estimated that worldwide food production must be increased by 70 % by the year 2050 to fulfill the need of expanding population and growing consumption of food (Godfray et al., 2010). In this age of technology, biotechnology has opened up new horizons in the field of science. It is a viable option, which can provide improved genotypes that can survive under changing climate. Advancements in fields of genomics, stress biology and bioinformatics can help in development of stress tolerant crops. There are multiple approaches like transformation, mutagenesis and proteome profiling in practice to adopt better traits of agronomic importance. In this review we will focus on molecular biology applications for crop improvement like allele mining, gene pyramiding, linkage and association mapping, genetic engineering (GE) or recombinant DNA technology, Molecular Breeding (MB), Marker assisted back cross (MABC) and Marker assisted recurrent selection (MARS), Genome wide selection (GWS) and Next Generation Sequencing (NGS).

Plant tissue culture in crop improvement

Plant tissue culture is an enabling in vitro technology from which many novel techniques have been developed to assist plant breeders. Changes induced by plant tissue culture are known as somaclonal variations. Pieces of plant tissues will slowly divide and develop into colorless mass of the cells called Callus. Callus is the first step in the formation of new plant from the plant tissues (Jain, 2001).

Major things required for the plant tissue culture are the plant tissues (explants), medium containing organic and inorganic compounds on which the plant could grow and develop further and a high amount of growth hormones particularly Auxin and cytokine. Sterile conditions are mandatory to make tissue culture successful. Different techniques in plant tissue culture like micro propagation may offer certain advantages over traditional plant breeding techniques. This tool enables us to understand the vast abilities of plants as a totipotent cell (Fitter and Krikorian, 1981; Brown and Thorpe, 1995). Plant tissue culture has been exploited to create genetic variability from which crop plants can be improved. Tissue culture in association with molecular techniques have been used to transfer desirable both commercially and genetically traits. Ornamental, clonally propagated crop industries are working massively and hence dramatically increase the crop cultivars. Chromosomal variations induced by tissue culture are observed in many crops. Molecular and transposable variations are also present. A number of serious attempts are being made to produce crops by introducing somaclonal variations. Hence a large amount of cytoplasmic and nuclear genetic alterations are made to bring about phenotypic variations. A new type of hybrid plants and clones are being made with the improved traits. This is considered the safest technique to produce plants with desired traits. A large number of clones are required to produce desired results on large scale.

Major advantages or impacts of plant tissue culture on crops are being mentioned in detail below. Crops used to produce from this technology facilitate the interspecific and intergeneric crosses to overcome physiological based self-incompatibility (Brown and Thorpe, 1995; Azam et al., 2013). A vast variety of crops has been recovered through IVF via pollination of pistils and self and cross pollination of ovules. Agricultural crops like tobacco, clover, corn, ,canola, Cole, poppy, cotton etc. the use of delayed pollination, distant hybridization, pollination with abortive and irradiated pollen and physical and chemical treatment of host ovary have been used to implied haploidy. Embryo culture is another kind used to make crops valuable. Orchids, roses, bananas are being formed by embryo culture. In-vitro selection for salt tolerance is commonly occurring as temporary adaptation. Cells are being able to store extra salts in the vacuoles and survive by adjusting the osmotic pressure. This results in the production of salt tolerant halophytes and well adapted to high salt environment and become unable to grow without salt. Tobacco salt tolerant cell lines are produced. Different other varieties like stress, drought, and heat tolerant varieties are also successfully formed. In vitro propagation via meristem, cell tissue and organ culture, organogenesis and somatic embryogenesis are presented. These technologies could easily simplify breeding programs and overcome some important economical and agronomic traits that would never be produced from conventional ways of plant breeding and plant improvement (De Filippis, 2013; Ali et al., 2014a). Different growth conditions and media requirements are different for different crops. Biopharming of plants with notable advantages in plant cost and safety is regarded as the platform for the production of a vast variety of recombinant proteins and a number of potentially crucial drugs (Brown, 1989). The method of plant tissue culture plays a dominant role in the second green revolution in which plant biotechnology is considered to make desirable crops. The applications of various tissue culture approaches to crop improvement are;

·Breeding & biotechnology

·Wide hybridization

·Haploidy

·Somaclonal variation

·Micro propagation

·Synthetic seed

·Pathogen eradication

·Germplasm preservation

Plants are being grown in-vitro to produce bio-fuel. This is the most astonishing advancement in crop improvement. Yield and quality of the crops are massively increasing by using this technology widely. Increased nutrition and food safety are the basic points kept in mind before implanting tissue culture techniques.

