**Table 1.**

*Synthesis of transgenesis and cisgenesis results presenting the transfer of single or multiple resistance genes in order to improve biotic stress resistance in potato.*

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

#### **Figure 2.**

*The principal mechanisms of interaction between pathogens (bacteria, fungi and viruses) on the left and insect pests on the right with the potato host: the pathogens trigger two responses PTI (pathogen triggered immunity) and ETI (effector triggered immunity); in PTI the membrane proteins PRR recognize pathogen molecular patterns (PAMPs) and induce transcription factors (TFs) which activate immunity genes; in ETI effector molecules interact with specific resistance genes (R), but they can also interact with sensitivity genes (S) to inhibit PTI; insect pests interaction with its host is less understood but at first the pest interacts with leaf trichomes, glandular and/or non-glandular, mainly acting as a physical barrier; after wounding the leaf cells are inducing either tolerance responses like compensatory photosynthesis and delayed plant development, or resistance responses through synthesis of toxins like glycoalkaloids. Resistance mechanisms can activate HIPV (herbivore induced plant volatiles).*

of R genes are: *R1*, *R2* and *R3a*, *R3b*, originally identified in *S. demissum*; *Rpi-blb1* (RB), *Rpi-blb2*, *Rpi-blb3* from *S. bulbocastanum*; *Rpi-vnt1.1* and *Rpi-vnt1.2* from *S. venturii*; *Rpi-mcq 1 from S. mochiquense* [6, 64], etc. R genes were also delivered into potato varieties as gene stacks. In Europe BASF Company petitioned for the release of potato Fortuna resistant to late blight (*Pi*) after stacking of two R genes: *Rpi-blb1* and *Rpi-blb2*, obtained after a long effort of breeding, but unfortunately, this cultivar was never marketed [6]. The Simplot's second generation Innate® potato which besides reduced browning and bruising, also carries R genes and hence is resistant to late blight (*Pi*), was approved for cultivation in USA [65], and for cultivation and consumption in Canada [66]. One important research project was developed in Netherland between 2006 and 2015 on Durable Resistance in potato against *Phytophthora* (DuRPh) at Wageningen University and Research Centre [64]. The aim of this project was to identify and clone new durable resistance genes from potato wild relatives and transfer them as single or stalked genes into varieties by cisgenesis using marker assisted selection (MAS). Through this project a great deal of data has been accumulated and cisgenic varieties resistant to late blight were produced but these will require some more backcrosses to be released as resistant and productive varieties [64]. Still cisgenesis is considered as GM in Europe. A successful cisgenic approach was applied in Africa, where highland varieties were transformed with an efficiency of 75% using three Rpi genes: *Rpi-blb1*, *Rpi-blb2* and *Rpi-vnt1.1* (**Table 1**) [34]. R genes that improve resistance to other pathogens were also discovered: *Rx1* and *Rx2* (from *S. tuberosum* ssp*. andigena* and *S. acaule*, respectively), that confer resistance to potato virus X (PVX) [67]; *Gro1–4* from *S. spegazzinii,* confer resistance to root cyst nematode *Globodera rostochinensis* (**Table 1**) [41]. Another strategy for resistance to

a broad spectrum of pathogens is overexpression of a single gene located upstream in signalling cascades and thus regulates large number of defense-responsive genes. There are many examples of successful engineered plants using different constructs to overexpress trans- and endogenous genes in crops, including potato. Overexpression of these upstream signalling genes and defense-related genes can lead to a constitutive expression of resistance phenotype. In plant disease resistance, a vital role is played by small G-proteins and subsequent cellular responses to pathogens such as bacteria, fungi and viruses [52]. A number of G-proteins have been transferred to different plant species including potato where stable overexpression of *AtRop1* (DN-*AtRop1*) increased resistance to *Pi* infection (**Table 1**) [32]. An important breakthrough is the continuous research identifying new molecular markers linked to resistance genes or more recently QTLs (quantitative trait loci) such are: AFLP, RFLP, SSR, RAPD and their maps available for potato breeding [68]. At International Potato Center a continuous effort, as mentioned above, aims to store genetic diversity and improve it for the benefit of the next generations and efficient alleviation of underdeveloped nations' poverty. Several other genes were also cloned and transferred into potato crop for improvement of resistance to: PVY (*eIF4E-1* variant *Eva1*) and *Pi* – host induced gene silencing (HIGS) (**Table 1**) [33, 48]. The aim of the latest strategy is to achieve more durable resistance than R genes, but this also uses gene constructs that fall under GM rules [6].

#### *2.1.2 Insect resistant potato crop*

Insects are also a plague for potato production but the most difficult to control is the voracious Colorado potato beetle (CPB). It is estimated that 75% of potato production can be lost by pests if left uncontrolled [69]. CPB develop on potato crop, larvae and adults eat leaves and are able to completely skeletonize the plants. During development, the three stages of instar larvae consume around 40 cm<sup>2</sup> of potato leaves [70]. Plant breeding and biotechnology were not able to release a variety resistant to CPB without GM technology. Wild potato relatives are a reservoir of resistance traits as it was discussed for pathogens. Two natural host plant resistances are known: glandular trichomes and specific glycoalkaloids, the leptines I and II [71]. Detailed knowledge on the interaction between potato and resistant relatives with the voracious beetle are still scarce (**Figure 2**). Another interesting mechanism of resistance was discovered [72], the hypersensitive reaction of plants to CPB egg masses and egg drop. Any breakthrough into the physical, physiological and molecular mechanisms of resistance will fasten the progress of resistance breeding using biotechnology. The main strategy of genetic engineering to induce resistance to CPB was based on bacterial toxin from *Bacillus thuringiensis* (*Bt*), a bacterium also used in integrated pest management by spraying bacterial suspensions in the field. The technology is very specific for a certain species of pest, because *Bt* not only has a large repertoire of the *cry* genes that produce the protoxins involved in pest induced mortality, but the toxin is formed only in the gut of feeding pests and would not affect non-targeted beneficial insects [71]. The first success was introducing by gene transfer the *cry3a* gene into potato cv. Russet Burbank to protect it from CPB attack [73]. The GM variety with resistance to CPB was approved for human consumption and was commercially available in USA between 1996 until 2001, proving to control the beetle in the field without any unwanted effects on the cultivar [74]. NewLeaf™ potato, developed by Monsanto, containing *cry3a* proved to supress CPB populations at greater extent as insecticides or sprays based on formulations from *Bt* bacteria containing CRY3A protein [71]. In the next years, after the first success with cry3a, other *cry* genes have been optimize and transferred into potato: *cry3Ca1*, *cry1*, *cry3Bb1* [71] (**Table 1**).

#### *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

Coombs *et al* [75] combined leptines, glycoalkaloids considered as toxic to CPB, with glandular trichomes and *Bt*-*cry3a* to obtain transgenic potato host plants resistant to CPB. In that way the main problem of *Bt* potato, the development of resistance, could be also managed [75]. To date, there are no *Bt* potato on the market, as discussed in public acceptance of GM potatoes. Recent studies have been focusing on RNAi technology, including direct spraying of dsRNA in the field [71]. The first success with dsRNA used in a transgenic approach [76], lead to long or short double stranded RNA used to target a specific gene at posttranscriptional level determining mRNA fragmentation and hence silencing the gene. This proof of concept brought about a growing interest for the use of RNAi technology for controlling the CPB pest [57]. Moreover, non-transgenic alternatives were developed including dsRNA spraying on the plants [59, 77], but in this year (2021), resistance development in CPB populations after dsRNA foliar-delivery in potato has been already observed [78]. Sequence of CPB transcriptome can assist in the identification of new target genes for RNAi that can be used to control this pest [79]. To date, 24 target genes with important roles in cellular functions were silenced using RNAi, as reviewed by Balaško e*t al* [71]. Knockdown of those genes affect insect morbidity and mortality. There were also different delivery methods of dsRNA into CPB, like the use of bacteria, liposomes and nanocarriers, all of them able to protect and deliver dsRNA [77]. Moreover, other improvements for CPB control were the xenobiotic transcription factor Cap 'n' collar isoform C (CncC) that regulates the expression of multiple cytochrome P450 genes, and plays crucial roles in CPB insecticide resistance. The suppression of CncC by RNAi reduced imidacloprid resistance of CPB [80]. Ochoa-Campuzano *et al* [81] identified prohibitin, an essential protein for CPB viability, as Cry3Aa binding protein. Combination of feeding prohibitin dsRNA and treatment with Cry3Aa enhanced the toxic effect by threefold and CPB was killed faster with 100% mortality in five days. The molecular mechanisms of synergism between prohibitin, RNAi and Cry3Aa toxin are not understood, but this study proposes an interesting method, combining toxins derived from bacteria or other organisms with RNAi in order to improve efficiency of dsRNA in pest control. Moreover, recently targeted mutagenesis using CRISPR-Cas9 technology in CPB was demonstrated [60], a technology which holds great promise for the future.

#### *2.1.3 Gene transfer for resilience to abiotic stress*

Abiotic stresses such as drought, salt, high temperatures and extreme weather also limit potato yield around the world. With global climate change, abiotic stress is expected to be less predictable in the years to come and also affect pathogen attacks and pest effects on potato and other crops. The response of the plants to abiotic stresses involve generally the expression of inducible resistance genes. In particular, transcription factors (TFs) that control resistance genes are a key in gene regulatory networks that control the expression of many genes involved in stress responses [82]. Transgenesis uses genes for such TFs like WRKY, MYB or DREB, the last also used in potato crop (**Table 2**). Other genes that were engineered in potato are related to response of the plants to abiotic stress, like *StProDH1,* which is a key player in potato response to drought stress [93]. Through the manipulation of abscisic acid signal transduction after loss of function of cap-binding protein (CBP) [71], in cv. Désirée, a higher tolerance to drought was reported [92]. Through transgenic approach, potato lines with increased betaine aldehyde dehydrogenase, an enzyme for glycine betaine biosynthesis, which has important role in drought stress, has been able to induce drought tolerance in potato [88]. Transcriptome analysis, comparing control with drought stressed potato plants, has indicated many genes that are overexpressed


#### Solanum tuberosum *- A Promising Crop for Starvation Problem*

*amiRNA – artificial miRNA; APX - ascorbate peroxidase; BADH - betaine aldehyde dehydrogenase; CaPF1- pepper transcription factor belonging to the family of TFs ERF/AP2; CBF - C-repeat Binding Factor;DREB - dehydration responsive element binding protein; CodA - choline oxidase; GB – Glycinebetaine; HSc70 – heat shock cognate 70 gene; HvNHX2 – Hordeum vulgare vacuolar Na+/H+ antiporter; IbOr – Ipomeaea batata orange gene; ScTPS-Saccharomyces cerevisiae trehalose-6-phosphate synthase; SOD - superoxide dismutase; StEREBP1 – S. tuberosum ethylene responsive element binding protein 1; StnsLTP1 – S. tuberosum nonspecific lipid transfer protein 1; StProDH1 – S. tuberosum proline dehydrogenase 1; TPS1- yeast trehalose-6-phosphate synthase 1.*

#### **Table 2.**

*Examples of single or multiple resistance genes transfer to improve abiotic stress tolerance in potato.*

#### *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

or underexpressed during drought stress, with genes involved in processes like: intracellular water and ion homeostasis, membrane structural stability, and reconstruction of primary and secondary metabolism, and stress regulatory genes, as calcium ions, TFs and receptor protein kinases that are involved in stress response through signal transduction and metabolic pathways [112].

