A,Preliminary,Screening,of,Optimum,Medium,and,Symbiotic,Fungi,for,Paphiopedilum,dianthum

Cuiping CAO Man SHEN Yufeng XU Ruidong JIA Hong GE

Abstract ［Objectives］ This study aimed to screen the suitable medium and symbiotic fungi for tissue culture of Paphiopedilum dianthum and to establish a rapid propagation system of Paphiopedilum, so as to promote the development of planting industry and commercial application of Paphiopedilum.

［Methods］Using P. dianthum as an experimental materials, the optimal culture medium for P. dianthum was selected from DE medium, Harvais modified medium and (bark+DE) liquid medium. Then, six fungal species isolated from the original habitat soil of Paphiopedilum were co-cultured with P. dianthum on the selected optimal culture medium. By comparing the morphology, the growth amount and the physiological indexes, such as chlorophyll content, soluble sugar content and root vitality of tissue culture plantlets of P. dianthum co-cultured with the six fungal species, the suitable fungal strains for symbiosis with P. dianthum were screened out.

［Results］ In three kinds of culture media, P. dianthum grew normally. The growth rate was higher than that in the mediums containing DE, and there were no significant differences in the content of soluble sugar from the leaves of tissue culture plantlets, which were symbiotic with the six different fungi species. Among the six fungi species growing on the optimal medium, the growing rate of XH-A was the fastest, and that of YY001 was the slowest. The growth and physiological indexes of P. dianthum co-cultured with DY1 in DE medium were higher than those in the control group.

［Conclusions］DE medium was the optimal tissue culture medium for P. dianthum, and the optimal symbiotic fungus was DY1.

Key words Paphiopedilum dianthum; Symbiotic fungi; Tissue culture

Received: December 27, 2020  Accepted: February 22, 2021

Supported by Project of Construction of Advanced Horticulture Discipline Under Beijing Municipality)(2020); Key Laboratory of Biological and Genetic Improvement of Horticultural Crops under Ministry of Agriculture.

Cuiping CAO (1995-), female, P. R. China, Master, devoted to research about ornamental plant cultivation physiology.

*Corresponding author. E-mail: shenman@vip.sina.com.

Paphiopedilum is the most primitive group in Orchidaceae, as well as one of the most distinctive ornamental orchids［1-3］, which is deeply loved by flower lovers in the world［4-5］. Paphiopedilum dianthum belongs to the epiphytic orchids. It is a new species discovered by Chinese botanists Tang Jin and Wang Fazan in 1940, and is endemic to China［6］. The fruiting rate of wild P. dianthum after anthesis is extremely low. In recent years, it has been plundered by humans. In addition to the deterioration of its living environment due to natural or man-made reasons, the distribution area and number of P. dianthum have been drastically reduced and it is on the verge of extinction［5,7］. At present, P. dianthum is not only listed as a rare and endangered plant in China for protection, but also included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and is internationally protected［8-11］. So far, with the continuous maturity of tissue culture technology, the propagation technology of orchids has also been continuously improved, which provides technical guarantee for the propagation of P. dianthum. In this study, with P. dianthum as an experimental material, its suitable tissue culture medium and symbiotic fungal species were screened for the establishment of a rapid propagation system for tissue culture Paphiopedilum, so as to carry out in-depth research on Paphiopedilum and its relationship with symbiotic fungi, and use symbiotic fungi to promote seed germination and seedling growth and promote the development and commercial application of Paphiopedilum.

Materials and Methods

Experimental materials

In this experiment, pollution-free in-vitro propagated culture plantlets of P. dianthum with 4 true leaves and basically the same root growth and 6 kinds of symbiotic fungi derived from the in-situ soil of Paphiopedilum were all provided by Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science. The test numbers and genus and species names of the 6 fungi are shown in Table 1. The tested symbiotic media were DE medium, modified Harvais medium (referred to as cHa medium)［12］ and (bark+DE) liquid medium (referred to as bark medium). Among them, the bark liquid medium formula (per bottle) was bark 30 g+liquid DE medium 55 ml.

Experimental methods

Screening of media

P. dianthum plantlets were transferred into DE medium, cHa medium, and bark medium, and cultured in a tissue culture room under conditions of temperature (22.5±1) ℃, relative humidity 40%, light intensity 1 500 Lx, and light duration 9 h/d. Ten flasks of plantlets were cultured for each medium according to 2-3 P. dianthum plantlets per flask.

