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A Simple in Vitro Method to Study Tuber Growth of Solanum tuberosum L.
A Simple in Vitro Method to Study Tuber Growth of Solanum tuberosum L. B. SATTELMACHER and H. MARSCHNER lnstitut flir Pflanzenernwrung der Universitat Hohenheim, 7000 Stuttgart 70, Fruwirthstr. 20 Received April 29, 1985 . Accepted May 6, 1985
Abstract A simple in vitro method is described which allows the study of effects of mineral nutrient supply on growth rate of the tuber. Single node cuttings carrying axillary tubers are cultured under semi-sterile conditions in cooled ( + 1 0C) nutrient solutions. Tuber growth is monitored volumetrically and mineral nutrient uptake calculated from the nutrient solution taken up by the cuttings. Key words: Potato, tuber growth, in vitro.
Introduction
The influence of mineral nutrients on potato tuber growth has been the subject of many studies. Especially nitrogen (Watson, 1963; Moorby, 1967; Dyson and Watson, 1971) but also phosphate (Prummel and van Barnau-Sijthoff, 1984), and potassium (Boyd, 1970) have a pronounced influence on tuber growth. Plant nutrients may reveal their effects on tuber growth by influencing the shoot growth (Moll, 1981), by affecting the phytohormone balance in roots, shoots and tubers (Krauss and Marschner, 1976; Sattelmacher and Marschner, 1977) or by influencing the tuber growth directly (Koda and Okazawa, 1983). However, in studies with intact plants it is difficult, or even impossible, to distinguish between direct and indirect effects of mineral nutrient supply on tuber growth. This holds true for example for the negative correlation between potassium and starch content in potato tubers (Forster and Beringer, 1983; Lindauer and Haeder, 1984). For causal explanations in vitro experiments are therefore desirable. Standard procedures for cultivation of potato tubers in vitro (Koda and Okazawa, 1983) require sterile material and are therefore time-consuming and laborious. In the present paper a simple method is described to cultivate potato cuttings carrying axillary tubers under semi-sterile conditions. The system used is similar to the one described for cultivating wheat ears (Donavan and Lee, 1977). Materials and Methods Well sprouted potato tubers (cultivar Achat) were planted into 51 pots filled with about 3~ 1 of peat moss. The tubers were placed on top of perforated aluminium foil covering the peat moss completely. The tuber was covered completely with gravel (Fig. 1.1). The pots were watered every two days. The holes in the aluminium foil allowed the roots to grow into the peat
J. Plant Physiol.
Vol. 121. pp. 23-27 (1985)
24
B. SATIELMACHER and H. MARSCHNER
2'---_ _-'
3L-_ _ -'
20"C
1"C 4L-_ _....J
5
L--_----'
Fig. 1: Scheme for the production and cultivation of tuberized stem cuttings. For explanation see text. moss (Fig. 1.2). After the onset of tuberization (Fig. 1.3) the gravel was poured off and all the tubers were removed (Fig. 1.4). Thereafter the third fully developed leaf was cut off, and the node was covered with Kleenex tissue wetted with saturated calcium sulphate solution. In order to prevent evaporation and to keep the node dark the Kleenex was covered with aluminium foil. Approximately ten days after removal of the tubers a swelling of the axillary buds was observed (Fig. 1.5). When the tubers had reached a diameter of approximately 2 cm, the plants were decapitated leaving just the second fully developed leaf, i.e. the leaf above the nodal tuber. The stem was cut 10 cm below the nodal tuber and all leaves and nodes of the lower stem were removed. The lower stem segment was sterilized superficially with a 5 % hypochlorite solution, and the cuttings were transfered under aseptical conditions to small sterile glass vessels containing 80ml nutrient solution. The vessels were closed with a cotton plug (Fig. 1.6). Thereafter, the cuttings were allowed to grow in a growth chamber at 20/15 °C day and night temperature, respectively. Light intensity at plant height was 250Wm- 2 • The nutrient solutions were cooled to + 1 °C in a water bath in order to reduce microbial activity to a minimum.
