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Scientia Horticulturae 191 (2015) 25–30

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

pplication of growth models to evaluate the microenvironmentalonditions using tissue culture plantlets of Phalaenopsis Sogo YukidianV3’

hiachung Chen ∗

epartment of Bio-industrial Mechatronics Engineering, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan

r t i c l e i n f o

rticle history:eceived 19 January 2015eceived in revised form 1 May 2015ccepted 5 May 2015

eywords:rowth modelissue culture

a b s t r a c t

Phalaenopsis, an important pot plants worldwide, are propagated with a tissue culture technique. The aimof the present study was to validate growth models for Phalaenopsis plantlets with measured data. Effectof temperature and light irradiance on the growth characteristics was investigated by total weight, totalleaf area and weight ratio of shoot to root weight for Phalaenopsis Sogo Yukidian ‘V3’. Relationship betweenculture days and total weight and leaf area of plantlets with was analyzed by nonlinear regression analysis.A four-parameter logistics model for growth rate was selected used as the growth index for further study.The relationships between environmental factors and the total weight, total leaf area of plantlets and the

lantletshalaenopsis

weight ratio of leaf to root were evaluated by multiple regression analysis. These environmental factorsall had a significant effect on the growth characteristics. The optimal microclimate conditions for plantletsculture of Phalaenopsis Sogo Yukidian ‘V3’ determined from the regression results were light irradiance40–60 �mol m−2 s−1, light temperature 29–32 ◦C and dark temperature 22–25 ◦C. The results can be usefulinformation for propagating Phalaenopsis plantlets.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

In 2014, 80 million units of orchids were produced in Taiwan.ost were Phalaenopsis. Commercial Phalaenopsis is propagated

rom tissue culture. The quality and quantity of orchid plantletseeds to be improved continuously.

Orchid tissue culture plantlets are cultivated in a small asepticulture vessel. The air exchange between inside and outside air isimited because of the need to isolate microorganisms. Water andomposition of the medium in the vessel are the main source ofutrition. In the vessels, the aerial environment contains very highelative humidity. From the investigation of the microclimate insideessel, the CO2 concentration is decreased in the light period andncreased in the dark period (Chen, 2007).

The growth of plantlets inside vessels is affected by the internal

icroclimate, such as air temperature and humidity, light qual-

ty and quantity (Hsu and Chen, 2009; George and Davies, 2008;ujiwara and Kozai, 1995; Kozai, 2010). In the culture room, all

∗ Correspondence to: Department of Bio-industrial Mechatronics Engineering,ational Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan.el.: +886 4 22857562; fax: +886 4 22857135.

E-mail address: ccchen@dragon.nchu.edu.tw

ttp://dx.doi.org/10.1016/j.scienta.2015.05.007304-4238/© 2015 Elsevier B.V. All rights reserved.

vessels are placed on horizontal shelves. The practical way to con-trol the internal microclimate of the vessel is to modify the outsideenvironment of the culture room. Yao et al. (2007) proposed amodel to describe the microclimate inside a glass jar with medium.However, only a simulation result was presented. Models for airtemperature, relative humidity and photosynthetic photon flaxdensity (PPFD) was developed and validated with measured data(Chen, 2003, 2004, 2005).

The effect of photosynthesis and other physiological activitieson plantlets is of interest. Pospisilova et al. (1997) mentioned irra-diance, CO2 concentration, medium sucrose concentrations andgrowth regulators as factors. The highest net photosynthetic ratewas found with 25 ◦C and 226 �mol m−2 s−1 light irradiance. Limet al. (1992) observed the effect of sugar, light intensity and CO2concentrations on the growth and mineral uptake of Dendrobiumplantlets. The nitrate uptake was increase with increased lightintensity, and CO2 enrichment could not enhance the growth andion uptake. Yamagishi (1988) examined the effect of the culturetemperature on the growth characteristics of in vitro bulblets ofLilium japonicum Thunb and found that higher bulblets height and

sugar uptake at 20 ◦C than 15 ◦C or 26 ◦C. Nguyen et al. (1999)observed the effect of different CO2 concentrations and light inten-sities on the photosynthetic characteristics of coffee plantletsin vitro. Maximal dry mass, leaf area and photosynthetic rate were

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ound at low light intensity (150 �mol m−2 s−1) and high CO2oncentration (1400–1450 ppm). Cui et al. (2000) suggested thatncreased difference in light period (18 ◦C) and dark period (26 ◦C)emperature and high intensity (210 �mol m−2 s−1) increased thehoot weight, root weight and total fresh weight of Rehmannialutinosa plantlets.

