Improvement of Ex Vitro Desiccation through Elevation of CO2 Concentration in the Atmosphere of Culture Vessels during In Vitro Growth

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  • 1 Department of Horticulture, College of Aburaihan, University of Tehran, Pakdasht, Tehran, Iran
  • | 2 Department of Plant Sciences, University of California, Davis, CA 95616

Acclimatization of in vitro plantlets is one of the key steps in successful tissue culture propagation. Gaseous atmosphere during in vitro culture can influence the rate of ex vitro acclimation of the plantlets produced. In the current study, effects of elevated CO2 concentration on the leaf water loss dynamic responses of in vitro–produced walnut leaves during ex vitro desiccation were investigated. Elevated CO2 concentration in the headspace of culture vessels caused a considerable decrease in stomatal aperture. Although the traits related to stomatal size were not influenced by CO2 elevation, the number of small stomata was increased, and the number of large stomata was decreased at elevated CO2 concentration. Higher CO2 concentration resulted in a lower transpiration rate and a higher relative water content (RWC) during ex vitro desiccation. This improvement was due to decreased stomatal aperture during the first phase of water loss. Osmotic potential (ψs) was decreased under an elevated CO2 concentration, but no influence was observed on the concentration of compatible solutes. In conclusion, increasing the CO2 concentration of culture vessel headspace can be an efficient tool for improving acclimation of in vitro–grown walnuts without negative effects on plantlet growth.

Abstract

Acclimatization of in vitro plantlets is one of the key steps in successful tissue culture propagation. Gaseous atmosphere during in vitro culture can influence the rate of ex vitro acclimation of the plantlets produced. In the current study, effects of elevated CO2 concentration on the leaf water loss dynamic responses of in vitro–produced walnut leaves during ex vitro desiccation were investigated. Elevated CO2 concentration in the headspace of culture vessels caused a considerable decrease in stomatal aperture. Although the traits related to stomatal size were not influenced by CO2 elevation, the number of small stomata was increased, and the number of large stomata was decreased at elevated CO2 concentration. Higher CO2 concentration resulted in a lower transpiration rate and a higher relative water content (RWC) during ex vitro desiccation. This improvement was due to decreased stomatal aperture during the first phase of water loss. Osmotic potential (ψs) was decreased under an elevated CO2 concentration, but no influence was observed on the concentration of compatible solutes. In conclusion, increasing the CO2 concentration of culture vessel headspace can be an efficient tool for improving acclimation of in vitro–grown walnuts without negative effects on plantlet growth.

Conventional in vitro propagation can result in production of plants with low survival capacity (Crane and Hughes, 1990). Variable and often insufficient CO2 concentration, high relative humidity (RH), and accumulation of ethylene or other gases in the headspace of the culture containers can cause problems for plantlets during and after in vitro propagation (Hazarika et al., 2004; Kozai, 2010; Lamhamedi et al., 2003; Pospisilova et al., 2007). Headspace of tissue culture vessels is characterized by high RH and low vapor pressure deficit (VPD). Low VPD, through its impact on stomatal conductance and transpiration rate, can influence water relations of in vitro plantlets (Fuchigami et al., 1981; Grout and Aston, 1977; Nogues et al., 1998). Long-term exposure to high RH decreases the closing ability of stomata because of low foliar abscisic acid (ABA) levels (stomatal malfunctioning). As a result, the capacity of leaves to control water loss decreases when plants are exposed to conditions of increased evaporative demand (Aliniaeifard and van Meeteren, 2013, 2016; Aliniaeifard et al., 2014; Fanourakis et al., 2013; Rezaei Nejad and Van Meeteren, 2005, 2007). The consequence of low water conservation capacity of in vitro plants can be high mortality of plantlets after transferring to ex vitro conditions (Crane and Hughes, 1990; Shim et al., 2003). Various methods such as ventilation (Cui et al., 2000; Shim et al., 2003), lids permeable to water vapor (Ghashghaie et al., 1992), and others (Cha-um et al., 2003, 2010; Tanaka et al., 1992) have been used to decrease the RH in the headspace of culture vessels. However, growth retardation, low efficiency, and contamination have been reported as negative side effects of those techniques (Sallanon and Maziere, 1992).

Photosynthesis of in vitro plantlets is often restricted by low CO2 concentration, low light irradiance and presence of high sugar concentration in the medium (Kubota, 2002). Tissue culture media are typically supplemented with sugar as the source of carbon to maintain a positive carbon balance (Kwa et al., 1995). On the other hand, an increase in CO2 concentration in the headspace of culture vessels during darkness and decreased CO2 during light periods have been reported (Fujiwara et al., 1987; Thomas, 1999). Low CO2 concentration and high sugar levels limit the activity of ribulose bisphosphate carboxylase during most of the photoperiod (Donnelly and Vidaver, 1984; Kilb et al., 1996). Therefore, plantlets are forced to develop heterotrophy or photomixotrophy under in vitro conditions (Fujiwara et al., 1987; Kozai et al., 1997).

Low CO2 concentration in the atmosphere surrounding the leaf results in low internal CO2 concentration in the substomatal cavity (Ci), producing a signal to open the stomata. Conversely, high CO2 concentration in the atmosphere surrounding the leaf results in high Ci, causing closure of the stomata (Allaway and Mansfield, 1967; Uehlein et al., 2003). Observations in tobacco and grapevine plantlets showed stomata closed in response to high CO2 (During and Stoll, 1996; Posposilova et al., 1999). It has been shown that elevated CO2 concentration can enhance the effect of ABA on stomata closure (Pospisilova et al., 2000).

Low in vitro CO2 concentration combined with high RH in the headspace of culture vessels appears to promote an enduring open stomatal state in in vitro–produced plants (Hazarika, 2006). As a result, the water retention capacity of their leaves during ex vitro acclimation is inhibited. Because low water retention is thought to be due largely to high stomatal transpiration during ex vitro acclimation, the aims of this study were 1) to alter stomatal morphology during in vitro growth; 2) to determine the water loss characteristics of in vitro–generated leaves during ex vitro desiccation and; 3) to determine if increased CO2 concentration during in vitro development can increase the water retention capacity of leaves during ex vitro acclimatization.

