The establishment of a slow-growth conservation system in vitro for two wild lily species

There are abundant resources of wild lily in China. To achieve a sustainable use for these resources, a slow-growing preservation system should be well established. Therefore, to get an optimal storage condition, tube seedlings of Lilium davidii and Lilium longiflorum were used as explants cultured on nine different media at 24 and -2°C separately, which included: 1/4 or full Murashige and Skoog medium (MS) with 3, 6 or 9% sucrose, 1/4 or 1/2 MS with 1.0 or 3.0 mg/L abscisic acid (ABA). During the storage, the spout growth, and multiplication rate were measured every month. Results show that the growth restriction degrees for both species were related to the four factors (MS content, the concentration of sucrose and ABA, temperature). The inhibitory effect was more obvious at -2°C. At 24°C, the medium should be replaced every three to four months; otherwise the contamination rate would rise with the extension of storage time. At -2°C, the 1/4 MS with 9% sucrose or 3.0 mg/L ABA (M3A) were more effective, especially the latter one. The M3A played the greatest inhibitory effect on height growth of the two lily species, on which both species had been conserved on the original medium for more than 15 months. The tube seedlings conserved for 15 months could turn to normal plantlets after re-growth for one month which showed no obvious difference in morphology. 87.5% plantlets planted in peat could survive. In conclusion, the promising conservation condition was M3A at -2°C which was suitable for mid-long term preservation of lily germplasm resources.