A crop growth model is useful to describe the crop growthndex in the culture period. The effect of growth conditions or cli-

ate factors could be observed with the parameters of the growthodel. Many crop models have been proposed to describe the

rowth characteristics of horticultural crops (Thornley and Joshson,990). Tei et al. (1996) found that the Gompertz model was theest fit for lettuce and the exponential linear equation for red beetnd onion. Karadavut et al. (2010) compared the fitting ability ofrowth models for leaf growth data of maize and found that theogistic model was the adequate model. Fernamdes et al. (2014)roposed the Gompertz model to describe the growth curves ofoffee fruit.

The growth models for plantlets grown in vessels are limited.iu and Kozai (1997) developed a potato plantlet growth modely assuming that the dry weight of a plantlet at the end of oneay consisted of the dry weight of the previous day, the car-on accumulation from the daily net photosynthetic rate and thebsorption of sugar and minerals from the medium. However, onlywo datasets measured at days 8 and 15 were used for validationf the model and important parameter values were not considered.or the growth model for Oncidium plantlets (Chen, 2012), the gainn carbon source for Oncidium plantlets was assumed as the photo-ynthesis rate during the light period and the absorption of sugarrom the medium. The loss of the carbon source was due to theespiration during the dark period. Three growth equations wereroposed and the four-parameter logistic equation was the bestquation.

Many studies have investigated about the micropropagation ofhalaenopsis plantlets. Tokuhara and Mii (1993) described the mul-iplication method and the medium compositions. Hahn and Paek2001) reported that high photosynthetic photon flux density andigh CO2 concentration promoted photosynthesis of Phalaenop-is and three other orchids with photoautotrophic culture thaneterotrophic culture. Park et al. (2002) described the rapid propa-ation technique of Phalaenopsis plantlets from floral stalk-derivedeaves. Sinha et al. (2007) used inflorescence-axis thin sections asxplants for micropropagation of Phalaenopsis. Balilashaki et al.2014) reported the micropropagation technique of Phalaenop-is by using flower stalk nodes and leaves. Cha-um et al. (2010)ested the effects of temperature and relative humidity duringn vitro acclimatization. The leaf growth model was proposed andnfluencing factors of temperature, light intensity and fertilizationoncentration were evaluated for Phalaenopsis plants cultured in 9-m pots (Chen and Chien, 2012). However, interaction among theseactors affecting the growth of Phalaenopsis plantlets is limited.

In the previous literatures, the effect of factors on growth char-cteristics has been studied. The growth characteristics on fixedulture days were measured and then these data sets were usuallyvaluated by analysis of variance (ANOVA). However, growth char-cteristics of plantlets such as fresh weight and leaf area are affectedy different environmental factors during all culture days. Compar-

son the growth conditions on fixed days only may be inadequateo evaluate the influencing factors. Factors affecting the growth onll culture day should be studied by regression analysis.

Regression analysis has been proposed by plant biotechnolo-ists to evaluate the related quantitative treatment on the growthharacteristics of plantlets (Compton, 1994; Ibanez et al., 2003;

orenzo and Garcia-Borroto, 2008; Mize and Chun, 1988). Gomest al. (2010) used the multiple linear regression analysis to studyhe effect of the different factors on the multiplication rate of Arutus

rae 191 (2015) 25–30

unedo L. (strawberry tree). Faria et al. (2004) adopted polynomialregression equations to compare the effect of different concentra-tions of sucrose on the plant of in vitro of Dendrobium nobile. Hsuand Chen (2009) studied the effect of light spectrum on the growthcharacteristics of in vitro of Phalaenopsis.

