CYTOMETRIC ANALYSES OF SUGAR BEET (BETA VULGARIS L.) PLANTS REGENERATED FROM UNFERTILIZED OVULES CULTURED IN VITRO

Magdalena Tomaszewska-Sowa
Department of Agricultural Biotechnology, University of Science and Technology, Bydgoszcz, Poland

ABSTRACT

The results of cytometric analyses of 413 sugar beet plants obtained by regeneration from unfertilized ovules in vitro are presented. The formation of shoots was induced on different MS media varying in the contents and composition of phytohormones. The majority of plants (353) were analyzed only once during the in vitro stage and 60 – twice, during the in vitro stage and also after planting in the field. The analyses of the plants during the in vitro stage revealed that 32.7% were haploids, 45.8% – diploids and 21.5% – mixoploids. The highest percentage of haploid plants was achieved using media containing 4.4 µM BAP and 0.44 µM NAA and 2.2 µM BAP. The repeated analyses of plants (during the in vitro stage and also after planting) show during the in vitro stage the population consisting of 55% haploids and 45% diploids. Repeated tests demonstrated that during the period between the tests some of the haploids underwent a spontaneous diploidization since their percentage was reduced from 55% to 30%. This process was observed only for the plants derived from the media containing kinetin with the addition of NAA or 2.4-D. Observations of regenerants pointed to a vast diversity of phenotypes between haploids and diploids, which can be regarded to as a characteristic feature of plants with the DNA level of 1C and 2C.

Key words: diploids plants, flow cytometry, growth regulators, haploid plants, in vitro culture, ovules, ploidy, sugar beet.

INTRODUCTION

Sugar beet (Beta vulgaris L.) is an important economic crop and hence many efforts to improve sugar beet varieties. Traditional breeding methods, which proved to be too expensive and time consuming, can be supplemented with modern biotechnological methods, e.g. the latest achievements of in vitro culture.

Research procedures performed in numerous laboratories aim at improving and optimizing sugar beet regeneration methods, introducing desired features by using transformation techniques and production of homozygous lines from heterozygous material just over one generation. The most frequently used methods of obtaining haploids are the ones applying anther culture or isolated microspores. However, numerous attempts to obtain Beta vulgaris haploids with the induced androgenesis method did not succeed [15,25]. Haploid sugar beet plants were obtained only by in vitro cultures of unfertilized ovules and ovaries [2,4,10,16,23,28]. Successive studies performed by numerous laboratories concentrated on optimizing culture conditions to increase the method efficiency and stability [12,13,14]. The experiments presented here aimed at the improvement of plant regeneration systems from sugar beet ovules by the application of a two-phase, i.e. liquid-solid culture method. Research hypothesis assumed in obtaining from unfertilized ovules cultured in vitro the greatest number of haploid plants. The efficiency was evaluated by measurements of DNA content in regenerants by flow cytometry and assessment of the haploids-to-diploids ratio.

MATERIALS AND METHODS

Ovule explants of fertile, diploid and monogerm sugar beet plants (Beta vulgaris L.) (line 0170, kindly provided by the Polish company Kutnowska Hodowla Buraka Cukrowego) cultivated under field conditions in the Plant Breeding and Acclimatization Institute in Bydgoszcz, were used for the experiments. The unfertilized ovules for in vitro cultures were taken from three-fourths closed flower buds situated above flowers at the stage of anthesis (Fig. 1A). At this stage an embryo sac is already functionally differentiated; it contains egg apparatus, with the egg cell and two synergids, polar nuclei and antipods. Ovules were cultured in MS [24] liquid medium supplemented with 4.4 µM BAP (Fig. 1B). The ovules were cultured for 12 weeks. Then groups of 60 differentiating explants (Fig. 2A) were transferred to the modified MS media for shoot induction and multiplication (Fig. 2B), (Table 1).

The regenerated shoots were isolated and transferred onto fresh media. After 12 weeks of growth the shoots were excised and transferred from the proliferation to MS rooting medium supplemented with 14.8 µM IBA and 0.049 µM 2iP (Fig. 2C). All cultures were incubated at 24°C under cool white fluorescent light at 2000 lux under an 16 h light/8h dark cycle. Rooted plantlets (Fig. 2D) were transplanted into pots containing sterile soil and perlite (Fig. 2E) and kept under a plastic cover until new leaves developed. After several weeks of acclimatization in the greenhouse, the plants were planted in the field (Fig. 2F). The experiment was performed in the Plant Breeding and Acclimatization Institute in Bydgoszcz over 2005–2007.

