In Vitro Cell.Dev.Biol.Plant (2007) 43:370381 DOI 10.

1007/s11627-007-9062-5

PHYSIOLOGY

Regulating plant tissue growth by mineral nutrition

Randall P. Niedz & Terrence J. Evens

Received: 30 December 2006 / Accepted: 20 June 2007 / Published online: 20 July 2007 / Editor: T. J. Jones # The Society for In Vitro Biology 2007

Abstract The objective of this study was to determine if the growth of sweet orange (Citrus sinensis (L.) Osbeck cv. Valencia) nonembryogenic callus could be regulated and controlled via the mineral nutrient components of the medium. The 14 salts comprising Murashige and Skoog (MS) basal medium were subdivided into five component groups. These five groups constituted the independent factors in the design. A five-dimensional hypervolume constituted the experimental design space. Design points were selected algorithmically by D-optimality criteria to sample of the design space. Growth of the callus at each design point was measured as % increase of fresh weight at 14 d. An analysis of variance was conducted and a response surface polynomial model generated. Model validation was conducted by mining the polynomial for design points to two regionsMS-like growth and MS+25% growth and comparing callus growth to predicted growth. Five of the eight selected MS-like points and three of the six MS+25% growth points validated, indicating regions within the design space where growth was equivalent to MS, but the salt combinations were substantially different from MS, and a smaller region where growth exceeded MS by greater than 25%. NH4NO3 and Fe were identified as important factors affecting callus growth. A second experiment was conducted where NH4NO3 and Fe were varied, thus creating a two-dimensional slice through the region of greatest callus growth and provided increased resolution of the response.
R. P. Niedz (*) : T. J. Evens Agricultural Research Service, US Horticultural Research Laboratory, 2001 South Rock Road, Ft. Pierce, FL 34945-3030, USA e-mail: rniedz@ushrl.ars.usda.gov

Keywords Callus . Sweet orange . Citrus . Salts . Response surface

Introduction The basic components of plant tissue culture media are the mineral nutrients. How rapidly a tissue grows and the extent and quality of morphogenetic responses are strongly influenced by the type and concentration of nutrients supplied. Early research by Gautheret (1939), Heller (1953), White (1942), Hildebrandt et al. (1946), and Nitsch and Nitsch (1956) culminated in the development of Murashige and Skoog (MS) medium by Murashige and Skoog (1962). The potential benefits of optimizing the nutrient component of culture media for a particular response are well documented across a wide range of species and applications. For example, the concentration of NH4+ and NO3 affects numerous in vitro responses including the development of somatic embryos (Reinert et al. 1967; Halperin and Wetherell 1965; Meijer and Brown 1987; Leljak-Levanic et al. 2004; Elkonin and Pakhomova 2000; Poddar et al. 1997), the plating efficiency of protoplasts (Attree et al. 1989), the efficiency of plant recovery after ovule culture (McCoy and Smith 1986), shoot regeneration (Leblay et al. 1991), regulation of growth and biomass of bioreactor-grown plantlets (Sivakumar et al. 2005), and controlling the rate of root initiation on shoot cultures (Hyndman et al. 1982). The effects of some other mineral nutrients include the induction or suppression of shoot-tip necrosis by varying Ca2+ levels (Sha et al. 1985); reduction of vitrification by Ca(NO3)24H2O or by reducing the level of NH4NO3 (McLaughlin and Karnosky 1989); increasing shoot regeneration by varying the Na2SO4 level (Chandramu et al. 2003); AgNO3 acted

MINERAL NUTRITION

371

synergistically with 6-benzylaminopurine to increase shoot regeneration in thin cell layer explants (Carvalho et al. 2000); and increased levels of sodium molybdate increased the percentage of responding cultures and the number of shoot buds from cultured embryos (Chauhan and Kothari 2004). The interaction of the mineral nutrients and plant growth regulators (PGR) is a particularly intriguing relationship. Preece (1995) argued that PGR partially compensate for nutrient imbalances and that, by correcting the imbalance, PGR can be reduced or eliminated, as Gomez and Segura (1994) and Kothari et al. (2004) observed for NAA for shoot regeneration of Juniperus and finger millet, respectively. For additional information, see the review by Ramage and Williams (2002) on the effects of mineral nutrition on morphogenesis. Because there are 13 mineral elements essential for plant growth (Epstein and Bloom 2005), the experimental determination of optimal nutrient levels is complex. This complexity illustrates why the revised medium developed by Murashige and Skoog (1962) was an important development; although MS medium is not optimal for many tissues, many tissues will grow on it to some degree; hence, MS medium represents a starting point to begin the process of improving a response. A medium where there is no response cannot be optimized as the direction of improvement is unknown. This is why many media developed for specific applications are derivatives of MS. A number of approaches for estimating the correct balance of mineral nutrients have been developed. Hildebrandt et al. (1946) tested various salt combinations and their effect on tobacco and sunflower callus growth in a set of elegant experiments using his triangulation method and six 3-salt combinations. Callus growth was increased 63 and 162% for tobacco and sunflower by reformulating the salt levels in Whites (1942) basal medium. Murashige and Skoog (1962) increased callus growth fourfold over Whites basal medium by varying the level of each nutrient over several levels of the other nutrients. De Fossard et al. (1974) introduced the broad spectrum approach as a universal approach to defining the type of media formulation for any new or unresolved tissue culture situation. The broad spectrum experiment is a 43 factorial with 81 treatment combinations; the four factors are broad groupings of media componentsmineral, auxin, cytokinin, and organic growth factors (e.g., amino acids and vitamins) including sucrose. The original intent of the broad spectrum experiment was to include sucrose as a component separate from the organic growth factors, but the resulting 53 factorial would have required an impractical 243 treatment combinations. Margara (1977 and 1978) devised a set of eight media for identifying the approximate mineral nutrient formulation suitable for particular situation; the media varied in total N,NH , NO , Cl, K+, and Ca2+. Spaargaren 3 4

