journa l h om epa ge: www.elsev ier .com/ locate /sc ihor t i
re-acclimatization in the greenhouse: An alternative to optimizinghe micropropagation of gerbera
ean Carlos Cardosoa, Mônica Lanzoni Rossib, Isadora Bonfante Rosalemc,aime A. Teixeira da Silvad,∗
Departamento de Desenvolvimento Rural, CCA/UFSCar, Rod. Anhanguera, km 174, CEP 13600-970, C.P.153 Araras, SP, BrazilLaboratory of Histopatology and Structural Biology of Plants, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, Statef Sao Paulo, BrazilDepartamento de Produc ão Vegetal, ESALQ, Universidade de São Paulo, BrazilP.O. Box 7, Miki-cho Post Office, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan
r t i c l e i n f o
rticle history:eceived 28 March 2012eceived in revised form 14 October 2013ccepted 18 October 2013
eywords:erbera jamesonii
n vitro rooting and elongationucrose concentrationrowth conditions
a b s t r a c t
Rooting and elongation are the two stages of the micropropagation of gerbera (Gerbera jamesonii)‘BMC101’ that use the greatest laboratory area that reduce production capacity and increase productioncosts. In this study, we developed an efficient method to pre-acclimatize gerbera plants in the green-house under photoautotrophic conditions by intercropping different stages of plants rooted in vitro in agreenhouse using natural light in small tunnels. Three different concentrations of sucrose (0, 2 and 4%)in culture media were tested as were two different growth conditions (laboratory growth chamber andgreenhouse tunnels). Those plantlets grown in the greenhouse showed a significant increase (56.5%) inleaf number, leaf diameter (10.8%), rooting percentage (62.5, 93.8 and 100% with 0, 2 and 4% sucrose,respectively), root number (92%) and fresh (32.7%) and dry weight (43.4%), than those cultivated in lab
n vitro and ex vitro developmenttomata
conditions after culture for 30 days in vitro in rooting medium, which consisted of half-strength MSsalts, 100 mg L−1 myo-inositol and 0.5 mg L−1 indole-3-butyric acid. When acclimatization was complete,plantlets grown in sucrose-free rooting medium under greenhouse conditions were well acclimatizedand showed good ex vitro development in paper-pot culture. An increase in sucrose concentration in theculture medium (0–4%) led to a 92% increase in leaf stomatal density, but only in plants grown under
laboratory conditions.
. Introduction
Gerbera or Transvaal flower (Gerbera jamesonii Bolus) is a mem-er of the Asteraceae family and can be propagated by seeds ory asexual methods (Kanwar and Kumar, 2008). Commercial cul-ivars are developed mainly for the production of cut flowers, andn vitro micropropagation using shoot tips as explants is the prin-ipal method for the commercial propagation of these species dueo the large number of plantlets produced in a reduced area andime and the genetic stability of plantlets obtained from this systemBhatia et al., 2009).
High micropropagation costs are still a major problem for tissueulture laboratories. The development of technologies for low-cost
issue culture is considered to be a priority in agriculture, hor-iculture, floriculture and forestry in many developing countriesIAEA-TECDOC, 2004; Purohit et al., 2011). Consequently, plant
micropropagation techniques are considered to be a sustainabletechnology for UNESCO projects in Africa and the Caribbean. How-ever, it is necessary to reduce plant production costs associatedwith this technology (Brink et al., 1998).
The major part of production costs is attributed to factors suchas laboratory maintenance, especially human labour, energy costsand the area occupied by plant cultures. Moreover, the low rate ofmultiplication, problems with microbial contamination, a low rateof rooting in some species and loss of plantlets during acclimati-zation contribute to an increase in these costs (Kozai et al., 2005;Xiao et al., 2011). Artificial lighting with fluorescent cold light bulbsaccounts for more than 65% of energy costs associated with plantmicropropagation and is thus one of the factors associated withhigh costs of plantlet production in this system (Xiao and Kozai,2004), and even though several novel lighting systems are avail-able that can reduce those costs by as much as 66% (e.g. Wang et al.,2011), these are still far from being used extensively as lighting
systems.
In this context, the development of alternative systems thatresult in reduced costs and process optimization in one or morestages of plant micropropagation are of fundamental importance
o increase the competitiveness of micropropagated plantlets pro-uced in commercial laboratories. Among the alternatives toonventional micropropagation in lab conditions are photoau-otrophic and photomixotrophic growth, which are characterizedy in vitro culture in conditions favourable to photosynthesis inn environment with higher light intensity and CO2 enrichmenthan culture medium without sucrose (photoautotrophic) or with aeduction in the concentration of sucrose (photomixotrophic). Thehotoautotrophic system used for the micropropagation of plants
s also called photosynthetic, inorganic or sugar-free micropropa-ation (Kozai et al., 2005).