Flow cytometry was used for the determination of the regenerated plants ploidy level. Cell nuclei were extracted from the youngest leaves of 413 plants, 353 of them being examined only once during the in vitro stage, and 60 – twice during the in vitro stage and after planting in the field. Fragments of tissue were chopped with a sharp razor blade in a Petri dish in the presence of lysis buffer containing: 100 mM Tris, 2mM MgSO₄, 85 mM NaCl, 1 ml Triton X-100 and 0.001% fluorochrome dye: 4.6-diaminoino-2-phenylindol (DAPI). After chopping, 1 ml of buffer was added and the mixture was filtered through a 50-µm nylon filter. The DNA content was measured in a Partec CA II flow cytometer (Partec, Münster, Germany). The control material, constituting a standard DNA concentration level for beet cells, was derived from leaves of the Arthur diploid variety. Analyses were performed in the Laboratory of Molecular Biology and Cytometry of the Department of Plant Genetics and Biotechnology, the University of Technology and Life Sciences in Bydgoszcz.

RESULTS AND DISCUSSION

Cytometric evaluation in order to establish the ploidy level of regenerated sugar beet plants revealed that of the 413 plants examined during in vitro stage, 32.7% were haploids, 45.8% were diploids and 21.5% – mixoploids (Fig. 3). The highest level of haploid plants was obtained with media containing 4.4 µM BAP and 0.44 µM NAA (79.4%) and 2.2 µM BAP alone (52.8%). The biggest number of diploids was regenerated with media involving 2.2 µM KIN and 0.44 µM NAA (87.9%), and 2.2 µM KIN and 0.44 µM 2.4-D (80.9%). The remaining plants obtained with the latter phytohormones were characterized by a haploid chromosome number, whereas mixoploids did not occur (Table 2). The majority of sugar beet plants regenerated by Gośka [11] consisted of haploids (77.7–100%). Diploids accounted for 0 to 11.1% and mixploids for 0 to 14.3%. Similar results were presented by Bossoutrot and Hosemans [2], Doctrinal et al. [5] and Lux et al. [23]. These high ploidy level fluctuations as well as the occurrence of mixoploids, observed among sugar beet regenerants [17], may have resulted from the presence of growth regulators. Auxins and cytokinins present in medium affect the process of mitosis during the differentiation of the callus as well as during the formation of the shoots. The reasons for obtaining such a high percentage of haploids as reported by Gośka [11] should be related to low auxin and cytokinin concentrations in the initial medium. Evans and Gamborg [7] demonstrated that tobacco plants (Nicotiana ssp.) regenerated from media with the highest concentrations of phytohormones were characterized by higher ploidy levels. The regeneration of ovules described in this experiment occurred mainly by indirect organogenesis. Callus is the tissue taking part in this process and it is the tissue which undergoes the highest variability when it comes to DNA concentration. The presence of diploids and mixoploids among the regenerants from ovules may as well result from endoreduplication or from the formation of meristems during organogenesis from cells of different ploidy. Rearrangements within the genome also occur as a result of cell cycle disturbances. These effects may result from the activities performed by the components within the media, mainly phytohormones, used at subsequent culture stages, from the genotype characteristics of the mother plants (the plants from which the explants were isolated), the differentiation level of the explant and its age, the method of regeneration and the numbers of transfers to fresh media [9,20].

The genetic stability of the regenerates was evaluated for 60 plants based on repeated analyses of the ploidy levels. In the population examined the number of haploids in the in vitro stage accounted for 55%, diploids – for 45%. Repeated measurements showed that during the period (five months) between the two evaluations some individuals characterized by a haploid genome underwent a spontaneous diploidization since the number of haploids within the population decreased from 55% to 30% (Table 3). Worthy of note, the change occurred only in plants regenerated on media containing kinetin in three different concentrations, combined with NAA and 2.4-D. Within this group 15 of 16 plants (93.8%) changed the DNA concentration level from haploid to diploid. Considering both stages of development – in vitro and ex vitro – in case kinetin was applied, almost exclusively diploid plants (22 out of 23examined, i.e. 95.6%) were generated. In case the cytokinin BAP was added to the medium as the only growth regulator as well as with the additional use of 0.44 µM NAA, the haploids created did not undergo diploidization. The spontaneous diploidization process obviously results from factors in the culture medium, probably growth regulators (auxins added in large quantities to the rooting medium), but also the number of subcultures and culture length contributed to diploidization. The spontaneous diploidization process, occurring during the regeneration of sugar beet plants from ovules, was also reported by Gośka [11]. This author obtained, depending on the genotype, between 2 and 10% diploid from originally haploid plants. The heterogeneity of the DNA level in hypocotyl and cotyledon cells was noted in the in vitro plant material. The polysomatic effect may lead to the regeneration of polyploid or mixoploid plants [27]. Flow cytometry helped discovering polysomatic cells in sugar beet organs: hypocotyls [17], petioles, leaves [22,30] and cotyledons [18]. One may refer the changes in the genome size to the endoreduplication process, which leads to raising the DNA content without changing the number of the chromosomes. In case of plants with a small genome, some cells undergo a spontaneous poliplodization [26]. In case of other varieties this process is most often induced by the components of the medium. Phytohormones present in the medium influence the mitotic cell division during the differentiation and regeneration and thus stimulate the formation of polyploid cells. Changes like this have been described for Arabidopsis [8], corn [21] and tomato [1].

