Размещено на http://www.allbest.ru/

Размещено на http://www.allbest.ru/

Optimization of nutrient medium composition by the mathematical design of experiment for shoot tip development in four grapevine genotypes

Virus elimination in grapevine may be achieved by heat therapy and shoot tip culture (Gribaudo et al., 1994; Regner et al., 1995; Anaclerio et al., 1999). Nepoviruses may easily be eradicated by heat therapy at 36°C for four weeks and by shoot tip culture while closteroviruses is much more difficult to achieve, and the size of shoot tip explants is of crucial importance (Regner et al., 1995). Grapevine plants were obtained from shoot tip explants containing one or two leaf primordia and not more than 0.3 mm long (Bini, 1976; Blaich, 1984; Duran-Vila et al., 1988; Maekawa et al., 1993; Regner et al., 1995).

Genotypes of the genus Vitis differ in their ability to uptake macro-element ions from the soil and their optimum concentrations in shoots and roots which are needed for plant growth (Scienza et al., 1986). Various concentrations of MgSO₄ and KH₂PO₄ in liquid and solid media were optimum for plant growth of four grapevine genotypes (Slenco et al., 2001). For development of shoots from shoot tip explants of different grapevine genotypes, media containing N6-benzyladenine (BA) and different concentrations of macro-elements were used (Novak and Juvova, 1983; Harris and Stevenson, 1982; Gribaudo et al., 1994; Maekawa et al., 1993).

This report attempted to optimize component concentrations levels of the nutrient medium using a mathematical design of experiments in order to improve shoot tip survival rate and to increase shoot growth from the explants. Optimized media for shoot tip development may be used for sanitation of grapevine plants from viruses in meristem cultures.

Plant material

The experiments were based on four grapevine genotypes: the root stock Riparia x Rupestris `Kober 5 BB' and cultivars released by the Institute for Vine and Wine `Magarach': `Podarok Magaracha' (interspecific cross of a Vitis vinifera L. and a Franco-American hybrid), `Zhemchug Magaracha' and seedless `Sverkhrannii bessemyannyi' (intraspecific crosses of Vitis vinifera L.).

Shoot tip culture and conditions

Green shoots collected from a mature vine released from dormancy or from field-grown plants in the beginning of the vegetation period were used as a source of explants. Shoots, about 1.0-1.5 cm long, were surface sterilized in 70% (v/v) ethanol for 20 s and then in 1% sodium hypochlorite with 0.1%. Tween 20 for 15 min, and then rinsed four times in sterile water. Shoot tips with several leaf primordia (0.5-0.8 mm long) were excised under a stereomicroscope and cultured on different medium versions solidified with 7 g l^-1 Difco agar. The medium versions were supplemented with 0.5 mg l^-1 BA, and the pH was adjusted to 5.6-5.8 with NaOH prier to autoclaving at 103 kPa for 25 min. The compositions of the media used in experiments are indicated in `Results and discussion'. All shoot tip cultures were maintained in culture tubes with 22 mm in diameter containing 5 ml of culture medium under a photon flux density at the culture surface of 55 ?mol m-2 s-1 provided by cool white fluorescent tubes with a 16-h photoperiod at 27^oC.

Plant growth and development

To obtain rooted plants, shoots with well-developed leaves grown from shoot tips were divided into single buds with one leaf and established on solid medium for rooting. The thick basal portion of the arms was not used for rooting. Vitrified shoots and leaves sometimes developed on medium with BA. In such cases, only shoots with two or three leaves were established on the rooting medium. The rooting medium composition was described by Zlenko et al. (1995) and Slenko et al. (2001) containing: 308 mg l^-1 NH4NO3, 922 mg l^-1 KNO3, 597 mg l^-1 MgSO4 7H2O, 122 mg l^-1 KH2PO4, 331 mg l^-1 CaCl2 (MS), 1/2 Fe-EDTA and 1/2 micro-elements MS, 20 mg l^-1 myo-inositol, 0.1 mg l^-1 thiamine (MS), 0.5 mg l^-1 nicotinic acid (MS), 0.2 mg l^-1 pyridoxine, 0.2 mg l^-1 indole-3-acetic acid (IAA), 10 g l^-1 sucrose and 7 g l^-1 Difco agar. Subsequent in vitro propagation of plants was by dividing them into single-bud explants with one leaf and establishing the latter on the same rooting medium (Slenko et al., 2001) except for the presence of 0.1 mg l^-1 IAA.

Mathematical design of experiment and statistical analysis

Evaluation of results and calculation of the overall quality criterion.

