Micropropagation of Banana: Reversion, Rooting, and Acclimatization of Hyperhydric Shoots

in HortScience

Hyperhydricity is a physiological disorder impacting plant growth and multiplication and acclimatization of regenerated plantlets. We report the use of calcium nitrate for reversion and acclimatization of banana ‘Grand Naine’ hyperhydric shoots cultured on Murashige and Skoog medium containing agar or gellan. Although 100% rooting of hyperhydric shoots occurred at all concentrations of calcium nitrate, only 50% rooting was recorded in the absence of calcium nitrate. Electrolyte leakage decreased significantly in the reverted banana tissues compared with the hyperhydric tissues. Histochemical staining for reactive oxygen species indicated that reverted banana tissues possess lower levels of both hydrogen peroxide (H2O2) and superoxide (O2-) than do hyperhydric tissues. Rooting, growth, and survival of the reverted banana plantlets were significantly influenced by calcium nitrate concentrations as well as the type of gelling agent. Reverted banana plantlets in medium containing calcium nitrate (0.5–1 g·L−1) were acclimatized with 100% survival in a growing substrate of peatmoss and vermiculite (1:1).

Abstract

Hyperhydricity is a physiological disorder impacting plant growth and multiplication and acclimatization of regenerated plantlets. We report the use of calcium nitrate for reversion and acclimatization of banana ‘Grand Naine’ hyperhydric shoots cultured on Murashige and Skoog medium containing agar or gellan. Although 100% rooting of hyperhydric shoots occurred at all concentrations of calcium nitrate, only 50% rooting was recorded in the absence of calcium nitrate. Electrolyte leakage decreased significantly in the reverted banana tissues compared with the hyperhydric tissues. Histochemical staining for reactive oxygen species indicated that reverted banana tissues possess lower levels of both hydrogen peroxide (H2O2) and superoxide (O2-) than do hyperhydric tissues. Rooting, growth, and survival of the reverted banana plantlets were significantly influenced by calcium nitrate concentrations as well as the type of gelling agent. Reverted banana plantlets in medium containing calcium nitrate (0.5–1 g·L−1) were acclimatized with 100% survival in a growing substrate of peatmoss and vermiculite (1:1).

Banana (Musa spp. AAA; Musaceae) is an economically important fruit crop in tropical and subtropical countries, and it ranks as the largest fruit crop produced worldwide (FAOSTAT, 2017). The plant is vegetatively propagated using corms and suckers, allowing for the spread of diseases. There has been a shift toward cyclic replacement with new plantation because the yield starts to decline after 3 to 5 years and then declines rapidly after 10 to 15 years (Singh et al., 2011). To overcome disease spread through vegetative propagation and to meet the commercial demand, plant tissue culture techniques have been routinely used for propagation. Several reports have reviewed the biotechnological improvements and progress in banana tissue culture, thereby highlighting clonal mass propagation through direct and indirect regeneration pathways (Deepika et al., 2018; Rout et al., 2000; Strosse et al., 2004). Developing efficient protocols for banana tissue culture is the foundation for producing high-quality and pathogen-free planting materials and reducing production costs.

Hyperhydricity has been described as a physiological disorder of tissue-cultured plants whereby the hyperhydric propagules become translucent due to excessive hydration of tissues and exhibit glassy morphology (Dewir et al., 2014; Kevers et al., 2004). The limited aeration and ethylene accumulation and the high relative humidity inside the tissue culture container create an unsuitable environment for plant growth and induce physiological abnormalities such as hyperhydricity (Chakrabarty et al., 2006; Dewir et al., 2005, 2014; Rojas-Martinez et al., 2010). Moreover, cyclic subcultures and prolonged exposure to cytokinins, such as 6-benzylaminopurine (BAP) and thidiazuron, induce hyperhydricity (Dewir et al., 2018; Ivanova and Van Staden, 2011). As a consequence of the plant response to these in vitro stress conditions, the cell metabolism is altered and the production of reactive oxygen species (ROS) is increased (Balen et al., 2009; Franck et al., 1995; Tian et al., 2014) due to changes in the activity of antioxidant enzymes in hyperhydric tissues (Chakrabarty and Datta, 2008; Dewir et al., 2006; Gao et al., 2017a). Increasing evidence suggests a close link between oxidation stress and hyperhydricity (Chakrabarty et al., 2006; Dewir et al., 2006, 2014; Tian et al., 2014), ultimately resulting in plant malformation and malfunctioning. Several approaches, including modifications to the growth medium and improved aeration in the culture container, have been attempted to alleviate or eradicate hyperhydricity in plant tissue culture (Dewir et al., 2014; Hazarika, 2006). Although the majority of studies have focused on the prevention of hyperhydricity, few studies have investigated the reversion of hyperhydric propagules (Gao et al., 2017a, 2017b; Hassannejad et al., 2012; Reyes-Vera et al., 2008; Soundararajan et al., 2017).