The phenotypic variation of haploids and diploids was observed during in vitro culture (Fig. 4), as well as during growth in the field (Fig. 5). Haploid plants were characterized by narrower, more elongated leaf blades, longer petioles and smaller but longer roots when compared to diploids. These plants were more difficult to induce root formation and acclimatization to ex vitro conditions. They were also more susceptible to harsh conditions, experienced during field growth, and their reaction to winter storage was poor. Warzecha and Śliwińska [29] used those distinct phenotype differences between haploids and diploids in their research, performing a phenotypic analysis of corn seeds (Zea mays L.) effectively, which was based on morphological markers of the endosperm and embryo, marking accurately 80% of their seeds as haploid. The variability induced by in vitro culture may concern not only morphological differences but also those of physiological, cytological and molecular nature. Morphological changes mainly involve the habit of a plant, its colour or leaf shape as well as the dry matter content [19]. Physiological modifications mainly concern the plant reaction to biotic or abiotic stress, e.g. oxidative stress or tolerance to salinity [3,6].

The innovative aspect of the present experiments was the application of a two-phase culture: during the first stage regeneration was performed using a liquid medium supplemented with 4.4 µM BAP, at the following step a solid media with different hormone compositions were used. Differentiating tissues can be successively extracted and transferred onto regenerative media, eliminating one of the major limitations, which is the necessity to obtain ovules only during the short period of blooming, which, in turn, increases the amount of material ready for analysis when compared with the material extracted from single-ovule cultures. In addition, the ovules can be stored in liquid culture for months, prolonging the period for analyses. The approach to a cytometric evaluation of the ploidy level of the regenerates allows a very efficient identification of haploid plants while using only a small amount of plant tissue, thus eliminating the necessity of desterilization of the culture.

CONCLUSIONS

1. As the cytometric analysis demonstrated, over 30% of the plants regenerated by in vitro gynogenesis were haploid.

2. 25% of haploid plants in field conditions have undergone a process of spontaneous diploidization.

3. Phenotypic variation between haploid and diploid plants was found during in vitro culture and growth in the field.

4. An innovative method of a two-phase culture significantly increased the efficiency and effectiveness of in vitro culture of sugar beet (Beta vulgaris L.) from unfertilized ovules.

ACKNOWLEDGEMENTS

I would like to thank Professor Anna Majewska-Sawka, VitroGen Bydgoszcz, for her assistance in developing this publication, Professor Elwira Śliwińska, Institute of Plant Genetics and Biotechnology, University of Technology and Life Sciences in Bydgoszcz, for her assistance with cytometric analyses and Professor Norbert Keutgen, Department of Plant Physiology and Basics of Plant Biotechnology, University of Technology and Life Sciences in Bydgoszcz, for their advice on the development of the manuscript.

REFERENCES

1. Bino R.J., de Vries J.N., Kraak H.L., van Pijlen J.G., 1992. Flow cytometric determination of nuclear replication stages in tomato seeds during priming and germination. Ann. Bot. 69, 231-236.

2. Bossoutrot D., Hosemans D., 1985. Gynogenesis in Beta vulgaris L.: From in vitro culture of unpollinated ovules to the production of doubled haploid plants in soil. Plant Cell Rep. 4, 300-303.

3. Cassells A.C., Curry R.F., 2001. Oxidative stress and physiological, epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers. Plant Cell Tiss. Org. Cult. 64, 145-157.

4. D'Halluin K., Keimer B., 1986. Production of haploid sugar beets (Beta vulgaris L.) by ovule culture. [In:] Genetic Manipulation in Plant Breeding, W. Odenbach, O. Schieder (eds), de Gruyter Berlin, New York, 307-309.

5. Doctrinal M., Sangwan R.S., Sangwan-Norreel B.S., 1989. In vitro gynogenesis in Beta vulgaris L.: effects of plant growth regulators, temperature, genotypes and season. Plant Cell Tiss. Org. Cult. 17, 1-12.

6. Duncan R.R., 1997. Tissue culture-induced variation and crop improvement. Adv. Agron. 58, 201-240.

7. Evans D.A., Gamborg O.L., 1982. Chromosome stability of cell suspension cultures of Nicotiana ssp. Plant Cell Rep. 1, 104-107.

