Introduction

Calla lily, Zantedeschia spp. ‘Southern Light’, is a bulbous plant belonging to the genus Zantedeschia. It is native to Southern Africa and mainly distributed in highland from 1,000 to 2,000 meters with cool temperatures in South Africa, Zimbabwe, and Nigeria (Letty, 1973). It has been widely used as an ornamental plant because it has a unique leaf shape like flowers. Especially, the colored calla has a lot of different colors such as yellow, red, and purple, and is attracting attention as a cut flower, a pot flower, and a garden plant. In the early 1990s, the calla industry grew rapidly, with New Zealand's exports of cut flower increasing by 750% in five years, and efforts to produce bulbs throughout the year have been attempted (Clemens & Eddie Welsh, 1993).

In Korea, colored calla lily had been attempted to introduce from the mid of 1980s, but it was difficult to grow them throughout the country because of frequent occur rence of root rot above 28°C during summer (Funnell, 1993). In the southern part, the rot disease caused severe losses in calla lily production, resulting in discontinuation of production of cut flower of calla lily. On the other hand, In addition, efforts to import and cultivate bulbs are faced with the challenge of not only the high price of importing bulbs (4,000 ~ 5,500 KRW per 2-year-old bulb) but also royalties. Therefore, it is required to establish a mass proliferation system for producing, selling and exporting calla lily. However, it takes at least 2 to 4 years for seed propagation to produce flowering bulbs (Funnell, 1993), during which high temperature can cause rotting disease or dormancy that can adversely affect large-scale propagation. Due to these problems, seed propagation of the calla lily is not suitable for mass proliferation (Ko et al., 2003). Therefore, establishing a large-scale propagation system through virus-free in vitro culture can solve problems such as rotting and dormancy caused by high temperature, as well as enabling large-scale propagation at a time.

In New Zealand and the Netherlands, large-scale proliferation of calla lily through tissue culture had been attempted for a long time. Currently, they are producing large quantities of calla lily plantlets by tissue cultures through OEM in Southeast Asia and selling them to the domestic market at around 1,500 KRW per a plant. In Korea, various studies have been carried out for the mass propagation and stable supply of calla lily plants through tissue culture.

Although shoot primordia from tissue culture can be obtained, they lack value as flowering bulbs without proper roots developed. The purpose of this study was to establish a mass proliferation system of calla lily through tissue culture and to identify proper hormone conditions for early multiple root development on primordia, which would enable to provide large quantity of calla lily bulbs to farmers at low price.

Materials and Methods

Calla lily ‘Black Star’ was imported from New Zealand March 2010. Bulb diameter, length, and weight were 3.1 cm, 1.6 cm, and 18g. Number of axillary buds and bulbs was 7.2 and 1.4.

Size of flower was 5.9 cm and cut flower length was 45 cm. Plants were examined for viral infection at the Korean National Quarantine Station, and then they were planted in gardening soil and grown in a greenhouse at Sangi univeristy, Wonju, Republic of Korea. The gardening soil was formulated with pH 5.5 ~ 7.0, ≤ 1.2 ds/m of electrical conductivity, ≤ 500 mg / L of available phosphate, ≤ 600 mg/L of ammonium (NH₄-N), ≤ 45 ± 5% of water content, and ≤ 70± 5% of water holding capacity. When the axillary buds were about 3 ~ 5 cm, lateral bud with 0.5 cm bulb was removed and used for the experiment. The cut buds were sterilized with a solution containing 5% sodium hypochlorite and tween 20 for 10 minutes on rotating shaker, followed by sterilization with 70% EtOH for 1 minute, and washed with sterilized distilled water for 3 to 4 times. After washing the outermost leaves were removed, the sterilized buds were placed on MS medium supplemented with plant growth regulators.

All media used in this experiment were MS basal medium supplemented with 3% sucrose and 0.8% plant agar, and plant growth regulators as needed. The pH was adjusted to 5.6 ± 0.1 and MS media were sterilized for 15 minutes in an autoclave. Axillary buds were cultured in an incubator set at ambient conditions of 16hr/8hr day/night, light intensity 2,000 Lux, and temperature 25±1°C.

Results and Discussion

Effects of plant growth regulators on callus and shoot induction

In this experiment, the effect of each plant growth regulator on the development of callus and multiple shoots from the axillary buds of calla lily was investigated. Axillary buds of ‘Black Star’ were placed on MS media supplemented with 0, 1, 3, or 5 mg/L of NAA, 2,4-D, BA, or zeatin, and multiple shoot and callus formation were examined after 40-day culture (Table 1).

As a result, 3.0 mg/L of BA induced the highest 63.3% multiple shoot induction from the axillary buds (Table 1). On the other hand, auxins, 2,4-D and NAA induced callus formation, but not shoot primordia. This result is consistent with a previous report in which 2,4-D was suitable for callus formation, but not for shoot induction of calla lily ‘Southern Light’ (Ko et al., 2003). A similar result was also reported from a study of multiple shoot induction of Easter lily (Lilium longiflorum Thunb.) through tissue culture, in which multiple shoots were induced by the combination of cytokinin (kinetin) and auxin (NAA) but not by auxin (NAA) alone (Yazawa, 1978).

