Potato Research 39 (1996) 2 5 3 - 258

Potato micropropagation: Growth of cultivars in solid and liquid media A. D E L. A V I L A , S.M. P E R E Y R A and J.A. A R G O E L L O Laboratorio de Fisiologfa Vegetal, Facultad de Ciencias Agropecuarias. Departamento de Producci6n Vegetal, Universidad Nacional de C6rdoba, C.C. N~509, 5000 C6rdoba, Argentina Accepted for publication: 14 March 1996 Additional keywords: Solanum tuberosum L., nitrogen content, sugar content, Spunta, Kennebec, Huinkul, nutrient availability

Summary The effect of liquid and solid media on the growth of micropropagated potato shoots (cvs Spunta, Kennebec and Huinkul) was studied in relation to the availability of nutrients and water in the culture medium. Nitrogen and sugar assimilation in the shoot and the water content of the shoots were evaluated. The water content of the plant tissues was not affected by the physical state of the medium. The liquid medium caused a greater accumulation of dry matter in the shoot without changing assimilate partition. The liquid medium increased shoot growth without affecting the number of nodes. The higher growth rate of shoots in lfquid medium was correlated with increases in their organic nitrogen and sugar contents, indicating that nutrient assimilation was favoured in liquid media.

Introduction Micropopagation is a rapid multiplication technique used to obtain high quality potato seed tubers (Bryan et al., 1981), one of its main objectives being to optimize in vitro shoot growth. Considerable research has been done on the nutritional (Wetherell & Dougall, 1976: Dougall, 1981: Avila et al., 1994) and hormonal (Singha, 1982) aspects of culture media preparation: however, the physical state of the culture media and its effects on the mechanisms that regulate explant growth have received less attention (Singha, 1982). It has been observed that liquid or low-agar concentration media increase the growth of certain species cultivated in vitro (Singha, 1982: Debergh, 1983: Rossel et al., 1987). Low-agar concentration media increased root and shoot growth in apples cultivated in vitro ( R o m b e r g e r & Tabor, 1971: Singha, 1982). Similarly, liquid media increased the growth of cv. Spunta two-fold (Rossel et al., 1987); however, the growth-mechanisms were not elaborated upon. Previous findings indicate that the improved growth in species cultivated in liquid or low-agar concentration media is caused by greater availability of water (Bouniols, 1974: Debergh, 1983) and of nutrients (Singha, 1982; Debergh, 1983). This increased availability may be induced by a lower resistance to diffusion and closer contact between the explant and the culture medium (Romberger & Tabor, 1971: Hammerschlag, 1982: Singha, 1982; Pierik, 1990); however, these effects have not been fully proven.

Potato Research 39 (1996)

253

A. DE L. AVILA, S.M. PEREYRA A N D J.A. A R G U E L L O

The aim of the present work was to evaluate the effects of liquid and solid media on the growth of micropopagated potato cultivars, as related to a) nutrient assimilation, in terms of nitrogen and sugar content in the shoot; and b) water availability, measured as water potential in the culture medium and the water content of the shoot. Material and methods

Plant material. Cvs Kennebec, Spunta, and Huinkul were cultured from nucleus plants obtained from virus-free tubers provided by the Facultad de Ciencias Agropecuarias, Universidad Nacional de C6rdoba, Argentina. Plantlets grown from nodal cuttings with one axillary bud were used in culture techniques according to Hussey & Stacey (1981) and Avila et al. (1994). Nutrient media. The media for nodal cuttings contained Murashige & Skoog (1962) mineral salts and were either solid, in 0.7% agar (Sigma), or liquid. They were supplemented with 100 mg of inositol, 0.4 mg of thiamine-HCl, 2.0 mg of Capantothenate, and 30 g of sucrose per litre (Tovar et al., 1985). Eight ml of liquid or solid medium, pH 5.7-5.8, were placed in glass tubes (diameter 25 mm, length 150 mm), which were sterilized at 4.9 mPa for 30 min. Nodal-cutting culture. Plantlets (8-10 nodes) were cut into nodal sections with a single axillary bud, using sterile techniques in a laminar-flow cabinet, and one explant was placed in each tube. Culture conditions. Cultures were maintained for 30 days in a growth chamber at 20_+1 ~ and exposed to light from cool-white and Gro-Lux (Sylvania) fluorescent tubes for 16 h day-1. Light intensity was 47.5 laE m-2 s-I of photosynthetically active radiation. Growth. nutrient assimilation and water availability. Growth was evaluated after 30 days, by recording root and shoot dry weight (mg), shoot length (mm). number of nodes, and dry matter partition into shoot and root (%). Nutrient availability was evaluated after 30 days by measuring the nitrogen and sugar content (mg) in the shoot. Organic nitrogen content was determined following the procedure of Capo et al. (1955) and sugar content by the technique of Yemm & Willis (1954). The availability of water in the culture media was evaluated by water potential (~w) using a thermocouple psychrometer. The water content of the shoot was expresed as percentage of water on fresh weight basis. Experimental design and statistical analysis. Experiments were repeated at least three times, and each experiment comprised 10 replicate tubes (10 plantlets) of each cultivar arranged in a randomized design. Analysis of variance was performed on the data; means were compared using Tukey's test and correlations were performed using Pearson's coefficient. 254