Hyperhydricity is a common problem in plant tissue culture that hinders growth, multiplication, and acclimatization of regenerated plantlets (Debergh et al., 1992; Pospisilova et al., 2007). Losses of up to 60% in cultured shoots or explants have been reported due to hyperhydricity in commercial plant micropropagation (Piqueras et al., 2002; van den Dries et al., 2013). Consequently, hyperhydricity can limit the success and efficiency of micropropagation by decreasing the quantity and quality of the tissue-cultured plantlets and increasing the cost of production (Dewir et al., 2014; Gao et al., 2017a; Hazarika, 2006; Kozai et al., 1997). In this study, the effects of calcium nitrate on the reversion of hyperhydric banana ‘Grand Naine’, a commercially important dessert banana of the Cavendish subgroup, and the survival and acclimatization of the reverted plantlets under greenhouse conditions were investigated.

Materials and Methods

Plant material.

Shoot tips of banana (Musa ×paradisiaca L. ‘Grand Naine’) were cyclically subcultured four times (4 weeks per culture cycle) for multiplication on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% sucrose and supplemented with 3 mg·L−1 6-benzylaminopurine (BAP) and 1 mg·L−1 Kinetin (Fig. 1A). The medium was gelled with 0.2% gellan (Dephyte, Hannover, Germany). The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C and 118 kPa for 15 min. The cultures were incubated for 4 weeks at 25 ± 1 °C during a 16-h photoperiod at 25 μmol·m−2·s−1 photosynthetic photon flux density (PPFD) provided by cool white fluorescent tubes. Ten percent of the proliferated shoots showed symptoms of hyperhydricity during the fifth re-culture. These hyperhydric shoots were used as the plant material for the reversion experiments (Fig. 1B).

Fig. 1.
Fig. 1.

Reversion, rooting and acclimatization of hyperhydric banana ‘Grand Naine’ shoots. (A) Normal multiple shoots from the fourth subculture in Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (3 mg·L−1) and Kinetin (1 mg·L−1). (B) Hyperhydric shoots obtained from the fourth subculture. (C) In vitro reversion and rooting of the hyperhydric shoots in agar (8 g·L−1)-solidified MS medium supplemented with calcium nitrate (0.5 g·L−1) after 3 weeks in culture. (D) Reverted plantlets after 4 weeks of acclimatization.

Citation: HortScience horts 54, 8; 10.21273/HORTSCI14036-19

Reversion of hyperhydric banana shoots.

The hyperhydric shoots were cultured on MS medium supplemented with calcium nitrate [Ca (NO3)2] at different concentrations (0, 0.25, 0.50, 0.75, and 1 g·L−1) and 3% (w/v) sucrose. The media were solidified using 0.8% (w/v) agar-agar (Dephyte) or 0.2% (w/v) gellan. There were four replicates per treatment. Each replicate represented a culture of five individual shoots, resulting in a group of 20 shoots per treatment. All other culture conditions were as previously described. After 3 weeks of culture, the following parameters were recorded for each explant: rooting percentage, number of roots, root length (seedlings were washed and the longest root was measured), number of leaves, shoot length, and fresh weight.

Determination of chlorophyll content.

Chlorophyll was extracted overnight at 5 °C with 5 mL of dimethyl-formamide and determined according to the methods of Moran and Porath (1982) using a double-beam ultraviolet/visible spectrophotometer (Libra S80PC; Biochrom, Cambridge, UK) at 663 nm and 647 nm. Chlorophyll concentration is expressed as mg·g−1 fresh weight of leaves.

Microscopic observation of stoma.

