Introduction: Micropropagation of plants in in vitro condition with high relative humidity causes plants to wilt by transfer to in vivo condition. High relative humidity is one of the main factors disrupting stomatal function in the stress condition, darkness and the presence of ABA. Therefore, any factor that can affect the closure of the stomatal in in vitro, can lead to increased success in adaptation and transfer of tissue culture plants to in vivo condition. Embryogenesis is a technique in which bipolar structures are formed from somatic cells. During the cell differentiation process, the explant responds to endogenous stimuli and, as a result, directs the cell program. The induction and growth of somatic embryos are affected by the composition of the medium, especially sugar and osmotic conditions. In the present study, the effect of osmotic conditions was investigated on somatic embryo number, relative growth rate, photosynthetic pigments and proline of octoploid asparagus (Asparagus officinalis L.) embryogenesis.
Materials and Methods: Asparagus officinalis L., a native Iranian octoploid, was used in this project. The seeds were first washed with water and then immersed in 70% alcohol for 30 seconds. Seed disinfection was performed with 2% sodium hypochlorite for 15 minutes. The seeds were then washed three times with sterile distilled water and then dried with filter paper and placed on culture media. The B5 liquid medium containing 2 mg L^-1 2, 4-D was used for induction of somatic embryogenesis. Sucrose with two concentrations of 40 and 60 gL^-1, polyethylene glycol (PEG) with three concentrations of 30, 15 and 60 gL^-1, abscisic acid (ABA) of 5 and 10 μM, 4 and 6 dSm^-1 of salinity using NaCl were used to apply osmotic stress to asparagus somatic embryos. The cultured media were kept in a growth chamber with 3000 lux light at 25± 2°C for 16 hours. Four weeks after culture, embryos were examined and recorded for regenerative capacity.
Results and Discussion: According to the results, the highest number of spherical embryos (3.16) was related to PEG60 g, which was not statistically significantly different from 6 dS m^-1 NaCl (2.16). The highest number of bipolar embryos (13.16) was related to 6 dS m^-1 NaCl. PEG 60 g with 6.16 showed the highest number of bipolar embryos. The highest relative growth rate was observed in PEG 15 g (11.37 mg day^-1) and 4 dS m^-1 of NaCl (11.23 mg day^-1). The lowest relative growth rate was recorded in 10 μM ABA (3.47 mg day^-1). Based on the comparison results, the highest mean of chlorophyll a (0.077 mg FW^-1) was related to 60 g PEG and the lowest (0.028 mg FW^-1) was related to sucrose 60 g. The highest amount of chlorophyll b (0.103 mg kg^-1FW) was related to 60 PEG g and the lowest amount (0.01 and 0.007 mg kg^-1 FW) was related to 10 μM ABA. The highest amount of total chlorophyll (0.180 mg kg^-1 FW) was related to PEG 60 g and the lowest amount (0.0199 mg kg^-1 FW) was related to 10 μM ABA. The highest amount of carotenoids (0.054 and 0.047 mg kg^-1 FW) were related to PEG 60 g and sucrose 40 g, respectively. The highest amount of anthocyanin (0.184 mg kg^-1 FW) was obtained in 10 μM ABA and the highest amount of proline (67.66 μmol g^-1 FW) was obtained in 5 μM ABA.
Conclusion: Finally, salinity of 6 dS, PEG 60 g and ABA at a concentration of 5 μM with the least stressful effect compared to sucrose, had the greatest effect on the growth and development of asparagus somatic embryos which is recommended in the production stages of clones in asparagus. By applying high concentrations of polyethylene glycol due to osmotic pressure and stress, the production of oxygen free radicals increases and chloroplast structure destruction occurs. By inhibiting the biosynthesis of new chlorophylls and converting carotenoids to anthocyanins, it reduces the content of chlorophyll, carotenoid and increases anthocyanin. The in vitro response of asparagus somatic embryos to increased osmotic pressure with PEG is to increase the concentration of chlorophyll and anthocyanin. Increasing the osmotic pressure of the medium with sucrose led to an increase in proline in somatic embryos. Proline synthesis increases with increasing stress, which indicates the embryos effort to increase resistance to stressful conditions.