INTRODUCTION
There are rich resources of wild lily species in China (Fu et al., 2002).In recent years, lily resources' habitats have been suffering more and more serious destruction, and many wild species have become endangered species.So, germplasm conservation is very important as a source of genetic variation for breeding, research, and to prevent rare species from becoming extinct.Currently, the main ways of lily germplasm protection are the use of seed storage, field planting and tissue culture.Using seeds for preservation would break up unique genetic combinations, and the features would show segregation.Growth cycle from seed to flowering is too long, in addition to Lilium formolongi, normally takes two to four *Corresponding author.E-mail: gxjia@bjfu.edu.cn.Tel: +86 135 2161 5634.
years.Field planting is costly and vulnerable to natural environment.Collections of bulb crops are usually maintained in the field or greenhouse by yearly planting, harvesting and storing the bulbs, leading to high investments of labor and space and risking losses caused by diseases (Towill, 1988;Withers, 1991).The maintenance of a germplasm collection in vitro has several advantages over storage of bulbs, as it requires small amounts of space and reduces the risk of losses caused by diseases (Towill, 1988).Since 1975, Henshaw and Morel proposed the preservation of plant germplasm in vitro for the first time; this technology had been highly valued (Villalobos and Engelman, 1995).
There are three types of in vitro conservation: normal in vitro culture preservation, cryopreservation and slow growth in vitro conservation.At room temperature (normal preservation), germplasm materials need frequent subculture (Peschke et al., 1992).In order to solve this problem, the slow growth conservation and cryopreservation (that is, storage in liquid nitrogen, -196°C) have been developed.The basic work to achieve longterm preservation is the establishment of in vitro regeneration system.The greatest advantage of these two preservation methods is maintaining the material at a state of continued growth, while some material can be removed for other research (Mantell and Smith, 1983).Lily meristems have been successfully cryopreserved, by slow freezing (Bouman and De Klerk, 1990) and vitrification (Matsumoto et al., 1995).However, cryopreservation requests high experiment technique, and the system is complicated operation.
Low temperatures are often used to create conditions of minimal growth (Grout, 1991;Withers, 1991).In the commercial production of bulbs, the temperature of -2°C is commonly used to store lily bulbs (Beattie and White, 1993).Bulbs of Asiatic hybrids, Oriental hybrids and Lilium longiflorum could be stored in moist peat at temperatures between -1.5 and -2°C for one year (Beattie and White, 1993;Boontjes, 1983).Little research has been done in lily conservation in vitro.Right now, Bonnier andVan Tuyl (1997), andChen et al. (2006) had carried out some relevant research.The preservation effects were different on different species and cultivars.Collections in vitro (Asiatic hybrids, Oriental hybrids) survived 28 months of storage under 25 and -2°C, whereas at -2°C, L. longiflorum and Lilium henryi survived six months of storage, but died during prolonged storage (Bonnier and Van Tuyl, 1997).Storage at a lower temperature than -2°C would further minimize growth conditions and would therefore increase maximum storage duration.However, freezing tolerance of lily should be increased to reduce the risk of freezing injury (Bonnier, 1997).Freezing tolerance can be increased by cold-acclimatization, abscisic acid, partial dehydration, or low atmospheric pressure (Halloy and Gonzales, 1993;Hincha, 1994;Lång et al., 1994;Mantyla et al., 1995).In general, -2°C is the lowest temperature for lily preservation.
The adjustment of medium contents (the type and composition of the base medium, regulators.carbon source type and content) can prolong the storage time.Lowering the content in mineral elements of culture medium [1/2 or 1/4 Murashige and Skoog medium (MS)] and/or high sucrose concentration (6 to 9%) gave reduction in sprout growth of lily, thus extending the storage duration (Bonnier and Van Tuyl, 1997;Chen et al., 2006).In other crops, such as the protocorms of Cymbidium sp., Dendrobium chrysanthum and ochreatum, high sucrose concentrations allowed the conservation at low temperature (Homes et al., 1982;Tandon and Sharma, 1986).
The growth inhibitors also play some effects on slow growing.-5pyrimidinemethanol) is a substituted pyrimidine with potent plant growth regulatory activity, specifically inhibiting a series of oxidations in plant tissue.The potato microplants in vitro could be persisted through a 16months culture period (Debabrata et al., 2001).The addition of mannitol significantly reduced the growth of Colocasia esculenta and Xanthosoma brasiliense shoots (Staritsky et al., 1985).However, the growth of lily shoots cultured in medium supplemented with 10 to 50 gL -1 mannitol or 10 to 40 mgL -1 trimethy1ammonium (CCC) was not significantly restricted (Chen et al., 2006).
ABA is a growth retardant which was used to reduce the growth of shoots of potato, grape, persimmon and date plum (Westcott, 1981;Roca et al., 1982;Li et al., 1992;Ai and Luo, 2004).However, these authors indicated that ABA was detrimental to some varieties.For Lilium oriental hybrids 'Siberia', in vitro shoots could be conserved in the media supplemented with 1.0 to 3.0 mgL -1 ABA for more than nine months at normal temperature of 20±1°C (Chen et al., 2006).
Up to now, the method of slow-growth conservation in vitro has been used to conserve germplasm of potato (Solanum tuberosum) (Roca, 1975), sweet potato (Ipomoea btatas) (Sigueńas, 1987), Andean root and tuber crops (Toledo et al., 1994), and lily (Lilium L.) genotypes (Asiatic hybrids, Oriental hybrids, L. longiflorum and L. henryi) (Bonnier and Van Tuyl, 1997); therefore, it is a very promising method.However, this method also has some problems yet to be resolved.On one hand, there are rich lily resources in China, and some resources are endangered, it is necessary to preserve resources in vitro.Furthermore, different genotypes had different requirements on storage conditions.Right now, there was no report about the effect of MS content and ABA at -2°C on lily conservation.In this paper, the MS content, the concentration of sucrose and ABA combined with low temperature (-2°C) and normal temperature (24°C) were used for Lilium davidii and L. longiflorum.The effects of culture conditions on bud growth and further survival rate after planting in soil were investigated.

Establishment of in vitro regeneration system
L. davidii and L. longiflorum were used for experiment with bulb scales between 14 and 16 cm.Clean scales were rinsed with tap water for 40 min, then surface-sterilized for 30 s in alcohol, immersed in 20%(v/v) NaOCl for 15 min, washed three times with sterile water in the clean benches.Bulb scale segments of about 5 × 5 mm were cultured on MS-medium (Murashige and Skoog, 1962) with 3% sucrose, 6 gl -1 purified agar (Sigma), 1.5 mgl -1 6benzylaminopurine (6-BA) and 0.2 mgl -1 naphthaleneacetic acid  (NAA) at a pH of 5.8 in Erlenmeyer flask with a volume of 80 ml.
The bottle was wrapped around with a polyethylene film.The scale segments were cultured with their abaxial side on the medium at 24°C with 16-h day light at an intensity of 1500 lx.After nine to 12 weeks, regenerated plantlets were excised and given various treatments in Erlenmeyer flask with a volume of 80 ml containing 40 ml medium for in vitro conservation experiments.

