Evidence that genes from the male parent may influence the morphology of potato dihaploids

A number of recent studies have provided evidence that potato dihaploids (S. tuberosum) contain and express DNA from the male (dihaploid inducer) parent, S. phureja. The importance of this for breeding programmes that use dihaploid potatoes is to some extent dependent upon whether the S. phureja DNA influences dihaploid morphology. In the present study, 21 characters were used to compare the morphology of six dihaploids with those of their parents: S. tuberosum (cvs `Pentland Dell' and `Pentland Crown') and S. phureja (IVP48). Characteristics of S. phureja were found in all of the dihaploids examined. In principal component analyses, dihaploids formed intermediate groupings positioned between those of the parents, although much closer to S. tuberosum. It is concluded there is evidence that DNA originating from the dihaploid inducer can affect the morphology of potato dihaploids. Implications of the findings are discussed. Received: 26 November 1995 / Accepted: 9 February 1996     

A comparison of tissue culture response between related tetraploid and dihaploid S. tuberosum genotypes

The response to tissue culture of a series of related, agronomically useful, dihaploid (2n=2x=24) and tetraploid (2n=4x=48) S. tuberosum genotypes was assessed by regenerating shoots from leaf explants. Dihaploid genotypes that showed superior responses to their tetraploid parents were identified. Large differences in tissue culture response were also found between dihaploid genotypes derived from the same tetraploid parents. These results indicate that it should be possible to select agronomically useful dihaploid genotypes with good tissue culture responses for use in genetic manipulation experiments. Possible factors determining tissue culture response in S. tuberosum are discussed.     

The use of RAPD markers for the detection of gene introgression in potato

Randomly Amplified Polymorphic DNAs were employed to demonstrate that potato dihaploids generated after interspecific pollination of a tetraploid Solanum tuberosum cultivar (Pentland Crown) by Solanum phureja dihaploid inducer clones could not be of parthenogenetic origin. Of six different 10-mer oligonucleotides, four generated products from total potato dihaploid genomic DNAs which were not derived from the S. tuberosum parent. Gel electrophoresis and Southern analysis indicated that these amplified bands originated from S. phureja. The results are discussed in the context of recent cytological and molecular evidence which demonstrates that potato dihaploids are aneusomatic (Clulow et al. 1991) and emphasises this approach as a general methodology for the detection of alien gene introgression in both natural and cultivated plant populations.     

Methods and strategies for detecting Solanum tuberosum dihaploids in interspecific crosses with S. phureja

The frequency of dihaploid production following the interspecific pollination of Solanum tuberosum clones with Solanum phureja has been investigated. Seventeen hybrid combinations were tested and the crosses differed significantly in the number of seed produced, the proportions that were viable and the proportions that were dihaploid. The overall frequency of dihaploid production was 62%. The dominant embryo spot marker of the pollinator (IVP 48) in conjunction with seed diameter was used to classify the seed samples. Number of days taken to germinate was also recorded. It was therefore possible to examine retrospectively the effect of selecting sub-sets of the seed based on the presence or absence of the embryo spot, seed size and germination time. A seed selection system based on the absence of the embryo spot in conjunction with seed greater than 1.25 mm in diameter resulted in 93.6% of the potential dihaploids being identified.     

A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

The Role of Polyphenol Oxidase and Peroxidase in Potato Tuber Resistance to Soft Rot Caused by Erwinia carotovora

Tubers from somatic hybrids produced by protoplast fusion between Solanum brevidens, a diploid, non-tuber-bearing wild species, and a tetraploid S. tuberosum showed resistance to decay caused by soft rot Erwinia. Tubers of the S. tuberosum fusion parent and potato cultivar Russet Burbank are susceptible to bacterial soft rot. Tubers of somatic hybrids indicated higher levels of activities of polyphenol oxidase and peroxidase than tubers of the parental line of S. tuberosum and cultivar Russet Burbank. This is true for intact tubers and also for injured or inoculated tubers. Polish commercial potato cultivars indicated a higher susceptibility to soft rot than somatic hybrids. However, there were some differences in susceptibility to soft rot between Polish commercial potato cultivars, only slight differences were observed in the activities of the polyphenol oxidase and peroxidase between Polish cultivars. A relation between soft rot resistance and the activity of each enzyme was not found for intact, injured or inoculated tissue of commercial cultivars. On the contrary, the activities of both enzymes were significantly higher in the periderm than in the medullary tissue of somatic hybrids, the parental line and the commercial cultivars.     