For scanning electron microscopy, samples (4 mm2) were obtained from the leaves and fixed in glutaraldehyde (2.5%) for 24 h at 4 °C. Then, they were postfixed in osmium tetraoxide (1% OsO4) for 1 h at room temperature (Harley and Ferguson, 1990). Samples were dehydrated by passing them through increasing concentrations of acetone (30% to 100%). Samples were air-dried until the critical point and sputter-coated with gold. Images were obtained using a JEOL JSM T330A scanning electron microscope (JEOL, Tokyo, Japan).

Histochemical analysis of ROS.

Detection of superoxide (O2) and hydrogen peroxide (H2O2) were visualized as blue coloration of nitroblue tetrazolium (NBT) and red–brown coloration of 3, 3-diaminobenzidine (DAB). Cross and longitudinal leaf discs were vacuum-infiltrated with 10 mm of potassium phosphate buffer (pH 7.8) containing 0.1% (w/v) NBT (Sigma-Aldrich, Steinheim, Germany) according to the methods of Ádám et al. (1989) or 0.1% (w/v) DAB (Fluka, Buchs, Switzerland). NBT-treated and DAB-treated samples were incubated in daylight for 20 min and 2 h, respectively, and subsequently cleared in 0.15% (w/v) trichloroacetic acid in ethanol: chloroform at 4:1 (v/v) for 1 d (Hückelhoven et al., 1999). Cleared samples were washed with water and placed in 50% glycerol before evaluation. Discoloration of stem discs resulting from NBT or DAB staining was quantified using a ChemiImager 4000 digital imaging system (Alpha Innotech Corp., San Leandro, CA).

Electrolyte leakage.

Measurements were performed as described by Szalai et al. (1996) and Whitlow et al. (1992). Leaf discs of hyperhydric tissues and recovered tissues were placed individually in 25 mL of deionized water (Milli-Q 50; Millipore, Bedford, MA). Flasks were shaken for 20 h at ambient temperature to facilitate electrolyte leakage from injured tissues. Initial electrical conductivity (EC) measurements were recorded for each vial using an Acromet AR20 EC meter (Fisher Scientific, Chicago, IL). Flasks were then immersed in a hot water bath (Fisher Isotemp, Indiana, PA) at 80 °C for 1 h to induce cell rupture. The vials were again placed in an Innova 2100 platform shaker for 20 h at 21 °C. Final conductivity was measured for each flask. The electrolyte leakage percentage was calculated as follows: (initial conductivity/final conductivity) × 100.

Acclimatization.

Banana plantlets at the five-leaf stage were transplanted to culture pots (diameter, 5 cm) filled with a mixture of sterilized peatmoss and perlite (1:2). Each treatment had three replicates, and each replicate was represented by 20 plantlets. The plantlets were covered with a clear plastic film during the first 15 d of culture in a shade-controlled greenhouse and watered with 1 g·L−1 of solution containing 19N–8.3P–15.7K water-soluble fertilizer (Rosasol; Rosier, Moustier, Belgium). The environment of the greenhouse was adjusted to a temperature of 27 ± 2 °C, 60% to 70% relative humidity, and 100 µmol·m−2·s−1 PPFD. After 4 weeks of acclimatization, the following parameters were recorded for each plantlet: survival percentage, root length (seedlings were washed and the longest root was measured), shoot length, and fresh weight.

Experimental design and statistical analysis.

All experiments had a completely randomized design. All data were subjected to an analysis of variance and Duncan’s multiple range test using SAS statistical software (version 8.1; SAS Institute, Cary, NC).

Results and Discussion

Reversion and rooting of hyperhydric banana shoots.

BAP is a commonly used cytokinin for micropropagation of Musa sp. (Bairu et al., 2008; Escalona et al., 2003; Hui et al., 2013; Vuylsteke, 1989). However, in this study, hyperhydricity (10%) was recorded during the fourth subculture (Fig. 1B) of ‘Grand Naine’ multiple shoots in MS medium fortified with BAP (3 mg·L−1) and Kinetin (1 mg·L−1). High BAP concentrations and/or cyclic subcultures on BAP-enriched media have been reported to induce hyperhydricity in Musa sp. (Buah et al., 1999; Jafari et al., 2011) and other plant species, including Fragaria ×ananassa (Barbosa et al., 2013) and Thymus daenensis (Hassannejad et al., 2012). BAP has been associated with the rapid formation of N-glucosides, and its accumulation may enhance severe alterations in in vitro cultures (Bairu et al., 2007; Valero-Aracama et al., 2010).

