ISSN 10683674, Russian Agricultural Sciences, 2010, Vol. 36, No. 5, pp. 331–333. © Allerton Press, Inc., 2010. Original Russian Text © A.S. Lukatkin, 2010, published in Doklady Rossiiskoi Akademii Sel’skokhozyaistvennykh Nauk, 2010, No. 5, pp. 10–12.
PLANT INDUSTRY
Use of Maize Callus Cultures for Assessing Chilling Stress Resistance1 A. S. Lukatkin N.P. Ogarev Mordovian State University, Saransk, 430005 Russia email:
[email protected] Received June 9, 2009
Abstract—The production of maize callus lines and their use for assessing chilling injury are described. The substantial influence of physiological factors (type of explant, age of plant and culture) on the stress resistance of cells is shown. It is concluded that maize callus cultures can be used as a model object when studying stress resistance at the cellular level. Key words: maize, callus cultures, resistance, chilling stress DOI: 10.3103/S1068367410050046 1
In recent years the cell and tissue culture method has been being used increasingly for studying the effect of various extreme environmental conditions (abiotic and biotic) on plants. The use of cell cultures as exper imental objects when investigating plant injury mech anisms has a number of advantages, making it possible to assess cell physiology parameters; in this case, cell cultures and protoplasts are a rather homogeneous system in which many problems associated with inter cellular regulatory systems are eliminated [1–4]. The use of cell cultures for these purposes requires a thorough study of the characteristics of the lines obtained since the response of cells to an external fac tors changes under the influence of heterogeneity of the callus culture related to epigenetic characteristics of the initial cells of the explant [5] as well as the culturing conditions [3, 6, 7]. It is necessary to ascertain exactly the contribution of internal and external factors to the cellular stress response. In this case, special attention was devoted to revealing the dependence of the degree of chilling injury of cells on physiological characteristics (initial explant, age of plants and culture). METHODS
Variety Bukovinskaya 2 maize (Zea mays L.) plants served as the material for the investigations. Sterile plants, which were used for subsequent explantation, were grown from surfacesterilized seeds on hormone free, agarized Murashiga–Skoog (MS) nutrient medium containing 0.8% agar and 15 g/l sucrose under dark or light (illuminance 5000 lx) conditions. We isolated segments of the coleoptile, leaves, and root of size 1–2 cm and growing point of size 1–8 mm and 1 The work was supported by the Federal Education Agency (Ana
lytical Departmental Target Program “Development of the Sci entific Potential of High School,” project no. 2.1.1/624).
placed them on MS medium containing 1–2 mg/l 2,4dichlorophenoxyacetic acid (2,4D) and 0.5 mg/l 6benzylaminopurine (BAP). Petri dishes with explants were placed under dark conditions (temperaturecontrolled cabinet, temper ature 25°C). After 3–4 weeks, the primary callus was transplanted to fresh nutrient medium containing 1 mg/l 2,4D and 0.5 mg/l BAP (or without BAP). The response of cells to chilling stress was determined after 2–6 weeks of growth of the secondary callus. For this purpose, the calli were held for 24 h in a refriger ating chamber at 1°C; the control calli were held for the same time in the temperaturecontrolled cabinet at 25°C. After chilling, we determined the viability of cells of the control and chilled variants on the basis of reduction of triphenyl tetrazolium chloride (TTC) [8]. For this, the callus was placed in 1 ml of a 0.8% TTC solution in a 0.05 M sodium phosphate buffer (pH 7.5) and after 24 h the optical density of an alcohol extract from calli was determined on an SF46 spectropho tometer at wavelength 485 nm. The degree of chilling injury of calli of different lines was assessed on the basis of the viability of cells after chilling: the higher the percent of preservation of the ability of cells to reduce TTC after chilling, the less the injury. The experiments were conducted in three and fourfold analytical replications, and each experiment was repeated 4–6 times. The tables give the average values from all experiments with standard errors. RESULTS AND DISCUSSIONS The most suitable for maize culturing was MS medium with 1 mg/l 2,4D, on which we observed the formation of callus on various explants (except the root). Callus growth noticeably slowed with increase of auxin concentration. The addition of even small amounts of cytokinins (0.5 mg/l BAP) to the optimal
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LUKATKIN
Table 1. Dependence of callogenesis of explants on plant age Primary callus Explant
Transplant culture
age (days) after planting 3
Coleoptile Leaf Root Growing point
4
5
6
3
+ + + – + + + – – + – – – – – +++ +++ +++ +++ ++
4
5
6
– – – ++
– – – ++
– – – ++
Note: +++ very intensive callogenesis; ++ intensive; + weak; –there was no callus formation and growth.
Table 2. Dependence of the degree of chilling injury of callus tissue on initial explant (according to reduction of TCC) Reduction of TCC, optical density units
Explant
control Coleoptile Leaf Growing point
chilling
0.112 ± 0.004 0.069 ± 0.002 0.410 ± 0.006 0.098 ± 0.004 0.190 ± 0.007 0.056 ± 0.002
% of viable cells after chilling 61.6 ± 6.2 23.9 ± 4.2 29.5 ± 6.1
Table 3. Dependence of viability of plant callus cells on cul ture age Culture age, weeks 2.0 2.5 5.0 6.5
Reduction of TTC, optical density units control
chilling
% of viable cells after chilling
0.160 ± 0.002 0.180 ± 0.003 0.150 ± 0.003 0.680 ± 0.030
0.088 ± 0.004 0.079 ± 0.004 0.059 ± 0.002 0.132 ± 0.021
55.0 ± 8.5 43.9 ± 4.2 39.3 ± 5.4 19.4 ± 1.9
concentration of auxins (1 mg/l 2,4D) led to the for mation of slowgrowing calli, which quickly necro tized and died; callogenesis didn’t occur in the variant with 2 mg/l 2,4D and 0.5 mg/l BAP. The addition of BAP probably inhibited it due to the high cytokinin status of the young maize plants. Induction of callogenesis strongly depended on the lighting conditions. With the use of plants grown in the dark, callus formed almost on all explants (except the root). In plants grown under 5000lx illuminance, cal logenesis was noted only on isolated growing points; all other explants (leaf, stem, and root) were not able to form callus tissue. Accelerated differentiation of cells in light probably hinders the process of dediffer entiation.
