*3.3. The Effect of Different Treatments on Seedling Survival Percentage of S. aralocaspica*

Only the seedlings without the growth of bacteria and browning can be used as the source of plant material. The available sterile seedlings were obtained after the seeds were sterilized and cultured for 20 days. There were significant differences in the number of available seedlings obtained from different types of disinfectants (*p* < 0.001), ethanol time (*p* < 0.001) and seed type (*p* < 0.001) (Table 6).


**Table 6.** A mixed model ANOVA on the seedling survival percentage of *Suaeda aralocaspica*.

Seedlings grown from brown seeds had a higher survival percentage than that from black seeds. The seedlings sprouted after sterilization with HgCl2 were short, and most browning deaths cannot be included in the surviving available seedlings. Compared with NaClO, the HgCl2 treatment significantly reduced the seedling survival percentage for brown seeds of *S. aralocaspica*. The survival percentage of seedlings produced by brown seeds in treatment H7 was only 10%. When NaClO was used as the main disinfectant, the highest survival percentage of seedlings from brown seeds in treatments N4 and N7 was 46.67%, and the survival percentage was 43.33% in treatments N3 and N10. Under the treatment of two secondary disinfectants, the survival percentage of seedlings from black seeds was low. The highest survival percentage was only 13.33% under the NaClO treatment n10 and 10% under the HgCl2 treatment h10 (Table 7).

**Table 7.** Seedling survival percentage of *Suaeda aralocaspica* cultured with different treatments after 20 days incubation.


Different lowercase letters in each column indicate significant differences among different treatments; different uppercase letters indicate significant difference between dimorphic seeds treated with the same secondary disinfectant.

## **4. Discussion**

Although the ecology, physiology, and molecular biology of *Suaeda* species have been studied extensively, there is no culture system in vitro for further study of the molecular mechanism. The effective acquisition of high-quality sterile explant material is the key to the subsequent tissue culture [19]. Our study takes the first step of this process by comparing the sterilization effects of different disinfectants and their effects on seed germination percentage.

Compared with the brown seeds, the black seeds were not easily contaminated by microorganisms, which might be due to the protective effect of the black and dense seed coat on the surface of the black seeds. 75% ethanol needs to be used with other disinfectants, for using it solely has an incomplete and unsatisfactory sterilization effect [21,22], and 0.1% HgCl2 has a good sterilizing effect because Hg2+ can combine with negatively charged proteins to desaturated bacterial proteins and inactivate enzymes. NaClO solution is much milder than mercury chloride and is often used to sterilize tissue culture explants [20–28]. When the same disinfectant is used, the contamination rate will decrease, and the death rate will increase with the extension in sterilization time. Under natural conditions or abiotic stresses, the germination percentages of brown seeds of *S. aralocaspica* were much higher than that of black seeds [7,9,15], which was consistent with the germination results after our sterilization treatment. The seed type had a significant effect on the three evaluation indexes of germination percentage, bacterial growth percentage, and survival percentage. When treating explants, different sanitizer and sterilization time was used for sterilization, and the effect was obviously different. The results showed that with the prolongation of ethanol infiltration time, the browning number of seedlings from brown seeds of *S. aralocaspica* increased and the survival percentage decreased.

In our study, mercury chloride and sodium hypochlorite were used to disinfect with 75% ethanol. Sodium hypochlorite has strong oxidation, and long sterilization time means it is easy to cause plant browning. When 3.6% NaClO was used as the main disinfectant, N8 had the best comprehensive effect on brown seeds, which were soaked in 75% ethanol for 60 s, and then sterilized with 3.6% NaClO for 8 min, and finally inoculated into a pH 8.0 MS medium. Black seeds grew well under treatment 6 (n6), which was disinfected with 75% ethanol for 1 min and then treated with 3.6% NaClO for 3 min. Although mercury chloride can be effectively sterilized, it also has strong toxicity, causing irreversible browning damage to plants [19,20,22]. When 0.1% HgCl2 was used as the main disinfectant, H7 had the best comprehensive effect on brown seeds (75% ethanol for 1 min + 0.1% HgCl2 for 5 min + pH 8.0 MS). h6 had the best comprehensive effect on black seeds, which was 75% ethanol for 1 min + 0.1% HgCl2 for 1 min + pH 6.0 MS.

It was found that a large number of browning seedlings appeared on the 8th day of culture, and the whole germination and growth process was completed on the 7th day. Therefore, the culture time can be shortened to 7 days to reduce energy and costs. Some studies found that adding anti-browning agents, such as vitamin C, activated carbon to the medium, or improving the activity of polyphenol oxidase and the antioxidant system enzyme Mars could effectively inhibit seedling browning [29–31]. In this experiment, we did not take special measures to prevent seedling browning, which could be optimized in further research.