Salt stress caused by soil salinization is an increasing threat to agriculture worldwide [113]. Different factors lead to the continuous salinization of the soil, mainly different agricultural practices such as irrigation and some fertilization procedures. The mechanisms that are involved in salt stress response are cellular and physiological: e.g. different cellular signalling, various ion transport, water management and specific gene expression which are involved in growth, development and survival [113]. Researchers are working on halophytes, plants that are adapted to salty soil, to get new insights on plant responses to salt stress. In the case of potato, as presented in **Table 2**, there are transgenic strategies which proved their utility in obtaining salt tolerance, either alone or in combination with other stress factors. Potato plants adapt to salinity stress through different mechanisms like osmotic adjustment by accumulating compatible solutes in the cytosol, decrease leaf water potential leading to reduced cell turgidity and growth retardation and tuber yield loss. One of the most important compatible solute is proline, which was accumulated in cv. Désirée 3.5 fold and 11 fold at 100 and 200 mM NaCl, respectively [114]. However the proline effects on salt tolerance need additional studies because foliar application of proline has no effect on salt tolerance of plants [115]. Potato is adapted to cool weather mostly preferring temperate zone. The vegetative part of plants grow properly at 20–25°C temperature, while tubers develop better at 15–20°C. The response of potato plants to high temperature varies across the cultivars, one example being the commercial cv. Russet Burbank, which exhibit maximum rates of photosynthesis at 24 to 30°C and a reduction of photosynthetic activity only at or above 35°C [116]. Global warming and drought are expected to drastically reduce the potato productivity, but with biotechnology heat tolerant potato was successfully obtained (**Table 2**). Plants exhibit different strategies to cope with high temperature stress involving physiological, morphological and molecular levels. At molecular level heat stress increase the activity of heat stress TFs (HSFs), which trigger the accumulation of heat shock proteins (HSPs). HSPs are known to govern heat stress response (HSR) and acquired thermo-tolerance through their role as molecular chaperones [117]. In a genome wide study 27 *StHSFs* in the *Solanum tuberosum* genome were identified [118], which have diverse regulatory functions during stress. Underlining the molecular mechanism of how heat stress induces HSFs trimerization, their activation and synthesis of HSPs is still underway. Elucidation of the mechanisms of heat stress response may offer new insights that will be useful in breeding new heat resilient cultivars with sustained or even enhanced potato crop productivity and quality in response to climate change.

#### *2.1.4 Multiple stress factors*

In nature, generally multiple stresses act on crops at the same time and all of them contribute to noticeable losses in production. Nowadays, there is knowledge about various genes that contribute to both biotic and abiotic stress response and resistance/tolerance. The effects of abiotic stress on potato crop under climate change is detailed in a recent review [117]. Molecular and genomic analysis revealed transcriptionally regulatory pathways involved in modulation of stress responsive genes. As mentioned above TFs are playing a crucial role, particularly in multiple stress response of plants [119]. Examples of TFs that activate stress responsive genes are AP2/ERF, containing AP2/ERF binding domain, a large superfamily that divides in AP2, ERF and RAV [120]. This family of genes participate in developmental processes. AP2 family is involved in regulation of development, together with ERF protein family. Based on the differences in DNA box-binding ability of the single AP2/ERF domain, the ERF family is divided in ERF and CBF/DREB (C-repeat Binding Factor/Dehydration Responsive Element-Binding) (**Table 2**). ERF proteins are mainly involved in inducing disease resistance in a negative or positive mode of action. Gangadhar *et al* [121] have identified 95 genes involved in heat tolerance in potato, eleven of them being associated with multiple stress tolerance, like drought, salt and heat. Prolamins are a group of plant storage proteins that represent useful factors implicated in controlling both abiotic and biotic stress-response in plants. The plant non-specific lipid transfer protein, nsLTP, is involved in phospholipid transfer but also various other biological functions as seed storage, lipid mobilization, cuticle synthesis, somatic embryogenesis and pollen tube adhesion [110]. Transgenic potato lines over-expressing *StnsLTP1* acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of ROS and regulated expression of stress-related genes (**Table 2**) [110]. Another example is the use of TF *StWRKY1*, which successfully induced resistance to *Pi* and improved tolerance to water scarcity. This experiments prove the role of TFs and in particular WRKY in regulating both biotic and abiotic stress resistance thereby modulating plant basal defense networks and thus playing a significant role for potato crop improvement.

#### **3. Use of cell fusion between potato crop and its wild relatives for resilient potato**

Over the past fifty years the introgression of new traits from wild *Solanum* species have mainly achieved by using classical breeding methods. The number of wild species that could be integrated into potato breeding is quite limited because of sexual incompatibility and endosperm balance number (EBN), although there are techniques other than sexual crosses, such as manipulations of ploidy levels [122], breeding 2n gametes or using bridging species to integrate genes from wild *Solanum* species into modern cultivars [123]. Through sexual crosses the main source of resistance genes is still *S. demissum*, more than half of the modern cultivars contain introgressions from this species [123]. The main limitations of the potato classical breeding are tetraploidy and heterozygosity, which make breeding very complex and time-consuming [124]. Moreover, when genes from an incompatible wild species have to be exploited, as was in the case of *S. bulbocastanum*, the use of a bridging species was applied to produce new cultivars which took 49 years and then only one resistance gene (*Rpi-blb2*) against late blight was integrated into potato gene pool (cvs. Bionica and Toluca) [125]. Nowadays, somatic hybridization through protoplast fusion is a well refined and routinely used method in order to create *Solanum* hybrids with different useful properties [126, 127]. Plant protoplasts are naked somatic cells from which the cell wall has been removed by enzymatic digestion, therefore these cells can be used for gene transfer, somatic hybridization [128], and more recently for targeted mutagenesis and genome research. Protoplasts are still totipotent and they are able to regenerate new cell wall, divide to form new cell colonies, microcalluses, calluses and finally new plants. This protoplast technology proved to be very efficient in potato crop and is a reliable and useful way to regenerate large numbers of somatic hybrids (SHs) with distinct genetic backgrounds [129–131]. Among the agronomical important crops, potato was the first used in protoplast culture and somatic hybridization [132, 133], which opened

#### *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

the way for free gene transfer from potato wild relatives into potato crop [134]. Leaf mesophyll cells of *in vitro*-grown plants were used to isolate protoplasts [135], then the obtained fused products were cultured in VKM medium [136], followed by shoot development on the MS13K medium [137]. Recently, selection of SHs (*S. tuberosum* + *S. chacoense*) based on callus growth tagged with *gfp* has been also observed [138]. Different methods are available for protoplast fusion, but only two are generally used: electrofusion and PEG (polyethylene glycol) induced fusion [128]. Electrofusion is the most widely used method since its discovery in 1979 [139], and it consists in first instance of protoplast agglutination induced by the use of an alternating current (AC) field, the so-called dielectrophoresis [140]. In the second phase the agglutinated aligned protoplasts are induced to fuse by using direct current (DC) square wave pulses with a high intensity (2000 V cm−1) and very short duration (10–100 μs) [141]. PEG-induced fusion generally has a similar efficiency as electrofusion, especially after applying calcium solution washing step [128]. Immediately after fusion or after the plants have been regenerated, the obtained SHs are subject to different analysis, such as cytological (flow cytometry, chromosome counts, chloroplasts counts in guard cells, FISH - fluorescence in situ hybridization and GISH - genomic *in situ* hybridization), molecular: isozyme, molecular markers (e.g. RAPD, RFLP, ISSR - inter simple sequence repeat, SSRsimple sequence repeat, AFLP - amplified fragment length polymorphism, and DArT-diversity array technology) [8, 129], phenotypic changes (e.g. foliage, stem, leaf, flower and tuber traits) and pollen fertility. Due to their stability and universality SSR markers are the most widely used [129, 130]. Recently, the application of DaRT made it possible to find out the composition of the SHs genome between potato and *S. x michoacanum*, which demonstrated the presence of both parents genome in hybrid plants, and provided evidence for late blight resistance trait transfer from wild relatives into SHs [142]. SHs are also analysed for cytoplasm types (haplotype of chloroplast/mitochondria: W/α, T/β, W/γ, W/δ and S/ε) [143], based on organelle segregation after fusion and organellar genome-specific markers as described by Lössl *et al* [144]. Finally, SHs are examined for the presence of target traits under field or controlled conditions eventually being tested for phenotype and tuber qualities in the field [8, 145]. Somatic hybridization through protoplasts fusion, which circumvents pre- and post-zygotic crossing barriers, can be successfully used to insert resistance into potato (**Table 3**) [143]. It has a greater potential for self-generating biodiversity in numerous nuclear and cytoplasmic genome combinations than sexual hybridization [184]. It also provides an opportunity for initiating recombination events between parental genomes. Moreover, homeologous recombinations (recombination between similar but not identical DNA molecules), can also be increased, that might increase the integration of valuable traits, by inducing a DNA repair deficiency, for instance, mismatch repair deficiency (MMR) [145, 175, 185]. MMR was successfully induced by *Agrobacterium*-mediated transfer of *AtMSH2* gene in antisense orientation or a dominant negative gene into *S. chacoense* [185], followed by somatic hybridization with potato tetraploid variety Delikat through electrofusion. Resistance to Colorado potato beetle (CPB) was more common in MMR deficient somatic hybrid plants [175]; MMR was also responsible for greater diversity and a novel trait tolerance to drought stress [180]. Since 1980s, different wild *Solanum* species have been hybridized with potato using protoplast fusion, and many of them express various valuable traits, including resistance to viruses [186], bacteria [187], fungi [188], insect pests [175] or tolerance to abiotic stresses (**Table 3**) [181]. Furthermore, multiple resistance can be also transferred from wild relatives into the potato gene pool [130] and even SHs with multiple parent lines can be produced, as in the case of the tri-species somatic hybrids [178].




#### **Table 3.**

*The most important somatic hybrids with proved resistance to pathogens, pests and tolerant to abiotic stresses and the methods applied for their analysis.*

One of the most economically valuable SH was obtained by fusion between the incompatible *S. bulbocastanum* species and cultivated tetraploid potato [189], which highlighted the advantages of somatic hybridization in potato genome improvement, because the SHs were highly resistance to *Pi* in the laboratory and a field under intense disease pressure. After back-crossing of these SHs with potato cultivars the resistance to this disease was not lost. Subsequently, RB gene involved in durable resistance was isolated, which is located on chromosome VIII [190]. Transgenic plants with RB, were regenerated after *Agrobacterium*–mediated gene transfer and proved durable resistant [191]. Since then, *S. bulbocastanum* demonstrated several times its value as a resource of durable resistance genes against late blight, therefore it has been an increasing interest in transferring the resistance traits of this species to cultivated potato [154, 192]. RB gene was the first durable resistance gene described for late blight, but soon many other genes were discovered both in *S. bulbocastanum* and other wild species. To date, there are four characterized resistance genes in *S. bulbocastanum*: *Rpi-blb1* (formerly RB), *Rpiblb2*, *Rpi-blb3* and *Rpi-bt1* [190, 193–196]. In addition, late blight resistance from other sources was also accessed by generation of interspecific SHs with the wild species *S. pinnatisectum* [163], *S. tarnii* [129], *S. cardiophyllum* [130] and more recently *S. x microachanum*, a wild diploid derived from a spontaneous cross between *S. bulbocastanum* and *S. pinnatisectum* [160]. These newly produced SHs were also tested in the field and were resistant after two or three years of assessment, therefore they are suitable for introducing in breeding. *S. stenotonum* is an exquisite source of resistance to bacterial wilt caused by *Ralstonia solanacearum*, and all of the SHs obtained by fusion of potato protoplasts with this wild species were as resistant as the wild parent line [197]. Similarly, *S. chacoense* was explored for molecular markers associated with bacterial wilt resistance, and for introgression of resistance into the potato gene pool [148]. A very successful approach involved the transgenic induction of MMR deficiency in a high leptine-producing accession of *S. chacoense*, followed by somatic hybridization, because large number of generated plants exhibited both antixenosis and antibiosis against CPB [175]. Recently, by using gene specific markers four *Pi* resistance genes: *Rpi-blb1, Rpi-blb3, R3a* and *R3b* were identified in *S. bulbocastanum* and derived SHs with potato cvs. Delikat and Rasant. The genes were present also in BC1 and BC2 progenies and resistance to late blight

