Abscisic acid increases the viability of cryopreserved shoot tips of Lilium pumilum by regulating the osmotic stress and programmed cell death

Lilium pumilum plays an important role in resistance breeding of lily because of its versatile value and strong stress resistance. It is of great significance to study the conservation of its resources. To improve its vitality after cryopreservation, the effect of abscisic acid (ABA) on the viability of cryopreserved shoot tips and its mechanism were studied based on the establishment of cryopreservation-vitrification protocol of Lilium pumilum. When 25 μM abscisic acid was added into the preculture medium, the survival rate after cryopreservation was increased by 22.73% and the regeneration rate by 11.48%, reaching 93.64% and 63.33%, respectively. Adding exogenous abscisic acid increased the contents of endogenous osmotic regulatory substances proline, soluble sugar and soluble protein and reduced the activities of caspase-3-like and caspase-9-like enzyme that characterize programmed cell death (PCD). This suggests that the addition of abscisic acid during preculture phase of cryopreservation improved viability of shoot tips by affecting osmotic stress and programmed cell death. A simple and efficient cryopreservation procedure for the shoot tips of Lilium pumilum was established. Exogenous abscisic acid increased the viability of shoot tips by regulating osmotic stress and programmed cell death.


Introduction
Lilium pumilum is an important ornamental, medicinal and edible bulbous plants of the genus Lilium, found in mountains, grass slopes or exposed rocks at an altitude of 400-2600 m in northern China, eastern Siberia, Korea and Mongolia (Macrae 1998;Tang et al. 2014).It is an excellent wild germplasm resource with wide distribution and strong resistance with drought and salt tolerance (Jin et al. 2014;So et al. 2022), so it plays an important role in resistance breeding of lily.
At present, there is a lack of long-term effective preservation methods for the germplasm resources of L. pumilum.Cryopreservation is an ideal technology for long-term preservation of germplasm resources using liquid nitrogen (LN 2 ) at low temperature (− 196 °C or liquid nitrogen vapor from − 165 to − 190 °C) (Panis 2019).Under these extreme cold conditions, the metabolism of plant cells basically stops, so plant materials can be preserved for a long time (Engelmann 2009;Wang et al. 2018).The vitrification method is more suitable for tissue culture cryopreservation (Kaviani 2011).It refers to a high concentration and viscosity of cryoprotectant is used to osmotic dehydrating plant cells, and the intracellular water is transformed into an amorphous glass structure without cells damaged by ice crystals when the temperature is reduced to extremely low temperatures (Fahy et al. 1984).It has been widely used because of its simple operation, high freezing rate and wide adaptability (Wang et al. 2014).The embryos, callus, suspended cells, protoplasts and shoot tips of more than 200 plants have been preserved by cryo-vitrification (Panis 2019).The shoot tips are more suited for plant germplasm cryopreservation than cell suspensions and callus.They have a higher level of genetic stability and grow into plants that are identical to the original plants (Li et al. 2019;Normah et al. 2019).The aim of this study is to establish a cryo-vitrification procedure for shoot tips of L. pumilum with high viability.
During vitrification cryopreservation, osmotic stress, cold stress, cryoprotectant toxicity and drastic temperature change may cause plant cells or tissues damage (Uchendu et al. 2010).Osmotic and low temperature stress not only change cell membrane structure (Farooq et al. 2009), enzyme function (Kubien et al. 2003) and osmotic regulation substances (Popko et al. 2010), but also promote the excessive generation of reactive oxygen species (ROS).It will lead to oxidative stress and plant cell structure damage (Halliwell 2006).In addition, The excess ROS is associated with programmed cell death (PCD) occurring in plant vitrification cryopreservation, and PCD may be another important factor that contributes to reduced vitality after cryopreservation (Jiang et al. 2019).
Abscisic acid (ABA) plays a key role in plant response to abiotic stress as one of the five major hormones in plants (Vishwakarma et al. 2017).In salt, drought and low temperature stress and so on, ABA synthesis is significantly induced, and a series of signaling pathways are initiated to activate the plant stress resistance mechanism (Chen et al. 2020).Previous studies showed that ABA can enhance the osmotic resistance and antioxidant enzyme activities in plants by promoting the synthesis of osmotic regulatory substances (Finkelstein 2013;Roychoudhury et al. 2013).In addition, exogenous ABA inhibited the PCD occurrence in barley aleurone layer cells and anthers to protect seedlings growing in variable environments (Wang et al. 1996(Wang et al. , 1999)).Relevant research shows that exogenous ABA down-regulates the pro-apoptotic protein gene CsBAX and up-regulates the anti-apoptotic protein gene CsBI-1 expression at 4 °C or 12 °C, preventing the PCD occurrence and increasing the cold resistance of cucumber seedlings (Talanova et al. 2017).However, the specific mechanism by which ABA regulates PCD remain unclear.The same is true in cryopreservation studies.
But some studies shows that adding ABA in the preculture period can effectively improve the survival rate of plant materials after cryopreservation.For example, exogenous ABA increased the survival rate of cell embryos of spruce (Picea glauca × engelmannii) (Kong and von Aderkas 2011), tissue cultures of Rubus humulifolius (Edesi et al. 2020), Ginkgo biloba cells (Lu et al. 2009) and Chrysanthemum × grandiflorum shoot tips (Kulus 2018) after cryopreservation.However, little has been reported about the effective mechanism of ABA in cryopreservation.The relationship between ABA and PCD in cryopreservation has not been reported.
In this study, the cryo-vitrification procedure of L. pumilum was established firstly, and then ABA was added and the survival rate of shoot tips was observed.The changes of osmotic regulatory substances and the activities of Caspase-3-like and caspase-9-like enzymes were measured to explore the mechanism of ABA.The aim is to provide more ways to improve the vitality after cryopreservation.