A photoautotrophic micropropagation system was successfullystablished for different species of herbaceous plants such asanana (Nguyen and Kozai, 2001), including ornamentals such asymbidium, an orchid (Teixeira da Silva et al., 2007), as well asoody plants such as Macadamia (Cha-um et al., 2011) and Cas-
anea (Sáez et al., 2012), and in most cases, it improved the growthharacteristics of acclimatized plants.
The objective of this study was to grow gerbera at rooting andlongation in vitro stages under photoautotrophic conditions (i.e.,ugar-free medium) and then to grow plantlets in greenhouse con-itions with high light intensity in a bid to increase acclimatizationnd/or post-acclimatization growth performance.
. Materials and methods
.1. Plant material
Two gerbera cultivars were used for this experiment, ‘BMC101’nd ‘AL101’, both are used as cut flowers with white and orangenflorescences, respectively. However, due to the volume of infor-
ation and similarities between the results obtained, we only showhe data obtained with ‘BMC101’.
Plantlets were derived from the in vitro culture of shoot tipsnd maintained at the multiplication stage, in similar conditionsescribed by Cardoso and Teixeira da Silva (2012) with auto-laved MS (Murashige and Skoog, 1962) medium supplementedith 30 g L−1 of sucrose, 0.1 g L−1 of myo-inositol, 0.5 mg L−1 6-
enzyladenine and solidified with 6.0 g L−1 of agar (Algagel, Brazil).he pH was adjusted to 5.7. This process was undertaken to obtainufficient quantities of plants for the elongation stage and rootingf plantlets. In the final multiplication stage, approximately 500lantlets were obtained, from which 250 with similar morpholog-
cal characteristics were selected for the experiment.
.2. In vitro rooting and elongation
The rooting and elongation medium (REM) consisted of half-trength MS medium (macro- and micronutrients) supplementedith 100 mg L−1 myo-inositol and 0.5 mg L−1 indole-3-butyric acid
IBA). The pH was adjusted to 5.8 with NaOH or 0.1 N HCl beforeolidification with 6.0 g L−1 agar (Algagel, Brazil). The medium wasutoclaved at 121 ◦C and 1 kg cm−2 for 20 min. Sucrose, whicherved as the carbon source in REM, was tested at three concen-rations: 0, 2 and 4% (w/v), with 0% being the photoautotrophicondition. Approximately 25 mL culture medium was placed in00-mL flasks 13 cm in height and 6.6 cm in diameter and cappedith transparent polypropylene. This culture system was selected
ince it did not show any apparent effects caused by the expecteduild-up of ethylene.
Plantlets from the multiplication stage were subcultured in REM
ontaining 2 or 3 leaves/plant, 3–4 cm tall and mean fresh weightf 0.32 g. After culture, flasks were placed in two environmentalonditions: (a) a growth chamber in the laboratory at 25 ± 1 ◦C,hotosynthetic photon flux density (PPFD) = 25 �mol m−2 s−1
lturae 164 (2013) 616–624 617
provided by 40 W cool white lamps (Philips Electronics N.V.,Netherlands) and a 16-h photoperiod; (b) a greenhouse at 25 ± 5 ◦C,PPFD = 100 �mol m−2 s−1 and a 12-h photoperiod, i.e., naturalgrowing conditions in April, 2011. These specific conditions bothin vitro and in the greenhouse were based on extensive two-yeartrials (unpublished data).
The experiment was conducted in a 3 × 2 factorial design, withthree different concentrations of sucrose and two different grow-ing conditions, with five replicates (bottles) per treatment, eachcontaining eight plantlets.
For SEM, after dehydration in 100% ethanol, portions of leaveswere critical point dried using liquid CO2 in CPD/030 Blazers equip-ment (Lichenstein, Germany), mounted on metal supports (stubs)and sputter coated with 20 nm gold. Samples were viewed witha Zeiss LEO 435 VP scanning electron microscope (Cambridge,England) operated at 20 kV at NAP/MEPA-ESALQ-USP.
2.4. Plantlet acclimatization
Twenty plantlets obtained from each treatment were acclima-tized under greenhouse conditions in coconut fibre and vermiculite(5:1), which served as the substrate, in 54-cell plastic trays (Eco-vaso, Jaguariúna, Brasil). This procedure was repeated twice andthe repetitions were conducted in a randomized complete blockdesign. The plants were maintained in tunnels covered with green-house plastic (Nortene, Barueri, Brazil) for 15 days to maintain highair humidity (90 ± 5%) and to reduce the incidence of natural sun-light (130 �mol m−2 s−1 PPFD) on the plantlets. Thereafter, plantswere removed from the tunnels and cultured for 15 days under280 �mol m−2 s−1 PPFD at 25 ± 5 ◦C. The photoperiod in April, atthe time the experiment was conducted, decreased from 11.8 to11.2 h of light, at the start and end of the experiment, respectively.Plants were ferti-irrigated daily according to horticultural and cli-mate conditions described by Ludwig et al. (2008) with a solutioncontaining (in mg L−1): 142.0 NO3
Plantlet development was evaluated by height, number ofleaves/plant (LN) and number of roots (RN)/plant, leaf diame-ter (LD; cm), percentage of rooted plants, fresh weight (FW) and
618 J.C. Cardoso et al. / Scientia Horticulturae 164 (2013) 616–624
Table 1Aspects of in vitro development of plantlets of gerbera cv. BMC101 (Gerbera jamesonii) growth after 30 days in different growth conditions and sucrose concentration inculture medium.