#### *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

was maintained along good tuber traits [146]. The resistance gene pool of wild *Solanum* species can also be used to combat abiotic stresses like salt, drought and frost. For example SHs originating from fusion between potato and *S. bertaultii* are tolerant to salt stress [182]. Freezing is another abiotic factor, which decrease the yield of potato and SHs of *S. tuberosum* (+) *S. malmeanum* proved to be tolerant to frost [181]. Furthermore, SHs of potato and *S. chacoense* show different level of drought and salt tolerance beside resistance to CPB [184]. Interspecific somatic hybridization gave good results but the intraspecific somatic hybridization proved to be also suitable for potato improvement. Starting in the 1990s, somatic hybridization was used to study different dihaploid lines of potato generated by crossing with *S. phureja* [198] or pollen and anther *in vitro* culture. The results of the protoplast fusion of two dihaploid potato lines were at first not very promising, but the restoration of tetraploids from two dihaploid lines with valuable yield and resistance traits soon proved to be a valuable approach for potato breeding. Furthermore, resistance to nematodes, viruses (PVY) and *Phytium* bacterial diseases were achieved by intraspecific protoplast fusion [169, 199]. The intraspecific hybridization has a finite repository, but as long as this area is not exploited, it is worth considering. During interspecific somatic hybridization two obstacles may occur: (1) transfer of too much exotic, wild genetic material along with the desirable gene(s) from the wild species; and (2) genetic imbalance which lead to somatic incompatibility. These limitations result either in abnormal growth and development of the SHs, and/or regeneration of infertile plants. In order to reduce the wild imprint, the introgressive hybridization is followed by one or multiple back-crosses of the somatic hybrids with cultivars. The purpose of these cross-hybridization processes is on the one hand to eliminate the undesirable part of the wild genome, on the other to retain the target traits inherited from the wild parents and to restore the agronomic valuable cultivars, with high yield and adequate tuber quality [129, 130, 139]. Several experiments proved that, the above mentioned disadvantages could be eliminated through multiple back-crosses. Somatic hybrids of cultivated potato and *S. tarnii* were resistant to late blight and PVY, and these valuable traits were successfully transferred to BC1 progenies, which also presented good tuber yield and quality [129]. Multiple years of field evaluations of *S. etuberosum* + *S. tuberosum* and progenies showed stable transmission and expression of PLRV and PVY resistances in three BC1, BC2 and BC3 and two BC1 and BC2 generations, respectively [171]. Furthermore, late blight resistance can be transferred through breeding from tetraploid somatic hybrids (*S. × michoacanum* + *S. tuberosum* and autofused *S. × michoacanum*) to common varieties [142]. Bacterial wilt resistance was transferred to advanced progenies of somatic hybrids between *S. commersonii* and cultivated potato, and three highly resistant clones (BC1 and BC2) were selected as breeding materials [170]. In the case of potato there are many reports of symmetric interspecific somatic hybridization between diploid wild species and potato dihaploid lines [127]. The main problem with the majority of these hybrids was the infertility, which made difficult the restoration of valuable cultivar. For this reason symmetric somatic hybridization between tetraploid potato cultivars and diploid wild species became more popular [200]. The expected results after tetraploid with diploid protoplast fusion are hexaploid SHs, but among them aneuploid or mixoploid hybrids are often regenerated [131]. Genetically, the hybrids may be unstable and usually eliminate chromosomes from the wild species during the next stage of tissue culture, as occurred in the case of potato and *S. bulbocastanum* hybrids, but, after two back-crosses with cultivated potato, many of them re-stabilize at tetraploid level [131, 145]. Theoretically hexaploid or near hexaploid SHs of potato will tend to eliminate the wild species chromosomes and maintain only a few alien chromosomes or introgress some genes from the wild

parent. Chromosome elimination in some interspecific somatic hybrids of potato largely depends on the phylogenetic relationship, type of genome: A, B, C, D and P [201], cell cycle synchronization after fusion and the two parent chromosomes interaction during mitosis [202]. Asymmetric somatic hybrids can be a result of the ordinary symmetric fusion or can be induced by fragmenting the donor species DNA by using the donor-recipient method [203]. Production of asymmetric somatic hybrid plants aroused interest of breeders, because with controlled chromosome transfer the restoration process of cultivars is faster and easier [204]. Usually, the donor protoplasts are treated with sub-lethal doses of ionizing irradiation, such as gamma, X rays [205, 206] or UV irradiation [207], in order to induce double-strand breaks and hence partial genome elimination [208]. In addition to irradiation, chemical agents can be used to induce chromosome elimination, such as restriction endonucleases, spindle toxin or chromosome condensation agents [209]. With applying these methods, asymmetric potato hybrids with some wild *Solanum* species [210] and intergeneric somatic hybrids were successfully produced [211, 212]. Another possible limitation of somatic hybridization is the production of somatic hybrids with resistant traits, but with decreased tuber yield and/or quality (misshaped tubers). Various solutions exists to overcome these disadvantages: use of haploidization and intra-specific hybridization of dihaploid potato lines [198], or the use of somatic fusion in which tetraploid potato cultivars are fused with sexually incompatible diploid wild species, when the resulted hexaploids are most of the time fertile and are crossable with other tetraploid cultivars [129–131]. Somatic hybridization produced a large number of somatic hybrids in potato some of them being integrated into pre-breeding and then breeding programs. The advantage of somatic hybridization is the transfer of multiple resistance genes, although it is difficult to control the genes transferred into the crop from its wild relative. It was thought that asymmetric fusion will allow better control on the genetic material to be transferred but soon it was demonstrated that only a low amount of donor DNA is eliminated and there is no correlation between the dose of radiation and DNA fragmentation. Nowadays, new strategies can be applied to better control the genetic fate of the SHs. Molecular markers can be used to select the traits or genes of interest [145], selection pressure like pathotoxins can be applied to increase the number of resistant SHs to a certain pathogen, *etc*. Moreover, there is a new opportunity to use all the genomic tools to get more insights into the complexity of the SHs and better understand the complex interaction between six genome forced together by artificial fusion in one cell. The main advantages of this biotechnological tool is its status as non-transgenic in Europe (directive 2001/18/EC, annex 1B) and its acceptance by consumers.

#### **4. The new biotechnological techniques (NBT) and their success in improving potato crop**

In the last decade, new plant breeding technologies (NPBT) have been developed to address plant breeding for important traits of current days. Those technologies were refinements of transgenesis and ended up with such advancements as leaving no foreign DNA in the new modified plants. In a review published by Lusser *et al* [213] the techniques used for NPBT were zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs). In the same year a new NPBT was discovered and became the preferred alternative for plant genome editing, the clustered regulatory interspaced palindromic repeats or CRISPR [214]. CRISPR, a natural system used by bacteria and archaea to fight bacteriophages and foreign genetic fragments, has emerged as one of the most powerful and promising

#### *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

genome editing techniques shaping the future of biotechnology [215]. CRISPR-Cas method is based on a short single-guide RNA (sgRNA), with a 20 bp guide sequence complementary to a target region in recipient genome, a promoter and a sgRNA scaffold, which in combination with a Cas9 nuclease [214], can induce mutations in a target region of choice. Cas9 or an alternative nuclease induce double strand breaks (DSBs), that are repaired by the cell's own repair mechanism, either through nonhomologous end joining (NHEJ) or homologous recombination (HR) [214]. NHEJ is an error prone and often leads to random-sized inserts or deletions (indels), which may cause a knockout of gene function. In potato the first results using CRISPR-Cas9 have shown mutations by using *Agrobacterium*-mediated stable transformation. The targeted genes were: gene encoding an Aux/IAA protein, the *StIAA2*, in a double haploid potato cultivar [216] and the *ALS* gene in both diploid and tetraploid potato [217]. More recently, TALEN and CRISPR-Cas9 were stably introduced targeting *ALS* and using a geminivirus-mediated guide, to facilitate designed mutations [218]. Because of its simplicity and cost efficiency CRISPR-Cas9 was adopted for many plant species [219], including potato as a tetraploid where targeted multialleles mutagenesis was achieved [220]. Traits such as: improved resistance to cold-induced sweetening, herbicide tolerance, processing efficiency, modified starch quality and self-incompatibility have been targeted in potato using CRISPR/Cas9 and TALEN editing technologies in diploid and tetraploid clones [221]. Potato varieties with knockout mutations in all alleles of the *VInv* (vacuolar invertase gene) through precise genome engineering were also produced [222]. This was accomplished by transiently expressing transcription activator-like effector nucleases (TALENs) designed to bind and cleave specific DNA sequences in the *VInv* locus. The doublestranded breaks (DSBs) created by the TALENs were repaired by NHEJ, which introduced indel (insertion/deletion) mutations that compromised *VInv* gene function. Due to the high levels of heterozygosity in the potato genome, the task of simultaneously targeting multiple alleles required careful TALEN design and optimization [223]. In contrast to previous RNAi work, TALENs achieved complete knockout lines without incorporating foreign DNA. As a result, the new potato lines have significantly lower levels of reducing sugars and acrylamide in heat-processed products [224]. In another attempt CRISPR-Cas9 was successfully applied to reduce browning of potato silencing *PPO* gene [225]. Increase resistance to late blight was obtained by mutating S (sensitivity gene) genes *StDND1* and *StCHL1* [226]. CRISPR-Cas13a was used to increase resistance to PVY, and it induced resistance to all strains of the virus [51], while RNAi confronted with many drawbacks because of the virus genetic evolution (**Table 1**) [50]. Although, the successfully edited plants by using CRISPR-Cas are deposited in Plant Genome Editing Database (PGED) [227], to date (2021-04-31) there is no registry for potato.

#### **5. Acceptance by consumer and combinatorial biotechnology**

Potato biotechnology has developed potato varieties with one or multiple genes, which resist one or multiple biotic and/or abiotic stresses. Unfortunately, the continuous debate and consumer lack of trust affect the GM cultivation in the field and specifically the adoption of GM plants in the food chain. There were success stories about genetically engineered potato crop, some of them were deregulated and had a short time of field cultivation. One of the examples that presents the fate of GM potato is the Monsanto potato story. Monsanto has developed GM potatoes with insect resistance (IR) and virus resistance (VR). In 1995, Monsanto received US government approval for Cry3A *Bt* potato, resistant to CPB. 600 ha were planted with this transgenic potato in USA. Another GM potato with resistance to potato

leaf roll virus (PRLV) was approved in 1998 and a variety resistant to PVY in 1999. Moreover the *Bt* trait was stacked with PRLV and/or PVY resistance. From 1995 to 1998 the area with GE (genetically engineered) potato increased to 20,000 ha representing 3.5% of total area of potato crop in USA. But, in 2000 the area planted with GE potato declined sharply, a decline attributed to lack of acceptance by some consumers, the fast-food chain refusal of GE potato use and the incapacity of potato industry to test and segregate GE from non-GE potato. In these conditions, growers were concerned that their GE potato will no more be purchased by their buyers. The farmers, on the other hand, were purchasing a new insecticide for CPB and other pests rather than using GE varieties. In 2001 Monsanto decided to close its potato division [69, 228, 229]. Another story is about Amflora potato in Europe. After authorization procedure and favourable scientific opinions the European Commission approved the cultivation of BASF Amflora starch potato in 2010. This was the first GE plant approved for cultivation in EU in 12 years. The Amflora potato was not intended to be authorised for food, only for industrial use in starch production and its by-products as feed. Many member states were reacting against the GE potato authorization. In 2013, the EU General Court annulled the authorization of Amflora potato. In 2012 the BASF Company decided to move it's headquarter in USA (North Carolina) and halted the production of Amflora potato from EU market. Although, new breeding technologies and particularly CRISPR-Cas technology does not leave any foreign DNA into targeted mutagenized crops, EU has decided to consider edited crops under GM low in 2018, but there is hope that these modified crops will be accepted for cultivation and commercialization in the near future. The acceptance of modified crops by consumers varies from one country to another, depending on culture, history, environmental pressure *etc*., but it seems that the benefits of transgenic and editing methods will at the end extend at scientific level, because cost benefits, CO2 reduction and reduction on pesticides use will override the consumers unscientific doubts. But until then there are other effective biotechnological tools that are not considered as GMOs, for instance, somatic fusion and production of somatic hybrids as presented above can also address many resistance traits and be included in breeding. We have proposed a new strategy of biotechnological results integration in potato breeding called combinatorial biotechnology and already gave some good examples for the SHs of potato varieties (4x) with the diploid wild species *S. bulbocastanum* and *S. chacoense* [145, 180]. For instance in the case of somatic hybrids potato + *S. cahacoense*, presented above, transgenesis using *AtMSH2* gene, somatic hybridization, molecular analysis and stress selection were combined. For further integration in breeding somatic hybrids have to be back-crossed with cultivars and embryo rescue will be applied for BCs regeneration. Moreover, to remove the transgenes another strategy has to be applied as: gene segregation, RNAi or CRISPR-Cas. Finally, these genotypes with very interesting traits: resistance to CPB (antibiosis and antixenosis), tolerance to drought and salt would be integrated in breeding. The adoption of these biotechnological tools coupled with new knowledge on potato genomics and phenome's will most probably change the ways how the biotechnology is integrated in potato breeding for resilient potato, which is indispensable in today's challenging agriculture.

