Lycium ruthenicum, one of the most important drought and salt resistant desert pioneer species, is a perennial thorny shrub of Lycium ruthenicum in Solanaceae. In the world, many Lycium ruthenicum species are found in South America, South Africa, southwest North America, Mediterranean, Asia, and there is a native species of Lycium ruthenicum in Australia (Levin et al., 2007). In China, it is mainly distributed in special environments, such as Tibet, Helan Mountain in Ningxia, Eastern Qinghai, Northern Shanxi, Northern Xinjiang, Neiling, river bed and sand beach, etc. and it is resistant to low temperature (Liu et al., 2018).

Wild Lycium ruthenicum is rich in nutrients, as well as Calcium, Magnesium, Copper, Zinc, Manganese, Iron, Lead, Nickel, Cadmium, Cobalt, Chromium, Potassium, Sodium, Lithium, Phosphorus, Titanium, Vanadium, Aluminum, Boron, Silicon and other metal elements (Qi et al., 2019). Lycium ruthenicum is also rich in anthocyanins, which has antioxidant and anti-aging medicinal value. Lycium ruthenicum is a combination of economic value, ecological value, medicinal value and health value. How to fully and reasonably develop and utilize Lycium ruthenicum resources is an important issue that needs urgent attention (Hao et al., 2016).

Plant tissue culture technology is an asexual propagation technology based on cell totipotency. It has the advantages of small space occupation, no regional and seasonal restrictions, short culture cycle, rapid propagation in a short time, no variation, maintaining all genetic characteristics of female parent, low investment and high economic benefits. Once it comes out, it was rapidly applied in flowers, crops and forest seedling production. So far, researchers have used different Lycium ruthenicum tissues as explants to induce callus, including twigs (Hao et al., 2005), seeds (Chen et al., 2018), tender stems (Sun et al., 2016), mature leaves (Sun et al., 2016; Yang et al., 2016), cotyledons (Wang et al., 2016), hypocotyls (Zheng et al., 2017). With the help of plant tissue culture technology, the cultivation method of regeneration seedlings of Lycium ruthenicum was established to produce large-scale seedlings of Lycium ruthenicum, so as to meet the needs of high quality, high yield and industrialization of Lycium ruthenicum.

RAPD, SRAP, cpDNA and SSR were used to analyze the genetic diversity and structure of Lycium ruthenicum (Zhang et al., 2001; Liu et al., 2012; Chen et al., 2014b; Chen and Zhong, 2014). Liu analyzed the genetic diversity of 14 endangered wild Lycium ruthenicum populations in Northwest China, and found that the genetic diversity frequency of Lycium ruthenicum in this region was very high using sequence related amplified polymorphic markers, which was of great significance for the protection and breeding of endangered species (Liu et al., 2012). Zhao used 18 pairs of simple sequence repeat (SSR) markers to analyze the genetic diversity and population structure of 139 Lycium ruthenicum materials (Zhao et al., 2010). Chen found that the differentiation time of different lineages of Lycium ruthenicum was consistent with the rapid uplift period of Qinghai Tibet Plateau and the formation and expansion time of desert in northern China (Chen et al., 2014b). Chen identified 1913 up-regulated genes and 536 down-regulated genes in Lycium ruthenicum by RNA sequence analysis (Chen et al., 2014a). Liu used virus induced gene silencing (VIGS) technology to silence the endogenous genes of Lycium ruthenicum for gene function analysis (Liu et al., 2014). Xue cloned and obtained the key flowering gene FT (Flowering Locus T) of Lycium ruthenicum and Lycium barbarum. They are mainly expressed in leaves, which is helpful for the follow-up study of related functional genes (Xue et al., 2015).

In this study, callus was directly induced from sterile seedlings of Lycium ruthenicum, and regenerated plants were obtained quickly and effectively, which greatly shortened the time of tissue culture, and laid the foundation for the next step of rapid obtaining Lycium ruthenicum plants.

1 Results and Analysis

1.1 Obtaining differentiated seedlings of wild Lycium ruthenicum

Firstly, the seeds of Lycium ruthenicum were disinfected, and then the sterilized seeds were transferred to the MS ordinary medium plane. After about 14 days, the aseptic seedlings grew into seedlings, and the aseptic seedlings with good growth were selected for subsequent experiments. Then, the well-developed sterile seedlings of Lycium ruthenicum were transferred to MS-1 (callus induction medium), waiting for the growth and development of callus (Figure 1A; Figure 1B; Figure 1C).

After about 21 days, the callus grew rapidly and grew to a callus with a diameter of about 10 mm, which could be peeled off and placed on MS-2 (induction differentiation medium) (Figure 1D); then the callus continued to grow and grew to a diameter of about 15 mm about 30 days after peeling off (Figure 1E), and then grew to a diameter of about 20 mm about 40 days after peeling off. In addition, the state of callus changed from hard green to soft yellow green (Figure 1F); then about 60 days after peeling from callus, about 3 subcultures (each subculture cycle is about 20 days), differentiated buds appeared (Figure 1G).