CO2 enrichment can be achieved by growing the explants in tissue culture containers sealed with gas-permeable films, direct supply of CO2 in the vessels by forced ventilation, or using CO2-generating chemicals in containers (Kozai, 1991; Solarova and Pospisilova, 1997; Xiao et al., 2011). In the current study, to prevent problems due to ventilation (Sallanon and Maziere, 1992), increased CO2 was achieved by CO2-generating chemicals isolated from the medium. Persian walnut (Juglans regia L.) was chosen for this study because of 1) difficulties in its propagation through vegetative reproduction (Aviles et al., 2010); 2) the commercial importance of mass propagation of high quality, disease-free, and uniformly multiplied cultivars with desirable traits through in vitro propagation (Payghamzadeh and Kazemitabar, 2011) and; 3) difficulties in acclimatization after in vitro production.

Material and Methods

Plant materials and growth conditions.

Microshoots of Persian walnut (Juglans regia L. cv. Chandler) (20 ± 2 mm length) were selected from previously established cultures; transferred every 3–4 weeks to fresh medium; cultured on the DKW (Driver–Kuniyuki Walnut) medium (Driver and Kuniyuki, 1984) supplemented with IBA (0.01 mg·L−1), BAP (1 mg·L−1), and sucrose (3%); and solidified with Gelrite (2.2 g·L−1). Four vessels for each treatment and two explants per vessels were used. Vessels were 65 mm in diameter and 85 mm in height (≈350 mL), and each vessel contained 50 mL of DKW medium; therefore, the volume of headspace atmospheric space was 300 cm−3. Plantlets (nodal explants with leafy part) were grown under photomixotrophic conditions (DKW medium supplemented with 3% sucrose). Medium pH was adjusted to 5.5 before autoclaving for 20 min at 121 °C. Work was conducted in a growth chamber with 100 µmol·m−2·s−1 light intensity at 25 ± 2 °C under 16/8 h light/dark cycle for 30 d, without further transfer of the plant material. Growth chamber supplemented with fluorescent lamps with 15 cm vertical distance between the lamps and culture vessels.

Increasing CO2 in the headspace of culture vessels.

Plantlets were exposed to two different CO2 concentrations in the headspace of culture vessels: a) vessels with 3 mL of mixed NaHCO3 and Na2CO3 solution (3 M) in the ratio of 73/27 (v/v), respectively. The mixed solution, autoclaved and placed into 4-mL vials, released 40 ppm/m3 CO2 in to the atmosphere of the vessel headspaces (Solarova and Pospisilova, 1997). This is the CO2-releasing potential of the mixed solution; however, when and how it releases depends on many factors and is not possible to calculate, and b) vessels without the CO2-releasing solution which were considered as control plantlets. Concentration of CO2 in the headspace atmosphere of the culture vessels can also be influenced by some plant metabolic processes (e.g., photosynthesis and respiration); therefore, in many previous publications, the exact amount of CO2 in time have not been mentioned (Joshi et al., 2009; Mitra et al., 1998; Suthar et al., 2009). Work stages are presented in Fig. 1.

Fig. 1.
Fig. 1.

Work stages for CO2 elevation in the atmosphere of the vessel headspaces. (A) Microshoots were cultured in the culture vessels. (B) Vials containing 3 mL of mixed NaHCO3 and Na2CO3 solution (3 M) in the ratio of 73/27 (v/v), respectively, were placed in the vessels. (C) The samples were incubated and grown at 25 ± 2 °C under 16/8 h light/dark cycles for 30 d.

Citation: HortScience horts 52, 7; 10.21273/HORTSCI11922-17

Vegetative characteristics.

At the end of the experiment, the microshoots were removed from the culture medium and vegetative characteristics were measured. Shoot lengths were measured with a ruler, fresh shoot weights were taken, and then leaf areas were scanned and analyzed using ImageJ (U.S. National Institutes of Health; Bethesda, MD; http://imagej.nih.gov/ij/). Dry weights of shoots were obtained by placing the samples in an oven at 70 °C for 48 h. Specific leaf area (SLA), leaf water content per unit area (LWCA), leaf mass area, and leaf water content (LWC) were measured according to the equations of Aliniaeifard et al. (2016). The relative chlorophyll (Chl) content was measured with a portable leaf chlorophyll meter (SPAD; Konika Minolta).

Stomatal morphology.

To investigate the differences in stomatal morphology between CO2-treated shoots and control shoots, the lower epidermis of the second lateral leaflets from the apex (adaxial surface) of five tissue-cultured shoots for each treatment were coated by a thin layer of nail polish. After a few minutes, a strip of transparent sticky tape was applied on the dried polish. Sticky tapes were peeled from leaves and along with nail polish were mounted on microscopic slides. Images of 200 stomata taken from these epidermal strips using a light microscope (model Olympus) in combination with ImageJ (U.S. National Institutes of Health, Bethesda, MD; http://imagej.nih.gov/ij/) were used to measure the stomatal length, stomatal width, pore length, pore width, stomatal area, ratio of stomatal length to stomatal width, and stomatal density. (The accuracy of the scale that was used for measuring stomatal traits was 1 µm.)

Stomatal response to desiccation.

The weighing of detached leaves at room temperature under controlled conditions was used to study the effect of desiccation on transpiration rate and RWC. The leaves were detached from shoots and used immediately under 50 μmol·m−2·s−1 irradiance at room temperature and 50% RH, resulting in 1.24 kPa VPD. For leaf desiccation, after measuring leaf area, leaves were placed upside down on a balance and gravimetrically weighed every 5 min for a duration of 90 min. Transpiration rate was calculated according to Aliniaeifard et al. (2014) based on the following equation:
DE1

To determine the RWC, the leaves were dried for 48 h at 70 °C. The RWC during the desiccation period was calculated according to Slavik (1974).

Leaf ψs and compatible solutes determination.

For determination of ψs, leaves were cut into small segments, placed into Eppendorf tubes perforated with four small holes, and immediately frozen in liquid nitrogen. After being encased individually in a second intact Eppendorf tube, they were allowed to thaw for 30 min and centrifuged at 15,000 g for 15 min at 4 °C. The collected supernatant was used for ψs determination. Osmolarity (c) was assessed with a vapor pressure osmometer (Osmomat 030-gonatec) and converted from mosmoles kg−1 to MPa according to the Van’t Hoff equation (Martinez et al., 2004).

