Abstract
This study investigated the effects of different sucrose, ventilation, and paclobutrazol (PBZ) treatments on the growth of potato plantlets cultured in modified temporary immersion culture vessels. Temporary immersion culture vessels were modified to enable the plantlets to be cultured on semi-solid growth medium and provided with forced ventilation. The plantlet responses to two sucrose concentrations (15 g⋅L−1 and 30 g⋅L−1) in combination with two forced ventilation treatments (5 min/2 h and no ventilation) and three PBZ concentrations (0, 0.2, and 0.4 mg⋅L−1) were studied. Shoot growth was severely inhibited by PBZ in the growth media, whereas leaf formation was promoted by ventilation irrespective of the PBZ concentration. In nonventilated vessels, it is noteworthy that PBZ was able to increase the number of leaves formed in plantlets grown on medium supplemented with 30 g⋅L−1 sucrose, but not on medium with 15 g⋅L−1 sucrose. In growth media without PBZ, a high percentage of potato plantlets was able to produce secondary axillary shoots when provided with 30 g⋅L−1 sucrose. However, their ability to produce secondary shoots was reduced when PBZ was included in the growth medium, particularly those with 15 g⋅L−1 sucrose. Plantlets grown in ventilated culture vessels combined with 30 g⋅L−1 sucrose in the growth medium produced more than three times more shoots than the other treatments. Under ex vitro conditions, plantlets that had been grown in ventilated vessels had fewer leaf deaths, and the inclusion of PBZ in the growth media further reduced the number of dead leaves. Findings of this study showed that PBZ had a key role in the responses of potato plantlets to ventilation and sucrose treatments, as well as their tolerance to ex vitro conditions. The modified temporary immersion system can be used for the in vitro culture of potato plantlets on semi-solid medium and provide forced ventilation to improve their growth.
Potato (Solanum tuberosum L.) is the fourth most important food crop after wheat, rice, and corn, and it is the largest noncereal food crop worldwide (Zhang et al., 2017). With the conventional setup of an in vitro propagation system, the headspace of culture vessels is characterized by high relative humidity, high ethylene levels, and low CO2 concentrations (Fujiwara and Kozai, 1995). Consequently, plantlets produced under these conditions tend to possess poor morphological, anatomical, and physiological characteristics. These plantlets usually have a low chance of surviving in ex vitro conditions during the acclimatization phase unless changes to the environmental conditions are made. Several types of natural and forced ventilation designs have been introduced to improve the environmental conditions of the headspace of culture vessels (Armstrong et al., 1997; Heo and Kozai, 1999; Xiao et al., 2005, 2011; Zobayed et al., 2004).
In our previous study, we developed a novel forced ventilation system by modifying temporary immersion system culture vessels (Plantima®; A-Tech Bioscientific Co., Ltd., Taipei, Taiwan) to allow semi-solid growth media to be used in place of liquid media. Forced ventilation was provided to the vessels using the original air pump controller, which can also be used to adjust the ventilation frequency and duration. Findings of the study showed significant improvements in the in vitro growth of Protea cynaroides explants (Wu et al., 2018). This forced ventilation system has the following advantages: the original temporary immersion system is readily available, which eliminates the need to build a separate system, and it can be easily converted from a temporary immersion system to one that can be used to culture plants on semi-solid media with three types of ventilation setups (i.e., forced ventilation, natural ventilation, or no ventilation). Depending on the type of ventilation needed, the inlet and outlet openings located on the side of the culture vessel can be connected through silicone tubes to the original air pump controller (forced ventilation), attached with microfilters only (natural ventilation), or completely sealed (no ventilation). Other advantages are that the forced ventilation frequency and duration can be adjusted using the original system controller, and that the modifications are reversible. The morphological and physiological characteristics of P. cynaroides microshoots were improved when grown in these modified culture vessels with forced ventilation (Wu et al., 2018).
PBZ is a type of triazole plant growth regulator that inhibits cell elongation (Desta and Amare, 2021). It restricts stem growth by blocking gibberellin biosynthesis, and it is commonly used for a wide range of crops in vitro, such as apple (Kepenek and Karoglu, 2011), orchids (Gimenes et al., 2018), stevia (El-Said and El-Fadl, 2017), and sugarcane (Valdés et al., 2017). In addition to its dwarfing effect, PBZ has been shown to increase the chlorophyll content and delay leaf senescence (Desta and Amare, 2021; Kumar et al., 2012). Moreover, PBZ has been found to increase the tolerance of plants to environmental stresses such as drought, heat, and ultraviolet radiation (Orabi et al., 2010).
