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In vitro multiplication of statice plantlets using sugar-free media
Scientia Horticulturae 109 (2006) 71–77 www.elsevier.com/locate/scihorti
In vitro multiplication of statice plantlets using sugar-free media Yulan Xiao a,b,*, Toyoki Kozai b b
a Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan Yangtze Delta Region Institute of Tsinghua University, Jiashan, Zhejiang 314100, China
Received 9 May 2005; received in revised form 21 February 2006; accepted 24 February 2006
Abstract Clumps of statice (Limonium latifolium) plantlets grown photomixotrophically were used as explants and cultured for 25 days on a sugar-free modified Murashige and Skoog (MS) medium in Magenta-type vessels with the number of air exchanges of the vessel (NAE) being 3.8 h1, at a photosynthetic photon flux (PPF) of 100 mmol m2 s1 and a CO2 concentration of 1500 mmol mol1 in the culture room. A factorial experiment was conducted with three levels of 6-benzylaminopurine (BA) concentration, namely 0, 0.25 and 0.5 mg L1, and two types of supporting material, agar and Florialite (a porous material). The control treatment was a photomixotrophic culture using a sugar- and BA (0.25 mg L1) containing agar medium in the vessel with NAE of 0.2 h1, at a PPF of 50 mmol m2 s1 and a CO2 concentration of 400 mmol mol1 in the culture room. Leaf area, chlorophyll concentration and net photosynthetic rate were greater in the sugar-free medium treatment with a BA concentration of 0.25 mg L1 and Florialite than those in the control treatment. The number of shoots and dry weight per clump in the sugar-free medium treatment were comparable to those in the control treatment. Among the sugar-free medium treatments, the number of shoots increased with increasing BA concentration, however, the leaf area, dry weight, chlorophyll concentration and net photosynthetic rate decreased with increasing BA concentration. The use of Florialite significantly enhanced the growth and root induction as well as net photosynthetic rate, compared with the treatments that use agar. These results indicated that sugar-free medium micropropagation could be commercially applied to the multiplication of statice plantlets. # 2006 Elsevier B.V. All rights reserved. Keywords: Shoot multiplication; CO2 concentration; Net photosynthetic rate; Chlorophyll concentration; BA; Supporting material
1. Introduction The attractive color and longevity of statice, a perennial herb and a cutting flower, makes it an ideal candidate for sale, both as a fresh and dry flower, in the flower market. Micropropagation of statice plantlets is conventionally carried out in four stages. First, a small bud is taken from a mother plant, sterilized, and aseptically cultured in vitro to produce propagules or explants. Next, the propagules are grown to form a clump of shoots; this clump is subdivided into several small clumps and repropagated for several generations in a multiplying medium at the ‘‘multiplication stage.’’ Then, an individual shoot from the clump is placed in a rooting medium. Finally, the rooted plantlets are first transferred to a greenhouse for acclimatiza-
Abbreviations: BA, 6-benzylaminopurine; MS, Murashige and Skoog; NAE, the number of air exchanges of the vessel; Pn, net photosynthetic rate; PPF, photosynthetic photon flux; SFM treatment, sugar-free medium treatment * Corresponding author: Tel.: +86 573 4291108; fax: +86 573 4291108. E-mail address: [email protected] (Y. Xiao). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.02.029
tion, and then transplanted into the field. Thus, the addition of sugar in the medium as an energy and/or carbon source is a requisite for plantlet growth. However, in conventional micropropagation, the loss of plantlets due to microbial contamination and poor growth of plantlets in vitro (Kozai, 1990; Lees, 1994) as well as a low percent survival during the ex vitro acclimatization (Desjardins et al., 1995; Kozai and Zobayed, 2000) is a problem that needs to be solved. Photoautotrophic micropropagation using a sugar-free medium and leafy explants, i.e. sugar-free medium micropropagation, in which plantlets utilize CO2 in the air as an energy and/or carbon source, has several advantages over conventional micropropagation. The advantages include minimized microbial contamination, enhanced photosynthesis, growth and rooting in vitro and increased percent survival ex vitro. A number of reports have been published on the enhanced growth and photosynthesis of plantlets in vitro by using the sugar-free media, and by increasing photosynthetic photon flux (PPF) and the CO2 concentration in the vessel. These reports include the studies by Kozai and Iwanami (1988a) on carnation, Kirdmanee et al. (1995) on Eucalyptus, Niu and Kozai (1997)
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on potato, Nguyen et al. (1999) on coffee and Kubota and Kozai (2001) on tomato. The explants used in these studies were mostly leafy nodal cuttings. When leafy nodal cuttings are used, the number of unfolded leaves of a plantlet determines the multiplication rate of the plantlet. A few studies using sugar-free media have involved the shoot multiplication. These include the studies by Nguyen and Kozai (2001) on banana (Musa spp.) and Erthurk and Walker (2000) on sugarcane, in which shoots excised from a clump were used as explants, because leafy nodal cuttings were not available in these plant species. In this case, the number of shoots per plantlet determines the multiplication rate of the plantlet. When compared with the plantlets grown on a sugarcontaining medium, it was observed that the plantlets grown on a sugar-free medium had a lower dry weight, but a higher multiplication rate in the case of banana, and a lower multiplication rate in the case of sugarcane. The gelling agents, agar and phytagel, were used as the supporting material in both experiments. The supporting material is an important factor for plantlet growth in vitro. Afreen-Zobayed et al. (1999) compared the growth of sweet potato plantlets cultured photoautotrophically in vitro with five different types of supporting material: agar, gellan gum, vermiculite, cellulose plug and Florialite (Nisshinbo Industries Inc., Tokyo, Japan), which is a mixture of vermiculite and cellulose fibers. They observed that sweet potato plantlets exhibited the greatest growth when Florialite was used as the supporting material. Cytokinins such as 6-benzylaminopurine (BA) are a class of plant hormones that play an essential role in plant morphogenesis and have an influence on the formation of shoots and their relative growth rate (Debi et al., 2005). Cytokinins are commonly used in conventional micropropagation to increase the multiplication rate. In the case of photoautotrophic micropropagation, leafy nodal cuttings exhibit rapid growth on the sugar-free medium due to the high net photosynthetic rate; thus, a high multiplication rate can be easily achieved, therefore, cytokinins are not a requisite for the multiplication of leafy nodal cuttings. However, the effects of cytokinins on shoot multiplication in the sugar-free medium have not been determined when shoot clumps are used as explants, as in the case of statice plantlets. The objectives of this study were (1) to investigate the effects of BA concentration and the type of supporting material on growth and multiplication of statice plantlets cultured on a sugar-free medium and (2) to assess the possibility of shoot multiplication of statice plantlets grown on a sugar-free medium based on a comparison with the plantlets grown on a sugar-containing medium. 2. Materials and methods 2.1. Plant material, treatments and culture conditions Clumps each having two to three leafy microshoots, excised from photomixotrophically grown statice (Limonium latifolium) plantlets, were used as explants and cultured for 25 days