Treatments
Tube seedlings of L. davidii and L. longiflorum were used as explants cultured on two groups of media (Tables 1 and 2) and the fifth medium was the control.The sprout growth was measured before treatment.Table 1 shows five combinations of basal media and sucrose concentrations.The materials were divided into two groups placed on the same media.One-half of the tube seedlings were placed at 24°C (16 h/d, 1500 lx).The other one-half of the tubes were placed at 2°C for four weeks acclimatization, and then transferred to -2°C in the dark.45 plantlets were used for each treatment with three replications.Five combinations of basal media plus different concentrations of ABA are listed in Table 2.The tubes were placed at 2°C for four weeks acclimatization, and then transferred to -2°C in the dark.45 plantlets were used for each treatment with three replications.

Sprout growth
The seedling height was measured every month.At normal temperature, the increment of sprout growth was obvious in the first three months, while under the low temperature, it was not conspicuous.The majority of sprouts had reached the top of the tube at normal temperature after five to six months.Therefore, we selected two periods of three and six months for detailed analysis at 24°C.The data were analyzed using the SPSS13.0.

Growth after storage
The bulblets on nine different media at -2°C after 15 months storage were placed on restoration medium (MS+NAA 1.0 mgL -1 + 30 gL -1 Sucrose + 6 gL -1 Agar) at 24°C with 16-h day light at an intensity of 1500 lx for two weeks, then they were taken from Erlenmeyer flask and planted in moist peat at 20°C in the greenhouse (Bonnier and Van Tuyl, 1997).The bulblets stored at 24°C were first placed at 4°C for six weeks to break dormancy (Langens-Gerrits et al., 2003), then planted in moist peat at 20°C in the greenhouse.The survival rate was measured.

The effects of basal media and sucrose concentrations on the preservation at room temperature (24°C) and low temperature (-2°C)
After 3 months storage at 24°C, the growth of two species was restricted to varying degrees by adjusting basal medium and sucrose concentration.There were no apparent external morphological changes for two species.
The influences of various media on two species were different (Tables 3 and 4).The growth of seedling cultured in medium supplemented with 3% sucrose (the control) was not apparently restricted.Lowering the content of mineral elements (1/4 MS) and/or adding high sucrose concentrations (6 to 9%) had an obvious restraint effect.The average increment of treatment groups was less than that of control group and the difference was very The values are mean ± SD; different lower-case and capital letters in the same line represent the significant difference at p < 0.05 and p < 0.01 respectively.The values are mean ± SD; different lower-case and capital letters in the same line represent the significant difference at p < 0.05 and p < 0.01 respectively.significant.The inhibition effects increased using high sucrose concentrations (6 to 9%) (Tables 5 and 6).The combination of 1/4 MS nutrients and 9% sucrose gave the highest reduction in sprout growth.The increment of L. davidii was 6.5 mm for three months storage.The bulblets swelled significantly, but seldom propagated small bulblets.For L. longiflorum, the increment was 1.9 mm, meanwhile, the seedlings propagated many small bulblets during the storage.Besides, the bulb enlargement would occur.
Compared with the conservation at 24°C, the sprout growth of treatments at -2°C was more significantly reduced (Tables 3 and 4).Through the variance analysis, the effects of different sucrose concentration were significant.However, the effects were not similar for two species (Tables 5 and 6).At -2°C, the inhibitory effect for L. davidii was more obvious with 9% sucrose, which was significant with media with 3 and 6% sucrose.While for L. longiflorum, the effects of media with 6 and 9% sucrose were notable with the media plus 3% sucrose.(Tables 5  and 6) With the extension of storage, the sprout grew slowly.
The results indicate that the appropriate concentration of sucrose was 9% for L. longiflorum, while 6 to 9% for L. davidii (Table 8).However, the combination of low nutrient and high sucrose could play an important role in restriction conservation.At -2°C, the sprout growth was only 14.7 mm for L. davidii and 8.8 mm for L. longiflorum after six months by using 1/4 strength MS medium with 9% sucrose (Tables 7 and 8), it was significantly reduced.The promising preservation medium was ¼ MS with 9% sucrose for L. davidii, while ¼ or full MS with 9% sucrose for L. longiflorum (Table 7).For long-term preservation of lily germplasm resources, 1/4MS with 9% sucrose may be in common use.