Screening of symbiotic strains

A 0.9 cm hole puncher was used to take agar blocks of the 6 strains that grew well on the PDA plate. They were placed in the center of the P. dianthum tissue culture bottles at approximately the same distance from each plantlet. After sealing with a sealing film, they were placed in the tissue culture room for cultivation.

Determination of related indexes

Five flasks of tissue-cultured P. dianthum plantlets were randomly selected for the observation of leaf number, root number, fresh weight, maximum leaf length, maximum leaf width and maximum root length on 0 d (the initial cultivation) and 30 d (the final cultivation) respectively, and the change rate of each index was calculated. The plant height, maximum leaf length, maximum leaf width and maximum root length were all measured with a ruler in cm. For the calculation of the rate of change, with the fresh weight index as an example, the formula was: average fresh weight change rate (%)=(Final weight-Initial fresh weight)/Initial fresh weight×100%, and the change rates of other indexes could be deduced by analogy.

Three flasks of tissue-cultured P. dianthum plantlets were randomly selected, and the physiological indexes under each experimental treatment were measured. The chlorophyll content was determined by the 95% ethanol direct extraction method［13］. The anthrone method［14］ was adopted to determine the soluble sugar content of leaves. The TTC method［15］ was applied for qualitative and quantitative determination of root vitality.

Statistics and analysis

WPS Excel and SPPS 26 software were used for experimental data processing and analysis of variance, and Origin 2017 software was used for graphing.

Results and Analysis

Effects of different media on the growth and physiological indexes of P. dianthum

The tested P. dianthum was transferred into and cultured on the media of different formulas, which was cultured for 30 d. The survival rate of the tissue culture plantlets was 100%, and the growth status was normal. The change rate was calculated by comparing the difference between the initial values (0 d of cultivation) and the final values (30 d of cultivation) of the observation indexes in the 10 groups of P. dianthum. A negative value indicated a reduction rate, that is, the final value of the growth index of P. dianthum was less than the initial value; and a positive value indicated a growth rate, that is, the final value of the growth index of P. dianthum was greater than the initial value.

It can be seen from Table 2 that the total number of leaves, total number of roots and fresh weight of P. dianthum were on the increase, indicating that these three media could not only promote the production of new leaves and roots, but also promote plant growth.

Fresh weight is one of the important indicators to describe plant growth, and it can characterize plant growth rate and other conditions. The fresh weight growth rates in the three media in descending order were: DE>(DE+bark)>cHa. The growth rate of the fresh weight of P. dianthum cultivated on DE medium was 3.03 times that on cHa, and 1.14 times that on (bark+DE). There were significant differences in the impact. It can be seen that P. dianthum grew better in the medium containing DE.

However, in our test, it was found that the maximum root length of P. dianthum in the DE medium decreased, indicating that DE was not conducive to root elongation; and in the (bark+DE) liquid medium, the maximum leaf length of P. dianthum decreased, indicating that (bark+DE) was not conducive to leaf growth.

In general, the growth rate of the total number of roots of P. dianthum in the DE medium and the growth rate of the maximum leaf length in the (bark+DE) liquid were less than those in the cHa medium, while other growth indexes were all higher than those in the cHa medium.

Plant roots are active absorption organs and synthetic organs. Root vitality, i.e., root growth and metabolism level, directly affects the growth and nutritional status of plants aboveground part, which is one of the important physiological indexes of plant growth［12］. P. dianthum was cultured on three media for 30 d, and there were no significant differences in soluble sugar content and root vitality (Table 3). However, when cultivated in the (bark+DE) medium, the root vitality of P. dianthum was 1.28 times that on DE medium, indicating that the bark components in the medium had a positive effect in enhancing root vitality.

The level of chlorophyll content reflects the ability of plants to produce organic matter through photosynthesis, and is closely related to the growth status of Paphiopedilum plants［12］. When cultivated in (bark+DE), P. dianthum had a significant difference in chlorophyll content from that cultivated on the DE and cHa media, and its content was the lowest. The chlorophyll contents of P. dianthum cultured on DE and cHa were both higher, and the difference was not significant.

Cultivated in 3 kinds of media, the chlorophyll contents of P. dianthum determined were in order of DE>cHa>(DE+bark). Therefore, we believe that DE medium is suitable for tissue culture of P. dianthum.

Comparison of the growth of different strains on the optimal medium for P. dianthum

In order to test whether the DE medium that was most suitable for the growth of P. dianthum plantlets was also suitable for the growth of the 6 tested symbiotic strains, the 6 strains were respectively inoculated on the blank DE medium. After 9 d of cultivation, the growth of the 6 strains was observed and compared (Fig. 1).

