Keywords

9.1 Introduction

9.1.1 Abiotic Stresses and Cultivation of Plants

Crop plants are adversely affected by abiotic stress conditions. Major abiotic stresses which affect plants include heat, cold, salinity, drought, flooding/submergence (anoxia), excess light, and chemical toxicities. Abiotic stresses profoundly affect growth and yield of plants. Drought or water stress is among the most important constraints in obtaining higher crop yields worldwide. Water stress affects plants in several ways. During vegetative stages, water stress reduces leaf expansion, photosynthesis, height of plant, and leaf area. Leaf rolling and leaf tip drying are the primary symptoms resulting from drought stress. Cell enlargement is severely affected by water stress. Decrease in water potential closes stomata and decreases transpiration (Kallarackal et al. 1990). The reproductive processes in plants are particularly sensitive to drought stress. Rice is most sensitive to drought for 10 days before flowering to the end of flowering (Yoshida et al. 1981). The water stress at flowering stage inhibits panicle exertion and spikelet filling. This causes high sterility, leading to decreased yield in rice (Ekanayake et al. 1989). The salt stress (mainly sodium chloride) results in Na+ toxicity as well as physiological water stress to the plants. Estimates show that more than a third of all the irrigated land in the world is presently affected by salinity (Sahi et al. 2006). This is exclusive of the regions classified as arid and desert lands (which comprise 25 % of the total land of our planet). Salt affected lands occur in all climatic regions from deserts to tropical belts of Africa, Latin America, and Polar regions. The loss of farmable land due to salinization is directly in conflict with the needs of the world population. Excess salt levels affect enzyme activities and photosynthesis. The unfavorable Na+ and Na+/K+ ratios adversely affect the grain yield of crops under salt stress conditions (Kaya et al. 2013). Temperature stress is observed when ambient temperature is below (low temperature stress) or above (high temperature stress) the optimal levels. These temperature extremes are injurious to plant growth and development as plants have evolved to grow in a narrow temperature regime (Singh and Grover 2008). In recent years, there has been a general increase in extreme events including floods, droughts, and heat episodes. The man-made changes in the climate of the earth due to the multifarious activities linked to development have become the focus of scientific attention (Grover et al. 2003). The most imminent of climatic changes of the earth is the increase in the atmospheric temperatures due to increased levels of CO2 and other greenhouse gases. In certain places, climatic extremes such as droughts, floods, timing of rainfall, and melting of snow have also shown erratic trends. This is adversely affecting agriculture through its direct and indirect effects on crops (Grover et al. 2013).

9.2 Genetic Improvement of Plants Against Abiotic Stresses

From the preceding discussion, it is amply clear that crop production can be appreciably enhanced if proper management practices to save plants from such stressful conditions are devised. In simple terms, the crop production is an outcome of G × E equation where G refers to the genotype; E, the environment (El-Soda et al. 2014). It is relatively difficult, expensive, and hence impractical to change the environmental variables for obtaining optimal growth of crops. On the other hand, it is relatively inexpensive if the G factor (i.e., the genotype) can be suitably altered enabling plants to successfully grow, reproduce, and set seeds under stressful conditions. Crops have been genetically altered and improved for a host of different agronomic traits in the long history of the development of agriculture. Plant breeders have been greatly successful in enhancing the yields of major crops employing the conventional Mendelian tools like selection, hybridization, and progeny analysis. However, the response of crops against abiotic stresses is very complex because it involves multiple genes. The application of conventional plant breeding methods has proven difficult for genetic improvement of plants against abiotic stresses. In recent years, newer breeding tools like ‘molecular breeding’ have shown promising results in genetic improvement of plants against salt stress, flooding stress etc. (Jenks et al. 2007). The nonconventional tools of genetic improvement like ‘transgenic tools’ are becoming very significant for genetic improvement of crops against abiotic stresses.

