Callus tissue was induced on root tips of in vitro cultured seedlings of Allium commutatum Guss. on MS medium supplemented with 3 % sucrose, 0.8 % agar, 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.6 μM kinetin. Pieces of developed calli were transferred to MS medium with different concentrations of 2,4-D (0.02, 0.05 and 0.1 μM) and kinetin (0.02, 0.1 and 0.5 μM) or without them. After five weeks of cultivation, callus proliferation and differentiation as well as adventitious shoot and root regeneration were analysed. The best callus proliferation and adventitious root induction were achieved on MS medium containing 0.1 and 0.5 μM kinetin; addition of 2,4-D had no significant influence. Higher concentrations of kinetin also favoured higher incidence of meristemoids. Adventitious shoot development was noticed only on three media tested

Branka Pevalek-Kozlina

Marija Edita Šolić

Marija Vujević

Mirjana Pavlica

English

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Acta Bot. Croat. 58, 57-64, 1999 CODEN: ABCRA 25 ISSN 0365-0588Dedicated to Prof. dr. Mercedes Wrischer on the occasion of her 70th birthday.UDC 581.19:582.57SHOOT AND ROOT REGENERATION FROM CALLUS TISSUE OF Allium commutatum Cuss.Marija V ujević1*, Branka Pevalek-Kozlina1, Mirjana Pavi.ica2, Marija Edita Šolić31 University of Zagreb, Faculty of Science, Department o f Botany, Rooseveltov trg 6, 10000 Zagreb, Croatia2 University of Zagreb, Faculty of Science, Department o f Molecular Biology, Rooseveltov trg 6, 10000 Zagreb, Croatia3 Institute “Planina i More”, Franjevački Put 1, 28300 Makarska, CroatiaCallus tissue was induced on root tips of in vitro cultured seedlings of Allium commutatum Guss. on MS medium supplemented with 3 % sucrose, 0.8 % agar, 4.5 pM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.6 pM kinetin. Pieces of developed calli were transferred to MS medium with differ­ent concentrations of 2,4-D (0.02, 0.05 and 0.1 pM) and kinetin (0.02, 0.1 and 0.5 pM) or without them. After five weeks of cultivation, callus proliferation and differentiation as well as adventitious shoot and root regeneration were analysed. The best callus proliferation and adventitious root induction were achieved on MS medium containing 0.1 and 0.5 pM kinetin; addition of 2,4- -D had no significant influence. Higher concentrations of kinetin also favoured higher incidence of meristemoids. Adventitious shoot development was noticed only on three media tested.Key words: Allium commutatum, callus culture, differentiation, regenerationIntroductionThe genus Allium comprises up to 700 species, including some that are economi­cally important such as A. cepa L, A. sativum L„ A. ampeloprasum L. and A. poirum* Corresponding author: E-mail: mvujevic@zg.biol.pmf.hrACTA BOT. CROAT. VOL. 58, 1999 57VujEVic M., Pevalek-Kozlina B., Pavlica M., Solió M. E.L. Some of them are important medicinal plants due to their antibiotic, antitumor and antithrombic effects on animal cells (Ayabe et al. 1995), while some have frequ­ently been used as bioassay organisms (Pavlica et al. 1998, Rank and N ielsen 1997).A. commutation Guss. is a typical Mediteranean species. So far it has been used in cytogenetic investigations of callus cultures (Pavlica and Pevalek-Koz­lina 1999) and karyological investigations of natural populations (Besendorfer et al. 1997). The aim of this study was to induce callus tissue formation and to investigate the influence of 2,4-D and kinetin on regeneration ability in A. com- mutatum Guss.Material and methodsRoot tips of in vitro cultivated seedlings of A. commutatum were used as ini­tial explants for initiation of callus culture. The seedlings were grown from seeds collected in their natural habitat (Makarska, Croatia).The sterilisation of seeds was carried out with 2 % (w/v) water solution of the chlorine product Izosan-G (99 % sodium dichloroisocyanurate dihydrate, Pliva, Zagreb) for 5 min. After three rinses in sterile distilled water (5 min each), seeds we­re treated with 6 % solution of hydrogen peroxide for 5 min. After three washes in sterile distilled water (5 min each), the seeds were