Results and Discussion Both source and concentration of nitrogen have a pronounced effect on tuber growth (Tab. 1). Compared to NH~03, glutamine is a much better form of nitrogen for the tuber growth in the system described. Apart from this, leaf longevity was reduced and malformation of tubers increased (knobby tubers and tuber regrowth) if f. Plant Physiol. Vol. 121. pp. 23-27 (1985)
Tuber growth in vitro
25
Table 1: Tuber fresh weight and nutrient solution uptake as influenced by nitrogen form and concentration (Duration of the experiment = 10 days, n = 8). Glutamine concentration (mM)
(g)
Tuber fresh weight
Uptake of Nutrient solution (mild)
9 18 36
11.6± 1.1 16A± 1.3 14.0±1.9
10.6± 1.7 12.0±2.5 8.1±2.7
8.5±3.1
8.5±3.9
glutamine concentration was increased above 18 mM or if ~N03 was the sole nitrogen source. Whereas in the in vitro system with wheat ears optimal grain growth requires addition of 3-4% sucrose to the nutrient solution (Waters et aI., 1984), addition of sucrose (2; 4, and 6 %) has negative effects on tuber growth, and leaf longevity was reduced. Based on these results sucrose was omitted in further experiments and the nutrient solution described by Waters et al. (1984) was used with minor modifications: Glutamine: 18 mM; CaS04: 0.6 mM, K2 S04: 1.1 mM, KH2 P04: 1.6 mM; MgS04: 3 mM, Fe-citrate (28 % Fe) 0.02 mM; H 3B0 3: 5 p.M; MnS04: 0.1 mM; (NH4)~07024: 0.001 p.M; ZnS04: 0.05 p.M; CuSO,: 0.5 p.M. The nutrient solution (80 ml per vessel) was renewed at least once a week. The uptake of the nutrient solution was recorded. Tuber growth rate was calculated from tuber volume increment. For this purpose tuber length and diameter was determined and tuber diameter calculated by assuming the tuber to be a rotary ellipsoid. Table 2: Mineral nutrient content of potato leaves, stems, and axillary tubers after growing for three weeks in semi-sterile culture. For composition of the nutrient solution, see above. Sample
N
P
Leaves Stems Tubers
2046 3.01 4.05
1.03 0040 0.63
K
Ca
Mg
2.97 3.58 2.54
1.91 1.64 0.14
0.66 0.32 0.15
(% in dry weight)
In Tab. 2 the mineral element composition of the cuttings after three weeks in vitro culture is shown. Compared to average data from intact potato plants, especially in the leaves, the concentration of phosphorus and potassium is high and low, respectively. Corresponding modifications in composition of the nutrient solution are therefore advisable. It appears necessary to state that the nutrient uptake by the cuttings is a purely passive process mediated by transpiration-driven influx of nutrient solution into the open xylem vessels of the stem base. This has both advantages and disadvantages. As advantage the uptake rate of the nutrient solution reflects the uptake rate of the individual nutrients. As disadvantage, the composition of the nutrient solution has to be carefully adjusted to the demands of the cuttings.
J Plant Physiol. Vol. 121. pp. 23-27 (1985)
26
B.
SATIELMACHER
and H.
MARSCHNER
Leaf longevity is a limiting factor in the described in vitro system. Even under medium glutamine concentrations leaf longevity seldom exceeds three weeks. It was therefore attempted to improve leaf longevity by the addition of kinetin as it has been succesfully used for soybean explants grown in a nutrient solution (Neumann and Nooden, 1982). However, in potato cuttings kinetin had no effect at low concentration and at higher concentrations (5 and 10 mg/l) leaf senescence was even enhanced (Tab. 3). Whether lower kinetin concentrations or cytokinins such as zeatin could prolong leaf longevity remains an open question. Tuber growth rate is relatively low in the described system (Tab. 3) if compared with data from intact plants (Engels, 1983). However, net assimilation rate (NAR) calculated from volume increment by assuming a tuber dry matter content of 20 % leads to a five times higher NAR than that given by Watson (1947) as optimum for field grown plants. It is assumed, therefore, that the small leaf area of the single node cuttings used in our system, cannot meet the carbohydrate demands of the axillary tubers. Shortage of assimilates could also be responsible for early leaf senescence due to excessive retranslocation of assimilates from the leaf and the stem segment to the tuber. Such a speculation is supported by the observed formation of big medullary cavities in the stem segments carrying axillary tubers. It therefore appears possible to increase both tuber growth rate and leaf longevity by addition of low sucrose concentrations to the nutrient solution. Table 3: Tuber growth rate and nutrient solution uptake as influenced by kinetin. Kinetin concentration (ppm)
o 1
5 10
(cm 3 /d)
Tuber growth rate
Uptake of nutrient (mild)
Leaf longevity (d)
0.25±0.06 0.32±0.02 0.17±0.02 0.20±0.01
60.0±14 45.0±22 3S.0± 12 42.0±15
21 21 14
7
Early leaf senescence could, however, also be the result of high endogenous ABA levels as has been reported for potato leaves after tuberization (Krauss and Marschner, 1982). In the past, stem cuttings have been used to study effects of daylength (Ewing and Wareing, 1978) and other factors related to tuberization in potato (Kahn and Ewing, 1983). It is suggested, that the system described here, can be used to study the effects especially of mineral nutrient supply on tuber growth rate. Modifications in the concentration of the nutrient solution as mentioned above, are advisable to increase both tuber growth rate and leaf longevity. Acknowledgements The authors thank Mr. H. Bremer and Mrs. E. Gorgus for skillful technical assistance.