Then the differentiated buds continued to grow for about 20 days (Figure 1H), and the well-developed buds were stripped off and inserted into MS ordinary medium to induce rooting (Figure 1I); after the buds continued to grow for about 20 days, they would grow into seedlings, and each seedling had 3-4 roots, which could be used for the next transplanting. The following is the specific time of the changes of tissue cell state of Lycium ruthenicum (Table 1).

1.2 Seedling refining and transplanting of Lycium ruthenicum

Firstly, the seedlings of Lycium ruthenicum with good growth condition were selected, the cap of culture bottle was opened, and the seedlings were adapted to normal temperature and light for 2~3 days. Then put 1:2 ratio of fine sand and nutrient soil matrix into the flowerpot, and add appropriate amount of farmyard manure, mix and fit. Soaked the flowerpot in water to make it wet from bottom to top, then selected the seedlings that grew well after refining, selected 2 seedlings to plant in one pot, and covered them with plastic film to keep moisture. After waiting for 3 days, opened a corner of the preservative film, opened half of it in 3 days, and removed all the preservative film in 3 days. When the seedlings survived and developed well, divided them into pots, one plant in each pot, and do a good job in water and fertilizer management. The survival rate of Lycium ruthenicum seedlings was over 85%.

2 Discussion

This study provided a rapid method to obtain regeneration seedlings of Lycium ruthenicum. Callus was directly obtained from the sterile seedlings of Lycium ruthenicum. The specific explant part was the contact surface between the root and stem of Lycium ruthenicum and the upper surface of the culture medium. It was particularly easy to produce callus, and grew rapidly. The callus state changed from green to yellow green, from hard to soft, and in a short period of time the callus would germinate multiple buds, then grew into seedlings, and obtained multiple differentiated plants within a period of time. Therefore, the callus differentiation efficiency of Lycium ruthenicum was higher. Compared with the traditional callus induction through the leaves, stems and roots of seedlings, it greatly shortened the time of tissue culture, greatly reduced the cultivation cost of Lycium ruthenicum, and effectively improved the cultivation efficiency. The survival rate of regenerated seedlings was as high as 85%.

By adding 0.5~1.0 mg NAA and 1.5~2.5 mg 6-BA to each liter of MS medium, it was easier to obtain differentiated callus, and the callus formation rate was up to 90%. The rooting rate could be as high as 97% without adding any hormone, and the root growth rate was faster. Each seedling grew at least three roots, and the roots were strong and the root hairs were dense. In this study, the method of rapid regeneration of Lycium ruthenicum had the characteristics of good genetic stability, low cost, short seedling period, high reproduction coefficient and simple operation.

3 Materials and Methods

3.1 Disinfection of seeds and obtaining of aseptic seedlings

The seeds of Lycium ruthenicum were soaked in 70 v% (volume percent) alcohol for 3~7 min, then disinfected with 0.1 wt% (mass percent) mercuric chloride solution for 10~20 min, and then washed with sterile water for 2~3 times. Sterilized seeds were placed in MS medium, and aseptic seedlings could be obtained after 8~12 days of germination.

3.2 Callus induction

The aseptic seedlings were transplanted into MS-1 medium (MS+0.5~1 mg/L NAA+1.5~2.5 mg/L 6-BA+30 g/L sucrose+8 g/L agar, pH 5.2) to induce callus. The culture conditions were as follows: temperature 23℃~27℃, light 12 h/D, medium pH 5.0~5.5, culture 35~45 D.

3.3 Obtain of adventitious buds

The callus of step 3.2 was separated from the explants and placed in MS-2 medium (MS+0.5~1 mg/L NAA+0.5~1.4 mg/L 6-BA+30 g/L sucrose+6 g/L agar, pH 5.2) to induce adventitious buds. The culture conditions were as follows: temperature 23℃~27℃, light 14 h/D, medium pH 5.0~5.5, proliferation culture for 3~5 generations.

3.4 Root induction

The adventitious buds of step 3.3 were transplanted into the MS medium to induce rooting, and the regeneration seedlings of Lycium ruthenicum were obtained; the culture conditions were as follows: temperature 23℃~27℃, light 14 h/D, culture time 17~22 D. The obtained rooting seedlings were transferred into the greenhouse for at least 2 weeks.

Authors’ contributions

Wang like is the executor of the experimental design and research; Wang like and Zhong Huiping complete the data analysis and write of the first draft of the paper; Mao shanguo, Yang Ping, Wu Yunwen, He Ying and Chen Quanzhan participate in the experimental design and the analysis of the experimental results; Wang like is the designer and director of the project, guiding the experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu Province, China (No. BK20150087), the Natural Science Research of Jiangsu Province Colleges and Universities, China (No. 15KJB180009), and the Jiangsu Province college students innovation and entrepreneurship training program provincial general project (201911460055y).

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