Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

Abiotic stresses, such as drought, salinity, extreme temperatures, chemical toxicity and oxidative stresses, are serious threats to agriculture besides their deteriorative impact to the environment. Drought and salinity are the most important environmental stress factors that limit food production worldwide, and may cause a serious salinization on more than 50% of all arable lands by the year 2050 (Wang et al. 2003). In China, almost half of the land is arid or semi-arid and the crop production is strongly affected by drought and salinity seasonally even if in the irrigated farm land.

Potato is regarded as a moderately salt-sensitive (Ahmad and Abdullah 1979), and drought-sensitive crop compared with other crops from the field experiments (Salter and Goode 1967; van Loon 1981). Drought always influences development and growth of stem, root and tuber (Ojala et al. 1990), and reduces number of tubers and yield in potato (Cavagnaro et al. 1971).

Complex reaction of plant in response to drought and salinity stresses is a quantitative character which genetic control includes the functions of many genes and biochemical-molecular mechanisms. These responses lead to a wide variety of biochemical and physiological changes such as the accumulation of various organic compounds of low-molecular weight, collectively known as compatible solutes or osmolytes, synthesis of late-embryogenesis-abundant (LEA) proteins, and activation of several detoxification enzymes (Bajaj et al. 1999).

Using transgenic approaches to enhance drought and salinity tolerance has been thoroughly reviewed lately in plants (Apse and Blumwald 2002; Rontein et al. 2002; Wang et al. 2003; Chen and Murata 2008; Kolodyazhnaya et al. 2009) and potato (Byun et al. 2007). It is generally accepted that drought and salinity tolerance could be increased through transgenic approaches by incorporating genes involved in stress protection into plants that lack them. Transgenic potato plants with improved tolerance to drought and salinity stress have been produced using various genes which have been summarized in Table 22.1 and briefly discussed below.

Table 22.1 Genes overexpressed in transgenic potato plants for drought and salinity tolerance
Full size table

Trehalose is a non-reducing disaccharide of glucose. A plant that produces trehalose is often highly tolerant to desiccation stress. Goddijn et al. (1997) engineered trehalose biosynthesis in potato by introducing the otsA and otsB genes from Escherichia coli, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. Jeong et al. (2001) introduced GPD gene for glyceraldehydes-3-phosphate dehydrogenase from the oyster mushroom (Pleurotus sajor-caju) into potato and obtained transgenic potato plants with enhanced salinity tolerance. Ambard-Bretteville et al. (2003) suppressed FDH gene encoding for formate dehydrogenase in transgenic potato plants which formate levels are increased. Thus, the suppression resulted in accumulation of praline in response to osmotic stress. Turhan (2005) developed transgenic potato plants with higher salinity tolerance by expressing oxo gene which enhanced synthesis of oxalate oxidase for catabolizing oxalic acid. The dehydration-responsive element (DRE) is essential for regulating dehydration-responsive gene expression (Yamaguchi-Shinozaki and Shinozaki 1994). The transformation of plants using regulatory genes is an attractive approach for producing dehydration-stress tolerant plants. The overexpression of DREB1A gene for DRE-binding protein from Arabidopsis in transgenic potato showed that the tolerance to salt-stress was increased in proportion to its copy number of the gene in tetrasomic tetraploid potato (Behnam et al. 2006). Oxidative stress is a major damaging factor for plants exposed to environmental stresses. Tang et al. (2006) obtained transgenic potato plants with increased tolerance to multiple environmental stress due to the overexpressed both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. Glycine betaine (GB) is a common compatible solute in many different organisms including higher plants. Many plant species can accumulate GB in response to drought and salinity. Ahmad et al. (2008) and Zhang et al. (2011) showed that the transgenic potato plants were more tolerant to drought and salinity stress because of overexpressing codA and betaine aldehyde dehydrogenase (BADH) genes for GB synthesis.

This chapter, based on our research, presented the case study of enhancing drought and salinity tolerance in transgenic potato plants expressing BADH gene from spinach. Roles of transgenic potato in sustainable crop production and its biosafety concerns were also addressed.