Plant materials
The first generation of tissue-culture plantlet of L. pumilum were provided by the Lab of National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing.

Shoot tips for cryopreservation
Under aseptic condition, bulbs were cut off from the first generation of tissue-culture plants.And the scales were peeled off and cut into about 1 cm 2 as explants.The explants were inoculated on induction medium, MS (Murashige and Skoog 1962) supplemented with 2.0 mg L −1 of 6-benzylaminopurine (6-BA), 0.2 mg L −1 of 1-naphthalene acetic acid (NAA), 30 g L −1 of sucrose, and 6.5 g L −1 of agar, and the pH5.8.Then the bulblets were induced and then transferred to rooting medium on MS, supplemented with 30 g L −1 of sucrose, 6.5 g L −1 of agar for rooting, and the second generation of tissue-culture plants were obtained.After that, the plants as explants were continue sub-cultured every 2 months until third and fourth generations plants were obtained.
The outer scales of bulblets from 2 months old tissueculture plants from the second generations were stripped and only the inner1-2 layers scales enclosing the shoot tips were retained.The explants were incubated in a growth room under a 14/10 h day/night photoperiod at 25 ± 2 °C, with cool-white fluorescent lamps (Philips, India) with a light intensity of 40 μ mol m −2 s −1 .

Basic cryo-vitrification procedure for shoot tips
On the basis of consulting a large number of literatures, combined with the preliminary research of our research group, the basic vitrification procedure was designed.The following steps were carried out continuously: (1) Pre-culture: The shoot tips of 2 months old plants from the 2nd generation were inoculated on induction medium and adding 0.3 M of sucrose for 4 days at 4 °C in dark.
(2) Loading: After pre-cultured, the 2 mm long shoot tips were gain under aseptic condition and stereoscopic microscope.The 10 shoot tips were put into a 1.5 ml frozen-resistant centrifuge tube with 1 ml Loading solution (MS liquid medium with 0.4 M of sucrose and 2 M of glycerin) and treated for 20 min at 25 °C.(3) PVS2 dehydration: The Loading solution was removed by pipette gun and 1 ml of precooled PVS2 solution at 0 °C (MS liquid medium with 0.4 M of sucrose, 30% of glycerol, 15% of ethylene glycol and 15% of DMSO) was added, and treated for 100 min at 0 °C.(4) LN 2 frozen-thawing: The PVS2 solution was replace by 1 ml of fresh one, then the centrifuge tube holed shoot tips and PVS2 solution was put into LN 2 quickly, storage at least for 1 h.The centrifugal tube was removed from LN 2 storage tank and was put in a water bath at 37 °C quickly.(5) Unloading: After thawing, the PVS2 solution in the tube was sucked out by pipette gun and the shoot tips were washed twice with the unloading solution (MS liquid medium with 1.2 M of sucrose) at 25 °C, each time 10 min.

The optimization of key steps of cryo-vitrification procedure
The optimized parameters (if any) of the previous step was taken, and the parameters in the basic procedure were adopted in subsequent steps, and the optimized parameters for this step was determined.