It can be seen from Fig. 1 that the 6 strains could grow normally in DE medium, and the growth rates were moderate. Among them, XH-a showed the fastest growth rate and YY001 had the slowest growth rate (Fig. 1-b, Fig.1-c).

The colonies of such 3 strains as XH011, XH023 and XH-a grew on the DE plates in contact with the medium and were thick in the middle and thin at the edge. The colonies continued to grow radially in the later stage, producing aerial hyphae, forming concentric rings. From 7 to 9 d, they grew all over the petri dishes. The growth rates were moderate (Fig. 1-a, Fig. 1-b, Fig. 1-f).

Observing other three strains YY001, DY1 and DY2 (Fig.1-c, Fig. 1-d, Fig. 1-e) cultured on DE plates, it was found that the colonies grew in contact with the medium in the early stage, with neat edges, and were thick in the middle and thin at the edge. In the later stage, aerial hyphae were produced, which were very obvious, and the colonies were white. They grew all over petri dishes in the period of 12-20 d. The growth rates were relatively slow.

Screening of strains suitable for symbiosis with tissue-cultured seedlings of P. dianthum

Comparison of growth rates of P. dianthum cultivated with different strains

It can be seen from Table 4 that P. dianthum was inoculated to the DE medium with the 6 strains, respectively. After 30 d of co-cultivation with the 6 strains of P. dianthum, in the control group (CK group) without symbiotic fungi, the indexes including the total number of leaves, total number of roots and fresh weight were on the increase, and the YY001 group, DY1 group, XH023 group and XH-a group inoculated with the symbiotic fungi were the same as the CK group, indicating that these 4 species of fungi could co-grow well with P. dianthum on DE medium.

In the YY001 group, XH023 group and DY1 group, the growth rates of 5 determined indexes were higher than that of the CK group, while the XH-a group only had 4 higher ones. The fresh weight growth rates of P. dianthum ranked as CK>YY001>XH023>DY1>DY2>XH-a>XH011.

In addition to fresh weight, plant height is also an important index to measure the growth of tissue culture P. dianthum plantlets［10,12］. In this study, the order of the plant height growth rates of tissue culture P. dianthum plantlets was: DY1 group>XH023 group>XH-a group>YY001 group>CK group>DY2 group>XH011 group.

Therefore, comprehensively from the growth rates of the growth indexes, the strains suitable to be co-cultured with P. dianthum were DY1>XH023>YY001>XH-a>CK>DY2>XH011.

It can be seen that DY1 was the most suitable strain for symbiotic tissue culture with P. dianthum, followed by XH023, XH-a, and YY001, while DY2 and XH011 were not suitable for symbiosis with P. dianthum on DE medium.

Comparison of physiological indexes of P. dianthum cultivated with different strains

P. dianthum and symbiotic fungi were cultured on DE medium for 30 d, and the root vitality, soluble sugar and chlorophyll contents were determined (Table 5).

Table 5 shows that the root vitality and soluble sugar content of P. dianthum showed unobvious differences, while the differences in chlorophyll content were relatively significant. Compared with the CK group, the chlorophyll content, the root vitality and the soluble sugar content of P. dianthum were higher in DY1 group.

The soluble sugar content is an important nutrient in the plant, which provides energy for the growth of plants. The soluble sugar content of P. dianthum in the DY2 group was the highest, but the chlorophyll content and root vitality were significantly lower than those in the CK group, and the root vigor was the lowest. The chlorophyll content of P. dianthum in the YY001 group was the highest, and the root vitality in the XH023 group was the highest, but their soluble sugar contents were both significantly lower than that in the CK group. For P. dianthum in other groups inoculated with the symbiotic fungi, at least two physiological indexes were lower than those in the CK group.

Therefore, it can be considered that the symbiotic strain DY1 was most suitable for tissue culture plantlets of P. dianthum.

Comparison of growth status of P. dianthum cultivated with different strains

In order to further observe the effects of the inoculated symbiotic fungi on the growth of tissue culture P. dianthum plantlets, the agar blocks of each strain were co-cultured with the tissue culture plantlets (DE medium) for 30 d, and the morphological changes were observed. The root system of P. dianthum co-cultured with DY1 strain for 30 d was taken and observed.