2 Enhancement of Drought and Salinity Tolerance in Transgenic Potato by Expressing BADH Gene

To ensure their own survival and prosperity of their offspring, plants have evolved a range of strategies to cope with various abiotic stresses. One common mechanism is the accumulations of compatible solutes including certain polyols, sugars, amino acids, betains and related compounds. Glycine betaine (GB) is one of the most important of osmolytes (Chen and Murata 2008). Many plant species accumulate betaine in response to drought and salinity, thus adapt arid and saline areas (Rhodes and Hanson 1993). In higher plants, GB is synthesized by conversion from choline to GB through a two-step oxidation via the intermadiate betaine aldehyde (Hanson and Scoff 1980). The relevant enzymes are choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) (Sakamoto and Murata 2000). Transgenic plants of various species have been produced, which tolerance to drought and salinity has been enhanced because they have elevated levels of GB by expressing CMO or BADH transformed with the corresponding genes (Sakamoto and Murata 2000).

The fact that many important crops, such as rice, potato and tomato, are betaine-deficient has inevitably led to the proposal that it might be possible to increase drought and salinity tolerance by genetic engineering of GB synthesis (McCue and Hanson 1990). In this chapter, the spinach BADH gene was transformed into potato under the control of the constitutive expression promoter CaMV 35S and stress-inducible expression promoter rd29A respectively, and the resultant transgenic potato plants gained the ability resistant to drought and salinity stresses (Zhang et al. 2009, 2011).

2.1 Isolation of BADH Gene and Plasmid Construction

The 1,556 bp cDNA of BADH gene (GenBank accession AY156694) was isolated from spinach using reverse transcription-polymerase chain reaction (RT-PCR) method (Zhang et al. 2004). The sequence analysis showed that the BADH cDNA contains 1,494 bp open reading frame (ORF) encoding a protein of 497 amino acids. The nucleotide sequence of BADH cDNA shared 99.87% identity with BADH gene which was previously cloned from spinach (GenBank accession M31480) (Weretilnyk and Hanson 1990). The nucleotide sequence at position of 781–813 nt encodes the deca-peptide Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys-Ser-Pro and at position of 992–994 nt encodes cysteine (Cys) related to the function of enzyme activity, and the deca-peptide and Cys are highly conserved among general dehydrogenase (Weretilnyk and Hanson 1989).

The 824 bp of rd29A promoter was amplified from Arabidopsis thaliana genome by the PCR technique (Zhang et al. 2005). Sequence analysis showed that the cloned fragment shared 99.39% identity with reported rd29A promoter (GenBank accession D13044) (Yamaguchi-Shinozaki and Shinozaki 1993) and contained several cis-acting elements including dehydration responsive element (DRE) and ascorbic acid (ABA) responsive element (ABRE) (Shinozaki and Yamaguchi-Shinozaki 1997). The result from transgenic potato plants showed that the expression of β-glucuronidase (GUS) gene under control of the rd29A promoter was induced by drought, salinity, low temperature and ABA (Zhang et al. 2005).

The plant expression vectors pBIBB and pBIrB were constructed by fusing BADH gene with the constitutive expression promoter CaMV 35S in plasmid pBI121 and the stress-inducible expression promoter rd29A in plasmid pBIrd (Fig. 22.1) (Si et al. 2007; Zhang et al. 2005). The expression vectors were then introduced into Agrobacterium tumefaciens strain LBA4404 by freeze-thaw method (Hofgen and Willmitzer 1988) and proved by the enzyme digestion and PCR amplification.

Fig. 22.1
figure 1

Schematic diagram of the expression vectors pBIBB (a) and pBIrB (b). RB right border, LB left border, NOS-pro nopaline synthase promoter, NOS-ter nopaline synthase terminator, NPTII neomycin phosphotransferase II gene, 35 S the constitutive expression promoter CaMV 35S, rd29A the stress-inducible expression promoter rd29A, BADH betaine aldehyde dehydrogenase gene. Hind III, BamH I, EcoR I, Sac I restriction endonuclease recognition sites

Full size image

2.2 Potato Transformation and Molecular Analysis

Microtubers of potato cultivar Gannongshu 2 were used as the receptor for Agrobacterium-mediated transformation performed as described previously (Si et al. 2003). Green shoots were produced directly from surface of the transformed microtuber slices after 4 weeks cultured in MS medium (Murashige and Skoog 1962) containing 1 mg/L indole-3-acetic acid (IAA), 0.2 mg/L gibberellic acid (GA3), 0.5 mg/L 6-benzyladenine (BAP) and 2 mg/L zeatin riboside (ZR) supplemented with 75 mg/L kanamycin and 400 mg/L carbenicillin. Roots were formed in about 10 days when green shoots transferred to MS medium supplemented with 50 mg/L kanamycin and 200 mg/L carbenicillin. The plantlets with well-developed roots were propagated for further molecular analysis. PCR and Southern blot analysis showed that BADH gene has been integrated into genome of potato (data not shown). Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants transformed with BADH gene driven by the promoter rd29A (Fig. 22.2), while was not induced in the transgenic plants driven by the promoter CaMV 35S as shown in Fig. 22.3 (Zhang et al. 2009; Zhang et al. 2011).