Rooting and growth parameters (root length, number of leaves, shoot length, chlorophyll content, and fresh weight) of the hyperhydric shoots were significantly improved by the addition of calcium nitrate in the culture medium (Table 1; Fig. 1C). Although 100% rooting occurred at all concentrations of calcium nitrate, only 50% was recorded in the control experiments. The highest values of rooting and growth were obtained on gellan-solidified medium supplemented with 0.75 g·L−1 calcium nitrate. High rooting and growth were also observed in agar-solidified medium supplemented with 0.5 g·L−1 calcium nitrate. The type of gelling agent also significantly affected the number of roots, root length, and number of leaves; however, shoot length, chlorophyll content, and fresh weight were not significantly affected. The interaction effect for the type of gelling agent and calcium nitrate significantly influenced the number of roots and leaves and the chlorophyll content (Table 1). A previous report by Buah et al. (1999) indicated that the type of gelling agent influenced the growth of banana shoots, mainly due to the physical properties of the medium (i.e., water potential). Moreover, the hardness of gellan-solidified medium decreases when calcium is reduced from 80 to 40 mg·L−1, but it is unaffected in the agar-solidified medium (Cameron, 2001). De Klerk et al. (2017) proposed that chelating compounds excreted by plant tissues liquefy the gellan-solidified medium. Therefore, variations in the growth of banana shoots (Table 1) could be attributed to the enhanced water availability and nutrient uptake in gellan-solidified medium compared with that in agar-solidified medium.

Table 1.

Effects of calcium nitrate on rooting and growth of hyperhydric banana ‘Grand Naine’ shoots after 3 weeks in culture.

Table 1.

Calcium is associated with several attributes, such as membrane structure and function, ion uptake, interactions with growth regulators, and enzymatic activation (via calmodulin) (Malavolta et al., 1997). The structural function of calcium is characterized by its use in the synthesis of new cell wall, particularly the middle lamellae that separate newly divided cells (Taiz and Zeiger, 2006). Calcium deficiency is well-known in the hyperhydric tissues of Dianthus caryophyllus (Kevers and Gaspar 1986) and Petunia hybrida (Zimmerman et al., 1988). Machado et al. (2014) demonstrated that the addition of calcium chloride (1.32 g·L−1) to the MS culture medium reduced hyperhydricity in Lavendula angustifolia shoots from 23% and 30% to 6% and 1.3% in the first and second subcultures, respectively. Similar findings in Cydonia oblonga (Singha et al., 1990) and Solanum tuberosum (Sha et al., 1985) indicated that increases in calcium improve plant growth and reduce or eliminate deformities such as hyperhydricity and shoot tip necrosis in cultures.

Supplementation of growth media with calcium nitrate improved the chlorophyll content in the reverted banana shoots (Table 1). A decrease in the intensity of the chlorophyll pigment in the hyperhydric shoots of Fragaria ×ananassa (Barbosa et al., 2013), Thymus daenensis (Hassannejad et al., 2012), and Vanilla planifolia (Sreedhar et al., 2009), compared with that in normal shoots has been reported. This decrease in chlorophyll concentration may be due to fewer chloroplasts in the hyperhydric leaves or the damaging effects of hyperhydricity on thylakoid membranes (Chakrabarty et al., 2006; Marschner and Possingham, 1975). The malformed nonfunctional stomata is a common abnormality in hyperhydric shoots (Apóstolo and Llorente, 2000; Barbosa et al., 2013; Gribble et al., 1996; Olmos and Hellin, 1998). Our results indicated the presence of widely open deformed stomata in the hyperhydric banana shoots (Fig. 2B), thus indicating abnormal functioning of stomata compared with that in normal and reverted shoots (Fig. 2A and C). Unlike the typical elliptical-shape cells found in normal and reverted banana shoots, the stomata in hyperhydric shoots are nearly round, with deformed guard cells (Fig. 2B). Guard cell deformity could be due to the excessive water absorption leading to turgidity, consequently changing the cell wall structure and elasticity (Fontes et al., 1999).

Fig. 2.
Fig. 2.

Scanning electron microscopy of stomata in the leaves of banana ‘Grand Naine’ shoots after 3 weeks in culture. (A and B) Normal and hyperhydric shoots [fourth subculture in MS medium supplemented with 6-benzylaminopurine (3 mg·L−1) and kinetin (1 mg·L−1)]. (C) Reverted shoots [cultured in agar (8 g·L−1)-solidified MS medium supplemented with calcium nitrate (0.5 g·L−1)].