9.3 Births and Growth of Transgenic Technology

Molecular biology science was born essentially in the middle of the twentieth century. The experiments leading to (1) elucidation of DNA structure and proposition of the double helix model of this molecule and (2) the discovery of the mechanisms underlying DNA replication and processes involved in its functioning (i.e., transcription leading to formation of RNA from DNA and translation leading to synthesis of proteins from mRNA) have brought revolutionary changes in our understanding of the living cells. In the beginning of 1980s, the art of producing transgenic plants was crafted (Galun and Breiman 1997). The production of transgenic plants provides a way to modify the genetics of plants so that superior types can be bred in a relatively short term. The gene transfers take place between the sexually compatible individuals in conventional and molecular plant breeding approaches. As against this, gene transfer is practiced across a wider platform of individuals overcoming the barriers of sexual compatibility in the transgenic approach (Grover et al. 1999). For instance, transgenic plants have been produced for increased insect resistance by incorporating a gene from bacteria and for increased cold resistance incorporating a gene from fish using the methods of plant genetic engineering (Duman and Wisniewski 2014; Ibrahim and Shawer 2014). Since 1980, substantial progress has been made in transgenic plant production science. Transgenic plants with improved resistance to insect infestations using the most celebrated principle of Bt gene technology has been widely practised in a host of different crops (Peferon 1997). Significant success has also been obtained in breeding herbicide and virus resistance in crops by rDNA technology (Chen and Lin 2013). In recent years, transgenic plants have been produced to improve nutritional quality and to change the physiological and developmental aspects of plants (Zhu et al. 2007). To sum up, the approach of transgenic plant production is a new and, highly effective arsenal in the hands of agricultural scientists to enhance crop yields.

9.4 Production of Abiotic Stress Tolerant Transgenic Crops

Coming back to abiotic stresses, question we now address is how to engineer crops against abiotic stresses like salt, drought, and adverse temperatures. While much of the success in transgenic experiments has come for insect resistance and herbicide tolerance as mentioned above, several attempts have also been made to genetically engineer plants against abiotic stresses in the past 25 years. Scientists have explored a host of different transgenes for enhancing resistance of crops against salt, drought, and high and low temperatures. Several genes have enabled production of transgenic rice against drought, salt, flooding, and temperature extremes (Table 9.1). Likewise, host of plant species including model species like Arabidopsis and tobacco and crops like tomato and wheat have been genetically transformed for different abiotic stresses. It can be safely argued that results obtained from these experiments are of somewhat mixed nature. On the one hand, there are definite reports showing that plants have been successfully produced with enhanced abiotic stress tolerance by transgenic methods. On the other hand, there is poor success in field application of abiotic stress tolerant transgenic crops in spite of all the intensive efforts. Most of the success reported till this day is from laboratory-based experiments (Grover et al. 2013). It is therefore important to revisit these experiments and address what we possibly lack in our efforts in producing abiotic stress tolerant crops at the level of field cultivation.

Table 9.1 Protocol details involved in genetic engineering for increased abiotic stress tolerance in rice
Full size table

The success in production of transgenic plants is principally governed by three critical inputs: (a) the availability of the effective gene, (b) relevant techniques for transferring the transgene inside the genome of the desired trans-host species, and (c) regulated expression of the transgene in the trans-host (Grover et al. 2001). The transfer of genes in genetic engineering experiments is largely a random event. Experiments have shown that the level of expression of the transcripts/proteins from the transgenes in the trans-hosts is highly variable. A great deal of effort is therefore needed to select the progenies that optimally express the trans-protein. To do so, it is important that methods of genetic transformation must yield larger populations of the transformed progenies. The progenies need to be adequately screened to select transgenic types with the optimal levels of gene expression. For this input, it is critical that gene transfer technologies are suitably optimized for effective biotechnological applications.

9.5 Methods of Genetic Transformation of Crops

The transfer of genes in destined plant cells is a critical step in rDNA technology. Since 1970s, several techniques have been optimized to deliver the desired genes to plant cells (Anami et al. 2013). Among these, Agrobacterium tumefaciens has been the major workhorse for plant scientists for the gene transfer. For a large number of plant species, this is the most-promising method of gene delivery. When Agrobacterium infects a wound site, a portion of its DNA (called transfer/T-DNA) is mobilized and gets integrated into the chromosome of the host cells which begin to proliferate, leading to the formation of tumor-like growth. The genes transferred from the bacterium to the plant are carried on the extrachromosomal circular plasmids called tumor-inducing (Ti) plasmids (Tzfira and Citovsky 2006). For the Agrobacterium to be an effective vehicle for DNA transfer, the tumor-inducing gene of Ti plasmids is removed through a process called ‘disarming’ (Simpson et al. 1986). The bulk of the genetic transformations with Agrobacterium were initially shown to work with dicot plant species. In recent years, there has been significant success in genetic transformation of monocot species like rice, corn, and wheat by Agrobacterium (Ji et al. 2013).