for multiplication. The average leaf area, and fresh and dry weight per clump were 647 108 mm2, 461 92 mg and 23 8 mg, respectively. Three clumps were transplanted in each Magenta-type vessel (370 mL, Verde Co., Ltd., Japan) containing 70 mL MS solution with the pH adjusted to 5.7 before autoclaving. This experiment included six sugar-free medium (SFM) treatments: AL, AM, AH, FL, FM and FH, where A and F indicate agar and Florialite substrate and L, M and H indicate three levels of BA concentration, 0, 0.25 and 0.5 mg L1, respectively; and one sugar-containing (30 g L1) medium treatment with a BA concentration of 0.25 mg L1 and agar as the control (Table 1). Ten replications (10 vessels) were used for each treatment. The experiment was conducted twice. The sugar-free explants were cultured on the sugar-free modified MS medium under the following conditions: PPF (50 mmol m2 s1 from days 0 to 7 and 100 mmol m2 s1 from days 8 to 25), CO2 concentration (1500 mmol mol1 in the culture room) and the number of air exchanges of the vessel (NAE: 0.2 h1 from days 0 to 3, 1.8 h1 from days 4 to 7, 2.7 h1 from days 8 to 17, 3.8 h1 from days 18 to 25). NAE was measured by using the method of Kozai et al. (1986) and varied by increasing the number of gas-permeable filter disks (10 mm in diameter, pore size 0.5 mm, Milli-Seal, Nihon Millipore, Ltd., Tokyo), which were attached to the holes on the lid of the vessel (Fig. 1). The control explants were cultured on the sugar-containing MS medium under the following conditions: PPF of 50 mmol m2 s1, CO2 concentration of 400 mmol mol1 in the culture room, and NAE of 0.2 h1. Throughout the culture period, the air temperature and relative humidity in the culture rooms were maintained at 25 1 8C and 80 5%, respectively. The photoperiod was 16 h per day supplied with cool white fluorescent lamps. 2.2. Measurement, calculation and statistical analysis The CO2 concentrations inside and outside the vessels were measured once every 5 days by a gas chromatograph (GC-9A, Shimadzu Co., Ltd., Kyoto, Japan). This measurement was performed when the CO2 concentrations in the culture vessels and the culture room were stable during the photoperiod. Table 1 Description of treatments Treatment code
Sugar (g/L)
Supporting material
BA (mg/L)
Control ALa AM AH FL FM FH
30 0 0 0 0 0 0
Agar Agar Agar Agar Florialite Florialite Florialite
0.25 0 0.25 0.5 0 0.25 0.5
a
The first letters A and F denote agar and Florialite, respectively. The second letters L, M and H denote three levels of BA concentration, 0, 0.25 and 0.5 mg L1, respectively.
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Fig. 1. Effects of two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1) on the growth and multiplication of statice plantlets cultured in Magenta-type vessels containing sugar-free medium on day 25. The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium. For treatment codes, refer to Table 1.
The net photosynthetic rate, Pn (mmol h1 per clump), was calculated in accordance with the method of Fujiwara et al. (1987) using the following formula: Pn ¼
K N VðCout Cin Þ E
where K is the conversion factor of CO2 from volume to moles (0.0407 mol L1 at 26 8C), N the number of air exchanges of the vessel (h1), V the air volume of the vessel (L), Cin and Cout the CO2 concentrations (mmol mol1) inside and outside the vessel under steady-state conditions during the photoperiod and E is the number of clumps per vessel. The chlorophyll concentration was measured by the method of Moran (1982). The appropriate mass of leaves was soaked in 3 mL N,N-dimethylformamide solution for 36 h at 2 8C and absorption of light was measured with a spectrophotometer (U1100, Hitachi, Co., Ltd., Japan) at wavelengths 664, 647 and 603 nm. The chlorophyll concentration including chlorophyll a and b on a fresh weight basis (mg g1) were calculated by the formula of Moran (1982). The number of shoots, leaf area, fresh and dry weight per clump and chlorophyll concentration were measured on day 25.
The relationship between the supporting materials (SM) and BA concentrations in the SFM treatments was examined with an analysis of variance (ANOVA). The relationship between these variables among all the treatments, including the control treatment, was examined with the least significant difference (L.S.D.) test. 3. Results 3.1. Growth and multiplication The results of the plantlet growth and multiplication on day 25 are summarized in Table 2 and in Figs. 1 and 2. Among all the treatments, the dry weight was greatest in the FL treatment, although there was no significant difference in dry weight among the FL, FM and the control treatments. Further, it was lowest in the AH treatment. The leaf area was greatest in the FL treatment, and fresh weight was greatest in the control treatment. The growth values in the SFM treatments varied with the BA concentration levels and the types of supporting material. The number of shoots per clump was greatest in the FH among all the treatments, although there was no significant
Table 2 Effects of two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1) on leaf area, fresh weight (FW), dry weight (DW) and net photosynthetic rate (Pn) of statice per clump (PC) and per shoot (PS) on day 25 cultured in vitro on sugar-free medium Treatment code
Leaf area of PC (mm2)
FW of PC (mg)
DW of PC (mg)
Pn of PC (mmol h1)
Leaf area of PS (mm2)
FW of PS (mg)
DW of PS (mg)
Control AL AM AH FL FM FH
5742 1190c1 6938 1148b 6124 1212c 5328 1311d 8180 904a 7066 1047b 6366 845c
5059 1193a 4074 875bc 3859 916cd 3236 753e 4561 712b 4186 705b 3646 494d
286 71a 248 68b 235 62b 198 44c 301 54a 282 49a 232 37b
48.4 10.2e 20.8 2.3b 17.7 1.6c 13.8 1.7d 25.4 2.7a 22.3 1.8ab 16.4 3.1c
486 135cd 988 192ab 663 216bc 324 114d 1275 407a 777 209ab 401 145cd
121 25bc 228 52a 162 38ab 83 25c 247 75a 194 50a 104 28c
10 3cd 25 5ab 15 3bc 5 1d 32 8a 17 4b 8 2d
ANOVA2 SM BA SP BA
* * NS
* * NS
* ** *
** ** **
* ** *
NS ** **
** ** *
The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium; NS, * and ** indicate nonsignificant or significant at 5 or 1% level of probability, respectively. 1 Mean S.D. separation within columns by least significant difference (L.S.D.) test at 5% level. For treatment codes, refer to Table 1. 2 Analysis of variance was applied to six sugar-free medium treatments with two types of supporting material (SM) and three levels of BA concentration.
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Fig. 2. Number of shoots (A) and chlorophyll concentration (B) of statice plantlets in vitro with sugar-free medium on day 25 as affected by two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1). The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium. For treatment codes, refer to Table 1. Vertical bars represent S.E.
difference in the number of shoots per clump among the FH, FM and the control treatments. Further, it was lowest in the AL, although there was no significant difference in the number of shoots per clump between the AL and FL treatments (Fig. 2A). Among the SFM treatments, the number of shoots increased with increasing BA concentration; but the leaf area, fresh and dry weight and chlorophyll concentration decreased with increasing BA concentration (Table 2; Fig. 2A). The percent loss of the plantlets due to contamination was 0% in all the SFM treatments and 10% in the control treatment. Growth of the shoots for the next generation was greatest in the FL treatment, in which leaf area, and fresh and dry weight per shoot were, respectively, 2.6, 2.0 and 3.2 times higher, compared with those in the control treatment, followed by the AL and FM treatments (Table 2).