Most studies have investigated the effects of PBZ on potato microtuber formation (Kianmehr et al., 2012; Simko, 1993, 1994; Suharjo et al., 2019). Information regarding the response of potato plantlets to ventilation and sucrose in vitro is lacking (Kubota and Kozai, 1992; Mohamed and Alsadon, 2010). To the best of our knowledge, no studies have investigated how potato plantlets grown in different ventilation and sucrose treatments are affected by exogenous PBZ concentrations. In the present study, we used the modified temporary immersion vessels described to culture potato plantlets on semi-solid media and provide forced ventilation under photomixotrophic conditions. This study aimed to determine the growth of potato plantlets in response to ventilation, sucrose, and PBZ in modified temporary immersion vessels.
Materials and Methods
Plant material and culture conditions.
Potato plantlets that were maintained on half-strength Murashige and Skoog, medium (Murashige and Skoog, 1962) without growth regulators were used in this study. Explants with two nodes were prepared with all apical buds, leaves, and roots removed. The explants were cultured on full-strength Murashige and Skoog medium supplemented with two sucrose concentrations, 15 g⋅L−1 and 30 g⋅L−1, and three PBZ concentrations, 0, 0.2, and 0.4 mg⋅L−1. The pH of all growth media was adjusted to 5.8 before adding 9 g⋅L−1 agar. Growth medium (250 mL) was dispensed into modified Plantima® (A-Tech Bioscientific Co., Ltd, Taiwan) temporary immersion culture vessels and autoclaved at 121 °C and 104 kPa for 30 min. Ten explants were planted in one culture vessel with five vessels per treatment. The culture vessels were placed in a growth room with the temperature adjusted to 26 °C±1 °C and a photoperiod of 16 h. Lighting was provided by white fluorescent tubes at an intensity of 80 μmol⋅m−2⋅s−1 photosynthetic photon flux density (PPFD). After 28 d in culture, potato plantlets were transplanted to perlite in plastic trays and placed in ex vitro conditions. The light, temperature, and relative humidity conditions were 150 μmol⋅m−2⋅s−1 PPFD, 27 °C±2 °C, and 55%, respectively. The plantlets were not covered, and no humidity-intensifying procedures were used to increase the relative humidity around the plantlets. The plantlets were watered every day and remained in ex vitro conditions for 35 d.
Ventilation condition.
Plantima® culture vessels have a removable partition that divides them into an upper section where explants are grown and a lower section where liquid growth media are housed. There are also inlet and outlet openings on each culture vessel that are positioned on the sides of the lower and upper sections, respectively. The inlet opening is connected via silicone tubes to an air pump, which, when turned on, causes the liquid medium to temporarily rise to immerse the explants. The outlet opening allows the air to escape the culture vessel. In this study, Plantima® culture vessels were modified by removing the partition inside the vessel. As a result, each vessel was left with a single growing space in which the potato explants were cultured on semi-solid growth media. Two ventilation treatments were used in this study: forced ventilation and nonventilation. In the forced ventilation treatment, air was pumped every 2 h for 5 min through silicone tubes via a sterilant solution (1000 ppm Cu++), and then through the inlet opening into the culture vessel. The forced pressure inside the Plantima® vessel escapes through the outlet opening, thus renewing the air inside. A 0.22-μm microfilter was attached to the inlet and outlet openings to maintain sterility. In the nonventilated treatment, explants were grown in sealed culture vessels throughout the duration of the study.
Statistical analysis.
A completely randomized design was used. During the in vitro propagation stage, data regarding the shoot length, number of leaves, percentage of plantlets forming secondary axillary shoots, and the number of secondary axillary shoots formed were collected. The percentage of leaf death in each treatment after transplanting to ex vitro conditions was also recorded. An analysis of variance and Duncan’s multiple range test were used to determine statistical differences between treatments. Data analysis was performed using SAS software (SAS Institute, Cary, NC).
Results
In vitro shoot growth and leaf formation.
