Leena Pietilä

Enhanced production of dihaploid lines via anther culture of tetraploid potato (Solanum tuberosum L. SSP. Tuberosum) clones

A total of 1000 anther-derived plants was regenerated from tetraploid potato (Solanum tuberosum L.) genotypes. Capacity to undergo androgenesis was analysed in 41 potato cultivars and 7 clones grown either in the greenhouse or in the field. Of the 48 genotypes, 33 produced embryos and 23 regenerated shoots from embryos. Anther-derived plantlet production was determined in genotypes 86110, Agria, Calgary, Escort, Helios, Idole, JO 0982 JO 1432, Kainuun Musta, Kardal, KE48, Matilda, Nicola, Petra, Pito, Rustica, Stirling, Torridon, Ute, Van Gogh, Vebeca, Vento and White Lady. The highest number of shoots (24 shoots/100 anthers) was obtained from cv. Calgary, when anthers were isolated from field-grown donor plants. Incubating anthers at 28 C, rather than at 20 C or 24 C, enhanced embryo production in four genotypes tested. However, shoot production was improved only in cv. Pito cultured at 28 C. When anthers of cv. Petra were cultured at 28 C for four weeks, followed by reduction of culture temperature to 24 C, a high rate of shoot production was recorded (14 shoots/100 anthers). The ratio between dihaploids and tetraploids varied among the anther-derived plants of the different genotypes. The number of dihaploids was highest in potato clone JO1432 (100%) and in cv. Calgary (93%) and lowest in cvs. Pito (21%) and Torridon (6%).

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.

Glycoalkaloid aglycone accumulations associated with infection by Clavibacter michiganensis ssp. sepedonicus in potato species Solanum acaule and Solanum tuberosum and their interspecific somatic hybrids

Solanum acaule Bitt., a wild potato species, is closely related to cultivated potato (Solanum. tuberosum L.). Incorporation of desirable traits from allotetraploid [2n=4x=48, 2 endosperm balance number (EBN)] S. acaule (acl) into autotetraploid (2n=4x=48, 4EBN) S. tuberosum (tbr) is difficult due to incongruity boundaries. In this study, three hybrid combinations, each with a specific genome constitution, were produced through protoplast fusion: (1) hexaploid 2x acl (+) 4x tbr, (2) tetraploid 2x acl (+) 2x tbr, and (3) hexaploid 4x acl (+) 2x tbr hybrids. In terms of glycoalkaloid aglycones, the hybrids produced demissidine, tomatidine and solanidine, similarly to the S. acaule parental species, but S. tuberosum synthesised only solanidine. Inoculations with Clavibacter michiganensis ssp. sepedonicus (Cms), which is the causal agent of bacterial ring rot in potato, yielded significantly lower total glycoalkaloid aglycone accumulation both in S. acaule plants and in interspecific hybrids in comparison with the corresponding mock-inoculated plants. However, in S. tuberosum the aglycone levels were either higher or unchanged as a result of infection by Cms. To incorporate the desirable traits of the interspecific somatic hybrids into 4EBN S. tuberosum, sexual backcrosses were carried out. The hexaploid 4x acl (+) 2x tbr hybrids with the hypothetical 4EBN showed the greatest capacity to undergo backcrosses with S. tuberosum.

The fruit of Ullucus (Basellaceae), an old enigma

Summary The fruit of the cultivated ulluco (Ullucus tuberosus Loz.) has been referred to in the literature either as non-existing or as a berry, seldom as a dry fruit. The fruit of this species as well as the genus is described here for the first time, and is shown to be a dry and indehiscent nutlet of an obovate shape and bilaterally symmetrical cross-section. The thin testa of the seed is immediately inside the thickened fruit wall. The embryo is strongly curved and encircles the relatively scanty perisperm.

Production and characterization of "second generation" somatic hybrids derived from protoplast fusion between interspecific somatohaploid and dihaploid Solanum tuberosum L.

Protoplasts were fused to produce somatic hybrids between a triploid (2n=3x=32-34) interspecific somatohaploid betweenSolanum brevidens Phil. andS. tuberosum L., and a dihaploid (2n=2x=24) anther-derived line ofS. tuberosum cv. Van Gogh. A total of 265 plants were regenerated from protoplast fusion derived calli and their hybridity was verified using fusion partner specific RAPD markers. These “second generation” somatic hybrids were aneuploid pentaploids (2n=5x=51-65) with a 2C DNA content ranging from 3.36 to 4.43 pg, which corresponded to the sum of the 2C values of each of the fusion partners (somatohaploid: 2.22 pg; and the dihaploid line of cv. Van Gogh: 1.87 pg). Most of the “second generation” somatic hybrids were vigorous, but variable in morphology. They were extremely resistant to PLRV and they had tolerance to PVY infection derived from the somatohaploid fusion partner. Even though most of the “second generation” hybrids tuberized, the tuber morphology was variable and most were poorly shaped. InErwinia soft rot resistance tests, the tubers showed higher level of resistance than the tetraploidS. tuberosum cultivars, the dihaploidS. tuberosum fusion partners and the hexaploid somatic hybrids betweenS. brevidens andS. tuberosum. The “second generation” somatic hybrids were all male sterile and failed to produce berries or seeds.

