Introduction

Colored Zantedeschia (calla lily) is perennial reproduce asexually herbaceous bulb and the genus belongs to Zantedeschia Spreng of Araceae (Singh et al. 1996). They have large and showy flower spathes with white-spotted leaves and are generally cultured as ornamental plants and cut flowers. Colored Zantedeschia propagated by division in spring. Small rhizomes that is overwintered in pots under cover can be literally cut up into sections, and each of them has a visible bud. Large overwintered clumps in the garden can be separated by lifting the plant before there are much top growth, and chopping through the roots with a spade and dividing into smaller sections. However, large-scale production of the plants is limited by tuber propagation, which is the traditional reproduction method as low propagation coefficient. That is because the plants will be hurt during division propagation, and the wound can be easily infected by some fungal and bacterial infection during the cultivation process. In recent years, tissue culture has become a major approach to overcome the shortage of traditional reproduction method in colored Zantedeschia seedling production.

In recent years, some researches on tissue culture of Zantedeschia, and they gained the plantlets in vitro successfully (Chang et al. 2003; Fan et al. 2005; Wang et al. 2005; Wu et al. 1999). And the researches have mostly focused on hormones (Huang et al. 2010; Janowska 2013; Jiang et al. 2010; Kozłowska et al. 2007), environmental control of tissue culture (Jao et al. 2005; Lu et al. 2014; Que et al. 2009; Veatch-Blohm et al. 2012) and cultivation technology of transplant and training (Arhip et al. 2015; Han et al. 2015; Li et al. 2011). However, there were many problems during the cultivation process, such as high pollution rate, low propagation officiency, unsatisfactory rooting effect, and lower transplanting survival rate, and etc. (Li et al. 2001; Zhou and Wu 2006).

Sugar-free culture had a positive function on improving the situation of avoiding microbial pollution in tissue culture (Kozai et al. 1997). Many researchers emphasized that there were obvious accelerations on the growth of Oncidium aloha callus (Huang and Li 2010 ) and rice plantlets ( Seko and Nishimura 1996) under sugar-free or less sugar culture. Other researchers also found that different sugar sources (Liu et al. 2013) and sugar concentrations had significant impact on the plantlets vegetative growth (Ibrahim and Arafa 1994) of Dendrobium casiflake (Xiu et al. 2012) and Narcissus (Chow et al. 1992). And sugar was still necessary for Oncidium explants in vitro under CO₂ enrichment (He et al. 2003). Only few researches have focused on the effect of environmental control and sugar-free on the growth of colored Zantedeschia. Qu et al. (2004) studied t he effect of CO₂ concentration as the single factor on the growth of colored Zantedeschia in vitro.

Based on the previous studies, this study investigated the effects of different sugar concentrations on the growth of colored Zantedeschia in vitro under certain CO₂ concentration and explored the decreasing level of sugar concentration, as well as the possibility on utilization of sugar-free that maintains the normal growth of colored Zantedeschia in vitro.

Materials and Methods

Materials, treatments and culture conditions

The tubers with buds of colored Zantedeschia (Z.antedeschia elliotiana, provided by Henan Linying Zitian Flower Seedlings Limited Company.) were induced to form rootless shoots as follows: shoots were micropropagated in vitro in 100 mL erlenmeyer flasks containing 40 mL Murashige and Skoog (MS; Murashige and Skoog 1962) medium with 2.0 mg·L^-1 6-benzyladenine (6-BA), 0.5 mg·L^-1 1-naphthaleneacetic acid (NAA) and 20 g·L^-1 sugar. All media were adjusted to pH 5.3. All the treatments were kept at 25 ± 1℃ under white fluorescent lamps at a light intensity of 45 μmol·m^-2·s^-1 with a 12 h photoperiod. Rootless shoots with two leaves, and uniform height (2.5 cm) were chosen as the test materials after 30 d.