(2) Loading time The optimal parameters for preculture were adopted, then the shoot tips were put into Loading solution for 20, 40, 60 or 80 min at 25 °C, respectively.
(3) PVS2 solution treatment time The optimal parameters for preculture and Loading were adopted, then the Loading solution was removed by pipette gun and precooled PVS2 solution (MS liquid medium with 0.4 M of sucrose, 30% of glycerol, 15% of ethylene glycol and 15% of DMSO) at 0 °C was added, and treated for 40, 60, 80, 100 or 120 min at 0 °C, respectively.

Survival rate of cryopreserved shoot tips
The 2,3,5-triphenyltetrazolium chloride (TTC) method was used to assess the survival of shoot tips.The shoot tips were placed on petri dish with 2 ml 0.8% (w/v) of TTC and 50 mM of PBS, pH 7.4, and incubated for 30 min at 37 °C water bath in the dark.The number of stained shoot tips were counted.

Recovery rate of cryopreserved shoot tips
After LN 2 stored and thawed, shoot tips were inoculated on the recovery medium (MS liquid medium with 30 g L −1 of sucrose and 6.5 g L −1 of agar) at 25 ± 2 °C in the dark.
After 2 weeks of culture, when the shoot tips turned green and obviously elongated, the recovery rate was counted.

Adding appropriate exogenous ABA
ABA of 0.132 g was dissolved in 50 ml distilled water and configured into 0.01 mol/l ABA solution.When 1.25 ml ABA solution was added to 500 ml MS medium that sterilized at high temperature but not solidified, and a medium containing 25 µmol/l ABA could be obtained.
The different concentrations of ABA at 25, 50, 75, 100 μM were added into the preculture medium respectively at preculture step.The following steps are processed using the optimal procedure, then the concentration with the highest shoot tips survival rate after cryopreservation was used as the optimal ABA adding concentration.
The group adding optimal ABA concentration in preculture medium was marked (Pre + ABA) and the group without adding ABA marked (Pre-ABA).

Proline determination
The method of Abraham (Abraham et al. 2010) was slightly improved.The shoot tips of 0.1 g was placed into a 1.5 ml Survival rate % =Number of shoot tips stained red∕number of total incubated tested shoot tips × 100% recovery rate % = number of regenerated shoot tips∕number of total culturecryopreservtedshoot tips × 100% centrifuge tube, 0.5 ml of 3% sulfosalicylic acid solution was added and ground to homogenate, then extracted in a boiling water bath for 10 min, and centrifuged at 3000 rpm for 5 min after cooling.The supernatant was the proline extract.The extract of 0.2 ml, 0.2 ml of glacial acetic acid and 0.2 ml of 2.5% acid ninhydrin reagent were added to the centrifuge tube in sequence, and then the centrifuge tube was heated in a boiling water bath for 30 min until the solution turned red.After cooling, it was centrifuged at 3000 rpm for 5 min.Then 0.4 ml of toluene was added into the centrifuge tube, shaken for 30 s, and stood for a while.The upper proline red toluene solution was gently absorbed into the colorimetric cup with a straw.The absorbance value was measured at the wavelength of 520 mm.