Pn was positive and increased with time in the sugar-free medium treatments; however, it was negative and decreased with time in the control during the photoperiod throughout the experiment (Fig. 4). Pn was greatest in the FL treatment, although there was no significant difference in Pn between the FL and FM treatments, and it was lowest in the control among all the treatments. During days 0–25, Pn increased from 0.1 to 25.4 mmol h1 per clump in the FL treatment and from 0.1 to 22.3 mmol h1 per clump in the FM treatment, while it decreased from 3.7 to 48.4 mmol h1 per clump in the control. Among the SFM treatments, a significant difference in Pn on day 25 was observed between the plantlets cultured with Florialite and those cultured with agar when the same BA concentration was used (Table 2; Fig. 4). 3.3. Chlorophyll concentration
3.2. CO2 concentration and Pn The CO2 concentration inside the vessel during the photoperiod decreased with time in all the SFM treatments. On the contrary, it increased with time in the control treatment. As a result, it was lowest in the FL treatment, and highest in the control among all the treatments (Fig. 3).
Fig. 3. Time courses of CO2 concentration inside the vessel and in the culture room. For treatment codes, refer to Table 1. Vertical bars represent S.E.
Among all the treatments, the plantlet chlorophyll concentration was greatest in the FL treatment being 2.5 times that of the control. An increase in BA concentration decreased the chlorophyll concentration in the SFM treat-
Fig. 4. Time courses of net photosynthetic rate (Pn) of statice plantlets at multiplication stage. For treatment codes, refer to Table 1. Vertical bars represent S.E.
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ments. Significant differences in the chlorophyll concentration were observed between the plantlets cultured with different BA concentrations when the same supporting material was used, and between the plantlets cultured with different supporting materials when the same BA concentration was used (Fig. 2B). 4. Discussion 4.1. Growth and multiplication The results clearly showed that statice plantlets grown on sugar-free medium in the FM treatment with a BA concentration of 0.25 mg L1 and Florialite achieved a high multiplication rate with enhanced growth, although there were no significant differences in the number of shoots and dry weight between the FM and the control treatments. Leaf area and chlorophyll concentration of the FM plantlets were 1.2 and 1.7 times higher than those of the control plantlets, although fresh weight of the control plantlets was 1.2 times that of the FM plantlets (Table 2). This result was consistent with those of Mosaleeyanon et al. (2004) who observed that the plantlets cultured in vitro on a sugar-containing medium had reduced leaf area and chlorophyll concentration. Clearly, the plantlets cultured under sugar-free medium micropropagation conditions were healthier than those cultured under conventional micropropagation conditions. In addition, leaf area, and fresh and dry weight per shoot for the next generation in the FM treatment were, respectively, 1.6, 1.6 and 1.7 times higher than those in the control (Table 2). In general, the intrinsic quality of the plantlet, produced in vitro, is of the utmost importance for the successful regeneration of plantlets in any of the micropropagation protocols (Singh and Syamal, 2001). Moreover, the beneficial effect of using sugar-free medium on rooting has been clearly demonstrated in this study; root formation was observed in the SFM treatments from day 5; however, no root formation was observed in the control until the harvest, furthermore, there were a mass of callus at the control shoot base that might increase the plantlet weight. Similarly, the plantlet growth, especially the root growth, was observed to be greater in sugar-free medium than in sugar-containing medium in several species such as potato (Kozai et al., 1988b), Coffea arabusta (Nguyen et al., 1999) and China fire (Xiao and Kozai, 2004). It can be considered that sugar-free medium micropropagation would enable to merge the multiplication and rooting stages into one combined stage and thus simplify the micropropagation process. An appropriate BA concentration in the medium is necessary for balancing both the multiplication rate and quality of the shoots (Pierik et al., 1982; Hempel, 1985). A high BA concentration in the medium could lead to a higher multiplication rate, however, it resulted in a poor quality of the plantlets in the AH and FH treatments. On the contrary, the plantlet relying only on its endogenous hormones could produce high quality shoots, however, it resulted in a lower multiplication rate in the AL and FL treatments.
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The type of supporting material was found to strongly affect on the growth and multiplication rate. The use of Florialite significantly increased the number of shoots, leaf area, and dry weight, and enhanced root induction, compared with the use of agar (Table 2; Fig. 1). Growth promotion by using Florialite was probably due to the fact that a porous material with high air porosity generally maintained a higher dissolved oxygen concentration around the plantlet base than a gelled supporting material like agar (Aitken-Christie et al., 1995; Fujiwara and Kozai, 1995), thus consequently enhanced root and shoot induction of the statice plantlets in this study. 4.2. CO2 concentration and Pn The study revealed that statice plantlets cultured in vitro had the photosynthetic ability at the multiplication stage. The CO2 concentrations inside the vessels during the photoperiod were lower in all the SFM treatments than in the culture room (approximately 1500 mmol mol1) even on the first day, and it gradually showed a significant decrease with time (Fig. 3), indicating a positive Pn and an increase in Pn of the plantlets from the beginning of culture. In addition, the CO2 concentrations in the FL, FM and AL treatments on day 25 were 220, 328 and 351 mmol mol1, respectively, these were approximately 1200 mmol mol1 lower than in the culture room, becoming a limiting factor for photosynthesis during the culture period in these treatments. Therefore, it is considered that increasing CO2 concentration inside the vessel could promote the photosynthesis of in vitro plantlets. A high CO2 concentration inside the vessel can be achieved by increasing the number of air exchanges of the vessel, and by increasing the CO2 concentration in the culture room. On the contrary, the CO2 concentrations inside the vessel in the control treatment during the photoperiod were 10, 15, 22, 33, 60 and 128 times higher than those in the culture room (approximately 400 mmol mol1) on days 0, 5, 10, 15, 20 and 25, respectively, indicating that the CO2 evolution rate of the plantlets was much higher than the CO2 uptake rate of the plantlets on the sugar-containing medium, resulting in a negative Pn throughout the culture period. The high CO2 concentration in the vessel in the control treatment probably resulted from the CO2 produced by the callus at the shoot base (Zobayed et al., 1999), in addition to that produced by the respiration of plantlet. A combination of the low NAE, and the negative Pn throughout the culture period also made a contribution to that. This observation is in agreement with the findings of Nguyen and Kozai (2001) on banana and Xiao et al. (2003) on sugarcane. The negative Pn in the control was probably due to the sugar-containing medium, which suppressed photosynthesis of plantlets in vitro (Desjardins et al., 1995), because plantlets cultured in the sugar-containing medium depend more on the sugar in the medium than on the CO2 in the air, resulting in a low or negative Pn (Kubota and Kozai, 2001). On the other hand, unlike Pn observed in the present study, Lian et al. (2002) observed that statice plantlets showed a positive Pn even under photomixotrophic conditions during the
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rooting stage, which indicated that the photosynthetic characteristics of statice plantlets were different in the multiplication and rooting stages. Similarly, Serret et al. (1996) observed that Gardenia jasminoides plantlets had more developed photosynthetic characteristics during the root induction period than those during the shoot multiplication period. 4.3. Chlorophyll concentration The chlorophyll concentration plays an important role in the absorption of light during photosynthesis. In the present study, it was observed that the chlorophyll concentration of the plantlets was influenced by a few factors, including the presence or the absence of sugar in the medium, BA concentration and the type of supporting material. As shown in Fig. 2, the presence of sugar in the medium reduced the chlorophyll concentration. This, subsequently, resulted in the lowest Pn value in the control (Fig. 4). Capellades et al. (1991) and Azcon-Bieto (1983) observed that the presence of sugar in the medium was attributed to large deposits of starch in the chloroplasts. The carbohydrate accumulation in the chloroplasts may cause a feedback inhibition of photosynthesis, resulting in the decrease in Pn (Hdider and Desjardins, 1994). In addition, the increase in BA concentration led to a decrease in the chlorophyll concentration and Pn (Figs. 2 and 4). A higher BA concentration probably stresses on growth and development of the plantlets, and in some cases, lead to the physiological or morphological disorders such as hyperhydricity and mutation (George, 1996), which may cause a decrease in chlorophyll concentration and a subsequent decrease in the Pn. Moreover, the use of Florialite significantly increased the chlorophyll concentration and Pn. The plantlets cultured with Florialite showed a higher chlorophyll concentration, compared with those cultured with agar (Fig. 2). Similarly, Guimaraes et al. (1999) observed that the chlorophyll concentration was influenced by soil conditions. Previous researchers (AfreenZobayed et al., 1999; Nguyen et al., 1999; Xiao et al., 2003) also observed that the use of Florialite significantly promoted photosynthesis of the plantlets. However, the reason for the increased chlorophyll concentration when Florialite was used as the supporting material is still not clear. A further research is required for a complete understanding of the relationship between chlorophyll concentration and the supporting material. 5. Conclusion In comparison with the multiple shoots produced by conventional micropropagation using sugar-containing medium with BA concentration of 0.25 mg L1 and agar, the multiple shoots produced by using sugar-free medium with BA concentration of 0.25 mg L1 and Florialite (a porous supporting material) resulted in comparable multiplication rate, similar dry weight, higher chlorophyll concentration, higher net photosynthetic rate, better developed rooting system, better shoots and less microbial contamination.
