Response of cotyledon explants of Capsicum annuum L. cv. kujawianka to chosen plant growth regulators in in vitro culture

Shoot buds originated directly on cotyledon explants of Capsicum annuum L. cv. Kujawianka, when Linsmaier and Skoog medium was enriched with BAP (2 mg/l). Kinetin (2 mg/l) or kinetin with IAA (1 mg/l + 1 mg/l) induced indirect shoot buds regeneration from callus. Rooting was obtained with explants cultivated on a medium containing NAA (0,5 mg/l). Occurrence of the early stages of differentiation was proved at the histological level.


INTRODUCTION
Plant tissue culture in vitro is widely used for micropropa gation of plants. Many plants of economic and horticultural value are propagated successfully using this technique.
Organogenesis in vitro is a complex and little understood process. Generally organogenesis is obtained from various ex plants via callus formation, but some are developing directly from the original explant cells. Direct differentiation of shoot buds has been observed in several plant species. In Solanum surratense shoot buds were formed on stem explants (Gupta and Chandra, 1982), while in Allium species (Rauber and Grunewaldt, 1988) and Myriophyllum heterophyllum (Kane and Albert, 1989) they formed on leaf explants. Shoot buds were induced directly on cotyledon explants of Albizia spp (Tomar and Gupta, 1988) and hypocotyl explants of Capsi cum frutescens (Subhash and Christopher, 1988).
Capsicum annuum L., the red pepper, belongs to Solanaceae family. It is an important vegetable plant which has also medicinal value. The fruit of red pepper is rich in ascorbic acid, vitamins Bi, B2, E, carotenoids, pro vitamin A, organic acids (e.g. citric acid) and capsaicin. The application of in vitro techniques for the production of a large number of plantlets of Capsicum annuum could be very important economi cally. Untill now the histological aspects of organogenesis in red pepper have been mentioned briefly (Agrawal et al" 1989), but without detailed description of the cellular events and the anatomical changes in the initial explants.
Our preliminary studies (data unpublished) allowed us to establish the most efficient medium compositions for orga nogenesis on cotyledon explants of Capsicum annuum cv. Ku-Abbreviations: BAP, 6-benzylaminopurine; NAA, 2-naphthalene acetic acid; IAA, indole acetic acid. jawianka. The present work aimed at studying histological changes in the cotyledon explants and to observe sequence of the events leading to regeneration of the shoot buds.

MATERIALS AND METHODS
Seeds of Capsicum annuum L. cv. Kujawianka were ob tained from the seed company Przedsiębiorstwo Nasiennictwa Ogrodniczego i Szkółkarstwa in Kraków. The seeds were soaked in tap water for 24 hours, surface-sterilized with aqueous 0.1% HgCh for 5 min and then treated with 0.5% H2SO4 for 2 min. They were rinsed with distilled water (4 times 5 min.) and then placed in culture tubes containing 5 ml of 0.7% agar medium with 2% glucose. Germination took place in darkness.
Cotyledon explants from two-week-old seedlings were transferred onto the Linsmaier & Skoog medium (1965), with their abaxial surfaces uppermost. The basal medium contain ing casein hydrolysate (400 mg/1), thiamine (0.4 mg/1) and meso-inositol (100 mg/1) was enriched with BAP, NAA, IAA and kinetin in various concentrations and combinations. The pH was adjusted to 5.8. The explants were cultured in a growth chamber at 25 ± 2°C in continuous light (white, neu tral fluorescent tubes; intensity ca. 1000 lx) for four weeks.
For histological studies cotyledon explants were fixed in FAA (formaldehyde 5%, acetic acid 5%, 70% ethanol 90%). at regular seven-day intervals. They were dehydrated through a graded series of ethanol and embedded in paraffin. Sections (about 15 pm) were stained with safranin and fast green and mounted in Canada balsam after dehydration.