The development of shoot tips after 50 days was evaluated using the following criteria: survival rates (%), shoot length (cm) and leaf development by means of a 1-5 point numerical scale. The trait `leaf development' enters as the subjective (visual) evaluation of cytokhinin-induced vitrification and leaf lobation scored in points: 1 - very high vitrification and lobation or no leaf development at all, 2 - high vitrification and lobation, 3-4 - medium vitrification and lobation, and 5 - no lobation with the typical leaf shape of each cultivar.

Survival rates of shoot tips may be high but shoot tips may fail to develop into shoots with leaves. Therefore an overall quality criterion was needed calculated as follows (Zlenko et al., 1995). Since each trait (variables: Ix1,2,... i; IIx1,2,... i.... nx1,2,... i) varies to a larger or smaller extent depending on the medium composition, variation of the traits must be reduced to certain limits which reflected the usefulness of a trait, i.e. a more useful trait provided a wider scale of variation and the scale of variation of a less useful trait is narrower. Variation of traits (variables: It1,2,... i; IIt1,2,... i.... nt1,2,... i) on different medium versions within limits determined by us and the overall quality criterion (o.q.c.) were calculated using the following equations:

o.q.c. = It · IIt... ·... nt (3)

nutritious mathematical grapes genotype

where Ixmax; IIxmax....... nxmax and Ixmin; IIxmin....... nxmin are extreme values of traits; xi is the value being estimated of a trait; M is a random number determined depending on the usefulness of a trait, e.g. if M = 10, ti varies over a range of 0.1-1.0 and if M=2, ti varies over a range of 0.5-1.0. In this paper M = 10 for the traits of all processes.

Mathematical design of experiment. To calculate component concentrations in medium enabling optimum shoot development from shoot tips, a mathematical design basing on the random balance method was used (Hartmann et al., 1977). Since it is practically impossible to do a full polyfactorial experiment if too many factors are involved, each at three or two levels (such factors were component levels in numerous medium versions used in the experiment). Algorithms of multiple curvilinear stepwise regression were applied. The influence of the factors was evaluated based on the significance of the members of regression equations describing processes. Stepwise optimization of each process was calculated using the steepest ascent method of Box-Wilson. Component concentrations in media were used to increase the overall quality criteria of shoot development from shoot tips in four grapevine genotypes, therefore, optimum medium for each process was determined. This mathematical design used in the experiment was described in the previous study (Zlenko et al., 1995).

Statistical analysis. Each medium version for each cultivar was in 15 replications. Confidence limits of average values of the traits were calculated with significant at P<0,05. Deviations (variation coefficient V with significant at P<0,05) of shoot tip development from average values were not more than 23% for survival rates (%), 31% for the shoot lengths (cm), 34% for leaf development and 29% for the overall quality criteria (conventional units).

Calculation of regression equations describing shoot tip development in four grapevine genotypes and individually in rootstock `Kober 5BB'

Shoot growth in grapevine is affected by various nitrogen-containing salts presented in the nutrient medium and their concentrations (Villegas et al., 1992). Therefore, not only nitrogen sources of the MS formulation (NH4NO3 and KNO3) but also some other substances [(NH4)2SO4 and NH4Cl], KCl and K2SO4 as a source of the K⁺ ion were used in the media (Table I).

Meaning of factors expressed as natural and coded variables. Experiment focused on optimizing medium for shoot tip development in four grapevine genotypes: `Kober 5 BB', `Podarok Magaracha', `Zhemchug Magaracha' and "Sverkhrannii bessemyannyi"

These substances were applied at levels leading to the content of nitrogen in the form of ammonium or nitrate and the content of the K⁺ ion equal to those in the macro-elements of the full- and the half-strength MS medium. High cytokinin levels induced development of malformed shoots with subsequent poor rooting (Slenko et al., 2001), so a low level of BA(0.5 mg l^-1) in the experiments were needed. To determine factor (component) levels and factor interactions influencing the process, the random balance method was used. The experiment included 18 factors, each at three or two levels and expressed in natural units (mg l^-1 or g l^-1) and in the coded form (Table I). Design of the experiment using the random balance method is shown in Table II, with variables in the coded form. Green shoots collected from a nature vine released from dormancy were used as a source of shoot tip explants. Taking into consideration characteristics of shoot tip development (survival rates, shoot length and leaf development scored in points) after 50 days in culture on each of 20 medium versions, the overall quality criterion (see `Material and methods') for each of the four genotypes and the average value of the overall quality criteria were calculated (Table II).