The main highlights of this study include: (1) statice plantlets could be cultured on sugar-free medium in the vessel to produce multiple shoots by providing a PPF of 100 mmol m2 s1, a CO2 concentration of 1500 mmol mol1 in the culture room and the number of air exchanges of the vessel of 3.8 h1, (2) the use of Florialite as supporting material increased the chlorophyll concentration and net photosynthetic rate, and enhanced the root growth, and (3) an appropriate BA concentration in the sugar-free medium for shoot multiplication was found to balance the multiplication rate and plantlet quality. Together, this study demonstrated a reasonable shoot multiplication rate using sugar-free media. It can be considered that the sugar-free medium micropropagation is an effective method for producing a large number of high quality plantlets. Not only leafy nodal cuttings but also shoot clumps could be used as explants for rapid propagation of plantlets in sugar-free medium micropropagation. Acknowledgements The authors are grateful to China Scholarship Council for the financial support and the members of Laboratory of Environmental Control Engineering, Faculty of Horticulture, Chiba University, Japan, for their kind help and support during the experiment. References Afreen-Zobayed, F., Zobayed, S.M.A., Kubota, C., Kozai, T., Hasegawa, O., 1999. Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically. In Vitro Cell. Dev. Biol. 35, 470–474. Aitken-Christie, J., Kozai, T., Takayama, S., 1995. Automation in plant tissue culture. General introduction and overview. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 1–18. Azcon-Bieto, J., 1983. Inhibition of photosynthesis by carbohydrates in wheat leaves. Plant Physiol. 73, 681–686. Capellades, M., Lemeur, L., Debergh, P., 1991. Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro. Plant Cell Tissue Org. Cult. 25, 21–26. Debi, B.R., Taketa, S., Ichii, M., 2005. Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa). J. Plant Physiol. 162, 507–515. Desjardins, Y., Hdider, C., Riek, J., 1995. Carbon nutrient in vitro regulation and manipulation of carbon assimilation in micropropagation system. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 441–465. Erthurk, H., Walker, P.N., 2000. Effects of rooting period, clump size, and growth medium on sugarcane plantlets in micropropagation during and after transformation to photoautotrophy. Trans. ASAE 43, 499–504. 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. Agric. Met. 43, 21– 30. Fujiwara, K., Kozai, T., 1995. Physical microenvironment and its effects. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 319–369. George, E.F., 1996. Plant Propagation by Tissue Culture. Part 2 in Practice, second ed. Butler & Tanner Ltd., Frome, Somerset, pp. 744–745.
Y. Xiao, T. Kozai / Scientia Horticulturae 109 (2006) 71–77 Guimaraes, T., Fonts, G., Rezende, P.C., Pereira, P.R., 1999. Relations among chlorophyll contents determined by a portable meter and nitrogen forms in leaves of tomatoes cultivated in two soil types. Bragantia Campinas 58, 209–216. Hempel, M., 1985. The influence of micropropagation on progeny plants. Acta Hort. 167, 263–272. Hdider, C., Desjardins, Y., 1994. Effects of sucrose on photosynthesis and phosphoenol-pyruvate carboxylase cativity of in vitro cultured strawberry plantlets. Plant Cell Tissue Org. Cult. 36, 27–33. Kirdmanee, C., Kitaya, Y., Kozai, T., 1995. Effects of CO2 enrichment and supporting material in vitro on photoautotrophic growth of Eucalyptus plantlets in vitro and ex vitro. In Vitro Cell. Dev. Biol. 31, 144–149. Kozai, T., Fujiwara, K., Watanabe, I., 1986. Fundamental studies on environments in plant tissue culture vessels. 2. Effects of stoppers and vessels on gas exchange rates between inside and outside of vessels closed with stoppers. J. Agric. Met. 42, 119–127. Kozai, T., Iwanami, Y., 1988a. Effects of CO2 enrichment and sucrose concentration under high photon fluxes on plantlet growth of carnation (Dianthus caryophyllus L.) in tissue culture during the preparation stage. J. Jpn. Soc. Hort. Sci. 57, 279–288. Kozai, T., Koyama, Y., Watanabe, I., 1988b. Multiplication of potato plantlets in vitro with sugar free medium under high photosynthetic photon flux. Acta Hort. 230, 121–127. Kozai, T., 1990. Environmental control and automation in micropropagation. In: Karube, I. (Ed.), Automation in Biotechnology. Elsevier Science Publishers, Amsterdam, The Netherlands, pp. 279–304. Kozai, T., Zobayed, S.M.A., 2000. Acclimatization. In: Spier, R. (Ed.), Encyclopedia of Cell Technology. John Wiley & Sons, New York, pp. 1–12. Kubota, C., Kozai, T., 2001. Growth and net photosynthetic rate of tomato plantlets during photoautotrophic and photomixotrophic micropropagation. Hort. Sci. 36 (1), 49–52. Lees, R.P., 1994. Effects of the light environment on photosynthesis and growth in vitro. In: Lumsden, P.J., Nicholas, J.R., Davies, W.J. (Eds.), Physiology, Growth and Development of Plants in Culture. Kluwer Academic Publishers, Dordrecht, pp. 31–46.