RESULTS
Regeneration of shoot buds, almost 100%, was obtained on media supplemented with 1 mg/1 kinetin and 1 mg/1 of IAA or 2 mg/1 BAP. The best regeneration of roots (100%) was ob served on a medium with 0.5 mg/1 NAA. To compare the ef-   fects of two cytokinins, explants cultured on a medium with 2 mg/1 of kinetin were also analysed, though in this case shoot buds regeneration occurred with lower frequency.
Observations of several slides, made from explants fixed at regular intervals, allowed us to follow the histological changes during the formation of the leaves, roots and callus. In the first week of culture on a medium containing 2 mg/1 BAP some increase of parenchymal cell was observed at the distal cut end of the explants and in contact with the medium surface some increase of parenchymal cells was observed. Accompanying frequent divisions of the epidermal cells were revealed by the presence of large nuclei, intensely stained with safranin ( Fig. 1).
As a result of these divisions the epidermis folded. Both anticlinal and periclinal divisions were observed in the subepidermal layer. Parenchymal cells, adjoining the dividing cells, contained several starch grains (Fig. 2). Following all the changes, meristemoids formed and then gave rise to shoot buds and leaf primordia. After two weeks of culture, shoot buds and several large leaf primordia appeared ( Fig. 3 and 4). Some of them had a large strand, consisting of tracheary ele ments with spiral wall thickenings. Groups of meristematic cells often occurred within the parenchyma of the explants (Fig. 7). After four weeks of culture, small quantities of cal lus and leaflets could be detected macroscopically on the dis tal end of the explant. On the proximal end there was only a small volume of callus, formed as early as the first week of culture.
Some of the epidermal cells divided after one week of cul ture, differentiated glandular hairs. After two weeks of cul ture, the hairs were already fully developed.These hairs were composed of a short one-, or two-celled stalk and multicellu lar spherical head. They appeared on the whole epidermal sur face covering newly regenerated structures (Fig. 8).
Divisions of the parenchyma and callus cells in the region between the regenerated shoot buds and the cotyledon explant led to the formation of elongated cells with large nuclei, dis tinctly different from the neighbouring cells. These cells were arranged in strands running from the explant vascular bundle towards the forming shoot buds (Fig. 9). Then the cells dif ferentiated into tracheary elements with spiral wall thicken ings, identical to those in the vascular bundle of the explant. These elements were a continuation of the vascular tissue of the explant in the direction of shoot buds. Differentiation of the vascular tissue within the forming leaf primordia occured basally, while the vascular bundle of the explant elongated into the apical direction. This mode of joining the vascular tissue of the explant and the leaf primordia resembles the dif ferentiation pattern of the shoot vascular system in vivo.
Apart from the differentiating vascular tissue, connecting shoot buds to the explant, tracheary elements were observed, single or in groups. The tracheary elements differentiated also  from the elongated cells, arranged in parallel rows (Fig. 10) or in whirled systems (Fig. 11). Both systems occurred inde pendently, usually in the callus tissue of the explant. When the elongated cells formed rows, the differentiation of tra cheary elements was linear. Immature elements were observed at the end of each row. When the arrangement of elongated cells was whirled, tracheary elements differentiated both in side and outside the whirls. The cell walls of all tracheary ele ments in the callus had spiral, sparse and slightly lignified thickenings ( Fig. 12; Fig. 4). The average length of the ele ments was 92.2 pm. Cotyledon explants, grown on a basal medium supple mented with 1 mg/1 kinetin and 1 mg/1 IAA produced high amounts of callus at both their cut ends during the first week of culture (Fig. 5). Shoot buds were formed from the areas of the meristematic cells, appearing in the callus tissue of twoweek-old explants. Besides, cells with thickened walls ar ranged in periclinal rows were frequently observed on the cal lus surface. The whole pattern resembled the periderm (Fig.  15).
Several tracheary elements, differentiating from the elong ated cells, were arranged in rows and whirls located among the callus cells. These elements were irregularly shaped and the structure of their secondary wall varied considerably re garding its pattern. Elements with spiral, reticulate, scalari form and pitted type of secondary wall thickenings were found (Fig. 13). All tracheary elements were strongly thick ened and lignified, and their average length was 30.9 pm. After four weeks of culture, rosettes of leaves could be ob served (Fig. 16).
Regeneration of shoot buds from the explants cultured on the medium with 2 mg/1 kinetin occurred indirectly (Fig.6), via callus, similarly to the regeneration under the influence of kinetin together with IAA (see above). Histological analysis  revealed that the callus cells located in the vicinity of the re generated structures contained several starch grains. The sur face of the shoot buds was covered by an epidermis with hairs composed of multicellular head and pedicle. Tracheary elements with spiral wall thickenings, elongated (54.8 pm) and slightly lignified, were observed in the callus (Fig. 14). Histological analysis of the explants, cultured on medium with 0,5 mg/1 NAA, indicated that roots differentiated both directly from explant tissues (Fig. 17) or via the callus tissue (Fig. 18). In both cases rhizogenesis occurred adjacently to the tracheary elements.

DISCUSSION
The anatomical studies proved that two cytokinins, applied in equal concentrations, stimulated the process of shoot bud regeneration in two ways. In the presence of BAP, the shoot buds formed directly from the explant tissues, in the primary phase of the culture. These results are compatible with the findings of Agrawal et al. (1989). Kinetin induced at first cal lus formation, then shoot buds differentiated from the callus.
Addition of auxin (IAA) to the media containing kinetin caused some increase in the indirect regeneration of the shoot buds. Indirect shoot buds regeneration appeared only in the final phase of culturing, what may be explained as acquiring by callus the capacity to regenerate during the culture period (Barciela, Vietez, 1991). According to the previous studies on red pepper, carried out by Ochoa and Garcia (1990), the pro cess of rhizogenesis is stimulated mainly by auxins. Our studies confirm these observations. Several hairs, composed of multicellular head and pedicle, were observed on shoot buds and leaves regenerated on media containing cytokinins. They were similar to the glandular hairs covering the stems and the abaxial leaf surface of plant in vivo (Somos, 1984). It may be assumed that the cytokinins which initiated multiple divisions within the epidermal layer at the beginning of the culture period, also induced divisions in the epidermal layer, giving rise to these hairs.
Tracheary elements in the callus always differentiated from elongated cells. It seems interesting that these elongated cells were arranged in single rows or whirls.           Kurczyńska (1986) observed similar whirls of cambial cells, covering the stem of Fraxinus excelsior, near the adventitious buds. She interpreted this arrangement on the basis of the cir cular flux of auxin.
The requirement of initial cell divisions for the differentia tion of tracheary elements is still an open question. Fukuda and Komamine (1980) reported that during the culture of Zin nia leaf mesophyl (the system of isolated cells) cell divisions were not a prerequisite for tracheary elements formation. However, Phillips (1987) pointed out that in the culture of ex plants from tubers of Helianthus tuberosus (multicellular sys tem) cell divisions were necessary for the differentiation of tracheary elements. Our studies, carried'out on the multicellu lar system, provide evidence for the latter hypothesis.
Our results show that the combination of auxin and cyto kinin caused formation of smaller tracheary elements than cy tokinin alone. Aloni (1992) concluded that in vivo high levels of auxins in the stem close to the young leaves induced sev eral small vessels, though with a lower concentration of au xins differentiation occurred more slowly and there were fewer and larger vessels.