Fig. 22.2
figure 2

Northern blotting analysis of BADH gene expression in the transgenic potato transformed with BADH gene under the control of the stress-inducible expression promoter rd29A. Plants were grown in vermiculite in 3 L pots in a greenhouse under natural light at 25 °C, and watered and fertilized weekly with a complete nutrient solution. When the plants reached 10 cm in height, they were treated with NaCl and polyethylene glycol (PEG, MW 6,000), respectively. NaCl treatment was begun at a concentration of 50 mM and increased stepwise by 50 mM every day until the final concentration, 500 mM, was reached. PEG treatment was conducted with 15% PEG solution once a day for 10 days. Lane C untransformed potato plant, Lane 1 and 2 untreated transgenic potato plants, Lane 3 and 4 transgenic potato plants subjected to NaCl and PEG treatments for 5 days, respectively, Lane 5 and 6 transgenic potato plants 3 days after being subjected to NaCl and PEG treatments for 10 days, respectively. Each lane in electrophoresis contained the similar 30 μg RNA sample stained with ethidium bromide. This figure was reproduced from Zhang et al. (2011)

Full size image
Fig. 22.3
figure 3

Northern blotting analysis of transgenic potato plants transformed with BADH gene under the constitutive expression promoter CaMV 35S. Treatment was same as Fig. 22.2. Lane 1 untransformed potato plant, Lane 2–6 transgenic potato plants 3 days after being subjected to NaCl and PEG treatments for 10 days, respectively. Each lane in electrophoresis contained the similar 30 μg RNA sample stained with ethidium bromide. This figure was reproduced from Zhang et al. (2009)

Full size image

2.3 BADH Activities and Relative Electrical Conductivities of the Transgenic Plants

The analysis of BADH activity demonstrated that it could be detected in the transgenic potato plants transformed with BADH gene driven by the promoter CaMV 35S, but the enzyme activity could not be detected in the untransformed control plants. The activity of BADH varied from 2.1 to 11.5 U among different transgenic individuals. The BADH activity and relative electrical conductivity on the transgenic potato leaves were highly negatively related (y  =  −3.7738x  +  57.083, r  =  0.989**) (Zhang et al. 2009).

The activities of BADH in the transgenic potato plants transformed with BADH gene driven by the promoter rd29A were rather low when they were not stressed, but increased greatly 3 days after the treatment with NaCl and PEG had applied. The BADH activities varied between 10.8 and 11.7 U and varied a little among the different transgenic plant lines. The relative electrical conductivities among the transgenic plants were 17.4–19.6% under NaCl and PEG stress, much less than those among the control plants (45.6%). The low conductivities showed that the cell membranes of the transgenic plants were less injured than those of the control plants under NaCl and PEG stress. A significant negative linear relationship between the relative electrical conductivity (y) and BADH activity (x) was observed, which could be represented by a function of y  =  −2.2083x  +  43.329 (r  =  0.9495), revealing that BADH activity was positively related to protection of cell membrane permeability (Zhang et al. 2011).

2.4 Drought and Salinity Tolerance in Transgenic Potato Plants

The growth of the transgenic potato plants in vitro was normal and better than the untransformed plants under NaCl and PEG stresses. Plant height increased 0.41–1.0 cm and fresh weight per plant increased 10–35% for the transgenic potato plants transformed with BADH gene under the control of the promoter CaMV 35S compared with the control potato plants (Zhang et al. 2009). For the transgenic potato plants with the promoter rd29A, plant height of the transgenic plants increased 0.4–0.9 cm and fresh weight per plant increased 17–29% compared with the control potato plants as shown in Fig. 22.4 and 22.5 (Zhang et al. 2011).

