Overview of Research on Tissue Culture and Rapid Propagation Technology of Pholidota

Miao ZHANG Long CHEN Hua ZHU Qiaoling SONG Lirong YANG

Abstract The genus  Pholidota  has good medicinal value, and is often over-excavated by humans. Coupled with its low natural reproduction rate,  Pholidota  is almost endangered. This paper summarized the tissue culture and rapid propagation technology of  Pholidota  in recent years, aiming to provide key technical support for resource protection and development of  Pholidota  and preliminary foundation and technical support for follow-up related research.

Key words  Pholidota ; Tissue culture; Explant; Rapid propagation technology

Received: September 1, 2021  Accepted: October 31, 2021

Supported by The Basic Ability Improvement Project of Young and Middle-aged Teachers in Guangxi Universities (2019KY0349); Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicines (GKJZ ［2014］ 32); Collaborative Innovation Center of Zhuang and Yao Ethnic Medicines (GKKY ［2013］ 20); Ethnic Medicine Resources and Application Engineering Research Center of Guangxi Zhuang Autonomous Region (GFGGJH ［2020］ 2605); "The Eighth Batch of Guangxi Specially-employed Expert Projects" (GRCTZ ［2019］ 13); Youth Fund Project of Guangxi University of Traditional Chinese Medicine (2019QN012).

Miao ZHANG (1991-), female, P. R. China, research assistant, PhD, devoted to research about identification and development of traditional Chinese medicine and ethnic medicine.

*Corresponding author. E-mail: 390508271@qq.com.

The genus  Pholidota  belongs to the Orchidaceae family. It has about 30 species, which are distributed in tropical and subtropical areas in the south of Asia, to Australia and the Pacific islands in the south. There are 14 species in China, produced in southwestern China and southern China to Taiwan (Flora of China).  Pholidota  plants have high medicinal and ornamental value［1］.  Pholidota chinensis  Lindl. can be used to treat dizziness and headache［2］, and  Pholidota cantonensis  Rolfe. can treat sore throat, cough, phlegm, pneumonia,  etc. ［3］.  Pholidota  plants are epiphytic herbs, and their fruit is capsules with very many seeds. However, the seeds are small and have no endosperm, resulting in a low natural reproduction rate. In addition,  Pholidota  has good medicinal value and is often over-excavated by humans, making  Pholidota  almost endangered. Therefore, plant tissue culture technology with the advantages of fast reproduction speed, short cycle, artificial control of cultivation conditions and convenient management is an important choice for the rapid propagation method of the genus  Pholidota .

The research of plant tissue culture began in 1920, when the German botanist Haberlandt first proposed the concept of totipotency of cells, and it has been in a stage of rapid development since the 1960s［4］. Plant tissue culture, also known as  in vitro  culture, can induce calli, adventitious buds, adventitious roots and finally form complete plants, based on the totipotency of plant cells, using isolated organs (such as stem tips, leaves, flowers,  etc. ), tissues (such as pith cells, cortex, endosperm,  etc. ) or cells (such as spores, somatic cells,  etc. ) and protoplasts, under sterile and appropriate medium and temperature conditions. As a result, it has become possible to break through the technical bottleneck of asexual and rapid propagation of  Pholidota  through plant tissue culture technology, and success has been achieved in other species, such as  Bletilla striata  (Thunb.) Reichb. f. and  Dendrobium nobile  Lindl［5-6］. Rao  et al. ［7］ used tissue culture technology to increase the reproduction coefficient of  Pinellia ternata  and shortened the reproduction cycle of  P. ternata .

This paper summarized the tissue culture and rapid propagation techniques of  Pholidota  in recent years, with an attempt to provide key technical support for resource protection and development of  Pholidota  and preliminary foundation and technical support for follow-up related research.

Types of Explants

The genus  Pholidota  belongs to the Orchidaceae family, and its fruit is capsules, and a single capsule contains a large number of tiny seeds［8］. Although the seeds of  Pholidota  have no endosperm, and one end of the seeds is slightly blunt, and has seed holes, which are conducive to the entry of various nutrients and help germination. At present, researches on tissue culture and rapid propagation technology of  Pholidota  plants all use seeds as explants［9-10］, which have a high germination rate on a suitable medium, and the sterilization of  Pholidota  capsules is simple and the pollution rate is low.