Characterisation of somatic hybrids between Solanum tuberosum and its frost-tolerant relative Solanum commersonil

 Somatic hybrids between three dihaploid Solanum tuberosum (2n=2x=24) genotypes and the frost-tolerant, diploid, relative Solanum commersonii (2n=2x=24) were analysed for variation in morphological traits, fertility and frost tolerance. The somatic hybrids were more vigorous than their parents and in many ways resembled a tetraploid S. tuberosum. All of the hybrids flowered profusely, although the male fertility was largely dependent on the S. tuberosum genotype used. In one hybrid combination all plants were both male- and female-fertile, while in the other two combinations the majority of plants were male-sterile but female-fertile. The somatic hybrids showed an increase in direct frost tolerance when compared with the dihaploid S. tuberosum parents, and to a varying extent had gained the capacity to cold acclimate. These somatic hybrids will be used in breeding programmes involving repeated cycles of anther culture and somatic hybridisation. Received: 20 May 1997 / Accepted: 12 June 1997     

RAPD markers for confirmation of somatic hybrids in the dihaploid breeding of potato (Solanum tuberosum L.)

Summary The applicability and reliability of RAPD markers were evaluated for an examination of the possible use of RAPD markers to confirm hybridity of somatic hybrids between dihaploids of potato (Solanum tuberosum L.). Most of the primers examined detected polymorphism among either tetraploids or dihaploids, and polymorphism was easily detected even among closely related clones. Most of the examples of polymorphism were confirmed as being the result of amplification from the nuclear genome by a comparison of patterns generated by PCR of clones that carried the same cytoplasm. All the bands of dihaploids were transmitted stably to the respective hybrids. In the absence of primers that generated complementary polymorphic bands for both parents, a mixture of two appropriate primers, each of which generated a band specific to one parent, permitted the simple confirmation of hybridity. Hybridity of all the fusion-derived regenerants of 32 fusion combinations was unequivocally confirmed, a result that suggests that RAPD analysis could be universally applicable to the confirmation of hybridity in the dihaploid breeding of potato.     

Interspecific somatic hybrids between wild potato Solanum acaule Bitt. and anther-derived dihaploid potato (Solanum tuberosum L.)

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition. Received: 19 January 1998 / Revision received: 27 March 1998 / Accepted: 20 April 1998     

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy

Summary The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5–7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9–14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% – 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.     

Application of somatic hybrids between dihaploids of potato Solanum tuberosum L. and wild diploid species from Mexico in breeding: Generation and backcrossing of dihaploids of somatic hybrids

The efficiency of an original approach to involvement of the valuable genetic pool of wild diploid potato species from Mexico is estimated. The essence of this method is in generation of dihaploids (2n = 2x = 24) of tetraploid somatic hybrids (2n = 4x = 48) followed by backcrossing with dihaploids of Solanum tuberosum. A haploid producer, S. phureja IvP35, was used to generate ten dihaploids of S. tuberosum + S. pinnatisectum, all of which crossed with fertile S. tuberosum dihaploids and developed plump viable seeds. This gives the possibility of an efficient introgression of the genes valuable for breeding from wild species to the bred plants at a diploid level, which has several advantages compared with the corresponding procedure at a tetraploid level. A part of the dihaploids produced was compatible (the pollen tubes reached the ovary) with diploid and tetraploid forms of S. pinnatisectum; however, no viable seeds were developed. The attempt to generate the dihaploids of S. tuberosum + S. bulbocastanum somatic hydrides using the haploid producer S. phureja IvP35 was unsuccessful.     

Rare instances of haploid inducer DNA in potato dihaploids and ploidy-dependent genome instability

In cultivated tetraploid potato (Solanum tuberosum), reduction to diploidy (dihaploidy) allows for hybridization to diploids and introgression breeding and may facilitate the production of inbreds. Pollination with haploid inducers (HIs) yields maternal dihaploids, as well as triploid and tetraploid hybrids. Dihaploids may result from parthenogenesis, entailing the development of embryos from unfertilized eggs, or genome elimination, entailing missegregation and the loss of paternal chromosomes. A sign of genome elimination is the occasional persistence of HI DNA in some dihaploids. We characterized the genomes of 919 putative dihaploids and 134 hybrids produced by pollinating tetraploid clones with three HIs: IVP35, IVP101, and PL-4. Whole-chromosome or segmental aneuploidy was observed in 76 dihaploids, with karyotypes ranging from 2n = 2x − 1 = 23 to 2n = 2x + 3 = 27. Of the additional chromosomes in 74 aneuploids, 66 were from the non-inducer parent and 8 from the inducer parent. Overall, we detected full or partial chromosomes from the HI parent in 0.87% of the dihaploids, irrespective of parental genotypes. Chromosomal breaks commonly affected the paternal genome in the dihaploid and tetraploid progeny, but not in the triploid progeny, correlating instability to sperm ploidy and to haploid induction. The residual HI DNA discovered in the progeny is consistent with genome elimination as the mechanism of haploid induction.