Crop improvement by Genetic Engineering

For many decades, Gene transfer between unrelated species of plants has been playing a very crucial role in crop improvement. By transforming genes many useful traits like resistance to insects, stress and disease has been transferred to many crop varieties from non-cultivated plants. Recombinant DNA methods and many other methods are in use for transformation of genetic information. Genetic engineering is a DNA recombination technique that has made possible gene transfer between dissimilar genera or species (Ali et al., 2011a; Ali et al., 2011b). Genetic engineering is an exceptional way of breeding as compared to conventional breeding (Ahmad et al., 2015a). It is a way of extending genetic base. Secondly, as it avoids the problem of linkage drag associated with the conventional breeding it is more effective and it is less time consuming. Till now, many genetic engineered crops have been developed and commercialized that result in improved production efficiency, increased market focus, and enhanced environmental conservation. Such crops include longer post-harvest storage tomatoes, insect resistant cotton and maize, virus resistant potato, herbicide resistant soybean and canola, and many other (Dunwell, 2000; Akhtar et al., 2014; Amin et al., 2014; Dar et al., 2014; Tariq et al., 2014; Khan et al., 2015; Puspito et al., 2015). To improve crops through genetic engineering, an efficient transformation system is required. Currently there are different approaches that are used to transform different crops.

Gene transfer through hybridization

Plant breeding and intraspecific gene transfer

In 19^th century plant breeding began with discoveries of how plant traits are inherited. Plant breeding could be carried out by selection of plants with attributes of interest and manipulating into cross fertilization. Improved variety with desired characteristics is formed when a cultivated variety is back crossed with a wild variety (Goodman et al., 1987; Khan et al., 2015).

Interspecific gene transfer

In 20^th century, plant breeders used inter-specie hybridization for gene transfer from a non-cultivated plant species other convertible crop species. For example Avina sativa (oat) and Beta vulgaris (sugar beet) has been transformed and resulted in increased yields 25-30% and sugar beet nematode resistance respectively (Sharma and Gill, 1983).

Gene transfer by non-sexual methods

As plant cells, tissues and organs can be cultured in vitro so transfer of genes between plants is possible by non-sexual methods. Non-sexual gene transfer methods depend on ability to produce in certain plant species fully differentiated plants from non-sexual organs and tissues. Stems, pieces of leaves and different undifferentiated clumps of cells in culture can be used as starting material for regeneration. In some species, even a single somatic cell can be used. Cell fusion methods and recombinant DNA techniques for gene transfer have been used from many years. Here we will discuss some gene transfer techniques that are used for crop improvement (Goodman et al., 1987; Jahangir et al., 2014; Butt et al., 2015).

Cell fusion/ protoplast fusion

Methods to prepare large number of single plant cells without their cell walls (protoplast) were developed in 1960s. Fusion could be induced among protoplasts of various plant cells by using electroporation technique and certain chemicals and liposomes. Callus tissue produced from somatic hybrid when grown in vitro. In certain species a whole plant can be regenerated from this callus tissue. Sexually incompatible species could have their choromsomes combined by the use of cell fusion method. This method is of little importance for commercial use in agriculture because of its limitations.

Gene transfer by manipulating DNA directly

In 1940s, methods for transferring DNA directly from one organism to another organism developed as DNA established as a chemical base of genetic inheritance. Non-sexual DNA transfer techniques make possible manipulations that are outside the repertory of breeding and cell fusion techniques. Genes can be obtained from plant, animal, bacterial and viral sources and injected in crops. Tissue specificity, timing and level of gene expression is under control and it can be modified by gene modification into new host. These methods provide the source of diversity and allow controlling the expression of genes (Qamar et al., 2015).

Agrobacterium-mediated gene transfer

Agrobacterium tumefaciensisis a plant-pathogenic bacterium that holds ability to transfer some part of its own genetic material into other plant species by a simple process called transformation. The genes encoded in a region of Ti plasmid called T-DNA. This causes tumorous growth called “crown gall” disease in plants. This bacterium is modified in lab and it transfers gene of interest into plants without causing symptoms of disease. The Agrobacterium system is appealing because of the easy protocol that is associated with minimum cost in terms of equipment and also the resulting transgenic plants have simple copy insertion (Hansen and Wright, 1999; Tariq et al., 2014; Aaliya et al., 2016). Many very efficient vectors are designed that contain extra copies of virulence genes and are mutated that increases the level of expression of virulence genes (Hamilton et al., 1996). For successful results we should test many parameters like feeder cells, infiltration of bacteria, agrobacterium strains etc. (Hansen and Wright, 1999).