Proline concentration of leaves was spectrophotometrically measured as described by Bates et al. (1973). Free proline content (µg·g−1 DW) was determined from a standard curve prepared with five standard concentrations (0–200 µg·mL−1) of L-proline (Bates et al., 1973; Meloni et al., 2004).

Glycinebetaine concentration (µg·g−1 DW) was estimated according to Grieve and Grattan (1983). The absorbance was determined at 365 nm with a spectrophotometer (Lambda 25-ultraviolet/VIS spectrometer). Reference standards of GB (50–200 μg·mL−1) were prepared in 2 M sulfuric acid (Grieve and Grattan, 1983).

Statistical analysis.

Data of stomatal morphology, compatible solutes, and vegetative traits were subjected to ANOVA with P ≤ 0.05 considered as not significant. For stomatal characteristics, data obtained from one leaf were considered not independent, and a paired t test was used to find significant differences (P ≤ 0.05) between CO2-treated plantlets and control plantlets. For the statistical analysis of transpiration rate (E) and RWC, data were fitted using nonlinear regression with one-phase exponential decay, E = (E0 − Bottom)*exp(−K*X) + Bottom, where E0 is E at time zero, K is the slope of the curve, Bottom is E when it reaches a plateau, and X is time. An F test was used for comparing the parameters of the fitted curves. The change of E as a function of RWC was fitted using a sigmoidal dose–response curve with a variable slope (E = Bottom + {(Top − Bottom)/[1 + 10(RWC50 − RWC).Slope]}) and an F test was used for comparing the parameters of the fitted curves. GraphPad Prism 5 for Windows (GraphPad software, Inc. San Diego, CA) was used for analyzing the data.

Results

Stomatal morphology.

No significant changes in stomatal size were observed because of exposure of leaves to high CO2 concentration in culture vessels (Table 1). However, stomatal aperture in leaves grown under high CO2 was 34% narrower than stomatal aperture in controls and stomata were closed in leaves grown under high concentration of CO2 (Table 1; Fig. 2). Huge heterogeneity in stomatal size was observed under both treatments (Fig. 3). However, growing plantlets under elevated CO2 increased the number of small-sized stomata (≤150 μm2), while number of stomata larger than 500 μm2 were decreased by high CO2 concentration (Fig. 2).

Table 1.

Stomatal features of walnut tissue-cultured plantlets (cv. Chandler) developed under two CO2 concentrations. Micropropagated shoots were grown in jars with a CO2-releasing solution in the headspace of the culture vessels (CO2) or without the CO2-releasing solution as control plantlets (C). The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

Table 1.
Fig. 2.
Fig. 2.

Stomata on abaxial surface of in vitro–grown walnut (cv. Chandler) leaves developed under ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars (A) without CO2-releasing solution as control plantlets or (B) with CO2-releasing solution in the headspace of culture vessels. The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

Citation: HortScience horts 52, 7; 10.21273/HORTSCI11922-17

Fig. 3.
Fig. 3.

Variation in stomatal size on leaves of in vitro–grown walnut (cv. Chandler) microshoots exposed to ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

Citation: HortScience horts 52, 7; 10.21273/HORTSCI11922-17

Stomatal response to ex vitro desiccation.

Transpiration rate (E) at the start of ex vitro desiccation was decreased by 47% in leaves grown in vitro under high CO2 concentration (Fig. 4A; top in Table 2). Span (difference between E0 and bottom) of the E curve in CO2-treated plantlets was one-third the span of the E curve for control plantlets (Table 2).

Fig. 4.
Fig. 4.

(A) Leaflet transpiration rate, (B) relative water content and (C) transpiration rate as a function of RWC during 1.5 h of ex vitro desiccation of leaves excised from tissue cultured walnut microshoots (cv. Chandler). In vitro–formed leaves were grown under either ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, the leaves of plantlets were detached and placed with their abaxial side up on a balance in an environment with 50% relative humidity, 21 °C, and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

Citation: HortScience horts 52, 7; 10.21273/HORTSCI11922-17

Table 2.

Parameters of curve fitting for transpiration rate (E) during 1.5 h of ex vitro leaf desiccation in tissue cultured walnut plantlets (cv. Chandler) exposed to two CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, the leaves of microshoots were detached and placed with their abaxial side up on a balance in an environment with 50% relative humidity 21 °C, and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

Table 2.

Exposure of shoots to high CO2 concentration during in vitro growth significantly influenced the RWC when leaves were exposed to ex vitro desiccation (Fig. 4B). Half-value of RWC in CO2-treated plantlets was reached 20 min after desiccation, whereas the half-value of RWC in control plantlets occurred only 15 min after desiccation. In addition, the RWC of CO2-treated shoots plateaued at 35%, vs. 10% for controls (bottom in Table 3). The span of the RWC curve for leaves exposed to elevated CO2 was 33% smaller than in controls (Table 3). These results indicate that elevated CO2 concentration in the headspace during in vitro culture can increase water retention capacity during ex vitro acclimatization.

Table 3.

Parameters of curve fitting for relative water content during 1.5 h ex vitro desiccation of leaves from tissue cultured walnut microshoots (cv. Chandler) exposed to ambient and elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, detached leaves were placed with their abaxial side up on a balance in an environment with 50% relative humidity, 21 °C and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

Table 3.

E × RWC curve for leaves exposed to high CO2 concentrations in vitro was considerably lower than the E × RWC curve for controls (Fig. 4C). The RWC50 (RWC at which E is midway between the top and bottom of the E × RWC curve) of the fitted curve was 81% for the leaves treated with CO2 and 58% for controls (Table 4).

Table 4.

Parameters of curve fitting for E × RWC during 1.5 h ex vitro desiccation of leaves from tissue cultured walnut microshoots (cv. Chandler) exposed to ambient and elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, detached leaves were placed with their abaxial side up on a balance in an environment with 50% relative humidity, 21 °C and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

Table 4.

Vegetative characteristics.

No significant changes in vegetative traits were observed because of CO2 concentration in the headspace of culture vessels (Table 5).

Table 5.

Effects of elevated CO2 concentration in the headspace of culture vessels on vegetative characteristics, ψS (MPa) and compatible solutes of walnut tissue cultured leaves (cv. Chandler). Micropropagated shoots were grown in jars with CO2-releasing solution in in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

Table 5.

Leaf ψS and compatible solute determination.

