Effect of CO2 Concentration on Sugar-free Micropropagation of Chrysanthemum

SONG Yue-dong, MA Ming-jian

School of Agricultural and Food Engineering; Shandong University of Technology; Zibo, 255049, China E-mail:songyuedong68@qq.com

Abstract: the growth rate of sugar-free micropropagation of chrysanthemum was observed in our own tissue culture system. The effects of different CO2 concentration on growth characteristic of chrysanthemum plantlets were analyzed. The results showed it was efficient to promote the growth of sugar-free tissue culture of chrysanthemum appropriately enhancing CO2, the growth rate of the plantlets was significantly enhanced when the CO2 concentration was 1600�mol.mol-1. Key words: sugar-free micropropagation; liquid culture; CO2 con-centration

Due to confinement of a conventional vessel, plant is-sues can not realize gas exchange with the outside. As a result, the CO2 concentration in the culture vessel is low in the daytime and too high at night and there are also the problems of weak illumination and high humidity. All these limit the photosynthesis of plants [1~3], and force the plants to turn to heterotrophic growth or mixotrophic growth. Exogenous sugar must be added in the culture medium to provide a carbon source needed for growth, whereas sugar will generate photosynthetic inhibiting effect [4]. In order to change the situation, sugar-free micropropagation emerged and became a new research subject of micropropagation workers in every country [5-6]. This research adopted an open space micropropagation system developed by our task force, analyzed the adjustment of CO2 concentration under sugar-free culture condition, studied its effect on chrysanthemum growth and provided a theoretical basis for realizing sugar-free large-space micropropagation. 1 Materials and method 1.1 Materials

The chrysanthemum micropropagation plantlets culti-vated by our research center were chosen as the test mate-rials. 1.2 Method

The bottle chrysanthemum plantlets with consistent growth state are chosen and cut into sections. Each section has two internodes and is inoculated in the sugar-free mi-

cropropagation system developed by us. This system adopts 1/2MS liquid culture, without any hormones; the matrix is pearlite. The pearlite is put into a �3cm 5cm high nutrition bowl and the space of plant rows is 5cmx5cm. Meanwhile, a conventional method is used to inoculate 20 bottles of micropropagation plantlets as a control group (CK). The culture medium is MS, without any hormones. Cane sugar concentration is 20gl-1. The culture vessels are 350ml transparent glass bottles, filled with 50ml of culture solution. 3 plantlets are inoculated in each culture bottle. The control group (CK) is also put into the open space mi-cropropagation system and cultivated.

CO2 concentration is set to be: 700, 1000, 1300, 1600, 1900�mol.mol-1 respectively. The concentration is regu-lated from 9:00 to 15:00. The CO2 concentration in other time rests with the natural state of the system. Each treat-ment group has 25 plants and is measured three times.

During cultivation, the temperature is controlled at 24-28 in the daytime and 18-20 at night; the relative humidity is maintained at 70-80% in the daytime and 90-95% at night; and illumination intensity is 3500LX and illumination period is 12h.d-1. After 21d’s cultivation, the growth indexes of each treatment group are measured. Seven plants are randomly taken from each treatment group and control group and measured three times. 2 Result and analysis 2.1 Effect of CO2 concentration on root system

Root number and length were related to CO2 (Figure 1). The number of roots in each of the five treatment groups was greater than the number of roots in the control group, more than 3 times (1600�mol.mol-1) the number of roots in the control group at most, and more than two times (1900�mol.mol-1) in the least. With the increase of CO2 concentration, the number of roots increased, too, but when concentration reached 1900�mol.mol-1, the number of roots was less than the value when concentration was 1600�mol.mol-1, indicating it is not that higher CO2 con-centration is better. With the increase of concentration, root length increased. When concentration was 1600�mol.mol-1, root length was largest, more than two times that of the control group. After that, with the increase of concentration,

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root length decreased. However, the research discovered that although the root number and length of the control group were low, the roots were thick and strong and only they were dark and did not have branches and lacked root hair. In comparison, the roots in the five treatment groups

were fair and clear and densely distributed with fine root hair and also had a great many branches. This indicates the roots in the control group had weak or no absorption ca-pacity. 2.2 Effect of CO2 concentration on leaves

A B Figure 1 Effect of CO2 on root A number of roots B length of roots

The leaf properties of chrysanthemum micropropagation plantlets were related to CO2 (Table 1). With the increase of CO2 concentration, the leaf area of the plants increased, too, but when concentration was 1900�mol.mol-1, the leaf area of plants was smaller than 1600�mol.mol-1, while variance analysis had no difference. When concentration was 1600�mol/mol, the leaf area of plants even reached or ex-ceeded 3.1 times the leaf area in the control group. Even the least leaf area (700�mol.mol-1) in a treatment group was 1.5 times the leaf area in the control group. As for the number of leaves, all treatment groups did not have differ-ence from the control group. This result is different from the report of the document [7].

