Jagesh K. Tiwari1, Poonam1, A.K. Srivastava2*, B.P. Singh1 & T.K. Bag2
1Central Potato Research Institute, Shimla – 171 001 Himachal Pradesh, India
2Central Potato Research Station, Shillong, Meghalaya, India
*Corresponding author: A.K. Srivastava; email: firstname.lastname@example.org
Gene transfer is the basis for almost all crop improvement including potato. Wild species of potato harbors myriad of genes providing resistance against biotic and abiotic stresses. Breeders have been tempted to utilize them for incorporating valuable genes ranging from resistance to grain yield, and produce quality. But many sources of useful genes cannot be included in crop improvement programme in potato due to tetrasomic inheritance, asexual mode of propagation, a heavy load of deleterious recessive alleles and varying levels of sexual and EBN (endosperm balance number) incompatibilities. Gene transfer through transformation, though possible, requires identification, isolation and cloning of the concerned genes. Somatic hybridization through targeted protoplast fusion between two dihaploids of tetraploid potatoes, or between a dihaploid and a wild diploid Solanum species across sexual/ EBN barriers, or between two monoploids of diploid/ dihaploid potatoes offers great opportunities for targeted whole genome manipulation vis-à-vis complementation. The technology is slowly being functionally integrated into the potato breeding program in India at CPRI, Shimla by developing parental lines with increased level of resistance against late blight and Potato virus Y.
Keywords Potato, protoplast fusion, somatic hybrid.
Gene transfer is the basis for almost all crop improvement including potato. Conventionally, this is achieved through sexual hybridization; this rather limits the range of species from which gene flow can occur into a crop species. Wild species have contributed remarkably to the success of latter; they allowed the crops to retain their commercial status. As a result plant breeders have sought to utilize an increasing number of wild species as a source of valuable genes ranging from disease resistance to grain yield, and produce quality. But many sources of useful genes cannot be included in crop improvement programme primarily because of sexual incompatibilities (Gopal, 2006). Genetic transformation, a focused and direct gene transfer approach require identification, isolation and cloning of the concerned genes. Further it is expensive and technically most exacting, although it may represent the ultimate strategy. However, some characters of interest may be governed by two or more and yet unknown genes; transfer of such characters through genetic transformation may pose many difficulties. Finally transfer of cytoplasmic organells, viz., chloroplast and mitochondria may often be desired objectives; this, however is not possible through genetic transformation, while it can readily be achieved by somatic hybridization.
The wild Solanum spp. is reproductively isolated from cultivated potatoes and also not crossable due to difference in ploidy number and endosperm balance number (EBN) (Spooner and Salas, 2006). Hence, protoplast fusion or somatic hybridization is the technique that enables to transfer agronomically important traits by bypassing such sexual barriers, besides the conventional and recombinant- DNA technologies approaches. Despite these crossingbarriers, many researchers have used this technique and subsequently produced somatic hybrids with cultivated potato. Production of hybrid plants through the fusion of protoplasts of two different plant species/varieties is called somatic hybridization, and such hybrids are called somatic hybrids (Sharma et al. 2011). Therefore, somatic hybridization can be resorted to only when the following two criteria are satisfied: i) isolation of protoplast in large quantity and ii) totipotency of the isolated protoplasts.
The techniques of somatic hybridization involve the following four steps: i) isolation of protoplast, ii) fusion of protoplasts of desired species/varieties, iii) selection of somatic hybrid cells, and iv) culture of hybrid cells and regeneration of hybrid plants from them (Figure 1). A brief, elementary consideration of these steps is presented below:
Protoplast isolation and culture
Somatic hybrid plants are the result of a multi step process. The first one is isolation of viable protoplasts and efficient plant regeneration. Out of many published protocols, the ones most often employed were those presented by Binding et al. (1978) and Haberlach et al. (1985). In general, three-week-old in vitro plants are used to isolate mesophyll protoplasts. In vitro plants are cultivated in the dark (covered with dark-black muslin cloth) in a culture room at 20ºC for 48h under a 16h photoperiod prior to protoplast isolation to integrate cell cycles.