Exposure of walnut microshoots to elevated CO2 resulted in a 28% decrease in leaf ψSs) relative controls (Table 5) even though the concentrations of proline and glycinebetaine in these leaves were unaffected (Table 5).

Discussion

In our study, increasing the CO2 concentration in vitro resulted in the improvement of water retention capacity during ex vitro desiccation. Low CO2 concentration in the headspace of culture vessels is thought to be the main factor limiting photosynthesis in plants produced in vitro. Increasing the CO2 concentration in vitro can lead to an increase in the carboxylase and a decrease in the oxygenase activity of ribulose-1,5-bisphosphate carboxylase oxygenase, resulting in improved carbon assimilation (Reddy et al., 2010; Rybczynski et al., 2007). The low net photosynthetic rate, and consequently low growth rate of plantlets after transplanting, is usually due to low CO2 concentration in tightly closed cultivation vessels (Pospisilova et al., 2007). In the current study, no significant differences in vegetative growth characteristics were observed between high CO2 and control plantlets. This could be due to limited duration (30 d) of exposure to high CO2 concentration. Although improved content of Chl a and Chl a/b ratio and photosynthesis following exposure of in vitro plants to elevated CO2 has been previously reported (Pospisilova et al., 2000), we did not find a significant change in chlorophyll content (SPAD) (Table 5).

In the present study, elevated CO2 concentration in the vessel headspace altered stomatal functioning and improved ex vitro acclimatization of walnut plantlets. Similarly, stomatal conductance was decreased in tobacco plantlets treated with ABA and acclimated under elevated CO2 concentration (Pospisilova et al., 2000). Furthermore, decreased stomatal conductance and improved plant water status were observed in tobacco plantlets after transplantation under elevated CO2 concentration (Posposilova et al., 1999). Also, improved stomatal function was observed in Dianthus Caryophyllus L. leaves grown under CO2 applied by forced ventilation. This corresponded with increased K+ concentration in the guard cells and increased free ABA content (Majada et al., 1997).

Generally, heterotrophic plantlets show abnormal leaf anatomy and inadequate water control (Brainerd and Fuchigami, 1982; Galzy and Compan, 1992) and the transpiration rate at the beginning of acclimatization of in vitro plantlets is commonly very high but decreases after adaptation to ex vitro conditions (Chaari-Rkhis et al., 2011; Hazarika, 2006; Posposilova et al., 1999). However, if water loss can be controlled by environmental conditions and initial stomatal function improved, then survival and growth during the acclimatization period can be improved. Therefore, the objective of this article was to study the effect of elevated CO2 concentration in culture vessel headspace on growth rate, water relations and stomatal function, all of which potentially influence the acclimatization phase and mortality of walnut tissue culture plantlets during the transfer to ex vitro condition.

Elevated CO2 concentration in the headspace of culture vessels increased leaf stomatal density relative to controls (Table 1). Less functional stomata with wide apertures have been reported previously in leaves of plants produced under high RH and low CO2 concentrations (Aliniaeifard and van Meeteren, 2013, 2014, 2016; Zobayed et al., 1999). Stomatal function at a high CO2 concentration may be mediated by an increased K+ concentration in the guard cells and the free ABA content of leaves (Majada et al., 1997; Pospisilova et al., 2000). Low foliar ABA content has been suggested as a cause of stomatal malfunctioning and high water loss in high RH-grown plants (Arve et al., 2013; Fanourakis et al., 2011; Rezaei Nejad and Van Meeteren, 2007; Santamaria et al., 1993). This is because of the limited plant ability to produce ABA owing to low evaporative transpiration rate (i.e., in vitro condition) (Aliniaeifard and van Meeteren, 2013, 2014). RH in closed in vitro containers is kept close to 100%; thus, the plantlets are never exposed to evaporative difference which is necessary to induce the synthesis of ABA (Santamaria et al., 1993). Accumulation of ABA in leaves developed under elevated CO2 concentration has been also reported (Majada et al., 1997; Pospisilova et al., 2000). In agreement with our results, stomatal closure in response to high CO2 concentration was also observed in tobacco and grapevine plantlets (During and Stoll, 1996; Posposilova et al., 1999). In this study, the number of small-sized stomata increased in response to high CO2 (Figs. 2 and 3). Formation of more small stomata with narrow apertures resulted in decreased transpiration rate and, as a result, improved control of water loss during desiccation. It has been shown that stomatal morphology correlates with stomatal responsiveness to desiccation and that smaller stomata have a more rapid response to closing stimuli (Hetherington and Woodward, 2003). High transpiration rates during desiccation of the tissue-cultured plantlets can be due to stomatal abnormalities (Brainerd and Fuchigami, 1982; Grout and Aston, 1977; Hazarika, 2006), reduced leaf epicuticular wax (Grout and Aston, 1977; Shackel et al., 1990; Sutter, 1988), and high stomatal density (Desjardins et al., 1987; Hazarika, 2006). Our previous study (unpublished data) showed that the role of cuticular transpiration is much lower than the role of stomatal transpiration during ex vitro desiccation of in vitro–produced leaves. In the current study, the E of CO2-treated walnut plantlets was almost half that of the control plantlets (Fig. 4A), resulting in higher RWC during ex vitro desiccation (Fig. 4B).

The first phase of water loss during ex vitro desiccation depends on the original leaf stomatal density and aperture. Since the stomatal density of walnut in in vitro leaves was not significantly increased by high CO2 treatment, stomatal aperture was the main determinant of water loss during the first phase of acclimatization (span in Tables 2 and 3; top in Tables 2 and 4). Stomatal functionality, another key determinant of water loss during desiccation (Aliniaeifard et al., 2014), was not a determinant factor for water loss in the current study (slope in Tables 2 and 4).

RWC50 of the E × RWC curve was 81% for the leaves of CO2-treated plantlets and 58% for control plantlets. This indicates that the ability of in vitro CO2-treated leaves to conserve their water content during ex vitro desiccation is superior to that of the control leaves (38% for CO2-treated leaves and just 7% in control leaves). Similarly, RWC of Pfaffia glomerata (Spreng.) was increased when vessel atmosphere was enriched with high CO2 (Saldanha et al., 2013).