Sucrose (g L−1) Growth conditions Height (cm) Leaf number LD (cm) Rooting percentage Root number
0 Laboratory 4.86 b 2.31 b 1.11 aA 62.50 bB 0.63 cBGreenhouse 5.05 b 4.06 a 0.97 bB 100.0 aA 1.63 cA
20 Laboratory 6.38 a 4.00 a 1.10 aB 93.75 aA 1.19 bBGreenhouse 6.05 a 5.06 a 1.25 aA 100.0 aA 2.13 bA
40 Laboratory 6.21 a 2.81 ab 0.96 bB 100.0 aA 1.75 aBGreenhouse 6.06 a 4.69 a 1.26 aA 100.0 aA 2.44 aA
Numbers followed by the same letter in the columns are not significantly different by Tukey’s test at 5% (*) and 1% (**) probability. The values are means of five replicationswith eight explants per treatment. Data were obtained 28 days after inoculation of explants. LD – leaf diameter.
dtoRemlamtwds
3ra
bvsaret
3
3
iimft
raL
tutt
ry weight (DW; g) of shoots and roots, and microscopic evalua-ions of the stomatal density (SD) and the diameter of the osteolef plantlets grown for 30 days after culture and incubation onEM containing different concentrations of sucrose and in bothnvironmental culture conditions. SD means resulted from theeasurement of four areas (0.2 cm2) from three images of each
eaf processed per treatment (four leaves/treatment), totalling 12nalyzed images/treatment. Also, the increase in plantlet DW wasonitored throughout the experimental incubation period. For
his, five additional flasks with eight plantlets for each treatmentere evaluated every 6 days for a total of 30 days. For DW (g)ata, the shoots and roots were placed in an oven (37 ◦C) until DWtabilized.
At the acclimatization stage, evaluations were performed after0 days of growth of gerbera plantlets in paper pots. The survivalate (%), height (cm), leaf number and the leaf diameter (cm) werelso evaluated.
Data on mean height, leaf and root number, leaf diameter, num-er and size of osteole were compared by two-way ANOVA. Meanalues were compared by Tukey’s test ( = 0.05). The statisticaloftware Assistat 7.6 (Silva and Azevedo, 2006) was used for thenalysis. Regression analysis was also performed to establish cor-elations between developmental stages and treatments, and tostablish DW curves over time. The experiments were repeatedwice.
. Results
.1. In vitro growth and development
Sucrose concentration and growth conditions affected then vitro development of gerbera plantlets. Plantlet height was onlynfluenced by sucrose concentration when added to the culture
edium at the elongation and rooting stages (Table 1). Most leavesormed in greenhouse conditions, regardless of sucrose concentra-ion used, or in laboratory conditions when 2% sucrose was used.
Under laboratory conditions, a higher sucrose concentrationeduced plantlet LD unlike greenhouse conditions, where there was
positive correlation between sucrose concentration and plantletD (Table 1).
Rooting was observed in 100% of plants at the end of the cul-
ivation period in the laboratory, but only when 4% sucrose wassed, and under greenhouse conditions, at all sucrose concentra-ions. The RN of plantlets also increased as sucrose concentration inhe culture medium increased under both culture conditions, and
RN was higher in greenhouse than in laboratory conditions at allsucrose concentrations (Table 1).
The addition of sucrose to the culture medium at 2 or 4% aswell as growth under greenhouse conditions increased the total FWand DW of plantlets. Under photoautotrophic conditions (0 g L−1
sucrose) there were no differences in the total FW and DW ofplantlets cultured under laboratory and greenhouse conditions(Table 2). The DW was only influenced by sucrose concentrationin the culture medium. The difference observed in total FW ofplantlets obtained, which was larger in greenhouse conditions,occurred only in response to a considerable increase in root FWof these plants (Fig. 1A – shaded area A). In contrast, the accu-mulation of root and shoot DW contributed to total DW (Fig. 1B –shaded areas A and B). The differences in DW growth of shoots androots between lab and greenhouse conditions were observed in thefirst 6 days after the establishment of the experiment in treatmentswith sucrose, and increased over time, but only in these treatments(Fig. 2). The pH and EC of the media decreased considerably in thosetreatments with high values of total FW and DW. The reduction inpH values was larger in laboratory than in greenhouse conditions.An opposite trend was observed for EC values (Table 2).