It was found that compared with the CK group, the tissue culture plantlets of P. dianthum with symbiotic fungi formed more new roots (Fig. 2-a, Fig. 2-b), on which the root hairs were vigorous and appeared obvious white, and the morphology of the root hair area was significantly different from other areas. After qualitative inspection of root vitality, the color of the root tip turned red, indicating that the root vitality was strong (Fig. 2-c). It indicated that the symbiotic fungus DY1 could promote the formation of new roots of P. dianthum to a certain extent.

However, as the time of symbiotic culture increased, P. dianthum and hyphae directly or indirectly absorb the nutrients in the medium, and to a certain extent, they will also form a competitive relationship. As the amount of hyphae continued to increase, hyphae climbed to the walls of culture bottles and the leaves of the

issue cultured P. dianthum plantlets (Fig.4-a), and even twined or overwhelmed the plantlets (Fig. 4-b). It can be seen from Fig. 4 that the hyphae of the symbiotic fungi not only twined the leaves of the plantlets, but also nearly completely covered the root system of P. dianthum and tightly attached to the root system.

Discussion and Discussions

Almost all Orchidaceae plants live in symbiosis with fungi, which accompanies the entire life history of orchid plants［16-17］. The symbiosis of orchids and fungi is specific, and it is related to the seed germination, seedling differentiation and growth and development of adult plants of orchids. It has always been a research hotspot of scientists［17-19］. In the studies of Orchidaceae mycorrhiza fungi, most of the fungi that coexist with mycorrhizae of orchids are Sebacinales, Ceratobasidiaceae and Tullasnellaceae of "Rhizoctonia"［20］. Studies have also found that other basidiomycetes other than "Rhizoctonia" can also form mycorrhizae with Orchidaceae plants［21］. The nutrient exchange between Orchidaceae plants and mycorrhize fungi is mainly the mycorrhiza fungi supplying inorganic salts and organic compounds required by Orchidaceae plants. Orchidaceae mycorrhiza fungi are different from other mycorrhiza fungi in that they cannot obtain organic nutrients from the roots of hosts. They must obtain nutrients from the surrounding environment and then decomposed into small molecular carbohydrates such as glucose for the absorption and utilization by themselves and Orchidaceae plants［22］.

In the tissue culture study of Paphiopedilum plants, Yang et al.［24］, Zeng et al.［25-26］, Ding et al.［27］ established an in-vitro rapid propagation system of P. henryanum, P. hirsutissimum, and P. appletonianum without symbiotic fungi, laying a foundation for the propagation of Paphiopedilum plants. However, compared with symbiotic culture with symbiotic fungi, the above method provides conditions for easy access to nutrients and growth, while symbiotic culture is more similar to the symbiosis of orchids and fungi under natural conditions, can induce better growth of Paphiopedilum seedlings, and has been proved to be beneficial to seedling growth［18］.

In the process of tissue culture breeding, Paphiopedilum seedlings and symbiotic fungi promote each other and restrict each other. On the one hand, Paphiopedilum seedlings obtain nutrients through fungi, and the fungi quickly grow and reproduce along the Paphiopedilum root system; and on the other hand, Paphiopedilum obtains some nutrients from the culture medium, which limits the excessive growth of fungi. Based on the phenomena observed in experiments, we believe that in the study of tissue culture breeding of Paphiopedilum and its symbiotic fungi, the amount of inoculated fungi should also be considered. In this study, the amount of symbiotic fungi inoculated was a 0.9 cm in diameter agar block containing certain fungus. But we found that some fungi grew too fast, and hyphae climbed the bottle walls, twined the plant leaves, and even caused the plant leaves to fall and die. Therefore, the next step is to further optimize the inoculum amount of the symbiotic strains screened out above. In the screening of suitable fungal amount, it is possible to consider setting a certain gradient of fungal concentration for the strains that have been screened, and use the method of injecting fungal liquids into the medium to analyze the effects of different fungal amounts on tissue culture plantlets of P. dianthum.

The symbiosis mechanism between Paphiopedilum seedlings and fungi still remains unexplained. The 6 strains used in this study were sourced from the in-situ soils of P. armeniacum, P. hirsutissimum and P. micranthum. Under natural conditions, these strains live in symbiosis with the three species of Paphiopedilum, which provide nutrients for plant growth and development. In this study, these strains were co-cultured with the tissue culture plantlets of P. dianthum, and it was found that some fungus species promoted the growth of P. dianthum, such as DY1, while some fungus species inhibited the growth of P. dianthum, such as XH011. However, whether the symbiosis mechanism of these fungi with P. dianthum is different from that with P. hirsutissimum and P. armeniacum remains to be further studied.

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