Compatible solutes (proline and glycinebetaine) and tissue water content were not influenced by in vitro CO2 (Table 5). However, foliar ψs was decreased by the CO2 treatment (Table 5). ψs is directly influenced by sucrose content of the culture medium (de Paiva Neto and Otoni, 2003) and interferes with the water content of in vitro cultivated plant tissues (Cha-um et al., 2011). Sugar is normally used as a source of carbon to maintain positive carbon balance in the tissue culture process (Kwa et al., 1995). It appears that in the current study, the presence of both CO2 gas and sugar as carbon sources (photomixotrophic system) resulted in a decrease in leaf ψs in the CO2-treated plantlets. Several studies have shown that in vitro growth under photoautotrophic conditions and a CO2-enriched atmosphere can lead to a greater increase in biomass accumulation than under photomixotrophic conditions (Badr et al., 2011; Kozai, 2010; Saldanha et al., 2013; Solarova and Pospisilova, 1997). However, vegetative characteristics in the current study were not influenced by elevated CO2 concentration. Similarly, the presence of sugar in the medium combined with high CO2 concentration in the atmosphere of culture vessels did not result in improved growth of fern (Kwa et al., 1995). Short exposure time to the high CO2 and also low light intensity could be reasons for the lack of growth improvement in present study.

Despite of an increase in compatible solutes, no significant treatment differences were found in proline or glycinebetaine content (Table 5). Content of compatible solutes such as proline can be increased by reducing in RWC or by structural damage in plant leaves (Taylor, 1996). In the current study, LWC also was not altered by high CO2 concentration (Table 5).

Improved ex vitro acclimatization due to elevated CO2 concentration in tissue culture vessels has been shown in several plant species (Buddendorf-Joosten and Woltering, 1994; Carvalho et al., 2002; Pospisilova et al., 2000; Solarova and Pospisilova, 1997). In the present study employing leaves of in vitro–grown walnut microshoots we found exposure to increased CO2 concentration resulted in generation of more small-sized and fewer large-sized stomata and found no negative effects on plantlet vegetative characteristics. Alterations in stomatal traits led to improved resistance to ex vitro desiccation. This improvement was mainly due to decrease in the first phase of water loss during the dynamic response of the leaf to ex vitro desiccation. Therefore, increasing CO2 concentration during in vitro culture of walnuts can be a useful tool to reduce ex vitro water loss in a species that is particularly sensitive to desiccation during ex vitro acclimation.

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  • Bates, L., Waldren, R. & Teare, I. 1973 Rapid determination of free proline for water-stress studies Plant Soil 39 205 207

  • Brainerd, K. & Fuchigami, L. 1982 Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA, and CO2 J. Exp. Bot. 33 388 392

    • Search Google Scholar
    • Export Citation
  • Buddendorf-Joosten, J. & Woltering, E. 1994 Components of the gaseous environment and their effects on plant growth and development in vitro J. Plant Growth Regul. 15 1 16

    • Search Google Scholar
    • Export Citation
  • Carvalho, L., Santos, P. & Amancio, S. 2002 Effect of light intensity and CO2 concentration on growth and the acquisition of in vivo characteristics during acclimatization of grapevine regenerated in vitro Vitis 41 1 1 6

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Chanseetis, C., Chintakovid, W., Pichakum, A. & Supaibulwatana, K. 2011 Promoting root induction and growth of in vitro macadamia (Macadamia tetraphylla L. ‘Keaau’) plantlets using CO2-enriched photoautotrophic conditions Plant Cell Tissue Organ Cult. 106 435 444

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Mosaleeyanon, K., Kirdmanee, C. & Supaibulwatana, K. 2003 A more efficient transplanting system for Thai neem (Azadirachta siamensis Val.) by reducing relative humidity Sci. Asia 29 189 196

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Ulziibat, B. & Kirdmanee, C. 2010 Effects of temperature and relative humidity during in vitro acclimatization, on physiological changes and growth characters of Phalaenopsis adapted to in vivo Austral. J. Crop. Sci. 4 750 756

    • Search Google Scholar
    • Export Citation
  • Chaari-Rkhis, A., Maalej, M., Drira, N. & Standardi, A. 2011 Micropropagation of olive tree (Olea europaea L.) cv. ‘Oueslati’ Turk. J. Agr. For. 35 403 412

    • Search Google Scholar
    • Export Citation
  • Crane, J. & Hughes, H. 1990 Medium overlays for improved hardening of micropropagated potatoes HortScience 25 794 795

  • Cui, Y.Y., Hahn, E.J., Kozai, T. & Paek, K.Y. 2000 Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro Plant Cell Tissue Organ Cult. 62 219 226

    • Search Google Scholar
    • Export Citation
  • de Paiva Neto, V.B. & Otoni, W.C. 2003 Carbon sources and their osmotic potential in plant tissue culture: Does it matter? Sci. Hort. 97 193 202

  • Desjardins, Y., Gosselin, A. & Yelle, S. 1987 Acclimatization of ex vitro strawberry plantlets in CO2 enriched environments and supplementary lighting J. Amer. Soc. Hort. Sci. 112 846 851

    • Search Google Scholar
    • Export Citation
  • Donnelly, D.J. & Vidaver, W.E. 1984 Pigment content and gas exchange of red raspberry in vitro and ex vitro J. Amer. Soc. Hort. Sci. 109 177 181

  • Driver, J.A. & Kuniyuki, A.H. 1984 In vitro propagation of Paradox walnut rootstock Hort. Sci. 19 507 509

  • During, H. & Stoll, M. 1996 Stomatal patchiness of grapevine leaves. I. Estimation of non-uniform stomatal apertures by a new infiltration technique Vitis 35 65 68

    • Search Google Scholar
    • Export Citation
  • Fanourakis, D., Carvalho, S.M., Almeida, D.P. & Heuvelink, E. 2011 Avoiding high relative air humidity during critical stages of leaf ontogeny is decisive for stomatal functioning Physiol. Plant. 142 274 286

    • Search Google Scholar
    • Export Citation
  • Fanourakis, D., Heuvelink, E. & Carvalho, S.M. 2013 A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidity J. Plant Physiol. 170 890 898

    • Search Google Scholar
    • Export Citation
  • Fuchigami, L., Cheng, T. & Soeldner, A. 1981 Abaxial transpiration and water loss in aseptically cultured plum J. Amer. Soc. Hort. Sci. 106 519 522

  • Fujiwara, K., Kozai, T. & Watanabe, I. 1987 Measurements of carbon dioxide gas concentration in closed vessels containing tissue cultured plantlets and estimates of net photosynthetic rates of the plantlets J. Agr. Met. 43 21 30