The enzymatic solutions of 1% cellulose ‘Onozuka’ RS (Yakult Pharmaceuticals, Tokyo) and 0.5% macerozyme R 10 (Yakult) are used for cell wall degradation. Young leaf tissues are minced in a petri dish (Ø = 90 × 15mm) containing protoplast digestion solution (PDS) (10ml PDS g-1 tissue) followed by incubation in the dark at 25ºC for 16h without shaking. After incubation, 0.3M KCl (filter sterilized) is added in a 1:1 ratio (PDS:KCl) followed by gentle shaking of PDS containing released protoplast. Subsequently, suspension is filtered through a 41µm nylon sieve and collected in centrifuge tubes. For purification of protoplasts the filtrates are centrifuged at 60 RCF for 5 min and then the pellets are resuspended in 9ml of 0.6M sucrose (filter sterilized) followed by 1 ml of 0.3M KCl layer onto it and centrifuged as above. Live protoplasts (green) are recovered from sucrose: KCl interface, diluted with 5ml of 0.3M KCl, centrifuged as above and finally suspended in 200–400µl of 0.5M Mannitol, depending upon the isolation, to a final density of 1 × 106 protoplast ml-1. Protoplasts are then counted by Hemacytometer.
Protoplast fusion and obtaining somatic hybrids
Among several known agents inducing protoplast fusion, the two most often used are polyethylene glycol (PEG induced fusion) and a pulse of electric field (electrofusion). Both methods are equally good and the choice is mostly the matter of personal preferences and technical possibilities. The electrofusion protocol typically involves fusion in a 3.2mm microslide using the Electro Cell Manipulator. The purified protoplasts are aligned at 30–50 Vcm-1 (1 MHz alternating current) for 30s and electrofused by one direct-current pulses at 850–1,250 Vcm-1 for 60µs with a 10s post-fusion AC field for compacting the fusion products. Protoplast fusion is relatively inefficient and nonspecific so the post-fusion mixture contains both original fusion components as well as the products of homo-and hetero-fusion. Consequently it is necessary to select heterokaryons. When genetic markers required for mass selection are not available, manual selection or automatic cell sorting are possible alternatives. These selection systems, applicable to any combination of parental lines, are usually better suited for creation of plants potentially useful in breeding.
Selection of somatic hybrid cells
Hybridity of plants regenerated from heterokaryons must be confirmed by the presence of DNA or expression of genes from both fusion components. The former can be achieved by any of the known types of molecular markers. Restriction fragment length poly-morphism (RFLP) markers utilizing oligonucleotide or single copy species-specific repetitive probes have been extensively used for confirming hybridity of somatic fusion product. PCR based markers like RAPD and SSR are also utilized by many workers. The presence of one genome fragment in the hybrid cells can be detected by genomic in situ hybridization (GISH). Because Solanum chromosomes are small, classical karyological analysis is difficult. Chromosomes can be counted but their origin cannot be determined. Karyotyping often supplements other methods such as RAPD and RFLP. The total amount of DNA determined by flow cytometry, although correlated with chromosome number, gives only an indirect proof of hybridity. It means that the loss of lesser amount of DNA from asymmetric hybrid can not be detected. Another indication of ploidy level is the number of chloroplasts present in stomatal guard cells.
Culture of hybrid cells and regeneration of hybrid plants
Electrofused products are immobilized on thin-layer sodium alginate (2.8%) matrices and grown in VKM liquid medium (Binding & Nehls 1977) supplemented with glucose (90mgml-1) at 25oC in the dark. Following the cell wall development, regenerating microcalli, are transferred onto MS13K solid medium (Behnke 1975) at 20oC under a 16h photoperiod for macrocalli and shoot development. Newly regenerated shoots from calli are cultured on MS medium for subsequent growth and multiplication. Characterization of the regenerants is based on regeneration of first shoot per callus followed by subsequent multiplication for further molecular and phenotypic studies.
Examples of potato somatic hybrids
Resistance trait transferred
|Recipient (Common potato)S. tuberosum spp. tuberosum||Donor (Wild spp.)