(1996) proposed that the ideal nutrient media for biological growth would have the same elemental composition as the cell, tissue, or organism being grown. Monteiro et al. (2000), Nas and Read (2004), Bouman (2001), and Staikidou et al. (2006) tested a variation of Spaargarens concept by successfully improving in vitro growth by devising culture media where the mineral nutrient levels and/or proportions matched the those of the cultured organism. The objective of this study was to efficiently characterize the mineral nutrient requirements to accurately predict and regulate the growth of plant tissue in vitro.

Materials and Methods Experimental approach. The basic strategy was to (1) create a n-dimensional experimental design space where each dimension was defined by a specific MS salt or group of MS salts; (2) grow the tissue on a set of treatment combinations represented as points contained within or on the surface of the n-dimensional design space; (3) generate a prediction equation that describes tissue growth through the n-dimensional design space; (4) test the prediction equation by growing callus at points not included in the original design but contained within the n-dimensional design space and comparing the actual growth to the predicted growth; and (5) explore a lower dimensional region of the n-dimensional design space using factors identified by the analysis of variance (ANOVA) as important. The specific steps were as follows: 1. The 14 salts comprising MS basal medium were subdivided into five component groups (Table 1). These five groups constituted the independent factors in the design and the five dimensions that would be studied.
Table 1. The five factors used to construct the five-dimensional design space, their component MS salts, and concentration range expressed as MS levels Factors Group 1 Group 2 Group 3 (mesos) MS Salts NH4NO3 KNO3 CaCl22H2O KH2PO4 MgSO4 MnSO4H2O ZnSO47H2O CuSO45H2O KI CoCl26H2O H3BO3 Na2MoO42H2O FeSO47H2O Na2EDTA Range 0.51.5 0.51.5 0.51.5

Group 4 (micros)

0.54

Group 5 (Fe)

0.54

372

NIEDZ AND EVENS

Table 2. Five-factor design including 23 model points, 10 lack-of-fit points, and 11 replicated points, including MS (points 4446) for pure error estimation Treatment design pointsa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
a

Factor 1 NH4NO3 0.50 1.36 0.50 1.44 1.50 0.50 0.62 0.97 1.50 1.50 0.50 1.50 0.62 1.50 1.50 0.50 0.50 1.50 0.95 0.50 1.50 1.50 1.50 0.50 1.06 1.38 1.50 1.50 0.50 1.01 0.50 1.50 0.50 1.01 1.50 0.50 0.90 0.50 1.50 1.38 0.50 1.50 1.50 1.00 1.00 1.00

Factor 2 KNO3 1.50 0.50 1.50 1.50 1.50 0.50 0.60 1.03 0.50 0.50 1.50 0.50 0.62 0.50 1.50 1.43 1.50 1.50 0.80 0.50 1.04 0.50 1.04 0.50 1.50 0.62 0.50 1.50 1.50 1.50 1.50 1.50 0.50 1.50 1.19 1.50 1.11 0.50 0.50 0.62 1.24 1.50 1.19 1.00 1.00 1.00

Factor 3 mesos 1.50 0.67 0.88 0.52 1.50 0.62 1.50 0.50 1.50 0.50 0.88 0.50 1.38 1.50 1.50 1.50 0.50 0.50 0.84 1.50 1.05 0.50 1.05 0.50 1.50 1.50 1.50 0.50 0.50 0.95 1.50 1.50 1.50 0.95 0.50 1.50 1.05 0.50 0.52 1.50 0.50 0.50 0.50 1.00 1.00 1.00

Factor 4 micros 0.50 0.50 2.11 4.00 4.00 3.57 0.50 4.00 4.00 0.50 2.11 0.50 3.57 4.00 4.00 1.80 4.00 0.50 1.24 0.50 4.00 4.00 4.00 4.00 2.45 4.00 0.50 0.50 4.00 0.50 4.00 0.50 4.00 0.50 1.67 4.00 3.79 0.50 3.25 0.93 0.50 4.00 1.67 1.00 1.00 1.00

Factor 5 Fe 2.36 4.00 0.50 3.79 4.00 3.57 3.94 0.50 0.50 0.50 0.50 0.50 0.50 0.50 4.00 4.00 4.00 2.34 2.07 0.50 0.50 4.00 0.50 0.50 0.50 3.57 4.00 2.34 4.00 4.00 0.50 0.50 4.00 4.00 4.00 0.50 2.84 4.00 1.24 0.93 0.50 0.50 4.00 1.00 1.00 1.00

% Fresh wgt. Increase 855 206 488 434 459 655 310 447 204 319 450 349 415 198 651 786 641 592 705 347 242 635 257 458 322 612 389 600 641 478 237 71 830 515 518 354 803 280 496 350 525 227 594 588 648 634

Design points 143 were randomly assigned to blocks as follows: Block 1 (points 115); Block 2 (points 1629); Block 3 (points 3043). Additionally, one MS point was run with each block (points 4446).