#### **6. Conclusions**

There are many tools in potato biotechnology which could be applied to improve potato resistance to biotic and abiotic stresses and to increase the quality of potato tubers for different application as food, feed, industrial use of even medicinal applications (**Figure 3**). These tools coupled with the new knowledge of genomics

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

**Figure 3.**

*Starch, protein and other valuable compound content of a potato raw tuber (detailed on the left (mg)) (modified data for cv. Russet Burbank https://www.researchgate.net/publication/265480176\_27\_).*

and phenomics will be more and more accepted in improving potato crop for actual and future agriculture. Combinatorial biotechnology that in our opinion will use all advantages of potato genome manipulation, tissue culture techniques, and next generation biotechnologies along with genome, transcriptome and metabolome research will contribute to resilient potato crop.

#### **Acknowledgements**

I. M. was supported by funds from the Young Researcher Grants 2019-2020 (GTC-2020).

## **Author details**

Elena Rakosy-Tican\* and Imola Molnar Plant Genetic Engineering Group, Department of Molecular Biology and Biotechnology, Faculty of Biology and Geology, Babeş-Bolyai University, Cluj-Napoca, Romania

\*Address all correspondence to: arina5744@yahoo.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Barrell PJ, Meiyalaghan S, Jeanne ME et al. Applications of biotechnology and genomics in potato improvement. Plant Biotech J. 2013; 11:907-920. DOI: 10.1111/ pbi.12099

[2] Bradshaw JE. Potato Breeding: Theory and Practice**.** Dordrecht: Springer International Publishing; 2021. 563 p. DOI 10.1007/978-3-030-64414-7

[3] Zaheer K, Akhtar MH. Potato production, usage, and nutrition–a review. Crit Rev Food Sci Nutr. 2016; 56:711-721. DOI: 10.1080/10408398. 2012.724479

[4] Kehoe MA, Jones RAC. Improving Potato virus Y strain nomenclature: Lessons from comparing isolates obtained over a 73-year period. Plant Pathol. 2016; 65: 322-333. DOI: 10.1111/ ppa.12404

[5] Vreugdenhil D, Bradshaw J, Gebhardt C, et al. editors. Potato Biology and Biotechnology. Advances and Perspectives. 1st ed. (2007) Amsterdam: Elsevier; 2007. 856 p.

[6] Halterman D, Guenthner J, Collinge S, et al. Biotech potatoes in the 21st century: 20 years since the first biotech potato. Am J Potato Res. 2016; 93:1-20. DOI: 10.1007/s12230-015-9485-1

[7] CIP Position Paper [Internet]. 2015. Available from: https://cip-cgiar-bucket1. s3.amazonaws.com/wp-content/ uploads/2018/05/Biotechnology-Position-Paper-18Nov15.pdf [Accessed: 2021-04-15]

[8] Thieme R, Rakosy-Tican E. Somatic cell genetics and its application in potato breeding. In: Chakrabarti SK, Xie C, Tiwari JK, editors. The Potato Genome. AG Cham: Springer International Publishing; 2017. p. 217-269. DOI: 10.1007/978-3-319-66135-3\_13

[9] Jansky S. Breeding for disease resistance in potato. Plant Breeding Reviews. 2000; 19: 69-155. DOI: 10.1002/9780470650172.ch4

[10] Christou P. Plant genetic engineering and agricultural biotechnology 1983-2013. Trends Biotechnol. 2013; 31:125-127. DOI: 10.1016/j.tibtech.2013.01.006

[11] Stiekema WJ, Heidekamp F, Louwerse JD, et al. Introduction of foreign genes into potato cultivars Bintjeand Désirée using an *Agrobacterium tumefaciens* binary vector. Plant Cell Rep. 1988; 7:47-50. DOI 10.1007/BF00272976

[12] Sheerman S, Bevan MW. A rapid transformation method for *Solanum tuberosum* using binary *Agrobacterium tumefaciens* vectors. Plant Cell Rep. 1988; 7:13-16. DOI: 10.1007/BF00272967

[13] Rakosy-Tican L, Aurori CM, Dijkstra C, et al. The usefulness of reporter gene *gfp* for monitoring *Agrobacterium*-mediated transformation of potato dihaploid and tetraploid genotypes. Plant Cell Rep. 2007; 26:661- 671. DOI: 10.1007/s00299-006-0273-8

[14] Directive 2001/18/EC of the European Parliament and of the Council [Internet]. 2001. Available from: https://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=CONSL EG:2001L0018:20080321:EN:PDF [Accessed: 2021-04-02]

[15] Evaluation of new breeding technology (EFSA) [Internet]. 2011. Available from: https://www.efsa.europa. eu/en/topics/topic/gmo [Accessed: 2021-04-03]

[16] Spooner DM, Hijmans RJ. Potato systematics and germplasm collecting, 1989-2000. Am J Potato Res. 2001; 78: 237-268. DOI: 10.1007/BF02875691

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

[17] Visser RGF, Bachem CWB, de Boer JM, et al. Sequencing the potato genome: Outline and first results to come from the elucidation of the sequence of the World*'*s third most important food crop. Am J Potato Res. 2009; 86:417-429. DOI: 10.1007/s12230-009-9097-8

[18] De Block M. Genotype-independent leaf disc transformation of potato (*Solanum tuberosum*) using *Agrobacterium tumefaciens*. Theor Appl Genet. 1988; 76(5):767-74. DOI: 10.1007/BF00303524

[19] Gimenez-Ibanez S. Designing disease-resistant crops: From basic knowledge to biotechnology. Mčtode Science Studies Journal. 2021; 11:47-53. DOI: 10.7203/metode.11.15496

[20] Dong OX, Roland PC. Genetic engineering for disease resistance in plants: Recent progress and future perspectives. Plant Physiology. 2019; 180:26-38. DOI: 10.1104/pp.18.01224

[21] Potato Genome Sequencing Consortium. Genome sequence and analysis of the tuber crop potato. Nature. 2011; 475:189-195. DOI: 10.1038/ nature10158

[22] Aversano R, Contaldi F, Ercolano MR, et al. The *Solanum commersonii* genome sequence provides insights into adaptation to stress conditions and genome evolution of wild potato relatives. Plant Cell. 2015; 27:954-968. DOI: 10.1105/tpc.114.135954

[23] Leisner CP, Hamilton JP, Crisovan E, et al. Genome sequence of M6, a diploid inbred clone of the high-glycoalkaloidproducing tuber-bearing potato species *Solanum chacoense*, reveals residual heterozygosity. The Plant Journal. 2018; 94:562-570. DOI: 10.1111/tpj.13857

[24] Lorenc-Kukuła K, Jafra S, Oszmiański J, et al. Ectopic expression of anthocyanin 5-*O*-Glucosyltransferase in potato tuber causes increased resistance to bacteria. J Agric Food

Chem. 2005; 53(2):272-281. DOI: 10.1021/jf048449p

[25] Almasia NI, Bazzini AA, Esteban Hopp H, et al. Overexpression of *snakin*-1 gene enhances resistance to *Rhizoctonia solani* and *Erwinia carotovora* in transgenic potato plants. Mol Plant Pathol. 2008; 9(3):329-338. DOI: 10.1111/j.1364-3703.2008.00469.x

[26] Bidondo LF, Almasia N, Bazzini A, et al. The overexpression of antifungal genes enhances resistance to *Rhizoctonia solani* in transgenic potato plants without affecting arbuscular mycorrhizal symbiosis. Crop Protection. 2019; 124: 104837. DOI: 10.1016/j.cropro.2019.05.031

[27] Jones JDG, Witek K, Verweij W, et al. Elevating crop disease resistance with cloned genes. Phil Trans R Soc B. 2014; 369:20130087. DOI: 10.1098/ rstb.2013.0087

[28] Jo KR, Kim CJ, Kim SJ, et al. Development of late blight resistant potatoes by cisgene stacking. BMC Biotechnology. 2014; 14:50. DOI: 10.1186/1472-6750-14-50

[29] Listanto E, Riyanti EI, Santoso TJ, et al*.* Genetic stability analysis of *RB* gene in genetically modified potato lines tolerant to *Phytophthora infestans*. Indonesian J Agric Sci. 2015; 16(2):51-58. DOI: 10.21082/ijas.v16n2.2015.pp.51-58

[30] Zhu S, Li Y, Vossen JH, et al. Functional stacking of three resistance genes against *Phytophthora infestans* in potato. Transgenic Res. 2012; 21:89-99. DOI: 10.1007/s11248-011-9510-1

[31] Bouwmeester K, Han M, Blanco-Portales R, et al. The *Arabidopsis* lectin receptor kinase LecRK-I.9 enhances resistance to *Phytophthora infestans* in *Solanaceous* plants. Plant Biotechnol J. 2014; 12(1):10-6. DOI: 10.1111/pbi.12111

[32] Zhang Z, Yang F, Na R, et al. AtROP1 negatively regulates potato resistance to

*Phytophthora infestans* via NADPH oxidase-mediated accumulation of H2O2. BMC Plant Biol. 2014; 14:392. DOI:10.1186/s12870- 014-0392-2.44

[33] Jahan SN, Asman AK, Corcoran P, et al. Plant-mediated gene silencing restricts growth of the potato late blight pathogen *Phytophthora infestans*. Journal of Experimental Botany. 2015; 66: 2785-2794. DOI: 10.1093/jxb/erv094

[34] Ghislain M, Byarugaba AA, Magembe E, et al*.* Stacking three late blight resistance genes from wild species directly into African highland potato varieties confers complete field resistance to local blight races. Plant Biotechnol J*.* 2019; 17:1119-1129. DOI: 10.1111/ pbi.13042

[35] Webi EN, Kariuki D, Kinyua J, et al. Extreme resistance to late blight disease by transferring 3 R genes from wild relatives into African farmer-preferred potato varieties. Afr J Biotechnol. 2019; 18(29):845-856. DOI: 10.5897/AJB2019. 16856

[36] Osusky M, Osuska L, Kay W, et al. Genetic modification of potato against microbial diseases: *in vitro* and *in planta* activity of a dermaseptin B1 derivative, *MsrA*2. Theor Appl Genet. 2005; 111:711- 722. DOI: 10.1007/s00122-005-2056-y

[37] Goyal RK, Hancock REW, Mattoo AK, et al. Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses. PLoS ONE. 2013; 8(10):e77505. DOI: 10.1371/journal.pone.0077505

[38] Buziashvili A, Cherednichenko L, Kropyvko S, et al. Obtaining transgenic potato plants expressing the human Lactoferrin gene and analysis of their resistance to phytopathogens. Cytol Genet. 2020; 54:179-188. DOI: 10.3103/ S0095452720030020

[39] Zhu W, Bai X, Li G, et al. SpCYS, a cystatin gene from wild potato (*Solanum*  *pinnatisectum*), is involved in the resistance against *Spodoptera litura*. Theor Exp Plant Physiol. 2019; 31:317- 328. DOI: 10.1007/s40626-019-00148-8

[40] Koropacka KB. Molecular contest between potato and the potato cyst nematode *Globodera pallida*: modulation of Gpa2-mediated resistance [thesis]. Wageningen: Wageningen University; 2010.

[41] Paal J, Henselewski H, Muth J, et al. Molecular cloning of the potato Gro1-4 gene conferring resistance to pathotype Ro1 of the root cyst nematode *Globodera rostochiensis*, based on a candidate gene approach. The Plant Journal. 2004; 38:285-297. DOI: 10.1111/j.1365-313X. 2004.02047.x

[42] Green J, Wang D, Lilley CJ, et al. Transgenic potatoes for potato cyst nematode control can replace pesticide use without impact on soil quality. PLoS ONE. 2012; 7(2):e30973. DOI: 10.1371/ journal.pone.0030973

[43] Missiou A, Kalantidis K, Boutla A, et al. Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol Breed. 2004; 14:185-197. DOI: 10.1023/ B:MOLB.0000038006.32812.52

[44] Bukovinszki A, Diveki Z, Csanyi M, et al. Engineering resistance to PVY in different potato cultivars in a markerfree transformation system using a 'shooter mutant' *A. tumefaciens.* Plant Cell Rep. 2007; 26:459-465. DOI: 10.1007/s00299-006-0257-8

[45] Dusi AN, Lopes de Oliveira C, de Melo PE, et al. Resistance of genetically modified potatoes to Potato virus Y under field conditions. Pesq Agropec Bras Brasília. 2009; 44(9):1127-1130. DOI: 10.1590/S0100-204X2009000900009

[46] Rakosy-Tican E, Aurori CM, Dijkstra C, et al. Generating marker free transgenic potato cultivars with a hairpin *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

construct of PVY coat protein. Romanian Biotech Lett. 2010; 15(1suppl):63-71.