Soluble sugar determination
The method of Wang (Wang et al. 2004) was slightly improved.The shoot tips of 0.01 g was put into a 1.5 ml centrifuge tube, and 1 ml of distilled water was added and ground to homogenate.The centrifuge tube was placed in a water bath at 95 °C for 10 min, cooled, and centrifuged at 8000 rpm for 10 min at 25 °C.The extract of 200 µl, 200 µl of double distilled water, 100 µl of anthrone-ethyl acetate solution and 1 ml of concentrated sulfuric acid were added into the centrifuge tube, and then put the centrifuge tube in a water bath at 95 °C for 10 min.After cooling, the absorbance values of blank tube and measurement tube were measured at 620 nm.

Soluble protein determination
The method of Wang (Monreal et al. 2007) was slightly improved.The shoot tips of 0.02 g was put into a 1.5 ml centrifuge tube, and 0.5 ml of phosphate buffer containing 1%PVP and 50 mM with pH7.8 was added and ground to homogenate.Then it was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was taken as enzyme solution.0.5mI of the enzyme solution was put into a 10 ml centrifuge tube, and 5 ml of Coomassie brilliant blue G-250 solution was added, mixed and shaken, and the absorbance value at 595 nm was measured after 10 min.

Caspase-3-like enzyme and caspase-9-like enzyme activity determination
The activities of caspase-3-like protease and caspase-9-like were detected by apoptotic enzyme activity detection kit (Beyotime Institute of Biotechnology, Shanghai, China).The specific test operation was carried out according to the kit instruction.

Statistical analysis
The experiments were conducted in a completely randomized block design.The cryopreservation experiment was repeated three times, with 10 shoot tips each time.The physiological and biochemical indexes were measured in triplicate, and the test results were expressed as the mean and standard deviation (SD).SPSS 26.0 (Version 26.0 SPSS Inc., Chicago, IL, USA) was used for data statistics.A oneway ANOVA followed by least significant difference (LSD) tests was used to detect significant differences at P < 0.05.Microsoft Excel 2019 software (Microsoft Corp., Richmond, CA, USA) was used for chart drawing.

The shoot tips for cryo-vitrification
The scales of bulb from the first generation tissue-culture plants of L. pumilum (Fig. 1A) were inoculated on the induction medium (Fig. 1B), and the buds were induced from the scales explants about 1 month later (Fig. 1C).After about 2 weeks, the buds developed into bulblets (Fig. 1D).The bulblets were transferred to the rooting medium (Fig. 1E).After 15 days, the bulblets were rooted (Fig. 1F).It is the source of continued cultivation for the second generation of tissue-culture plants.

The optimal cryo-vitrification procedure
The survival rate of the shoot tips precultured with various concentration sucrose after cryo-vitrification was obviously different.It gradually increased with the increase of the concentration of sucrose in the preculture medium.The sucrose concentration was 0.5 M, the shoot tip survival rate reached the maximum value of 68.89%, and then decreased (Fig. 2A).
The preculture time also had a great influence on the survival rate of shoot tips.With the increase of preculture time expansion, the survival rate gradually increased, and the highest survival rate was 70.91% after 7 days culture (Fig. 2B).Thus, preculture of shoot tips with 0.5 M of sucrose for 7 days was optimal.
The precultured shoot tips need to be loaded with loading solution to achieve protective osmotic dehydration.The survival rate of shoot tips with loading 20 min reached the maximum value of 70.91% (Fig. 2C).So the optimal loading time was 20 min at 25 °C.
When the shoot tips was treated in PVS2 solution for 40-60 min at 0 °C, the survival rate did not change significantly.However, when the time was extended to 80 min, the survival rate began to significantly increase, and reached the highest at 100 min with 71.82%, which was not significantly different from that at 80 min (Fig. 2D).In order to reduce the toxic of PVS2 solution on the shoot tip, the optimal PVS2 time was set to 80 min.