Another main factor observed in this experiment was theincrease in DW of shoots and roots in greenhouse condi-tions (100 �mol m−2 s−1) compared with laboratory conditions(25 �mol m−2 s−1). The differences in shoot DW between these twoconditions were 18.4, 21.7 and 34.8% after 30 days of culture in 0, 2and 4% sucrose, respectively although the most evident differencesbetween greenhouse and laboratory conditions were the percent-age increases in root DW, 30.61, 271.84 and 148% by 30 days afterculture.
3.2. Microscopic analysis
When in vitro plantlets were grown under laboratory conditions,there was a significant increase in SD after the addition of sucroseto the culture medium relative to photoautotrophic culture (i.e., 0%sucrose). However, in greenhouse conditions, only small variationswere observed in SD, demonstrating that the addition of sucroseto the medium was largely responsible for the increase in SD ingerbera leaves (Table 3, Fig. 3C–H). In the laboratory, sucrose at 2or 4% resulted in a larger stomatal opening while in the greenhouse
there was a significant effect between sucrose concentration andosteole diameter (Table 3, Fig. 3C–H).
The arrangement of tissues obtained in vitro was similar to theleaves of greenhouse gerbera plants, with an organized and defined
J.C. Cardoso et al. / Scientia Horticulturae 164 (2013) 616–624 619
Table 2pH and EC of the culture medium, fresh and dry weight of plantlets of gerbera (Gerbera jamesonii) cv. BMC101 growth after 30 day in different growth conditions and sucroseconcentration in culture medium.
Sucrose (g L−1) Growth conditions Culture medium Total fresh weight (g) Dry weight
pH EC (mS cm−1) Total (g) (%)
0 Laboratory 4.75 2.7 0.72 aB 0.046 aC 6.52 cGreenhouse 5.31 2.4 0.84 aB 0.055 aC 6.54 c
20 Laboratory 4.22 1.5 1.39 bA 0.100 bB 7.26 bGreenhouse 4.66 0.6 1.99 aA 0.147 aB 7.45 b
40 Laboratory 4.16 0.8 1.65 bA 0.126 bA 7.61 bGreenhouse 4.97 0.3 2.28 aA 0.206 aA 9.05 a
Numbers followed by the same letter in the columns are not significantly different by Tukey’s test at 5% (*) and 1% (**) probability. The values are means of five replicationswith eight explants per treatment. Data were obtained 28 days after inoculation of explants. EC – electrical conductivity, CV – coefficient of variation.
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25 30 35 40
Sucrose (g L-1)
Fres
h w
eigh
t (g)
SH LB SH GH RT LB RT GH Total LB Total GH
A
0
0.05
0.1
0.15
0.2
0.25
0 10 20 30 40
Sucrose (g L-1)
Dry
wei
ght (
g)
SH LB SH GH RT LB RT GH Total LB Total GH
A
B
Fig. 1. Fresh and dry weight of plantlets of gerbera (Gerbera jamesonii) cv. ‘BMC101′ growth for 30 days in culture medium with different sucrose concentrations and underdifferent growth conditions. SH – shoots, RT – roots, LB – laboratory, GH – greenhouse. (A) Increase in root fresh and dry weight in gerbera plantlets grown in greenhouseconditions relative to laboratory conditions. (B) Increase in shoot dry weight in gerbera plantlets grown under greenhouse conditions relative to laboratory conditions.
R² = 0.77*
R² = 0.87*
R² = 0.91*
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
35302520151050
Time (days)
Dry
wei
ght o
f sho
ots
(g)
Lab 0 Lab 20 Lab 40 GH 0 GH 20 GH 40
R² = 0.76*
R² = 1.00**
R² = 0.89*
R² = 0.98**
R² = 0.95**
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
35302520151050
Time (days)
Dry
wei
ght o
f roo
ts (g
)
Lab 0 Lab 20 Lab 40 GH 0 GH 20 GH 40
Fig. 2. Accumulation of dry weight (g) of shoots (left) and roots (right) in plantlets of gerbera (Gerbera jamesonii) cv. ‘BMC101’ growth in vitro in media REM with differentconcentrations of sucrose (0, 20 and 40 g L−1) and under laboratory (Lab) and greenhouse (GH) environmental conditions. The data were the result of means of five repetitions(flasks) with eight plantlets each per treatment. ns – non-significant, *significant at P < 0.05, **significant at P < 0.01.
620 J.C. Cardoso et al. / Scientia Horticulturae 164 (2013) 616–624
Table 3Analysis of stomata before acclimatization and gerbera plant development after 30 days of acclimatization in greenhouse conditions.