Significant interaction effects between ventilation and sucrose × PBZ concentrations were present in all the measured parameters (Table 1). Except for the percentage of plantlets forming secondary axillary shoots, all other parameters showed highly significant interaction effects (P ≤ 0.001). These results showed that the growth of potato plantlets is affected by changes in the PBZ concentration under different sucrose and ventilation treatments. Overall, the inhibitory effect of PBZ on the growth of axillary shoots that emerged from the nodal explants was strikingly evident (Fig. 1). The addition of PBZ in the growth media significantly reduced shoot length, regardless of the type of ventilation and sucrose treatments used. In growth media containing no PBZ, significant differences in shoot length were found between plantlets that were ventilated, with plantlets cultured in 15 mg⋅L−1 sucrose longer than those in 30 g⋅L−1 sucrose. However, similar shoot lengths were observed in plantlets in the nonventilated treatments (Fig. 1). These results also revealed that plantlets grown in both ventilated treatments produced significantly longer shoots than those cultured in nonventilated culture vessels, irrespective of the sucrose concentration. However, with the addition of 0.2 or 0.4 mg⋅L−1 PBZ in the growth media, plantlets provided with 30 g⋅L−1 sucrose produced significantly longer shoots than those in the 15 g⋅L−1 sucrose treatments under both ventilation conditions (Fig. 1). In other words, the sucrose concentration became the deciding factor for shoot growth when plantlets were under the inhibitory effects of PBZ.
The effect of the paclobutrazol (PBZ) concentration on the length of shoots and number of leaves produced by potato nodal explants grown in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on the length of shoots and number of leaves produced by potato nodal explants grown in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on the length of shoots and number of leaves produced by potato nodal explants grown in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
Interaction effects of ventilation, sucrose concentration, and paclobutrazol (PBZ) concentration on the vegetative growth of potato plantlets and their response in ex vitro conditions.
In terms of the number of leaves formed, the results showed the overwhelming stimulatory effect of ventilation on the formation of leaves (Fig. 1). A significantly higher number of leaves was produced by plantlets in the ventilated treatments compared with those that were not ventilated, regardless of the PBZ concentration. In growth medium with no PBZ, plantlets grown under ventilated conditions produced almost three times more leaves than those in nonventilated treatments. However, the striking effects of PBZ in combination with 30 g⋅L−1 sucrose on promoting leaf formation were clearly demonstrated for plantlets in the nonventilated treatments. This stimulatory effect was particularly evident with the 0.4 mg⋅L−1 PBZ treatment, which was associated with twice as many leaves forming on plantlets that were cultured with 30 g⋅L−1 sucrose compared to those grown with 15 g⋅L−1 sucrose (Fig. 1). On the contrary, the number of leaves that emerged from plantlets cultured in the nonventilated plus 15 g⋅L−1 sucrose treatment remained consistently low with all PBZ concentrations.
In vitro secondary axillary shoot formation.
In addition to forming leaves, some of the shoots that emerged from the nodal explants were found to produce secondary axillary shoots. The results showed that the concentration of sucrose added to the growth medium was the most important factor affecting the percentage of plantlets producing secondary shoots (Fig. 2). Overall, a higher percentage of plantlets grown in 30 g⋅L−1 sucrose formed secondary shoots compared to those grown in 15 g⋅L−1 sucrose. A lower percentage of plantlets produced secondary shoots when grown on media supplemented with PBZ. Furthermore, 30 g⋅L−1 sucrose alone could not counteract the inhibitory effects of PBZ on secondary axillary shoot formation, as shown by the decrease from almost 80% to less than 30% with the nonventilated plus 30 g⋅L−1 sucrose treatment. The provision of both ventilation and 30 g⋅L−1 sucrose was necessary to ensure a high percentage of plantlets that produced secondary shoots across all PBZ concentrations. Although the addition of 0.4 mg⋅L−1 PBZ also led to a significant reduction in the ventilated plus 30 g⋅L−1 sucrose treatment, 70% of plantlets produced secondary shoots in contrast to only ≈10% with the 15 g⋅L−1 sucrose treatments. Regarding the number of secondary axillary shoots produced, the highest number of shoots emerged from plantlets grown in the ventilated plus 30 g⋅L−1 sucrose treatment without PBZ; this number was more than three times more than that of plantlets cultured in the other treatments (Fig. 2). In fact, except for the ventilated plus 30 g⋅L−1 sucrose treatment, all plantlets cultured in the other treatments produced fewer than three secondary shoots with any of the concentrations of PBZ. These results showed that the ventilated plus 30 g⋅L−1 sucrose treatment is the adequate combination for inducing potato plantlets to produce secondary axillary shoots and promote the formation of a high number of secondary shoots.
