Analysis of Growth and Physiological Variations Among Cyclocarya Species of Different Genotypes in a Clonal Seed Orchard

Background: Cyclocarya paliurus (C. paliurus) is a woody species that has many medical benets to human health. Geographical species has been formed among different natural forests of C. paliurus, but gradually decrease or extinct by human’s excessively exploitation in recent years. So, it is worthy to conserve this native and valuable species in China. Environmental factors affected plant growth, and seed is the only way for breeding offspring among Cyclocarya species, herein, we assess plant growth of six genotypes (FJ, JX, TG, WF, JH, AJ) from 2017 to 2019 year and physiological variations from April to October in 2018 during the construction of a clonal seed orchard. Results: The survival rate of plant reached 100% in a C. paliurus seed orchard. Plant height and basal diameter varied in different genotypes. Plant of six genotypes had different changes in water content and total soluble sugar content from April to October, Ca content reached maximum among the four detected mineral content, four kinds of antioxidant enzyme activity was in order of SOD (cid:0) PPO (cid:0) POD (cid:0) CAT, and the highest content of phytochemical was total avonoid. Plant growth and physiological changes during growth period was signicantly correlated with environment factors by correlation analysis. Conclusions: Plants of ex situ conservation adapted changeable environmental factors by physiological changes and showed the differentiation of plant growth in a clonal seed orchard. This would provide a foundation for ex situ conservation management in Cyclocarya species and selection of suitable cultivation provenance. in plant height and basal diameter. (2) Difference in WC and TSS content among 6 genotypes varied from April to October. Four kinds of antioxidant enzyme and mineral element content was in order with SOD (cid:0) PPO (cid:0) POD (cid:0) CAT, Ca (cid:0) K (cid:0) Mg (cid:0) Na, respectively. Total avonoid accumulation in leaf was the highest, then was polysaccharides, and nally was total triterpenoid. (3) Both of plant growth and physiological index had relation with environmental factors (mean temperature, precipitation, sunshine hours) and various physiological index also related with each other by relation analysis. These results suggested that plants of 6 genotypes had a better adaptation in CSO by physiological changes during growth period and different genotypes varied in plant growth and nutrient accumulation. Therefore, results from this study provide a foundation for ex situ conservation management of Cyclocarya species and selection for breeding programs.


Background
Forest is one of the major natural resources and has important role on economy, ecology and social. With the rapid development of human population and industry, forest resource has undergone massive destruction mainly due to over-exploitation, overgrazing, unsustainable practices, forest res and environment-unfriendly development projects (Glikson et al. 2012, Karthikeya et al. 2007), which is much beyond the regenerative ability of forest (Bahu et al. 2015). These lead to the decrease of forest diversity and the extinction of many forest resources. Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus) is a native and unique species distributed in highland of sub-tropical areas in China (Fang et al. 2006). During the long history of evolution and development, the geographical differentiation is formed among Cyclocarya species ) and natural forests sporadic distributed in China.
As a material for food industry and medicinal value, C. paliurus has been demonstrated to possess a myriad of human health bene ts like anticancer, antimicrobial, antihyperlipidemic, antioxidant and anti-in ammatory, which is primarily due to biological activities of various phytochemical in leaf (Xiong  Xie et al. 2012Xie et al. , 2013. For obtaining these bene ts, natural forests of C. paliurus were damaged by human's activity to meet the huge demand of leaf production in the past years. And the lower regeneration of natural forest could further cause this valuable species to be extinct in the future. Now, natural forest of C. paliurus is now protected with different conservation status: critically endangered, server convention and convention (http://www. iplant. cn). Therefore, it is very urgent and important to establish an effective way of conserving Cyclocarya species.
Ex situ conservation is an effective way for preserving the extinct or endangered plant species in order to rescue the natural heritage of plant biodiversity (Pritchard et al. 2010, Corlett 2016. A clonal seed orchard (CSO), one of ex situ conservation strategy, often cultivated trees collected from a single provenance or multiple provenances to achieve genetic gain (Chaloupkova et al. 2019, Giertych 1975, Zobel et al. 1958). In C. paliurus, isolation and diverse biological activities of phytochemical from leaf was one of numerous researches about C. paliurus (Peng et al. 2019, Cao et al. 2017, Zhu et al. 2015, another were mainly focused on the increase of phytochemical accumulation in leaf by improving cultivated conditions (Deng et al. 2019a, 2019b, Liu et al. 2018a, 2018b. However, researches about conserving Cyclocarya species were very few. Up to date, seed is the only way for breeding offspring of Cyclocarya species, and CSO would be a suitable way for conserving Cyclocarya species. However, environmental factors affect on plant adaptation (Enßlin et al. 2011;Cao et al. 2018), which could be impact on the successful construction of CSO among Cyclocarya species.
Hence, based on the collection of seed from different species of C. paliurus, this study investigated growth variation in physiological changes during the process of CSO construction, and analyzed the relation between environmental factors in the CSO and plant growth. This could be bene t to assess their adaptation in the ex situ conservation and make a solid foundation for selecting superior family for breeding new species and suitable cultivation species.