Sucrose (g L−1) Growth conditions Before acclimatization After acclimatization
Stomata density (/mm2) Osteole diameter (�m) Survival (%) Number of leaves/plant Plantlet height (cm) LD (cm)
0 Laboratory 225.2 bA 2.41 bA 80 bB 6.2 bB 8.9 bB 4.06 bBGreenhouse 243.0 aA 2.59 cA 100 aA 12.2 aA 15.0 aA 5.46 aA
20 Laboratory 408.9 aA 6.53 aA 100 aA 11.6 aA 14.8 aA 5.78 aAGreenhouse 221.2 aB 3.66 bB 100 aA 8.2 abA 13.1 aA 5.46 aA
40 Laboratory 432.5 aA 6.50 aA 100 aA 12.2 aA 14.5 aA 5.90 aAGreenhouse 250.2 aB 6.03 aA 100 aA 6.6 bB 12.7 aA 5.48 aA
Numbers followed by the same letter in the columns are not significantly different by Tukey’s test at 5% (*) and 1% (**) probability. Before acclimatization: The values arem s of 20
m(
3
apcs(tt
FB4fl
eans of 12 analyzed images/treatment. After acclimatization: The values are mean
esophyll in the two types of parenchyma (palisade and spongy)Fig. 4).
.3. Plantlet acclimatization
The plants obtained from in vitro culture in both laboratorynd greenhouse conditions were successfully acclimatized inlastic trays with a coconut fibre-based substrate. There was nolear trend for the number of leaves/plant but plant height was
ignificantly lower in LB0 treatment than in other treatmentsTable 3). Except for those plants obtained in treatment LB0, allhe treatments showed good shoot development during acclima-ization (Table 3; Fig. 3I and J). In addition, those plants obtained
ig. 3. Effects of sucrose concentration on in vitro growth and acclimatization of gerbera) Sucrose at 0, 2 and 4% (w/v) under laboratory (A) and greenhouse conditions (B) (abov% (w/v) sucrose, respectively. (F–H) Leaf stomata under greenhouse conditions at 0, 2 aowering in the greenhouse. (A and B) Bars = 4.0 cm, (C–H) bars = 20 �m.
plantlets per treatment and the experiment was repeated twice. LD – leaf diameter.
in treatment GH0 had similar development to those developed inmedia with sucrose (GH20 or GH40).
Regarding the development after acclimatization, the plantsobtained in all treatments showed similar development and hadvery similar vegetative and reproductive morphological featuresas the donor plants (Fig. 3K).
4. Discussion
4.1. Sucrose in medium increases the in vitro development of
plantlets
In this study, the addition of sucrose to medium promoted sig-nificantly increases in all shoot and rooting parameters evaluated,
(Gerbera jamesonii) cv. ‘BMC101’ in laboratory and greenhouse conditions. (A ande, shoots; below, roots). (C–E) Leaf stomata under laboratory conditions at 0, 2 andnd 4% (w/v) sucrose, respectively. (I and J) Acclimatized plantlets. (K) Adult plants
J.C. Cardoso et al. / Scientia Horticulturae 164 (2013) 616–624 621
Fig. 4. Transverse sections (light microscopy) of gerbera (Gerbera jamesonii) cv. ‘BMC101’ leaves grown in vitro. (A–C) Plantlets grown in the greenhouse at 0, 2 and 4% (w/v)s (w/vs
matcrlwwstc(meCarfedttfwvacaSpit
(tiwutrpa
ucrose, respectively; (D–F) plantlets grown in laboratory conditions at 0, 2 and 4%tomata; t – trichome. Bars = 100 �m.
ainly under laboratory conditions, including plantlet height, leafnd root number, and rooting percentage of gerbera. Interestingly,he addition of sucrose also increased the total FW and DW, and per-entages of DW in laboratory and greenhouse conditions. Similaresults were obtained for Dendrobium, in which increasing plant-et height, root number and FW were observed in MS medium
ith 3 and 6% sucrose (Faria et al., 2004) and for Juglans regia,hich showed a large increase in shoot and root mass when 4%
ucrose was added to the medium (Vahdati et al., 2004). In con-rast, when sucrose was added to medium, regardless of growthonditions, shoot and root performance of macadamia was reducedCha-um et al., 2011). In Coffea arabusta, the addition of sucrose to
edium also resulted in poor development of plantlets (Nguyent al., 2001). Mitra et al. (1998) did not observe differences inhrysanthemum plantlet development between photoautotrophicnd photomixotrophic growth conditions. The contrasts in theseesults can be explained, in part, by the differences in methods usedor photoautotrophic micropropagation (Kozai et al., 1997; Purohitt al., 2011; Xiao et al., 2011), although too few studies have beenedicated to determine the best PPFD requirements of in vitro cul-ures under photoautotrophic and photomixotrophic conditions. Inhe studies conducted on macadamia (Cha-um et al., 2011) and Cof-ea (Nguyen et al., 2001), the authors used porous culture vessels
ith vermiculite as support, enriched CO2 conditions and forcedentilation, maintaining or increasing the percentage of rootingnd root quality. Forced ventilation under CO2-enrichment andonstant PPFD stimulated rooting in Cymbidium, but only when
rockwool substrate was used (Teixeira da Silva et al., 2007).habanpour et al. (2011) also observed a small reduction in theercentage of rooting of gerbera as the concentration of sucrose
ncreased from 3 to 4% in laboratory conditions (25 ◦C, 16-h pho-operiod and 67.5 �mol m−2 s−1 PPFD).
