Keywords: cactuses, vitroculture, hyperhydria

Vol. 19, issue 2, 9, pp.351-357 THE INITIATION OF ECHINOCACTUS MIHANOVICHII, ECHINOPSIS CHAMAECEREUS F. LUTEA AND AYLOSTERA HELIOSA VITROCULTURES Teodora Iuliana VIDICAN 1 *, Dorina CACHIŢĂ-COSMA 2, J u lieta-emilia ROMOCEA 1 1 University of Oradea, Romania 2 Vasile Goldis Western University, Arad, Romania ABSTRACT. The multiplication of cacti is difficult, especially in chlorophyllien-deficient species. To facilitate the multiplication of cacti species: Echinocactus mihanovichii (red cactus) Echinopsis chamaecereus f. lutea (yellow cactus) and Aylostera heliosa (green cactus) by vitrotechniques, we used spheroid buds as explants, taken from adult stems. This explants were inoculated on a basic medium culture with Murashige-Skoog (1962) macroelements, end Fe EDTA, Heller (1953), end microelements. Added in the medium were the vitamins: HCl thiamine, HCl pyridoxine and nicotinic acid, each 1 mg / l, 1mg / l meso-inositol, to which - depending on the organized experimental variant growth regulators were added, such as: 1 mg / l benzyladenine (BA), or 1 mg / l β-indolil-butyric acid (AIB), or a mixture - in equal parts - among them. The vitrocultures evolution was observed for a period of 9 days. The reaction of explants cultivated in vitro was different depending on the species with which was worked and on the nature of growth regulator present in the culture medium. Finally, inoculums of Echinocactus mihanovichii and those of Echinopsis chamaecereus f. lutea showed a caulogenesis phenomena; in time, the initial explant presented necrosis. Aylostera heliosa explants were better adapted to in vitro micropropagation, but they generated callus, largely hyperhydric. The largest morphogenesis, in general, has been manifested in the growing medium with a mixture of BA and AIB, 1mg / l of each. In general, risogenesis was absent. Keywords: cactuses, vitroculture, hyperhydria INTRODUCTION In the plant and flower world, cactuses occupy a special place, having an interesting look, with various sizes and colors (Grinţescu, 1985), which attract collectors. Colored forms of cactus are genetic mutations (Copăcescu, 1), which - in general - have a low content of chlorophyll. But, generally, the cactus stems are assimilated one by one. In the assimilated parenchyma, in the chloroplasts of these plants, the ratio of chlorophyll a / chlorophyll b is much lower than in normal leaves of plants (Kalishev, 1). Those cactuses which were the subject of our research in practice are vegetative multiplied by grafting (Shemorakov, 3). Multiplying chlorophylldeficient cacti is slow, and non-cost efficient. From this point of view, the in vitro multiplication - especially on these cactuses - is goal achieving (Corneanu and Corneanu, 1996). This paper is a comparative study, in terms of the response regime of explants consisting from regenerated buds on reclaimed cactuses grafted on Hylocereus triangularis rootstocks, grown in the greenhouse (fig. 1), to a vitroculture. The grafts consisted from the following species of cacti: Echinocactus mihanovichii (red cactus), Echinopsis chamaecereus f. lutea (yellow cactus) and Aylostera heliosa (green cactus). Explants were cultured on aseptic complex medium, with the addition of various growth regulators. MATERIALS AND METHODS For in vitro cultures initiation, we prelevated caulinar buds from Echinocactus mihanovichii (Fig. 1), Echinopsis chamaecereus f. lutea (fig.1b) or Aylostera heliosa (fig.1c), stem, and grafted them on Hylocereus triangularis roatstocks. The plant material (juvenile Echinocactus mihanovichii and Echinopsis chamaecereus f. lutea, or young stalks of Aylostera heliosa) was sterilized through its submersion in 96º ethylic alcohol, for one minute, then the material was covered by a.8% hypochlorite sodium solution, mixed with tap water(1:2); added to the disinfectant solution was - as a surfactant - a few drops of Tween. During sterilization, vegetative material was shaken. After minutes, the disinfectant agent was removed and the plant material was washed down with distilled sterile water, performing five consecutive rinsings, each of them for five minutes. Next, the plant material was deposited in Petri aseptic capsules with sterilized filter paper prior to drying the stove, deposited, in the laminar flow horizontally chimney hood with sterile air(cachiţă,4). Explants were about 1cm in length,.5 cm thick, and with a diameter of.5-1.5 cm, depending on the area from which the fragments were collected and the species of cacti that has been worked on. In the Aylostera heliosa cactus, cross sections have been applied, resulting in rounded portion in fragments of about 1 cm (fig.2b). *Correspondence: Teodora Iuliana Vidican,University of Oradea, email: iuliateodora68@yahoo.com Article received: September 9; published: November 9