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Lian, M.L., Murthy, H.N., Paek, K.Y., 2002. Culture method and photosynthetic photon flux affect photosynthesis, growth and survival of Limonium ‘Misty Blue’ in vitro. Sci. Hort. 95, 239–249. Moran, R., 1982. Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol. 69, 1376–1381. Mosaleeyanon, K., Cha-um, S., Kirdmanee, C., 2004. Enhanced growth and photosynthesis of rain tree (Samanea saman Merr.) plantlets in vitro under a CO2-enriched condition with decreased sucrose concentrations in the medium. Sci. Hort. 103, 51–63. Nguyen, T.Q., Kozai, T., 2001. Growth of in vitro banana (Musa spp.) shoots under photomixotrophic and photoautotrophic conditions. In Vitro Cell. Dev. Biol. 37, 824–829. Nguyen, T.Q., Kozai, T., Nguyen, K.L., Nguyen, U.V., 1999. Effects of sucrose concentration, supporting material and number of air exchanges of the vessel on the growth of invitro coffee plantlets. Plant Cell Tissue Org. Cult. 58, 51–57. Niu, G., Kozai, T., 1997. Simulation of the growth of potato plantlets cultured photoautotrophically in vitro. Trans. ASAE 40 (1), 255–260. Pierik, R.L.M., Steegmans, H.H.M., Verhaegh, J.A.M., Wouters, A.N., 1982. Effect of cytokinin and cultivar on shoot formation of Gerbera jamesonii in vitro. Neth. J. Agric. Sci. 30, 341–346. Serret, M.D., Trillas, M.I., Matas, J., Araus, J.I., 1996. Development of photoautotrophy and photoinhibition of Gardenia jasminoides plantlets during micropropagation. Plant Cell Tissue Org. Cult. 45, 1–16. Singh, S.K., Syamal, M.M., 2001. A short pre-culture soak in thidiazuron or forchlorfenuron improves axillary shoot proliferation in rose micropropagation. Sci. Hort. 91, 169–177. Xiao, Y., Lok, Y., Kozai, T., 2003. Photoautotrophic growth of sugarcane in vitro as affected by photosynthetic photon flux and vessel air exchanges. In Vitro Cell. Dev. Biol. 39, 186–192. Xiao, Y., Kozai, T., 2004. Commercial application of a photoautotrophic micropropagation system using large vessels with forced ventilation: plantlet growth and production cost. Hort. Sci. 39 (6), 1387–1391. Zobayed, S.M.A., Armstrong, J., Armstrong, W., 1999. Cauliflower shootculture: effects of different types of ventilation on growth and physiology. Plant Sci. 141, 209–217.
In vitro multiplication of statice plantlets using sugar-free media Yulan Xiao a,b,*, Toyoki Kozai b b
a Faculty of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan Yangtze Delta Region Institute of Tsinghua University, Jiashan, Zhejiang 314100, China
Received 9 May 2005; received in revised form 21 February 2006; accepted 24 February 2006
Abstract Clumps of statice (Limonium latifolium) plantlets grown photomixotrophically were used as explants and cultured for 25 days on a sugar-free modified Murashige and Skoog (MS) medium in Magenta-type vessels with the number of air exchanges of the vessel (NAE) being 3.8 h1, at a photosynthetic photon flux (PPF) of 100 mmol m2 s1 and a CO2 concentration of 1500 mmol mol1 in the culture room. A factorial experiment was conducted with three levels of 6-benzylaminopurine (BA) concentration, namely 0, 0.25 and 0.5 mg L1, and two types of supporting material, agar and Florialite (a porous material). The control treatment was a photomixotrophic culture using a sugar- and BA (0.25 mg L1) containing agar medium in the vessel with NAE of 0.2 h1, at a PPF of 50 mmol m2 s1 and a CO2 concentration of 400 mmol mol1 in the culture room. Leaf area, chlorophyll concentration and net photosynthetic rate were greater in the sugar-free medium treatment with a BA concentration of 0.25 mg L1 and Florialite than those in the control treatment. The number of shoots and dry weight per clump in the sugar-free medium treatment were comparable to those in the control treatment. Among the sugar-free medium treatments, the number of shoots increased with increasing BA concentration, however, the leaf area, dry weight, chlorophyll concentration and net photosynthetic rate decreased with increasing BA concentration. The use of Florialite significantly enhanced the growth and root induction as well as net photosynthetic rate, compared with the treatments that use agar. These results indicated that sugar-free medium micropropagation could be commercially applied to the multiplication of statice plantlets. # 2006 Elsevier B.V. All rights reserved. Keywords: Shoot multiplication; CO2 concentration; Net photosynthetic rate; Chlorophyll concentration; BA; Supporting material
1. Introduction The attractive color and longevity of statice, a perennial herb and a cutting flower, makes it an ideal candidate for sale, both as a fresh and dry flower, in the flower market. Micropropagation of statice plantlets is conventionally carried out in four stages. First, a small bud is taken from a mother plant, sterilized, and aseptically cultured in vitro to produce propagules or explants. Next, the propagules are grown to form a clump of shoots; this clump is subdivided into several small clumps and repropagated for several generations in a multiplying medium at the ‘‘multiplication stage.’’ Then, an individual shoot from the clump is placed in a rooting medium. Finally, the rooted plantlets are first transferred to a greenhouse for acclimatiza-
Abbreviations: BA, 6-benzylaminopurine; MS, Murashige and Skoog; NAE, the number of air exchanges of the vessel; Pn, net photosynthetic rate; PPF, photosynthetic photon flux; SFM treatment, sugar-free medium treatment * Corresponding author: Tel.: +86 573 4291108; fax: +86 573 4291108. E-mail address: [email protected] (Y. Xiao). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.02.029
tion, and then transplanted into the field. Thus, the addition of sugar in the medium as an energy and/or carbon source is a requisite for plantlet growth. However, in conventional micropropagation, the loss of plantlets due to microbial contamination and poor growth of plantlets in vitro (Kozai, 1990; Lees, 1994) as well as a low percent survival during the ex vitro acclimatization (Desjardins et al., 1995; Kozai and Zobayed, 2000) is a problem that needs to be solved. Photoautotrophic micropropagation using a sugar-free medium and leafy explants, i.e. sugar-free medium micropropagation, in which plantlets utilize CO2 in the air as an energy and/or carbon source, has several advantages over conventional micropropagation. The advantages include minimized microbial contamination, enhanced photosynthesis, growth and rooting in vitro and increased percent survival ex vitro. A number of reports have been published on the enhanced growth and photosynthesis of plantlets in vitro by using the sugar-free media, and by increasing photosynthetic photon flux (PPF) and the CO2 concentration in the vessel. These reports include the studies by Kozai and Iwanami (1988a) on carnation, Kirdmanee et al. (1995) on Eucalyptus, Niu and Kozai (1997)
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on potato, Nguyen et al. (1999) on coffee and Kubota and Kozai (2001) on tomato. The explants used in these studies were mostly leafy nodal cuttings. When leafy nodal cuttings are used, the number of unfolded leaves of a plantlet determines the multiplication rate of the plantlet. A few studies using sugar-free media have involved the shoot multiplication. These include the studies by Nguyen and Kozai (2001) on banana (Musa spp.) and Erthurk and Walker (2000) on sugarcane, in which shoots excised from a clump were used as explants, because leafy nodal cuttings were not available in these plant species. In this case, the number of shoots per plantlet determines the multiplication rate of the plantlet. When compared with the plantlets grown on a sugarcontaining medium, it was observed that the plantlets grown on a sugar-free medium had a lower dry weight, but a higher multiplication rate in the case of banana, and a lower multiplication rate in the case of sugarcane. The gelling agents, agar and phytagel, were used as the supporting material in both experiments. The supporting material is an important factor for plantlet growth in vitro. Afreen-Zobayed et al. (1999) compared the growth of sweet potato plantlets cultured photoautotrophically in vitro with