Citation: HortScience horts 54, 8; 10.21273/HORTSCI14036-19

Histochemical analysis of ROS.

Histochemical staining for ROS, including O2 and H2O2, were visualized as blue and brown colorations, respectively. NBT or DAB staining and quantification indicated that the recovered banana tissues possess lower levels of both H2O2 and O2 compared with those in hyperhydric tissues (Figs. 3 and 4). The excessive water accumulation in plant tissue, which is the most characteristic symptom of hyperhydricity, generates aeration stress that depletes oxygen levels and limits its diffusion in cells. Therefore, it has been proposed that hyperhydric tissues can be under hypoxic stress (Franck et al., 1998, 2004; Gribble et al., 1996, 1998; Kevers and Gaspar, 1986; Kevers et al., 2004; Olmos et al., 1997). Increased levels of ROS involving the superoxide and hydroxyl free radicals as well as hydrogen peroxide have been observed in hyperhydric tissues of Dianthus chinensis (Gao et al.,2017a, 2017b), Malus sp. (Chakrabarty et al., 2006), and Mammillaria gracilis (Balen et al., 2009). Several reports suggested that oxidative stress, an important damaging factor in hyperhydricity induction, may be responsible for many metabolic changes in hyperhydric tissues such as lipid peroxidation and, consequently, membrane injury, protein degradation, enzyme inactivation, and DNA damage (Chen and Ziv, 2001; Dewir, 2005; Dewir et al., 2006; Franck et al., 1995, 1998; Olmos et al., 1997).

Fig. 3.
Fig. 3.

Histochemical analysis of reactive oxygen species in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture. (A) Superoxide and (B) hydrogen peroxide.

Citation: HortScience horts 54, 8; 10.21273/HORTSCI14036-19

Fig. 4.
Fig. 4.

Quantification of reactive oxygen species in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture. (A) Superoxide and (B) hydrogen peroxide.

Citation: HortScience horts 54, 8; 10.21273/HORTSCI14036-19

Electrolyte leakage.

Electrolyte leakage was significantly decreased in the reverted banana tissues compared with that in hyperhydric tissues (Fig. 5). Cellular membrane dysfunction due to stress increases permeability and the release of ions, which can be readily measured based on the efflux of electrolytes (Dewir et al., 2015a, 2015b). Cell wall properties and composition are considered some of the most important factors affecting the development of anomalous morphology in hyperhydric tissues (Dewir et al., 2014). Different researchers have shown modifications in the cell wall constituents of hyperhydric tissues, mainly cellulose, lignin, and pectins (Kevers et al., 1987; Majada et al., 2000; Olmos et al., 1997; Saher et al., 2005a, 2005b) and their mechanical properties (Kevers et al., 1987; Komali et al., 1998). Hypolignification has been attributed to the decrease in enzyme activities, as reported for Origanum vulgare (Andarwulan and Shetty, 1999) and Prunus avium (Phan and Hegedus, 1986). Electrolyte leakage has been used to quantify damage to cell membranes in hyperhydric Saintpaulia ionantha (Dewir et al., 2015b). Foyer et al. (1994) observed a higher rate of solute leakage in hyperhydric leaves compared with that in the control, indicating marked deterioration of the membrane. Our results indicated that banana shoots cultured on calcium nitrate–perhydric shoots, indicating the protective role of calcium nitrate against oxidative stress.

Fig. 5.
Fig. 5.

Electrolyte leakage in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture.

Citation: HortScience horts 54, 8; 10.21273/HORTSCI14036-19

Acclimatization and survival of the reverted banana plantlets.