    • Search Google Scholar
    • Export Citation
  • Galzy, R. & Compan, D. 1992 Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro. Plant Cell Tissue Organ Cult. 31 239 244

    • Search Google Scholar
    • Export Citation
  • Ghashghaie, J., Brenckmann, F. & Saugier, B. 1992 Water relations and growth of rose plants cultured in vitro under various relative humidities. Plant Cell Tissue Organ Cult. 30 51 57

    • Search Google Scholar
    • Export Citation
  • Grieve, C. & Grattan, S. 1983 Rapid assay for determination of water soluble quaternary ammonium compounds Plant Soil 70 303 307

  • Grout, B. & Aston, M. 1977 Transplanting of cauliflower plants regenerated from meristem culture. I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration Hort. Res. 17 1 7

    • Search Google Scholar
    • Export Citation
  • Hazarika, B.N. 2006 Morpho-physiological disorders in in vitro culture of plants Sci. Hort. 108 105 120

  • Hazarika, B.N., Parthasarathy, V.A. & Nagaraju, V. 2004 Influence of in vitro preconditioning of Citrus sp. microshoots with sucrose on their ex vitro establishment Indian J. Hort. 61 29 31

    • Search Google Scholar
    • Export Citation
  • Hetherington, A.M. & Woodward, F.I. 2003 The role of stomata in sensing and driving environmental change Nature 424 901 908

  • Joshi, N., Dave, A., Vyas, S. & Purohit, S. 2009 Growth and shoot proliferation in Chlorophytum borivilianum Sant. et Fernand. in vitro under different carbon dioxide environment Indian J. Biotechnol. 8 323 327

    • Search Google Scholar
    • Export Citation
  • Kilb, B., Wietoska, H. & Godde, D. 1996 Changes in the expression of photosynthetic genes precede loss of photosynthetic activities and chlorophyll when glucose is supplied to mature spinach leaves Plant Sci. 115 225 235

    • Search Google Scholar
    • Export Citation
  • Kozai, T. 1991 Photoautotrophic micropropagation In Vitro Cell. Dev. Biol. Plant 27 47 51

  • Kozai, T. 2010 Photoautotrophic micropropagation environmental control for promoting photosynthesis Prop. Ornam. Plants 10 188 204

  • Kozai, T., Kubota, C. & Jeong, B.R. 1997 Environmental control for the large-scale production of plants through in vitro techniques Plant Cell Tiss. Org. Cult. 51 49 56

    • Search Google Scholar
    • Export Citation
  • Kubota, C. 2002 Photoautotrophic micropropagation: Importance of controlled environment in plant tissue culture Proc. Intl. Plant Prop. Soc. 52 906 913

    • Search Google Scholar
    • Export Citation
  • Kwa, S.H., Wee, Y.C., Lim, T.M. & Kumar, P.P. 1995 Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichment J. Exp. Bot. 46 1535 1542

    • Search Google Scholar
    • Export Citation
  • Lamhamedi, M.S., Chamberland, H. & Tremblay, F.M. 2003 Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimatization Physiol. Plant. 118 554 561

    • Search Google Scholar
    • Export Citation
  • Majada, J.P., Fal, M.A. & Sanchez-Tames, R. 1997 The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L In Vitro Cell. Dev. Biol. Plant 33 62 69

    • Search Google Scholar
    • Export Citation
  • Martinez, J.P., Lutts, S., Schanck, A., Bajji, M. & Kinet, J.M. 2004 Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L? J. Plant Physiol. 161 1041 1051

    • Search Google Scholar
    • Export Citation
  • Meloni, A.D., Gulotta, M.R., Martinez, C.A. & Oliva, M.A. 2004 The effects of salt stress on growth, nitrate reduction and proline and glycinebetaine accumulation in Prosopis alba Braz. J. Plant Physiol. 16 1 39 46

    • Search Google Scholar
    • Export Citation
  • Mitra, A., Dey, S. & Sawarkar, S. 1998 Photoautotrophic in vitro multiplication of the orchid Dendrobiom under CO2 enrichment Biol. Plant. 41 1 145 148

    • Search Google Scholar
    • Export Citation
  • Nogues, S., Allen, D.J., Morison, J.I. & Baker, N.R. 1998 Ultraviolet-B radiation effects on water relations, leaf development, and photosynthesis in droughted pea plants Plant Physiol. 117 173 181

    • Search Google Scholar
    • Export Citation
  • Payghamzadeh, K. & Kazemitabar, S.K. 2011 In vitro propagation of walnut—A review Afr. J. Biotechnol. 10 290 311

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    • Search Google Scholar
    • Export Citation
  • Pospisilova, J., Synkova, H., Haisel, D. & Semoradova, S. 2007 Acclimation of plantlets to ex vitro conditions: Effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review) Acta Hort. 748 29 38

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    • Export Citation
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Contributor Notes

We would like to thank Iran National Science Foundation (INSF), Center of Excellence of Walnut Improvement and Technology of Iran, and University of Tehran for their supports.

Corresponding authors. E-mail: kvahdati@ut.ac.ir or aliniaeifard@ut.ac.ir.

  • View in gallery

    Work stages for CO2 elevation in the atmosphere of the vessel headspaces. (A) Microshoots were cultured in the culture vessels. (B) Vials containing 3 mL of mixed NaHCO3 and Na2CO3 solution (3 M) in the ratio of 73/27 (v/v), respectively, were placed in the vessels. (C) The samples were incubated and grown at 25 ± 2 °C under 16/8 h light/dark cycles for 30 d.

  • View in gallery

    Stomata on abaxial surface of in vitro–grown walnut (cv. Chandler) leaves developed under ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars (A) without CO2-releasing solution as control plantlets or (B) with CO2-releasing solution in the headspace of culture vessels. The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

  • View in gallery

    Variation in stomatal size on leaves of in vitro–grown walnut (cv. Chandler) microshoots exposed to ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). The samples were incubated at 25 ± 2 °C under 16/8 h light/dark cycles.