[47] Tabassum B, Nasir IA, Khan A, et al. Short hairpin RNA engineering: In planta gene silencing of potato virus Y. Crop Protection. 2016; 86:1-8. DOI: 10.1016/j.cropro.2016.04.005

[48] Duan H, Richael C, Rommens C. Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1. Transgenic Research. 2012; 21:929-938. DOI: 10.1007/s11248-011- 9576-9

[49] Arcibal E, Morey Gold K, Flaherty S, et al. A mutant eIF4E confers resistance to potato virus Y strains and is inherited in a dominant manner in the potato varieties Atlantic and Russet Norkotah. Am J Potato Res. 2015; 93(1):64-71. DOI: 10.1007/s12230-015-9489-x

[50] Pooggin MM. RNAi-mediated resistance to viruses: a critical assessment of methodologies. Curr Opin Virol. 2017; 26:28-35. DOI: 10.1016/j. coviro.2017.07.010

[51] Zhan X, Zhang F, Zhong Z, et al. Generation of virus-resistant potato plants by RNA genome targeting. Plant Biotech J. 2019; 17:1814-1822. DOI: 10.1111/pbi.13102

[52] Osmani Z, Sabet MS, Nakahara KS, et al. Identification of a defense response gene involved in signaling pathways against PVA and PVY in potato. GM Crops & Food. 2021; 12(1):86-105. DOI: 10.1080/21645698.2020.1823776

[53] Naimov S, Dukiandjiev S, de Maagd RA. A hybrid *Bacillus thuringiensis* deltaendotoxin gives resistance against a coleopteran and a lepidopteran pest in transgenic potato. Plant Biotechnol J. 2003; 1:51-57. DOI: 10.1046/j.1467-7652.2003.00005.x

[54] Davidson MM, Takla MFG, Jacobs JME, et al. Transformation of potato (*Solanum tuberosum*) cultivars with a *cry1Ac9* gene confers resistance to potato tuber moth (*Phthorimaea operculella*). New Zealand J Crop Hort Sci. 2004; 32(1):39-50. DOI: 10.1080/01140671.2004.9514278

[55] Outchkourov NS, de Kogel WJ, Schuurman-de Bruin A, et al. Specific cysteine protease inhibitors act as deterrents of western flower thrips, *Frankliniella occidentalis* (Pergande), in transgenic potato. Plant Biotech J. 2004; 2:439-448. DOI: 10.1111/j.1467-7652. 2004.00088.x

[56] Zhu F, Xu J, Palli R, et al. Ingested RNA interference for managing the populations of the Colorado potato beetle, *Leptinotarsa decemlineata*. Pest Manag Sci. 2011; 67:175-182. DOI:10.1002/ps.2048

[57] Zotti MJ, Smagghe G. RNAi technology for insect management and protection of beneficial insects from diseases: lessons, challenges and risk assessments. Neotrop Entomol*.* 2015; 44:197-213. DOI: 10.1007/s13744-015- 0291-8

[58] Zhang J, Khan SA, Hasse C, et al. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science. 2015; 347:991- 994. DOI:10.1126/science.1261680

[59] Hussain T, Aksoy E, CalisKan MEC, et al. Transgenic potato lines expressing hairpin RNAi construct of moltingassociated EcR gene exhibit enhanced resistance against Colorado potato beetle (*Leptinotarsa decemlineata*, Say). Transgenic Res. 2019; 28:151-164. DOI: 10.1007/s11248-018-0109-7

[60] Gui S, Taning CNT, Wei D, et al. First report on CRISPR/Cas9-targeted mutagenesis in the Colorado potato beetle, *Leptinotarsa decemlineata*. J Insect Physiol. 2020; 121:104013. DOI: 10.1016/j.jinsphys.2020.104013

[61] Sharma HC, Sharma KK, Crouch JH. Genetic transformation of crops for

insect resistance: Potential and limitations. Crit Rev Plant Sci. 2004; 23(1):47-72. DOI: 10.1080/073526 80490273400

[62] Du J, Verzaux E, Chaparro-Garcia A, et al. Elicitin recognition confers enhanced resistance to *Phytophthora infestans* in potato. Nat Plants. 2015; 1(4):15034. DOI: 10.1038/nplants.2015.34

[63] Kawchuk LM, Hachey J, Lynch DR, et al. Tomato *Ve* disease resistance genes encode cell surface-like receptors. Proc Natl Acad Sci *USA.* 2001; 98:6511-6515. DOI: 10.1073/pnas.091114198

[64] Haverkort AJ, Boonekamp PM, Hutten R, et al. Durable late blight resistance in potato through dynamic varieties obtained by cisgenesis: scientific and societal advances in the DuRPh project. Potato Res. 2016; 59:35-66. DOI 10.1007/s11540-015-9312-6

[65] Waltz E. USDA approves nextgeneration GM potato. Nature biotechnology. 2015; 33(1):12-13. DOI: 10.1038/nbt0115-12

[66] Ridler K. Canada approves three types of genetically engineered potatoes [Internet]. 2017. Available from: https:// www.ctvnews.ca/health/canadaapproves-three-typesof-geneticallyengineered-potatoes-1.3531998 [Accessed: 2021-04-22]

[67] Bendahmane A, Kohm BA, Dedi C, et al. The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato. Plant J. 1995; 8:933-941. DOI: 10.1046/j.1365-313x.1995. 8060933.x

[68] Danan S, Veyrieras J-B, Lefebvre V. Construction of a potato consensus map and QTL meta-analysis offer new insights into the genetic architecture of late blight resistance and plant maturity traits. BMC Plant Biology. 2011; 11:16. DOI: 10.1186/1471-2229-11-16

[69] James C. (2011) Global Status of Commercialized Biotech/GM Crops, Vol 44. New York: ISAAA; 2011.

[70] Ferro DN, Logan JA, Voss RH, et al. Colorado potato beetle (Coleoptera: *Chrysomelidae*) temperature-dependent growth and feeding rates. Environ Entomol. 1985; 14:343-348. DOI: 10.1093/ ee/14.3.343

[71] Balaško MK, Mikac KM, Bažok R, et al. Modern techniques in Colorado potato beetle (*Leptinotarsa decemlineata* Say) control and resistance management: History review and future perspectives. Insects. 2020; 11(9):581. DOI: 10.3390/ insects11090581

[72] Balbyshev NF, Lorenzen JH. Hypersensitivity and egg drop: A novel mechanism of host plant resistance to Colorado potato beetle (Coleoptera: *Chrysomelidae*). J Econ Entomol. 1997; 90:652-657. DOI: 10.1093/jee/90.2.652

[73] Perlak FJ, Stone TB, Muskopf YM, et al. Genetically improved potatoes: Protection from damage by Colorado potato beetles. Plant Mol Biol. 1993; 22:313-321. DOI: 10.1007/BF00014938

[74] Grafius EJ, Douches DS. The present and future role of insect-resistant genetically modified potato cultivars in IPM. In: Romeis J, Shelton AM, Kennedy GG, editors. Integration of Insect-Resistant Genetically Modified Crops within IPM Programs. 1st ed. Dordrecht: Springer; 2008. p. 195-221. DOI: 10.1007/978-1-4020-8373-0\_7

[75] Coombs JJ, Douches DS, Li W, et al. Combining engineered (Bt-cry3A) and natural resistance mechanisms in potato for control of Colorado potato beetle. J Am Soc Hortic Sci. 2002; 127:62-68. DOI: 10.21273/JASHS.127.1.62

[76] Baum J, Bogaert T, Clinton W, et al. Control of coleopteran insect pests through RNA interference. Nat Biotechnol. 2007; 25:1322-1326. DOI: 10.1038/nbt1359

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

[77] Joga MR, Zotti MJ, Smagghe G, et al. RNAi efficiency, systemic properties, and novel delivery methods for pest insect control: what we know so far. Front Physiol*.* 2016; 7: 553. DOI: 10. 3389/ fphys. 2016. 00553

[78] Mishra S, Dee J, Moar W, et al. Selection for high levels of resistance to double stranded RNA (dsRNA) in Colorado potato beetle (*Leptinotarsa decemlineata* Say) using non-transgenic foliar delivery. Sci Rep. 2021; 11:6523. DOI: 10.1038/s41598-021-85876-1

[79] Schoville SD, Chen YH, Andersson MN, et al. A model species for agricultural pest genomics: The genome of the Colorado potato beetle, *Leptinotarsa decemlineata* (Coleoptera: *Chrysomelidae*). Sci Rep. 2018; 8:1-18. DOI: 10.1038/s41598-018-20154-1

[80] Gaddelapati SC, Kalsi M, Roy A, et al. Cap'n'collar C regulates genes responsible for imidacloprid resistance in the Colorado potato beetle, *Leptinotarsa decemlineata*. Insect Biochem Mol Biol. 2018; 99:54-62. DOI: 10.1016/j.ibmb.2018.05.006

[81] Ochoa-Campuzano C, Martínez-Ramírez AC, Contreras E, et al. Prohibitin, an essential protein for Colorado potato beetle larval viability, is relevant to *Bacillus thuringiensis* Cry3Aa toxicity. Pestic Biochem Physiol. 2013; 107:299-308. DOI: 10.1016/j.pestbp.2013. 09.001

[82] Singh KB, Foley RC, Oñate-Sánchez L. Transcription factors in plant defense and stress responses. Curr Opin Plant Biol. 2002; 5:430-436. DOI: 10.1016/S1369-5266(02)00289-3

[83] Youm JW, Jeon JH, Choi D, et al. Ectopic expression of pepper *CaPF1* in potato enhances multiple stresses tolerance and delays initiation of *in vitro* tuberization. Planta. 2008; 228:701-708. DOI: /10.1007/s00425-008-0782-5

[84] Dou H, Xv K, Meng Q, et al. Potato plants ectopically expressing *Arabidopsis thaliana* CBF3 exhibit enhanced tolerance to high-temperature stress. Plant Cell Environ. 2015; 38:61-72. DOI: 10.1111/pce.12366

[85] Trapero-Mozos A, Morris WL, Ducreux LJM, et al. Engineering heat tolerance in potato by temperaturedependent expression of a specific allele of HEAT-SHOCK COGNATE 70. Plant Biotechnol J. 2018; 16:197-207. DOI: 10.1111/pbi.12760

[86] Behnam B, Kikuchi A, Celebi-Toprak F, et al. *Arabidopsis rd29A::DREB1A* enhances freezing tolerance in transgenic potato. Plant Cell Rep. 2007; 26:1275-1282. DOI: 10.1007/ s00299-007-0360-5

[87] Stiller I, Dulai S, Kondrak M, et al. Effects of drought on water content and photosynthetic parameters in potato plants expressing the *trehalose-6 phosphate synthase* gene of *Saccharomyces cerevisiae*. Planta. 2008; 227:299-308. DOI: 10.1007/s00425-007-0617-9

[88] Zhang N, Si HJ, Wen G, et al. Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach. Plant Biotechnol Rep. 2011; 5:71-77. DOI: 10.1007/s11816-010- 0160-1

[89] Kondrak M, Marincs F, Antal F, et al. Effects of yeast trehalose-6 phosphate synthase 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions. BMC Plant Biol. 2012; 12:74. DOI: 10.1186/1471-2229-12-74

[90] Pal AK, Acharya K, Vats SK, et al. Over-expression of *PaSOD* in transgenic potato enhances photosynthetic performance under drought. Biol Plant. 2013; 57(2):359-364. DOI: 10.1007/ s10535-012-0277-x

[91] Iwaki T, Guo L, Ryals JA, et al. Metabolic profiling of transgenic potato tubers expressing *Arabidopsis* dehydration response element-binding protein 1A (DREB1A). J Agric Food Chem. 2013; 61:893-900. DOI:10.1021/ jf304071n

[92] Pieczynski M, Marczewski W, Hennig J, et al. Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato. Plant Biotechnology Journal. 2013; 11:459-469. DOI: 10.1111/pbi.12032

[93] Li S, Zhang N, Zhu X, et al. Enhanced drought tolerance with artificial microRNA-mediated *StProDH1* gene silencing in potato. Crop Sci. 2020; 60(3):1462-1471. DOI: 10.1002/csc2. 20064

[94] Hmida-Sayari A, Gargouri-Bouzid R, Bidani A, et al. Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants. Plant Sci. 2005; 169(4):746-752. DOI: 10.1016/j.plantsci.2005.05.025

[95] Bayat F, Shiran B, Belyaev DV, et al. Potato plants bearing a vacuolar Na+/H+ antiporter HvNHX2 from barley are characterized by improved salt tolerance. Russ J Plant Physiol. 2010; 57(5):696-706. DOI: 10.1134/S1021443710050134

[96] Wang L, Liu Y, Li D, et al. Improving salt tolerance in potato through overexpression of AtHKT1 gene. BMC Plant Biol. 2019; 19(1):357. DOI: 10.1186/s12870-019-1963-z

[97] Liu M, Li Y, Li G, et al. Overexpression of StCYS1 gene enhances tolerance to salt stress in the transgenic potato (*Solanum tuberosum* L.) plant. J Integrative Agri. 2020; 19(9):2239-2246. DOI: 10.1016/S2095-3119(20)63262-2

[98] Lee HE, Shin D, Park SR, et al. Ethylene responsive element binding protein 1 (*StEREBP*1) from *Solanum tuberosum* increases tolerance to abiotic stress in transgenic potato plants.