Vidican T.I., C a c h i ţă-cosma D., Romocea J.E. Fig. 1 Chlorophyllien-deficitar cactus grafted on rootstocks of Hylocereus triangularis (where: A - red cactus, Echinocactus mihanovichii; B - yellow cactus, Echinopsis chamaecereus f. lutea, and C - cactus green, Aylostera heliosa; a graft; b caulinar buds; c rootstocks) The explant and its inoculation was done in the perimeter aseptic laminar flow horizontally chimney hood with sterile air, in use. To increase the explants growth, we used, the basic (MB) medium culture, consisting of: Murashige-Skoog (1962) macroelements and Fe-EDTA, Heller (1953) microelements, mineral mixture in which the following vitamins were added: HCl pyridoxine, HCl thiamine and nicotinic acid (1 mg / l each), 1 mg / l meso-inositol, g / l sucrose and 7 g / l agar-agar; the ph of culture medium was adjusted to the value of 5.8, prior to autoclaving it. To the described basic medium culture (MB), we added growth regulators, as follow: - V variant - MB, without growth regulators, controls group; - V 1 variant - MB supplemented with 1mg / l BA (benzyladenine); - V 2 variant - MB supplemented with 1mg / l AIB (acid β-indolil-butyric); - V 3 variant - MB supplemented with mixture of 1mg / l BA and 1mg / l AIB. Vial sterilization with culture media was done by autoclaving it at 121ºC, for 3 minutes. The vials were made from glass and had a capacity of 15 ml, in each container was placed 5 ml of the culture medium. The explants inoculation was performed after the cooling and solidification of the medium culture. After inoculation, the culture tubs were covered with disinfected polyethylene sheets, in advance - with 7 o ethanol the sheets were immobilized, at the vials mouth, with elastic rings. The regime in the growth chamber consisted of: illuminating cultures with white light, emitting from fluorescent tubes, as 16 hours light/24h photoperiod; light intensity was 17 lucks; the temperature near the culture vessels was between -24ºC. Inoculums reaction was monitored for 9 days. From 3 to 3 days to assess the progress of the explants and to determine the survival percentage of inoculums; observed aspects in the witness batch (V, explant inoculated on the basic medium, without growth regulators) were considered as reference. In figure 3A they are presented as a histogram, the data on the percentage of infected explants, from inoculi coming from the three species of cacti, and inserted histograms in figure 3 (BD) were illustrated (reporting to a number of 1 inoculated bottles) percentage of inoculums survivors at 3, 6 and 9 days after in vitro cultures initiation. A. B. Fig. 2 Schematic representations of explanting method of fragments that were inoculated on aseptic medium culture, where: A - buds of Echinocactus mihanovichii and Echinopsis chamaecereus f. lutea; B spheroid rounded portions of Aylostera heliosa, sections from young stems (fig. 1) 352 Vol. 19, issue 2, 9, pp. 351-357

The initiation of Echinocactus mihanovichii, Echinopsis chamaecereus f. lutea and Aylostera heliosa vitrocultures RESULTS AND DISCUSSIONS Five days after the explants inoculation (Fig. 3), at the witness batch, all three species of cacti that have been experienced, the percentage of infected inoculi was very high, compared to the other side in the experimental variants. The presence of growth regulators in the medium culture seems to influence the fungal mycelium extension in a negative way especially to the variant V 3, in all of the three species. The monitoring of inoculated explants during experiments, gave us the opportunity to see the evolution of its in function of nature of species according to the growth regulators present in the culture medium. Thus, in the 3-day after inoculation, the survival percentage of Echinocactus mihanovichii (fig.3b) at explants grown on culture medium suitable to the witness batch (V o ) was 7%, while all of the others culture medium was at 8%; at Echinopsis chamaecereus f. lutea, the medium variant with the addition of 1 mg / l BA (V 1 ) - this parameter was increased by 1% over the marked values to the witness batch (V o ), and with a % increase in variant V 3 (MS basic medium with the addition of 1 mg / l BA + 1 mg / l AIB), over the recorded figures in this parameter have grown similar inoculums on medium without growth regulators (V o ). Instead, the cactus Aylostera heliosa survival percentage - the medium variant with the addition of 1 mg / l BA - was decreased by 5% comparatively to that which was recorded from the witness batch, while the basic MS medium with the addition of 1 mg / l AIB (V 2 ), the survival percentage of inoculums was almost equal to that marked witness variant(v ), the mixed batch with BA plus AIB (V 3 ), this parameter increased by 15% compared to the explants lot grown on a medium culture without growth regulators. A. B. INFECTION PERCENTAGE, in a 5 day from the "in vitro" cultures initiation Echinocactus m. Echinopsis ch. Aylostera h. 1 % 8 6 SURVIVAL PERCENTAGE, in the 3- day from the "in vitro" cultures 1 % initiation 8 6 C. D. SURVIVAL PERCENTAGE, in the 6-day from the "in vitro" cultures initiation % 1 8 6 SURVIVAL PERCENTAGE, in the 9-day from the "in vitro" cultures initiation % 1 8 6 Fig. 3 A - Explants evolution of Echinocactus mihanovichii, Echinopsis chamaecereus f. lutea and Aylostera heliosa, grown on medium culture, without growth regulators(variant V ), or on the culture medium with 1 mg / l BA (V 1) or on the culture medium with 1 mg / l AIB (V 2), or cultivated in vitro on a culture medium with 1 mg / l BA plus 1 mg / l AIB (V 3), (where: A - percentage of infested inoculums and B-D survival percentage of inoculums from a total of 1 inoculated vials). Vol. 19, issue 2, 9, pp.351-357 353