Methods plant materials and site description
Seeds of C. paliurus from six genotypes were collected from natural forests in late October 2015 according to the method described by Deng et al. (2014) and information about six genotypes was seen in Fig.1. The collected seed were rst subjected to removing wings, followed by exogenous gibberellin A3 (GA3) treatment, and then strati cation treatment using a method described by Fang et al. (2006). After 5 months, the germinated seedlings were Extraction and analysis of phytochemical content Extraction was obtained using an ultrasonic-assisted method with a slight modi cation (Liu et al. 2018c). Each sample (about 1.0g) was added to 20mL 75% ethanol, then centrifuged at 25℃, 11000g for 15min after heating at 70℃ for 60min with ultrasonic cleaner (KQ-800DE, China), and nally extraction was obtained.
Total avonoid content was determined by method described by Liu et al. (2018c), then calculated by the standard rutin curve and expressed as milligrams rutin equivalent per gram of dry mass (mg/g).
Total triterpenoid content was assessed by using the Folin-Ciocalteu colorimetric method and then expressed as milligrams gallic acid equivalent per gram of dry mass (mg/g). Analysis of water content WC in leaf was different with growth periods and genotypes. WC change was similar from June to October among FJ, AJ, TG, JX and WF. WC of JH gradually decreased and had the highest content on June (Fig.2).

Analysis of total soluble sugar content
Plants of different genotypes varied in TSS content during growth period. TSS content of FJ and JH had the highest value on August, of which change was opposite with that of AJ. TSS content of JX and TG gradually increased and reached maximum on October, which was contrary with that of WF (Fig.3).
Analysis of mineral content K, Ca, Na and Mg were contained in leaves among 6 genotypes, the highest content was Ca (≥6.0mg/g), and then was K(≥3.0mg/g), nally was Mg and Na (≤2.0mg/g) (Fig.4).

Analysis of antioxidant enzyme activity
Four kinds of antioxidant enzyme activity was in order of SOD PPO POD CAT. However, every antioxidant enzyme of different genotypes had different activity in growth period (Fig.5).
All of genotypes had the highest SOD activity on April and its activity was in order with AJ FJ JX WF JH TG. Among six genotypes, SOD activity of FJ gradually decreased from April to October and similar change exit among FJ, JX and TG. SOD activity among WF, AJ and JH rstly decreased, then increased and nally decreased. The different peak of POD activity was observed among six genotypes, both of JX and FJ had more than 3.0U/(g·min) on June, TG and JH had the highest activity on April and on October, respectively. There was similar change among FJ, JX, TG and JH, but AJ had an opposite change with WF.
CAT activity of JX and FJ was lower than 0.5U/(g·min) from April to October. Similar change was found between TG and JH, but its activity of TG was higher than that of JH. AJ and WF reached maximum on August and on June, respectively, and the peak was more than 2.0 U/(g·min).

Analysis of phytochemical accumulation
High content of total avonoid and polysaccharides (≥40mg/g) was detected, but total tritenpenoid content was found in lower amount (≤3.0mg/g) in ex situ conserved plants. The change of their content varied with growth period and genotypes (Fig.6).
Total avonoid content of FJ rstly decreased and then increased from April to October, which was similar with that among JX, WF and AJ. However, there was no signi cant difference from June to October in JH and in TG.
Polysaccharides content of FJ reached 100.16mg/g on April, compared with that, its content decreased by 50.52%, 57.29% and 20.39% from June to October, respectively. Other ve genotypes had the highest content of polysaccharides on October , there was similar change between WF and AJ, between JH and TG.
The highest content of total triterpenoid reached on August among FJ, JX, JH and TG; and their changes were similar, but were contrary with that of WF and AJ. AJ had no signi cant difference in triterpenoid content from June to October, similar results were observed in WF and in JX.