By using this method, genes for insect and disease resistance has been transferred. This is the most suitable method of non-sexual gene transfer and there are many useful crops that are tested and are good candidates for agriculture use. By recombinant DNA technique many plant and bacterial genes that encodes enzymes has been engineered that makes plant crops tolerant to broad spectrum and environmentally safer herbicide. For this bacterial gene is engineered in such a way that its enzyme is insensitive to herbicide and then transfer it to plant. This can also do by engineering plant so that they express genes that detoxify herbicide. Genes obtained from Bacillus thuringienesis has been engineered and transfer to plants that act as insecticides (Zameer et al., 2015; Shahid et al., 2016).

Biolistic transformation

Biolistic transformation is the process of delivery of micro projectiles that are of tungsten or gold coated with DNA and push into the target cells by acceleration. Acceleration provided by electric charge, CO₂, gun powder and by gases and DNA can introduced into and tissue. This method has some limitations e.g. it reveals a complex pattern of transgene integration, the delivery of long fragment DNA is challenging and it is more expensive in terms of equipment (Hansen and Wright, 1999).

Microinjection

The microinjection technique is a direct physical approach, for introducing substances under microscopic control into defined cells without damaging them (Neuhaus and Spangenberg, 1990). By means of micropipettes, DNA solution is introduced into plant protoplasts. Microinjection can be used with crop species from which whole plant can be obtained from single transformed cells.

Mutagenesis and crop improvement

Mutational breeding is powerful tool for raising plant varieties with desired traits with equally beneficial to food crop as well horticulture. About 2,000 plant varieties with induced mutation have been cultivated commercially (Maluszynski, 2001; Ahmad et al., 2015b; Rizwan and Akhtar, 2015). Mutations are the source of changes in the genome either permanent or temporary. Spontaneous mutation is occurring naturally with very low frequencies of 10^-6 due to transposable elements which move into genome and cause alteration in DNA sequence (Wessler, 2006). Induced mutation are caused by either chemical mutagens or other agents like UV radiation, X rays α- particles and β particles The main purpose of mutation breeding technology is the development of new and desired variation(s) through breeding program for crop improvement. Induced mutations can play an important role in the conservation and preservation of crop biodiversity. Induced mutations and related advance technologies are important not only for increasing the genetic diversity of crops but also are an important source of additional biodiversity enhancement of neglected and local crops (Hussain et al., 2012; Roychowdhury and Tah, 2013). In this approach, mutants with desired traits were selected in the M1 or M2 generation after treatment with mutagens and then released as new variety for cultivation after evaluation and trials. Those were not selected as cultivators, can be used in cross breeding program for the desired allele. (Roychowdhury and Tah, 2013). Mutational breeding shows great potential over genetic engineering because of some economic issues developing countries this technology may not readily be operational, especially in the developing countries. Other major problem is the regulation and positional insertion of introduced gene (Jain, 2010; Masood et al., 2015).

According to the FAO / IAEA Mutant Varieties Database (http://www-mvd.iaea.org), there are 1,357crop species which are officially released mutant cultivars, 490 mutant varieties of ornamental and decorative plants were mainly developed in seed propagated plant species (1,284 entries), whereas vegetative propagated crops are represented by only 73 varieties. Among the cereals (869 mutant varieties), rice (333) ranks first, followed by barley (261), bread wheat (147), maize (49), durum wheat (25), and others (54). Most of the rice mutant varieties (67.6 %) were released as ‘direct mutants’ (Roychowdhury and Tah, 2013).