Recovery culture of LN 2 frozen-thawed shoot tips
The shoot tips (Fig. 3A) were cryo-vitrificated adopting the optimal procedure and inoculated on recovery medium (Fig. 3B).After 1 week of culture in the dark, the surviving shoot tips turned green (Fig. 3C) and the dead shoot tips turned brown (Fig. 3D).After 2 weeks of culture under dark conditions, the regeneration rate of shoot tip was 51.85% and significant elongation of surviving shoot tip was observed (Fig. 3E).After 2 months of culture under normal light conditions, the shoot tip could develop into a complete plant (Fig. 3F).

The effect of shoot tips from different generations plants on survival rate
The different shoot tip from the 2-4 generations plants were cryopreservated using the optimal procedure of cryo-vitrification.There was no significant difference in the survival rate of LN 2 frozen-thawed shoot tip (Fig. 4).It indicating that the L. pumilum was subcultured 2-4 times had no significant effect on the viability of the cryopreservated shoot tips.These materials all can be used for cryo-vitrification.

Effect of exogenous ABA on viability of shoot tips after cryo-vitrification
Addition exogenous ABA at different concentrations into the preculture medium had significant effected on shoot tips survival rate after LN 2 frozen-thawed.With the increase of ABA concentration, the survival rate was increased at first, then decreased and increased again.The ABA concentration was 25 μmol/l with the highest survival rate of shoot tips, 93.64%, which was significantly higher than that of the control group (0 μM of ABA) (Fig. 5).So adding 25 μmol/l of ABA in preculture is suitable.
There was a significant difference between the recovery rates of shoot tips after LN 2 frozen-thawed with ABA adding or not.Adding ABA had a positive effect with the recovery rates 63.33%, 11.48% higher than control 51.85% (Fig. 6).But plantlets morphological differences were not observed between adding ABA group (Pre + ABA) and without ABA (Pre − ABA) (Fig. 7).

Effect of exogenous ABA on content of osmotic regulating substances in shoot tips of cryo-vitrification
Compared with the control group, exogenous ABA obviously increased the proline content of shoot tips in each periods and reached the maximum value at PVS2 and thawing periods with significantly different from that of the control, loading and unloading period were relatively low and no significant difference from that of the control.But the change trend of proline content during cryo-vitrification was similar to that in control group (Fig. 8A).
The exogenous ABA obviously increased the soluble sugar content of shoot tips in each periods and reached the maximum value in PVS2 and thawing period.The soluble sugar content in preculture, PVS2 and thawing period was significantly higher than that in the control group.The change trend of soluble sugar content during cryo-vitrification was slightly different from that in control group (Fig. 8B).
The effect of exogenous ABA on the soluble protein content was similar to the soluble sugar content.The soluble protein content was significantly higher than that of the control group in death period.The different was significant except unloading period.The change trend of soluble protein content during cryo-vitrification was same as that in control group (Fig. 8C).

Effect of exogenous ABA on apoptotic enzymes activities in shoot tips of cryo-vitrification
The exogenous ABA obviously decreased the activity of caspase-3-like enzyme of shoot tips in each periods and the difference reached significantly lever compared with the control, except that in preculture period.But the change trend of the enzyme activity during cryo-vitrification was same as that in control group (Fig. 9A).
The exogenous ABA had same effect on the activity of caspase-3-like enzyme and caspase-9-like.It obviously decreased The activity of caspase-9-like enzyme in each periods and had same change trend as that in control group.The only difference was that the activity of caspase-9-like enzyme reached different significantly lever compared with the control in the PVS2, thawing and unloading period three latter stages (Fig. 9B).