Fig. 22.4
figure 4

Height of transgenic potato plants 3 days after 10 days of NaCl and PEG treatment had been completed, respectively. Plants were grown in vermiculite in 3 L pots in a greenhouse under natural light. Treatment was same as Fig. 22.2. The data are the mean  ±  standard error (SE) from three replicates. C nontransgenic potato plant, 1–4 transgenic potato plant lines. This figure was reproduced from Zhang et al. (2011)

Full size image
Fig. 22.5
figure 5

Fresh weight per plant of transgenic potato plants 3 days after 10 days of NaCl and PEG treatment had been completed, respectively. Treatment was same as Fig. 22.2. Plants were grown in vermiculite in 3 L pots in a greenhouse under natural light. The data are the mean  ±  standard error (SE) from three replicates. C nontransgenic potato plant, 1–4 transgenic potato plant lines. This figure was reproduced from Zhang et al. (2011)

Full size image

When exposed to various degree of NaCl stress (0%, 0.3% and 0.6%) for 2 months, the leaves of transgenic potato plant still stayed green, while the leaves of nontransgenic potato plant became yellow and wilting under 0.3% NaCl stress. A better growth performance was still observed in transgenic plants when they grew under the condition supplemented with 0.6% NaCl in comparison to nontransgenic control (Fig. 22.6), demonstrating that the transgenic plants with BADH gene acquired higher tolerance to NaCl stress than that of nontransgenic ones (Li et al. 2007).

Fig. 22.6
figure 6

Comparison on growth of the transgenic potato plants transformed with BADH gene and nontransgenic potato plants under stress of NaCl concentration of 0%, 0.3% and 0.6% for 2 months respectively. (a–c) nontransgenic potato plants under stress of NaCl concentration of 0%, 0.3% and 0.6% respectively, (d–f) transgenic potato plant line GN-4 under stress of NaCl concentration of 0%, 0.3% and 0.6% respectively. This figure was reproduced from Li et al. (2007)

Full size image

The result suggested that the BADH gene can be used to improve the drought and salinity tolerance of important crops that are betaine-deficient through genetic engineering.

3 Role of Transgenic Potato in Sustainable Potato Production

With increasing population, higher demand for economic development and rapid urbanization, the global potentials for food will continue to grow; furthermore, climate change is leading to production uncertainties. Against this backcloth, a range of transgenic crops, that are herbicide-tolerant, insect-resistant, drought and other forms of stress-tolerant, or higher yielding, have been developed, and a few are now being grown in many parts of the world. The area of transgenic crops grown globally has increased from 2 to 134 million hectares in 2009 since wide-scale planting started in 1996 (Park et al. 2011). The principal transgenic crops are soya bean, maize, cotton and canola, which are modified for agronomic input traits such as herbicide tolerance and/or insect resistance (Bacillus thuringiensis-Bt). Recent work has focused on the use of biotechnology to produce abiotic stress-tolerant and nutritionally enhanced food and feed with a range of new events being predicted by 2015 (Newell-McGloughlin 2008).

Potato is an easy-to-grow plant and can provide more nutritious food faster and on less land than any other food crop, and in almost any habitat. A shift towards the use of potato in convenience foods, such as potato chips and French fries, has been recorded in developed nations, but in developing economies the majority of the potato crop is still used for direct consumption. Today, potatoes are grown worldwide and more than a billion people consume them on a daily basis. To support this demand, a lot of varieties have been developed (Mullins et al. 2006).

It generally requires 10–15 years to develop a single potato cultivar through traditional breeding (Byun et al. 2007). It also relies on utilizing existing genetic stocks, whose quantities are limited (Bajaj et al. 1999). In addition, the cultivated species Solanum tuberosum is autotetraploid which has a highly complicated quantitative inheritance pattern, and is difficult to hybridize sexually with the related relative wild species Solanum. The most obvious advantage of using transgenic approaches for crop improvement is that genes from any organism can be utilized, whereas in conventional breeding the gene pool is restricted to closely related plant species. Thus, a large variety of genes are available for transfer into plants to confer a specific desirable effect (Bajaj et al. 1999).

Park et al. (2011) deemed that transgenic crops are a potential ‘tool’ giving options for ongoing sustainable development if the growing world population is to be adequately fed, both in terms of quantity and quality, without further compromising the environmental services that the planet provides, and elucidated the considerable contribution of transgenic crops in relation to the three traditional pillars of sustainability, i.e. economically, environmentally and socially (Park et al. 2011 and references therein).