The aim of the present study was to validate growth models forPhalaenopsis plantlets with measured data. The parameter of theadequate model was then used to evaluate the effect of environ-mental factors. The experimental design included seven levels oflight/dark temperature and three levels of photosynthetic photonflux density (PPFD).

2. Materials and methods

2.1. Plant materials

Samples were multiplicated plantlets of Phalaenopsis (SogoYukidian ‘V3’). Plantlets were transplanted into 550 ml conical glassvessels with 120 ml medium in each vessel. Ten plantlets were cul-tured per flask. Vessels were sealed with a rubber stopper that hada hole with permeable film to provide ventilation.

The root medium was composed of 1/2 MS nutrients (Murashigeand Skoog, 1962), supplemented with 200ml l−1 coconut liquid6.5 g l−1 agar, 10 g l−1 sucrose, 10 mg l−1 6-benzylaminopurine (BA)and 5.0 mg l−1 d-naphthaleneacetic acid (NAA).

Typical figures of shoot proliferation and rooting is shown inFig. 1.

2.2. Experimental design

The cultures were incubated in a photoperiod of 14 h light and10 h dark. A total of thirty-six culture vessels were placed on threelayers of shelves in a growth chamber, 12 vessels per shelf. Follow-ing are the seven experimental designs:

1. Light temperature 32 ◦C, dark temperature 26 ◦C, and three irra-diance levels (PPFD): 20, 40 and 60 �mol m−2 s−1.

2. Light temperature 32 ◦C, dark temperature 23 ◦C, three irradi-ance levels.

3. Light temperature 28 ◦C, dark temperature 22 ◦C, three irradi-ance levels.

4. Light temperature 25 ◦C, dark temperature 20 ◦C, three irradi-ance levels.

5. Light temperature 23 ◦C, dark temperature 19 ◦C, three irradi-ance levels.

6. Light temperature 20 ◦C, dark temperature 18 ◦C, three irradi-ance levels.

7. Light temperature 25 ◦C, dark temperature 25 ◦C, three irradi-ance levels.

2.3. Measurement of the growth parameters

The adequate time for good quality plantlets of Phalaenopsisranged from 63 to 75 days. The growth characteristics were mea-sured at day 15, 30, 40, 50, 60 and 72 days. Two culture vesselswere taken out to measure the growth characteristics of plantletsat the time of each interval. The total fresh weight, total leaf areaand ratio of leaf weight to the root weight were measured. The ratioof total leaf weight to total root weight (WR) served as a criterionto evaluate the quality of Phalaenopsis plantlets.

The fresh weight of roots and leaves was measured by using ofan electronic balance (Metter PM 400, HP, Palo Alto, CA). The leaf

area was determined with use of an area meter (LI-300 area meter,L2-COR, Inc., Lincoln, NE).

C. Chen / Scientia Horticultu

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y = 6.790 + 28.42861 + 2.507 exp(−0.09863t)

R2 = 0.979, s = 0.955(7)

Fig. 1. The typical figures of shoot proliferation and rooting.

.4. Statistical analysis

Two growth characteristics, total fresh weight and total leaf areaere quantitative criteria to evaluate the growth rate of plantlets.

he distribution of total fresh weight or leaf area, and culture daysas analyzed by using of Sigma Plot v 12.0 (SPSS Inc., Chicago, IL)

o evaluate the fitting ability of these nonlinear models. The crite-ia to compare the fitting performance of growth models were theoefficient of determination R2 and standard error of the estimatedalue s.

.5. Model development

Two growth models were used (Thornley and Joshson, 1990).

. The monomolecular equation

y = A1 + B1exp(−C1t) (1)

where A1, B1 and C1 are constants.

rae 191 (2015) 25–30 27

2. The four-parameter logistic equation

y = y2 + A2

1 + B2exp(−C2t)(3)

where y2, A2, B2 and C2 are constants.