five different types of supporting material: agar, gellan gum, vermiculite, cellulose plug and Florialite (Nisshinbo Industries Inc., Tokyo, Japan), which is a mixture of vermiculite and cellulose fibers. They observed that sweet potato plantlets exhibited the greatest growth when Florialite was used as the supporting material. Cytokinins such as 6-benzylaminopurine (BA) are a class of plant hormones that play an essential role in plant morphogenesis and have an influence on the formation of shoots and their relative growth rate (Debi et al., 2005). Cytokinins are commonly used in conventional micropropagation to increase the multiplication rate. In the case of photoautotrophic micropropagation, leafy nodal cuttings exhibit rapid growth on the sugar-free medium due to the high net photosynthetic rate; thus, a high multiplication rate can be easily achieved, therefore, cytokinins are not a requisite for the multiplication of leafy nodal cuttings. However, the effects of cytokinins on shoot multiplication in the sugar-free medium have not been determined when shoot clumps are used as explants, as in the case of statice plantlets. The objectives of this study were (1) to investigate the effects of BA concentration and the type of supporting material on growth and multiplication of statice plantlets cultured on a sugar-free medium and (2) to assess the possibility of shoot multiplication of statice plantlets grown on a sugar-free medium based on a comparison with the plantlets grown on a sugar-containing medium. 2. Materials and methods 2.1. Plant material, treatments and culture conditions Clumps each having two to three leafy microshoots, excised from photomixotrophically grown statice (Limonium latifolium) plantlets, were used as explants and cultured for 25 days
for multiplication. The average leaf area, and fresh and dry weight per clump were 647 108 mm2, 461 92 mg and 23 8 mg, respectively. Three clumps were transplanted in each Magenta-type vessel (370 mL, Verde Co., Ltd., Japan) containing 70 mL MS solution with the pH adjusted to 5.7 before autoclaving. This experiment included six sugar-free medium (SFM) treatments: AL, AM, AH, FL, FM and FH, where A and F indicate agar and Florialite substrate and L, M and H indicate three levels of BA concentration, 0, 0.25 and 0.5 mg L1, respectively; and one sugar-containing (30 g L1) medium treatment with a BA concentration of 0.25 mg L1 and agar as the control (Table 1). Ten replications (10 vessels) were used for each treatment. The experiment was conducted twice. The sugar-free explants were cultured on the sugar-free modified MS medium under the following conditions: PPF (50 mmol m2 s1 from days 0 to 7 and 100 mmol m2 s1 from days 8 to 25), CO2 concentration (1500 mmol mol1 in the culture room) and the number of air exchanges of the vessel (NAE: 0.2 h1 from days 0 to 3, 1.8 h1 from days 4 to 7, 2.7 h1 from days 8 to 17, 3.8 h1 from days 18 to 25). NAE was measured by using the method of Kozai et al. (1986) and varied by increasing the number of gas-permeable filter disks (10 mm in diameter, pore size 0.5 mm, Milli-Seal, Nihon Millipore, Ltd., Tokyo), which were attached to the holes on the lid of the vessel (Fig. 1). The control explants were cultured on the sugar-containing MS medium under the following conditions: PPF of 50 mmol m2 s1, CO2 concentration of 400 mmol mol1 in the culture room, and NAE of 0.2 h1. Throughout the culture period, the air temperature and relative humidity in the culture rooms were maintained at 25 1 8C and 80 5%, respectively. The photoperiod was 16 h per day supplied with cool white fluorescent lamps. 2.2. Measurement, calculation and statistical analysis The CO2 concentrations inside and outside the vessels were measured once every 5 days by a gas chromatograph (GC-9A, Shimadzu Co., Ltd., Kyoto, Japan). This measurement was performed when the CO2 concentrations in the culture vessels and the culture room were stable during the photoperiod. Table 1 Description of treatments Treatment code
Sugar (g/L)
Supporting material
BA (mg/L)
Control ALa AM AH FL FM FH
30 0 0 0 0 0 0
Agar Agar Agar Agar Florialite Florialite Florialite
0.25 0 0.25 0.5 0 0.25 0.5
a
The first letters A and F denote agar and Florialite, respectively. The second letters L, M and H denote three levels of BA concentration, 0, 0.25 and 0.5 mg L1, respectively.
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Fig. 1. Effects of two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1) on the growth and multiplication of statice plantlets cultured in Magenta-type vessels containing sugar-free medium on day 25. The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium. For treatment codes, refer to Table 1.
The net photosynthetic rate, Pn (mmol h1 per clump), was calculated in accordance with the method of Fujiwara et al. (1987) using the following formula: Pn ¼
K N VðCout Cin Þ E
where K is the conversion factor of CO2 from volume to moles (0.0407 mol L1 at 26 8C), N the number of air exchanges of the vessel (h1), V the air volume of the vessel (L), Cin and Cout the CO2 concentrations (mmol mol1) inside and outside the vessel under steady-state conditions during the photoperiod and E is the number of clumps per vessel. The chlorophyll concentration was measured by the method of Moran (1982). The appropriate mass of leaves was soaked in 3 mL N,N-dimethylformamide solution for 36 h at 2 8C and absorption of light was measured with a spectrophotometer (U1100, Hitachi, Co., Ltd., Japan) at wavelengths 664, 647 and 603 nm. The chlorophyll concentration including chlorophyll a and b on a fresh weight basis (mg g1) were calculated by the formula of Moran (1982). The number of shoots, leaf area, fresh and dry weight per clump and chlorophyll concentration were measured on day 25.
The relationship between the supporting materials (SM) and BA concentrations in the SFM treatments was examined with an analysis of variance (ANOVA). The relationship between these variables among all the treatments, including the control treatment, was examined with the least significant difference (L.S.D.) test. 3. Results 3.1. Growth and multiplication The results of the plantlet growth and multiplication on day 25 are summarized in Table 2 and in Figs. 1 and 2. Among all the treatments, the dry weight was greatest in the FL treatment, although there was no significant difference in dry weight among the FL, FM and the control treatments. Further, it was lowest in the AH treatment. The leaf area was greatest in the FL treatment, and fresh weight was greatest in the control treatment. The growth values in the SFM treatments varied with the BA concentration levels and the types of supporting material. The number of shoots per clump was greatest in the FH among all the treatments, although there was no significant
Table 2 Effects of two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1) on leaf area, fresh weight (FW), dry weight (DW) and net photosynthetic rate (Pn) of statice per clump (PC) and per shoot (PS) on day 25 cultured in vitro on sugar-free medium Treatment code
Leaf area of PC (mm2)
FW of PC (mg)
DW of PC (mg)
Pn of PC (mmol h1)
Leaf area of PS (mm2)
FW of PS (mg)
DW of PS (mg)
Control AL AM AH FL FM FH
5742 1190c1 6938 1148b 6124 1212c 5328 1311d 8180 904a 7066 1047b 6366 845c
5059 1193a 4074 875bc 3859 916cd 3236 753e 4561 712b 4186 705b 3646 494d
286 71a 248 68b 235 62b 198 44c 301 54a 282 49a 232 37b
48.4 10.2e 20.8 2.3b 17.7 1.6c 13.8 1.7d 25.4 2.7a 22.3 1.8ab 16.4 3.1c
486 135cd 988 192ab 663 216bc 324 114d 1275 407a 777 209ab 401 145cd
121 25bc 228 52a 162 38ab 83 25c 247 75a 194 50a 104 28c
10 3cd 25 5ab 15 3bc 5 1d 32 8a 17 4b 8 2d
ANOVA2 SM BA SP BA
* * NS
* * NS
* ** *
** ** **
* ** *
NS ** **
** ** *
The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium; NS, * and ** indicate nonsignificant or significant at 5 or 1% level of probability, respectively. 1 Mean S.D. separation within columns by least significant difference (L.S.D.) test at 5% level. For treatment codes, refer to Table 1. 2 Analysis of variance was applied to six sugar-free medium treatments with two types of supporting material (SM) and three levels of BA concentration.