Biochem Biophys Res Commun. 2007; 353(4):863-868. DOI: 10.1016/j.bbrc. 2006.12.095

[99] Tang Li, Kwon S-K, Kim S-H, et al. Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature. Plant Cell Rep. 2006; 25:1380-1386. DOI: 10.1007/s00299-006- 0199-1

[100] Movahedi S, Tabatabaei BES, Alizade H, et al. Constitutive expression of *Arabidopsis DREB1B* in transgenic potato enhances drought and freezing tolerance. Biol Plant. 2012; 56:37-42. DOI: 10.1007/s10535-012-0013-6

[101] Bouaziz D, Pirrello J, Amor HB, et al. Ectopic expression of dehydration responsive element binding proteins (StDREB2) confers higher tolerance to salt stress in potato. Plant Physiol Bioch. 2012: 60:98-108. DOI: 10.1016/j. plaphy.2012.07.029

[102] Bouaziz D, Pirrello J, Charfeddine M, et al. Overexpression of *StDREB1* transcription factor increases tolerance to salt in transgenic potato plants. Mol Biotechnol. 2013; 54:803-817. DOI: 10.1007/s12033-012-9628-2

[103] Sievers N, Muders K, Henneberg M, et al. Establishing glucosylglycerol synthesis in potato (*Solanum tuberosum* l. cv. Albatros) by expression of the *ggpPS* gene from *Azotobacter vinelandii.* J Plant Sci & Mol Breed. 2013; 2:1. DOI: 10.7243/2050-2389-2-1

[104] Ahmad R, Hussain J, Jamil M, et al. Glycinebetaine synthesizing transgenic potato plants exhibit enhanced tolerance to salt and cold stresses. Pak J Bot. 2014; 46(6):1987-1993

[105] Shahzad R, Harlina PW, Cong-hua X, et al. Overexpression of potato transcription factor (StWRKY1) conferred resistance to *Phytophthora* 

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

*infestans* and improved tolerance to water stress. Plant Omics. 2016; 9(2):149-158. DOI: 10.21475/poj.160902.p7649x

[106] Muñiz García MN, Cortelezzi JI, Fumagalli M, et al. Expression of the Arabidopsis *ABF4* gene in potato increases tuber yield, improves tuber quality and enhances salt and drought tolerance. Plant Mol Biol. 2018; 98:137- 152. DOI: 10.1007/s11103-018-0769-y

[107] Lehretz GG, Sonnewald S, Lugassi N, et al. Future-proofing potato for drought and heat tolerance by overexpression of Hexokinase and SP6A. Front Plant Sci. 2021; 11:614534. DOI: 10.3389/fpls.2020.614534

[108] Ahmad R, Kim MD, Back KH, et al. Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses. Plant Cell Rep. 2008; 27:687-698. DOI: 10.1007/ s00299-007-0479-4

[109] Ahmad R, Kim YH, Kim MD, et al. Simultaneous expression of choline oxidase, superoxide dismutase and ascorbate peroxidase in potato plant chloroplasts provides synergistically enhanced protection agains various abiotic stresses. Physiol Plant. 2010; 138:520-533. DOI: 10.1111/j.1399-3054. 2010.01348.x

[110] Gangadhar BH, Sajeesh K, Venkatesh J, et al. Enhanced tolerance of transgenic potato plants over-expressing non-specific lipid transfer Protein-1 (StnsLTP1) against multiple abiotic stresses. Front Plant Sci. 2016; 7:1228. DOI: 10.3389/fpls.2016.01228

[111] Cho K-S, Han E-H, Kwak S-S, et al. Expressing the sweet potato orange gene in transgenic potato improves drought tolerance and marketable tuber production. C R Biologies. 2016; 339:207- 213. DOI: 10.1016/j.crvi.2016.04.010

[112] Gong L, Zhang H, Gan X, et al. Transcriptome profiling of the potato (*Solanum tuberosum* L.) plant under drought stress and water-stimulus conditions. PLoS ONE. 2015; 10(5): e0128041. DOI: 10.1371/journal. pone.0128041

[113] van Zelm E, Zhang Y, Testerink C. Salt tolerance mechanisms of plants. Ann Rev Plant Biol. 2020; 71:403-433. DOI: 10.1146/annurev-arplant-050718-100005

[114] Fidalgo F, Santos A, Santos I, et al. Effects of long-term salt stress on antioxidant defense systems, leaf water relations and chloroplast ultrastructure of potato plants. Ann Appl Biol. 2004; 145:185-192. DOI: 10.1111/j. 1744-7348. 2004.tb00374.x

[115] Odemis B, Caliskan ME. Photosynthetic response of potato plants to soil salinity. Turk J Agric Nat Sci. 2014; 2:1429-1439.

[116] Dwelle RB, Kleinkopf GE, Pavek JJ. Stomatal conductance and gross photosynthesis of potato (*Solanum tuberosum* L.) as influenced byirradiance, temperature and growth stage. Potato Res. 1981; 24:49-59. DOI: 10.1007/ bf02362016

[117] Dahal K, Li X-Q, Tai H, et al. Improving potato stress tolerance and tuber yield under a climate change scenario – A current overview. Front Plant Sci. 2019; 10:563. DOI: 10.3389/ fpls.2019.00563

[118] Tang R, Zhu W, Song X, et al. Genome-wide identification and function analyses of heat shock transcription factors in potato. Front Plant Sci. 2016; 19(7):490. DOI: 10.3389/fpls.2016.00490

[119] Schenk PM, Kazan K, Wilson I, et al. Coordinated plant defense responses in *Arabidopsis* revealed by microarray analysis. Proc Natl Acad Sci USA. 2000; 97:11655-11660. DOI: 10.1073/pnas. 97.21.11655

[120] Bouaziz D, Charfeddine M, Jbir R, et al. Identification and functional characterization of ten *AP2/ERF* genes in potato. Plant Cell Tiss Organ Cult. 2015; 123:155-172. DOI: 10.1007/ s11240-015-0823-2

[121] Gangadhar BH, Yu JW, Sajeesh K, et al. A systematic exploration of high-temperature stress-responsive genes in potato using large-scale yeast functional screening. Mol Genet Genomics. 2014; 289:185-201. DOI: 10.1007/s00438-013-0795-z

[122] Jansky S. Breeding, genetics and cultivar development. In: Singh J, Kaur L, editors. Advances in potato chemistry and technology. Burlington: Academic Press; 2009. p. 27-62. DOI: 10.1016/B978-0-12-374349-7.00002-7

[123] Ross H. Potato breeding-problems and perspectives. Berlin: Springer; 1986.

[124] Muthoni J, Kabira J, Shimelis H, et al. Tetrasomic inheritance in cultivated potato and implications in conventional breeding. Aust J Crop Sci. 2015; 9(3): 185-190

[125] Haverkort AJ, Struik PC, Visser RGF, et al. Applied biotechnology to combat late blight in potato caused by *Phytophthora infestans*. Potato Res. 2009; 52:249-264. DOI: 10.1007/s11540- 009-9136-3

[126] Thieme R, Darsow U, Gavrilenko T, et al. Production of somatic hybrids between *S. tuberosum* L. and late blight resistant Mexican wild potato species. Euphytica. 1997; 97:189-200. DOI: 10.1023/A:1003026125623

[127] Rokka VM. Protoplast technology in genome manipulation of potato through somatic cell fusion. In: Li X-Q, Donnelly D, Jensen TJ, editors. Somatic genome manipulation—advances, methods and applications. New-York: Springer; 2015. p. 217-235. DOI: 10.1007/978-1-4939-2389-2\_10

[128] Davey MR, Anthony P, Power BJ, et al. Plant protoplasts: status and biotechnological perspectives. Biotechnol Adv. 2005; 23(2):131-171. DOI: 10.1016/j.biotechadv.2004.09.008

[129] Thieme R, Rakosy-Tican E, Gavrilenko T, et al. Novel somatic hybrids (*Solanum tuberosum* L. + *Solanum tarnii*) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight. Theor Appl Genet. 2008; 116:691-700. DOI: 10.1007/s00122-007-0702-2

[130] Thieme R, Rakosy-Tican E, Nachtigall M, et al. Characterization of the multiple resistance traits of somatic hybrids between *Solanum cardiophyllum* Lindl. and two commercial potato cultivars. Plant Cell Rep. 2010; 29:1187- 1201. DOI: 10.1007/s00299-010-0905-x

[131] Rakosy-Tican E, Thieme R, Nachtigall M, et al. The recipient potato cultivar influences the genetic makeup of the somatic hybrids between five potato cultivars and one cloned accession of sexually incompatible species *Solanum bulbocastanum* Dun. Plant Cell Tiss Organ Cult. 2015; 122:395-407. DOI: 10.1007/s11240-015-0777-4

[132] Shepard JF, Totten RE. Mesophyll cell protoplasts of potato. Isolation, proliferation and plant regeneration. Plant Physiol. 1977; 60:313-316. DOI: 10.1104/pp.60.2.313

[133] Puite KJ, Roest S, Pijnacker LP. Somatic hybrid potato plants after electrofusion of diploid *Solanum tuberosum* and *Solanum phureja*. Plant Cell Rep. 1986; 5:262-265. DOI: 10.1007/ BF00269817

[134] Wenzel G. Biotechnology in potato improvement. In: Gopal J, Khurana SM, editors. Handbook of potato production, improvement and postharvest management. New York: The Haworth Press Inc; 2006. p. 109- 146. DOI: 10.1017/S0014479707005133

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

[135] Binding H, Nehls R, Schieder O, et al. Regeneration of mesophyll protoplasts isolated from dihaploid clones of *Solanum tuberosum* L. Plant Physiol. 1978; 43:52-54. DOI: 10.1111/ j.1399-3054.1978.tb01565.x

[136] Binding H, Nehls R. Regeneration of isolated protoplasts to plant *Solanum dulcamara* L. Z Pflanzenphysiology. 1977; 85:279-280. DOI: 10.1016/ S0044-328X(77)80255-9

[137] Behnke M. Regeneration in Gewebekulturen einiger dihaplider *Solanum tuberosum*-Klone. Z Pflanzenzücht. 1975; 75:262-265

[138] Rakosy-Tican E, Aurori A. Green fluorescent protein (GFP) supports the selection based on callus vigorous growth in the somatic hybrids *Solanum tuberosum* L. + *S. chacoense* Bitt. Acta Physiol Plant. 2015; 37:201. DOI: 10.1007/s11738-015-1946-0

[139] Senda M, Takeda J, Abe S, et al. Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol. 1979; 20:1441-1443. DOI: 10.1093/oxfordjournals.pcp.a075944