The effect of the paclobutrazol (PBZ) concentration on the percentage of plantlets forming secondary axillary shoots and the number of axillary shoots formed in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on the percentage of plantlets forming secondary axillary shoots and the number of axillary shoots formed in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on the percentage of plantlets forming secondary axillary shoots and the number of axillary shoots formed in vitro under different ventilation and sucrose treatments after 28 d in culture. Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
Plantlet response to ex vitro conditions.
On growth medium with no PBZ, more than 30% of leaves on plantlets that were cultured without ventilation withered and died when transplanted to ex vitro conditions (Fig. 3). This was approximately twice the number of leaf deaths compared to those cultured in the ventilated treatments. The results also showed that, except for the ventilated plus 15 g⋅L−1 sucrose treatment, the percentage of leaf death decreased significantly when the plantlets were grown in medium supplemented with 0.2 mg⋅L−1 PBZ (Fig. 3). For the ventilated plus 15 g⋅L−1 sucrose treatment, a significant decrease only occurred when cultured in the 0.4 mg⋅L−1 PBZ concentration. For plantlets grown in the nonventilated plus 15 g⋅L−1 sucrose treatment, although the leaf death percentage was significantly reduced in the 0.2 mg⋅L−1 PBZ concentration, the leaf death rate was still 21%, which was significantly higher than that of all the other treatments with PBZ. Interestingly, even when cultured in medium supplemented with 0.4 mg⋅L−1 PBZ, the leaf death percentage remained at ≈21%. This is an indication of the limits of PBZ to delay leaf senescence in nonventilated potato plantlets when cultured on growth medium supplemented with a lower sucrose concentration (15 g⋅L−1). In contrast, for plantlets cultured in ventilated culture vessels and in growth medium with a comparatively higher sucrose content (30 g⋅L−1), the percentage of leaf death was already reduced to less than 10% with the 0.2 mg⋅L−1 PBZ concentration (Fig. 3). Within the same ventilation treatment, the influential role of the sucrose concentration on the percentage of leaf death was particularly striking when plantlets were cultured in growth medium containing PBZ. This was evidenced by the leaf death percentage always being lower when plantlets had been cultured on growth medium supplemented with 30 g⋅L−1 sucrose than with 15 g⋅L−1 sucrose (Fig. 3). Even for plantlets that were not ventilated but were cultured in 30 g⋅L−1 sucrose, the leaf death percentage decreased to ≈15%, which was comparable to those of the ventilated treatments.
The effect of the paclobutrazol (PBZ) concentration on leaf death (%) of plantlets under different ventilation and sucrose treatments in ex vitro conditions. Data presented are the 3-week average (second, third, and fourth week). Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on leaf death (%) of plantlets under different ventilation and sucrose treatments in ex vitro conditions. Data presented are the 3-week average (second, third, and fourth week). Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
The effect of the paclobutrazol (PBZ) concentration on leaf death (%) of plantlets under different ventilation and sucrose treatments in ex vitro conditions. Data presented are the 3-week average (second, third, and fourth week). Different letters indicate significant differences between treatment means using Duncan’s multiple range test at P ≤ 0.05.
Citation: HortScience 57, 11; 10.21273/HORTSCI16833-22
Discussion
In growth media with no PBZ, the shoot length was significantly longer when cultured with 15 g⋅L−1 sucrose than with 30 g⋅L−1 sucrose in the ventilated culture vessels (Fig. 1). Our results are not in agreement with those reported by Mohamed and Alsadon (2010), who found that the height of potato plantlets grown under similar sucrose concentration (10 g⋅L−1) was shorter than that of those in 30 g⋅L−1 sucrose. This may be attributable to the difference in the type of ventilation used, which directly affected the level of water loss from the medium, because they used natural ventilation in which air exchange occurred through microporous vents on the culture vessels. According to Goncalves et al. (2007), a higher level of water loss occurs in ventilated vessels, which may lead to changes in the concentration of additives as well as changes in growth medium characteristics. As a result, the optimal sucrose concentration for plantlet growth may be lower in ventilated culture vessels. It is probable that the forced ventilation used in the present study led to higher water loss than that in naturally ventilated vessels, which resulted in the 15 g⋅L−1 sucrose concentration becoming more optimal for shoot growth rather than the 30 g⋅L−1 concentration. Furthermore, the results also suggest that the sucrose concentration has a role in the extent to which shoot growth is inhibited by PBZ. This was demonstrated by the shoot length being significantly longer within the same ventilation treatments in 30 g⋅L−1 sucrose than in 15 g⋅L−1 sucrose with both 0.2 and 0.4 mg⋅L−1 PBZ concentrations. At 15 g⋅L−1, which is half the commonly used sucrose concentration, the dwarfing effect of PBZ was further enhanced, thus leading to an even more significant reduction in shoot length in both ventilation treatments. These results indicate that the relatively higher carbon source provided by 30 g⋅L−1 sucrose is able to reduce the dwarfing effects of PBZ. Increasing the sucrose concentration in the presence of PBZ in the growth medium also improved the growth of date palm (Awadh et al., 2019) and the length of gladiolus cormels (Nagaraju et al., 2002) in vitro.
