Monohaploid Potato Research Articles

Chromosome pairing and potential for intergeneric recombination in some hypotetraploid somatic hybrids of Lycopersicon esculentum (+) Solanum tuberosum

Three somatic hybrids resulting from protoplast fusions of a diploid kanamycin-resistant line of tomato (Lycopersicon esculentum) and a dihaploid hygromycin-resistant transformant of a monohaploid potato (Solanum tuberosum) line were used for a cytogenetic study on chromosome pairing and meiotic recombination. Chromosome counts in root-tip meristem cells revealed two hypotetraploids with chromosome complements of 2n = 46 and one with 2n = 47. Electron microscope analyses of synaptonemal complex spreads of hypotonically burst protoplasts at mid prophase I showed abundant exchanges of pairing partners in multivalents involving as many as eight chromosomes. In the cells at late pachytene recombination nodules were found in multivalents on both sides of pairing partner exchanges, indicating recombination at both homologous and homoeologous sites. Light microscope observations of pollen mother cells at late diakinesis and metaphase I also revealed multivalents, though their occurrence in low frequencies betrays the reduction of multivalent number and complexity. Precocious separation of half bivalents at metaphase I and lagging of univalents at anaphase I were observed frequently. Bridges, which may result from an apparent inversion loop found in the synaptonemal complexes of a mid prophase I nucleus, were also quite common at anaphase I, though the expected accompanying fragments could be detected in only a few cells. Most striking were the high frequencies of first division restitution in preparations at metaphase II/anaphase II, giving rise to unreduced gametes. In spite of the expected high numbers of balanced haploid and diploid gametes, male fertility, as revealed by pollen staining, was found to be negligible.

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.

Orientation of diplochromosomes in mitoses of potato (Solatium tuberosum L.) callus cultures

The mitotic behaviour of diplochromosomes was investigated in early calli from leaf segments of a monohaploid potato (x = 12) with the aid of the fluorescent plus Giemsa technique. The sister chromosomes of the diplochromosomes do not separate during metaphase because the inner chromatids remain connected. Consequently the diplochromosomes orient in three stable configurations during metaphase. In the case in which sister chromosomes become auto-oriented, the outer chromatids face one pole and the inner chromatids the opposite pole (U-shaped configuration), or the outer and inner chromatid of each sister chromosome face different poles (alternate configuration). Less frequently the sister chromosomes become oriented to different poles (C-shaped configuration). Linear orientation is not observed. At least until the onset of telophase only the inner chromatids originating from U-shaped configurations may remain paired, but they fall apart before the next metaphase. The mitoses with diplochromosomes give rise to two cells with double the number of chromosomes.Key words: potato, diplochromosomes, mitosis, orientation, polyploidization.

Chromosomes in somatic hybrids between Nicotiana plumbaginifolia and a monoploid potato

Leaf mesophyll protoplasts of the monohaploid potato (Solanum tuberosum L.) clone H7322 were fused with callus protoplasts of nitrate reductase deficient (NR−) mutants Cnx 20 and NA 36 of Nicotiana plumbaginifolia. Somatic hybrid lines were selected for nitrate reductase proficiency. All callus lines tested appeared to be stable for the retention of the potato chromosome carrying the compensating NR gene when grown for over 1.5 years in the absence of nitrate. Shoots were regenerated from six different fusion lines of Cnx 20 + H7322 24 months after fusion. Chromosomal analysis in callus cultures revealed that in both fusion combinations 40–120 N. plumbaginifolia chromosomes were present, as were 9–20 potato chromosomes. Cells with 17 potato chromosomes in combination with a relatively small number (31) of N. plumbaginifolia chromosomes were found in one line. Preferential loss of species-specific chromosomes was not observed. Analysis of regenerating tissue from three lines of Cnx 20 + H7322 revealed that after 24 months of culture intra- and intergeneric translocations, fragments and deletions were present. Elimination of the potato and N. plumbaginifolia chromosomes had taken place before and after genome doubling.

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.

Ploidy variability in greenhouse cultured and in vitro propagated potato (Solanum tuberosum) monohaploids (2n=x=12) as determined by flow cytometry

The DNA distributions of 23 different monohaploid potato clones were investigated by flow-cytometric measurements. All monohaploid clones differed in DNA distribution but none of them contained only monoploid cells in the leaves. All were highly stable on the monohaploid level for 2-3 years. Investigation of the influence of different factors on the DNA distribution in leaf cells showed that the material derived from in vitro shoot tip propagation contained a lower proportion of polyploidized cells than greenhouse grown plants. With protoplast isolation the enzyme treatment of in vitro cultured plant material induced a striking shift of DNA distribution towards the lower C-value whereas the mechanical purification steps caused a selective loss of monoploid nuclei. Seasonal influence on the DNA patterns could be detected.

Protoplast fusion of monohaploid (2n = x = 12) potato clones: Identification of somatic hybrids using malate dehydrogenase as a biochemical marker

The isolation, fusion and successful culture of protoplasts from monohaploid potato clones is described. These monohaploids were obtained from two diploid lines coded M9 and PD23-2. M9 was homozygous for malate dehydrogenase (MDH) (1 slowly moving band; banding pattern A) and PD23-2 was heterozygous (3 bands; banding pattern AB). The nine monohaploids X1,1–X1,9 obtained from M9 all showed banding pattern A and the 49 monohaploids X2,1–X2,49 from PD23-2 showed banding pattern A or B (the latter showed 1 fast moving band). Protoplasts of X1,1 with banding pattern A and of X2,1 with banding pattern B were fused. Some calli of the heterofusion experiment showed plant regeneration. Based on MDH tests these regenerants could be characterized as somatic hybrids. From the results presented it can be concluded that MDH can be used as a biochemical marker in fusion experiments with monohaploids and that plant regeneration ability can be increased by fusion of different plant clones.

Morphology and vigour of monohaploid potato clones, their corresponding homozygous diploids and tetraploids and their heterozygous diploid parent

To study the joint effects of homozygotization and polyploidization in potato, the performance has been examined of five potato genotypes at three (x, 2x, 4x) and two genotypes at two (x, 2x) ploidy levels. Six out of the seven genotypes studied were compared with their heterozygous diploid parental clone. In this way comparisons could be made between i) the heterozygous diploid and its monohaploid derivatives, ii) three or two ploidy levels per genotye and iii) homozygous di- and tetreploids and their heterozygous diploid source.

Behaviour of chromosomes in potato leaf tissue cultured in vitro as studied by BrdC-Giemsa labelling

Cells of leaf explants of a monohaploid potato (Solanum tuberosum) were stimulated to mitosis on a medium with 5-bromodeoxycytidine during a period of 7 days. The cells cycled with mono- or diplochromosomes which showed differential staining of the sister chromatids and sister chromatid exchanges by the fluorescent plus Giemsa technique after two rounds of BrdC incorporation. Through the staining pattern the course of the first three cell cycles could be traced and the duration of the cycles estimated. Polyploidisation was enhanced by selective stimulation of polyploid cells and by endoreduplication of G2-phase cells. The percentage of polyploid mitoses increased from 10 to 70.