Discussion
The effect of cryopreservation by vitrification depends on various factors, such as the genotype and physiological state of plant materials, and the parameters of preservation procedure, including preculture, loading, PVS2 dehydration, LN 2 freezing, thawing, unloading and recovery culture and so on.
Most studies showed that the 0.3 M sucrose in preculture medium for 1-5 days and loading 20 min were more effective for the cryopreservation of Lilium (Chen et al. 2011;Bouman et al. 2003;Yin et al. 2014).In this study, a similar conclusion was obtained.The shoot tips of L. pumilum precultured with 0.3-0.7 M sucrose concentration for 4-10 days and loaded 20 min all gain the same good effect on shoot tips of cryopreservation by vitrification.However, the dehydration time of PVS2 varied greatly among Lilium plants.The optimal dehydration time of PVS2 for Lilium lancifolium was 90-120 min (Chen et al. 2011) and for the shoot tips of Lilium Oriental hybrids for 3-4 h (Yin et al. 2014).In this study, the shoot tips of L. pumilum treated 80-120 min in PVS2 solution achieved the same good effect.The physiological and biochemical state of tissue culture plants may change during continuous subculture process, which will affect the viability of materials of cryopreservation.The survival rate of the shoot tips of the tissue cultured seedlings of L. lancifolium after 1-4 subgenerations is significantly different, and the survival rate of the second generation of tissue cultured seedlings is significantly higher than that of the other three generations (Shen et al. 2014).But this study results showed that the shoot tips of L. pumilum after 2-4 subgenerations there was no significant difference in the viability before and after cryo-vitrification.However, too long in vitro and too many times of subculture may lead to genetic variation (Coelho et al. 2020).Therefore, it is necessary to avoid using the tissue cultures materials with excessive subculture times for cryopreservation.
A large amount of osmotic regulatory substances such as proline and soluble sugar are accumulated in plants under osmotic stress and cold stress so as to improve the stress resistance of plants (Bashir et al. 2014).The cryopreservation of potato shoot tips showed that the soluble sugar content was increased by sucrose and cold pretreatment and the survival rate of shoot tips was increased (Folgado et al. 2014).This study showed that the contents of three osmotic regulatory substances (proline, soluble sugar and soluble protein) were increased during each period of cryo-vitrification compared with the control group, especially after PVS2 treatment and LN 2 frozen-thawed.It is possible that both osmotic and low temperature stress in those two periods were more serious, which promoted the accumulation of osmotic regulatory substances in shoot tips.
As an important endogenous hormone in plants, ABA plays an important role in plant response to high salt, drought and cold stress (Bakht et al. 2013;Yang et al. 2012;Zhang et al. 2010).Under drought stress, ABA can increase the content of proline, thereby enhancing the stress tolerance of plants (Kaur and Asthir 2020).In addition, some studies showed that the exogenous addition of ABA in preculture medium is beneficial to the survival of the materials after cryopreservation (Edesi et al. 2020;Kong and Von 2011;Kulus 2018;Lu et al. 2009).ABA promoted the overexpression of ASKIN gene during the period of garlic cryopreservation and recovery culture, which may be related to plant resistance to cold stress and osmotic stress (Xing et al. 2022).Exogenous addition of 2 mg/l ABA during the cryopreservation preculture period could significantly increase the endogenous soluble sugar content and improved the survival rate of Ginkgo biloba callus (Lu et al. 2009).In this study, the addition of 25-100 μM of ABA in preculture medium could increase the shoot tips viability.And 25 μM of ABA had the best effect, which could increase the shoot tip viability from 70.91 to 93.64%.Adding 25 μM ABA in preculture medium significantly increased the contents of endogenous proline, soluble sugar and soluble protein in shoot tips, thereby enhancing the tolerance to osmotic stress and improving the survival rate of shoot tips.Proline as an important osmoregulatory substance, plays an important role in protecting cell osmotic balance and subcellular structure stability.It can also act as an antioxidant and play an important role in regulating ROS balance in plant cells (Zhang et al. 2023).The soluble sugars are involved in osmoregulation and may play an important role in maintaining the stability of plant proteins (Zhao et al. 2006).
PCD is an active death process that often occurs when plants encounter abiotic stresses (Li et al. 2018;Wang et al. 2015).PCD occurred in some plant cryopreservation, such as pollen of Paeonia sufruticosa (Ren et al. 2020), protocorm-like bodies of Dendrobium nobile (Jiang et al. 2019), embryogenic callus of Agapanthus praecox (Zhang et al. 2015).This study the activities of caspase-3-like and caspase-9-like enzyme were measured, which can indicate PCD happened.Those enzyme activities gradually increased during the cryo-vitrification and reached the highest in LN2 frozen-thawed period, which was consistent with Jiang's research results (Jiang et al. 2019).But adding exogenous ABA in preculture medium could significantly reduce these two kinds of enzymes activity during the cryo-vitrification and improve the viability of shoot tips.Some researches results showed that ABA is associated with PCD during plant growth (Wang et al. 1996(Wang et al. , 1999)).However, there are few studies on the relationship between ABA and PCD under abiotic stress.The results of this study show that ABA increases shoot tips viability by inhibiting the occurrence of PCD during vitrification cryopreservation.The mechanism of action is worthy of further study.