Survival and growth of the reverted banana plantlets were significantly influenced by calcium nitrate as well as gelling agents used during in vitro rooting (Table 2). Furthermore, 100% survival was recorded for plantlets reverted in medium containing 0.5 to 1 g·L−1 calcium nitrate regardless of the solidifying agent. The reverted plantlets grown in medium lacking calcium nitrate and solidified with gellan or agar resulted in 43% and 83% survival, respectively. A low calcium nitrate concentration (0.25 g·L−1) resulted in 85% and 92% survival of plantlets reverted on gellan and agar, respectively. Therefore, solidifying the MS medium with agar during the reversion of hyperhydric shoots was more efficient than using gellan for the survivability of plantlets. Dehydration and death of hyperhydric plants during the acclimatization stage were mainly due to water loss through epidermal discontinuities and nonfunctional stomata (Apóstolo and Llorente, 2000; Gribble et al., 1996; Olmos and Hellin, 1998). Hyperhydric Simmondsia chinensis shoots exhibiting malformed nonfunctional stomata fail to survive acclimatization (Apóstolo and Llorente, 2000). Nonfunctional stomata, hypolignification, and epidermal discontinuity resulted in the loss of protection needed for acclimatization. Calcium nitrate proved effective for reversion and acclimatization of ‘Grand Naine’ hyperhydric shoots. All plantlets cultured in a medium containing calcium nitrate (0.5–1 g·L−1) were reverted and acclimatized (Fig. 1D). Previous studies reported varied percentages of reversion for hyperhydric shoots such as Dianthus chinensis (67% on medium containing 5 mg·L−1 silver nitrate) (Gao et al., 2017b) and Atriplex canescens (39.7% on vented Magenta vessels; pore size, 0.22 μm) (Reyes-Vera et al., 2008), indicating that reversion to normal morphology is influenced by the culture conditions and plant genotype.

Table 2.

Effects of calcium nitrate on survival and growth of the reverted banana ‘Grand Naine’ plantlets after 4 weeks of acclimatization in a greenhouse.

Table 2.

We concluded that 58% and 88% of the hyperhydric banana ‘Grand Naine’ shoots cultured in media lacking calcium nitrate and solidified gellan or agar, respectively, were estimated as losses because these shoots failed to either root or survive past the acclimatization stage. Moreover, 100% rooting of hyperhydric shoots occurred at all concentrations of calcium nitrate. Growth and survival of the reverted banana plantlets were significantly influenced by calcium nitrate concentrations as well as the type of gelling agent used. Reverted banana plantlets in medium containing calcium nitrate (0.5–1 g·L−1) were acclimatized with 100% survival.

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    • Search Google Scholar
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  • GaoH.XiaX.AnL.XinX.LiangY.2017bReversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culturePlant Sci.254111

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  • HuiA.V.BhattA.SreeramananS.KengC.L.2013Establishment of a shoot proliferation protocol for banana (ABB Group) Cv. ‘Pisang Awak’ via temporary immersion systemJ. Plant Nutr.36529538

    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
  • JafariN.OthmanR.Y.KhalidN.2011Effect of benzylaminopurine (BAP) pulsing on in vitro shoot multiplication of Musa acuminata (banana) cvBerangan. Afr. J. Biotechnol.1024462450

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    • Export Citation
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    • Export Citation
  • SaherS.Fernández-GarcíaN.PiquerasA.HellínE.OlmosE.2005bReducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: A hypoxia stress?Plant Physiol. Biochem.43573582

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  • SoundararajanP.ManivannanA.ChoY.S.JeongB.R.2017Exogenous supplementation of silicon improved the recovery of hyperhydric shoots in Dianthus caryophyllus L. by stabilizing the physiology and protein expressionFront. Plant Sci.8738

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Contributor Notes

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group NO (RGP-1438-012), and the Research Support & Services Unit (RSSU) for their technical support.

Corresponding author. E-mail: ydewir@hotmail.com or ydewir@ksu.edu.sa.

  • View in gallery

    Reversion, rooting and acclimatization of hyperhydric banana ‘Grand Naine’ shoots. (A) Normal multiple shoots from the fourth subculture in Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (3 mg·L−1) and Kinetin (1 mg·L−1). (B) Hyperhydric shoots obtained from the fourth subculture. (C) In vitro reversion and rooting of the hyperhydric shoots in agar (8 g·L−1)-solidified MS medium supplemented with calcium nitrate (0.5 g·L−1) after 3 weeks in culture. (D) Reverted plantlets after 4 weeks of acclimatization.

  • View in gallery

    Scanning electron microscopy of stomata in the leaves of banana ‘Grand Naine’ shoots after 3 weeks in culture. (A and B) Normal and hyperhydric shoots [fourth subculture in MS medium supplemented with 6-benzylaminopurine (3 mg·L−1) and kinetin (1 mg·L−1)]. (C) Reverted shoots [cultured in agar (8 g·L−1)-solidified MS medium supplemented with calcium nitrate (0.5 g·L−1)].

  • View in gallery

    Histochemical analysis of reactive oxygen species in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture. (A) Superoxide and (B) hydrogen peroxide.