Frost, Soft rot, Late blight, PVY
Bacterial wilt, Soft rot, Frost-tolerant
PLRV, PVY, Green peach aphid,
Late blight and PVY
Potato somatic hybrids at CPRI, Shimla
Potato somatic hybrid of Solanum tuberosum dihaploid C-13 (+) S. pinnatisectum clone ‘P 7’ was registered in the NBPGR, New Delhi (NBPGR Reg. No. INGR 11051) for its unique features: Interspecific potato somatic hybrids produced by protoplast fusion between dihaploid S. tuberosum L. and wild spp. S. pinnatisectum, tetraploid and male fertile, and resistance to potato late blight introgressed from S. pinnatisectum (Sarkar et al. 2011; Tiwari et al. 2011) (Figure 1). Second potato somatic hybrid of S. tuberosum dihaploid C-13 (+) S. etuberosum clone ‘E 1-3’ was registered in the NBPGR, New Delhi (NBPGR Reg. No. INGR 11050) for their unique features: Interspecific potato somatic hybrids produced by protoplast fusion between dihaploid S. tuberosum L. and wild spp. S. etuberosum, tetraploid and male fertile, and resistance to potato virus Y introgressed from S. etuberosum (Tiwari et al. 2010) (Figure 2).
i. Somatic hybrids can be produced between species, which cannot be hybridized sexually.
ii. Somatic hybrids can be readily used in breeding programme for transfer of resistance genes
iii. Hybrids can be produced even between such clones, which are completely sterile.
iv. Cytoplasm transfer can be done in one year, while back crossing may take 5-6 years.
v. Even where backcrossing is not applicable, cytoplasm transfer can be made using this approach.
vi. Mitochondria of one species can be combined with chloroplast of another species. This may be very important in some cases, and is not achievable by sexual means even between easily crossable species.
vii. Recombinant organelle genomes, especially of mitochondria, are generated in somatic hybrids. Some of these recombinant genomes may possess useful features.
i. Techniques for protoplast isolation, culture and fusion are very complicated.
ii. In many cases, chromosome elimination occurs from somatic hybrids leading to asymmetric hybrids. Such hybrids may be useful, but there is no control on chromosome elimination.
iii. Many somatic hybrids show genetic instability, which may be an inherent feature of some species combinations.
iv. Many somatic hybrids either do not regenerate or give rise to sterile regenerants. Such hybrids are useful for crop improvement. All interfamily somatic hybrids are genetically unstable and/or morphologically abnormal, while intergeneric and intertribal hybrids are genetically stable, but produce abnormal and/ or sterile plants
Somatic hybridization allows transfer of cytoplasmic organelle in a single generation and offer unique opportunities for combining mitochondria of one species and chloroplast of another species in a single hybrid. This capability may permit improvement of characteristics certain cytoplasmic male sterile line, which may lead to their commercial exploitation. In addition, even non-flowering and non-tuber bearing species can be utilized in breeding programme. The transfer of gene governing resistance to biotic and abiotic stresses is an important objective. In potato, this technique is already being used in commercial breeding programme of the Netherlands and Germany. In general, somatic hybrids have low pollen fertility, but they can often be used as female parents in backcrossing with one of fusion parents. It is likely that the approach of somatic hybridization will find grater applications in potato improvement for enabling transfer of useful genes from sexually incompatible species. In this context, it is important that the DNA segment carrying the desired gene from wild species is introgressed into the genome of cultivated potato and exhibits stable inheritance. The possible mechanissm for such introgression are homeologous pairing leading to crossing over, and intergenomic translocation. An understanding of the gene introgression mechanism may enable their enhancement using suitable treatment; this, in turn will enhance the opportunities for utilization of somatic hybrids in potato improvement.
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Tiwari, J.K., Poonam, Sarkar, D., Pandey, S.K., Gopal, J. and Kumar, S.R. 2010. Molecular and morphological characterization of somatic hybrids between Solanum tuberosum L. and S. etuberosum Lindl. Plant Cell, Tissue and Organ Culture 103:175–187.
Tiwari, J.K., Sarkar, D., Poonam, Sharma, S. and Gopal, J. 2011. Solanum tuberosum (+) S. pinnatisectum somatic hybrids: a new source of horizontal resistance to potato late blight in India. SOL Newsletter, pp. 4.