This experiment included four levels of sugar concentrations: 0 (sugar-free culture treatment), 10, 15 and 30 g·L^-1. And shoots were cultured in vitro in 500 mL erlenmeyer flasks on MS medium containing 0.2 m g·L^-1 6-BA, 0.5 mg·L^-1 NAA and 2 g·L^-1 Gellangum. All media were a djusted to pH 5.8. There were 10 shoots in each erlenmeyer flask. Each treatment was repeated three times. And the 30 g·L^-1 treatment was treated as control.

Measurement, calculation and statistical analysis

All the treatments were cultured for 60 days, and the following parameters were assessed: plant height, leaf number, maximum leaf length, leaf width (i.e., leaf expanded surface of the third leaf from the apex), root number, maximum root length, root vigor, shoot and root fresh/dry weight (FW/DW) (total and separately). Chlorophyll content of the third leaf from the apex was measured with a chlorophyll meter (MINOLTA SPAD-502, Japan) and expressed as the soil plant analysis development (SPAD) value. Root length was measured from the base of attachment of the root to the stem of the root tip of the longest root harvested from the plantlets. To measure DW, shoots and roots were dried separately at 105℃ for 30 min then at 60℃ for 48 h in a thermostat, or until constant weight. The root vigor of plantlet in vitro was detected by the triphenyl tetrazolium chloride (TTC) reduction methods described by Ryssov-Nielson (1975) and Trevors (1984).

Analysis of variance (ANOVA) was performed with DPS software (Refine Information Tech. Co., Ltd., China) and significant differences between means were determined by Duncan’s multiple range test (DMRT) at P ≤ 0.05.

Results

External morphological characteristics

The results of the effects on the growth of plantlets in vitro of colored Zantedeschia are shown in Table 1. The plant height, leaf length, leaf width and root length of plantlets in vitro increased as the sugar concentration increased under CO2 enrichment, and the above parameters were significantly higher compare to 0 g·L^-1, 10 g·L^-1 and 15 g·L^-1 treatments under the 30 g·L^-1 treatment. However, the root number and rooting rate of plantlets in vitro were the largest under the 0 g·L^-1 treatment (sugar-free culture treatment).

Fresh weight, dry weight and dry mass rate of plantlets in vitro

Table 2 showed that the fresh and dry weight of plantlet shoots under CO₂ enrichment increased with the rise of sugar concentrations. The fresh and dry weight of plantlet roots and total plantlets showed the same trend to that of shoots except the 0 g·L^-1 treatment, they all increased as the sugar concentration increased under CO₂ enrichment. The fresh and dry weight of plantlet root and total plantlet fresh weight under 0 g·L^-1 sugar were obviously higher than the 10 g·L^-1, 15 g·L^-1 and 30 g·L^-1 treatments, and the dry weight of total plantlet (114.3 mg) under 0 g·L^-1 sugar was the second highest only after 30 g·L^-1 sugar (123.5 mg) and there were no significant differences between the groups.

The dry mass rate of plantlets in vitro under CO₂ enrichment had a similar trend to the fresh and dry weight of plantlets in vitro, and the dry mass rate of plantlets root under 0 g·L^-1 sugar was also significantly higher than the others.

Expression patterns of leaf chlorophyll content (SPAD value)

The leaf chlorophyll content (SPAD value) of plantlets in vitro under 30 g·L^-1 sugar was significantly highest (33.50 a) among the treatments. However, there were no significant differences among the treatments of 0, 10 and 15 g·L^-1 (Fig. 1).

Root vigor of plantlets in vitro under <span class="chem-struct">CO₂</span> Enrichment

The root vigor of plantlets in vitro also had a similar trend to the dry mass rate of plantlets in vitro (Fig. 2). The root vigor of plantlets in vitro under the 0 g·L^-1 treatment was obviously higher (279.23 a) than the treatments of 10 g·L^-1 (261.32 c), 15 g·L^-1 (266.81 b) and 30 g·L^-1 (268.35 b). The root vigor of plantlets in vitro increased as the sugar concentration increased among the treatments of 0, 10 and 15 g·L^-1.

