according to the form and intensity of the nitrogen nutrition. Anthocyanin production in KNO3 treatment, for example, showed a 20-fold increase as compared to the casein hydrolysate treatment, the production of anthcyanin increased with the increase of nitrogen supplied to the solution.
The difference in the osmotic pressure of the cell sap showed that KNO3 treatment had the highest osmotic pressure, followed by casein hydrolysate treatment and control (app.
H, table 2). Measuring osmotic pressure of the liquid solution showed that solution media of KNO3 treatment had the lowest osmotic pressure, followed by casein hydrolysate and standard treatment. Since osmotic pressure results from the concentration of molecules in the solution, it can be stated that the nitrogen form alters the osmotic pressure of the plant cell and of the media.
The soluble protein content of different nitrogen treatments showed that the highest rate of protein was produced when casein hydrolysate was supplied to the system, followed by modified B5 having all three types of nitrogen (app. H, table 2; app. I). The lowest values were determined in the KNO3 treatment. These results did not show the same trend regarding dry weight proportions. It can be presumed that the protein content in the casein hydrolysate treatment is high, but the non-soluble protein substances are less as compared to KNO3 treatment, which has less protein content but a higher dry weight percentage.
During the realization phase, the activity of the inductive form of the enzyme NR was highest when KNO3 was the only source of nitrogen, followed by the control and casein hydrolysate treatment. Measurement of the constitutive enzyme NR showed an extremely small difference between different treatments (app. I).
use of radio active leucine gave valuable indication not only about the location of the cells which later formed rhizogenic and embryogenic centers stimulated through auxin application, but also about the quantitative accumulation of leucine containing proteins in different cells and structures within specific culture periods (app. A, fig. 7, 8, 10, 11).
4.2.1 Histological Observations During the Induction Phase of Somatic Embryogenesis (culture in B5 with 2,4-D)
The petiole section of a 6-to-8 weeks old carrot plant is a heart-shaped structure and shows 5 collenchyma supporting structures adjacent to the periphery of the epidermis (app. A, fig. 1). Under the cuticle is a single-lined epidermis cell layer, under which is a small, vacuolized 2-to-3-lined sub-epidermal layer of vacuolized cells. The proportion of the nucleus to the whole cell is greater than that of ground and epidermal cells (Schäfer et al., 1985). The ground tissue by itself is composed of highly vacuolized cells varying in size.
There are three or more collateral vascular bundles. The largest vascular bundle lies in the center, having xylem in the center and phloem tissues on the periphery. Glandular channels as a typical structure for plants belonging to Apiaceae are located between the conducting channels of vascular bundle and epidermis. There are three glandular channels in this phase of growth. Glandular channels are schizogen, secreting glands.
B5+ cultured carrot petiole explants show the following histological changes:
As little as 4 days after incubation of the petioles in an auxin-containing medium, one or more meristematic centers are formed on the periphery of the vascular bundle (app. A, fig.
2). Individual cells of these meristems distinguish themselves from adjacent cells of the ground tissue in form, size and stainability with Hematoxylin. Regarding the form, these cells are round in shape and have a better-defined geometrical form. They are smaller in size and possess more cytoplasm with an obvious larger nucleus.
This process remains a mere microscopic event even after 6 days of petiole culture, but after 8 days of culture, splitting of the petioles can be seen macroscopically. The formation
of rhizogenic centers can be better observed in culture condition with less concentration of auxin. Further growth of the rhizogenic meristems finally leads to the rupture of the epidermis and some times adventitious roots appear (app. K) if the petioles are kept for more than 18 days in an auxin-containing medium.
Under the epidermis, but with a lapse of 5 days, almost the same developmental pattern can be observed. As early as 10 days after incubation of the petioles in an auxin-containing culture, some originally vacuolised sub-epidermal cells accumulate more cytoplasm and start to divide and form meristematic centers called embryogenic centers, resembling rhizogenic centers, but with smaller, more round and more compact cells (app. K, fig. B, C). These structures remain under the epidermis and with in the course of time rupture the epidermis. Some sub-epidermal cytoplasm rich cells show a specific developmental mode in which the cytoplasm of a single cell divides up first into 2, then into 4 cells and more, and in this way forms the pre-embryos (app. A, fig. 5).
The idea behind using a radioactive isotope was to show the differentiation and the changes relating to possible changes in the pattern of protein synthesis of cultivated carrot explants during the induction phase of somatic embryogenesis. So that for a specific change e.g. different culture times, some specific protein spectrum or some specific pattern for the protein synthesis related to that particular culture time and the developmental stage could be detected. The chosen time intervals for petiole culture were after 5 hours, denoted as 0, after 7, 14, 19 and 21 days, denoted as t0, t7, t14, t19 and t21 respectively (app. A fig. 1-6).
Histological observations strongly suggest that the protein content of the plant cell in an auxin-containing medium increases 7 days after culture (t7) as compared to t0 (app. A, fig.
1, 2). In the next 7 days in t14 this trend continues (app. A, fig. 6).
On the basis of the histological observations and the determination of the absorption the rate of 14C-leucine, one can postulate that the accumulation of 14C-leucine and its incorporation in the protein synthesis during the induction phase increases. In these series of observations, it was evident too, that the embryogenic centers in all the observation periods, namely t7, t14, t19 and t21, were labelled (app A, fig. 7, 8, 9, 10, 11). The rhizogenic centers, by contrast showed a selective absorption pattern, so that it can be postulated that the protein synthesis in embryogenic centers is more intensive than in the rhizogenic centers (app. A, fig, 8, 9, 10, 11).
A short comparison between rhizogenic and embryogenic meristems follows:
Development of Rhizogenic Centers:
Time of appearance = around 7 days Location = near vascular bundles Cell form = rather long
Cell size = small
Stainability with Hematoxylin = all structures are dyed
Absorption of radioactive leucine = some meristems are labelled and others not
Development of Sub-Epidermal Cytoplasm Rich Cells and Embryogenic Centers:
Time of appearance = 10-12 days
Location = sub-epidermal cell layers between epidermis and vascular bundles Cell form = rather round
Cell size = small
Stainability with Hematoxylin = all structures are dyed Absorption of radioactive leucine = all meristems are labelled
4.2.2 Histological Observations During the Realization Phase of Somatic Embryogenesis (culture in B5 with out 2,4-D)
After the petioles were 14 days in an auxin-containing medium, they were sub-cultured in a medium without an exogenous auxin. Using a very high concentration of auxin for the induction, however, is usually inhibitory to development of the somatic embryos in advanced stages. In the hormone-free medium, development of globular staged somatic embryos followed; heart, torpedo stage and finally plantlets were formed.
Thus induction of carrot somatic embryogenesis requires a single hormone signal to induce a bipolar structure (app. J, fig, 3) capable of forming a complete plant upon transfer to a hormone-free medium.
4.3 Protein Spectrum and Pattern of Protein Synthesis in Cultured Petiole of