Potato Research 39 (1996)

POTATO MICROPROPAGATION

IN S O L I D A N D L I Q U I D M E D I A

Results and discussion All the explants showed marked increases in elongation and dry weight in liquid culture (Figs 1 & 2), concurring with similar observations on cv. Spunta (Rossel et al., 1987), and on other species (Singha, 1982: Tran Thanh Van & Trinh, 1978). However. the liquid medium did not increase the number of nodes (Fig. 1), as was observed in apple in vitro culture (Singha, 1982) in which an increased number of shoots were noted in liquid and Iow-agar concentration media. Table 1 shows that the solid medium did not significantly (P<0.05) affect either the water potential of the culture medium or the percentage of water in the shoot. This suggests that the effects of the medium are not related to its water availability. Contrary to these results, studies on other species (Bouniols, 1974: Hammerschlag, 1982: Debergh, 1983) found that high agar concentration media decreased water availability, limiting shoot growth. However, our work shows that the increase in

Spun~

~

H

Kennebec q

Hulnkul

~ - - 4

~

,4

~

-

-

~

Height (mm)

:,,qld

Soi!d ~

, i,.luid

NRNodes

Fig. 1. Shoot growth (height and number of nodes) of three cultivars propagated in liquid and solid media. Bar length indicates standar error of the mean (P<0.05%).

Spunta

~-

Kennebec ~

Huinkul __

_q 15

i 10

~.

~ - i

Soiia

- ~

Sond

-

~

Liquid

~

~

Solid

L I

Shoot dry weight mg)

0

9 a 8 Root dry weight (mgl

Fig. 2. Root and shoot dry matter content in three cultivars propagated in liquid and solid media. Bar length indicates standar error of the mean (P_<0.05% ). Potato Research 39 (1996)

255

A. DE L. AVILA,S.M. PEREYRAAND J.A. ARGUELLO Table I. Water potential (~w) in the culture media and percentage of water in the shoots of three cultivars propagated in liquid and solid media.

Liquid Solid LSD (0.05)

Media

Water in shoot (% of fresh weight)

Water potential (Mpa)

Spunta

Kennebec

Huinkul

0.89635 0.84716 0.085

87.6 87.6 0.9

88.7 89.2 1.2

86.7 87.1 1.1

a

a

0.8

'II 0.4

2

12

a

B_

0

,- 10 a |

a

6

0

o

4

a

ct -

Z

L,. m

/ SPUNTA

KENNEBEC

r/t

HUINKUL

Fig. 3. Nitrogen content (A), sugar content (B) and sugar/nitrogen ratio (C) in potato shoots propagated in liquid ([3) and solid (m) media. Different letters represent significant differences (P_<0.05%). 256

Potato Research 39 (1996)

POTATO MICROPROPAGATION IN SOLID A N D LIQUID MEDIA

Table 2. Correlation coefficients between shoot length, nitrogen content, and sugar content in shoots of three cultivars propagated in liquid and solid media. Parameters

Shoot length and nitrogen content Shoot length and sugar content Dry weight and nitrogen content Dry weight and sugar content