These growth equations are derived from the mechanicalbasic of the biological theory. The parameters of equations couldbe interpreted with biological meaning. The values of A1 andA2 represent the maximal growth characteristics for plantlets.The values of C1 and C2 express the growth rate under eachexperimental treatment. The relationship between environmentfactors such as light temperature, dark temperature and PPFDwith growth rate was analyzed by multiple regression (Raymond,2000). The significant factors then were determined by statisticalanalysis.

3. Results and discussion

3.1. Total weight of plantlets

Data for the relationship between culture days and the totalfresh weight for three temperatures and three levels of PPFD arepresented in Fig. 2.

The typical regression models are as follows:

1. 32/26 ◦C, 40 �mol m−2 s−1

a. The monomolecular equation

y = 8.191 + 194763.0 exp(−1.4857 × 10−6t)

R2 = 0.955, s = 1.5562(4)

b. The four-parameter logistical equation

y = 8.2910 + 33.4451 + 2.586(−0.06936t)

R2 = 0.988, s = 0.853(5)

2. 32/23 ◦C, 60 �mol m−2 s−1

a. The monomolecular equation

y = 6.191 + 23.8358 exp(−0.0222t)

R2 = 0.917, s = 1.656(6)

b. The four-parameter logistical equation

Fig. 2. The relationship between culture days and total fresh weight for three tem-peratures and three levels of photosynthesis photon flux density.

28 C. Chen / Scientia Horticulturae 191 (2015) 25–30

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ig. 3. The effect of light temperature on the growth rate of total weight at threeight levels.

. 28/22 ◦C, 20 �mol m−2 s−1

a. The monomolecular equation

y = 6.348 + 138519.8exp(−2.4833 × 10−6t)

R2 = 0.975, s = 1.143(8)

b. The four-parameter logistical equation

y = 6.5814 + 28.52241 + 2.396 exp(−0.06016t)

R2 = 0.990, s = 0.915(9)

In comparing R2 and s values, the four-parameter logisticalodel had better fitting ability than the monomolecular equation.

he parameter C2, growth rate of total weight, was used for furthertudy.

Effect of light and dark temperature on the growth rate (C2alue) is shown in Figs. 3 and 4. The effect of the temperature on therowth rate was difficult to observe visually. Relationship betweenhe growth rate and the temperature and light intensity were ana-yzed by regression analysis and tested by its t value. The regressionquation was obtained as follows:

y1 = 0.62960 + 0.010016Tl − 0.06226Td + 0.0031824T2l

+ 0.0059705T2 − 0.00019631Is − 0.0079231T · T

d l d

+ 0.0015750Tl · Is − 0.00016412Td · Is

R2 = 0.750, s = 0.00876

(10)

ig. 4. The effect of dark temperature on the growth rate of total weight at threeight levels.

Fig. 5. The relationship between culture days and total leaf area for three temper-atures and three levels of photosynthesis photon flux density.

where Tl is light temperature, Td is dark temperature; and Is is theirradiance level.

The factors that significantly affected the growth rate of totalweight were light and dark temperature, light intensity, inter-action of light temperature and illumination, interaction of darktemperature and illumination and interaction of light and darktemperature.

3.2. Total leaf area of plantlets

Typical relationship between culture days and the total leaf areain various experimental designs is presented in Fig. 5.

With nonlinear regression, the monomolecular equation pro-duced lower R2 values and higher s values. Only the C2 parameter ofthe four-parameter logistic equation values was adopted for furtheranalysis.

Relationship between the leaf growth rate and the light and darktemperature are shown in Figs. 6 and 7. The equation for the resultof regression analysis is as follows:

y2 = −0.09995 − 0.02624Tl + 0.03823Td + 0.0005361T2l

− 0.0008190T2d

+ 0.002825Is − 3.4013 × 10−5I2s

R2 = 0.859, s = 0.00765

(11)

Light and dark temperature and PPFD had a significant effect on

the growth of the leaf area. No interaction between light and darktemperature and PPFD could be found.

Fig. 6. The effect of light temperature on the growth rate of total leaf area at threelight levels.