Shoot tip culture on the medium version 20 containing MS mineral elements did not improve shoot development in any of the four genotypes (Table II). In this respect, root stock `Kober 5 BB' performed the best on the medium version 8, cvs. `Podarok Magaracha' and `Zhemchug Magaracha' did so on the medium version 6, and the version 11 was the most beneficial medium for cv. `Sverkhrannii bessemyannyi'. The highest value (0.58) of the average overall quality criterion for the four genotypes was achieved on the medium version 6.

Design of experiment using variables in the coded form (Table I) and results pertaining to shoot tip development in four grapevine genotypes (expressed as units of the overall quality criterion). The variation coefficient (V) for each medium version of each genotype is not more than 29% (p < 0.05)

Based on the average values of the overall quality criteria, regression equations for shoot tip development as functions of concentrations of substances in medium versions (Tables I and II) were calculated for the four genotypes (y₁) and individually for the root stock `Kober 5 BB' (y₂):

y ₁ = 0.151 - 0.177х₇·х₈ + 0.143х₇ - 0.037х₇·x₈·х₁₄ + 0.085х₁ - 0.056х₁·х₈ -

- 0.042х₈ (4)

y ₂ = 0.156 + 0.077х₄·х₇ + 0.175х₇·х₁₀ - 0.161х₇·х₈ - 0.052х₇·х₉ - 0.072х₈ +

+ 0.062х₁₀ + 0.048х₉ - 0.030х₄·х₈ (5)

with the following characteristics of the equations (Table III): the level of significance of y₁ is 0.001, the determination coefficient is 0.694, the standard error is 0.097; the level of significance of y₂ is 0.000, the determination coefficient is 0.977, the standard error is 0.065. The factor x₇ (CaCl₂) provided the most significant effect on both equations since the first members of the equations are of the highest significance. They also are characterized with the highest determination coefficients, i.e. make the largest contribution to the description of the process by means of the equation (Table III).

Table III. - improvement of statistical characteristics of regression equations (4 and 5) during sequential selection of equation members describing overall quality criteria of shoot tip development as a function of concentrations of substances in medium versions (Tables I and II).

Stepwise optimization of media for shoot tip development based on regression equations

Table IV shows stepwise optimization of macro-element concentrations in media for shoot tip development based on the regression equations 4 and 5.

Table IV. - Macro-element concentrations in media optimized based on regression equations with a view to improve shoot tip development in the four grapevine genotypes and individually in the root stock `Kober 5 BB'. In addition to the macro-elements indicated in this Table, each medium version contained (Slenko et al., 2001): Fe-EDTA and micro-elements MS medium, 100 mg l^-1 myo-inositol, 10 mg l^-1 thiamine, 5 mg l^-1 nicotinic acid; 0.2 mg l^-1 pyridoxine, 10 mg l^-1 glycine, 5 mg l^-1 para-aminobenzoic acid, 0.5 mg l^-1 BA, 30 g l^-1 sucrose and 7 g l^-1 Difco agar.

The optimization began (Table IV) by determining the optimum medium version for each of the process: macro-element concentrations in the medium version 6 (Table II) for shoot tip development of the four genotypes and macro-element concentrations in the medium version 8 (Table II) individually for the root stock `Kober 5 BB'. Green shoots collected from field-grown plants were used as a source of shoot tip explants. Table V shows shoot tip development after 50 days in culture on the media (Table IV) of the different optimization designs for each of the processes.

Shoot tip development in four grapevine genotypes after 50 days in culture on media (Table IV) containing macro-element concentrations calculated by using the regression equations (4) and (5). Deviations of shoot tip development (V) from average values (p < 0.05) were not more than 31% for the shoot length and 34% for leaf development.

The design of an experiment for four grapevine genotypes (equation 4) led to encouraging results, though they were not reliable enough (macro-element concentrations of the medium version 6 from Table II, entering as control) for shoot tip development in cvs. `Podarok Magaracha' (optimized medium version 3cv4, Table V, Fig. 1) and `Zhemchug Magaracha' (optimized medium version 2cv4, Table V). On the contrary, the results were disappointing for root stock `Kober 5 BB' and cv. `Sverkhrannii bessemyannyi' due to the design of experiment by means of equation (4) began by using macro-element concentrations of the medium version 6 from Table II which was optimum for cvs. `Podarok Magaracha' and `Zhemchug Magaracha' but not so either for root stock `Kober 5 BB' (for which macro-element concentrations of the medium version 8 from Table II, designated as 8 Table II in Table V, was optimum) or for cv. `Sverkhrannii bessemyannyi' (for which macro-element content of the medium version 11 from Table II, designated as 11 Table II in Table V, was optimum). In root stock `Kober 5 BB' (Table V) shoot tips performed the best on the medium version 2Kob achieved by stepwise optimization using the regression equation (5) particularly for this genotype. This medium version 2Kob contained 208 mg l^-1 NaH2PO4 H2O (Table IV). Macro-elements at 3/4 levels of the MS formulation medium, supplemented with 170 mg l^-1 NaH2PO4 H2O, were optimum for grapevine shoot tip development (Harris and Stevenson, 1982).