J. Plant Physiol. Vol.
121. pp. 23-27 (1985)
Tuber growth in vitro
27
References BoYD, D. A.: Pot. Symp. 9, 461-473 (1970). DONAVAN, G. R. andJ. W. LEE: Plant Science Letters 99,107-113 (1977). DYSON, P. W. and D. J. WATSON: Ann. Bio!. 69, 47 -63 (1971). ENGELS, CHR.: Dissertation University Hohenheim, 1983. EWING, E. E. and P. F. WAREING: Plant Physio!' 61, 348-353 (1978). FORSTER, H. and H. BERINGER: Z. Pflanzenernahr. Bodenk. 146, 572-582 (1983). KAHN, B. A. and E. E. EWING: Ann. Bot. 52, 861-871 (1983). KODA, Y. and Y. OKAZAWA: Jap. J. Crop. Sci. 52, 582-591 (1983). KRAuss, A. and H. MARSCHNER: Z. Pflanzenernahr. Bodenk. 139, 143-155 (1976). - - Potato Res. 25, 13 -22 (1982). LINDAUER, M. G. and H. E. HAEDER: Landw. Forsch. 40, 209-216 (1984). MOLL, A.: Z. Acker und Pflanzenbau 25, 457 -463 (1981). MOORBy,J.: Ann. Bot. 32,57-68 (1968). NEUMANN, P. M. and L. D. NOODEN: J. Plant Nutr. 6, 735-742 (1982). PRUMMEL, J. and P. A. VAN BARNAU-SIJTHOFF: Fertilizer Res. 5, 203-211 (1984). SATTELMACHER, B. and H. MARSCHNER: Physio!' Plant. 44, 65-68 (1977). WATERS, S. P., P. MARTIN, and P. T. LEE: J. Exp. Bot. 35,829-840 (1984). WATSON, D. J.: Ann. Bot. 11, 41-76 (1963).
J. Plant Physiol. Vol.
121. pp. 23-27 (1985)
Abstract A simple in vitro method is described which allows the study of effects of mineral nutrient supply on growth rate of the tuber. Single node cuttings carrying axillary tubers are cultured under semi-sterile conditions in cooled ( + 1 0C) nutrient solutions. Tuber growth is monitored volumetrically and mineral nutrient uptake calculated from the nutrient solution taken up by the cuttings. Key words: Potato, tuber growth, in vitro.
Introduction
The influence of mineral nutrients on potato tuber growth has been the subject of many studies. Especially nitrogen (Watson, 1963; Moorby, 1967; Dyson and Watson, 1971) but also phosphate (Prummel and van Barnau-Sijthoff, 1984), and potassium (Boyd, 1970) have a pronounced influence on tuber growth. Plant nutrients may reveal their effects on tuber growth by influencing the shoot growth (Moll, 1981), by affecting the phytohormone balance in roots, shoots and tubers (Krauss and Marschner, 1976; Sattelmacher and Marschner, 1977) or by influencing the tuber growth directly (Koda and Okazawa, 1983). However, in studies with intact plants it is difficult, or even impossible, to distinguish between direct and indirect effects of mineral nutrient supply on tuber growth. This holds true for example for the negative correlation between potassium and starch content in potato tubers (Forster and Beringer, 1983; Lindauer and Haeder, 1984). For causal explanations in vitro experiments are therefore desirable. Standard procedures for cultivation of potato tubers in vitro (Koda and Okazawa, 1983) require sterile material and are therefore time-consuming and laborious. In the present paper a simple method is described to cultivate potato cuttings carrying axillary tubers under semi-sterile conditions. The system used is similar to the one described for cultivating wheat ears (Donavan and Lee, 1977). Materials and Methods Well sprouted potato tubers (cultivar Achat) were planted into 51 pots filled with about 3~ 1 of peat moss. The tubers were placed on top of perforated aluminium foil covering the peat moss completely. The tuber was covered completely with gravel (Fig. 1.1). The pots were watered every two days. The holes in the aluminium foil allowed the roots to grow into the peat