Explants of ‘Black Star’ treated with cytokinin began to show milky white and soft callus formation on the cutting surface after 20-day culture, and then hard-looking callus began to show shoot primordia within 10 days. After 40- day culture, 53.3% of explants produced shoots with an average of 5.6 shoots per explant in MS media containing 3.0 mg/L zeatin. On the other hand, 63.3% of explants showed an average of 7.5 shoots per explant with 3.0 mg/L BA treatment (Table 1), which is consistent with previous reports in which the highest rate of shoot formation was observed at concentrations of 3.0 mg/L of BA in calla lily cultivars, 'Sunlight', 'Chiante', and 'Pink Persuasion' (Lee, 1996; Lee & Ko, 2005). To induce callus, low concentration of auxins was more effective than higher concentrations. In ‘Black Star’ callus formation was observed in 50% of explants treated with NAA 1.0 mg/L, 60% with 2,4-D 1.0 mg/L, 36.7% with BA 1.0 mg/L, and 40% with zeatin 1 mg/L, indicating that 1 mg/L of 2,4-D is the most suitable concentration for callus induction of ‘Black Star’.

Effects of mixed plant growth regulators on callus and multiple shoot formation

It was reported that the combination of NAA and BA was effective for plant regeneration from pedicel of amaryllis 'Dazzler' (Ko et al., 2005) and from leaf explants of chrysanthemum (Lee et al., 1999). Also, the mixed treatment of NAA and BA markedly increased shoot regeneration rate and number of shoots per explant from the leaves or stipules of strawberry (Choi et al., 1998). Based on these results, it was investigated whether a combination of NAA and BA was suitable for callus induction and shoot regeneration of 'Black Star'. Callus and multiple shoots were formed in all combinations of NAA and BA in 'Black Star' (Table 2; Fig. 1).

Callus induction tended to high on MS medium with 1:1 ratio of auxin and cytokinin. The highest callus induction of 83.3% was observed in treatment with 1.0 mg/L of both NAA and BA (Table 2). Auxin NAA alone also induced callus formation, but it failed to induce shoot regeneration as shown in both Table 1 and Table 2. The highest shoot regeneration of 100% was observed on MS medium with the combination of 1mg/L of NAA and BA, in which the average number of shoots per an explant was 14.7 with an average length of 4.1 cm (Table 2), indicating that this combination is most suitable not only for callus formation but also for shoot formation. Previously, it was reported that a number of shoots per explant were higher when callus was differentiated in other calla lily cultivars, ‘Chiante’ and ‘Pink Persuasion’ (Lee, 1996). In this study, however, callus formation prior shoot induction did not show clear correlation with multiple shoot formation (Table 2).

Effect of auxin on root induction from primordia of ‘Black Star’

It is known that shoots from tissue culture of calla lily did not have agricultural value without enough roots developed because they could rarely survive in soil. Therefore, although the condition for shoot regeneration from ‘Black Star’ was established through tissue culture, it is necessary to develop roots prior to transplanting to the soil. It was found that shoots of ‘Black Star’ generated 1 ~ 2 roots in 40 days, which is consistent with previous reports (Lee 1996). Because shoots with 1~2 roots were not healthy enough to produce proper small bulbs, it is required to develop decent roots from shoots before planting in soil. Since plant hormone auxin is well known for adventitious root induction from shoots, we examined whether auxin could be used for root induction from shoots. Three different auxins, NAA, IAA, and 2,4-D were used for root induction from shoots of ‘Black Star’. A long tap roots were developed first from shoots of ‘Blast Star’ on MS media containing 0.1, 0.5, 1.0, or 2.0mg/L of IAA or 2,4- D, and then multiple lateral roots were generated. Although both IAA and 2,4-D induced root development, IAA was more effective in root induction than 2,4-D. The highest root induction (average of 4.9 per explant) was observed in MS medium with IAA 1.0 mg/L (Table 3). Also, we expected that NAA could result in decent root development, but it failed to induce adventitious roots from shoots of calla lily ‘Black Star” (Table 3; Fig. 2).

NAA has long been used as a very effective root inducer in many other plant species, Cucurbita foetidissima and Asclepias syriaca (Lan & Paek, 2000), chrysanthemum (Lee et al., 1999), strawberry (Choi et al., 1998), cyclamen (Eun et al., 1995), Neoregeria carorinea 'Tricolor' (Joung, 1995), and lilies (Waichiro and Tadashi, 1989). However, NAA did not induce roots from shoots in calla lily ‘Black Star’ as in other plant species. This is not a first observation that NAA failed to induce root development in calla lily. Previously, it was reported that NAA also failed to develop adventitious roots from shoots in other calla lily cultivars ‘Sunlight’, ‘Chiante’, and ‘Pink Persuasion’ (Lee et al., 2005). We found the highest root induction from shoots treated with IAA 1.0 mg/L (Table 3), which was consistent with previous reports that the same concentration of IAA (1.0 mg/L) was most effective in root induction of ‘Sunlight’ and ‘Chiante’ (Lee, 1996). On the other hand, it was reported that IBA was more effective for root induction in calla lily ‘Pink Persuasion’ (Lee, 1996), suggesting that selection of proper auxin is important for better root induction of calla lily.

적 요

본 연구는 기내번식시 auxin과 cytokinin의 단용 또는 혼 용처리가 유색칼라 측아의 다아체분화에 미치는 영향을 알 아보고, 이를 통한 유색칼라의 대량증식 체계화를 위하여 수 행되었다.