Vidican T.I., C a c h i ţă-cosma D., Romocea J.E. Fig. 4 Explants evolution of Echinocactus mihanovichii, cultivated in vitro on basic Murashige-Skoog (1962) modified medium culture, by us, and with the adding of 1 mg / l BA and 1 mg / l AIB; morphologicol aspects: A - 3 days after inoculation ; B-C to 6 days after inoculation (B normal inoculum; C necrosis of inoculum over time), and D - 9 days after inoculation (where lt - leaflet transformed into prickle; ivi - initially viable inoculum; nii necrosis in time of initial inoculum, cm - culture medium; cn - caulinar neoformation) Worth mentioning is the fact that any experimental variation - this time - have not made noticed morphogenesis processes (risogenesis, caulogenesis or calusogenesis), but the inoculums have retained the original color and they have increased in volume, the best growth, showed by which ore inoculated on the basic MS culture medium with the addition of 1 mg / l BA + 1 mg / l AIB (V 3 ) (figures 4-6, position A). At in the 6 days after inoculation of the cactus explants, observations revealed that the survival percentage, while being compared to that achieved in the witness batch (V ), decreased (fig.3c). Thus, Echinocactus mihanovichii on suitable culture mediums, variants V 1 and V 2, decreased with %, and the V 3 variant with 15%. Echinopsis chamaecereus f. lutea on basic MS medium culture, both with the addition of 1 mg / l BA, and the variant V 2 - medium with the addition of 1 mg / l AIB, the survival percentage equaled to that of the witness, while the variant V 3 (MS basic medium culture with the addition of 1 mg / l BA + 1 mg / l AIB) values of this parameter exceeded 3% with that of its witness. At Aylostera 354 heliosa to variant V 1, all inoculum showed necrosis, while in V 2 and V 3 variants, the survival percentage was equal to that obtained witness batch V, respectively with 5%. Observations made at in the 9 days after initiation of the experiment (figure 3D), we have shown that Echinocactus mihanovichii on the basis medium culture MS, with the addition of 1 mg / l BA (V 1 ) and basic MS medium culture with the addition of 1 mg / l BA + 1 mg / l AIB (V 3 ), the survival rate exceeded 1% respectively with the parameter being evaluated to that of the witness batch group (V ), while the MS with the addition of 1 ml / l AIB (V 2 ), this parameter was decreased by 3%, compared to recorded values at regastrated similar explant inoculated inoculums and grown on medium culture without growth regulators, respectively the variant V o. Echinopsis chamaecereus f. lutea explants were grown at V 2 and V 3 variants. The survival rate at exceeded with 1%, %, respectively, the witness (V ), while the basic MS medium, with the addition of 1 mg / l BA ( V 1 ) all inoculums showed necrosis. Vol. 19, issue 2, 9, pp. 351-357