The effects of basal media and ABA concentrations on the preservation at low temperature (-2°C)
There were remarkable differences in lily preservation between different treatments of ABA and nutrient concentrations.After three months storage at -2°C, the growth of two species was restricted to varying degrees by adjusting basal medium and ABA concentrations, the difference between groups and control group was significant (Tables 9 and 10).The lower nutrient content combined with increasing concentration of ABA had apparent inhibiting effect on sprout growth.Moreover, for L. davidii, the effect was more significant on the low nutrient medium with 3.0 mgL -1 ABA.It was generally similar for L. longiflorum.Different treatments showed no obvious difference in morphology after storage.
As prolonging storage, the effect was more significant for the low nutrient medium and the high ABA concentration from 1.0 to 3.0 mg/L.Through the duncan tests, the results show that the nutrient medium and ABA played a parallel restraint role in sprout growth after three months conserved (Table 10).After six months storage, the change of plantlet height of L. longiflorum was smaller than that of L. davidii, the effects of ABA from 1.0 to 3.0 mg/L were not significant (Tables 11 and 12).For L. davidii, the restricted effects were very significant for different concentrations of ABA and the nutrient medium.The promising preservation medium was 1/4 MS with 3.0 mgL -1 ABA.

The effects of preservation for the two species
With the extension of storage duration, the restraining effect of low temperature on lily growth decreased, moreover, sprout growth had an increase, especially the control (Figure 1).The growth of L. davidii began to accelerate at four months, at six months it was more obvious; condition of 1/4 basal media with 9% sucrose could effectively retard the sprout growth of lily, the mean height was still maintained at about 20 to 30 mm, in addition to a few of leggy seedlings.The increment of L. longiflorum was smaller than that of L. davidii.Besides,  2B).The results show that ABA may be a promising growth retardant to preserve lily germplasm.

Growth after storage
The tube seedlings conserved for 15 months could turn to normal plantlets after re-growth for one month which showed no obvious difference in morphology compared with control.The mean re-growth percentage after 15 months storage at -2°C on 1/4 strength MS medium plus 3.0 mg/L ABA was 87.5% for L. davidii, and 62% for L. longiflorum.

DISCUSSION
Varying degrees of the growth restriction for both species were related to the four factors (MS content, the concentration of sucrose and ABA, temperature).
Compared with other conservation measures, storage at -2°C on 1/4 MS medium plus 3.0 mg/L ABA resulted in more apparently inhibitory effect.As to L. longiflorum, the data measurements and recodes were ended after nine months treatment, for the seedlings had grown to the top of the bottle.While L. davidii was growing rapidly, the control had grown to the mouth of the bottle after six months.Although the growth was accelerated in six months, the tube seedlings could still be preserved on the medium.Furthermore, the materials on 1/4 MS medium plus 3.0 mg/L ABA had the highest survival rate of regrowth after 15 months conservation at -2°C.Actually, results show that the lily genotypes managed to survived, even with some slight pollution during the preserving process, after the storage as long as 24 months under the condition of -2°C on 1/4 MS medium plus 3.0 mg/L ABA .At 24°C, bulb growth and leaf formation occurred during prolonged storage.Due to consumption, the medium should be replaced once around every three to four months, especially the media with high concentration sucrose that promoted the bulb growth and accelerated the consumption.After five to six months storage, sprouts in some tubes had reached the top of the tube and the contamination rate rose with the extension of storage duration.Therefore, at normal temperature, the method of adding high concentration of sucrose and reducing macro element was suitable for mid-short term preservation of lily germplasm resources and could promote bulb growth and propagation.
At -2°C, based on investigation, we found that the adaptability to temperature and preservation effect were different between genotypes.The spout length of L. davidii increased rapidly (Figure 1) since the fourth month at the temperature of -2°C, which can be explained by the fact that dormancy was broken at this temperature; nevertheless, it could still keep on being preserved in this condition.However, the growth of seedlings of L. longiflorum, was slow (Figure 2).In addition, Langens-Gerrits et al. (2001) found that dormancy of L. longiflorum 'Snow Queen' was induced in vitro at 15°C, declined with increasing culture temperature.Moreover, the duration of the cold treatment needed for complete removal of dormancy was as long as two weeks at 5°C in Snow Queen.Therefore, it might be due to low temperature (5 to 15°C) which induced dormancy of L. longiflorum.At the condition of -2°C it was still dormant, or its growth was effectively delayed.L. longiflorum was more restricted to low temperature than L. davidii (Table 3), which could reflect their different origins.L. davidii originates from mountainous areas of Southwestern China (Abdullah and Ahmad, 1980;Anderson, 1988) where it grows in climate with moderately cold winters and warm summers.Wet and dry seasons are obviously in this region.However, L. The nine media, including: 1, MS medium plus 6% sucrose; 2, MS medium plus 9% sucrose; 3, 1/4 MS medium plus 6% sucrose; 4, 1/4 MS medium plus 9% sucrose; 5, MS medium plus 3% sucrose (the control); 6, 1/2 MS medium plus 3% sucrose and ABA (1.0 mgL -1 ); 7, 1/2 MS medium plus 3% sucrose and ABA (3.0 mgL -1 ); 8, 1/4 MS medium plus 3% sucrose and ABA (1.0 mgL -1 ); 9, 1/4 MS medium plus 3% sucrose and ABA (3.0 mgL -1 ); sprout length was measured every month.Each data point is the average sprout length of 45 plantlets.