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Fig. 2. Number of shoots (A) and chlorophyll concentration (B) of statice plantlets in vitro with sugar-free medium on day 25 as affected by two types of supporting material (agar and Florialite) and three levels of BA concentration (0, 0.25 and 0.5 mg L1). The control is a photomixotrophic culture using sugar- and BA (0.25 mg L1) containing agar medium. For treatment codes, refer to Table 1. Vertical bars represent S.E.
difference in the number of shoots per clump among the FH, FM and the control treatments. Further, it was lowest in the AL, although there was no significant difference in the number of shoots per clump between the AL and FL treatments (Fig. 2A). Among the SFM treatments, the number of shoots increased with increasing BA concentration; but the leaf area, fresh and dry weight and chlorophyll concentration decreased with increasing BA concentration (Table 2; Fig. 2A). The percent loss of the plantlets due to contamination was 0% in all the SFM treatments and 10% in the control treatment. Growth of the shoots for the next generation was greatest in the FL treatment, in which leaf area, and fresh and dry weight per shoot were, respectively, 2.6, 2.0 and 3.2 times higher, compared with those in the control treatment, followed by the AL and FM treatments (Table 2).
Pn was positive and increased with time in the sugar-free medium treatments; however, it was negative and decreased with time in the control during the photoperiod throughout the experiment (Fig. 4). Pn was greatest in the FL treatment, although there was no significant difference in Pn between the FL and FM treatments, and it was lowest in the control among all the treatments. During days 0–25, Pn increased from 0.1 to 25.4 mmol h1 per clump in the FL treatment and from 0.1 to 22.3 mmol h1 per clump in the FM treatment, while it decreased from 3.7 to 48.4 mmol h1 per clump in the control. Among the SFM treatments, a significant difference in Pn on day 25 was observed between the plantlets cultured with Florialite and those cultured with agar when the same BA concentration was used (Table 2; Fig. 4). 3.3. Chlorophyll concentration
3.2. CO2 concentration and Pn The CO2 concentration inside the vessel during the photoperiod decreased with time in all the SFM treatments. On the contrary, it increased with time in the control treatment. As a result, it was lowest in the FL treatment, and highest in the control among all the treatments (Fig. 3).
Fig. 3. Time courses of CO2 concentration inside the vessel and in the culture room. For treatment codes, refer to Table 1. Vertical bars represent S.E.
Among all the treatments, the plantlet chlorophyll concentration was greatest in the FL treatment being 2.5 times that of the control. An increase in BA concentration decreased the chlorophyll concentration in the SFM treat-
Fig. 4. Time courses of net photosynthetic rate (Pn) of statice plantlets at multiplication stage. For treatment codes, refer to Table 1. Vertical bars represent S.E.
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ments. Significant differences in the chlorophyll concentration were observed between the plantlets cultured with different BA concentrations when the same supporting material was used, and between the plantlets cultured with different supporting materials when the same BA concentration was used (Fig. 2B). 4. Discussion 4.1. Growth and multiplication The results clearly showed that statice plantlets grown on sugar-free medium in the FM treatment with a BA concentration of 0.25 mg L1 and Florialite achieved a high multiplication rate with enhanced growth, although there were no significant differences in the number of shoots and dry weight between the FM and the control treatments. Leaf area and chlorophyll concentration of the FM plantlets were 1.2 and 1.7 times higher than those of the control plantlets, although fresh weight of the control plantlets was 1.2 times that of the FM plantlets (Table 2). This result was consistent with those of Mosaleeyanon et al. (2004) who observed that the plantlets cultured in vitro on a sugar-containing medium had reduced leaf area and chlorophyll concentration. Clearly, the plantlets cultured under sugar-free medium micropropagation conditions were healthier than those cultured under conventional micropropagation conditions. In addition, leaf area, and fresh and dry weight per shoot for the next generation in the FM treatment were, respectively, 1.6, 1.6 and 1.7 times higher than those in the control (Table 2). In general, the intrinsic quality of the plantlet, produced in vitro, is of the utmost importance for the successful regeneration of plantlets in any of the micropropagation protocols (Singh and Syamal, 2001). Moreover, the beneficial effect of using sugar-free medium on rooting has been clearly demonstrated in this study; root formation was observed in the SFM treatments from day 5; however, no root formation was observed in the control until the harvest, furthermore, there were a mass of callus at the control shoot base that might increase the plantlet weight. Similarly, the plantlet growth, especially the root growth, was observed to be greater in sugar-free medium than in sugar-containing medium in several species such as potato (Kozai et al., 1988b), Coffea arabusta (Nguyen et al., 1999) and China fire (Xiao and Kozai, 2004). It can be considered that sugar-free medium micropropagation would enable to merge the multiplication and rooting stages into one combined stage and thus simplify the micropropagation process. An appropriate BA concentration in the medium is necessary for balancing both the multiplication rate and quality of the shoots (Pierik et al., 1982; Hempel, 1985). A high BA concentration in the medium could lead to a higher multiplication rate, however, it resulted in a poor quality of the plantlets in the AH and FH treatments. On the contrary, the plantlet relying only on its endogenous hormones could produce high quality shoots, however, it resulted in a lower multiplication rate in the AL and FL treatments.