C. Chen / Scientia Horticulturae 191 (2015) 25–30 29

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Fig. 8. The contour plots for the total weight, leaf area and ratio of leaf to root weight

ig. 7. The effect of dark temperature on the growth rate of total leaf area at threeight levels.

.3. Ratio of leaf to root weight (WR)

No significant relationship could be found for the WR data andulture days. The WR values at 72 d with various treatments weresed for further analysis. The relationship between WR values and

ight or dark temperature was established by regression analysis.

WR = −0.70855 − 0.16980Tl + 0.28061Td + 0.0032624T2l

− 0.0060140T2d

− 0.0018041Is + 1.51786 × 10−5I2s

R2 = 0.919, s = 0.0202

(12)

WR was significantly affected by light and dark temperature andPFD. No interaction of the light temperature and illumination orhe dark temperature and illumination could be found.

.4. The effect of environmental factors on growth ofhalaenopsis plantlets

In this study, light and dark temperature and PPFD had signif-cant effects on the growth rate of total weight and leaf area ofhalaenopsis plantlets. The ratio of leaf to root weight was alsoffected.

The contour plots for total weight, leaf area and WR and theight and dark temperature at 40 �mol m−2 s−1 obtained from theegression analysis equations are shown in Fig. 8. The optimalemperature for total weight was 30–32 ◦C (light) and 21–25 ◦Cdark), for leaf area was 29–32 ◦C (light) and 20–25 ◦C (dark) andor WR was 29–32 ◦C (light) and 22–26 ◦C (dark). Phalaenopsislantlets have higher criteria in total weight and WR. However,

arger leaf area may not be required for good quality. Largereaf area could be a problem in the acclimation stage because ofhe increased of evaporation. Therefore, the optimal microclimateonditions for plantlet culture of Phalaenopsis Sogo Yukidian ‘V3’etermined from the regression results are as follows: light irra-iance 40–60 �mol m−2 s−1, light temperature 29–32 ◦C and darkemperature 22–25 ◦C.

Study of the effect of microclimate of vessels on the growthharacteristics of plantlets is limited. Cui et al. (2000) reported thatemperature had a significant effect on leaf area and total weight ofehmannia glutinosa plantlets. Yamagishi (1988) reported that theresh weight of L. japonicum bulblets was higher at 20 ◦C than 15 ◦Cnd 26 ◦C on the same culture days. Chen and Chien (2012) foundhat the leaf growth rate of Phalaenopsis plant cultivated in 9-cmots was affected by temperature, light intensity and fertilization

oncentrations. Vaz et al. (2004) observed the effect of temperaturen growth height, leaf number and dry weight of P. pusilla plantletsultured for 90 d and found these growth conditions increased at7 ◦C.

(WR) and light and dark temperature at 40 �mol m−2 s−1.

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The factors of light quality, nutrients, moisture and gas con-entrations that affect the growth have been reported in detailLumsden et al., 1994). However, the study of the effect of temper-ture and light irradiance on the growth characteristics of in vitrorown plantlets is limited. In many tissue culture laboratories, roomemperature is constantly maintained in the light and dark period.

e found that the growth characteristics of total fresh weight, leafrea and WR were highly affected by temperature and light irra-iance and proposed that the optimal microclimate conditions ofhalaenopsis Sogo Yukidian ‘V3’ plantlets.

. Conclusions

In this study, growth models are applied to validate the opti-al microenvironmental conditions for Phalaenopsis plantlets witheasured data. The Phalaenopsis Sogo Yukidian ‘V3’ was used as

he testing material. The relationship between culture days andotal weight and leaf area was analyzed by nonlinear regressionnalysis. The factors that affect the growth characteristics coulde observed for all culture days. These environmental factors allad a significant effect on the growth characteristics. The optimalicroclimate conditions for plantlet culture of Phalaenopsis Sogo

ukidian ‘V3’ determined from the regression results are light irra-iance 40–60 �mol m−2 s−1, light temperature 29–32 ◦C, and darkemperature 22–25 ◦C. The results can provide useful informationor propagation of Phalaenopsis plantlets.

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