J. Plant Physiol.
Vol. 121. pp. 23-27 (1985)
24
B. SATIELMACHER and H. MARSCHNER
2'---_ _-'
3L-_ _ -'
20"C
1"C 4L-_ _....J
5
L--_----'
Fig. 1: Scheme for the production and cultivation of tuberized stem cuttings. For explanation see text. moss (Fig. 1.2). After the onset of tuberization (Fig. 1.3) the gravel was poured off and all the tubers were removed (Fig. 1.4). Thereafter the third fully developed leaf was cut off, and the node was covered with Kleenex tissue wetted with saturated calcium sulphate solution. In order to prevent evaporation and to keep the node dark the Kleenex was covered with aluminium foil. Approximately ten days after removal of the tubers a swelling of the axillary buds was observed (Fig. 1.5). When the tubers had reached a diameter of approximately 2 cm, the plants were decapitated leaving just the second fully developed leaf, i.e. the leaf above the nodal tuber. The stem was cut 10 cm below the nodal tuber and all leaves and nodes of the lower stem were removed. The lower stem segment was sterilized superficially with a 5 % hypochlorite solution, and the cuttings were transfered under aseptical conditions to small sterile glass vessels containing 80ml nutrient solution. The vessels were closed with a cotton plug (Fig. 1.6). Thereafter, the cuttings were allowed to grow in a growth chamber at 20/15 °C day and night temperature, respectively. Light intensity at plant height was 250Wm- 2 • The nutrient solutions were cooled to + 1 °C in a water bath in order to reduce microbial activity to a minimum.
Results and Discussion Both source and concentration of nitrogen have a pronounced effect on tuber growth (Tab. 1). Compared to NH~03, glutamine is a much better form of nitrogen for the tuber growth in the system described. Apart from this, leaf longevity was reduced and malformation of tubers increased (knobby tubers and tuber regrowth) if f. Plant Physiol. Vol. 121. pp. 23-27 (1985)
Tuber growth in vitro
25
Table 1: Tuber fresh weight and nutrient solution uptake as influenced by nitrogen form and concentration (Duration of the experiment = 10 days, n = 8). Glutamine concentration (mM)
(g)
Tuber fresh weight
Uptake of Nutrient solution (mild)
9 18 36
11.6± 1.1 16A± 1.3 14.0±1.9
10.6± 1.7 12.0±2.5 8.1±2.7
8.5±3.1
8.5±3.9
glutamine concentration was increased above 18 mM or if ~N03 was the sole nitrogen source. Whereas in the in vitro system with wheat ears optimal grain growth requires addition of 3-4% sucrose to the nutrient solution (Waters et aI., 1984), addition of sucrose (2; 4, and 6 %) has negative effects on tuber growth, and leaf longevity was reduced. Based on these results sucrose was omitted in further experiments and the nutrient solution described by Waters et al. (1984) was used with minor modifications: Glutamine: 18 mM; CaS04: 0.6 mM, K2 S04: 1.1 mM, KH2 P04: 1.6 mM; MgS04: 3 mM, Fe-citrate (28 % Fe) 0.02 mM; H 3B0 3: 5 p.M; MnS04: 0.1 mM; (NH4)~07024: 0.001 p.M; ZnS04: 0.05 p.M; CuSO,: 0.5 p.M. The nutrient solution (80 ml per vessel) was renewed at least once a week. The uptake of the nutrient solution was recorded. Tuber growth rate was calculated from tuber volume increment. For this purpose tuber length and diameter was determined and tuber diameter calculated by assuming the tuber to be a rotary ellipsoid. Table 2: Mineral nutrient content of potato leaves, stems, and axillary tubers after growing for three weeks in semi-sterile culture. For composition of the nutrient solution, see above. Sample
N
P
Leaves Stems Tubers
2046 3.01 4.05
1.03 0040 0.63
K
Ca
Mg
2.97 3.58 2.54
1.91 1.64 0.14
0.66 0.32 0.15
(% in dry weight)
In Tab. 2 the mineral element composition of the cuttings after three weeks in vitro culture is shown. Compared to average data from intact potato plants, especially in the leaves, the concentration of phosphorus and potassium is high and low, respectively. Corresponding modifications in composition of the nutrient solution are therefore advisable. It appears necessary to state that the nutrient uptake by the cuttings is a purely passive process mediated by transpiration-driven influx of nutrient solution into the open xylem vessels of the stem base. This has both advantages and disadvantages. As advantage the uptake rate of the nutrient solution reflects the uptake rate of the individual nutrients. As disadvantage, the composition of the nutrient solution has to be carefully adjusted to the demands of the cuttings.