RNA Interference

RNA interference is an emerging tool in biotechnology for crop improvement. It has been widely used for increasing crop yield, resistance against biotic and Abiotic stresses and enriched nutrient fruits. RNAi includes the sequence specific gene silencing at post transcription level (Kamthan et al., 2015). Two major player of RNA interference are (endogenous) microRNA and exogenous, such as transgene, small interfering RNA (SiRNA). They are produced by the breakdown of dsRNA by the ribonuclease enzyme DICER or DICER like enzymes (DCL) (Bernstein et al., 2001; Hutvagner et al., 2001). Then a RNA induced silencing complex (RISC) is activated by the incorporation of these single stranded RNAs. RISC contains protein which has ribonuclease activity to degrade the mRNA and RNA binding domains (Hammond et al., 2000). RISC contains another important protein, argonaut that has been reported In Arabidopsis thaliana, makes the catalytic core of RISC and involved in slicing (Vaucheret, 2008). Activated RISC- RNA (antisense strand) than bind to target sequence specifically by complementary base pairing and degrade the mRNA (Williams et al., 2004). siRNAs can also regulate gene expression at transcription level by regulating the chromatin siRNA maintain the transcription rate at minimal level by controlling histone modification including the cytosine methyl transferase CHROMOMETHYLASE3 (CMT3) which keep the DNA into transcriptional inactive state (Ossowski et al., 2008).

The phenomena of RNA interference can be used for producing desirable traits. The process of RNAi can triggered by the entry Small siRNA into a cell by several different ways, such as by Agrobacterium mediated transfer, viral mediated dsRNA transfer bombardment or by infiltration (Sijen and Kooter, 2000). An RNAi vector is used to transform cell and produce stable dsRNA in vivo.

Biotic resistance

The strategy of RNA interference was firstly used to develop resistance against virus in plants. Mechanism of Pathogens derived resistance was developed in which expression of three different virus derived protein viral coat protein (CP) and replication-associated proteins (Reps) antisense and hpRNA was used for silencing to produce resistance against viruses (Shepherd et al., 2009). Transgenic Potato resistant against Spindle Tuber Viroid (PSTVd) infection has been developed which produce dsRNA against PSTVd sequences (Schwind et al., 2009). RNAi is also effective against DNA viruses. Resistance was developed in rice by using sequences from disease specific protein gene and CP gene from Rice Stripe Virus (Zhou et al., 2012). Mechanism of RNAi is also effective against bacterial diseases. Crown gall disease was managed by using RNAi against tumor formation gene in Arabidopsis thaliana (Escobar et al., 2001). In resistance management against fungal diseases fatty acid genes were targeted. Suppression of gene OsSSI2 in rice result into increase resistance to blast fungus Magnaporthe grisea and leaf blight bacterium Xanthomonas oryzae (Jiang et al., 2009).

Plant varieties resistant against pest and nematode also have been developed. For this insects are feed with dsRNA as dietary component, which result in the decrease expression of target gene. This strategy has been applied to corn plant to produce transgenic corn plant by targeting tubulin or vacuolar ATPase genes to develop western corn root warm resistance.

Abiotic stress

One of major problem that affect the crop yield and quality is drought. RNAi also gives solution to this problem. Activated C-kinase 1 receptor gene was targeted in transgenic rice plants to enhance drought tolerance (Li et al., 2009; Ali et al., 2016). A family of miRNAs miR393 shows expression in stress condition. Osa-miR319a as a transgene when over expressed in rice plant shows enhance tolerance against drought and salt stress (Zhao et al., 2007; Mohamed et al., 2015).

RNAi for Male Sterility

RNAi has also been used for generating sterility in seeds and producing hybrid seed. Genes that involved in pollen production can be targeted by RNAi. A male sterile tobacco line has been developed by targeting the expression of TA29, a gene necessary for pollen development (Mao et al., 2007). Male sterility is also generated by RNAi by controlling the Msh1 gene expression in tobacco and tomato that result rearrangements in the mitochondrial DNA that is associated with natural cytoplasm male sterility (Baum et al., 2007).

Modified metabolic pathways

Basic metabolic pathways of plants can be manipulated through RNAi to get nutritionally improved fruits and crops. Some improved plant varieties with target gene are summarized in Table 1 (Kamthan et al., 2015).

Next generation sequencing

which The term NGS is applied to detail all the latest sequencing technologies other than Sanger hold potential to sequence human genome at the cost of thousand dollars (Service, 2006).

Next-generation sequencing (NGS) technology is the cutting-edge technology for genome sequencing of several species. It has been proved an essential gadget for development of novel or atypical molecular markers and determining genes of agricultural importance (Edwards and Batley, 2010). Long drawn out and tedious clone-by-clone process has been replaced by NGS, this previous method was used for genome sequencing with the strategy of identifying the least redundant super-imposed clones, a physical genetic map of the crop to be sequenced is the prerequisite for carrying out these time-taking experiments (Ariyadasa and Stein, 2012). GS-FLX and Illumina HiSeq, are leading NGS methods for utilizing the whole genome shotgun (WGS) approach for sequencing of several crops as massive amounts of data is being generated in lesser time using these platforms. There are a number of companies that have made third generation sequencing technologies available to the market (Egan et al., 2012).