Significant improvements are often reported for the overall vegetative growth of plantlets cultured in forced-ventilated vessels (Xiao et al., 2007, 2011; Zobayed et al., 2004). Studies have shown increases in the number of leaves formed in gerbera (Xiao et al., 2005), jojoba (Mills et al., 2004), papaya (Teixeira da Silva, 2014), sweetpotato (Heo and Kozai, 1999), and walnut (Hassankhah et al., 2014) under ventilated conditions. Our results showed that forced ventilation improves leaf formation in potato plantlets across all treatments. However, these results are different than those reported by Kubota and Kozai (1992), who reported that similar numbers of leaves were found on potato plantlets cultured in forced ventilation and minimal ventilation treatments. It is possible that other factors, such as the use of different supporting materials or sucrose concentrations in their study, affected leaf formation. There are conflicting reports of the number of leaves formed by plants in response to PBZ. For Schfflera arboricola (Mazher et al., 2014) and Tabernaemontana coronaria (Youssef and Abd El-Aal, 2013), exposure to PBZ increased the leaf numbers, whereas the leaf numbers decreased for Allium cepa (Ashrafuzzaman et al., 2009), Arbutus unedo (Navarro et al., 2007), and Pentas lanceolata (Taha and Sorour, 2016). However, PBZ did not affect the number of leaves formed on plants such as Manihot esculenta (Abah et al., 2017). In the present study, the stimulatory effect of PBZ on promoting leaf formation of nonventilated potato plantlets was evident when 30 g⋅L−1 sucrose was used; however, the same effect was not observed with 15 g⋅L−1 sucrose (Fig. 1). Particularly, with 0.4 mg⋅L−1 PBZ, the impact of PBZ on the nonventilated plus 30 g⋅L−1 sucrose was that the number of formed leaves approached the number that formed with the ventilated treatments. Therefore, when potato plantlets are grown without ventilation, leaf formation can be increased by supplementing the growth medium with PBZ in combination with 30 g⋅L−1 sucrose. From these findings, it can be established that the concentration of sucrose in the growth medium is a crucial factor that determines the extent to which PBZ promotes leaf growth in nonventilated conditions.
The ability of potato plantlets to form new secondary axillary shoots was found to be affected primarily by the sucrose concentration rather than ventilation (Fig. 2). No studies have reported how ventilation, sucrose, or PBZ affect the ability of plantlets to form secondary shoots. Nevertheless, our results indicated that it is likely that 30 g⋅L−1 sucrose in the growth medium provided the required carbon source for the potato plantlets to produce new shoots, and that PBZ in general reduced their ability to form shoots. In terms of shoot numbers, Awadh et al. (2019) reported inconsistent results for date palm plantlets that varied according to sucrose and PBZ concentrations. One of the main advantages that is often stated with regard to plantlets cultured in ventilated vessels is their improved ability to survive in ex vitro conditions (Xiao et al., 2005, 2011; Zhang et al., 2009). Ventilation reduces the relative humidity and ethylene accumulation inside vessels, which improves morphological and physiological characteristics of plantlets. PBZ has also been shown to induce morphological changes in leaves and improve the environmental stress tolerance of plants (Desta and Amare, 2021). During our study, the percentage of leaf death that occurred in ex vitro conditions provides a good indication of the adaptability of the potato plantlets to ex vitro conditions. The results confirmed the beneficial effects of ventilation on improving the ability of plantlets to withstand ex vitro conditions, as shown by their significantly lower percentage of leaf deaths (Fig. 3). PBZ have been shown to enable plants to withstand water-deficit stress (Jungklang et al., 2017; Rady and Gaballah, 2012). Positive effects of PBZ on the ex vitro survival rate have been reported for Citrus (Hazarika et al., 2001), Dendrobium (Wen et al., 2013), and Prunus (Eliasson et al., 1994). In the present study, the addition of PBZ to the growth medium reduced leaf death in both ventilated and nonventilated treatments, with the lowest leaf death found for plantlets that were ventilated in the 0.4 mg⋅L−1 PBZ concentration. Similar findings were reported for grapevines by Smith et al. (1992), who scored the severity of leaf wilting using a scale after transplanting to ex vitro conditions. The results revealed a lower leaf wilt score for grapevine plantlets cultured on PBZ-enriched medium, and that the lowest score for leaf wilt was observed on plantlets cultured on the same medium in vessels that were naturally ventilated.