J Plant Physiol. Vol. 121. pp. 23-27 (1985)
26
B.
SATIELMACHER
and H.
MARSCHNER
Leaf longevity is a limiting factor in the described in vitro system. Even under medium glutamine concentrations leaf longevity seldom exceeds three weeks. It was therefore attempted to improve leaf longevity by the addition of kinetin as it has been succesfully used for soybean explants grown in a nutrient solution (Neumann and Nooden, 1982). However, in potato cuttings kinetin had no effect at low concentration and at higher concentrations (5 and 10 mg/l) leaf senescence was even enhanced (Tab. 3). Whether lower kinetin concentrations or cytokinins such as zeatin could prolong leaf longevity remains an open question. Tuber growth rate is relatively low in the described system (Tab. 3) if compared with data from intact plants (Engels, 1983). However, net assimilation rate (NAR) calculated from volume increment by assuming a tuber dry matter content of 20 % leads to a five times higher NAR than that given by Watson (1947) as optimum for field grown plants. It is assumed, therefore, that the small leaf area of the single node cuttings used in our system, cannot meet the carbohydrate demands of the axillary tubers. Shortage of assimilates could also be responsible for early leaf senescence due to excessive retranslocation of assimilates from the leaf and the stem segment to the tuber. Such a speculation is supported by the observed formation of big medullary cavities in the stem segments carrying axillary tubers. It therefore appears possible to increase both tuber growth rate and leaf longevity by addition of low sucrose concentrations to the nutrient solution. Table 3: Tuber growth rate and nutrient solution uptake as influenced by kinetin. Kinetin concentration (ppm)
o 1
5 10
(cm 3 /d)
Tuber growth rate
Uptake of nutrient (mild)
Leaf longevity (d)
0.25±0.06 0.32±0.02 0.17±0.02 0.20±0.01
60.0±14 45.0±22 3S.0± 12 42.0±15
21 21 14
7
Early leaf senescence could, however, also be the result of high endogenous ABA levels as has been reported for potato leaves after tuberization (Krauss and Marschner, 1982). In the past, stem cuttings have been used to study effects of daylength (Ewing and Wareing, 1978) and other factors related to tuberization in potato (Kahn and Ewing, 1983). It is suggested, that the system described here, can be used to study the effects especially of mineral nutrient supply on tuber growth rate. Modifications in the concentration of the nutrient solution as mentioned above, are advisable to increase both tuber growth rate and leaf longevity. Acknowledgements The authors thank Mr. H. Bremer and Mrs. E. Gorgus for skillful technical assistance.
J. Plant Physiol. Vol.
121. pp. 23-27 (1985)
Tuber growth in vitro
27
References BoYD, D. A.: Pot. Symp. 9, 461-473 (1970). DONAVAN, G. R. andJ. W. LEE: Plant Science Letters 99,107-113 (1977). DYSON, P. W. and D. J. WATSON: Ann. Bio!. 69, 47 -63 (1971). ENGELS, CHR.: Dissertation University Hohenheim, 1983. EWING, E. E. and P. F. WAREING: Plant Physio!' 61, 348-353 (1978). FORSTER, H. and H. BERINGER: Z. Pflanzenernahr. Bodenk. 146, 572-582 (1983). KAHN, B. A. and E. E. EWING: Ann. Bot. 52, 861-871 (1983). KODA, Y. and Y. OKAZAWA: Jap. J. Crop. Sci. 52, 582-591 (1983). KRAuss, A. and H. MARSCHNER: Z. Pflanzenernahr. Bodenk. 139, 143-155 (1976). - - Potato Res. 25, 13 -22 (1982). LINDAUER, M. G. and H. E. HAEDER: Landw. Forsch. 40, 209-216 (1984). MOLL, A.: Z. Acker und Pflanzenbau 25, 457 -463 (1981). MOORBy,J.: Ann. Bot. 32,57-68 (1968). NEUMANN, P. M. and L. D. NOODEN: J. Plant Nutr. 6, 735-742 (1982). PRUMMEL, J. and P. A. VAN BARNAU-SIJTHOFF: Fertilizer Res. 5, 203-211 (1984). SATTELMACHER, B. and H. MARSCHNER: Physio!' Plant. 44, 65-68 (1977). WATERS, S. P., P. MARTIN, and P. T. LEE: J. Exp. Bot. 35,829-840 (1984). WATSON, D. J.: Ann. Bot. 11, 41-76 (1963).
J. Plant Physiol. Vol.
121. pp. 23-27 (1985)