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Variation of agronomic traits of potato somaclones produced by meristem culture

Variation of agronomic traits of potato somaclones produced by meristem culture
Viive Rosenberg*, Marje Särekanno, Katrin Kotkas, Virge Vasar, Ann Ojarand Estonian Research Institute of Agriculture, Plant Biotechnological Research Centre EVIKA.
Saku, Teaduse 6A, Harjumaa, 75501, Estonia
Following a thermotherapy virus eradication procedure potato plantlets were multiplied as
meristem clones in vitro. The agronomic traits of meristem clones were compared in field
trials over three years. A total of 16 meristem clones of cultivar Agrie dzeltenie and 10 of
cultivar Juku were evaluated. There was a significant variation in the number and size of
tubers per plant amongst clones. The yield of clones of cultivar Agrie dzeltenie varied from
32.4 to 51.4 t ha-1 and for cultivar Juku from 27.4 to 33.5 and 6.1 t ha-1. Previous studies with
other varieties indicated that meristem clones might differ in starch content in tubers and
disease tolerance. It is possible to improve the agronomic traits of potato cultivars by
selecting meristem clones with suitable characteristics. This phenomenon can be used in seed
potato production programmes and in plant breeding.
Keywords: Estonia, meristem clones, potato, somaclonal variation, yield
*To whom correspondence should be addressed (E-mail:
The Republic of Estonia is one of the smallest states in the European Union with an area of 45.100 km2, and 1.4 million inhabitants. Estonia is located on the coast of the Baltic Sea in the temperate climate zone. The annual precipitation is 500-700mm and the vegetation period typically lasts 183 days, from April to September. The precipitation during vegetation period is 290-380mm. Annual sunshine hours ranges from 1600 to 1870 a year. About 50% of Estonia is covered by forest and wetlands. The area of arable land has decreased by 30% over the past 10 years to the present 870 000 ha. Intensive cultivation of potato in Estonia began in 1850. In 1952 the largest area (104 000 ha) was planted, and the highest production of 1 521 900 t was achieved in 1968. Presently potatoes is produced on ca 30 000 ha and total production is around 500 000 t. Potato varieties have been bred in Estonia for more than 80 years. There are 40 local varieties currently registered. In the past the crop was utilized as table potatoes and chips, for the production of ethanol and starch, and a remarkable amount of seed and ware potatoes was exported. Now mainly seed potato and table potato are produced for local consumption. The multiplication of potato seed material by tissue culture methods is used in Estonia since 1966. Numerous problems have been investigated, including cultivation of meristems, elimination of viruses, propagation of meristem plants, growth of the first generation seed tubers and effective utilization of seed material. Systems have been developed to eliminate virus diseases, for propagation of plants, and for growing the first generation seed tubers in field (Kotkas & Rosenberg, 1999). Previous investigations including more than 500 meristem clones from 35 potato varieties have shown that clones differ in yield capacity, dry matter content, resistance to diseases, intensity of flowering, number of stems and tubers per hill (Rosenberg et al., 2004; Rosenberg, 1995; Nielsen et al., 2002). This paper describes a project with potato meristem clones where important agronomic traits were evaluated under field conditions. Material and methods

Agrie dzeltenie, a cultivar of Latvian origin was released in 1962. It is an early to mid-
ripening table potato with very good culinary properties, susceptible to virus diseases, moderately resistant to Phytophthora infestans de Bary (potato late blight) and Streptomyces spp. (Common scab). By 1980 the variety had lost its yield potential. After virus eradication by EVIKA technology, and selection of meristem clones, this cultivar has become high-yielding again and is included in the Estonian variety-list since 1993. Juku is a new cultivar bred in Jõgeva Plant Breeding Institute in Estonia and released in 1997. It is a late variety with very good culinary properties for consumption and processing. It is tolerant to late blight and has a good resistance to Erwinia carotovora (wet rot). Meristem tissue of 0.2-0.3 mm in size was cut from apical or lateral buds of plants that had passed through 6-8 weeks of thermotherapy treatment. Usually 20-30 explants per variety were cut. The explants were cultivated on a modified Murashige-Skoog medium. The plants regenerated within 4-12 weeks. Fast-developed plants with stem-lengths of 8-12 exhibiting root development were selected. Regenerates with chlorotic or abnormal leaf or stem shape, that remained short, or did not develop any roots were eliminated from the tests. Thus a first selection was made at the primary meristem stage. The selected meristem plantlets were propagated in vitro by means of microcuttings. The ELISA-test was used for virus identification and only virus-free clones were selected for