Tissue Culture and Rapid Propagation Techniques of New Variety in Cymbidium hybridum

Liyuan YANG Xiangbei LI Shaobo ZHANG Xiaofei YANG Jinqiao PENG2, Rongjiao DONG Xinglong WU

Abstract ［Objectives］ This study was conducted to select media suitable for proliferation, differentiation and rooting of Cymbidium hybridum "Huangjinjia".

［Methods］ The lateral buds and protocorms of the new variety C. hybridum "Huangjinjia" were used as materials to explore the effects of different concentrations of 6-BA and NAA on protocorm proliferation and rooting.

［Results］ The optimal medium for protocorm propagation was 1/2 MS+6-BA 1.0 mg/L+NAA 0.5 mg/L +potato 50 g/L+sucrose 20 g/L, in which the protocorms multiplied easily and grew rapidly. The optimal medium for inducing adventitious buds was 1/2 MS+6-BA 1.5 mg/L+NAA 0.3 mg/L+sucrose 30 g/L+banana 25 g/L+apple 25 g/L+activated carbon 1.0 g/L, in which the induction rate of adventitious buds reached 335%. The optimal medium for rooting was 1/2 MS+NAA 0.5 mg/L+sucrose 25 g/L+banana 75 g/L+apple 25 g/L+activated carbon 1.0 g/L , in which the average root length was 3.0 cm, the average number of roots was 2.6, and plantlets had green leaves, thick roots and suitable plant height.

［Conclusions］ This study provides a theoretical basis and reference for the establishment of a rapid propagation system using lateral buds.

Key words Cymbidium hybridum ; Tissue Culture; Protocorm; Proliferation and differentiation

Received: March 8, 2020Accepted: May 8, 2020

Liyuan YANG (1966-), female, P. R. China, lecturer, devoted to research about plant cultivation and physiology.

*Corresponding author. E-mail: 1976246961@qq.com.

Cymbidium hybridum is a perennial herbaceous plant of Cymbidium in Orchidaceae. It is loved by people around the world and has great market potential due to its long flowering period and gorgeous and huge flowers［1-4］. C. hybridum can be propagated with seeds, but the conventional seed germination rate is very low, so it is difficult to reproduce and produce in large quantities. Moreover, most C. hybridum varieties are hybrids, which are unable to maintain the characteristics of their varieties, and have relatively weak ability to survive after plant division. It is difficult to carry out large-scale production to meet people餾 increasing demand, which largely limits the development of C. hybridum industry［5-7］. The use of plant tissue culture technology can obtain genetically stable plantlets in a short period of time to meet market demand［8-9］. The plant tissue culture technology of C. hybridum was carried out earlier at abroad. Morel［10］ successfully cultivated complete plants with the stem tip of the pseudobulb of C. hybridum . In the 20﹖h century, China began to carry out tissue culture of C. hybridum . Although the research started relatively late, there is still a distance from the level of countries at abroad. However, there are also many researches on the tissue culture technology of C. hybridum in China, and some breakthroughs have been made［5,11-13］.

C. hybridum "Huangjinjia" is a new variety bred from "Caishen" and "Yangguifei" by the Qixing Flower Planting Base in Jiangchuan District, Yuxi City, Yunnan Province, and Yunnan Agricultural University. This variety has a moderate plant type, a high bolting rate, and more flowers, which have larger petals of clear color, red tongue, round lips with a touch of red, and yellow flower edges. It is very popular in the market. In this study, the lateral buds and protocorms of "Huangjinjia" were used as materials to select media suitable for the proliferation, differentiation and rooting of C. hybridum "Huangjinjia", so as to establish a rapid propagation system using lateral buds.

Materials and Methods

Experimental materials

Plant materials

The lateral buds and protocorms of C. hybridum "Huangjinjia" (F1 of C. hybridum "Caishen" and "Yangguifei") were provided by the Qixing Flower Planting Base in Jiangchuan District, Yuxi City, Yunnan.

Concentration combinations of 6-BA and NAA

Experimental methods

Disinfection of explants

Moderately sized and intact lateral buds were selected from the mother plant. The outermost leaves were stripped off, and the upper leaves were cut flat. The buds were clean with detergent, rinsed three times with aseptic water, then wiped with 75% ethanol, and finally soaked in 50 mg/L chlorine dioxide disinfectant for 20 min with shaking. After taken out, they were wiped with 75% ethanol, and the leaves were stripped off one by one with a sterilized scalpel, leaving the exposed buds, which were put in 50 mg/L chlorine dioxide disinfectant, and soaked for 15 min with shaking. Finally, a high-temperature sterilized scalpel was used to carefully cut the tender buds off on an ultra-clean workbench. The bud tips were inserted and fixed into a medium with high-temperature sterilized forceps.