Explant Disinfection

Disinfection of explants is the first step in plant tissue culture, as well as a crucial step. Incomplete disinfection of explants may cause contamination, while excessive disinfection of explants may kill plant cells and made them fail to differentiate to form callus. Commonly used disinfectants include ethanol, sodium hypochlorite solutions, mercuric chloride solutions and hydrogen peroxide solutions. When Li  et al. ［9］ carried out tissue culture of  P. cantonensis , in which the capsules were sterilized with 0.1% HgCl2 solution for 7-9 min, the soaking time of 0.1% HgCl2 was investigated, and finally 8 min was determined as the best sterilization time. Wen studied  Pholidota  tissue culture［10］. They investigated the treatment of  P. chinensis  capsules with 0.1% HgCl2, 2% sodium hypochlorite and 10% hydrogen peroxide solution for 6, 8, 10 min, respectively, and the results showed that the sterilization treatment of 0.1% HgCl2 solution for 8 min was suitable for  P. chinensis  capsules with the lowest pollution rate. Lin  et al. ［11］ carried out 0.1% HgCl2 soaking and disinfection for 10 min, and some scholars［12］ adopted disinfection and soaking for 8 min. And they also achieved good results.

Selection of media

The nutrients required for seed germination are mainly derived from culture media. Different culture media contain different inorganic nutrients, organic nutrients, growth regulators and additives. Commonly used media include MS, Kassanis, WPM, N6, B5, KC medium,  etc. ; and the amounts of nutrients required for different growth stages of plants are also the same. During the rooting stage of plants, the amounts of macronutrients and sugars are reduced, because high osmotic potential is conducive to the operation of nutrients and hormones and to the occurrence and formation of roots.

Seed germination

For the germination of  P. chinensis  seeds, Wu  et al. ［12］ used MS as the basic medium and carried out orthogonal experiments to investigate the effects of different hormone combinations of 6-BA, KT, NAA, IAA and different concentration pairs of 6-BA, KT, NAA on seed germination of  P. chinensis . They screened out the optimal hormone concentration ratio for seed germination of  P. chinensis  seeds. On this basis, the seeds were respectively inoculated on the medium of basic medium MS+NAA 0.1 mg/L+KT 0.5 mg/L+peptone 1 g/L, which was then added with different concentrations of 6-BA, and the most suitable medium for seed germination of  P. chinensis  was determined as MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+KT 0.5 mg/L+peptone 1 g/L.   Li  et al. ［13］ investigated basic media MS, 1/2MS, B5, N6, VW and KC by adding different types and concentrations of cytokinins (BA, kinetin, TDZ), and finally determined that the best basic culture media were KC, 1/2VW and N6. Some scholars［11］ investigated different media formulations such as MS, MS+coconut juice 50 ml/g, MS+6-BA 1.0 mg/L+NAA 0.5 mg/L, MS+6-BA 10 mg/L+NAA 0.5 mg/L+coconut juice 50 ml/L, MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+ banana puree 50 g/L, MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+ activated carbon (AC) 1 g/L, and MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+coconut juice 50 ml/L was the best induction medium for  P. chinensis  seeds. Wen  et al. ［10］ used the single-factor comparison method to investigate the effects of five basic media MS, 1/2MS, N6, B5 and KC on aseptic sowing of seeds, and determined the best basic medium to be N6 medium.

Regarding the germination of  P. cantonensis  seeds, Liu  et al. ［9］ investigated the effects of MS and KC on the germination of  P. cantonensis  seeds, and finally determined that the seed germination medium MS+20 g/L sucrose+8 g/L agar could achieve a germination rate over 70%.  You［14］ added the basic medium MS with different combinations of BA (0.1, 0.5, 1.0 and 1.5 mg/L) and NAA (0.01, 0.05, 0.10 and 0.20 mg/L) to study the effects on seed germination. It was found that when using MS+0.5 mg/L BA+0.05 mg/L NAA+7 g/L agar, the germination rate was as high as 72%, and the obtained protocorm was colored green and clustered.

Subculture

The subculture stage is mainly to increase the multiplication of clumping sprouts. You［14］ studied the effects of zeatin (0.1, 0.5 and 1.0 mg/L), coconut juice (10, 30 and 50 ml/L) and 0.05 mg/L NAA on the tissue culture of  P. chinensis  on the growth of protocorm, and fount that the effect was best when using MS+0.1 mg/L ZT+30 ml/L coconut juice+0.05 mg/L NAA+7g/L agar. Liu  et al. ［9］ investigated 6 kinds of culture media, among which MS+50 g/L potatoes+20 g/L sucrose+8 g/L agar medium was beneficial to the differentiation of protocorm to plantlets, and the combination of low concentration 6-BA and NAA was suitable for protocorm proliferation and differentiation of buds.