Conclusion
The longest shoot length was found on potato plantlets cultured in the ventilated plus 15 g⋅L−1 sucrose treatment without PBZ, whereas the number of leaves formed was greater in a ventilated environment regardless of the sucrose or PBZ concentration. The percentage of plantlets that formed secondary shoots was higher with the ventilated plus 30 g⋅L−1 sucrose treatment, even when cultured in growth media supplemented with PBZ. The highest number of secondary axillary shoots was found for plantlets cultured in the ventilated plus 30 g⋅L−1 sucrose treatment without PBZ. In ex vitro conditions, plantlets grown without PBZ in ventilated culture vessels had a lower percentage of leaf death, whereas the opposite was true for treatments without ventilation. However, with the same ventilation and sucrose treatments with PBZ, the percentage of leaf death decreased for all treatments, but the leaf death percentage was always higher for plantlets cultured with the nonventilated plus 15 g⋅L−1 sucrose treatment. Overall, the best results were found for plantlets grown in modified temporary immersion vessels provided with ventilation and supplemented with 30 g⋅L−1 sucrose.
References
Abah, S.P., Nsofor, G.C., Okoroafor, U.E., Ezeji, L.A., Ben-Uchechi, I., Mbe, J.O. & Okocha, P.I. 2017 Effect of paclobutrazol and kinetins on the vegetative growth, root initiation and tuberization of Manihot esculenta Crantz Niger. Agric. J. 48 1 206 213
Armstrong, J., Lemos, E.E.P., Zobayed, S.M.A., Justin, S.H.F.W. & Armstrong, W. 1997 A humidity-induced convective throughflow ventilation system benefits Annona squamosa L. explants and coconut calloid Ann. Bot. 79 31 40 https://doi.org/10.1006/anbo.1996.0299
Ashrafuzzaman, M., Nasrul Millat, M., Razi Ismail, M., Uddin, M.K., Shahidullah, S.M. & Meon, S. 2009 Paclobutrazol and bulb size effect on onion seed production Int. J. Agric. Biol. 11 3 245 250
Awadh, H.A.A., Abdulhussein, M.A.A. & Almusawi, A.H.A. 2019 Effects of paclobutrazol and sucrose in date palm (Phoenix dactylifera L.) micropropagation via direct organogenesis Plant Arch. 19 1 1130 1134
Desta, B. & Amare, G. 2021 Paclobutrazol as a plant growth regulator Chem. Biol. Technol. Agric. 8 1 15 https://doi.org/10.1186/s40538-020-00199-z
El-Said, R. & El-Fadl, A. 2017 Effect of growth retardants on shoot and root development of stevia (Steviarebaudiana bertoni) plant grown in vitro J. Agric. Vet. Sci. 10 2 16 24 https://doi.org/10.9790/2380-1002011624
Eliasson, M.K., Beyl, C.A. & Barker, P.A. 1994 In vitro responses and acclimatization of Prunus serotina with paclobutrazol J. Plant Growth Regul. 13 137 142 https://doi.org/10.1007/BF00196377
Fujiwara, K. & Kozai, T. 1995 Physical microenvironment and its effects 319 369 Aitken-Christie, J., Kozai, T. & Smith, M.A.L. Automation and environmental control in plant tissue culture. Kluwer Academic Publishers Dordrecht, Switzerland
Gimenes, R., Pivetta, K.F.L., Mazzini-Guedes, R.B., Ferraz, M.V., Pereira, S.T.S., Santos, A.S., de Faria, R.T. & de Almeida, L.C.P. 2018 Paclobutrazol on in vitro growth and development of Zygopetalum crinitum orchid, and on seedling acclimatization Am. J. Plant Sci. 9 1029 1036 https://doi.org/10.4236/ajps.2018.95079
Goncalves, L.A., Geraldine, R.M., Picoli, E.A.T., Vendrame, W.A., de Carvalho, C.R. & Otoni, W.C. 2007 In vitro propagation of Herreria salsaparilha Martius (Herreriaceae) as affected by different sealing materials and gaseous exchanges Plant Cell Tissue Organ Cult. 92 243 250 https://doi.org/10.1007/s11240-007-9327-z