Selection of five factors was considered appropriate as this allowed us to both test the suitability of using n-dimensional design spaces for understanding a basic in vitro growth response and, two, the number of treatment combinations were small enough to be manageable.

2. A range, defined as a minimum and maximum multiple of those salts in MS medium, was selected for each of the five factors (Table 1). 3. Design points were selected to adequately sample the five-dimensional design space.

MINERAL NUTRITION Table 3. Two-factor design including ten model points and five additional lack-of-fit points and ten replicated points Treatment design points 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Factor 1 NH4NO3 0.42 0.25 0.25 0.38 0.42 0.58 0.63 0.58 0.38 0.38 0.75 0.50 0.75 0.58 0.63 0.25 0.75 0.25 0.75 0.75 0.38 0.25 0.25 0.75 0.58 Factor 2 Fe 3.21 2.14 1.07 2.67 1.07 3.21 2.67 1.07 2.67 1.61 3.21 2.14 2.14 1.07 1.61 3.21 1.07 3.21 3.21 2.14 1.61 2.14 1.07 1.07 3.21 % Fresh wgt. increase 505 906 683 781 806 419 666 845 646 1,129 342 686 758 850 799 747 848 612 618 638 903 872 803 832 575

373

vitro grown seedlings of Citrus sinensis (L.) Osbeck cv. Valencia. Seed were germinated in MS basal medium without PGR and supplemented with 3% (w/v) sucrose. One-centimeter epicotyl explants were excised from 15- to 21-d-old seedlings and placed onto MT medium (Murashige and Tucker 1962) supplemented with 2.5-M 2,4-dichlorophenoxyacetic acid (2,4-D), 1-M 6-benzylaminopurine (BA), and 100 mg l1 casein hydrolysate. The cultures were grown in a growth cabinet under low light (1520 mol m2 s1), provided by cool-white fluorescent lamps, constant 27C, and a 4-h photoperiod. After 6 mo. of selection, a rapidly growing callus designated Val01 was obtained. For maintenance, the 2,4-D concentration was reduced to 1 M. Experiments were initiated by first subculturing approximately 1 g of callus onto each treatment formulation (i.e., design point), using 10020 mm polystyrene culture dishes, for acclimation to the formulation. Preliminary experiments (data not shown) indicated that a single subculture cycle substantially reduced carry-over effects. After an acclimation cycle on each treatment formulation, approximately 1 g from the acclimated cultures was subcultured again onto each treatment formulation. Fresh weight was determined on day 14. Six duplicate plates (i.e., pseudoreplicates) were used per treatment in the five-factor experiment and eight in the two-factor experiment. Percent increase in fresh weight was calculated from the initial subcultured weight of the callus. Experimental design. The objective was to regulate the biomass accumulation by varying the basal salt composition. The initial five-factor experimental design was a 23model-point D-optimal response surface sufficient for modeling a quadratic polynomial. The design was augTable 4. ANOVA of % fresh weight increase of sweet orange callus in the five-factor experiment Term Overall modelF value (p value)* Lack-of-fitF value (p value)** R2 Adjusted R2 Predicted R2 Adequate precision*** % Fresh wgt. increasea 26.45 (<0.0001) 0.96 (0.5432) 0.9544 0.9183 0.7856 18.691

4. Sweet orange callus was grown on the salt combination specified at each design point. 5. Fresh weight growth, the primary response variable in this report, was collected after 14 d of growth. Dry weights were also recorded. 6. ANOVA 7. A polynomial equation was generated from the ANOVA model terms describing growth through the five-dimensional design space. 8. Additional design points were selected in the design space not included in the original design. Callus was grown on the salt formulations specified at these new points. The resulting fresh weight growth was then compared to the growth predicted to occur from the equation calculated in step #7. 9. A two-factor experiment based on the validated points identified in step #8 was conducted to further characterize growth in a region of the original five-dimensional design space.

Plant material and tissue. A 4-yr-old nonembryogenic cell line (Val01) was developed from epicotyl explants of in

*The F value for the overall model and the probability of obtaining a larger F value. The overall model is a reduced cubic and includes 25 terms (Table 5). **A p>0.05 indicates no additional variation that might be accounted for using a better model. ***Design-Expert recommends a value greater than four to ensure adequate predictions. a The measured response variable.

374 Figure 1. Five-factor model adequacy plots(a) BoxCox plot; (b) normal plot of residuals; (c) residuals vs. predicted plot; (d) outlier t value plot; (e) Cooks distance plot; (f) predicted vs. actual plot.