The initiation of Echinocactus mihanovichii, Echinopsis chamaecereus f. lutea and Aylostera heliosa vitrocultures Fig. 5 Explants evolution of Echinopsis chamaecereus f. lutea, cultivated in vitro on basic Murashige-Skoog (1962) modified culture medium, with an adding of 1 mg / l BA and 1 mg / l AIB; A in the 3-day after inoculation ; B-C observations made in the 6 days after inoculation (B normal inoculum; C necrosis of inoculum, over time) and D - observations made in the 9 days after inoculation (where lt - leaflet transformed into prickle; ivi - initially viable inoculum; nii necrosis, in time of the initial inoculum; cm - culture medium; cn - caulinar neoformation). After in the 6 days, Aylostera heliosa explant had not registered any changes in the survival percentage, compared with the assessments made at V 2 and V 3 variants. Of the three types of medium culture whose effects were tested in the present article, we observed that on the basis of the MS culture medium, with the addition of 1 mg / l BA + 1 mg / l AIB (V 3 ), from the explants have regenerated many bids respectively spheroid neostems, a phenomenon present in all three species of cacti that we used in our experiments, but predominantly at the vitrocultures of Aylostera heliosa. Viewing the fact that the explants of Echinocactus mihanovichii, regenerated neoformations of inoculums were red (fig.4b), while initially inoculums in certain areas had necrosis or became yellow-orange. Generally, the phenomenon of initial inoculum - in time - was present in all of the culture medium, even those who have grown in size and which have regenerated callus (fig.4c). Instead, in the Echinopsis chamaecereus f. lutea, the necrosis phenomenon of Vol. 19, issue 2, 9, pp.351-357 explants, occurred only at inoculums that have not generated bids (fig. 5C) instead inoculums remained viable and kept the yellow color and more neoformed light green buds (fig. 5C). At Aylostera heliosa explant, morphogenesis has been manifested through caulogenesis (neogenesis of numerous spheroidal green formations), but also by calusogenesis (fig.6b and C). In this species, at explants level regenerated an exuberant albino callus, especially on growing variants V 2 and V 3, it is due, probably, to growth regulator present in the medium, and predisposition of the species to form callus. However, the explants species that derived from the apical stem areas regenerated from the grafted level, were the most active with initial inoculums that remained viable for 9 days, the experiment lasted not showing any necrosis. Worth keeping in mind is that within the 9 days of vitroculture, none of the cacti species, used in experiments have shown risogenesis processes. 355

Vidican T.I., C a c h i ţă-cosma D., Romocea J.E. Fig. 6 Explants evolution of Aylostera heliosa. A - in the 3-day after inoculation; B - observations made in the 6 days after inoculation, E-C - observations made in the 9 days after inoculation (where lt - leaflet transformed into prickle; ivi - initially viable inoculum; nii - the original inoculum with an easy necrosis in final; cm - culture medium; cn calinar neoformation; hc - hyperhydric callus) CONCLUSIONS The initiation of Echinocactus mihanovichii, Echinopsis chamaecereus f. lutea and Aylostera heliosa vitrocultures is feasible on the culture medium modified by us, and with addition of BA and AIB, each in concentration of 1 mg / l. On this medium, the explant preleveted from the chlorofyllien-deficient cactus speciens, as Echinocactus mihanovichii and Echinopsis chamaecereus f. lutea, and regenerated colored buds ore increased. The monitoring was done at 6 days after the explants inoculation, on inoculum level, to note was an emphasis of pigmentation, and an increase of inoculums, and the number and size of buds. At Aylostera heliosa, the medium with BA or AIB, variations that were present, manifested the 356 Vol. 19, issue 2, 9, pp. 351-357

The initiation of Echinocactus mihanovichii, Echinopsis chamaecereus f. lutea and Aylostera heliosa vitrocultures phenomenon of calusogenesis, and regeneration of large number of buds. In none of the experimental variations during the 9 days of vitroculture, the risogenesis was absent. REFERENCES Cachiţă C.D., Deliu C., Rakosy-Tican L., Ardelean A., 4 Tratat de biotehnologie vegetală, vol. I, Ed. Dacia, Cluj-Napoca. Copăcescu S.V., 1 Cactuşii, monografie, Ed. Ceres, Bucureşti. Corneanu M., 1998 Studii citogenetice, fiziologice şi anatomo-morfologice la specii din familia Cactaceae cultivate in vitro. Teza de Doctorat, Universitatea "Babeş-Bolyai" Cluj-Napoca. Corneanu M., Corneanu G. C.,1996 Studii citogenetice la specii de Cactaceae cultivatein vivo şi in vitro. Cercet. Genet.Veget. Anim.IV: 193-199. Grinţescu I., 1985 Botanica (ediţia a II-a revizuită şi îmbunătăţită; coordonatori M. Andrei, N. Radulescu-Motru). Ed. Şt. şi Enciclop., Bucureşti. Heller R., 1953 Rescherches sur la nutrition minérale des tissus végétaux cultives in vitro. Ann.Sci. Nat. Bot. Veg. Ser.,II, pp. 1-5. Kalishev W., 1 Meet a new species, Cultivar 7(8), http://www.lapshin.org/cultivar/n18/class-e.htm Murashige T., Skoog F., 1962 A revised medium for rapid growth and biossays with tabacco tissue cultures, Physiol. Plant., 15, pp. 473 497. Shemorakov N., 3 Cultivar s classification by stem color, Cultivar 2(18), http://www.lapshin.org/cultivar/n7-8/indexe.htm Vol. 19, issue 2, 9, pp.351-357 357