In the subculture of  P. chinensis , Wen  et al. ［10］ selected N6 minimal medium, and performed orthogonal design using 2 factors (6-BA and NAA) and 3 levels (0.5, 1.0, 1.5 mg/L). They found that there were no significant differences in the effects of different phytohormone combinations on the growth of  P. chinensis  seedlings, and N6+1.5 mg/L+6-BA+0.5 mg/L NAA had a better effect. Lin  et al. ［11］ used MS added with 50 ml/L coconut juice as the basic medium, and carried out 6-BA (0, 0.5, 1.0, 2.0, 4.0 mg/L) and NAA (0, 0.1, 0.2, 0.4, 0.8 mg) /L) 2-factor 5-level tests. They found that MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+coconut juice 50 ml/L was the best proliferation medium. Li  et al. ［13］ used N6+6-BA 1.5 mg/L+NAA 0.5 mg/L medium for subculture. Wu  et al. ［12］ investigated the effects of different concentrations of NAA (0, 0.5, 1.0, 1.5 and 2 mg/L) on protocorm-induced proliferation, and determined that the optimal medium was MS+6-BA 1.0 mg/L+KT 0.5 mg/L+NAA 0.5 mg/L+peptone 1 g/L.

Rooting culture

Newly formed plantlets are often weak and small, and have no roots mostly, and the survival rate of plantlets after training and transplanting is low. The rooting culture of tissue-cultured plantlets mainly includes two methods: bottle rooting and filter paper bridge rooting. At present, the rooting method used in the genus  Pholidota  is mainly rooting in tissue culture bottles.

Liu  et al. ［9］ investigated the effects of adding activated carbon on the rooting of tissue-cultured plantlets when studying the tissue culture and rapid propagation technology of  P. cantonensis . The results showed that the tissue-cultured plantlets of  P. cantonensis  were easy to root, and the addition of activated carbon had little effect on it.

Li  et al. ［13］ studied the tissue culture technology of  P. chinensis  and found that the most suitable medium for rooting was KC+6-BA 2 mg/L+NAA5 mg/L+ coconut juice 50 ml+ activated carbon (AC) 500 mg/L or N6 basic medium+6-BA 0.5 mg/L+NAA 1.5 mg/L+ banana puree 75 g/L. Lin  et al. ［11］ investigated the effects of different concentrations of NAA (0, 0.1, 0.2, 0.4, 0.8 mg/L) on the rooting of  P. chinensis , and the results showed that 1/2MS+NAA 0.1 mg/L+AC 1 g/L was the best culture medium. Wen  et al. ［10］ used N6+0.5 mg/L 6-BA+1.5 mg/L NAA+75 g/L banana puree for rooting culture of  P. chinensis , and the rooting rate reached 85%.

Training and Transplanting

Plantlet training and transplanting is to select strongly-growing rooted plantlets for outdoor cultivation and transplant them to a loose and air-permeable substrate for field production after the plantlets have adapted to the external environment.

Lin  et al. ［11］ put rooted  P. chinensis  plants in the greenhouse for 4 d, and uncovered the bottles to continue training of the plantlets for 3 d. The obtained plants were washed to remove the medium, soaked with thiophanate-methyl 800 times dilution for 10 min, and transplanted in a substrate of perlite∶sand soil=1∶1 while keeping the air humidity above 75% and the ambient temperature at 18-28 ℃, and the survival rate reached over 85%. Xu  et al. ［15］ investigated the effects of different soil substrates, light intensity and growth temperature on the growth of  P. chinensis . The results showed that the cultivation effects of  Vnutrient soil∶Vwood chips=2∶1, Vnutrient soil∶Vmoss =2∶1 were better, and it was better to cultivate in a weak light environment avoiding direct sunlight, at a temperature of 25 ℃, with a water spray volume of 25 ml.

Wu  et al. ［12］ trained plantlets in culture bottles in the greenhouse under natural light for 5-7 d when training tissue culture plantlets of  P. chinensis  and air-dried the plants in a ventilated and cool place for 1 h after washing off the residual medium at the base of the tissue culture plants with clean water. The trained plants were transplanted into imported aquatic plant substrate, and a higher survival rate was achieved.

Common Problem Analysis

Browning refers to the phenomenon that culture materials release brown substances into their culture media, causing the culture media to gradually brown, and the culture materials brown or even die. The causes of browning mainly include improper plant material season and more phenolic substances contained in plants. The countermeasures are mainly obtaining materials in the season when plants are growing and adding antioxidants.