[140] Zimmermann U, Scheurich P. High frequency fusion of plant protoplasts by electric fields. Planta. 1981; 151:26-32. DOI: 10.1007/BF00384233

[141] Rakosy-Tican L, Turcu I, Lucaciu MC. Quantitative evaluation of yield and lysis in electrofusion of oat and wheat mesophyll protoplasts. Rom Agri Res. 1998; 9-10:27-33

[142] Smyda-Dajmund P, Sliwka J, Wasilewicz-Flis I, et al. Genetic composition of interspecific potato somatic hybrids and autofused 4x plants evaluated by DArT and cytoplasmic DNA markers. Plant Cell Rep. 2017; 35:1345- 1358. DOI: 10.1007/s00299-016-1966-2

[143] Lössl A, Adler N, Horn R, et al. Chondriome-type characterization of potato: mt α, β, γ, δ, ε and novel plastidmitochondrial configurations in somatic hybrids. Theor Appl Genet. 1999; 98:1-10. DOI: doi.org/10.1007/ s001220051202

[144] Lössl A, Götz M, Braun A, et al. Molecular markers for cytoplasm in potato: male sterility and contribution of different plastid-mitochondrial configurations to starch production. Euphytica. 2000; 116:221-230. DOI: 10.1023/A:1004039320227

[145] Rakosy-Tican E, Thieme R, König J, et al. Introgression of two broadspectrum late blight resistance genes, Rpi-Blb1 and Rpi-Blb3, from *Solanum bulbocastanum* Dun plus race-specific R genes into potato pre-breeding lines. Front Plant Sci. 2020; 11:699-716. DOI: 10.3389/fpls.2020.00699

[146] Rokka VM, Laurila J, Tauriainen A, et al. Glycoalkaloid aglycone accumulations associated with infection by *Clavibacter michiganensis* ssp. Sepedonicus in potato species *Solanum acaule* and *Solanum tuberosum* and their interspecific somatic hybrids. Plant Cell Rep. 2005; 23(10-11):683-691. DOI: 10.1007/s00299-004-0868-x

[147] Tek AL, Stevenson WR, Helgeson JP, et al. Transfer of tuber soft rot and early blight resistances from *Solanum brevidens* into cultivated potato. Theor Appl Genet. 2004; 109:249-254. DOI: 10.1007/s00122-004-1638-4

[148] Chen L, Guo X, Xie C, et al. Nuclear and cytoplasmic genome components of *Solanum tuberosum* + *S. chacoense* somatic hybrids and three SSR alleles related to bacterial wilt resistance. Theor Appl Genet. 2013; 126(7):1861-1872. DOI: 10.1007/s00122-013-2098-5

[149] Chen L, Guo X, Wang H, et al. Tetrasomic inheritance pattern of the pentaploid *Solanum chacoense* (+) *S. tuberosum* somatic hybrid (resistant to bacterial wilt) revealed by SSR detected alleles. Plant Cell Tiss Organ Cult. 2016; 127:315-323. DOI: 10.1007/ s11240-016-1051-0

[150] Wang H, Cheng Z, Wang B, et al. Combining genome composition and differential gene expression analyses reveals that SmPGH1 contributes to bacterial wilt resistance in somatic hybrids. Plant Cell Rep. 2020; 39:1235- 1248. DOI: 10.1007/s00299-020-02563-7

[151] Fock I, Collonnier C, Lavergne D, et al. Evaluation of somatic hybrids of potato with *Solanum stenotomum* after a long-term in vitro conservation. Plant Physiol Biochem. 2007; 45(3-4):209-215. DOI: 10.1016/j.plaphy.2007.02.004

[152] Ahn YK, Park TH. Resistance to common scab developed by somatic hybrids between *Solanum brevidens* and *Solanum tuberosum*. Acta Agric Scand Sect B. 2013; 63(7):595-603. DOI: 10.1080/09064710.2013.829867

[153] Thomson AJ, Taylor RT, Pasche JS, et al. Resistance to *Phytophthora erythroseptica* and *Pythium ultimum* in a potato clone derived from *S. berthaultii* and *S. etuberosum*. Am J Potato Res. 2007; 84:149-160. DOI: 10.1007/BF02987138

[154] Colton LM, Groza HI, Wielgus SM, et al. Marker-assisted selection for the broad-spectrum potato late blight resistance conferred by gene RB derived from a wild potato species. Crop Sci. 2006; 46:589-594. DOI: 10.2135/ cropsci2005.0112

[155] Iovene M, Savarese S, Cardi T, et al. Nuclear and cytoplasmic genome composition of *Solanum bulbocastanum* (+) *S. tuberosum* somatic hybrids. Genome. 2007; 50:443-450. DOI: 10.1139/g07-024

[156] Chen Q, Li HY, Shi YZ, et al. Development of an effective protoplast fusion system for production of new potatoes with disease and insect resistance using Mexican wild potato

species as gene pools. Can J Plant Sci. 2007; 88(4):611-619. DOI: 10.4141/ CJPS07045

[157] Chandel P, Tiwari JK, Ali N, et al. Interspecific potato somatic hybrids between *Solanum tuberosum* and *S. cardiophyllum*, potential sources of late blight resistance breeding. Plant Cell Tiss Organ Cult. 2015; 123:579-589. DOI: 10.1007/s11240-015-0862-8

[158] Espejo R, Cipriani G, Rosel G, et al. Somatic hybrids obtained by protoplast fusion between *Solanum tuberosum L. subsp*. tuberosum and the wild species *Solanum circaeifolium* Bitter. Rev Peru Biol. 2008; 15(1):73-78.

[159] Szczerbakowa A, Tarwacka J, Oskiera M, et al. Somatic hybridization between the diploids of *S*. x *michoacanum* and *S. tuberosum*. Acta Physiol Plant. 2010; 32:867-873. DOI: 10.1007/s11738- 010-0472-3

[160] Smyda P, Jakuczun H, Dębski K, et al. Development of somatic hybrids *Solanum* X *michoacanum* Bitter. (Rydb.) (+) *S. tuberosum* L. and autofused 4 X *S*. X *michoacanum* plants as potential sources of late blight resistance for potato breeding. Plant Cell Rep. 2013; 32:1231- 1241. DOI: 10.1007/s00299-013-1422-5

[161] Szczerbakowa A, Maciejewska U, Zimnoch-Guzowska E, et al. Somatic hybrids *Solanum nigrum* (+) *S. tuberosum*: morphological assessment and verification of hybridity. Plant Cell Rep. 2003; 21:577-584. DOI: 10.1007/s00299- 002-0555-8

[162] Polzerova P, Patzak J, Greplova M. Early characterization of somatic hybrids from symmetric protoplast electrofusion of *Solanum pinnatisectum* Dun. and *Solanum tuberosum* L. Plant Cell Tiss Organ Cult. 2011; 04:163-170. DOI: 10.1007/s11240-010-9813-6

[163] Sarkar D, Tiwari JK, Sharma S, et al. Production and characterization of

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

somatic hybrids between *Solanum tuberosum* L. and *S. pinnatisectum* Dun. Plant Cell Tiss Organ Cult. 2011; 107:427- 440. DOI: 10.1007/s11240-011-9993-8

[164] Tiwari JK, Poonam, Kumar V, et al. Evaluation of potato somatic hybrids of dihaploid *S. tuberosum* (+) *S. pinnatisectum* for late blight resistance. Potato J. 2013; 40 (2):176-179

[165] Tiwari JK, Luthra SK, Devi S, et al. Development of advanced back-cross progenies of potato somatic hybrids and linked ISSR markers for late blight resistance with diverse genetic base-First ever produced in Indian potato breeding. Potato Journal. 2018; 45:17-27.

[166] Tiwari JK, Rawat S, Luthra SK, et al. Genome sequence analysis provides insights on genomic variation and late blight resistance genes in potato somatic hybrid (parents and progeny). Mol Biol Rep. 2021; 48: 623-635. DOI: 10.1007/ s11033-020-06106-x

[167] Greplova M. Isolation, cultivation and fusion of protoplasts of *Solanum* genera [thesis]. Olomouc: Palacký University Olomouc; 2010

[168] Tarwacka J,

Polkowska-Kowalczyk L, Kolano B, et al. Interspecific somatic hybrids *Solanum villosum* (+) *S. tuberosum*, resistant to *Phytophthora infestans*. J Plant Physiol. 2013; 170(17):1541-1548. DOI: 10.1016/j. jplph.2013.06.013

[169] Nouri-Ellouz O, Gargouri-Bouzid R, Sihachakr D, et al. Production of potato intraspecific somatic hybrids with improved tolerance to PVY and *Pythium aphanidermatum*. J Plant Physiol. 2006; 163(12):1321-1332. DOI: 10.1016/j. jplph.2006.06.009

[170] Kim-Lee H, Moon JS, Hong YJ, et al. Bacterial wilt resistance in the fusion hybrids between haploid of potato and *Solanum commersonii*. Am J Potato Res. 2005; 82:129-137. DOI: 10.1007/ BF02853650

[171] Novy RG, Gillen AM, Whitworth JL. Characterization of the expression and inheritance of potato leafroll virus (PLRV) and potato virus Y (PVY) resistance in three generations of germplasm derived from *Solanum etuberosum*. Theor App Genet. 2007; 114:1161- 1172. DOI: 10.1007/s00122- 007-0508-2

[172] Novy RG, Alvarez JM, Corsini DL, et al. Resistance to PVY, PLRV, PVX, green peach aphid, Colorado potato beetle, and wireworm in the progeny of a tri-species somatic hybrid. Am J Potato Res. 2004; 81(1):77-78

[173] Gavrilenko T, Thieme R, Heimbach U, et al. Fertile somatic hybrids of *Solanum etuberosum* (+) dihaploid *Solanum tuberosum* and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y. Euphytica. 2003; 131:323-332. DOI: 10.1023/A:1024041104170

[174] Tiwari JK, Poonam Sarkar D, Pandey SK, et al. Molecular and morphological characterization of somatic hybrids between *Solanum tuberosum* L. and *S. etuberosum* Lindl. Plant Cell Tiss Organ Cult. 2010; 103: 175-187. DOI: 10.1007/s11240-010-9765-x

[175] Molnár I, Besenyei E, Thieme R, et al. Mismatch repair deficiency increases the transfer of antibiosis and antixenosis properties against Colorado potato beetle in somatic hybrids of *Solanum tuberosum* + *S. chacoense*. Pest Manag Sci. 2017; 73:1428-1437. DOI:10.1002/ps.4473

[176] Brown CT, Mojtahedi H, James S, et al. Development and evaluation of potato breeding lines with introgressed resistance to Columbia root-knot nematode (*Meloidogyne chitwoodi*). Am J Potato Res. 2006; 83(1):1-8. DOI: 10.1007/BF02869604

[177] Zhang LH, Mojtahedi H, Kuang H, et al. Marker-assisted selection of

Columbia root-knot nematode resistance introgressed from *Solanum bulbocastanum*. Crop Sci. 2007; 47:2021- 2026. DOI: 10.2135/cropsci2007.01.0003

[178] Novy RG, Alvarez JM, Sterret SB, et al. Progeny of a tri-species potato somatic hybrid express resistance to wireworm in eastern and western potato production regions of the US. Am J Potato Res. 2006; 83(1):126

[179] Kammoun M, Bouallous O, Ksouri MF, et al. Agro-physiological and growth response to reduced water supply of somatic hybrid potato plants (*Solanum tuberosum* L.) cultivated under greenhouse conditions. Agricultural Water Management. 2018; 203:9-19. DOI: 10.1016/j.agwat.2018.02.032

[180] Rakosy-Tican E,

Lörincz-Besenyei E, Molnár I, et al. New phenotypes of potato co-induced by mismatch repair deficiency and somatic hybridization. Front Plant Sci. 2019; 10:3. DOI: 10.3389/fpls.2019.00003

[181] Tu W, Dong J, Zou Y, et al. Interspecific potato somatic hybrids between *Solanum malmeanum* and *S. tuberosum* provide valuable resources for freezing-tolerance breeding. Research Square. 2021. DOI: 10.21203/rs.3.rs-307076/v1