Conclusion
The cryo-vitrification optimal procedure for shoot tips of L. pumilum is established with 70.91% survival rate and 51.85% regeneration rate, which provide a simple, efficient and long-term preservation method for germplasm resources of L. pumilum.It is as follows: (1) The 2 months old tissueculture plants from 2 to 4 subculture in vitro plantlets were precultured on induction medium with 0.5 M sucrose and 25 μM abscisic acid (ABA) for 7 days at 4 °C in the dark; (2) The 2 mm shoot tips gain from precultured plantlets and loading 20 min at 25 °C; (3) PVS2 solution treatment for 80 min at 0 °C; (4) The tube was removed from LN 2 storage and rapidly thawed in 37 °C water bath for 60 s; (5) Unloading solution washed twice at 25 °C, each 10 min.On this basis, Addition of 25 μM ABA in preculture medium improve the survival rate to 93.64% and regeneration rate to 63.33%.Exogenous ABA improve the osmotic stress resistance and decrease the occurrence of PCD by increase the content of.These results provide a basis for the application and further investigation of ABA in cryopreservation.

Fig. 1
Fig. 1 Tissue culture process of Lilium pumilum.A Scales from tissue-culture plants; B Shoot tips inoculated on induction medium; C The buds induced from the scales.D The buds developed into bulblets.E Bulblets inoculated on rooting medium.F Bulblets rooted.Bars = 1 mm

Fig. 2
Fig. 2 Effect of the different treatment of main steps of cryopreservation procedure on the survival rate of shoot tips.A Effect of sucrose concentration in preculture medium on the survival rate of shoot tips; B Effect of preculture time on the survival rate of shoot tips; C Effect

Fig. 3 Fig. 4 Fig. 5
Fig. 3 Regeneration of cryopreserved shoot tips of.A The shoot tips for cryopreservation; B LN 2 frozen-thawed shoot tips inoculated on recovery medium; C Surviving shoot tips after LN 2 frozen-thawed.

Fig. 6 Fig. 7 Fig. 8
Fig. 6 Effect of exogenous ABA on the recovery rate of shoot tips.Values represent the mean ± SD of three replicates, different letters indicate significant differences among the means (p < 0.05)

Fig. 9
Fig. 9 Effect of ABA on the caspase-like enzyme activity of shoot tips during cryo-vitrification.A The caspase-3-like enzyme; B the caspase-9-like enzyme.CK control group, Pr preculture treatment, L loading treatment, P PVS2 dehydration, LN liquid nitrogen freezing