inoculated in test tubes fil­led with MS medium (Murashige and Skoog 1962) with 3 % sucrose and 0.8 % agar.After two weeks, root tips (up to 0.3 cm long) were separated from seedlings and used as initial explants for callus induction on MS medium containing 4.5 pM 2,4-D and 4.6 pM kinetin.Pieces of developed calli (0.5 x 0.5 x 0.5 cm) were transferred to MS medi­um supplemented with different concentrations of 2,4-D (0.02, 0.05 and 0.1 pM) and kinetin (0.02, 0.1 and 0,5 pM) or without them. The pH value of all media tested was adjusted to 5.8 before autoclaving at 118 kPa and 120 °C for 15 minu­tes. Twelve explants per each treatment were inoculated.Ail cultures were incubated at 20 ± 2 °C under a 16 hour photoperiod (40 W fluorescent light, 80 pE~2s-1)' After five weeks in culture the increase of fresh callus weight was estimated. Callus morphology as well as adventitious shoot and root regeneration were examined as well.Samples of calli were also used for histological analysis. They were fixed in FAA (formalin, glacial acetic acid, 70 % ethanol, 1:1:18) and dehydrated in graded ethanol series (70 %, 80 %, 96 %), a mixture of ethanol and buthanol (2:1), a mix­ture of ethanol and buthanol (1:2) and buthanol. Afterwards, the callus was embed­ded in paraffin, then cut into 10-pm sections on a rotary microtom. The sections were stained with 0.05 % toluidine blue (10 seconds) and analysed under a light microscope. At least 100 sections per treatment were screened (Gerlach 1977).Results and discussion Seed sterilisation and germinationThe sterilisation procedure was satisfactory. The percentage of sterile cul­tures was 92.2 %. All inoculated seeds germinated.58 ACTA BOT. CROAT. VOL. 58. 1999DIFFERENTIATION AND REGENERATION IN ALLIUM COMMUTATUMCallus inductionSegments of root tips (0.3 cm long) were cut off and inoculated on MS medi­um supplemented with 4.5 pM 2,4-D and 4.6 pM kinetin. The development of callus tissue was noticed 7-10 days after inoculation in 92.2 % of explants. After 4-weeks incubation pieces of calli were transferred to MS medium supplement­ed with 15 different concentrations and combinations of 2,4-D and kinetin or without them (as shown in Tab. 1).Tab. I. The effect of growth regulators on adventitious organ induction in callus culture of Allium commutatum. Basal medium: MS with 3 % sucrose and 0.8 % agar (esti­mated after 5 weeks in culture).Growthregulator2,4-D Kinetin (pM) (pM)Callus TissueAdventitious shoots* Adventitious roots*0 <10 >10 0 <10 >100 0 91.7 8.3 0 50 16.7 33.30.02 0 100 0 0 63.6 9.1 27.30.05 0 100 0 0 58.4 25 16.60.1 0 100 0 0 58.4 33.3 8.30 0.02 100 0 0 66.7 25 8.30.02 0.02 100 0 0 25 50 250.05 0.02 100 0 0 58.4 8.3 33.30.1 0.02 91.7 8.3 0 25 8.3 66.70 0.1 100 0 0 33.3 0 66.70.02 0.1 100 0 0 0 40 600.05 0.1 91.7 8.3 0 0 12.5 87.50.1 0.1 100 0 0 9.1 27.3 63.60 0.5 100 0 0 33.4 8.3 58.30.02 0.5 100 0 0 41.6 41.7 8.30.05 0.5 100 0 0 50 0 500.1 0.5 100 0 0 16.7 50 33.3* The data are shown as percentage of callus cultures with induced adventitious organsCallus proliferationCallus proliferation was relatively slow and it was strongly affected by growth regulators, especially by kinetin (Fig.l). Hansen et al. (1995) reported typical slow growth of A. cepa callus also. The callus growth was better when higher concentrations of kinetin were added to the basal medium. The highest increase of callus fresh weight was observed on a medium supplemented with 0.1 pM kinetin. Callus development on MS medium with 0.02 pM or withoutACTA BOT. CROAT. VOL. 58, 1999 59V ujevió M., Pevalek-Kozi.ina B., Pavuca M , Solió M. E.kinetin was very poor. Although 2,4-D was important for callus induction (Yamada et al. 1971, Novak et al. 1986), it had no major influence on callus pro­liferation.2,4-D (pM)Fig. I . Increase of callus fresh weight estimated after five weeks of cultivation on media supplemented with different concentrations and combinations of 2,4-D and kinetin or without them.Fig. 2. A, Adventitious shoots and roots developed in callus tissue of Allium commuta- tum on MS medium with 0.1 pM 2,4-D and 0.02 pM kinetin; B-H, Sections through callus tissue