[182] Bidani A, Nouri-Ellouz O, Lakhoua L, et al. Interspecific potato somatic hybrids between *Solanum berthaultii* and *Solanum tuberosum* L. showed recombinant plastome and improved tolerance to salinity. Plant Cell Tiss Organ Cult. 2007; 91:179-189. DOI: 10.1007/s11240-007-9284-6

[183] Jbir-Koubaa R, Charfeddine S, Bouaziz D, et al. Enhanced antioxidant enzyme activities and respective gene expressions in potato somatic hybrids under NaCl stress. Biologia plantarum. 2019; 63:633-642. DOI: 63. 10.32615/ bp.2019.075

[184] Kumar A, Cocking EC. Protoplast fusion, a novel approach to organelle genetics in higher plants. Am J Bot. 1987; 74:1289-1303. DOI: 10.2307/2444164

[185] Rakosy-Tican L, Aurori A, Aurori CM, et al. Transformation of wild *Solanum* species resistant to late blight by using reporter gene *gfp* and *msh2* genes. Plant Breed Seed Sci. 2004; 50:119-128

[186] Thach NQ, Frei U, Wenzel W. Somatic fusion for combining virus resistance in *Solanum tuberosum*. L. Theor Appl Genet. 1993; 85:863-867. DOI: 10.1007/bf00225030

[187] Austin S, Lojkowska E, Ehlenfeldt MK, et al. Fertile interspecific somatic hybrids of *Solanum*: a novel source of resistance to *Erwinia* soft rot. Phytopathology. 1988; 78:1216-1220. DOI: 10.1094/Phyto-78-1216

[188] Mattheij WM, Eijlander R, De Koning JRA, et al. Interspecific hybridization between the cultivated potato *Solanum tuberosum* subspecies tuberosum L. and the wild species *S. ciraeifolium* subsp ciraeifolium Bitter exhibiting resistance to *Phytophthora infestans* (Mont.) de Bary and *Globodera pallida* (Stone) Behrens. I. Somatic hybrids. Theor Appl Genet1992; 83:459- 466. DOI: DOI: 10.1007/BF00226534

[189] Helgeson JP, Pohlman JD, Austin S, et al. Somatic hybrids between *Solanum bulbocastanum* and potato: a new source of resistance to late blight. Theor Appl Genet. 1998; 96:738-742. DOI: 10.1007/ s001220050796

[190] Song J, Bradeen JM, Naess SK, et al. Gene RB cloned from *Solanum bulbocastanum* confers broad spectrum resistance to potato late blight. Proc Natl Acad Sci USA. 2003; 100:9128-9133. DOI: 10.1073/pnas.1533501100

[191] Lozoya-Saldana H, Belmar-Diaz C, Bradeen JM, et al. Characterization of

*Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

*Phytophthora infestans* isolates infecting transgenic and somatic hybrid potatoes resistant to the pathogen in the Toluca Valley, Mexico. Am J Potato Res. 2005; 82:79

[192] Naess S, Bradeen J, Wielgus S, et al. Analysis of the introgression of *Solanum bulbocastanum* DNA into potato breeding lines. Mol Genet Genom. 2001; 265:694- 704. DOI: 10.1007/s004380100465

[193] Van der Vossen EAG, Sikkema A, Lintel Hekkert B, et al. An ancient R gene from the wild potato species *Solanum bulbocastanum* confers broadspectrum resistance to *Phytophthora infestans* in cultivated potato and tomato. Plant J. 2003; 36:867-882. DOI: 10.1046/j.1365-313x.2003.01934.x

[194] Oosumi T, Rockhold D, Maccree M, et al. Gene Rpi-bt1 from *Solanum bulbocastanum* confers resistance to late blight in transgenic potatoes. Am J Potato Res. 2009; 86:456-465. DOI: 10.1007/s12230-009-9100-4

[195] Lokossou AA, Park T-H, van Arkel G, et al. Exploiting knowledge of R/ Avr genes to rapidly clone a new LZ-NBS-LRR family of late blight resistance genes from potato linkage group IV. Mol Plan-Microbe Interact. 2009; 22(6):630- 641. DOI: 10.1094/MPMI-22-6-0630

[196] Orbegozo J, Roman ML, Rivera C, et al. Rpi-blb2 gene from *Solanum bulbocastanum* confers extreme resistance to late blight disease in potato. Plant Cell Tiss Organ Cult. 2016; 125(2):169-281. DOI: 10.1007/s11240-016-0947-z

[197] Fock I, Collonnier C, Luisetti J, et al. Use of *Solanum stenotomum* for introduction of resistance to bacterial wilt in somatic hybrids of potato. Plant Physiol Biochem. 2001; 39:899-908. DOI: 10.1016/S0981-9428(01)01307-9

[198] Rokka VM. Potato Haploids and Breeding. In: Touraev A, Forster BP, Jain SM, editors. Advances in haploid production in higher plants. Heidelberg: Springer; 2009. p. 199-208. DOI: 10.1007/978-1-4020-8854-4\_17

[199] Cooper-Bland S, DeMaine MJ, Fleming ML, et al. Synthesis of intraspecific somatic hybrids of *Solanum tuberosum*: assessments of morphological, biochemical and nematode (*Globodera pallida*) resistance characteristics. J Exp Bot. 1994; 45:1319-1325. DOI: 10.1093/ jxb/45.9.1319

[200] Helgeson JP, Haberlach GT. Somatic hybrids of *Solanum tuberosum* and related species. In: Altman A, Ziv M, Izhar S, editors. Plant biotechnology and in vitro biology in the 21st century, current plant science and biotechnology in agriculture, vol 36. Heidelberg: Springer; 1999. p. 151-154. DOI: 10.1007/978-94-011-4661-6\_35

[201] Gavrilenko T. Potato cytogenetics. In: Vreugdenhil D, editor. Potato biology and biotechnology: Advances and perspectives. Amsterdam: Elsevier BV; 2007. p. 203-216. DOI: 10.1016/ B978-044451018-1/50052-X

[202] Orczyk W, Przetakiewicz J, Nadolska-Orczyk A. Somatic hybrids of *Solanum tuberosum*— application to genetics and breeding. Plant Cell Tiss Organ Cult. 2003; 74:1-13. DOI: 10.1023/A:1023396405655

[203] Lakshmanan PS, Eeckhaut T, Deryckere D, et al. Asymmetric somatic plant hybridization: status and applications. Am J Plant Sci. 2013; 4:1-10. DOI: 10.4236/ajps.2013.48A001

[204] Grosser JW, Gmitter FG. Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell, Tissue Organ Cult. 2011; 104:343-357. DOI: 10.1007/s11240-010-9823-4

[205] Dudits D, Maroy E, Praznovszky T, et al. Transfer of resistance traits from carrot into tobacco by asymmetric

somatic hybridization: regeneration of fertile plants. Proc Natl Acad Sci USA. 1987; 84:8434-8438. DOI: 10.1073/ pnas.84.23.8434

[206] Oberwalder B, Schilde-Rentschler L, Ruo B, et al. Asymmetric protoplast fusion between wild species and breeding lines of potato—effect of recipients and genome stability. Theor Appl Genet. 1998; 97:1347-1354. DOI: 10.1007/ s001220051028

[207] Hall RD, Rouwendal GJA, Krens FA. Asymmetric somatic cell hybridization in plants. I. The early effects of (sub) lethal doses of UV and gamma irradiation on the cell physiology and DNA integrity of cultured sugarbeet (Beta vulgarris L.) protoplasts. Mol Gen Genet. 1992; 234:306-314.

[208] Gleba YY, Hinnisdaels S, Sidorov VA, et al. Intergeneric asymmetric hybrids between *Nicotiana plumbaginifolia* and *Atropa belladonna* obtained by gamma-fusion. Theor Appl Genet. 1998; 76(5):760-766. DOI: 10.1007/BF00303523

[209] Ramulu KS, Dijkhuis P, Famelaer I, Cardi T, Verhoeven HA. Cremart: a new chemical for efficient induction of micronuclei in cells and protoplasts for partial genome transfer. Plant Cell Rep. 1994; 13:687-691. DOI: 10.1007/ BF00231625

[210] Valkonen JPT, Xu Y-S, Rokka V-M, et al. Transfer of resistance to potato leaf roll virus, potato virus Y and potato virus X from *Solanum brevidens* to *S. tuberosum* through symmetric and designed asymmetric somatic hybridization. Ann Appl Biol. 1994; 124:351-362. DOI: 10.1111/j.1744-7348.1994.tb04139.x

[211] Wolters AMA, Koornneef M, Gilissen LJW. The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of *Solanum tuberosum* and *Nicotiana plumbaginifolia*. Curr Genet.

1993; 24:260-267. DOI: 10.1007/ BF00351801

[212] Ali SNH, Juigen DJ, Ramanna MS, et al. Genomic in situ hybridization analysis of a trigenomic hybrid involving *Solanum* and *Lycopersicon* species. Genome. 2000; 44:299-304. DOI: 10.1139/g00-114

[213] Lusser M, Parisi C, Plan D, et al. Deployment of new biotechnologies in plant breeding. Nat Biotechnol. 2012; 30:231-239 DOI: 10.1038/nbt.2142

[214] Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science*.* 2012; 337:816-821. DOI: 10.1126/science.1225829

[215] Kawall K. New possibilities on the horizon: Genome editing makes the whole genome accessible for changes. Frontiers in Plant Sci. 2019; 10: 525. DOI: 10.3389/fpls.2019.00525

[216] Wang S, Zhang S, Wang W, et al. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Rep. 2015; 34:1473-1476. DOI: 10.1007/ s00299-015-1816-7

[217] Butler NM, Atkins PA, Voytas DF, et al. Generation and inheritance of targeted mutations in potato (*Solanum tuberosm* L.) using the CRISPR/Cas system. PLoS ONE. 2015; 10(12):e0144591. DOI: 10.1371/journal.pone.0144591

[218] Butler NM, Baltes NJ, Voytas DF, et al. Geminivirus mediated genome editing in potato (*Solanum tuberosum* L.) using sequence-specific nucleases. Front Plant Sci. 2016; 7:1045. DOI: 10.3389/fpls.2016.01045

[219] Tang L, Zeng Y, Du H, et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol Genet Genomics. 2017; 292(3):525-533. DOI: 10.1007/s00438-017-1299-z

[220] Andersson M, Turesson H, Olsson N, et al. Genome editing in potato *Biotechnological Strategies for a Resilient Potato Crop DOI: http://dx.doi.org/10.5772/intechopen.98717*

via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant. 2018; 164:378- 384. DOI: 10.1111/ppl.12731

[221] Nadakuduti SS, Buell CR, Daniel F, et al. Genome editing for crop improvement – Applications in clonally propagated polyploids with a focus on potato (*Solanum tuberosum* L.). Front Plant Sci. 2021; 9: 1607. DOI: 10.3389/ fpls.2018.01607

[222] Voytas DF. Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol. 2013; 64:327-350. DOI: 10.1146/annurevarplant-042811-105552

[223] Draffehn A, Meller S, Li L, et al. Natural diversity of potato (*Solanum tuberosum*) invertases. BMC Plant Biol. 2010; 10: 271. DOI: 10.1186/1471- 2229-10-271

[224] Clasen BM, Stoddard TJ, Luo S, et al. Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnol J. 2015; 14(1):169-176. DOI: 10.1111/pbi.12370

[225] González MN, Massa GA, Andersson M, et al. Reduced enzymatic browning in potato tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein complexes delivery of the CRISPR/Cas9 system. Front Plant Sci. 2019; 10:1649. DOI: 10.3389/fpls.2019. 01649

[226] Kieu NP, Lenman M, WangES, et al. Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes. Scientific Reports. 2021; 11:4487. DOI: 10.1038/s41598- 021-83972-w

[227] Genome editing database [Internet]. Available from: http:// plantcrispr.org/cgi-bin/crispr/index.cgi [Accessed: 2021-04-30]

[228] Committee on Genetically Engineered Crops. Genetically

Engineered Crops: Experiences and Prospects [Internet]. 2016. Available from: http://nap.edu/23395 [Accessed: 2021-04-28]

[229] Fresh Fruit Portal [Internet]. 2021. Available from: https://www. freshfruitportal.com/news/2021/04/26/ usda-deregulates-simplots-geneticallyengineered-potato/ [Accessed: 2021-04-26]

Section 5 Potato Seeds

#### **Chapter 15**