further trials. Nodal cuttings were propagated in vitro and transplanted to plastic rolls for the production of the first generation tubers. Field trials The trials were conducted in the test field of Research Centre EVIKA in North-Estonia. The first generation tubers grown in the field were used for first year trials and every following year the second, third or fourth generation tubers was used respectively. The experimental design was a randomized block with four replications and with 40 tubers per plot. The same agro-technical procedures as in commercial fields were used in the trials. The crop was fertilized with 10-10-20 NPK at 500 t ha-1. The planting date depended on weather and varied from May 8th to 25th, and the harvesting date from September 2nd to 20th. Chemical weed control was applied when needed, and the crop was ridged two to three times during the growing period. No chemical disease or pest control was applied. The potato plants were monitored during the growing period. The rate and quality of emergence, beginning and intensity of flowering and infection with late blight was evaluated. The number of tubers per plant and the weight of tubers were determined at harvesting. The results were analyzed statistically using the dispersion analysis (Fisher’s criterion, F – test). Dispersion analyses were based on the mean yield of all meristem clones. LSD was calculated at the P = 5% based on Student’s theoretical criterion, T-test (Mead et al., 1993). Results and discussion
Seasonal yield results are presented in Table 1 and 2. The average yield and number of tubers for the three years trial period are summarized in Figures 1 and 2. The climate varied from year to year and trials were conducted on a different field each year. Despite of this the average yields were more or less on the same level in all years, namely 43.2; 40.6 and 40.1 t ha-1 for cultivar Agrie dzeltenie and 32.5; 31.4 and 28.3 for cultivar Juku. Comparing the yield of each clone with the mean yield of all meristem clones it is evident that meristem clone no 163 of Agrie dzeltenie produced the highest yield in all test years. The difference between the clone with the lowest yield (no 188) and that of the highest yield (no 163) was 19.1 t ha-1 on average. Differences in yield of meristem clones have been recorded in our previous studies as well. For instance, the average yield of meristem clones of cultivar Eba was 28.5, 44.5 and 47.5 t ha-1 (Rosenberg, 1995). The yield of ten meristem clones of Estonian cultivar Piret varied from 32.9 to 38.4 t ha-1 and that of cultivar Ants from 38.1 to 51.8 t ha-1 (Rosenberg et al., 2004). Considerable variations in yield capacity between meristem clones have been found for old as well as new cultivars. The yield deviation from the mean value of cultivar Agrie dzeltenie had the same tendency in all test years, but it was more irregular for Juku. Agrie dzeltenie is an early ripening cultivar and Juku is a late cultivar. The length of vegetative period, temperature and precipitation varied from year to year. Cultivars with shorter growing periods are more stable since the formation of tubers is faster and the plant reaches maximum yield before the onset of frosts. This may explain the lower yield of the late cultivar Juku. Meristem clones of the same cultivar differ in the length of their growing periods. Cultivars as well as meristem clones express different levels of tolerance to late blight. Pesticides were not applied in the trials to enable meristem clones evaluation for late blight tolerance. In Estonia serious late blight infections occur typically in two years out of ten, with some local cultivars exhibiting excellent tolerance against the disease. In recent years modern cultivars grown elsewhere in Europe have become popular in the Baltic region. These cultivars are often more susceptible to late blight and may have contributed to the distribution of the new races and mating types of potato late blight. The disease has become a serious problem in the Baltic in recent years. Late blight resistance of meristem clones was tested in vitro where the plants were inoculated with pure culture of P. infestans as well as in field conditions. Interesting results were obtained with the cultivar Bintje known for its susceptibility to late blight (Rosenberg et al., 2004). Four meristem clones were studied and one was found to be more tolerant to late blight than the others. Tolerance of in vitro plantlets was observed in the field where less infection was observed on the veins, leaves and tubers. Late blight tolerant clones were also superior to others in terms of yield and starch content. For cultivar Ants there was no clear correlation between the susceptibility to late blight and yield, probably because Ants is relatively tolerant to late blight and the infection did not affect the yield remarkably (Rosenberg et al., 2004). Data on the number of tubers per plant are represented in Table 2, and in Figures 1 and 2. High yield did not always correlate with number of tubers per plant. For cultivar Agrie dzeltenie the average number of tubers per plant varied between 