4 Biosafety of Transgenic Potato

Despite the growth and use of transgenic crops in many areas of the world, some governments, organizations and individuals still hesitate to acknowledge that transgenic crops provide economic and environmental benefits that are unobtainable in a timely manner via non-transgenic advances in plant breeding. Hall and Moran (2006) described some of the organizations that believe that there are unacceptable risks associated with the release of transgenic crops. Conner et al. (2003) and Nap et al. (2003) summarized the current status of environmental release of genetically modified (GM) crops around the globe. They provided an overview of the approaches used for regulating GM crop release into the environment and presented a detailed description of risk assessments and how they are performed, followed by a discussion of the perceived risks associated with the release of GM crops. Craig et al. (2008) summarized general features of risk assessments of GM crops, which provided an introduction to some of the main considerations made in the compilation and evaluation of risk assessments.

In order to alleviate some of the public concerns over the deployment of GM crops in agriculture, Conner and colleagues developed a novel transformation vector (intragenic vector) system (Conner et al. 2007; Barrell et al. 2010). Intragenic vector system is a gene transfer system composed of only DNA that originates from that host plant species (or related species to which it can be hybridised). Gene transfer using intragenic vectors will facilitate the well-defined genetic improvement of plants with all transferred DNA originating from within the gene pool already available to plant breeders. In this manner, genes can be introgressed into elite cultivars without the incorporation of any foreign DNA. The resulting plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. With gene transfer using intragenic vectors, there is no longer a clear biological distinction between traditional plant breeding approaches and development of GM crops (Conner et al. 2007). This opens up the possibility of using an intragenic vector system to create non-transgenic GM crops.

Many nontheological ethical objections to genetic engineering are associated with interfering with nature or natural evolution or the natural order of life. The intragenic vector system will help alleviate some of the ethical issues associated with transferring DNA across wide taxonomic boundaries, and will provide a socially acceptable and responsible way forward for the development of GM crops (Barrell et al. 2010).

For potato crop, genetic modification using intragenic vectors can therefore provide a valuable breeding tool. There are several groups improving potato traits, such as disease resistance, abiotic stress tolerance, pigmentation and processing quality, using intragenic/cisgenic approaches (Barrell et al. 2010). The intragenic vector system offers opportunities to accelerate the efficiency and extent of further potato improvement. Ongoing improvements can be expected in characteristics such as resistant to pests, diseases, herbicides, and environmental stress, as well as quality traits such as improved post-harvest storage, flavor, nutrition, shape and colour. Genetic manipulation of these characteristics will allow breeders to respond much more quickly to the market need for new and improved potato cultivars. The anticipated result is higher quality, blemish-free tubers with reduced chemical residues as demanded by the processors and consumers (Conner 2004).

The adoption rate of GM potato in agriculture will be become higher along with higher satisfaction of growers and benefits for the whole production chain result from best characters of GM potato. GM potato will be proved to be a promising solution for sustainable potato production, which could serve as one of powerful tools in combating famine and malnutrition in developing countries with increasing of world population in the future.

5 Conclusion

In the twenty-first century, food security has potential crisis in the world. Potato crop is grown worldwide and plays very important role in food security in developing countries. Since potato crop is introduced to China in the seventeenth century, it is steadily spread throughout the country. In order to overcome drought climate and backward production conditions in north China, the potato makes a greater contribution to solve food problem since it can be grown to yield relatively better production while other food crops is not in the arid and semi-arid areas.

Improvement of cultivated potato by traditional breeding method is slow and unpredictable due to its tetraploid genetics and the quantitative nature of inheritance. Genetic engineering provides a faster and more reliable means for potato crop improvement and these techniques are especially applicable to development of resistance to abiotic and biotic stresses such as drought, salt, cold, and pathogens (Byun et al. 2007).

In the chapter, the transgenic potato plants that are resistant to drought and salinity stresses are developed by transforming into potato with BADH gene from spinach. However, the transgenic potato plants have yet to be grown commercially since they must be performed assessment on field performance and biosafety. For practical applications, the useful stress-tolerant transgenic potato plants must give higher yield under stress and field conditions compared with the control plants. There is still much more work ahead in assessment of the transgenic potato plants under field performance.

Food insecurity is one of the most important social issues faced today. Strategies to address food insecurity must aim to increase agricultural productivity in order to tackle poverty, and must provide long-term improvements in crop yields to keep up with demands as the world’s population grows. Genetically enhanced plants provide one route to sustainable higher yields, either by increasing the intrinsic yield capability of the crop or by protecting them from biotic and abiotic constraints (Christou and Twyman 2004). In conclusion, genetic engineering has the potential to help increase production and productivity in agriculture. While there is no easy solution to improve potato crop for sustainable production and food supply, transgenic approach is an alternative way to meet the aim and contribute to safe food supply.