Development of several shoots after 50 days of shoot tip culture in cv. `Podarok Magaracha' on the medium version 3cv4 (Tables IV and V) (the explant removed from the culture tube, top view)

The addition of this substance stabilizes the pH of the medium during explant culture (Viskot and Bezdek, 1984). Callus was formed from shoot tips of cv. `Podarok Magaracha' on media optimized for shoot development in the root stock `Kober 5 BB' (Table V). Reliable differences were established in shoot tip development on MMS medium and on optimized medium versions for root stock `Kober 5 BB' (2Kob, Table V), cv. `Podarok Magaracha' (version 3cv4, Table V), cv. `Zhemchug Magaracha' (version 2cv4, Table V) and cv. `Sverkhrannii bessemyannyi' (macro-element content of the medium version 11 from Table II, designated as version 11 Table `II in Table V).

Within the genus Vitis, plants have genetically determined differences in the uptake of K⁺, Ca²⁺ and Mg²⁺ (Scienza et al., 1986). Grapevine genotypes differed in their needs for nitrogen levels of the soil for their growth: some genotypes were capable to grow well on the soils with low nitrogen levels (Murthy and Iygengar, 1997). Different root stock genotypes affected the levels of mineral substances (N, K and Fe) in the scion (Bavaresco, 2001) and induced different responses of the scion to soil nitrogen fertilization (Keller et al., 2001). Various levels of mineral substances found in different organs of the plant depending on the genotype and the levels of mineral substances in the soil or in the water culture may enter as an indirect proof of the fact that the grapevine genotypes used in our experiments needed different levels of mineral substances in their optimum media. The design of the experiment focused on medium optimization for one grapevine genotype did improve shoot tip development in the specific genotype while poorly-developed shoot tips were observed on the same medium in the other genotypes. Thus, macro-element content of the medium version 11 (Tables II and V) applied for cv. `Sverkhrannii bessemyannyi' enabled the growth of high-quality shoots and leaves while extremely poor shoot tip development was observed on that medium version in the remaining three genotypes. Our report in this paper and other results concerned with shoot tip culture and whole plant culture (Slenko et al., 2001) led to the conclusion that optimized mineral salts levels for a certain grapevine genotype may not be so for other genotypes, though, of course, some of a great diversity of grapevine cultivars and root stocks may be expected to grow well on this or that optimized medium.

Experiments focused on medium optimization for shoot tip culture by means of the mathematical design (Tables I-V) are time-consuming and required large numbers of explants. It can be seen from the two experiments (Tables II and V) that the four grapevine genotypes have different optimum concentration of macro-elements. Table VI shows media that proved to be best efficient in the two experiments. Optimization of the medium composition may be achieved sooner and with good though not universally the best results by culturing shoot tips of any other genotypes on seven media presented in Table VI to determine better medium version for each genotype. In some genotypes, shoot tip culture developed callus, the levels of thiamine, nicotinic acid and glycine presented in the media recommended should be reduced to 2 mg l^-1.

Table VI. - Compositions of nutrient media recommended for shoot tip development in various grapevine genotypes. In addition to the macro-elements indicated in the Table, each medium version contained other substances, indicated in Table IV.

Our findings lead us to the following conclusions:

1. Over the range of component levels in media (Tables I and II), macro-elements had a higher effect on grapevine shoot tip development compare to Fe-EDTA, micro-elements, vitamins and sucrose. In regression equations 4 and 5 describing shoot tip development in the four genotypes (y₁) and individually in the root stock `Kober 5 BB' (y₂) as a functions of macro-element concentrations in media, the members including the variable x₇ (factor CaCl2), based on the levels 166 mg l^-1 (1/2 MS), 331 mg l^-1 (MS) and 662 mg l^-1 (2 MS), were the most significant and had the largest determination coefficients (Table III).

2. Regression equation (5) describing shoot tip development in one genotype (`Kober 5 BB') as a function of macro-element concentrations in media has a larger determination coefficient (0.977) relative to regression equation (4) for the four genotypes as a whole (determination coefficient is 0.694) since different medium versions were optimum for different genotypes (Tables II and V). Stepwise optimization of the mineral composition of medium for grapevine shoot tip development should begin based on the optimum medium version for each genotype (Tables II and IV).