5.7 and 7.8 and the average mass per tuber varied between 81.4 and 115.0 g. Clone 163 had the highest yield, the number of tubers was 7.2 and the mass per tuber was 102.3 g. Clone 145 produced the highest number of tubers per plant (7.8) with an average mass of 81.5 g. Clone 164 with an average tuber mass of 115 g produced the smallest number of tubers. Clone 752 of cultivar Agrie dzeltenie was selected in 1987 and has demonstrated uniform canopy development, even maturation and high and stable tuber yield. The meristem clones that produced the higher yields in current study (163; 165; 99) were established in vitro in 1995. Clone no 145 were established in 1992. Meristem clones no 156 and 165 are the offspring of the same mother plant, clones 93 and 102 are also from the same mother plant. Our previous studies have shown no clear correlation between the yield capacity of a meristem clone and the characteristics of the mother plant. Offspring of the same mother plant can produce clones with high yield potential and clones with low yield potential (Rosenberg et al., 2004). The number of tubers per plant for cultivar Juku is presented in Table 2 and Figure 2. This cultivar does not produce large tubers, and tubers are not uniform, with deep-set eyes. The clones 101 and 272 have been selected for seed production. The selection criteria of meristem clones of cultivar Juku are the uniformity of tubers and the shallow eyes. The differences observed between meristem clones can be considered as somaclonal variations resulting from meristem culture. The frequency of point-mutations in meristem cultures can be higher than for intact plants. It is assumed that somaclonal variation can be increased by factors such as a high concentration of growth hormones in nutrient medium, lability of cultivar or exposure to heat treatment. The effect of the heat treatment on the frequency of somaclonal variations are presently under investigation. Somaclonal variation resulting from meristem culture has not been well documented yet. However, there are plenty of data about somaclonal variations initiated in callus culture and its utilization in potato breeding. In trials with plants regenerated from callus cultures of 14 cultivars, differences in haulm growth, tuber yield and ripening time were observed. Most of the somaclones exhibited negative traits in the early stages of development. Positive mutations were detected only on 1% of plants. The extent of variation in callus culture depended remarkably on cultivar (Thieme & Griess, 1996). Variations that occurred in callus culture have lead to new clones tolerant to late blight (Guseva & Komaletdinova, 1998). Conclusions
We have not detected the deviations from true-to-type morphological characteristics in our trials yet. However, meristem clones differ in the intensity of flowering, height of stems and in the uniformity of plants. Our experience has shown that the variability of meristem clones can serve as a valuable tool in improving the yield and other properties of potato cultivars. Acknowledgements
The experiments on which the present study was based were made within the Target Research project no 0442528s03 and grants no 6124 and 6132 of Estonian Science
GUSEVA, N., KOMALETDINOVA, F., 1998. Biotechnological methods and problems of
plant immunity. In: Biotechnology in Plant Breeding, Kaunas, 5-7. KOTKAS, K., ROSENBERG, V., 1999. Disease eradication and propagation of the initial seed potato material in Estonia. Potato Research 42, 577-583. MEAD, R., CURNOW, R. N. & HASTED, A. M., 1993. Statistical methods in agriculture and experimental biology. Second edition, Chapman and Hall, London, 70. NIELSEN, S. I., BÄNG, H., KOTKAS, K., KRISTENSEN, K., PALOUHTA, J.P:,TOLSTRUP, K., 2002. Stability of potato meristem clones. Nordic Council of Ministers, Copenhagen, 40 . ROSENBERG, V., KOTKAS, K., SÄREKANNO, M., TALVOJA, P., 2004. Research of Potato meristem clones Proceedings of EAPR Agronomy section meeting, Brasov, 2004, 109 – 119. ROSENBERG, V., 1995. Results, showing possibilities of meristem method for improving some characteristics of potato varieties. In: Current Issues in Plant Molecular and Cellular Biology. Kluwer Academic Publishers, The Netherlands, 423-426. THIEME, R., GRIESS, H., 1996. Somaklonale Variation des Krautes, der Vegetationslänge und des Ertrages bei Kartoffeln. Potato Research 39, 355-365. Table 1. The tubers yield (t ha-1) of different potato meristem clones of cultivars Agrie dzeltenie and Juku in 2000, 2001 and 2002 years dzeltenie
Table 2. The number of tubers per plant of different potato meristem clones of cultivars Agrie dzeltenie and Juku in 2000, 2001 and 2002 years Agrie dzeltenie



Decreto numero 146-86 (emitido el 27/10/1986) ley general de la administración publica (gaceta no.25088 del 29/11/1986) * - Considerando: que el creciente desarrollo de la actividad social y economica en nuestro pais, ha impuesto condiciones a la actividad estatal, que no conviene desatender. * - Considerando: que el gobierno de la republica, se ha empenado en la ejecución de los

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