  • View in gallery

    Quantification of reactive oxygen species in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture. (A) Superoxide and (B) hydrogen peroxide.

  • View in gallery

    Electrolyte leakage in the leaves of normal, hyperhydric, and reverted banana ‘Grand Naine’ shoots after 3 weeks in culture.

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  • GaoH.XuP.LiJ.JiH.AnL.XiaX.2017aAgNO3 prevents the occurrence of hyperhydricity in Dianthus chinensis L. by enhancing water loss and antioxidant capacityIn Vitro Cell. Dev. Biol. Plant53561570

    • Search Google Scholar
    • Export Citation
  • GaoH.XiaX.AnL.XinX.LiangY.2017bReversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culturePlant Sci.254111

    • Search Google Scholar
    • Export Citation
  • GribbleK.SarafisV.NailonJ.HolfordP.UwinsP.1996Environmental scanning electron microscopy of the surface of normal and vitrified leaves of Gypsophila paniculata (Babies Breath) cultured in vitroPlant Cell Rep.15771776

    • Search Google Scholar
    • Export Citation
  • GribbleK.TingleJ.SarafisV.HeatonA.HolfordP.1998Position of water in vitrified plants visualized by NMR imagingProtoplasma201110114

  • HarleyM.M.FergusonI.K.1990The role of the SEM in pollen morphology and plant systematics p. 45–68. In: D. Claugher (ed.). Scanning electron microscopy in taxonomy and functional morphology. Sys. Assoc. Spe. Clarendon Press Oxford

  • HassannejadS.BernardF.MirzajaniF.GholamiM.2012SA improvement of hyperhydricity reversion in Thymus daenensis shoots culture may be associated with polyamines changesPlant Physiol. Biochem.514046

    • Search Google Scholar
    • Export Citation
  • HazarikaB.N.2006Morpho-physiological disorders in in vitro culture of plantsScientia Hort.108105120

  • HückelhovenR.FodorJ.PreisC.KogelK.H.1999Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulationPlant Physiol.11912511260

    • Search Google Scholar
    • Export Citation
  • HuiA.V.BhattA.SreeramananS.KengC.L.2013Establishment of a shoot proliferation protocol for banana (ABB Group) Cv. ‘Pisang Awak’ via temporary immersion systemJ. Plant Nutr.36529538

    • Search Google Scholar
    • Export Citation
  • IvanovaM.Van StadenJ.2011Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphyllaPlant Cell Tissue Organ Cult.1041321

    • Search Google Scholar
    • Export Citation
  • JafariN.OthmanR.Y.KhalidN.2011Effect of benzylaminopurine (BAP) pulsing on in vitro shoot multiplication of Musa acuminata (banana) cvBerangan. Afr. J. Biotechnol.1024462450

    • Search Google Scholar
    • Export Citation
  • KeversC.GasparT.1986Vitrification of carnation in vitro: Changes in water content, intracellular space, air volume and ion levelsPhysiol. Veg.24647653

    • Search Google Scholar
    • Export Citation
  • KeversC.PratR.GasparT.1987Vitrification of carnation in vitro: Changes in cell wall mechanical properties, cellulose and lignin contentPlant Growth Regulat.55966

    • Search Google Scholar
    • Export Citation
  • KeversC.FranckT.StrasserR.J.DommesJ.GasparT.2004Hyperhydricity of micropropagated shoots: A typically stress-induced change of physiological statePlant Cell Tissue Organ Cult.77181191

    • Search Google Scholar
    • Export Citation
  • KomaliA.S.PelegM.GerhardsC.ShettyK.1998A study of the cell wall mechanical properties in unhyperhydrated shoots of oregano (Origanum vulgare) inoculated with Pseudomonas sp. by load deformation analysisFood Biotechnol.12209220

    • Search Google Scholar
    • Export Citation
  • KozaiT.KubotaC.Ryoung JeongB.1997Environmental control for the large-scale production of plants through in vitro techniquesPlant Cell Tissue Organ Cult.514956

    • Search Google Scholar
    • Export Citation
  • MachadoM.P.da SilvaA.L.L.BiasiL.A.DeschampsC.FilhoJ.C.B.ZanetteF.2014Influence of calcium content of tissue on hyperhydricity and shoot-tip necrosis of in vitro regenerated shoots of Lavandula angustifoliaMill. Braz. Arch. Biol. Technol.57636643