A dramatic uplift in the number of completely sequenced plants has been observed after replacement of traditional Sanger Sequencing by NGS. First completely sequenced plant was Arabidopsis thaliana, this project was carried out by the Arabidopsis Genome Initiative (Kaul et al., 2000). Rice genome sequencing was next to Arabidopsis thaliana (Yu et al., 2002; Project, 2005). The sequences of many valuable crop species e.g. grape, sorghum, maize and soybean have been studied since then using the traditional Sanger method and NGS (Varshney et al., 2009; DePristo et al., 2011; Van et al., 2013). Genome sequencing projects associated with sequencing of many other food and cash crops (e.g. banana, cotton, barley, wheat and oil palm) are in pipeline. The tomato genome sequence has been also published recently by exploiting NGS as well as Sanger technology (Consortium, 2012).

NGS has its applications in Genome Assisted Breeding that is an integrated approach for identification and selection of genetic variations (Varshney et al., 2005b). Molecular markers are used for the physical mapping and tracking of genes or quantitative trait loci for marker-assisted breeding (MAB) (Varshney et al., 2005a). Commercially available leading NGS techniques like Roche/454 (http://www.454.com/), Solexa/ Illumina (http://www.illumina.com/) and AB SOLiD (http://www3.appliedbiosystems.com/AB_Home/applicationstechnologies/SOLiDSystemSequencing/index.htm) have been proved superior to Sanger method. For example, sequencing can be multiplexed to a much greater extent by many parallel reactions at a greatly reduced cost (Hudson, 2008). Roche/454, Solexa and AB SOLiD find their applications predominantly in area of crop genetics and breeding. Roche/454 is better than Solexa and AB SOLiD because it can obtain longer sequence reads, maximum data output is higher for both Solexa and AB SOLiD (Gupta, 2008). Roche/454 is more costly than both the Solexa and AB SOLiD technologies in terms of cost per run.

NGS have found valuable applications in development of SNP-based markers, for characterization of de-novo sequence drafts of orphan-crops where no previous data is available e.g complex genomes of barley and wheat and for resequencing of those crops which already have been sequenced previously.an assembly of 11 700 and 8700 contigs was generated by using Wheat Roche/454 ESTs for two hexaploid wheat lines, later on compared with sequences for progenitor species of polyploidy wheat; 2500 contigs assemblies were assigned to one of the homologous wheat genomes and 1000 SNPs were discovered (http://www.intl-pag.org/17/abstracts/P03e_PAGXVII_144.html). The study indicates that NGS could be the lead tool for SNP identification in polyploidy crops (Cronn et al., 2008). Association mapping is done either by candidate gene sequence (CGS) or by whole genome sequencing. Both CGS and WGS based association mapping approaches can be elucidated well by NGS techniques (Nordborg and Weigel, 2008).

Approaches like Marker-assisted Breeding(MAS), designing of molecular markers for MARS, de-novo sequencing, resequencing of previously characterized crops, metagenomics , SNP haplotyping and epigenetic modifications have been taken to next level by using next generation sequencing approaches (Varshney et al., 2009). Assembly and analysis of data generated through Next generation sequencing is challenging because of limited availability of bioinformatics resources for this technology. In short speed of crop improvement has been shifted to a new pace because of HeliScope, Ion Torrent, single molecular real-time sequencing and Oxford Nanopore like techniques of third generation sequencing (van et al., 2014).

Bioinformatics tools in crop improvement

Bioinformatics resources in addition to different web databases are providing vast information about the genomic data that is largely required for the research purpose. Crop improvement through bioinformatics tools are more promising these days. With the passage of time the technology has been enhanced to surprising level. The bioinformatics is providing crucial information about the genomic data of crops and the sequence of many genes are being explored by this technology. This could possibly help us to sequence the corps which is economically important and the traits that are more beneficial. Whole genome comparisons are accelerating the rate of competent research.

