Introduction

Little attention has been paid to the effect of various rates of N, P, and K in the nutrient medium, all of which play a key role in induction of microtuberization (Wang and Hu 1985). N interacts with genotype (Sarkar and Naik 1998) and sugar (Ranalli 1997) in the nutrient medium. High concentration of N (60 L−1) inhibited coumarin-induced tuberization (Stallknecht and Farnsworth 1979; Wattimena 1983). Garner (1987) suggested a possible inhibitory effect on tuberization with an increase from 1x to 2x MS (Murashige and Skoog 1962) N concentration. In contrast, high N level did not influence cytokinin-induced microtuberization (Palmer and Smith 1969; Wang and Hu 1982; Estrada et al. 1986; Sarkar and Naik 1998).

Reducing N levels in nutrient medium free of growth regulators from 1x MS to 0.1x MS reduced tuber number (Garner 1987; Garner and Blake 1989). Cytokinin-induced nutrient medium with reduced N increased tuber number but decreased tuber weight (Sarkar and Naik 1998). Zarrabeitia et al. (1997) found that low N, 19–23 L−1, produced optimum results in micropropagation, and there was a “carry-over” effect of N content in the micropropagation medium on subsequent tuberization. A lower concentration of N, 23 L−1, induced earlier tuber initiation.

With respect to K and P, Naik and Sarkar (1998) reported K had a positive effect on microtuber size. Maximum microtuber size and harvest index were obtained with a nutrient medium concentration of 40 μM potassium. The impact of P on microtuberization has received little attention.

All of the previous reports evaluated one or two elements at the same time. There were no reports on the three macroelements evaluated simultaneously. Observations in our laboratory (unpublished data) suggested depletion of P and low levels of N and K in the Murashige and Skoog (1962) culture medium at harvest; hence, 2x and 3x MS P concentrations and 2x MS N and K concentrations were used in our experiment.

Materials and Methods

Plant Materials

Disease-tested plantlets of Nicola and Russet Burbank originated from the germplasm bank of “Laboratoire Agronomique EL BOURA” located in Taroudant, Morocco. Two methods of propagation were used depending on final use of the plantlets; either for micropropagation or for microtuberization (Radouani and Lauer 2015). Microprogation was made from single-middle-node cuttings on solid medium. Mother plantlets were 3 weeks old. These were derived from four subcultures made at three-week intervals. The MS nutrient medium was supplemented with NaH2PO4 (148 mg L−1), thiamine HCl (0.4 mg L−1), myo-inositol (100.0 mg L−1), and sucrose (30 g L−1). The solidifying agent was Phytagel (1.5 g L−1).

In vitro propagation of plantlets for microtuber production was done from whole plantlets. The plantlets were uniformly trimmed to four nodes excluding the shoot apex and basal node. The nutrient medium was the same as for micropropagation except for the lack of Phytagel. This medium was used during the “growth phase” defined as the time from initiation of the culture until tuberization. This phase was 5 weeks long.

The “tuberization phase” was 16 weeks long, after which tubers were counted and weighed. For the tuberization phase, the liquid medium for growth was removed and replaced with a similar nutrient medium, in which sucrose concentration was increased from 30 to 80 g L−1 and NaH2PO4 eliminated. Twelve NPK combinations were used including two concentrations of nitrogen, N1 = 841 and N2 = 1418 mg L−1; three concentrations of phosphorus, P1 = 39, P2 = 76.9 and P3 = 115 mg L−1; and two concentrations of potassium, K1 = 784 and K2 = 1518 mg L−1. All of these 12 nutrient media were supplemented with: thiamine HCl, 0.4 mg L−1; myo-inositol, 100 mg L−1; sucrose, 80 g L−1; and coumarin, 25 mg L−1. N1P1K1 represents levels of N, P and K found in standard MS media, and served as the control in this experiment.

The pH of all nutrient media was adjusted to 5.8 before autoclaving for 15 min at 121 °C and 1.5 kg/cm2.

Culture Vessels

Jars (650 ml volume) were used for solid medium and 250 ml Erlenmeyer flasks for liquid medium. The nutrient volume in jars for micropropagation was 60 ml during the first 5 weeks and 150 ml for microtuberization thereafter for 16 weeks. Jars were closed with plastic screw-top lids and flasks closed with cotton plugs topped with heavy-duty aluminum foil.

Culture Incubation Conditions

Three growth rooms, 5.20 m long, 3.95 m wide and 3.15 m high, were used. These rooms were equipped with shelves and fluorescent strip lights set to provide 85 μ mol m−2 s−1 PAR (photosynthetically active radiation) at the plant level on each shelf. A 16 h photoperiod was provided during the growth phase and total darkness during the tuberization phase. The vessels were gently agitated during the tuberization phase. Room temperature was set to 24 °C and controlled by an air conditioning system allowing a variation of ±1 °C from the programmed temperature. These aseptic rooms were equipped with an aseptic fresh air injector. The injector was programmed to inject air into the growth rooms every 2 h. The purification of air was done in three steps:

  1. 1.

    Prefiltration with Interpak H filter, 490/540/500 mm;

  2. 2.

    Irradiation with ultra violet light, TUV 15 W G15 T8; and

  3. 3.

    Final filtration with an MP99 filter (305 × 305 × 6 mm pore size, 0.12 μm).

Data Analysis

The experimental unit consisted of four Erlenmeyer flasks with four explants each. Tuber number (TTN), tuber weight (TTW), and stem and root weight (SRW) for each combination were recorded and statistically analyzed to identify the best nutrient medium for potato tuberization. The results for all traits were presented by cultivar and by interactions, if any.

Data were analyzed using SAS. Randomization was done using STATITCF software. Main effects were tested by ANOVA and mean were separated using the Student-Newman-Keuls test.

Results and Discussion

For both cultivars, N2P3K2 ranked first. Statistically, this combination was superior with respect to TTW for Russet Burbank and SRW for Nicola (Table 1). N1P1K1 almost always gave the lowest TTN, TTW, and SRW for Russet Burbank. Except for TTN for Nicola, N2P3K2 was significantly different from the control.

Table 1 Effect of 12 nitrogen, phosphorus and potassium nutrient media combinations on microtuberization of Nicola and Russet Burbank
Full size table

Increased NPK concentrations resulted in an increase of yield components and suggested that microtuberization of potato responds to a higher range of NPK concentrations. The three macroelements interact with each other and with cultivar. In this study the difference between N2P3K2 and N1P1K1 was more distinct for Russet Burbank than for Nicola. The observed interactions suggest that optimal concentrations could be expected to vary for different cultivars.

One could expect that larger microtubers might perform more consistently when planted directly in the field (Tovar et al. 1985 and Wiersema et al. 1987). Perhaps, under some conditions, larger microtubers could replace minitubers in seed potato programs. (Radouani and Lauer 2015).