  • View in gallery

    (A) Leaflet transpiration rate, (B) relative water content and (C) transpiration rate as a function of RWC during 1.5 h of ex vitro desiccation of leaves excised from tissue cultured walnut microshoots (cv. Chandler). In vitro–formed leaves were grown under either ambient or elevated CO2 concentrations. Micropropagated shoots were grown in jars with CO2-releasing solution in the headspace of culture vessels (CO2) or without CO2-releasing solution as control plantlets (C). For desiccation, the leaves of plantlets were detached and placed with their abaxial side up on a balance in an environment with 50% relative humidity, 21 °C, and 50 μmol·m−2·s−1 irradiance, resulting in 1.24 kPa vapor pressure deficit. The water loss of the leaves was recorded every 5 min for a duration of 90 min.

  • Aliniaeifard, S., Hajilou, J., Tabatabaei, S.J. & Sifi-Kalhor, M. 2016 Effects of ascorbic acid and reduced glutathione on the alleviation of salinity stress in olive plants Intl. J. Fruit Sci. 16 395 409

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  • Aliniaeifard, S., Malcolm Matamoros, P. & van Meeteren, U. 2014 Stomatal malfunctioning under low VPD conditions: Induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Physiol. Plant. 152 688 699

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  • Aliniaeifard, S. & van Meeteren, U. 2013 Can prolonged exposure to low VPD disturb the ABA signalling in stomatal guard cells? J. Exp. Bot. 64 3551 3566

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  • Aliniaeifard, S. & van Meeteren, U. 2014 Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognise the mechanism of disturbed stomatal functioning J. Exp. Bot. 65 6529 6542

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  • Aliniaeifard, S. & van Meeteren, U. 2016 Stomatal characteristics and desiccation response of leaves of cut chrysanthemum (Chrysanthemum morifolium) flowers grown at high air humidity Sci. Hort. 205 84 89

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  • Allaway, W.G. & Mansfield, T.A. 1967 Stomatal responses to changes in carbon dioxide concentration in leaves treated with 3-(4-chlorophenyl)-1, 1-dimethylurea New Phytol. 66 1 57 63

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  • Arve, L.E., Terfa, M.T., Gisleord, H.R., Olsen, J.E. & Torre, S. 2013 High relative air humidity and continuous light reduce stomata functionality by affecting the ABA regulation in rose leaves Plant Cell Environ. 36 382 392

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  • Aviles, F., Rios, D., Gonzalez, R. & Sanchez-Olate, M. 2010 Effect of culture medium in callogenesis from adult walnut leaves (Juglans regia L.) Chil. J. Agr. Res. 69 460 467

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    • Export Citation
  • Badr, A., Angers, P. & Desjardins, Y. 2011 Metabolic profiling of photoautotrophic and photomixotrophic potato plantlets (Solanum tuberosum) provides new insights into acclimatization Plant Cell Tissue Organ Cult. 107 13 24

    • Search Google Scholar
    • Export Citation
  • Bates, L., Waldren, R. & Teare, I. 1973 Rapid determination of free proline for water-stress studies Plant Soil 39 205 207

  • Brainerd, K. & Fuchigami, L. 1982 Stomatal functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA, and CO2 J. Exp. Bot. 33 388 392

    • Search Google Scholar
    • Export Citation
  • Buddendorf-Joosten, J. & Woltering, E. 1994 Components of the gaseous environment and their effects on plant growth and development in vitro J. Plant Growth Regul. 15 1 16

    • Search Google Scholar
    • Export Citation
  • Carvalho, L., Santos, P. & Amancio, S. 2002 Effect of light intensity and CO2 concentration on growth and the acquisition of in vivo characteristics during acclimatization of grapevine regenerated in vitro Vitis 41 1 1 6

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Chanseetis, C., Chintakovid, W., Pichakum, A. & Supaibulwatana, K. 2011 Promoting root induction and growth of in vitro macadamia (Macadamia tetraphylla L. ‘Keaau’) plantlets using CO2-enriched photoautotrophic conditions Plant Cell Tissue Organ Cult. 106 435 444

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Mosaleeyanon, K., Kirdmanee, C. & Supaibulwatana, K. 2003 A more efficient transplanting system for Thai neem (Azadirachta siamensis Val.) by reducing relative humidity Sci. Asia 29 189 196

    • Search Google Scholar
    • Export Citation
  • Cha-um, S., Ulziibat, B. & Kirdmanee, C. 2010 Effects of temperature and relative humidity during in vitro acclimatization, on physiological changes and growth characters of Phalaenopsis adapted to in vivo Austral. J. Crop. Sci. 4 750 756

    • Search Google Scholar
    • Export Citation
  • Chaari-Rkhis, A., Maalej, M., Drira, N. & Standardi, A. 2011 Micropropagation of olive tree (Olea europaea L.) cv. ‘Oueslati’ Turk. J. Agr. For. 35 403 412

    • Search Google Scholar
    • Export Citation
  • Crane, J. & Hughes, H. 1990 Medium overlays for improved hardening of micropropagated potatoes HortScience 25 794 795

  • Cui, Y.Y., Hahn, E.J., Kozai, T. & Paek, K.Y. 2000 Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro Plant Cell Tissue Organ Cult. 62 219 226

    • Search Google Scholar
    • Export Citation
  • de Paiva Neto, V.B. & Otoni, W.C. 2003 Carbon sources and their osmotic potential in plant tissue culture: Does it matter? Sci. Hort. 97 193 202

  • Desjardins, Y., Gosselin, A. & Yelle, S. 1987 Acclimatization of ex vitro strawberry plantlets in CO2 enriched environments and supplementary lighting J. Amer. Soc. Hort. Sci. 112 846 851

    • Search Google Scholar
    • Export Citation
  • Donnelly, D.J. & Vidaver, W.E. 1984 Pigment content and gas exchange of red raspberry in vitro and ex vitro J. Amer. Soc. Hort. Sci. 109 177 181

  • Driver, J.A. & Kuniyuki, A.H. 1984 In vitro propagation of Paradox walnut rootstock Hort. Sci. 19 507 509

  • During, H. & Stoll, M. 1996 Stomatal patchiness of grapevine leaves. I. Estimation of non-uniform stomatal apertures by a new infiltration technique Vitis 35 65 68

    • Search Google Scholar
    • Export Citation
  • Fanourakis, D., Carvalho, S.M., Almeida, D.P. & Heuvelink, E. 2011 Avoiding high relative air humidity during critical stages of leaf ontogeny is decisive for stomatal functioning Physiol. Plant. 142 274 286

