Introduction

The genus Lilium includes nearly 110 species that are dispersed in the northern hemisphere, Europe, South-East Asia, and North America (Comber 1949; De Jong 1974; Matthews 2007). Lilium is considered as one of the important commercial crop worldwide, due to its extensive use as cut flowers, potted plant, and as well as a garden plant. For these reasons it is ranked among top commercial plants in the world floriculture trade, and many breeding approaches have been practiced in various Lilium species that resulted into more than ten thousand cultivars (Younis et al. 2014). But, the genus Lilium is facing a serious threat of genetic erosion like other endangered plant species, due to habitat fragmentation, inbreeding, weaker immune system against pathogens and pests, and climate change. Also, the lily bulbs have to face different storage disease like, fusarium, viruses, gray mold and different physiological disorders are real threat to Lilium germplasm. This may leads to the loss of the bulbs, poor quality flowers and virus infection. Therefore, Lilium germplasm required careful attention for its long term conservation and preservation.

The genotypes resources of Lilium cannot be preserved in seed banks under low-temperature as they are propagated vegetatively. Also, using seeds as preservation material in Lilium is not recommended due to its unique and heterozygous combinations owing to that would lead to segregating its genetic combinations. Furthermore, to maintain bulbs in the field or greenhouse conditions by planting, harvesting and storing every year, require high inputs on labor, space and disease risk or extreme weather (Towill 1988; Withers 1991). Moreover, in vitro conservation (tissue culture) is also not possible as it is susceptible to contamination and also there are chances of soma-clonal variations (Scowcroft 1984).

Cryopreservation is a long-term low temperature storage technique to preserve any biological material (living cells and tissues) without deterioration for at least several thousands of years (Tsai and Lin 2012). Recently, cryopreservation approach has become one of the most effective techniques for long term storage of plant germplasm (Wang et al. 2012). In lilies, this technique was first time employed in Lilium speciosum, in which two steps freezing was followed for preservation of shoot tips (Bouman and de Klerk 1990). This approach exemplifies an appropriate method of the lily meristem and shoots tip preservation for long time. Several reports have proved that the meristem of Lilium have been preserved effectively by using vitrification procedure (Bouman and de Klerk, 1990; Yin et al. 2014). Other reports have indicated that many Lilium species have been successfully cryopreserved through encapsulation dehydration and vitrification methods (Chen et al. 2011; Yi et al. 2013; Yin et al. 2014). Dropletvitrification was first time successfully used for lily in which meristem from adventitious buds were cryopreserved (Chen et al. 2011).

The aim of this study was to establish a reliable, consistent and efficient protocol for long term cryopreservation of different Lilium germplasm.

Materials and Methods

Planting material

In this study, twenty two Lilium species were used. Among the species used, 7 belongs to section Sinomartagon, 4 to Leucolirion, 3 to Martagon, 2 to Lilium and other species belong to different section that are presented in Table 3.

Culturing of plant material

Murashige and Skoog (MS) medium was used as the basal medium. This medium contains sucrose 3% and pH was adjusted 5.8 then autoclave at 121℃ for 15 min. The preculturing was done at 23 in growth chamber maintained with 16 hrs photoperiod. Adventitious buds were produced on bulb scales after 30 days of culturing on MS medium supplemented with 0.1 mg • L⁻¹ IAA, 0.1 mg • L⁻¹ zeatin, 2.2 g • L⁻¹ phytagel and 3% sucrose. The bulb scales were sub cultured on MS medium with 0.2 mg • L⁻¹ zeatin, 0.15 mg • L⁻¹ IAA, 30 g • L⁻¹ sucrose, 2.7 g • L⁻¹ phytagel, and 1 g • L⁻¹ charcoal. For development of in vitro bulbs, MS medium supplemented with 0.2 mg • L⁻¹ zeatin, 0.15 mg • L⁻¹ IAA, 1 g • L⁻¹ charcoal, 2.7 g • L⁻¹ phytagel, and 7.5% sucrose.

Cold-hardening and pre-culture

Cold-hardening of bulb scales was carried at 4℃ for one week under 2000 lx light condition maintained for 16 hrs photoperiod at 23℃. Then, pre-culturing of apical shoot tips were carried out overnight in liquid MS medium supplemented with sucrose 0.3 M. Then, these were placed in 0.7 M sucrose for 10-12 hours under 2000 lx light conditions. Different steps followed in Lilium cryopreservation are presented in Fig 1.

Loading and dehydration process

The osmo protection of pre cultured shoot tips was done in loading solution at 23 for 40 and 60 mins. The loading solutions (LD1), having basal MS medium supplemented with 35% plant vitrification solution 3 (PVS3) were used (Table 1). The shoot tips were dipped in vitrification solution (PVS3) for 4-6 hrs. For droplet vitrification process, shoot tips were placed on sterilized aluminum foil strips having a droplet of vitrification solution. After this, these strips were immersed carefully in liquid nitrogen by using forceps. After immersion step, these strips were quickly moved into 2 mL cryo-tubes, which were plunged immediately in liquid nitrogen (Fig 2).