Hassankhah, A., Vahdati, K., Lotfi, M., Mirmasoumi, M., Preece, J. & Assareh, M.-H. 2014 Effects of ventilation and sucrose concentrations on the growth and plantlet anatomy of micropropagated Persian walnut plants Int. J. Hortic. Sci. Technol. 1 2 111 120 https://doi.org/10.22059/ijhst.2014.52781
Hazarika, B.N., Parthasarathy, V.A. & Nagaraju, V. 2001 Influence of in vitro preconditioning of citrus microshoots with paclobutrazol on ex vitro survival Acta Bot. Croat. 60 1 25 29
Heo, J. & Kozai, T. 1999 Forced ventilation micropropagation system for enhancing photosynthesis, growth and development of sweetpotato plantlets Environ. Control Biol. 37 1 83 92 https://doi.org/10.2525/ecb1963.37.83
Jungklang, J., Saengnil, K. & Uthaibutra, J. 2017 Effects of water-deficit stress and paclobutrazol on growth, relative water content, electrolyte leakage, proline content and some antioxidant changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink Saudi J. Biol. Sci. 24 7 1505 1512 https://doi.org/10.1016/j.sjbs.2015.09.017
Kepenek, K. & Karoglu, Z. 2011 The effects of paclobutrazol and daminozide on in vitro micropropagation of some apple (Malus domestica) cultivars and M9-rootstock Afr. J. Biotechnol. 10 24 4851 4859 https://doi.org/10.5897/AJB10.1456
Kianmehr, B., Parsa, M., Otroshy, M., Mohallati, M.N. & Morad, K. 2012 Effect of plant growth regulators during in vitro phase of potato microtuber production Agric. Technol. Thail. 8 5 1745 1759
Kubota, C. & Kozai, T. 1992 Growth and net photosynthetic rate of Solanum tuberosum in vitro under forced and natural ventilation HortScience 27 1312 1314 https://doi.org/10.21273/hortsci.27.12.1312
Kumar, S., Ghatty, S., Satyanarayana, J., Guha, A., Chaitanya, B.S.K. & Reddy, A. 2012 Paclobutrazol treatment as a potential strategy for higher seed and oil yield in field-grown Camelina sativa L. Crantz BMC Res. Notes 5 1 13 https://doi.org/10.1186/1756-0500-5-137
Mazher, A.A.M., Abdel-Aziz, N.G., El-Maadawy, E.I., Nasr, A.A. & El-Sayed, S.M. 2014 Effect of gibberellic acid and paclobutrazol on growth and chemical composition of Schefflera arboricola plants Middle East J. Agric. Res. 3 4 782 792
Mills, D., Zhou, Y. & Benzioni, A.A. 2004 Improvement of jojoba shoot multiplication in vitro by ventilation In Vitro Cell. Dev. Biol. Plant 40 396 402 https://doi.org/10.1079/ivp2004537
Mohamed, M.A.H. & Alsadon, A.A. 2010 Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets Scientia Hort. 123 295 300 https://doi.org/10.1016/j.scienta.2009.09.014
Murashige, T. & Skoog, F. 1962 A revised medium for rapid growth and bioassay with tobacco cultures Plant Physiol. 15 473 497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nagaraju, V., Bhowmik, G. & Parthasarathy, V.A. 2002 Effect of paclobutrazol and sucrose on in vitro cormel formation in gladiolus Acta Bot. Croat. 61 1 27 33
Navarro, A., Sanchez-Blanco, M.J. & Banon, S. 2007 Influence of paclobutrazol on water consumption and plant performance of Arbutus unedo seedlings Scientia Hort. 111 133 139 https://doi.org/10.1016/j.scienta.2006.10.014
Orabi, S.A., Salman, S.R. & Shalaby, M.A.F. 2010 Increasing resistance to oxidative damage in cucumber (Cucumis sativus L.) plants by exogenous application of salicylic acid and paclobutrazol World J. Agric. Sci. 6 3 252 259
Rady, M.M. & Gaballah, M.S. 2012 Improving barley yield grown under water stress conditions Res. J. Recent Sci. 1 6 1 6
