The effect of nitrogen sources on the growth response of Schisandra Chinensis (turcz.) baill in the in vitro culture

Schisandra is a valuable medical crop the vegetative reproduction of which by traditional methods is hindered, so the most modern and perspective method of genetically identical specimen replication is the clonal micropropagation method [1, 4, 7]. Korean and Chinese researchers conduct a large-scale work to create genetic collections and to develop different methods of this plant’s cultivation in in-vitro conditions. As a rule, such methods are reproduction models involving somatic embryogenesis induction through callus culture, or seeds are used as the original explants [6, 8, 9]. Such reproduction models produce genetically heterogenic vegetative offspring; therefore such technologies are not suitable for Schisandra varieties replication and its valuable forms.

The techniques we developed allow to successfully use apical and lateral buds as explants and to get in-vitro genetically homogenous micro-shoots suitable for rooting in artificial nutrition mediums [2, 5]. However, the speed of Schisandra shoot growth in the in-vitro culture is relatively slow that is mentioned by other authors [3]. As our work experience shows, the shift of growth regulators balance not always can radically resolve the issue of increasing proliferation activity. Moreover, changes of medium hormonal content can provoke shoot vitrification or increased callus formation. To this extent, it is perspective to search for the optimal balance of main mineral nutrients, in particular, sources of nitrogen as an effective regulator of Schisandra growth activity in the in-vitro culture.

Research methods

The research objects were Schisandra micro-shoots cultivated in the in-vitro conditions. We studied the effect of different concentrations of main nitrogen sources in the nutritious Quoirin&Lepoivre medium — ammonium nitrate (NH4NO3) and potassium nitrate (KNO3) in different proportions. For the purpose of control we used the full formula of the Quoirin&Lepoivre medium in the Standardi&Catalano modification [10] that contains 400 mg/l of NH4NO3 and 1800 mg/l of KNO3, as well as the medium variant fully devoid of main nitrogen sources (Table 1). We used 6-BAP in the concentration of 1 mg/l as the main growth regulator. Micro-shoots were cultivated in flasks with the volume of 250 ml, in the cultural room at the temperature of +22....25 °С, illumination intensity of 3000-3500 lx and photoperiod of 16/8 hours. The data were taken 30 days after cultivation, the number and the length of the formed shoots were taken into account.

Table 1

The proportion of main nitrogen sources in different experimental variants

Research Results

As experimental results have shown, the full removal of potassium nitrate from the medium’s content has a negative impact on the Schisandra micropropagation process in the in-vitro culture. The deficiency of this compound cannot be compensated by the presence of the only one ammonium form of nitrogen in the medium. The experimental variants containing the full concentration of potassium nitrate were distinguished by a relatively stable length of the forming shoots and did not have statistically significant differences with the increase of ammonium nitrate concentration from 0 to 400 mg/l (Figure 1).

Figure 1. The average length of Schisandra shoots formed in the conditions of different nitrogen supply.

Note: k1 — control 1, the medium does not contain any nitrogen sources; k2 — control 2, the full concentration of ammonium and nitrate forms of nitrogen in the medium.

As the data presented in Figure 1 show, the double decreasing of potassium nitrate concentration in the medium (to 900 mg/l) does not exert a significant influence over the processes of Schisandra shoots linear growth, while the quadruple one causes the depression of growth response the strength of which is comparable with control 1 that does not contain any nitrogen sources.

The most optimal proportions of ammonium nitrate and potassium nitrate are 400 mg/l and 1800 mg/l respectively, the average length of shoots is 15 mm. The results of the experimental data approximation by polynomial of degree 2 also confirm it as the optimum in this concentration range.

The evaluation of Schisandra micropropagation effectiveness according to the integral indicator of propagation coefficient (the average number of shoots per one explant), showed that index was inversely related to the values of the average shoot length (Figure 2).

Figure 2. The effect of different proportions of nitrogen sources in the medium on Schisandra in-vitro reproduction.

Note: k1 — control 1, the medium does not contain any nitrogen sources; k2 — control 2, the full concentration of ammonium and nitrate forms of nitrogen in the medium.

The general tendency for improvement of the micropropagation coefficient is preserved when potassium nitrogen concentration is increased from 0 to 1800 mg/l along with 400 mg/l of ammonium nitrate. Decrease of the content for two times or complete removal of the ammonium form from the macroelemental medium composition with preservation of high potassium nitrate level in the medium allows to significantly intensify the proliferative activity of Schisandra meristems.

Conclusions

By analysing the obtained results one can draw the conclusion of the selective sensitivity of Schisandra in the in-vitro culture to the nitrate form of nitrogen embedded as potassium nitrate. This nitrogen source is the optimal one at the stage of micropropagation, the decrease of its concentration suppresses the growth response of explants. On the contrary, ammonium nitrate is not the limiting factor for cultivation of this crop. In small concentration, it can stimulate elongation of Schisandra shoots along with optimal potassium nitrate dosages.