Morphological variation among clones of ulluco (Ullucus tuberosus, Basellaceae) collected in Southern Peru

Material of 229 clones of ulluco (Ullucus tuberosus), representing 10 accessions from markets in southern Peru and one from the wild in Bolivia, was analyzed with regard to morphological variation. The diploid chromosome number (2n = 24) was ascertained in 16 of the clones. Clones grown in different environments (e.g., long vs. short day) retained a more or less similar order of variation with respect to color of the tubers, stems, and leaves and the length/width ratio of the leaves. The shape of fully developed tubers also showed stable variation. Within a single experiment several other vegetative characters showed significant differences between accessions. Furthermore, many flower and inflorescence characters showed variation between accessions, some of them also indicating geographical variation between the Puno-Juliaca area and the Cuzco area. Considerable variation between the clones of an accession was evident, some accessions being clearly more variable than others. Our results show that ulluco is a crop plant that varies extensively even within a geographically limited area. Although repeatedly referred to in the literature as a purely asexual crop, ulluco was shown to be capable of sexual reproduction; this must have been and possibly still is an important source of genetic variation in the species.

Developmental abnormalities in the ovule and embryo sac of ulluco (Ullucus tuberosus) and their effect on seed set

SummaryUlluco (Ullucus tuberosus) is a vegetatively propagated crop plant that flowers abundantly but sets only few seeds. We examined if the low seed/ovule ratio can be explained by morphological abnormalities in ovules, embryo sacs or embryos. In 35 clones growing in the actual area of cultivation in Ecuador and in 5 clones grown in Finland flowers were analysed by clearing technique. About half of all analysed clones contained ovules and embryo sacs whose development was arrested, in two clones there were embryo sacs whose cell patterns were abnormal and in four clones there were double embryo sacs. There were both normal and abnormal ovules and embryo sacs in the same plant. Genetic load is suggested to be one of the causes for the low sexual fertility found in ulluco. It may be incapable of maintaining homeostasis of development of the ovule and embryo sac in a varying environment. The occurrence of developmental abnormalities should be studied in each clone intended for inclusion in traditional breeding programmes.

Seed set of ulluco (Ullucus tuberosus Loz.). Variation between clones and enhancement of seed production through the application of plant growth regulators.

SummaryUlluco, Ullucus tuberosus, is a tuber crop plant with a reduced ability to produce botanical seed. This paper discusses problems related to the botanical seed set of ulluco. Ten clones of this species were studied, and the number of seeds per plant was recorded. Enhancement of seed production was attempted by the application of three growth regulators; gibberellic acid, CCC, and a synthetic cytokinin, 6-benzylamine purine. Seed set ability was shown to vary between the clones, and the application of plant growth regulators did not enhance seed production. Variation in fruit weight was observed. Because of genetic variation between clones in their ability to set seed, it is proposed that there is a possibility of breeding ulluco using botanical seeds. However, methods for improving the yield of botanical seed need to be developed.

Seed of Ullucus tuberosus - an apomict or not.

SummaryThe paper deals with variations in enzyme pattern in the seeds of ulluco, Ullucus tuberosus. Six seeds per clone, four clones and three enzyme systems (malate dehydrogenase, glutamate oxaloacetate transaminase and 6-fosfoglucose dehydrogenase) were examined. There was variation in enzyme pattern between the seeds of a single clone. It is concluded that ulluco is not an apomictic species but a truly sexual one. Consequences of these findings for breeding programmes of ulluco are pointed out.

Pollination requirements for seed set in ulluco (Ullucus tuberosus)

SummaryPollination experiments were carried out on ulluco (Ullucus tuberosus), known as a very poor seed producer. All the clones used were diploids. The effect of self-vs. cross-pollination was studied in Experiment 1. Caged selfpollinated flowers produced no seeds. Different clones produced seeds in 0–8% of caged cross-pollinated flowers and in 5–19% of cross-pollinated non-caged flowers. Ulluco is thus basically an outbreeder.Experiment 2 studied whether seed set is limited by pollen availability. Total seed number per inflorescence number did not differ between plants treated with supplemental pollen and control plants; pollen availability thus did not limit seed set. The treated plants, however, produced more seeds in cross-pollinated than in other flowers on the plant, and the control flowers on treated plants produced fewer seeds than those on the control plants. In the treated plants, resources may have been allocated to artificially cross-pollinated flowers at the expense of other flowers. In some clones, seed set after artificial cross-pollination was at a level practicable for breeding work.