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The type of supporting material was found to strongly affect on the growth and multiplication rate. The use of Florialite significantly increased the number of shoots, leaf area, and dry weight, and enhanced root induction, compared with the use of agar (Table 2; Fig. 1). Growth promotion by using Florialite was probably due to the fact that a porous material with high air porosity generally maintained a higher dissolved oxygen concentration around the plantlet base than a gelled supporting material like agar (Aitken-Christie et al., 1995; Fujiwara and Kozai, 1995), thus consequently enhanced root and shoot induction of the statice plantlets in this study. 4.2. CO2 concentration and Pn The study revealed that statice plantlets cultured in vitro had the photosynthetic ability at the multiplication stage. The CO2 concentrations inside the vessels during the photoperiod were lower in all the SFM treatments than in the culture room (approximately 1500 mmol mol1) even on the first day, and it gradually showed a significant decrease with time (Fig. 3), indicating a positive Pn and an increase in Pn of the plantlets from the beginning of culture. In addition, the CO2 concentrations in the FL, FM and AL treatments on day 25 were 220, 328 and 351 mmol mol1, respectively, these were approximately 1200 mmol mol1 lower than in the culture room, becoming a limiting factor for photosynthesis during the culture period in these treatments. Therefore, it is considered that increasing CO2 concentration inside the vessel could promote the photosynthesis of in vitro plantlets. A high CO2 concentration inside the vessel can be achieved by increasing the number of air exchanges of the vessel, and by increasing the CO2 concentration in the culture room. On the contrary, the CO2 concentrations inside the vessel in the control treatment during the photoperiod were 10, 15, 22, 33, 60 and 128 times higher than those in the culture room (approximately 400 mmol mol1) on days 0, 5, 10, 15, 20 and 25, respectively, indicating that the CO2 evolution rate of the plantlets was much higher than the CO2 uptake rate of the plantlets on the sugar-containing medium, resulting in a negative Pn throughout the culture period. The high CO2 concentration in the vessel in the control treatment probably resulted from the CO2 produced by the callus at the shoot base (Zobayed et al., 1999), in addition to that produced by the respiration of plantlet. A combination of the low NAE, and the negative Pn throughout the culture period also made a contribution to that. This observation is in agreement with the findings of Nguyen and Kozai (2001) on banana and Xiao et al. (2003) on sugarcane. The negative Pn in the control was probably due to the sugar-containing medium, which suppressed photosynthesis of plantlets in vitro (Desjardins et al., 1995), because plantlets cultured in the sugar-containing medium depend more on the sugar in the medium than on the CO2 in the air, resulting in a low or negative Pn (Kubota and Kozai, 2001). On the other hand, unlike Pn observed in the present study, Lian et al. (2002) observed that statice plantlets showed a positive Pn even under photomixotrophic conditions during the
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rooting stage, which indicated that the photosynthetic characteristics of statice plantlets were different in the multiplication and rooting stages. Similarly, Serret et al. (1996) observed that Gardenia jasminoides plantlets had more developed photosynthetic characteristics during the root induction period than those during the shoot multiplication period. 4.3. Chlorophyll concentration The chlorophyll concentration plays an important role in the absorption of light during photosynthesis. In the present study, it was observed that the chlorophyll concentration of the plantlets was influenced by a few factors, including the presence or the absence of sugar in the medium, BA concentration and the type of supporting material. As shown in Fig. 2, the presence of sugar in the medium reduced the chlorophyll concentration. This, subsequently, resulted in the lowest Pn value in the control (Fig. 4). Capellades et al. (1991) and Azcon-Bieto (1983) observed that the presence of sugar in the medium was attributed to large deposits of starch in the chloroplasts. The carbohydrate accumulation in the chloroplasts may cause a feedback inhibition of photosynthesis, resulting in the decrease in Pn (Hdider and Desjardins, 1994). In addition, the increase in BA concentration led to a decrease in the chlorophyll concentration and Pn (Figs. 2 and 4). A higher BA concentration probably stresses on growth and development of the plantlets, and in some cases, lead to the physiological or morphological disorders such as hyperhydricity and mutation (George, 1996), which may cause a decrease in chlorophyll concentration and a subsequent decrease in the Pn. Moreover, the use of Florialite significantly increased the chlorophyll concentration and Pn. The plantlets cultured with Florialite showed a higher chlorophyll concentration, compared with those cultured with agar (Fig. 2). Similarly, Guimaraes et al. (1999) observed that the chlorophyll concentration was influenced by soil conditions. Previous researchers (AfreenZobayed et al., 1999; Nguyen et al., 1999; Xiao et al., 2003) also observed that the use of Florialite significantly promoted photosynthesis of the plantlets. However, the reason for the increased chlorophyll concentration when Florialite was used as the supporting material is still not clear. A further research is required for a complete understanding of the relationship between chlorophyll concentration and the supporting material. 5. Conclusion In comparison with the multiple shoots produced by conventional micropropagation using sugar-containing medium with BA concentration of 0.25 mg L1 and agar, the multiple shoots produced by using sugar-free medium with BA concentration of 0.25 mg L1 and Florialite (a porous supporting material) resulted in comparable multiplication rate, similar dry weight, higher chlorophyll concentration, higher net photosynthetic rate, better developed rooting system, better shoots and less microbial contamination.
The main highlights of this study include: (1) statice plantlets could be cultured on sugar-free medium in the vessel to produce multiple shoots by providing a PPF of 100 mmol m2 s1, a CO2 concentration of 1500 mmol mol1 in the culture room and the number of air exchanges of the vessel of 3.8 h1, (2) the use of Florialite as supporting material increased the chlorophyll concentration and net photosynthetic rate, and enhanced the root growth, and (3) an appropriate BA concentration in the sugar-free medium for shoot multiplication was found to balance the multiplication rate and plantlet quality. Together, this study demonstrated a reasonable shoot multiplication rate using sugar-free media. It can be considered that the sugar-free medium micropropagation is an effective method for producing a large number of high quality plantlets. Not only leafy nodal cuttings but also shoot clumps could be used as explants for rapid propagation of plantlets in sugar-free medium micropropagation. Acknowledgements The authors are grateful to China Scholarship Council for the financial support and the members of Laboratory of Environmental Control Engineering, Faculty of Horticulture, Chiba University, Japan, for their kind help and support during the experiment. References Afreen-Zobayed, F., Zobayed, S.M.A., Kubota, C., Kozai, T., Hasegawa, O., 1999. Supporting material affects the growth and development of in vitro sweet potato plantlets cultured photoautotrophically. In Vitro Cell. Dev. Biol. 35, 470–474. Aitken-Christie, J., Kozai, T., Takayama, S., 1995. Automation in plant tissue culture. General introduction and overview. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 1–18. Azcon-Bieto, J., 1983. Inhibition of photosynthesis by carbohydrates in wheat leaves. Plant Physiol. 73, 681–686. Capellades, M., Lemeur, L., Debergh, P., 1991. Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro. Plant Cell Tissue Org. Cult. 25, 21–26. Debi, B.R., Taketa, S., Ichii, M., 2005. Cytokinin inhibits lateral root initiation but stimulates lateral root elongation in rice (Oryza sativa). J. Plant Physiol. 162, 507–515. Desjardins, Y., Hdider, C., Riek, J., 1995. Carbon nutrient in vitro regulation and manipulation of carbon assimilation in micropropagation system. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 441–465. Erthurk, H., Walker, P.N., 2000. Effects of rooting period, clump size, and growth medium on sugarcane plantlets in micropropagation during and after transformation to photoautotrophy. Trans. ASAE 43, 499–504. 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. Agric. Met. 43, 21– 30. Fujiwara, K., Kozai, T., 1995. Physical microenvironment and its effects. In: Aitken-Christie, J., Kozai, T., Smith, M.A.L. (Eds.), Automation and Environmental Control in Plant Tissue Culture. Kluwer Academic Publishers, Dordrecht, pp. 319–369. George, E.F., 1996. Plant Propagation by Tissue Culture. Part 2 in Practice, second ed. Butler & Tanner Ltd., Frome, Somerset, pp. 744–745.
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