8. Galbraith D., Harkins K.R., Knapp S., 1991. Systemic endoploidy in Arabidopsis thaliana. Plant Physiol. 96, 985-986.

9. George E.F., 1993. Plant Propagation by Tissue Culture. Part 1. The Technology. Exegetics Ltd. Basingstoke, UK.

10. Gośka M., 1985. Sugar beet haploids obtained in the in vitro culture. Bull. Pol. Acad. Sci. Biol. Sci. 33, 31-33.

11. Gośka M., 1997. Haploidy i podwojone haploidy buraka cukrowego (Beta vulgaris L.) oraz możliwości ich wykorzystania w hodowli [Haploid and doubled haploid sugar beet (Beta vulgaris L.) and their use in breeding]. Monografie i Rozprawy Naukowe IHAR 2 [in Polish].

12. Gürel S., Gürel E., Kaya Z., 2000. Doubled haploid plant production from unpollinated ovules of sugar beet (Beta vulgaris L.). Plant Cell Rep. 19, 1155-1159.

13. Hansen A., Gertz A., Joersbo M., Andersen S.B., 1995. Short duration colchicine treatment for in vitro chromosome doubling during ovule culture of Beta vulgaris L. Plant Breed. 114, 515-519.

14. Hansen A., Gertz A., Joersbo M., Andersen S.B., 1998. Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture. Euphytica 101, 231-237.

15. Herrmann L., Lux H., 1988. Antherenkultur bei Zuckerrüben, Beta vulgaris var. altissima Döll. Arch [Kultury pylników buraka cukrowego Beta vulgaris var. altissima Döll]. Züchtungsforsch 18, 375-383 [in German].

16. Hosemans D., Bossoutrot D., 1983. Induction of haploid plants from in vitro culture of unpollinated beet ovules (Beta vulgaris L.). Z. Pflanzenzchtg 91, 74-77.

17. Jacq B., Tetu T., Sangwan R.S., De Laat A., Sangwan-Norreel B.S., 1992. Plant regeneration from sugarbeet (Beta vulgaris L.) hypocotyls cultured in vitro and flow cytometric nuclear DNA analysis of regenerants. Plant Cell Rep. 11, 329-333.

18. Jacq B., Tetu T., Sangwan R.S., De Laat A., Sangwan-Norreel B.S., 1993. Efficient production of uniform plants from cotyledon explants of sugar beet (Beta vulgaris L.). Plant Breed. 110, 185-191.

19. Joyce S.M., Cassells A.C., 2002. Variation in potato microplant morphology in vitro and DNA methylation. Plant Cell Tiss. Org. Cult. 70, 125-137.

20. Karp A., 1995. On the current somaclonal variation as a tool for crop improvement. Euphitica 85, 295-302.

21. Kowles R.V., Phillips R.L., 1985. DNA amplification patterns in maize endosperm nuclei during kernel development. Proc. Natl. Acad. Sci. USA 82, 7010-7014.

22. Krens F.A., Jamar D., 1989. The role of explant source and culture conditions on callus induction and shoot regeneration in sugar beet (Beta vulgaris L.). J. Plant Physiol. 134, 651-655.

23. Lux H., Herrmann L., Wetzel C., 1990. Production of haploid sugar beet (Beta vulgaris L.) by culturing unpollinated ovules. Plant Breed. 104, 177-183.

24. Murashige T., Skoog F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473-497.

25. Rogozińska J.H., Gośka M., 1976. Growth and differentiation of sugar beet anthers and callus. Bull. Acad. Pol. Sci. Biol. 24, 57-60.

26. Schubert V., Klatte M., Pecinka A., Meister A., Jasenecakova Z., Schubert I., 2005. Sister chromatidas are often incompletely cohesed in meristematic and endopolyploid interphase nuclei of Arabidopsis thaliana. Genetics 12, 1-32.

27. Śliwińska E., Zimny J., Drozdowska L., 2003. Niestabilność poziomu ploidalności podczas regeneracji transformowanego i nietransformowanego tytoniu (Nicotiana tabacum L.) [Ploidy level instability during regeneration of transformed and untransformed tobacco plants (Nicotiana tabacum L.)]. Biotechnologia 3, 260-266 [in Polish].

28. Van Geyt J., Spockmann Jr. G.J., Halluin K.D., Jacobs M., 1987. In vitro induction of haploid plants from unpollinated ovules and ovaries of the sugar beet (Beta vulgaris). Theor. Appl. Genet. 73, 920-925.

29. Warzecha E., Śliwińska E., 2006. Indukcja haploidów matecznych u kukurydzy. Haploidy i linie podwojonych haploidów w genetyce i hodowli roślin [Induction of maternal haploids in maize. Haploid and doubled haploid lines in plant breeding and genetics]. Instytut Genetyki Roślin PAN Poznań [in Polish].

30. Zhong Z., Smith H.G., Thomas T.H., 1993. Micropropagation of wild beet (Beta maritima) from inflorescence pieces. Plant Growth Regul. 12, 53-57.

Accepted for print: 02.11.2010

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