cultivated on MS medium with: B, 0.1 pM 2,4-D and kinetin with a well developed meristem; C-D, 0.1 pM 2,4-D and 0.5 pM kinetin with peripherally located meristematic region and central undifferentiated parenchy­mal cells (C) and tracheidal element formation (D); E, 0.02 pM 2,4-D and kinetin with procambial strands between differentiated tracheidal elements; F, 0.05 pM 2,4-D with tracheidal elements arranged in rows; G, 0.2 pM 2,4-D with longitu­dinal and transversal sections through adventitious roots with primitive vascular bundles; H, 0.1 pM 2,4-D and 0.02 pM kinetin with transversal sections through adventitious roots with developed vascular bundles. Pictures taken after 4 weeks in culture. (Bar = 0.1 mm)S -  adventitious shoot R -  adventitious rootM -  meristematic region P -  parenchymatic cellsPS -  procambial strand T -  tracheidal elementsVB -  vascular bundles60 ACTA BOT. CROAT. VOL. 58. 1999DIFFERENTIATION AND REGENERATION IN ALLIUM COMMUTATUMACTA BOT. CROAT. VOL. 58, 1999 61V ujevic M., Pevalek-Kozlina B., Pavlica M., Solic M. E.Callus morphologyAfter five weeks of cultivation on all media tested, A. commutatum callus tis­sue remained predominantly yellowish, slimy, clodded and loose. The callus tis­sue developed on media with higher concentrations of growth regulators was more compact. Partial callus browning was noticed on the medium with 0.5 pM kinetin and 0.1 pM 2,4-D. Media with 0.02 pM kinetin or without kinetin favoured the development of whitish coloured callus tissue. Appearance of greenish coloured callus was noticed on media supplemented with 0.02 and 0.1 pM kinetin, especially when combined with 0.02 pM and 0.05 pM 2,4-D.Shoot and root regenerationAdventitious roots and shoots were regenerated from callus tissue cultivated on MS medium without plant growth regulators (Tab. 1, Fig. 2.A). Addition of 2,4-D in combination with 0.02 pM or without kinetin decreased the number of regenerated adventitious roots. The promotive effect of kinetin was observed when it was added in higher concentrations (0.1 and 0.5 pM). Some of adventi­tious roots were green in their basal part. The synthesis of chlorophyll in basal parts of roots has already been reported by Fridborg (1971).Development of adventitious shoots was observed only on three media tested (Tab. 1). Dunstan and Short (1978), Phillips and Luteyn (1983) and Shahin and Kaneko (1986) have also reported low incidence of adventitious shoot deve­lopment from callus in the genus Allium.Shoot and root regeneration could be improved by supplementing the nutri­ent medium with N6-(2-isopentenyl)adenine (2iP) (Tanikawa et al. 1998) or picloram (Phillips and Luteyn 1983) as well as with some natural products like adenine, caseine hydrolyzate, coconut milk or yeast extract (Ayabe et al. 1995). Anyway, one should bear in mind the significant karyological variability in cal­lus cultures (Novak 1990; Seo et al 1995, 1996) before regenerating plants via the callus phase.Histological analysisHistological analysis of callus tissue revealed the presence of parenchyma­tous ground tissue on all media tested (Fig. 2.C). Some parenchymal cell clusters were surrounded with tracheidal elements.Individual tracheidal elements (Fig. 2.D) as well as tracheidal elements arranged in rows (Fig. 2.F) could be observed on all media tested. In callus tis­sue cultivated on media containing 2,4 -D, tracheidal elements were arranged in procambial strands (Fig. 2.E) or vascular bundles (Figs. 2.G, H)The formation of meristemoids (Fig. 2.B) from which adventitious roots and shoots will be differentiated was observed on MS medium supplemented with 0.1 pM kinetin. Meristematic cell regions were also found on media supple­mented with 0.02 pM kinetin in combination with 0.02 pM and 0.1 pM 2,4-D.Root primordia and well differentiated roots (Fig. 2.H) were observed in all callus tissues investigated. They were numerous on all media tested except on medium without 2,4-D and medium supplemented with 0.5 pM kinetin.62 ACTA BOT. CROAT. VOL. 58, 1999DIFFERENTIATION AND REGENERATION IN ALLIUM COMMUTATUMConclusionRoot tips of 4-weeks old Allium