"Vitrification" of tissue culture plantlets refers to the phenomenon that the leaves and tender shoots of test tube plantlets are crystal transparent or semi-permeable and hygrophanous during plant tissue culture. Vitrified plantlets are caused by physiological abnormalities in carbon and nitrogen metabolism and water during the growth process after bud differentiation is initiated.  Different crop types or varieties have different frequency of occurrence of vitrification. The countermeasures are usually to control the temperature and humidity during tissue culture, and add reasonable growth regulators.

Tissue culture seedling contamination mainly refers to the phenomenon that tissue culture plantlets cannot grow normally due to the infection of fungi and bacteria during the growth of tissue culture plantlets. Contamination often occurs during the tissue culture process, which is usually caused by improper operation or improper sterilization time or strength. Therefore, choosing right sterilizing agents and time can effectively improve the pollution problem.

References

［1］ WEN XP, LIN QQ, YANG CZ,  et al.  Protection and development and utilization of  Pholidota chinensis  resources for medicine and food［J］. Strait Pharmaceutical Journal, 2015, 27(6): 46-47. (in Chinese)

［2］ ZHANG M, ZHU H, CHEN L,  et al.  Study on pharmacognosy identification of  Pholidota chinensis  Lindl.［J］. Lishizhen Medicine and Materia Medica Research, 2018, 29(9): 2177-2179. (in Chinese)

［3］ LI B, LI SX, LIU YX,  et al.  Study on extraction technology and content determination of polysaccharides in  Pholidota cantonensis ［J］. Hunan Journal of Traditional Chinese Medicine, 2015, 31(7): 161-163. (in Chinese)

［4］ VASIL LK. 1972. Totipotency and embryogenesis in plant cell and tissue cultures［J］. Vitro, 8(3): 117-127.

［5］ CHEN C. Establishment of tissue culture and rapid propagation system of  Bletilla striata ［D］. Changsha: Hunan Agricultural University, 2016. (in Chinese)

［13］ CHEN X. Research on application progress of plant tissue culture technology in agricultural production［J］. Seed Science & Technology, 2017, 35(10): 123-124, 128. (in Chinese)

［6］ LIU XF. Optimization of the rapid propagation system of  Dendrobium candidum ［D］. Taiyuan: Shanxi University, 2017. (in Chinese)

［7］ RAO QQ, DUAN X, YE ZR,  et al.  Study on tissue culture and rapid propagation of  Pinellia ternata ［J］. Journal of Yangtze University: Natural Science Edition, 2018, 15(2): 50-53, 6. (in Chinese)

［8］ CHEN WH, CHEN HH. Orchid biotechnology［M］. Singapore: World Scientific Publishing Co.Pte.Ltd., 2007.

［9］ LIU BC, HUANG YZ, ZHAO YQ,  et al.  A preliminary report on aseptic seeding and rapid propagation of  Pholidota cantonensis  Rolfe［J］. Fujian Journal of Agricultural Sciences, 2014, 29(5): 461-464. (in Chinese)

［10］ WEN XP, LIN QQ, YANG CZ,  et al.  Preliminary report on aseptic seeding and tissue culture of  Pholidota chinensis  Lindl［J］. Heilongjiang Agricultural Sciences, 2015(8): 19-21. (in Chinese)

［11］ LIN HF, CHEN CM, SHANG W,  et al.  Study on aseptic sowing and rapid propagation technology for  Pholidota chinensis  Lindl. seed［J］. Journal of Anhui Agricultural Sciences, 2017, 45(8): 161-162, 166. (in Chinese)

［12］ WU YH, YU HX, LI J,  et al.  Effect of medium on germination of  Pholidota chinensis  Lindl seeds［J］. Agricultural Engineering, 2019, 9(3): 107-111. (in Chinese)

［13］ LI CH, LI KC. Propagation of  Pholidota chinensis  Lindl. seedlings and greenhouse production［J］. China Flowers & Horticulture, 2018(22): 34-38. (in Chinese)

［14］ YOU YF. Propagation experiment of  Pholidota cantonesis  Rolfe.［J］. Agricultural Engineering, 2018, 8(8): 113-118. (in Chinese)

［15］ XU QX, LI JX, ZOU ZL,  et al.  Study on artificial cultivation technique of  Pholidota chinensis  Lindl［J］. Jiangxi Chemical Industry, 2017(6): 104-107. (in Chinese)