Genome sequence projects of economically important crops have been completed and regarded as the gateway to further research. The database housing focused data set together in a compiled form in with rich annotations help to study gene families more precisely. Genome wise comparisons of different crops aid to point conserved regions among crops thus provide the common adaptive strategies by plants (Mochida and Shinozaki, 2010). After completing sequencing of the crops the data generated has been used to create modeled proteomic data which helped to figure the content of some gene families. Major events like gene duplication along with other abnormalities are being manipulated by the help of bioinformatics tools. Furthermore, advances in the technology and data acquisition sites ease the task to access crucial data necessary for the improvement in traits of crops. Hence the effective use of genetic data supports the sustainable improvement in crops. Different techniques like High through output sequencing generate a pile of data about the crops. Omics research is working on the prediction of candidate genes and consequently on the predicted functions (Lockhart and Winzeler, 2000). The recent accumulation of nucleotide sequences of the model organisms and other applied species like domestic animals and crops make the understanding more clear. Data upon transcriptomic and metabolomics also elucidated regulatory networks that are crucial against plant stressors. Hence various crops have been protected from biotic and abiotic stressors and yield has been restored. It is evident that bioinformatics tools are accelerating the speed of innovations and also improving different crops varieties of economic importance.

Nanotechnology in Crop Improvement

This is due to the nanotechnology that our food is expected to be better in taste and quality and other factors like increased shelf life with maximum nutrition .It is going to be the next amazing thing in agriculture. In future our food would in such a way that will have the ability to detect presence of contaminants and spoiling agents. Nanotechnology is a novel, explanatory, vast scientific technology that involves designing, development and application of materials at molecular level in nanometer scale. It is a broad spectrum emerging field of science which has examples in all field of science and agriculture is no exception (Ali et al., 2014b). There are many reports which have shown the involvement of Nano-particles or nanotechnology in crop improvement. Mostly used or studied Nano-particles are carbon and metal-oxide based particles. The positive effects observed by using these Nano-particles include enhanced germination, enhanced length of roots and shoots, and increased vegetative biomass of seedlings in many crops. In many crops including soybean, spinach and peanut enhancement in many physiological parameters have been observed such as photosynthetic activity and nitrogen metabolism. In 2009 it was reported that the germination of seed of tomato plant was enhanced by penetrance of carbon nanotubes (CNTs). The seed germination in this case was enhanced due to water uptake ability of CNTs.Ti02 Nano-particles have been known to enhance the growth of Spanish. The reason was that these Nano-particles have enhanced the Rubisco activase activity and have improved light absorbance. In 2010 it was reported that ZnO Nano-particles had retarded germination of seed in corn and rye grass. It was also reported that silicon Nano-particles when used in some plants have increased disease and stress resistance. Recently it was discovered that photosynthetic activity can be increased three times by SWCANT’S containing cerium Nano-particle. The use of magnetic fluid after exposing to magnetic field during germination of seed has shown a visible effect in increasing nucleic acid level. This was due to the process of regeneration of plant metabolism. Iron oxide was when used in pumpkin then increase root elongation was observed (Bombin et al., 2015). This was due to dissolution of iron. The genetic implications of Nano-particles-induced positive changes have been confirmed through decreased oxidative stress to spinach chloroplast under ultraviolet-B radiation by Nano-titanium dioxide. It was also observed in rice by transmission of fullerol through seeds for generations. Nano-particle also aided to change the genetic expression in potato and tomato through carbon nanotube.

Future perspective

As described previously the population of this planet is rapidly growing and in next two decades it is expected to cross the figure of 9 billion. So in the coming days it is going to be the greatest challenge to feed 9 billion people and to deal with hunger of such a huge population. The biggest hurdle is the rapidly changing climate with time. There is need to introduce better seeds which can survive under this changing climate and give maximum yield. Crop improvement is the prime element of agricultural advancements and there are still many areas to be worked on in the field of crop improvement. When talking about gene transfer or transfer of desirable traits to the target plant, in future there might be an option of complete chromosome transfer via microinjection and it can confer multigenic traits. NGS technology has made access to genomic resources of multiple plants and also to those lesser studied orphan crops. It will also facilitate the identification and confirmation of introgression lines for desirable traits. Crosses between distant relatives are promoted by novel embryo rescue techniques. Isozyme technology is emerging as a rational tool for various aspects of plant breeding.  Innovation in agricultural technologies is leading Molecular Farming into a new landscape but private sector companies and establishments are supposed to invest more resources to make it a successful idea that can provide higher productivity with lesser use of herbicides, insecticides and chemical fertilizers. These unforeseeable notions of future scientists will shift crops much toward the natural essence.

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