The increase in shoot and root DW in greenhouse conditionsusing natural light and high PPFD) in our study led us to concludehat in greenhouse conditions photosynthesis can be stimulated inn vitro plantlets. Alternatively, sucrose, when added to medium,
as more efficiently utilized by the greenhouse plantlets thannder laboratory conditions. The addition of sucrose increases
his stimulus to accumulate structural carbons, mainly in theoots. Hdider and Desjardins (1994) observed an increase inhosphoenolpyruvate carboxylase activity in media with sucrosend concluded that it is an important carboxylating enzyme in
) sucrose, respectively. e – epidermis; mc – mesenchyma; v – vascular tissues; s –
micropropagated plantlets. Fuentes et al. (2005) observed anincrease in photosynthetic parameters in coconut, includingRubisco activity and net photosynthesis, when the medium wassupplemented with 2.25% (w/v) sucrose at 40 or 400 �mol m−2 s−1
PPFD. These studies, which support the results obtained in ourexperiment, corroborate the notion that sucrose can improvephotosynthesis under different PPFDs, although major studieswill be required to prove this interaction. Deng and Donnelly(1993) showed that net photosynthesis rate was improved inmicropropagated Rubus idaeus plantlets growth in greenhouseconditions using similar treatments with or without CO2 enrich-ment. In coffee, net photosynthetic rates increased only in highlyforced ventilation, independently of the high or low stature ofPPFD (Nguyen et al., 2001), indicating that there are many factorsinvolved in different results obtained with each technique usedfor in vitro photoautotrophic culture.
In our experiment, we observed that the addition of sucrose tothe medium also promoted an increase in the uptake and betteruse of nutrients within that medium, observed by modifications inpH and a high reduction in EC compared with sugar-free medium,mainly in greenhouse conditions (Table 2). Kozai et al. (1991) alsoobserved an increase in nutrient uptake in photoautotrophic con-ditions (sugar-free media and high PPFD), but only in CO2-enrichedconditions, measured by the residual percentage of ions in themedium.
4.2. In vitro greenhouse growth improved gerbera plantletdevelopment without visible contamination
In vitro-grown gerbera plantlets established in greenhouse con-ditions with or without the addition of sucrose to the culturemedium were not visually contaminated, which is unexpectedin a less aseptic environment (a greenhouse) than a laboratory(Kubota and Tadokoro, 1999; Pospísilová et al., 1999). Microbialcontamination by fungi and bacteria can increase the costs of micro-propagation (Kozai et al., 1997; Leifert and Cassells, 2001; Purohitet al., 2011). Nagae et al. (1995) also observed no contaminationproblems in non-sterile conditions in the in vitro cultivation of
gerbera in sugar-free medium and in CO2-enriched conditions.
According to Kubota (2002), conventional photomixotrophicmicropropagation conditions are generally characterized by lowCO2 concentration during the photoperiod, low water vapour
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22 J.C. Cardoso et al. / Scientia H
ressure deficit, low air current speed and low PPFD, resulting inhe limited ability of plantlets and unfavourable environmentalonditions to photosynthesize (Nguyen and Kozai, 2005). Pho-oautotrophism, obtained by the growth of in vitro plantlets inigh PPFD, CO2-enrichment and increasing ventilation, presentsany advantages to micropropagated plantlets, including bio-
ogical, as well as in the production process, but also presentsome disadvantages such as the relative complexity and costs ofechniques required to establish a photoautotrophic system (Kozai,991; Kubota, 2001), including the maintenance of high artificialPFD (involving high energy costs) and maintaining high levelsf CO2 which requires additional laboratory space. In greenhouseonditions, the natural environment is rich in natural light (notnly high PPFD, but in light quality for photosynthesis, too) andan be used as an alternative to photoautotrophic propagationnder controlled environmental conditions, as described in mostxperiments related to the development of micropropagatedlants.