Discussion

The heterotrophic and mixotrophic growth of plantlets in vitro were obviously influenced by the concentration of sugar and CO₂ used as carbon source in traditional tissue culture (Yoon et al. 2009). The CO₂ concentration in the airtight container would decrease gradually with consumed by plantlets during tissue culture, the low CO₂ concentration couldn't meet the needs of plantlets photosynthesis at last, and it would restrain the growth of plantlets in vitro (Fujiwara et al. 1987; Kozai et al. 1986). Huang et al. (2010) found that the suitable sugar concentration for adventitious buds inducing culture in Oncidium was 4%, neither 3% nor 2%. When the concentration of sugar increased from 3% to 6%, the frequency of corm induction increased and significantly heavier corms of Watsonia were produced (Ascough et al. 2008). This study found that the plant height, leaf length, leaf width, root length, fresh and dry weight of shoot, and leaf chlorophyll content were the optimal in 30 g·L^-1 treatment. It indicated that sugar was benefit to the aerial part of plantlet growth in vitro of colored Zantedeschia, and the aerial part of the plantlet growth in 30 g·L^-1 treatment was better than other treatments. Hdider and Desjardins (1994) have found a similar result that sugar as a particular carbon metabolism could promote the growth of micropropagated plantlets. However, the root number, rooting rate and root vigor in 30 g·L^-1 treatment were lower than 0 g·L^-1 (sugar-free culture) treatment under CO₂ enrichment. This study suggested that exogenous sugar might disturb the normal sink-source relationship on the whole microplantlet basis, the disordered sink-source relationship partly restrained the photosynthetic capability, and the root development of plantlets in vitro was further affected by the poor photosynthetic capability. This opinion was similar to the conclusions of Hdider and Desjardins (1994), and the Desjardins et al. (1995).

It had been proved that photoautotrophic micropropagation was benefit to improve productivity by increasing the CO₂ concentration during production process (Kozai et al. 2005). And the photoautotrophic micropropagation had wide practical applications on ornamental and other horticultural crops (Norikane et al. 2013). Xiao and Kozai (2006) found that sugar-free medium micropropagation was an effective method for producing a large number of high quality statice plantlets. And 350 ± 5 0 μl·L^-1CO₂ enrichment could promote the growth of petunia, chrysanthemum and tomato plantlets in vitro (Qu et al. 2009). Qu et al. (2004) indicated that sugar-free culture was beneficial to colored Zantedeschia roots under 1000 - 1200 mg·L^-1CO₂ enrichment. And the increasing CO₂ content seemed necessary to increase the beet growth rate (Manderscheid et al. 2010). There were different adaptability and reaction among plant varieties under sugar-free culture. This research found that the root number, rooting rate, fresh and dry weight of root，and root vigor were the best in the 0 g·L^-1 (sugar-free culture) treatment, and we found that the sugar-free culture treatment of plantlets in vitro of colored Zantedeschia was effective to enhance the transplanting survival rate and shorten the seedling reviving time, and more stronger plantlets were produced by this method. The results also indicated that the sugar-free culture supplied with CO₂ could promote the photosynthesis and root development of colored Zantedeschia in vitro. This results are consistent with a previous study that showed that plantlets in vitro grew Photomixotrophically and could derive their carbon requirements from atmospheric CO₂ fixation (Yue et al. 1993). However, the fresh weight, shoot length, root length and leaf area of plantlets in vitro of colored Zantedeschia in the sugar-free culture treatment were poorer than that of other treatments, which was different from the conclusion of Nguyen et al. (1999). Poudel et al. (2008) found t hat there were significant difference on the growth and morphogenesis between different genotype grapes under the same external environmental condition. So it might be individual differences of genotype and physiological property among different plant varieties.

In general, this study indicated that the carbon sources varied to meet the growing requirement of the plantlets in vitro of colored Zantedeschia in different growing stages. The 30 g·L^-1 treatment was beneficial to the shoots growth, multiplication and seedling culture of the plantlets in vitro of colored Zantedeschia, and sugar-free culture was suitable for rooting culture and transplanting of the plantlets in vitro of colored Zantedeschia.