    • Search Google Scholar
    • Export Citation
  • MajadaJ.P.TadeoF.FalM.A.Sánchez TamésR.2000Impact of culture vessel ventilation on the anatomy and morphology of micropropagated carnationPlant Cell Tissue Organ Cult.63207214

    • Search Google Scholar
    • Export Citation
  • MalavoltaE.VittiG.C.de. OliveiraS.A.1997Avaliacao do estado nutricional das plantas: Principios e aplicacoes. 2nd ed. Piracicaba: Associacao Brasileira para Pesquisa da Potassa e do Fosfato

  • MarschnerH.PossinghamJ.V.1975Effects of K and Na on the growth of leaf discs of sugar beet and spinachZ. Pflanzenphysiol.75616

  • MoranR.PorathD.1982Chlorophyll determination in intact tissues using N,N-Dimethyl formamidePlant Physiol.6913701381

  • MurashigeT.SkoogF.A.1962A revised medium for rapid growth and bioassays with tobacco tissue culturesPhysiol. Plant.15473479

  • OlmosE.HellinE.1998Ultrastructural differences of hyperhydric and normal leaves from regenerated carnation plantsScientia Hort.7591101

    • Search Google Scholar
    • Export Citation
  • OlmosE.PiquerasA.Martinez-SolanoJ.R.HellinE.1997The subcellular localization of peroxidase and the implication of oxidative stress in hyperhydrated leaves of regenerated carnation shootsPlant Sci.13097105

    • Search Google Scholar
    • Export Citation
  • PhanC.T.HegedusP.1986Possible metabolic basis for the developmental anomaly observed in in vitro culture, called ‘vitreous plants’Plant Cell Tissue Organ Cult.68394

    • Search Google Scholar
    • Export Citation
  • PiquerasA.CortinaM.SernaM.D.CasasJ.L.2002Polyamines and hyperhydricity in micropropagated carnation plantsPlant Sci.162671678

  • PospisilovaJ.SynkovaH.HaiselD.SemoradovaS.2007Acclimation of plantlets to ex vitro conditions: Effects of air humidity, irradiance, CO2 concentration and abscisic acid (a review)Acta Hort.7482938

    • Search Google Scholar
    • Export Citation
  • Reyes-VeraI.PotenzaC.BarrowJ.2008Hyperhydricity reversal and clonal propagation of four-wing saltbush (Atriplex canescens, Chenopodiaceae) cultivated in vitroAustral. J. Bot.56358362

    • Search Google Scholar
    • Export Citation
  • Rojas-MartinezL.VisserR.G.F.de KlerkG.2010The hyperhydricity syndrome: Waterlogging of plant tissues as a major causePropag. Ornam. Plants10169175

    • Search Google Scholar
    • Export Citation
  • RoutG.R.SamantarayS.DasP.2000Biotechnology of the banana: A review of recent progressPlant Biol.2512524

  • SaherS.PiquerasA.HellinE.OlmosE.2005aPrevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: Implications of oxidative stressPlant Cell Tissue Organ Cult.81149158

    • Search Google Scholar
    • Export Citation
  • SaherS.Fernández-GarcíaN.PiquerasA.HellínE.OlmosE.2005bReducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: A hypoxia stress?Plant Physiol. Biochem.43573582

    • Search Google Scholar
    • Export Citation
  • ShaL.McCownB.H.PetersonL.A.1985Occurrence and cause of shoot-tip necrosis in shoot culturesJ. Amer. Soc. Hort. Sci.110631634

  • SinghH.P.UmaS.SelvarajanR.KarihalooJ.L.2011Micropropagation for production of quality banana planting material in Asia-Pacific. Asia-Pacific Consortium on Agricultural Biotechnology (APCoAB) New Delhi India

  • SinghaS.TownsendE.C.OberlyG.E.1990Relationship between calcium and agar on vitrification and shoot-tip necrosis of quince (Cydonia oblonga Mill.) shoots in vitroPlant Cell Tissue Organ Cult.23135142

    • Search Google Scholar
    • Export Citation
  • SoundararajanP.ManivannanA.ChoY.S.JeongB.R.2017Exogenous supplementation of silicon improved the recovery of hyperhydric shoots in Dianthus caryophyllus L. by stabilizing the physiology and protein expressionFront. Plant Sci.8738

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