3. Different concentrations of macro-mineral elements were optimum for shoot tip development in the four grapevine genotypes, irrespective of their specific origin (Vitis vinifera or interspecific hybrids, Table V).

The regression equations describing the pattern of mineral elements in optimized media for shoot tip cultures of different genotypes reflect their considerable heterogeneity as concerns best efficient concentrations of macro-elements in media. The proposed assessment of results by reducing the number of parameters characterizing a process to the overall quality criterion (equations 1-3) and the mathematical design of experiment may be used in physiological and agricultural research for optimization of processes affected by numerous factors. The optimized media (Table IV) may be used for growing of virus-free shoots in meristem cultures of different grapevine genotypes.

The authors wish to honour the memory of their teacher, Professor P.Ya. Golodriga, former director of the Institute for Vine and Wine `Magarach', and also wish to thank Dr. O. Adibekov for elaborating the mathematical design of their experiments.

References

1. ANACLERIO F., BORGO M. et COSMI T., 1999. Prove di risanamento vinale su biotipi di Vitis vinifera, Vignevini (Bologna), 26, 100-103.

2. BAVARESCO L., 2001. Portinnesto e nutrizione minerale della vite. Vignevini (Bologna), 28, 53-62.

3. BINI G., 1976. Prove di coltura `in vitro' di meristemi apicali di Vitis vinifera L. Rivista della Ortoflorofrutticoltura Italiana, №3, 289-296.

4. DURAN-VILA N., JUAREZ J. and ARREGUI J.M., 1988. Production of viroid-free grapevines by shoot tip culture. American Journal of Enology and Viticulture, 39, 217-220.

5. GRIBAUDO I., LENZI R. and MANNINI F., 1994. Grapevine clonal selection in Piedmont and Liguria: Virus eradication through meristem culture. Quaderni della Scuala di Specializzazione in Viticoltura ed Enologia (Univ. Torino), 18, 81-89.

6. HARRIS R.E. and STEVENSON J., 1982. In vitro propagation of Vitis. Vitis, 21, 22-32.

7. HARTMANN K., LEZKI E. and SCHДFER W., 1977. Trial design in study of technological processes. `World', Moscow.

8. KELLER M., KUMMER M. and CARMO VASCONCELOS M., 2001. Reproductive growth of grapevines in response to nitrogen supply and rootstock. Australian Journal of Grape and Wine Research, 7, 12-18.

9. MAEKAWA A., NAMBA I., TANAKA Y. and YAMASHITA H., 1993. Elimination of viruses by meristem culture. 2. Elimination of grapevine leafroll virus. Research Bulletin of the Plant Protection Service Japan, 29, 57-61.

10. MOREL G., 1975. Meristem culture techniques for the long-term storage of cultivated plants. In: Crop genetic resources for today and tomorrow. ed. Frankel O.H. and Hawkes J.G., p. 327-332.

11. MURASHIGE T. and SKOOG F.A, 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum, 15, 473-497.

12. MURTHY S.V.K. and IYENGAR B.R.V., 1997. Kinetic parameters of nitrogen absorption in varieties and rootstocks of grape (Vitis vinifera). Indian Journal of Plant Physiology, N 2, 2, 232-233.

13. NOVAK F.J. and JUVOVA Z., 1983. Clonal propagation of grapevine through in vitro axillary bud culture. Scientia Horticulturae, 18, 231-240.

14. REGNER F., BRANDT S., ROMANN H. und STADLHUBER A., 1995. In vitro-viruseliminierung bei reben (Vitis sp.). Mitteilungen Klosterneuburg, 45, 67-74.

15. SCIENZA A., FAILLA O. und ROMANO F., 1986. Untersuchungen zur sortenspezifischen mineralstoffaufnahme bei reben. Vitis, 25, 160-168.

16. SLENKO W.A., TROSCHIN L.P. und KOTIKOW I.V., 2001. Der einfluss der nдhrmedienzusammensetzung bei der in vitro-vermehrung verschiedener rebgenotypen. Mitteilungen Klosterneuburg, 51, 15-26.

17. VILLEGAS A., MAZUELOS C., CANTOS M. et TRONCOSO A., 1992. Influencia del nitrogeno sobre el desarrollo in vitro del portainjerto de vid 161-49. Suelo y Planta, N 2, 2, 529-539.

18. VYSKOT B. and BEZDEK M., 1984. Stabilization of the synthetic media for plant tissue and cell cultures. Biologia plantarum, 26, 132-143.

19. ZLENKO V.A., TROSHIN L.P. and KOTIKOV I.V., 1995. An optimized medium for clonal micropropagation of grapevine. Vitis, 34, 125-126.

Размещено на Allbest.ru