NIEDZ AND EVENS

mented to include ten additional points for lack-of-fit analysis for detecting additional signal (i.e., curvature) possibly not captured in the design. Eleven points were replicated for pure error estimation; this included one point, replicated twice, for MS medium, as it was a coordinate within the five-dimensional design space. The number of replicates was chosen based on the minimum required degrees of freedom for a statistically valid, pure error

estimation for the chosen experimental design. The design points selected in both experiments, including replicate points, were selected to achieve a statistical power >90%. The statistical power of this design provided a >99% chance of detecting an effect of two standard deviations for the main effects and two-way interactions and >94% for the quadratic terms. Thus, the complete design included 46 treatment points (Table 2). The experiment contained three

MINERAL NUTRITION Table 5. Significant terms with F value and p value in the ANOVA of % fresh weight increase of sweet orange callus in the five-factor experiment Significant ANOVA terms NH4NO3 NH4NO3 KNO3 NH4NO3 Mesos NH4NO3 Fe KNO3 micros KNO3 Fe MesosFe MicrosFe Mesos2 Fe2 (NH4NO3)2 Fe F value 62.71 13.91 9.72 5.68 30.26 13.23 40.01 27.00 11.09 48.42 20.61 p value (Prob>F) <0.0001 0.0010 0.0047 0.0254 <0.0001 0.0013 <0.0001 <0.0001 0.0028 <0.0001 0.0001

375

blocks based on the number of treatments that could be managed at one time; this removed any variation introduced from running the blocked treatments at different times. The second design included two factors and was a tenmodel-point D-optimal response surface sufficient for modeling a cubic polynomial. The purpose was to explore in additional detail the region of greatest growth in the fivedimensional design space. The design was augmented to include five additional points for lack-of-fit analysis. Eight points were replicated for pure error estimation. The complete design included 25 treatment points (Table 3). Data analysis. The ANOVA data were the means of the six or eight duplicate plates (i.e., pseudoreplicates) that were averaged together to arrive at the specific response for each treatment design point. Thus, the eleven replicated points in the first experiment were composed of 66 plates (i.e., 6 pseudoreplicates11 replicates), and the eight replicated points of the second experiment were composed of 64 plates (i.e., 8 duplicates8 replicates). For each experiment, the highest order polynomial model where additional model terms were significant at the 0.05 level was analyzed by ANOVA. Model adequacy tests as described by Anderson and Whitcomb (2005) and calculated by Design Expert 7 (Stat-Ease, Minneapolis, MN) were as follows: 1. BoxCox plotused to determine if the data require a power law transformation. A transformation is recommended, based on the best lambda value, which is found at the minimum point of the curve generated by the natural log of the sum of squares of the residuals (Box and Cox 1964; Myers and Montgomery 2002; Anderson and Whitcomb 2005). 2. Normality assumptiona normal probability plot of the internally studentized residuals was examined; the assumption is satisfied if the residuals plot closely along a line.

3. Constant variance assumptiona plot of the internally studentized residuals vs. predicted response value was examined; the assumption is satisfied if the points fall within the interval of 3 to +3 standard deviations (i.e., sigma), exhibit random scatter, and do not show a megaphone (<) pattern where the residuals increase with the predicted response. 4. Outlier t valuesa statistic calculated for each point; a point outside + 3.5 standard deviations is defined as an outlier and indicates either a problem with that point or with the chosen model. 5. Lack-of-fit testadditional design points were included in every experiment for this test. A significant lackof-fit indicates the model may not be capturing the entire signal in the observations. 6. Predicted vs. actual values plotpoints that are randomly scattered along and around a 45 line (i.e., perfect correlation) indicate the model appears to be unbiased when predicting new observations. 7. Cooks distancea statistic to identify points with unusually high influence on the fitted model (i.e. high leverage points), thus resulting in a model fitted more to the high leverage points than to the majority of points in the data set (Anderson and Whitcomb 2005). Myers and Montgomery (2002) suggest using a cutoff value of 1 to identify high leverage points.
Table 6. Reduced cubic polynomial generated from the ANOVA of the % fresh weight increase of sweet orange callus in the five-factor experiment % Fresh weight increase = +705.68 77.44 +11.43 2.61 2.14 16.30 41.47 35.98 +25.38 63.68 +45.26 +66.57 +60.62 +34.91 103.54 1.99 205.76 +181.94 38.24 +38.14

NH4NO3 KNO3 Mesos Micros Fe NH4NO3 KNO3 NH4NO3 mesos NH4NO3 Fe KNO3 micros KNO3*Fe *MesosFe MicrosFe (NH4NO3)2 Mesos2 Micros2 Fe2 (NH4NO3)2 Fe (KNO3)2 Fe Mesos3

Equation is reported in terms of coded factors. Coding normalizes the factor ranges by placing their low and high range value between 1 and +1.

376

NIEDZ AND EVENS

Table 7. Predicted design points for MS-like and MS(+25%) growth Solution # MS-2 MS-3 MS-5 MS-8 MS-10 MS-21 MS-24 MS-26 MS (+25%)-1 MS (+25%)-8 MS (+25%)-16 MS (+25%)-18 MS (+25%)-21 MS (+25%)-22 NH4NO3 0.59 0.53 0.5 1.49 0.51 1.38 1.27 1 0.5 0.5 0.53 0.5 0.5 0.5 KNO3 0.51 1.47 1.5 0.55 1.48 0.64 0.52 1 1.5 1.24 0.8 1.5 0.5 1.5 Mesos 0.61 0.91 1.49 0.53 0.52 1.45 0.52 1 1.15 1.21 1.19 0.98 0.89 0.71 Minors 3.56 2.03 0.54 3.98 3.96 3.99 3.94 1 1.19 4 4 0.5 4 1.04 Fe 3.57 0.78 1.05 3.62 3.97 3.42 1.57 1 2.77 2.99 2.84 2.49 3.24 2.14 Predicted growth 475758 461718 495760 544809 514775 533792 551812 519768 7901,070 7571,033 7381,007 7531,052 702982 704980 Observed growth 526 596 812 349 600 602 445 690 806 691 768 690 449 928

Rows colored italicized validated since the observed growth fell within the 95% predicted growth confidence interval.