Proliferation of protocorms

After the explants survived, dense protocorms formed around the explants through about 40 d of cultivation. Proliferation and cultivation of the protocorms could quickly provide more protocorms for subsequent experiments. The protocorms of the same size with good growth were selected, and a high-temperature sterilized scalpel was used to remove the attached medium. Different concentrations of 6-BA (0.5, 1.0, 1.5 mg/L) and NAA (0.5, 1.0 mg/L) were added into 1/2 MS, forming six different treatments, and then sucrose (20 g/L) and potato (50 g/L) were added into each medium for proliferation culture. The cultivation conditions were: temperature (23±2) ℃, relative air humidity 60%, light intensity 2 000 lx, and illumination time 12 h/d. If vitrification occurs, lowering the temperature can relieve it. Too-high relative air humidity will inhibit growth, and too low will easily breed bacteria to cause contamination. The cultivation time was 30 d, during which the protocorm proliferation was observed and recorded every day.

Induction of adventitious buds

Protocorms with good growth, emerald green color and a diameter greater than 3 mm were selected and inoculated on 1/2 MS medium, which was added with different concentrations of 6-BA (0.5, 1.0, 1.5, 2.0, 2.5 mg / L) and 0.3 mg/L NAA, followed by sucrose 30 g/L, banana 25 g/L, apple 25 g/L, potato 75 g/L and activated carbon 1 g/L. Induction culture was performed under suitable temperature, humidity and light conditions. Each culture medium was inoculated with 40 protocorms, and each treatment included 5 bottles. The cultivation time was 45 d. The growth of adventitious buds was observed and recorded every day. Finally, the number of adventitious buds was counted, and the average bud length and bud ratio were calculated.

Rooting culture

The plantlets that were induced and differentiated from protocorms, grew well and had a plant height of 3 to 4 cm, were selected. A sterilized scalpel was used to cut off protocorms and attached medium. The basic medium was 1/2 MS medium, which was added with different concentrations of NAA (0.1, 0.5, 1.0, 1.5, 2.0 mg / L), as well as activated carbon 1.0 g/L, apple 25 g/L, banana 75 g/L, potato 30 g/L and sucrose 25 g/L. Each culture medium was inoculated with 40 plantlets, and each treatment included 5 bottles. The plantlets were cultured for 40 d under suitable temperature, moderate and light conditions, during which the rooting condition was observed and recorded. The average number of roots and average root length were calculated.

Training and transplanting of plantlets

When the plants were about to grow to the mouth of the culture bottle, they were transferred to a larger culture bottle for cultivation. When the plants were 15 cm in length, they can be trained. The plants were transferred to greenhouse for about half a month. The temperature of the greenhouse was controlled at 28 ℃, and 50% of the sunshine was shielded. The purpose of plantlet training is to make seedlings grow stronger and adapt to the external environment more quickly to improve the survival rate of seedlings.

When transplanting the plantlets, the culture medium on the roots was rinsed away. The substrate was disinfected pine bark. After transplanting, the plants were watered with a sprayer once a day. The temperature was controlled at 25-30 ℃ and 50% of the sunshine was shielded. After half a month, pesticides were sprayed once a week to prevent various diseases and pests. After new roots grew out, foliage top dressing were carried out once a week.

Results and Analysis

Effects of different media on the proliferation of "Huangjinjia" protocorms

It can be seen from Table 2 that 6-BA and NAA were combined into 6 formulations, and the effect of T2 was relatively best, that is, the concentration of 6-BA was 1.0 mg/L and the concentration of NAA was 0.5 mg/L. With the conditions of T2, the protocorms had a large diameter, a tender green color, a fast proliferation rate, a large number, and a proliferation rate of 295%. The effect of T3 was second, and the number of protocorms multiplied was not as large as that of T2. The relatively worst treatment effects were observed in T1 and T6, which showed proliferative state of the protocorms far worse than T2, and the proliferation rates were only 135% and 150%, respectively. When the concentration of NAA was 0.5 or 1.0 mg/L, higher and lower concentrations of 6-BA slowed down the growth of protocorms and affected the state of protocorms, indicating that higher and lower concentrations of 6-BA both inhibited the proliferation of protocorms. When the concentration of 6-BA was the same and the concentration of NAA increased from 0.5 to 1.0 mg/L, the number of protocorms proliferated, the proliferation rate and the diameter all decreased, indicating that a higher concentration of NAA inhibited the proliferation of protocorms to a certain extent. The above results indicated that the optimal medium for protocorm propagation was 1/2 MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+potato 50 g/L+sucrose 20 g/L.