.3. Sucrose and growth conditions influence the control oftomatal density and osteole aperture
The addition of sucrose to media is considered to be one of thereatest causes of the loss of photosynthetic capacity in plantletsXiao et al., 2011), the latter occurring in part because sucrose inhe medium suppresses Rubisco activity (Hdider and Desjardins,995) and because of problems related to control of water loss dueo modifications in leaf morphology (Rezende et al., 2008), includ-ng the induction of dysfunctional stomata (Mohamed and Alsadon,010; Sáez et al., 2012). Stomatal density and osteole diameter in initro gerbera plantlets increased considerably in media with 2 and% sucrose, mainly under laboratory conditions (Fig. 3; Table 3).espite this, plantlet acclimatization was normal (Fig. 3I and J).nder greenhouse conditions, these effects were minimal. Stomatalensity is correlated with water loss capacity by transpiration andet photosynthetic rate in plants, and is influenced by environmen-al conditions including water stress, temperature and light (Xu andhou, 2008). Water deficit leads to an increase in stomatal densityZhang et al., 2006). This would explain the highest stomatal den-ity (432.5 mm−2) at the highest sucrose concentration used (4%)nder laboratory conditions (Table 3), because high sucrose con-entration reduces the osmotic potential of the culture medium,ut would not explain why, under greenhouse conditions (high
ight intensity), the stomatal density in leaves was lower than underaboratory conditions (low light intensity). High stomatal density
as observed in high, instead of, low light intensity as observedith Lycopersicon esculentum (Gay and Hurd, 1975) and Nicotiana
abacum (Voleníková and Tichá, 2001). However, this characteristiceems to be genotype-dependent, because some species showedn inverse correlation between light intensity and stomatal den-ity as was observed in different species of the Asteraceae familyMott and Michaelson, 1991; Siano et al., 2007), including gerbera,s observed in our experiment.
In sucrose treatments stomata were oval to spherical in shape.hese differences in stomatal anatomy (elliptical shape and func-ional stomata) were also observed when plantlets exposed toentilation and high PPFD were compared to those grown underhotomixotrophic conditions in culture medium with 3% sucrosespherical shape and dysfunctional stomata) (Sáez et al., 2012).n our study with gerbera, an increase in sucrose concentration
esulted in an increase in osteole diameter under low (laboratory)nd high (greenhouse) PPFD conditions, but in the laboratory high-st aperture was observed, but similar to greenhouse conditionsnly at 4% sucrose (Table 3).
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4.4. Plantlet acclimatization was independent of sucrose in themedium under greenhouse conditions
The acclimatization of gerbera ‘BMC101’ plantlets was 100% suc-cessful when the organic–mineral substrate mix was used. Gerberais normally an easy-to-root plant, but acclimatization is normallydifficult because water loss tends to be high from tender tissues,explaining why different authors observed only 50–70% acclimati-zation of plantlets in the greenhouse when plantlets were rootedunder laboratory conditions using conventional micropropagation(Kumar et al., 2004; Kumar and Kanwar, 2005; Sousa et al., 2006).However, Cardoso and Teixeira da Silva (2012) observed 100% suc-cessful acclimatization of plantlets.
The addition of sucrose to medium increased most character-istics evaluated in this experiment, including the DW (Table 2;Figs. 1 and 2), percentage of rooting 62.5 (0%), 93.8% (2%) and100% (4%) under laboratory conditions (Table 1). However, whensucrose-free medium was used, the rooted and acclimatizedplantlets that showed the best development were obtained onlyunder greenhouse conditions (Table 3; Fig. 3). Costa et al. (2009)also observed a higher survival of acclimatized banana (Musa sp.)plantlets that had been cultured under natural light conditionsin a greenhouse than in laboratory conditions. These authors alsoobserved that in laboratory conditions, high sucrose concentration(3%) was essential for the effective acclimatization of plantlets.
This diversity in results in the literature can be explained, at leastin part, by the establishment of different photoautotrophic abili-ties of plantlets grown under high PPFD relative to natural lightconditions (greenhouse) in sugar-free medium. Strawberry (Fra-garia × ananassa) plantlets grown in vitro had the largest rate ofphotosynthesis in culture medium containing 0 and 1% sucrose,but this rate decreased as sucrose concentration increased to 3 or5% (Hdider and Desjardins, 1994), partly because of the reductionin Rubisco activity caused by the high sucrose concentration in themedia (Hdider and Desjardins, 1995). In addition, better control(i.e., opening and closing) of stomata in sucrose-free media is asso-ciated with high PPFD (Sáez et al., 2012). Therefore, in this study ongerbera, under greenhouse conditions, there are most likely otherfactors that contributed to stomatal opening other than sucroseconcentration in the medium.
The greenhouse conditions promoted better use and uptake ofnutrients from the medium than laboratory conditions, as observedby a reduction of medium EC (Table 2). Kozai et al. (1991) alsoobserved an increase in nutrient uptake by in vitro strawberryplantlets at 200 �mol m−2 s−1 PPFD, in sugar-free medium and CO2enrichment.