8. R2, adjusted-R2, and predicted-R2calculated as follows: R2 =1SSresiduals/(SSmodel +SSresiduals) and is the fraction of overall variation explained by the model; adjusted-R2 is R2 adjusted for the number of terms in the model relative to the number of points in the design; predicted R 2 = 1 (PRESS/SS Total ) where PRESS is the predicted residual sum of squares. When selecting the best model, we maximized predicted-R2 (or minimized PRESS). 9. Adequate precisiona measure of signal to noise in the data; it compares the predicted values at the design points to the average prediction error. Ratios greater than 4 are preferred (Anderson and Whitcomb 2005). The software application Design-Expert (2005) 7 (StatEase) was used for experimental design construction, model evaluation, and all analyses.

Validation points. Points in the design space not included in the five-factor experiment were selected, callus was grown on these points, growth recorded, and the growth at these points was compared to the predicted growth. The two-factor design was constructed using information from these validated points. The purpose of validation points is to empirically assess the usefulness of the predictive capabilities of a proposed model.

that in vitro tissue growth could be regulated by alterations in the mineral nutrition. A summary of the ANOVA, lackof-fit test, three R2 statistics, and adequate precision statistic for % fresh weight increase is presented in Table 4. The best fitting model was a reduced cubic response surface obtained by stepwise regression. The data did not require transformation per the BoxCox analysis (Fig. 1a), and the remaining diagnostics were all within acceptable limits including the normality assumption (Fig. 1b), the constant variance assumption (Fig. 1c), no outlier t points (Fig. 1d), no points that exceeded a Cooks distance of one (Fig. 1e), and the predicted vs. actual value plot (Fig. 1f ). Additionally, the lack-of-fit test was not significant (p=0.5432) indicating that additional variation in the residuals could not be removed with a better model, the three R2 statistics were clustered with a difference less than 0.2, and the adequate precision statistic of 18.691 was greater than 4. The overall model was highly significant (p<0.0001), thus indicating significant factor effects on growth and was considered of sufficient quality to navigate the experimental design space and, therefore, useful for predicting new observations. The ANOVA revealed 11 significant terms; five of the terms had a p value<0.0001 (Table 5). The 19-term reduced cubic polynomial model is presented in Table 6. As the equation is reported in coded terms, the coefficients are directly comparable and provide information on how each term contributes to the shape of the callus growth response.

Results Analysis of five-factor. The growth response of the sweet orange callus as measured by % fresh weight increase ranged from 71855% (Table 2); this wide range suggested

To test the models predictive capabilities, we utilized the softwares numerical optimization routine that uses the desirability function of Derringer and Suich (1980). Two specific region(s) were selected for optimization in the fivedimensional design space; (1) the region where callus

MINERAL NUTRITION

377

Figure 2. (a) Five-factor response surface contour plot including the two predicted MS-equivalent growth contours. (b) Five-factor response surface plot. X and Y scales on both plots are MS levels.

Figure 3. Callus grown on MS salt formulation vs. callus grown on predicted point #22 salt formulation.

growth was equivalent to MS and (2) the region where callus growth exceeded MS by a minimum of 25%. We selected eight design points from the MS optimization solution set (including the actual MS formulation) and six design points from the MS (+25%) optimization solution set for validation. The points were selected by taking the minimum and maximum of each factor. These points and the resulting % fresh weight increase are listed in Table 7. Five of the eight observed MS points fell within the 95% prediction interval; three of the six MS (+25%) points fell

within the 95% prediction interval. MS(+25%) % fresh weight increase was 928%, the largest observed growth response. A two-dimensional slice through the NH NO and 4 3 Fe dimensions while holding the remaining three factors fixed at point #22 levels is shown in the contour plot (Fig. 2a); the response surface for this region is shown in Fig. 2b to more clearly visualize the shape of the response. MS-equivalent growth is delineated by the MS-623 contour lines illustrating where MS-equivalent growth occurs. The point #22 coordinate on the contour is 0.5

378 Figure 4. (a) Two-factor response surface contour plot of % fresh weight increase with treatment point locations and demarcation 800% contour of region of greatest growth. Blue points were replicated to obtain an estimate of pure error. (b) Two-factor response surface plot of % fresh weight increase. (c) Two-factor response surface plot of dry weight increase.