Effects of different media on the induction of adventitious buds of "Huangjinjia"

It can be seen from Table 3 that Y3 had the best effect, showing an induction rate reaching 335%, and the buds were emerald green, robust and grew evenly. The effects of Y2 and Y4 were second, with induction rates of 225% and 250%, respectively. The effects of Y1 and Y5 were significantly weaker. When the concentration of NAA was constant and the concentration of 6-BA was increased, the ability to induce adventitious buds also was gradually enhanced, but when the concentration of 6-BA exceeded 1.5 mg/L , the ability to induce adventitious buds was gradually weakened, which was reflected by decreased germination rate and slow and poor bud growth. Low concentrations of 6-BA had a greater influence on the average bud length. The average bud lengths of Y1 and Y2 were 1.3 and 2.6 cm, respectively. Higher concentrations of 6-BA led to an average bud length of more than 3.0 cm. The results showed that the optimal medium for inducing adventitious buds was: 1/2 MS+6-BA 1.5 mg/L+NAA 0.3 mg/L+﹕ucrose 30 g/L+banana 25 g/L+apple 25 g/L+activated carbon 1.0 g/L.

Effects of different culture media on the rooting of "Huangjinjia"

It can be seen from Table 4 that G2 had the best effect, and the plants had an average root length of 3.0 cm and an average root number of 2.6. And the plants grew best, with green leaves, thicker roots, and suitable height. The effects of G1 and G3 were second. In the G1 treatment, the root growth of plants was relatively slow, and the plantlets were weak. The roots of plants in G3 were relatively thin. When the concentration of NAA was greater than 1.0 mg/L, the rooting of the plants was inhibited, and the number of roots tended to increase first and then decrease. The plantlets in G4 and G5 grew the worst, with lighter colors and fewer roots, and were weaker. The results showed that the optimal medium for rooting of C. hybridum "Huangjinjia" was 1/2 MS+NAA 0.5 mg/L+sucrose 25 g/L+banana 75 g/L+apple 25 g/L+activated carbon 1.0 g/L.

Discussion and Conclusions

There are many kinds of explants that can be selected for C. hybridum  tissue culture, which can be divided into stem tip culture, lateral bud culture, leaf culture, root tip culture, etc. The medium is one of the important factors that determine the success or failure of plant tissue culture, and its main effect is to provide energy and nutrients to the isolated plant organs. There are dozens of media that can be used for tissue culture. There are also many media that can be used for plants in Orchidaceae. Commonly used basic media are MS medium, 1/2 MS medium, N6 medium, White medium, KC medium, etc. Zhang et al. ［14］ studied the in-vitro culture of C. hybridum using pollen, and selected 1/2 MS medium, which showed the highest germination rate in vitro . Zeng et al. ［15］ used 1/2 MS medium as the germination medium for hybrid seeds of C. hybridum and C. sinense, and achieved good results. Sun et al. ［16］ studied the effects of different culture media on the proliferation and differentiation of protocorms of the hybrid of C. goeringii and C. hybridum , and found 1/2 MS medium had a better effect, with a differentiation rate of 88%, which was significantly better than KC and B5 medium. Li et al. ［17］ successfully induced the differentiation of protocorms of the hybrid offspring of C. goeringii and C. hybridum using N6 medium. Zhu et al. ［18］ believed that 1/2 MS medium could promote the rapid propagation of C. hybridum . Plant growth regulators play an indispensable role in plant tissue culture. Although the amount added in the medium is small, the effect is very large. They can induce the formation of calli and the formation of adventitious buds and adventitious embryos, and promote rooting［19］. Li et al. ［20］ found that when the ratio of cytokinin/auxin in MS basic medium was 3.0, and the callus induction rate reached the highest. They study of Wei et al. ［21］ showed that adding 10%-20% of coconut juice and banana juice promoted the proliferation of orchid rhizomes. Meng et al. ［22］ found during the rooting culture of hybrid Cymbidium , adding 0.05 g/L of activated carbon to the medium achieved a good rooting state of test tube seedlings. This test was based on previous studies, and showed through the selection of different media that the effect was better when the 6-BA concentration was 1.0-1.5 mg/L and the NAA concentration was 0.1-0.5 mg/L.

In the process of establishing the plant tissue culture system of C. hybridum "Huangjinjia", it is of practical significance to find out the appropriate medium formula required for each growth period. The main factors that can affect the growth of orchids in the medium formulation include the type of basic medium, the type and concentration of additives, the type and concentration of growth regulators, and so on. In addition to these, the cultivation conditions are also very important, such as light, temperature and humidity, as well as the transplanting substrate, all of which affect the growth of C. hybridum .

In this study, the results showed the suitable medium for protocorm propagation was 1/2 MS+6-BA 1.0 mg/L+NAA 0.5 mg/L+potato 50 g/L+sucrose 20 g/L. The suitable medium for inducing adventitious buds was 1/2 MS+6-BA 1.5 mg/L+NAA 0.3 mg/L+sucrose 30 g/L+banana 25 g/L+apple 25 g/L+activated carbon 1.0 g/L. The medium suitable for rooting was 1/2 MS+NAA 0.5 mg/L+sucrose 25 g/L+banana 75 g/L+apple 25 g/L+activated carbon 1.0 g/L.

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