Correlation analysis among parameters
Plant height was signi cantly related with basal diameter, both of them were also related with AT, AP and AS (Tab.3).
Both of T and P seemed predominantly impact on various physiological index, (Fig.7). In conformity with CCA, both of T and P was signi cantly correlated with K, Ca, polysaccharides and total avonoid. S had correlation with POD and WC (Tab.4).
There was signi cant correlation between total triterpenoid and Mg, between Na and Ca, between K and SOD activity, between K and Na. WC had signi cant correlated with K, Ca and TT. Total avonoid was also related with Na, Ca and TP (Tab.4). The similarities are presented in cluster analysis (Fig.8). Environmental factors affected mineral uptake and distribution from soil to leaf in plants (Baghour et al. 2002(Baghour et al. , 2003, this was in agreement with our ndings that mineral content varied with growth period and had signi cant negative relation with temperature and precipitation (Tab.4, Fig.7).
Plants have developed antioxidant defense systems to adapt changeable environmental factors during growth period. In the enzymatic defense mechanism, SOD, in combination with CAT, PPO and POD, eliminated excessive H 2 O 2 and O 2 in tissue of plants (Burducea et al. 2019, Gill andTuteja 2010).
Results in our study also showed that SOD activity was the highest among the detected four enzymes and varied with genotypes and growth period (Fig.5).
These suggested that SOD might be the main enzyme to interfere and metabolize excess H 2 O 2 and O 2 at a high rate (Burducea et Hare 2002), our ndings also showed that variations in TP, TF and TT content were found from different genotypes and phytochemical accumulation was related with environmental factors during growth period (Fig.6, Fig.8).
According to our results, leaves of C. paliurus were rich in avonoid and polysaccharides, which was consistent with previous works ( Fig.8).
Between the four tested mineral elements and three kinds of phytochemical, Ca and Mg was signi cant positively correlated with polysaccharides and total avonoid without fertilization condition, respectively, which was disagreement with Deng et al. (2019b) who reported Ca and Mg had a signi cant negatively correlation with total avonoid accumulation under ve nitrogen fertilization levels (Deng et al. 2019b). This could be caused by nitrogen availability, which in uence nutrient balance and further affected phytochemical accumulation in C. paliurus (Deng et al. 2019b).

Conclusions
Plants of C. paliurus of six genotypes were conserved in a clonal seed orchards at Quanzhou, Fujian province. Variations in plant growth and physiological changes in leaf were analyzed and results showed that (1) plant growth among 6 genotypes was different in plant height and basal diameter. (2) Difference in WC and TSS content among 6 genotypes varied from April to October. Four kinds of antioxidant enzyme and mineral element content was in order with SOD PPO POD CAT, Ca K Mg Na, respectively. Total avonoid accumulation in leaf was the highest, then was polysaccharides, and nally was total triterpenoid. (3) Both of plant growth and physiological index had relation with environmental factors (mean temperature, precipitation, sunshine hours) and various physiological index also related with each other by relation analysis. These results suggested that plants of 6 genotypes had a better adaptation in CSO by physiological changes during growth period and different genotypes varied in plant growth and nutrient accumulation. Therefore, results from this study provide a foundation for ex situ conservation management of Cyclocarya species and selection for breeding programs.

Declarations
Authors' contributions YF designed the work plan and preparation of manuscript. JPH carried out the construction of a clonal seed orchard of C. paliurus and carried out mineral nutrient content determination, GXL measured water content and total soluble sugar content in the laboratory, ChQZh determined antioxidant enzyme activity, XYY performed phytochemical content measurement, XFL performed the statistical analysis.

Availability of data and materials
The data that support the ndings of this study are available from the authors (School of Resource and Environmental Science, Quanzhou Normal University China) upon reasonable requests and with permission from Quanzhou Normal University (China). T mean average temperature, P mean percipitation, S mean sunshine hours, WC mean water content, TSS mean total soluble sugar content, TT denoted total triterpenoid, TF denoted total avonoid, TP denoted polysaccharide.