Thawing, unloading and plant regeneration

The samples were placed in liquid nitrogen for 24-36 hrs. Then, the foil strips were plunged for 30 seconds in an unloading pre-heated solution (40℃) that has 0.8 M sucrose. After this second unloading solution (5 mL) was added at room temperature. Incubation of shoot tips was done for 30 min at room temperature to assist unloading step. This step facilitates in cleaning of shoot tips from vitrification solution. After thawing and unloading steps, shoot tips were transferred in regeneration MS medium supplemented with 0.2 mg • L⁻¹zeatin, 0.15 mg • L⁻¹ IAA, 15 mg • L⁻¹putrascine, 0.05 mg • L⁻¹GA₃, 2.2 g • L⁻¹ phytagel, and 30 g • L⁻¹ sucrose and cultured at 23℃ for 15 days under dark conditions. Then finally they were placed at 23℃ under 2000 lx light intensity maintained for 16 hrs photoperiod.

The survival rate was counted after every 2 weeks of cryopreservation. Then, the regeneration rate was counted after 7 to 8 weeks of cryopreservation by counting the number of shoot tips that were differentially swollen and green.

Morphological analysis

For comparison of morphological traits between cryopreserved and non-cryopreserved bulbs, various growth parameters were recorded. After breaking dormancy 4℃ bulbs were planted in greenhouse at Kyungpook National University? (National Agrobiodiversity Center) in 2012-2013.

Different characteristics like: flower, stigma and pollen colors, spots on flowers, flower’s direction etc.

Results and Discussion

The survival rate and regeneration rate in cryopreserved Lilium germplasm were recorded during year 2010-2012. The data are presented in Table 2 that depicted that in 2010, total 51 accessions were cryopreserved with total 486 treatments. The results demonstrated that 65.3% samples were survived and 57.3% plants were regenerated. During year 2011, 59 accessions were cryopreserved with total 608 sample treatments. Results showed that 66.4 and 58.3 percent survival and regeneration ratio respectively were counted. Almost similar trend of survival and regeneration percentage was observed in 2012 with total 50 accession cryopreserved (Table 2).

In three years, in total 160 accessions with 1521 sample treatments were conserved by using cryopreservation and 63.3% survival rate was recorded with 56.7% regeneration rate in different Lilium germplasm.

When regeneration rate in different Lilium species were compared it was found that L. hansonii section Martagon showed the highest regeneration (86%) (Table 3). Whereas, L. daviddii section Sinomartagon ranked second with 85% regeneration rate followed by L. regale section Leucolirion. The least regeneration rate 38% was observed in L. henryi (Table 3). Different Lilium hybrids were also cryopreserved and results depicted that Asiatic and Oriental hybrids showed 43 and 73 percent respectively regeneration rates.

To make cryopreservation more efficient, shoot tips are usually pre-cultured medium with sucrose to reduce the chances of desiccation and to improve cryo-tolerance. It is important to avoid intra-cellular freezing that can be possible in liquid nitrogen step (Sakai 1995). It was reported that preculturing in medium supplemented with sucrose (0.3 M) for 24-48 hrs was optimal to produce the maximum shoot regeneration rate in cryopreserved Lilium shoot tips (Matsumoto et al. 1995; Chen et al. 2011). In Lilium shoot tips, only preculture was not proved effective to induce liquid nitrogen tolerance, that necessitate the osmo-protection step by use of a loading solution (Matsumoto et al. 1995). In this study, the osmo protection of pre cultured shoot tips was done in loading solution at 23℃ for 40-60 mins.

The loading solutions, having basal MS medium supplemented with 35% plant vitrification solution were used that proved efficient in cryopreservation. This study results are consistent with other reports on Lilium (Matsumoto et al. 1995; Chen et al. 2011) as well as some other bulbous crops i.e. Allium (Kim et al. 2009; Kim et al. 2012), Galanthus (Maslanka et al. 2013). During cryopreservation, the cooling rate also plays a key role as the ultra-rapid freezing aids to avoid freezing of intracellular that leads to a vitrified state during freezing step (Fahy et al. 1984).

Morphological characteristics of non-cryopreserved and cryopreserved Lilium germplasm were recorded and presented in Table 4. The results showed that no changes were observed in growth and morphology of both germplasm except for a slight difference in plant height (Fig. 3). These results are similar with the finding of many researchers in different plant such as Doscorea floribunda (Ahuja et al. 2002), Potato (Schafer-Menuhar et al. 1997), and date palm (Fki et al. 2011). Chen and his co-workers demonstrated that during cryopreservation, the preserved meristems could produce true-to-type plants similar to the non-treated germplasm (Chen et al. 2011). It is important to note that callus phase before shoot formation should be avoided as it can increase the potential of genetic variability (Haskins and Kartha, 1980).

Conclusions

This technical report elaborates the cryopreservation protocol for of Lilium germplasm resources and this protocol can be adopted for meristem cryopreservation in other plant texa. This protocol can further be explored for its potential uses for virus eradication and long term cryopreservation of more Lilium germplasm. The morphological study of Lilium germplasm regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation. We this cryo-collection will be available and useful to curators or breeders of Lilium and this cryobank will also facilitate the conservation and international exchange of Lilium germplasm.