Cultivar Spunta

Kennebec

Huinkul

0.749":* 0.983** 0.986** 0.761"*

0.613" 0.216 0.469 0.409

0.949** 0.751"* 0.899** 0.528**

* P_<0.05, ** P<0.01

shoot growth in the liquid medium cannot be attributed to an increase in water availability. Total dry matter and soluble sugar content increased significantly in shoots cultured in liquid medium (Figs 2 & 3B), concurring with similar observations on Malus sp. (Singha, 1982) cultured in vitro in low-agar concentration media. It thus seems likely that sugar uptake by the explant is increased in liquid medium. AlthQugh dry matter accumulation was approximately two fold greater in the liquid than in the solid medium, dry matter partitioning was not affected. Shoot nitrogen content increased in liquid medium (Fig. 3A). These results indicate that increased shoot growth is related to increased assimilation of nitrogen and sugar in the culture medium (Table 2). Our results, suggest that nutrient assimilation is favoured in liquid media due to an increase in the nutrient incorporation rate. The influence of agar on the culture media could be explained by its effect on the reduced speed at which nutrients are diffused throughout the medium (Romberger & Tabor, 1971: Pierik, 1990). On the other hand, the sugar/nitrogen ratio was higher in liquid medium (Fig. 3C), suggesting that carbon metabolism is favoured over nitrogen metabolism. This, in turn, increases the carbon/nitrogen ratio, which could alter the physiological quality of the shoots cultivated in liquid medium, as observed in bean leaves in vivo (Pereyra & Trippi, 1992). This may diminish the regenerative capacity of the shoots in subsequent subcultures in liquid media.

Acknowledgements The work was supported by Consejo de Investigaciones Cientfficas y Tecnol6gicas de C6rdoba ( C O N I C O R ) and Secretarfa de Ciencia y T6cnica de la Universidad Nacional de C6rdoba (SECYT), Argentine Republic.

Potato Research 39 (1996)

257

A. DE L. AVILA, S.M. PEREYRA AND J.A. ARGOELLO

References Avila, A. de L., S.M. Pereyra, D.J. Collino & J.A. Argiaello, 1994. Effects of nitrogen source on growth and morphogenesis of three micropropagated potato cultivars. Potato Research 37: 161-168. Bouniols, A., 1974. Neoformation de bourgeons in vitro h partir de fragments de racime d'endive (Cichorium intybus L.): influence du degr6 d'hydratation des tissus, et ses cons6quences sur la composition en acides amin6s. Plant Science Letters 2: 363-371. Bryan, J.E., M.T. Jackson & N.J. Melendez, 1981. Rapid multiplication techniques for potatoes. International Potato Centre, Lima, Peru. Capo, B.G., G. Samuels, P. Lanrau, S. Alers & A. Riera, 1955. The method of foliar diagnosis as aplied to sugar cane. Agricultural Experimental Station, University of Puerto Rico, Bulletin 123. Debergh, P.C., 1983. Effects of agar brand and concentration on the tissue culture medium. Physiologia Plantarum 59: 270-276. Dougall, D.K., 1981. Media factors affecting growth. Experimental Botany 21: 277-280. Hammerschlag, F., 1982. Factors influencing in vitro multiplication and rooting of the plum rootstock myrabalan ( Prunus ceras(fera Ehrh.). Journal of American Society of Horticultural Science 107: 44-47. Hussey, G. & N.J. Stacey, 1981. In vitro propagation of potato (Solanum tuberosum L.). Annals of Botany 48: 787-796. Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobaco tissue cultures. Physiologia Plantarum 15: 473--497. Pereyra, S.M. & V.S. Trippi, 1992. Las relaciones C/N y PS/N durante el crecimiento y senescencia de hojas de Phaseolus vulgaris L. Agriscientia VII: 3-7. Pierik, R.L.M., 1990. Cultivo in vitro de las plantas superiores. Mundi-Prensa, Madrid, pp. 49-84. Romberger, J.A. & C. Tabor, 1971. The Picea abies shoot apical meristem in culture. I. Agar and autoclaving effects. American Jounal of Botany 58: 131-140. Rossel, G., G. D e Bertoldi & R. Tizzio, 1987. In vitro mass tuberisation as a contribution to potato micropropagation. Potato Research 30: 111-116. Singha, S., 1982. Influence of agar concentration on in vitro shoot proliferation of Malus sp. Almey and Pyrus communis Seckel. Journal of American Society of Horticultural Science 107: 657--660. Tran Thanh Van, K. & H. Trinh, 1978. Plant micropropagation: nonidentical and identical copies. In: K.W. Hughes, R. Henke & M. Constantin (Eds.). Propagation of higher plants through tissue culture. Technological Information Center, United States Department of Energy, USA, pp. 134--158. Tovar, P., R. Estrada, L. Schilde-Rentschler & J.H. Dodds, 1985. Inducci6n y utilizaci6n de tub6rculos in vitro de papa. CIP-Circular 13, pp. 12. Wetherell, D.F. & D.K. Dougall, 1976. Sources of nitrogen supporting growth and embryogenesis in cultured wild carrot tissue. Physiologia Plantarum 37: 97-103. Yemm, E.W. & A.J. Willis. 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochemistry Journal 57: 508-514.

258

Potato Research 39 (1996)