Simko, I. 1993 Effects of kinetin, paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light Plant Growth Regulat. 12 23 27 https://doi.org/10.1007/bf00144578
Simko, I. 1994 Effect of paclobutrazol on in vitro formation of potato microtubers and their sprouting after storage Biol. Plant. 36 1 15 20 https://doi.org/10.1007/bf02921262
Smith, E.F., Gribaudo, I., Roberts, A.V. & Mottley, J. 1992 Paclobutrazol and reduced humidity improve resistance to wilting of micropropagated grapevine HortScience 27 2 111 113 https://doi.org/10.21273/hortsci.27.2.111
Suharjo, U.K.J., Hasanudin, H., Pamekas, T., Pujiwati, H. & Vanturini, A. 2019 Promoting tuber formation in vitro with benzyl amino purine and paclobutrazol at different concentrations Akta Agrosia. 22 1 29 35 https://doi.org/10.31186/aa.22.1.29-35
Taha, A.M. & Sorour, M.A. 2016 Effect of paclobutrazol and its method of application on the growth of Pentas lanceolata plants J. Adv. Agric. Res. 21 4 686 699 https://doi.org/10.21608/jalexu.2016.195603
Teixeira da Silva, J.A. 2014 Photoauto-, photohetero- and photomixotrophic in vitro propagation of papaya (Carica papaya L.) and response of seed and seedlings to light-emitting diodes Thammasat Int. J. Sci. Technol. 19 1 57 71
Valdés, T.D., Ruvalcaba, L.P., Tafoya, F.A., Orona, C.A.L. & de Jesús Velázquez Alcaraz, T. 2017 Dose of paclobutrazol in the growth of sugarcane seedlings in vitro in the acclimatization stage Agric. Sci. 8 751 758 https://doi.org/10.4236/as.2017.88056
Wen, Z.Z., Lin, Y., Liu, Y.Q., Wang, M., Wang, Y.Q. & Liu, W. 2013 Effects of paclobutrazol in vitro on transplanting efficiency and root tip development of Dendrobium nobile Biol. Plant. 57 3 576 580 https://doi.org/10.1007/s10535-013-0319-z
Wu, H.-C., Guo, M.-L. & Chen, C.-M. 2018 Promotion of vegetative growth in force-ventilated P. cynaroides L. explants cultured in modified temporary immersion culture vessels HortScience 53 2 231 235 https://doi.org/10.21273/HORTSCI12513-17
Xiao, Y., He, L., Liu, T. & Yang, Y. 2005 Growth promotion of gerbera plantlets in large vessels by using photoautotrophic micropropagation system with forced ventilation Propag. Ornam. Plants 5 4 179 185
Xiao, Y., Niu, G. & Kozai, T. 2011 Development and application of photoautotrophic micropropagation plant system Plant Cell Tissue Organ Cult. 105 149 158 https://doi.org/10.1007/s11240-010-9863-9
Xiao, Y., Zhang, Y., Dang, K. & Wang, D. 2007 Growth and photosynthesis of Dendrobium candidum Wall. ex Lindl. plantlets cultured photoautotrophically Propag. Ornam. Plants 7 2 89 96
Youssef, A.S.M. & Abd El-Aal, M.M.M. 2013 Effect of paclobutrazol and cycocel on growth, flowering, chemical composition and histological features of potted Tabernaemontana coronaria Stapf plant J. Appl. Sci. Res. 9 11 5953 5963
Zhang, H., Xu, F., Wu, Y., Hu, H.-H. & Dai, X.-F. 2017 Progress of potato staple food research and industry development in China J. Integr. Agric. 16 12 2924 2932 https://doi.org/10.1016/S2095-3119(17)61736-2
Zhang, M., Zhao, D., Ma, Z., Li, X. & Xiao, Y. 2009 Growth and photosynthetic capability of Momordica grosvenori plantlets grown photoautotrophically in response to light intensity HortScience 44 3 757 763 https://doi.org/10.21273/hortsci.44.3.757
Zobayed, S.M.A., Afreen, F., Xiao, Y. & Kozai, T. 2004 Recent advancement in research on photoautotrophic micropropagation using large culture vessels with forced ventilation In Vitro Cell. Dev. Biol. – Plant 40 450 458 https://doi.org/10.1079/ivp2004558
