THE INITIATION OF ECHINOCACTUS MIHANOVICHII, ECHINOPSIS CHAMAECEREUS F. LUTEA AND AYLOSTERA HELIOSA VITROCULTURES

Headnote

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, 100mg / 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 90 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 (Grintescu, 1985), which attract collectors.

Colored forms of cactus are genetic mutations (Copacescu, 2001), 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, 2001).

Those cactuses which were the subject of our research in practice are vegetative multiplied by grafting (Shemorakov, 2003). Multiplying chlorophyll-deficient 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 96° ethylic alcohol, for one minute, then the material was covered by a 0.8% hypochlorite sodium solution, mixed with tap water(1:2); added to the disinfectant solution was - as a surfactant - a few drops of Tween 20. During sterilization, vegetative material was shaken. After 20 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(Cachita,2004).

Explants were about 1cm in length, 0.5 cm thick, and with a diameter of 0.5-1.5 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).

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), 100 mg / l meso-inositol, 20 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^sub 0^ variant - MB, without growth regulators, controls group;

- V^sub 1^ variant - MB supplemented with 1mg / l BA (benzyladenine);

- V^sub 2^ variant - MB supplemented with 1mg / l AIB (acid β-indolil-butyric);

- V^sub 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 121°C, for 30 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 70o 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 1700 lucks; the temperature near the culture vessels was between 20-24°C.

Inoculums reaction was monitored for 90 days. From 30 to 30 days to assess the progress of the explants and to determine the survival percentage of inoculums; observed aspects in the witness batch (V0, 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 100 inoculated bottles) percentage of inoculums survivors at 30, 60 and 90 days after "in vitro" cultures initiation.

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^sub 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 30-day after inoculation, the survival percentage of Echinocactus mihanovichii (fig.3B) at explants grown on culture medium suitable to the witness batch (V^sub o^) was 70%, while all of the others culture medium was at 80%; at Echinopsis chamaecereus f. lutea, the medium variant with the addition of 1 mg / l BA (V^sub 1^) - this parameter was increased by 10% over the marked values to the witness batch (V^sub o^), and with a 40% increase in variant V^sub 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^sub 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^sub 2^), the survival percentage of inoculums was almost equal to that marked witness variant(V^sub 0^), the mixed batch with BA plus AIB (V^sub 3^), this parameter increased by 15% compared to the explants lot grown on a medium culture without growth regulators.

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 (V3) (figures 4-6, position A).

At in the 60 days after inoculation of the cactus explants, observations revealed that the survival percentage, while being compared to that achieved in the witness batch (V^sub 0^), decreased (fig.3C). Thus, Echinocactus mihanovichii on suitable culture mediums, variants V^sub 1^ and V^sub 2^, decreased with 20%, and the V^sub 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^sub 2^ - medium with the addition of 1 mg / l AIB, the survival percentage equaled to that of the witness, while the variant V^sub 3^ (MS basic medium culture with the addition of 1 mg / l BA + 1 mg / l AIB) values of this parameter exceeded 30% with that of its witness. At Aylostera heliosa to variant V^sub 1^, all inoculum showed necrosis, while in V^sub 2^ and V^sub 3^ variants, the survival percentage was equal to that obtained witness batch V^sub 0^, respectively with 50%.

Observations made at in the 90 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^sub 1^) and basic MS medium culture with the addition of 1 mg / l BA + 1 mg / l AIB (V^sub 3^), the survival rate exceeded 10% respectively with the parameter being evaluated to that of the witness batch group (V^sub 0^), while the MS with the addition of 1 ml / l AIB (V^sub 2^), this parameter was decreased by 30%, compared to recorded values at regastrated similar explant inoculated inoculums and grown on medium culture without growth regulators, respectively the variant V^sub o^.

Echinopsis chamaecereus f. lutea explants were grown at V2 and V3 variants. The survival rate at exceeded with 10%, 40%, respectively, the witness (V^sub 0^), while the basic MS medium, with the addition of 1 mg / l BA ( V^sub 1^) all inoculums showed necrosis.

After in the 60 days, Aylostera heliosa explant had not registered any changes in the survival percentage, compared with the assessments made at V^sub 2^ and V^sub 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^sub 3^), from the explants have regenerated many bids respectively spheroid neo-stems, 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 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 V2 and V3, 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 90 days, the experiment lasted not showing any necrosis.

Worth keeping in mind is that within the 90 days of vitroculture, none of the cacti species, used in experiments have shown risogenesis processes.

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 60 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 phenomenon of calusogenesis, and regeneration of large number of buds.

In none of the experimental variations during the 90 days of vitroculture, the risogenesis was absent.

References

REFERENCES

Cachita C.D., Deliu C., Rakosy-Tican L., Ardelean A., 2004 - Tratat de biotehnologie vegetala, vol. I, Ed. Dacia, Cluj-Napoca.

Copacescu S.V., 2001 - Cactusii, monografie, Ed. Ceres, Bucuresti.

Corneanu M., 1998 - Studii citogenetice, fiziologice si anatomo-morfologice la specii din familia Cactaceae cultivate in vitro. Teza de Doctorat, Universitatea "Babes-Bolyai" Cluj-Napoca.

Corneanu M., Corneanu G. C.,1996 - Studii citogenetice la specii de Cactaceae cultivatein vivo si in vitro. Cercet. Genet.Veget. Anim.IV: 193-199.

Grintescu I., 1985 - Botanica (editia a II-a revizuita si Îmbunatatita; coordonatori M. Andrei, N. Radulescu-Motru). Ed. St. si Enciclop., Bucuresti.

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., 2001 - 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., 2003 - Cultivar's classification by stem color, Cultivar 2(18), http://www.lapshin.org/cultivar/N7-8/index-e.htm