The ability to form callus decreased in almost all explants with increasing plant age (Table 1). In this case, disks from older leaves and stem didn’t form it. Callogenesis occurred weakly on leaf and stem explants from relatively young plants, their growth ceased after transplanting primary calli to fresh nutri ent medium, and after 2–3 weeks of culture they fre quently necrotized and died. The growing point was the sole explant in which callogenesis practically didn’t depend on the age of the initial plant; however, the growth rate of the calli decreased somewhat with increasing plant age. The apical meristem, the size of which in maize is not greater than 0.2 mm, plays a determining role in the formation of callus on the growing point. It is bet ter to minimize the size of the explant for successful culturing. However, it was revealed in the experiment that callus didn’t form in the case of explanting a growing point with a size less than 1 mm. Coleoptile growth was observed upon explanting a section of the growing point with a size of more than 4 mm. Maxi mally rapid callus formation was on sections of the growing point with a size of 2–3 mm. Several methods are used for assessing the response of cells to stress: reduction of TTC [1, 6, 8–10], intra vital staining of cells [2, 3, 10], exosmosis of electro lytes [9, 10], and regrowth after stress [2, 6, 9] based on various physiological processes. We investigated the response of callus cultures to low temperatures because maize is a typical representative of chilling sensitive plants. In this case, viability of the cells was determined from the reduction of TTC as an indicator of the functional stage of mitochondria. The callus lines obtained on different explants differed in the ability to reduce TTC both in the control and after chilling (Table 2), which probably reflects the epige netic characteristics of cells in an in vitro culture. With respect to degree of preservation of viable cells after stress, the most resistant were calli from the coleoptile; other lines displayed a similar response and were sub stantially less chillresistant. A comparison of callus lines obtained from plants of various ages showed that cell survival after chilling stress has a tendency to increase with increasing age of the initial plant (data are not given). The age of the transplant culture had a great effect on the degree of injury. Maximum viability of cells after chilling stress was noted in the transplant culture at an age of 2 weeks, after which it progressively decreased (Table 3). Substantial chilling injury was found in cells of older calli in late growth stages. It is seen from the given data that sensitivity of cell cultures to low temper atures is determined to a considerable extent by both epigenetic (related to the initial explants) and physio logical characteristics of the culture. Thus, maize callus cultures can be used as a model object when studying stress resistance at the cellular level. However, to determine resistance, it is necessary
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to carefully assess the contribution of physiological (characterizing the properties of the initial plant and culture) and external factors to the development of cell response to an unfavorable factor. REFERENCES 1. Muhlbach, H.P. and Thiele, H., Response to Chilling of Tomato Mesophyll Protoplasts, Planta, 1981, vol. 151, no. 3, pp. 399–401. 2. DuPont, F.M., Staraci, L.C., Chou, B., Thomas, B.R., Williams, B.G., and Mudd, J.B., Effect of Chilling Temperatures upon Cell Cultures of Totamo, Plant Physiol., 1985, vol. 77, no. 1, pp. 64–68. 3. Pomeroy, M.K. and Mudd, J.B., Chilling Sensitivity of Cucumber Cotyledon Protoplasts and Seedlings, Plant Physiol., 1987, vol. 84, no. 3, pp. 677–681. 4. Sergeeva, L.E. and Mikhal’ska, S.I., Sunflower Cell Culture: An Experimental System for Studying the Effect of the Cadmium Ion, Fiziol. Biokhim. Kul’turn. Rast., 2005, vol. 37, no. 6, pp. 519–523. 5. Dias, S., Dolgikh, Yu.I., Shamina, Z.B., and Sheve lukha, V.S., Significance of Physiological and Genetic
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Factors in Induction of Embryogenic Callus in Various Maize Lines, Dokl. Rossel’khozakademii, 1994, no. 2, pp. 6–8. 6. Xin Zhanguo and Li, P.H., Abscisic AcidInduced Chilling Tolerance in Maize SuspensionCultured Cells, Plant Physiol., 1992, vol. 99, no. 2, pp. 707–711. 7. Lukatkin, A.S. and Geras’kina, A.V., Screening Cucumber Cell Cultures for Increased Chilling Resis tance, Biotekhnologiya, 2003, no. 3, pp. 65–73. 8. Enikeev, A.G., Vysotskaya, E.F., Leonova, L.N., and Gamburg, K.Z., On the Use of 2,3,5Triphenyl Tetra zolium Chloride for Assessing the Viability of Plant Cell Cultures, Fiziol. Rast., 1995, vol. 42, no. 3, pp. 423– 426. 9. Wu MinTze and Wallner, S.J., Heat Stress Responses in Cultured Plant Cells: Development and Comparison of Viability Tests, Plant. Physiol., 1983, vol. 72, no. 3, pp. 817–820. 10. Samygin, G.A., Volkova, L.A., and Popov, A.S., Com parison of Different Methods for Assessing Viability of Cells of Suspension and Callus Cultures, Fiziol. Rast., 1985, vol. 32, issue 4, pp. 813–818.
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