NIEDZ AND EVENS

NH NO and 2.14 Fe. From this information, we con3 4 cluded the following: 1. MS-equivalent growth is probably a larger region of the design space than the region of greatest growth. A large (or larger) region would explain the larger solution set and the greater number of validated points. A smaller region for the greatest growth would explain the smaller solution set (112 with six unique extreme points vs. 133 with eight unique extreme points). 2. A region exists within the five-dimensional design space where callus growth is significantly greater than MS. Validated point #22 had the largest increase in growth of the six validation points, exceeding MS growth by 44% and was visually distinct from MS (Fig. 3). 3. Iron has a large interactive effect on callus growth. Four of the six model terms where the p<0.0001 included Fe (Table 6). 4. Additional experimentation is required to better define this region. A single validating point provides little information on the size and characteristics of the region of greatest growth; additional exploration of the design space is required.

was set at its lowest level of 0.5 for two of the three points where growth exceeded 800%points #1 and #33. Fe was selected, as it was involved in six of the significant higher order terms (Table 6). Additionally, Fe was greater than 2 for each of the three points (#1, #33, and #37) where growth exceeded 800%. To determine the levels to fix the remaining three factors (Factor groups 2, 3, and 4), point #22 was selected as the location to build the twodimensional design space. Treatment point selection is shown in Fig. 4a. The growth response of the callus in the two-factor experiment ranged from 3421,129% (Table 3). A summary of the ANOVA, lack-of-fit test, three R2 statistics, and adequate precision statistic for % fresh weight increase is presented in Table 8. The best fitting model was a reduced quadratic response surface obtained by backward elimination. The data did not require transformation per the Box Cox analysis, and the residual and model diagnostics were all within acceptable limits, including the normality assumption, the constant variance assumption, no outlier t points, and the predicted vs. actual value plot. Additionally, the lack-of-fit test was not significant (p=0.5535), indicatTable 8. ANOVA of the response variable % fresh weight increase of sweet orange callus in the two-factor experiment Term Overall modelF value (p value) Lack-of-fitF value (p value) R2 Adjusted R2 Predicted R2 Adequate precision % Fresh wgt. increasea 11.35<0.0001 0.92 (0.5535) 0.6941 0.6330 0.5105 9.551

Analysis of two-factor. The strategy to further explore the region of greatest % fresh weight increase was to; (1) initiate the exploration around a previously validated MS (+25%) point and (2) explore a two-dimensional slice through the original five-dimensional design space. Constructing a two-dimensional design space required selecting two factors to vary and fixing the remaining three factors. NH NO and Fe (Factor groups 1 and 5) were selected as 3 4 the two factors to vary, as NH NO had the single largest 3 4 F value, and the MS (+25%) predicted point set NH NO 4 3 at or near its lowest level of 0.5 MS. This indicated that an increase in growth might be observed if the lower range of NH NO was extended downward. Reducing the lower 4 3 limit of the range for NH NO was also consistent with the 3 4 data obtained in the five-factor designthe NH NO level 3 4

*The F value for the overall model and the probability of obtaining a larger F value. The overall model is a reduced cubic and includes 25 terms (Table 5). **A p>0.05 indicates no additional variation that might be accounted for using a better model. ***Design-Expert recommends a value greater than four to ensure adequate predictions. a The measured response variable.

MINERAL NUTRITION

379

ing that additional variation in the residuals could not be removed with a better model, the three R2 statistics were clustered (difference less than 0.2), and the adequate precision statistic of 9.551 was greater than 4. The model was highly significant (p<0.0001), thus indicating significant factor effects on growth. The ANOVA revealed three significant terms with the Fe linear term having the largest effect on % fresh weigh increase (Table 9). The reduced quadratic polynomial model is presented in Table 10. The region of greatest fresh weight increase is a band running through the design plane defined by the 800% contour lines (Fig. 4a). The corresponding response surface visualizes the shape of the response and shows that fresh weight progressively declines as Fe levels increase; the decline increases as NH4NO3 levels increase (Fig. 4a and b).

Table 10. Reduced quadratic polynomial generated from the ANOVA of the % fresh weight increase of sweet orange callus in the two-factor experiment % fresh weight increase = +804.98 56.98 141.59 60.76 120.88 Equation is reported in terms of coded factors.

NH4NO3 Fe NH4NO3 Fe Fe2

Discussion A simple in vitro system was chosen to initiate this study for two reasons: one, our interest in better understanding the role of mineral nutrition in regulating and controlling in vitro growth and development, and two, a simple system seemed appropriate in characterizing the usefulness of an efficient experimental approach we thought would be useful in quantifying the inherent multivariate nature of in vitro systems. For example, a five-factor factorial where each factor is set at a low, medium, and high level would require 243 treatment combinations. The experimental design we used only required 33 treatment combinations (the remaining 10 were replicates for estimating pure error) to sample the same design space. The additional dissection of the five-dimensional design space required an additional 15 treatment combinations and was particularly helpful in further characterizing the response. This process of subdividing the larger design space into smaller and easily visualized components can continue until the precise region of interest is fully explored. We could have selected other two or three factor slices (e.g., KNO3 Minors) through the five-dimensional design space. The growth of citrus nonembryogenic callus tissue can be empirically studied, the effect of the mineral nutrients on growth measured, the mineral nutrient relationships on growth determined, and the resultant information then used
Table 9. Significant terms with F value and p value in the ANOVA of % fresh weight increase of sweet orange callus in the two-factor experiment Significant ANOVA terms NH4NO3 Fe Fe2 F value 4.36 29.63 6.61 p value (Prob>F) 0.0497 <0.0001 0.0182