Medi um code
longiflorum is a maritime plant of tropical nature growing in full sun with torrid summer and warm winter (Miller, 1993;Stoker, 1933).According to these facts, we can probably draw the conclusion that L. longiflorum is more sensitive to the low temperature, which gives rise to the growth stagnation during storage.Previously, Bonnier and Van Tuyl (1997) reported that the seedlings of L. longiflorum could be stored at -2°C for six months.Later, the damage was aggravating, and died during prolonged storage.However, our results show that L. longiflorum could be conserved for more than 15 months, though some plantlets died during storage.On the one hand, the acclimatization period was two weeks in Bonnier and Van Tuyl′s work, which was four weeks in our test.It suggests that the acclimatization period of four weeks could be sufficient to acclimatize bulbs to avoid further sprout growth of the bulblets, as well as reducing damage at low temperature.On the other hand, ABA as a growth inhibitor can enhance the ability to adapt to the stress of plantlets.Exogenous supply of ABA could inhibit sprout growth and leaf formation.Besides, ABA promotes tuberization and bulb formation in lily (Kim et al., 1994).Consequently, carbohydrate was placed into a storage organ which enhances the adaptability of seedlings to low temperature by osmotic stress.Another possible reason is that exogenous ABA may induce or enhance dormancy for some genotypes to a certain extent.ABA is considered to be the principal dormancy- inducing agent.For various seeds and buds, it has been shown that ABA plays a role in dormancy development (Hole et al., 1989;Suttle and Hultstrand, 1994).Furthermore, young bulblets were very sensitive to the hormone (Djilianov et al., 1994).
During storage, some in vitro seedlings presented in the shape change which showed a certain degree of instability, such as generating base bulbs, turning into purple color, which were the physiological changes produced by environmental stimulation under stress conditions, rather than genetic variation.The physiological changes can be reversed through recovery culture.The feasibility of preservation method was preliminarily validated at the protein level (Chen et al., 2006).However, due to the physiological status of different test materials, it is necessary to examine and analyze their preservation and regeneration of plant germplasm from molecular level, to get the optimal slowgrowth conservation in vitro technology.

Conclusion
It is concluded that the promising conservation conditions was the 1/4 MS with 3.0 mg/L ABA at -2°C which was suitable for mid-long term preservation of lily germplasm resources.

Figure 1 .
Figure 1.The effects of different media on the growth of L. davidii (A and B) stored at -2°C.

Table 1 .
The combinations of basal media and sucrose concentrations for the preservation at 24 and -2°C.
MS, Murashige and Skoog medium.

Table 2 .
The combinations of basal media and ABA concentrations for the preservation at -2°C.
MS, Murashige and Skoog medium.

Table 3 .
The effects of different basal media and sucrose concentrations on the sprouts growth of in vitro lilies after three months conservation at 24°C.

Table 4 .
The effects of different basal media and sucrose concentrations on the sprouts growth of in vitro lilies after three months conservation at -2°C.

Table 5 .
Duncan test of sucrose effect after three months conservation at 24°C.

Table 6 .
Duncan test of sucrose effect after 3 months conservation at -2°C.

Table 7 .
The effects of different basal media and sucrose concentrations on the sprouts growth of in vitro lilies after 6 months conserved at -2°C.

Table 8 .
Duncan test of different sucrose concentrations after 6 months conserved at -2°C.

Table 9 .
The effects of different basal media and ABA concentrations on the sprouts growth of in vitro lilies after three months conserved at -2°C.

Table 10 .
Duncan test of ABA and MS concentration effects after 3 months conserved at -2°C.

Table 11 .
The effects of different basal media and ABA concentrations on the sprouts growth of in vitro lilies after 6 months conserved at -2°C.

Table 12 .
Duncan test of ABA and MS concentration effects after six months conserved at -2°C.