Table 1 Effect of CO2 concentration on leaves

Treatment (CO2 concentration)

Leaves (number/strain) Leaf area(cm2/strain)

700�mol.mol-1 6.3±0.1a 2.90±0.04a 1000�mol.mol-1 6.5±0.2a 3.47±0.02b 1300�mol.mol-1 6.4±0.3a 4.80±0.01c 1600�mol.mol-1 6.3±0.2a 5.83±0.01d 1900�mol.mol-1 6.1±0.3a 5.51±0.03d

ck 6±0.1a 1.86±4e

Note: Different lower-case letters indicate Duncan’s significant test shows significant difference (P<0.05).

2.3 Effect of CO2 concentration on plant height, crown width, internode length and stem thickness

The relations of CO2 with plant height, crown width, in-

ternode length and stem thickness are shown in Figure 2. The crown width in each treatment group was larger than that in the control group and the crown width increased with the increase of concentration, but when concentration exceeded 1600�mol.mol-1, the crown width decreased, in-dicating it is not that higher concentration is better. With the increase of concentration, stem thickness increased. When concentration exceeded 1600�mol.mol-1, stem be-came thinner, similar to the change rule of crown width. The increase of CO2 concentration resulted in short and thick plants and short internodes. All treatment groups were different from the control group, but the difference among different concentrations is not very obvious. The reason is that after more CO2 was applied, the photosyn-thetic materials of the plants were sufficient, photosynthe-sis was enhanced and the plants became stronger. This in-dicates increase of CO2 in an open space may make plants stronger and raise the adaptability of the cultivated micro-propagation plantlets to natural environment. 2.4 Effect of CO2 concentration on plant weight

Plant weight was related to CO2 (Table 2). With the in-crease of CO2 concentration, both fresh weight and dry weight of the plant increased. When CO2 concentration reached 1600�mol.mol-1, weight reached maximum. After that, when concentration increased, plant weight decreased. This indicates excessive concentration is not good for plant growth. The fresh weight and dry weight in each treatment group were higher than those in the control group. The highest fresh weight (1600�mol.mol-1) was 2.62 times the

fresh weight in the control group, and the lowest (700�mol.mol-1) was 1.32 times. The effect in the aspect of dry weight was even more obvious, the highest (1600�mol.mol-1) is 3.1 times and the lowest (700�mol.mol-1) was 1.47 times. This is because the in-crease of CO2 concentration in an open space makes the

photosynthetic raw material of the plants sufficient and enhances photosynthesis of the plant, thus increasing the accumulated dry substance of the plants as manifested by obvious increase of plant weight compared to the control group.

A B

C D

Figure 2 Effect of CO2 concentration on culm height (A) , culm expansion (B), culm length (C) and culm diameter (D) Table 2 Effect of CO2 concentration on culm weight

Treatment(CO2 concentra-tion)

Total weight(mg/strains) Dry weight (mg/strains)

700�mol.mol-1 328.3±0.4a 24.9±0.2a

1000�mol.mol-1 457.6±0.1b 34.3±0.1b

1300�mol.mol-1 576.3±0.3c 46.7±0.3c

1600�mol.mol-1 649.8±0.1d 52.6±0.1d

1900�mol.mol-1 483.9±0.2e 38.8±0.4e

ck 247.9±0.3f 16.9±0.1f

Note: Different lower-case letters indicate Duncan’s significant test shows significant difference (P<0.05).

3 Discussion

CO2 is a raw material of plant photosynthesis. From the research on addition of CO2 to the open space sugar-free chrysanthemum micropropagation plantlets, we may see that the obtained plantlets were short and strong, similar to the naturally cultivated plantlets, and all growth indexes

ere completely different from conventional micropropa-

gation plantlets. The plantlets may be directly used for production in fields. The success of this research lays a practical foundation for industrial production of micro-propagation plantlets.

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