Apart from Agrobacterium, several other methods for genetic transformation have been devised. One such method is PEG-mediated DNA uptake where PEG (polyethylene glycol) and CaCl2 are used for stimulation of the DNA uptake process as well as its integration in the isolated protoplasts (Datta et al. 1992). Another method is a related technique of ‘electroporation’ where the isolated protoplasts are given a shock treatment electrically by giving a short, high voltage pulse, to make transient pores in the cell membranes. This enables the plasmid DNA molecules to pass through the pores and enter from culture medium to inside plant cells (Ji et al. 2013). Microprojectile bombardment or biolistics is another innovative way for DNA transfer (Ji et al. 2013). In this method, a microprojectile gun or DNA particle gun is used to deliver DNA directly into plant cells by shooting it through the cell. Microscopic particles of tungsten or gold are coated with plasmid DNA molecules and then shot into the target cells. In the process, DNA is transferred to the nucleus which then integrates into the plant genome. Among the various methods mentioned above, Agrobacterium-based approach stands out as the most favored technique for genetic transformation of crops because of its simplicity, reliability, and wider applicability.

9.6 Nanotechnology in Gene Transfer Experiments

Of late, principles of nanotechnology have been extensively applied in physical, biological, agricultural, and medical sciences in a highly beneficial manner. In textile industry, the use of nanotechnology includes minimizing loss of cellulose during processing of cotton into end product, coming out with 100 nm diameter fibers capable of being good absorbent of chemical input in agriculture and boosting the efficacy of enzymes required in cellulose–ethanol conversion (Lang 2013). The application of nanotechnology to produce nanosilica from rice husk has not only addressed the problem of rice husk disposal but also has come handy in making substances like glass and concrete (Awizar et al. 2013). Delivery systems for pests, nutrients, and plant hormones have been manufactured using nanotechnology tools (Sekhon 2014; Nair et al. 2010). Nanotechnology facilitates controlled release of agrochemicals allowing direct delivery of various macromolecules at the preconceived sites for enhancing plant disease resistance, nutrient utilization, and plant growth (Nair et al. 2010). Nanoencapsulation increases efficiency in terms of usage and safe handling of pesticides (Nair et al. 2010).

Lately, researchers have started applying nanotechnology in plant genetic engineering with noticeable results. Torney et al. (2007) demonstrated that a honeycomb mesoporous silica nanoparticle (MSN) system with 3 nm pores can act as an effective conduit for DNA and chemicals for their onward journey to segregated plant cells and intact leaves. Using MSN particles coated with a plasmid expressing green fluorescent protein (GFP) under CaMV35S promoter, the researchers showed transient expression in tobacco mesophyll protoplasts. Intact plant cells of tobacco cotyledons were also successfully transformed using gene gun after the mesopores of the DNA-coated MSN particles were capped by gold nanoparticles. Efficacy of this system for generating stable transgenic plants was established by raising callus from GFP bombarded immature maize embryos. Subsequently, the researchers generated transgenic tobacco plants harboring GFP gene driven by an inducible promoter which could be chemically triggered by addition of β-oestradiol. Transgenic plantlets were bombarded with β-oestradiol-loaded MSN particles having their pores capped by gold nanoparticles to prevent leaching. Presence of DTT as an uncapping agent in the germination medium allowed for the controlled release of the chemical inducer followed by expression of the GFP transgene. As the gold nanoparticles can prevent the molecules from moving out, the group, utilizing this trait, coated the β-oestradiol containing MSN particles with inducible GFP gene, capped the extremes with gold nanoparticles, and bombarded this system into wild type plants. Undoing the capping in presence of DTT resulted into controlled release of chemicals triggering gene expression in plants. Further diversification like increasing the probe size and multifunctionalization of these MSNs can help achieve target-specific delivery of proteins, nucleotides, and chemicals in plant cells. In addition, several biogenic species can be delivered together and released in a controlled fashion at the target sites by the use of MSN technology. This group proposed that nanotechnology can be applied to plant science to aid further investigation of plant genomics and gene function as well as improvement of crop species.

9.7 Future Generation Stress Tolerant Transgenic Plants

As discussed above, the production of abiotic stress tolerant transgenic plants is a highly desired commercial goal in present-day agriculture. To realize this goal, newer and more effective methods of genetic transformations are needed. The new discovery that using nanotechnology tools, gene transfers can be achieved in a controlled manner opens up new directions. Using this new method, it should be possible to transform wider range of species. Further, it should be possible to apply nanotechnology-based approach for genetically transforming those species which are not amenable to genetic transformation using Agrobacterium. By combining biotechnology and nanotechnology sciences, it should be possible to more effectively achieve goals like production of abiotic stress tolerant crops and plants with other desired agronomic traits.