    • Search Google Scholar
    • Export Citation
  • Fanourakis, D., Heuvelink, E. & Carvalho, S.M. 2013 A comprehensive analysis of the physiological and anatomical components involved in higher water loss rates after leaf development at high humidity J. Plant Physiol. 170 890 898

    • Search Google Scholar
    • Export Citation
  • Fuchigami, L., Cheng, T. & Soeldner, A. 1981 Abaxial transpiration and water loss in aseptically cultured plum J. Amer. Soc. Hort. Sci. 106 519 522

  • Fujiwara, K., Kozai, T. & Watanabe, I. 1987 Measurements of carbon dioxide gas concentration in closed vessels containing tissue cultured plantlets and estimates of net photosynthetic rates of the plantlets J. Agr. Met. 43 21 30

    • Search Google Scholar
    • Export Citation
  • Galzy, R. & Compan, D. 1992 Remarks on mixotrophic and autotrophic carbon nutrition of Vitis plantlets cultured in vitro. Plant Cell Tissue Organ Cult. 31 239 244

    • Search Google Scholar
    • Export Citation
  • Ghashghaie, J., Brenckmann, F. & Saugier, B. 1992 Water relations and growth of rose plants cultured in vitro under various relative humidities. Plant Cell Tissue Organ Cult. 30 51 57

    • Search Google Scholar
    • Export Citation
  • Grieve, C. & Grattan, S. 1983 Rapid assay for determination of water soluble quaternary ammonium compounds Plant Soil 70 303 307

  • Grout, B. & Aston, M. 1977 Transplanting of cauliflower plants regenerated from meristem culture. I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration Hort. Res. 17 1 7

    • Search Google Scholar
    • Export Citation
  • Hazarika, B.N. 2006 Morpho-physiological disorders in in vitro culture of plants Sci. Hort. 108 105 120

  • Hazarika, B.N., Parthasarathy, V.A. & Nagaraju, V. 2004 Influence of in vitro preconditioning of Citrus sp. microshoots with sucrose on their ex vitro establishment Indian J. Hort. 61 29 31

    • Search Google Scholar
    • Export Citation
  • Hetherington, A.M. & Woodward, F.I. 2003 The role of stomata in sensing and driving environmental change Nature 424 901 908

  • Joshi, N., Dave, A., Vyas, S. & Purohit, S. 2009 Growth and shoot proliferation in Chlorophytum borivilianum Sant. et Fernand. in vitro under different carbon dioxide environment Indian J. Biotechnol. 8 323 327

    • Search Google Scholar
    • Export Citation
  • Kilb, B., Wietoska, H. & Godde, D. 1996 Changes in the expression of photosynthetic genes precede loss of photosynthetic activities and chlorophyll when glucose is supplied to mature spinach leaves Plant Sci. 115 225 235

    • Search Google Scholar
    • Export Citation
  • Kozai, T. 1991 Photoautotrophic micropropagation In Vitro Cell. Dev. Biol. Plant 27 47 51

  • Kozai, T. 2010 Photoautotrophic micropropagation environmental control for promoting photosynthesis Prop. Ornam. Plants 10 188 204

  • Kozai, T., Kubota, C. & Jeong, B.R. 1997 Environmental control for the large-scale production of plants through in vitro techniques Plant Cell Tiss. Org. Cult. 51 49 56

    • Search Google Scholar
    • Export Citation
  • Kubota, C. 2002 Photoautotrophic micropropagation: Importance of controlled environment in plant tissue culture Proc. Intl. Plant Prop. Soc. 52 906 913

    • Search Google Scholar
    • Export Citation
  • Kwa, S.H., Wee, Y.C., Lim, T.M. & Kumar, P.P. 1995 Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichment J. Exp. Bot. 46 1535 1542

    • Search Google Scholar
    • Export Citation
  • Lamhamedi, M.S., Chamberland, H. & Tremblay, F.M. 2003 Epidermal transpiration, ultrastructural characteristics and net photosynthesis of white spruce somatic seedlings in response to in vitro acclimatization Physiol. Plant. 118 554 561

    • Search Google Scholar
    • Export Citation
  • Majada, J.P., Fal, M.A. & Sanchez-Tames, R. 1997 The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L In Vitro Cell. Dev. Biol. Plant 33 62 69

    • Search Google Scholar
    • Export Citation
  • Martinez, J.P., Lutts, S., Schanck, A., Bajji, M. & Kinet, J.M. 2004 Is osmotic adjustment required for water stress resistance in the Mediterranean shrub Atriplex halimus L? J. Plant Physiol. 161 1041 1051

    • Search Google Scholar
    • Export Citation
  • Meloni, A.D., Gulotta, M.R., Martinez, C.A. & Oliva, M.A. 2004 The effects of salt stress on growth, nitrate reduction and proline and glycinebetaine accumulation in Prosopis alba Braz. J. Plant Physiol. 16 1 39 46

    • Search Google Scholar
    • Export Citation
  • Mitra, A., Dey, S. & Sawarkar, S. 1998 Photoautotrophic in vitro multiplication of the orchid Dendrobiom under CO2 enrichment Biol. Plant. 41 1 145 148

    • Search Google Scholar
    • Export Citation
  • Nogues, S., Allen, D.J., Morison, J.I. & Baker, N.R. 1998 Ultraviolet-B radiation effects on water relations, leaf development, and photosynthesis in droughted pea plants Plant Physiol. 117 173 181

    • Search Google Scholar
    • Export Citation
  • Payghamzadeh, K. & Kazemitabar, S.K. 2011 In vitro propagation of walnut—A review Afr. J. Biotechnol. 10 290 311

  • Pospisilova, J., Haisel, D., Synkova, H., Catsky, J., Wilhelmova, N., Plzakova, S. & Prochazkova, D. 2000 Photosynthetic pigments and gas exchange of in vitro grown tobacco plants during ex vitro acclimation Plant Cell Tissue Organ Cult. 61 125 133

    • Search Google Scholar
    • Export Citation
  • Pospisilova, J., Synkova, H., Haisel, D. & Semoradova, S. 2007 Acclimation of plantlets to ex vitro conditions: Effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review) Acta Hort. 748 29 38

    • Search Google Scholar
    • Export Citation
  • Posposilova, J., Ticha, I., Kadlecek, P., Haisel, D. & Plzakova, S. 1999 Acclimatization of micropropagated plants to ex vitro conditions Biol. Plant. 42 481 497

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