4.5. In vitro greenhouse culture is an alternative to improvephotoautotrophic culture and reduce costs in micropropagatedplantlets
In vitro rooting under greenhouse conditions still has someadvantages over photoautotrophic culture with forced ventilation,high light intensity and CO2 enrichment in the culture environment(Zobayed et al., 2001; Kozai et al., 2005; Teixeira da Silva et al., 2006;Park et al., 2011). This is because it requires no additional area inthe laboratory and uses an environmental condition naturally richin light and CO2, thus reducing production area and energy costs,and improving the use of laboratory space for establishment andmultiplication stages. In conventional micropropagation, approxi-mately 50–80% of the growth room in a laboratory is used only forthe rooting stage. In this experiment, one of the greatest gains in
the use of pre-acclimatization in a greenhouse culture was the opti-mization of the laboratory area for the multiplication stage. Usingthe same type of flask (i.e., with an identical diameter), a mean of5 shoots explant−1 could be obtained (data not shown). Moreover,
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2.5:1 rooting: multiplication area ratio was required for gerberaicropropagation, as observed in our experiment. This resulted in
nly 28.6% of the functional area of a laboratory being used for mul-iple shoot induction while the remaining 71.4% could be used forooting shoots, hence limiting the area for multiplication, which ishe main focus of tissue culture labs.
Pre-acclimatization in a greenhouse is able to considerablymprove the commercial micropropagation and production of ger-era, while also reducing costs related to light energy. Underur experimental conditions, 40 W fluorescent lamps, 16-h pho-operiod and 30 days for rooting the shoots, a consumption of9.2 kW lamp−1 was observed. If 71.4% of the area is used for rootingerbera plantlets in the micropropagation stage, this represents aaving of 13.7 kW lamp−1 when connected for one period of growth30 days) using PAG culture, instead of laboratory conditions. Xiaond Kozai (2004) observed a 50% reduction in the culture period and
40% reduction in production costs of calla lily (Zantedeschia elliot-iana) plantlets when photoautotrophic culture was used, relativeo conventional micropropagation.
In vitro rooting under greenhouse conditions can be improved bypplying techniques that ameliorate the acclimatization of plants,ncluding the integration of photoautotrophic cultivation and these of mycorrhizal arbuscular fungi (Liu and Yang, 2008). In thisontext, in vitro culture in greenhouses can be one possible solutiono avoid unwanted contamination of plants with microorganismsuch as mycorrhizal fungi in commercial plant production labora-ories at key stages such as multiplication.
This study shows a new potential technique for using green-ouse conditions, together with a photoautotrophic system, tofficiently micropropagate gerbera. Photoautotrophic systemsave been developed for other ornamentals and other horticul-ural crops, including orchids (Epidendrum and Cymbidium) andanana (Teixeira da Silva et al., 2005a, 2005b) and SpathyphillumTeixeira da Silva et al., 2006) using a gas-permeable vessel, theitron.
Talavera et al. (2005) observed higher survival rate of plantlets,resh and dry weight, and number of leaves using natural light forn vitro cultivation of Cocos nucifera than those cultivated underaboratory conditions using artificial light. Costa et al. (2009) alsoested the use of natural light in greenhouse conditions and mea-ured anatomical and physiological modifications to banana (Musapp.). Their results about stomata density and leaf morphologynatomy, however, contradicted those obtained for gerbera in thistudy. Moreover, Costa et al. (2009) did not make in vitro and exitro growth measurements nor did they use sucrose-free media,aking the comparison with our experiment difficult. However,
hese experiments prove that more studies are required to betternderstand and to provide a functional and practical protocol forhe use of natural light for important commercial micropropagatedlants, as shown in this study for gerbera.
. Conclusion
The use of a greenhouse for the in vitro rooting and elonga-ion of gerbera provides a new potential environmental conditionhat associates this stage with the pre-acclimatization of ger-era. In addition, the conditions within this new technique, i.e.,re-acclimatization in a greenhouse, when associated with pho-oautotrophic culture (i.e., without sucrose) result in a similaruality of gerbera plantlets, following acclimatization, as those
erived from a sucrose-containing medium. The use of pre-cclimatization in a greenhouse can optimize the use of theroduction area in a laboratory and can reduce the electrical energyonsumption for gerbera micropropagation.
lturae 164 (2013) 616–624 623
Acknowledgements
J.C.C. acknowledges Vliet Flora Co. for infra-structural and finan-cial support to this research project and Prof. Dr. Adriana P.Martinelli for infra-structural support to microscopy analysis. I.B.R.thanks Prof. Dr. Paulo H. Viegas Rodrigues for educational supervi-sion in her final work in an agronomy course.
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