A large potato progeny population produced by crossing tetraploid cultivated clones to diploid Phureja lines displays rare instances of haploid inducer chromosomes, which are frequently damaged.     

Use of seedling progeny tests for genetical studies as part of a potato (Solanum tuberosum subsp. tuberosum) breeding programme

A diallel set of crosses, including selfs and some reciprocal crosses, was made between 15 parents chosen for their male fertility from those included in a tetraploid potato (Solanum tuberosum subsp. tuberosum) breeding programme at the Scottish Crop Research Institute. Seedling progeny tests were used to evaluate the progenies for non-race-specific resistance to late blight (Phytophthora infestans) in both foliage and tubers, quantitative resistance to the white potato cystnematode (PCN) (Globodera pallida) and the commercial worth of their tubers as judged by breeders' visual preference. No reciprocal differences were found. Comparisons of the selfs and crosses revealed inbreeding depression for breeders' preference, which varied among the parents from negligible to severe, whilst there were also statistically significant differences for foliage and tuber blight, but not for PCN. When the selfs were omitted from the combining ability analyses, large differences in general combining ability (GCA) were found for all four traits, and smaller differences in specific combining ability for tuber blight and breeders' preference. The only statistically significant correlation between GCAs for different traits was a favourable one of r = 0.56 between foliage and tuber resistance to late blight. It was concluded that prospects were good for simultaneously improving all four traits by multitrait genotypic recurrent selection.     

Somatic hybrids produced by protoplast fusion between S. tuberosum and S. brevidens: phenotypic variation under field conditions

Summary Phenotypic and flowering characteristics of hybrid plants generated by protoplast fusion between a tetraploid S. tuberosum line and diploid S. brevidens were assessed under field conditions. Hybrids were compared to both clonal parental material and protoplast-derived plants of each parent. Almost all of the hybrids were hexaploid. A wide range of variation in morphological characters was observed for hybrids and protoclones. Flowering was markedly reduced in protoclones. The majority of hybrids flowered, had viable pollen and set tubers. Tuber and pollen characteristics of hybrids produced from individual fusion calli also varied. The potential usefulness of fusion hybrids in potato improvement is discussed.     

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs

Summary The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A₁ plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

The effects of inbreeding on the parental values of potato dihaploids

Dihaploids obtained from a somatically chromosome-doubled dihaploid potato were crossed with Solanum phureja clones. To test the effect of inbreeding, measurements were made of their seed production and the tuber yield, tuber number and mean tuber weight of their offspring. On average, seed production of the second generation dihaploids was higher than that of the original dihaploid progenitor. Progeny tuber yield and its components were little different from those of the original dihaploid's progeny. Tuber flesh quality, as measured lack of blemishes, was better in the offspring of second generation dihaploids.
It is suggested that the negative effects of producing second generation dihaploids are minor compared with producing the first generation dihaploid from a tetraploid, because most deleterious recessives have already been unmasked. The results indicated residual variation within the original dihaploid which could be exploited for plant breeding purposes.
An indicator of inbred status, alternative to the inbreeding coefficient, is suggested which could be applied to both diploids and tetraploids.     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

Discovery of highly apomictic and highly amphimictic dihaploids in Allium tuberosum

 To discover highly apomictic and amphimictic Allium tuberosum diploids, we evaluated the degree of apomixis in three dihaploids (2n=16, 2x), KaD2, TeD1 and GMD1, derived from highly apomictic tetraploids. The degree of apomixis, calculated as the percentage of diploid seedlings in the progeny obtained after cross-pollination with tetraploid cultivars, was 96% in KaD2, 7% in TeD1 and 39% in GMD1. In addition to this general index of apomictic nature, two analytical indices were evaluated in KaD2 and TeD1. The degree of diplospory, calculated as the percentage of endoreduplicated embryo-sac mother cells, was 96% in KaD2 and 2% in TeD1. The degree of parthenogenesis, calculated as the percentage of ovules with the egg cell developing parthenogenetically, was 98% in KaD2 and 10% in TeD1. Among angiosperms with gametophytic apomixis, KaD2 is the first diploid apomict whose reproductive mode has been fully described by these three quantitative indices of apomictic nature. And TeD1 is the first highly amphimictic plant found in the A. tuberosum complex. Although TeD1 is poorly fertile, the present results encourage further screening trials for highly fertile, highly amphimictic dihaploids, which may be effective counterparts to KaD2 in diploid-level cross experiments to genetically analyze apomixis in A. tuberosum. Received: 4 December 1995 / Revision accepted: 8 May 1996