to regulate future growth to predictable levels. Reducing an in vitro response to an accurate predictive equation is directly useful for calculating the specific media formulations to achieve predefined levels of growth, but more importantly, it emphasizes the conceptual relativity of optimality. For example, given a growth response such as is depicted in Figs. 2 or 4, where does the callus grow best? This question in and of itself has no meaning, but requires a specific application to provide the appropriate context. Best is defined by the specifications required by the application. Thus, if the application is a maintenance formulation Point #22, where the callus grows 40% faster than MS, is probably not a good choice, as it would require more frequent subcultures. An aspect of this approach that we consider essential is the use of validation points. These are points generated from the prediction equation that are then empirically tested or validated. Validation becomes particularly important as the dimensionality of the design space increases. Given the complexity of working in a five-dimensional design space with a biological system, Point #22 would have been virtually impossible to find using a one-factor-at-a-time approach. By generating an estimate of the response through the five-dimensional design space, it became possible to locate Point #22. Of course, if the level of response is already contained among the design treatment points, then predicting other regions of the design space may not be required. For example, there were several treatments in the fivedimensional design space that exceeded MS by varying degrees. Mapping a response in a predefined region of interest not only provides information that is directly useable in an applied sense about how to control and optimize the response, but also contributes to the basic understanding of how the selected factors relate to the response or, as stated by George Box, what does what to what (Box et al. 2005). For example, the importance of iron and its interactions with the other factors in affecting callus growth is clear from these experiments. One problem of our experimental design is that we used salts as factors. The five factors included both single (Factors

380

NIEDZ AND EVENS De Fossard, R. A.; Myint, A.; Lee, E. C. M. A broad spectrum tissue culture experiment with tobacco (Nicotiana tabacum) pith tissue callus. Physiol Plant 30: 125130; 1974. Derringer, G. C.; Suich, R. Simultaneous optimization of several variables. J. Qual. Tech. 12:214219; 1980. Design-Expert 7 User Manual, Stat-Ease, Inc, Minneapolis, MN; 2005. Elkonin, L. A.; Pakhomova, N. V. Influence of nitrogen and phosphorus on induction embryogenic callus of sorghum. Plant Cell Tissue Org. Cult. 61:115123; 2000. Epstein, E.; Bloom, A.J. Mineral nutrition of plants: principles and perspectives. Sinauer Associates; 2nd edition, Sunderland, MA. 2005. Gautheret, R.J. Sur la possibilit de raliser la culture indefinite des tissues tubercule de carotte. Compt. Rend. Acad. Sci. Paris 208:118; 1939. Gomez, M. P.; Segura, J. Factors controlling adventitious bud induction and plant regeneration in mature Juniperus oxycedrus leaves cultured Cell. Dev. Biol. Plant 30:210218; 1994. Halperin, W.; Wetherell, D. F. Ammonium requirement for embryogenesis. Nature 205:519520; 1965. Heller, R. Researches on the mineral nutrition of plant tissues. Ann. Sci. Nat. Bot. Biol. Vg., 11th Ser. 14:1223; 1953. Hildebrandt, A. C.; Riker, A. J.; Duggar, B. M. 1946. The influence of the composition of the medium on growth of excised tobacco and sunflower tissues. Amer J Bot 33: 591597; 1946. Hyndman, S. E.; Hasegawa, P. M.; Bressan, D. A. The role of sucrose and nitrogen in adventitious root formation on cultured rose shoots. Plant Cell Tiss. Org. Cult. 1:229238; 1982. Kothari, S.L.; Agarwal, K.; Kumar, S. Inorganic nutrient manipulation for highly improved plant regeneration in finger milletEleusine coracana (L.) Gaertn. Cell. Dev. Biol. Plant 40:515519; 2004. Leblay, C.; Chevreau, E.; Raboin, L.M. Adventitious shoot regeneration from leaves of several pear cultivars (Pyrus communis L.). Plant Cell Tiss. Org. Cult. 25:99105; 1991. Leljak-Levanic, D.; Bauer, N.; Mihaljevic, S.; Jelaska, S. Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds. J. Plant Physiol. 161: 229236; 2004. Margara, J. La multiplication vegetative de la betterave (Beta vulgaris L.) en culture. Comptes Rendus Acadmies des Sciences (Paris) 282D: 10411044; 1977. Margara, J. Mise au point dune gamme de milieux minraux pour les conditions de la culture in vitro. Comptes Rendus de LAcadmie dAgriculture de France 64: 654661; 1978. McCoy, T. J.; Smith, L. Y. Interspecific hybridization of perennial Medicago species using ovule-embryo culture. Tag 71: 772783; 1986. McLaughlin, J.; Karnosky, D.F. Controlling vitrification in Larix decidua via culture media manipulation. Can. J. For. Res. 19:13341337; 1989. Meijer, E. G. M.; Brown, D. C. W. Role of exogenous reduced nitrogen and sucrose in rapid high frequency somatic embryogenesis in Medicago sativa. Plant Cell Tissue Org. Cult. 10:11 19; 1987. Monteiro, A. C. B. D.; Higashi, E. N.; Goncalves, A. N.; Rodriguez, A. P. M. A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis Sims. F-flavicarpa Deg.). Cell. Dev. Biol. Plant 36: 527531; 2000. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473 497; 1962. Murashige, T., Tucker, D.P.H. Growth factor requirements of citrus tissue culture. Proc. 1st Int. Citrus Symp., 3, 11551161; 1962. Myers, R. H.; Montgomery, D.C. Response surface methodology: process and product optimization using designed experiments, 2nd edition. New York, NY:John Wiley & Sons; 2002:7173.