commutatum Guss. seedlings grown in vitro were used as initial explants for callus tissue induction on MS medium supple­mented with 4.5 pM 2,4-D and 4.6 pM kinetin. Callus tissue was induced on 92.2 % of explants. Developed calli were transferred to MS medium supple­mented with different concentrations and combinations of 2,4-D and kinetin or without them.Higher concentrations of kinetin (0.1 and 0.5 pM) had a stimulating effect on fresh weight increase as well as on adventitious root induction. Histological analysis showed a high incidence of meristemoids in calli grown on MS medium with higher concentrations of kinetin.On the other hand, the addition of 2,4-D had no major influence on callus proliferation and differentiation.AcknowledgementThe research was supported by the Scietific Research Council of the Republic of Croatia (project No. 119116). The authors would like to thank Dr. Sibila Jelaska for providing the use of the Plant Tissue Culture Laboratory and also to Miss Dubravka Tianic and Mrs Ana-Marija Boljkovac for excellence technical assistance.ReferencesAyabe, M. Taniguchi, K., Sumi, S., 1995: Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.). Plant Cell Rep. 15, 17-21.Besendorfer V., Samardžija, M., Bušić M., Šolić M.-E., Papeš D., 1997: Distribution of heterochromatin and location of nucleolar organizing region of Allium commutatum Guss. Period. Biol. 99, 415-421.Dunstan, D. I., Short, K. C., 1978: Shoot production from onion callus tissue cultures. Sci. Hort. 9, 99-110.Fridborg, G., 1971: Growth and organogenesis in tissue culture of Allium cepa var. proliferum. Physiol. Plant. 25, 436-440.Gerlach, D., 1977: Botanishe Mikrotechnik, Georg Thieme Verlag, Stuttgart.Hansen, E. E„ Hubstenberger, J. F., Phillips, G. C., 1995: Regeneration of shoots from cell suspension-derived protoplasts of Allium cepa. Plant Cell Rep. 15, 8-11.Murashige, T„ Skoog, F., 1962: A revised medium for rapid growth and bios- say with tobacco tissue culture. Physiol. Plant. 15, 473-497.Novak, F. J., 1990: Allium tissue culture. In: Rabinowitch, H. D., Brewster, J. L. (eds.), Onions and allied crops, Vol. 1, 234-250. CRC Press, Inc., Boca Raton, Florida.Novak, F. J., Havel, L., Dolezel, J., 1986: Onion, garlic and leek (Allium species). In: Bajaj, Y. P. S. (ed.), Biotechnology in agriculture and forestry, Vol. 2, Crops 1, 387^108. Springer Verlag, Berlin.ACTA BOT. CROAT. VOL. 58, 1999 63V ujevic M„ P evalek-K ozlina B., Pavuca M„ Solió M. E.Pavlica, M., Vasilevsica, J. Papes, D., 1998: Genotoxicity of pentachlorophenol revealed by Allium chromosome aberration assay. Acta Biol. Cracoviensia Ser. Botanica 40, 85-90.Pavlica, M„ Pevalek-Kozlina, B., 1999: Cytological changes in callus cultures of Allium commutatum Guss. Phyton (in press).Phillips, G. S., Luteyn, K. J., 1983. Effects of picloram and other auxins on onion tissue cultures. J. Amer. Soc. Hort. Sci. 108, 948-953.Ra.nk, J., N ielsen, H., 1997: Screening of toxicity and genotoxicity in waste water by the use of the Allium test. Hereditas 121: 249-254.Seo, B. B„ Lee, S. H„ Pak, J. H„ Kim, I. S., Song, S. D., 1995: Karyological analysis of somaclonal variation in callus cells of Allium victoralis var. platy- phyllum. Kor. J. Plant Biol. 38, 321-328.Seo, B. B., Lee, S. H., Pak, J. H., K im, I. S., Song, S. D., 1996: Karyological variation of callus-derived regenerants in Allium victoralis var. platyphyllum. Kor. J. Plant Biol. 38, 321-328.Shahin, A. E., Kaneko, K., 1986: Somatic embryogenesis and plant regenera­tion from callus cultures of non bulbing onions. Hort. Sci. 21, 294-295.Tanikawa, T., Takagi, M., Ichii, M., 1998: Varietal differences in plant regener­ation from solid and suspension cultures in onion (Allium cepa L.). J. Japan. Soc. Hort. Sci. 67, 856-861.Yamada, Y., Yasuda, T., Koge, M., Sekiya, J., 1971: The interrelationship of 2,4-dichlorophenoxyacetic acid with molecular components of the cell dur­ing callus induction. Agr. Biol. Chem. 35, 99-104.64 ACTA BOT. CROAT. VOL. 58. 1999

Shoot and root regeneration from callus tissue of Allium commutatum Guss.

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