1, 2, and 5) and multiple salts (Factors 3 and 4). This introduces tremendous complexity into the design, as specific ion effects are confounded. Ion confounding is an inherent problem when experiments are designed using salts as factors. For example, factor 2 in the five-factor experiment was KNO3. Varying KNO3 actually varies both the K+ and NO ions simultaneously; hence, effects associated 3 with KNO3 cannot be attributed to K+ or NO, as the ion3 specific effects are confounded. To understand the effects of the mineral nutrients on particular in vitro responses, it is essential to treat the ions, and not the salts, as the factors. Niedz and Evens (2006) have recently reported a solution (and software) to the problem of ion confounding that allows ions rather than salts to be used as the independent factors. We are not aware of any studies, including this one, in plant mineral nutrition that do not exhibit ion confounding. Although our experimental design exhibited substantial ion confounding, the factor effects on callus growth are real; we are just unable to conclude anything about ionspecific effects from these experiments.

Acknowledgements We thank Mr. Eldridge Wynn for initiating and maintaining the cell line used in this study and his excellent work in setting up all the treatment combinations and careful data collection. We thank the folks at Stat-Ease for the extremely informative discussions on the various statistical aspects of this research.

References
Anderson, M. J.; Whitcomb, P. J. RSM simplified: optimizing processes using response surface methods for design of experiments. New York, NY: Productivity Press; 2005:120121, 226 228. Attree, S.M.; Dunstan, D. I.; Fowke, L. C. Initiation of embryogenic callus and suspension cultures, and improved embryo regeneration from protoplasts, of white spruce (Picea glauca). Can. J. Bot. 67: 17901795; 1989. Bouman, H. M. B. T. A. Development of new tissue culture media, using the relation between mineral composition of plant and medium. Acta Horticulturae 560: 373376; 2001. Box, G. E. P.; Cox, D. R. An analysis of transformations. J. Royal Stat. Soc. Series B 26:211; 1964. Box, G .E .P.; Hunter, J. S.; Hunter, W. G. Statistics for experimenters: design, innovation, and discovery, 2nd edition. John Wiley & Sons, Hoboken, New Jersey. 2005. Carvalho, M. H. C.; Bui,V. L.; Zuily-Fodil, Y.; Thi, A. T. P.; Tran, T. V. Efficient whole plant regeneration of common bean (Phaseolus vulgaris L.) using thin-cell-layer culture and silver nitrate. Plant Sci. 159: 223232; 2000. Chandramu, C.; Rao, D. M., Reddy, V. D. High frequency induction of multiple shoots from nodal explants of Vitex negundo L. using sodium sulphate. J. Plant Biotech. 5:107113; 2003. Chauhan, M.; Kothari, S. L. Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant Indian barley (Hordeum vulgare L.) Cell. Dev. Biol. Plant 40: 520527; 2004.

MINERAL NUTRITION Nas, M. N.; Read, P. E. A hypothesis for the development of a defined tissue culture medium of higher plants and micropropagation of hazelnuts. Sci. Hort. 101: 189200; 2004. Niedz, R. P.; Evens, T. J. A solution to the problem of ion confounding in experimental biology. Nature Methods 3:417; 2006. Nitsch, J. P.; Nitsch, C. Auxin-dependent growth of excised Helianthus tissues. Am. J. Bot. 43:839851; 1956. Poddar, K.; Vishnoi, R. K.; Kothari, S. L. Plant regeneration from embryogenic callus of finger millet Eleusine coracana (L.) Gaertn. on higher concentrations of NH4NO3 as a replacement of NAA in the medium. Plant Sci. 129:101106; 1997. Preece, J. Can nutrient salts partially substitute for plant growth regulators? Plant Tiss. Cult. Biotech. 1:2637; 1995. Ramage, C. M.; Williams, R. R. Mineral nutrition and plant morphogenesis. Cell. Dev. Biol. Plant 38:116124; 2002.

381

Reinert, J.; Tazawa, M.; Semenoff, S. 1967. Nitrogen compounds as factors of embryogenesis in vitro. Nature 216: 12151216; 1967. Sha, L.; McCrown, B. H.; Peterson, L. A. Occurrence and cause of shoot-tip necrosis in shoot cultures. J. Amer. Soc. Hort. Sci. 110: 631634; 1985. Sivakumar, G.; Kim, S. J.; Hahn, E. J.; Paek, K. Y. Optimizing environmental factors for large-scale multiplication of Chrysanthemum (Chrysanthemum grandiflorum) in balloon-type bioreactor culture. Cell. Dev. Biol. Plant 41:822825; 2005. Spaargaren, D. H. The design of culture media based on elemental composition of biological material. J Biotech. 45: 97102; 1996. Staikidou, I.; Selby, C.; Hanks, G.R. Development of a medium for in vitro culture of Galanthus species based on the mineral composition of bulbs. J. Hort. Sci. Biotech. 81:537545; 2006. White, P.R. Plant tissue cultures. Ann. Rev. Biochem. 11:615628; 1942.