Key words: acclimatization, CO 2 concentration, photosynthesis, reducing sugar, total sugar

Transcription

1 Plant Cell, Tissue and Organ Culture 62: , Kluwer Academic Publishers. Printed in the Netherlands. 219 Number of air exchanges, sucrose concentration, photosynthetic photon flux, and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro Yong-Yi Cui 1, Eun-Joo Hahn 1, Toyoki Kozai 2 & Kee-Yoeup Paek 1, 1 Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheong-ju , Korea; 2 Faculty of Horticulture, Chiba University, Matsudo, Chiba , Japan ( requests for offprints; Tel: ; Fax: ; paekky@cbucc.chungbuk.ac.kr) Received 27 June 2000; accepted in revised form 19 September 2000 Key words: acclimatization, CO 2 concentration, photosynthesis, reducing sugar, total sugar Abstract Rehmannia glutinosa plantlets were cultured for 4 weeks under different culture conditions to determine the optimum environment for in vitro growth and ex vitro survival. Plantlet growth increased with an increasing number of air exchanges of the culture vessel, exhibiting greatest shoot weight, total fresh weight, leaf area, and chlorophyll content at 4.4 h 1 of air exchanges. High sucrose concentration (30 g l 1 ) increased root weight but reduced shoot growth. Net photosynthetic rates of the plantlets were greatest when sucrose was not added to the medium. On the other hand, ex vitro survival of the plantlets was not influenced by sucrose concentration. In the experiment on difference in photoperiod and dark period temperatures (DIF) and photosynthetic photon flux (PPF), plantlet growth increased as DIF and PPF levels increased. Particularly, increasing PPF level had a more distinctive effect on plantlet growth than increasing DIF level. The interaction of DIF PPF was also significant, showing the greatest plantlet growth in positive DIF (+8 DIF) and a high PPF (210 µmol m 2 s 1 ). In conclusion, the results of this experiment suggest that increased number of air exchanges of the culture vessel, decreased sucrose concentration, and positive DIF in combination with high PPF level enhanced growth and acclimatization of Rehmannia glutinosa plantlets. Introduction Rehmannia glutinosa is in the Scrophulariaceae family and is one of the most common and important medicinal herb plants. It is a perennial herbaceous plant and its root is used in medicine (Zhu, 1981). Fresh or dried roots of Rehmannia are used mainly for hematologic conditions, sedation, insomnia and diabetes (Xu, 1988). Propagation by root tubers caused serious deterioration of the plant as a result of virus infection during vegetative propagation, which resulted in tuber yield reduction (Xu, 1988). To solve the problem, there had been attempts for in vitro approaches and Jiang and Mao (1979) first reported callus induction and plant regeneration from various organs such as root, bud and stem. There are many reports of in vitro propagation of Rehmannia glutinosa by meristem culture (Mao et al., 1983), clonal propagation (Shoyama et al., 1983), somatic embryogenesis (Chae and Park, 1993), and by shoot tip and root segment culture (Paek et al., 1995b). Paek et al. (1995a) found that growth and tuber yield of micropropagated Rehmannia glutinosa increased but sugar and catalpol contents declined slightly compared to conventionally propagated Rehmannia glutinosa. However, problems still remain in micropropagation of Rehmannia glutinosa since the acclimatization rate is low, requiring a complicate procedure and high production cost. To increase acclimatization rate in vitro environments ought to be improved, so that the plantlets can grow healthy without any physiological and morphological disorders such as stomatal malfunction and hyperhydricity. Kozai and Iwanami

2 220 (1988) reported a significant increase in carnation plantlet growth by increasing number of air exchanges, CO 2 concentration, and photosynthetic photon flux (PPF). These researchers suggested that in vitro plantlets photosynthesize if the growing environment is controlled similar to greenhouse or field conditions. Since then, studies have proved a remarkable growth of a variety of in vitro plantlets under high PPF, high CO 2 concentration, and increased number of air exchanges (Miyashita et al., 1996; Niu and Kozai, 1997; Kitaya et al., 1998). In the case of Rehmannia glutinosa, there have been few reports regarding increases in quality and quantity of the plantlets by improving in vitro culture environments. Paek et al. (1995a) compared field performance of in vitro propagated Rehmannia glutinosa and of conventionally propagated ones and reported a significantly higher rate of ex vitro growth in micropropagated plants than conventionally propagated plants. Subsequently, Seon et al. (1999) reported that in vitro growth and ex vitro survival of Rehmannia glutinosa increased by photoautotrophic culture. In this respect, it could be possible to obtain higher rates of in vitro growth and ex vitro survival of Rehmannia glutinosa plantlets with further experiments on optimum culture conditions. In this experiment, therefore, Rehmannia glutinosa plantlets were grown in various environments to determine optimum culture conditions for in vitro growth and acclimatization. Materials and methods Plant material Axillary buds of Rehmannia glutinosa (selected at the Korean Crop Research Institute) were surfacesterilized in a 0.5% sodium hypochlorite solution for 10 min and rinsed three times with sterile distilled water. Shoot tips attached with two leaf primordia were excised from the axillary buds then placed into polypropylene growth vessels ( mm, Osmotek, Israel) containing 50 ml of agar medium. The basal medium consisted of MS (Murashige and Skoog, 1962) major salts supplemented with 1 mg l 1 benzyladenine (BA) and 30 mg l 1 sucrose. The ph was adjusted to 5.8 with 0.1 N NaOH before the medium was solidified with 0.6% (m/v) Bacto agar. The shoots induced were transferred to propagation medium (MS medium supplemented with 1 mg l 1 BA, 0.3mgl 1 indole acetic acid, and 30 mg l 1 sucrose) for the experiment. Number of air exchanges of the culture vessel Four different sizes (0.8, 2.0, 3.8 and 12.6 cm 2 )ofgaspermeable microporous filters (Mill-Seal, Millipore, Tokyo, Japan; pore size 0.5 µm) were attached on each side of the culture vessel to vary the number of air exchanges of the culture vessels. The number of air exchanges was estimated to be 0 (control), 0.8, 1.5, 1.9 and 4.4 per hour, respectively according to the method described by Kozai et al. (1986). Cultures were maintained for 4 weeks in a growth chamber where air temperature, relative humidity and PPF were adjusted to 25 C, 70 ±5%, and 70 µmol m 2 s 1, respectively. Fluorescent and metal halide lamps were used for the illumination with a 16-h photoperiod. Air temperature, relative humidity and PPF were daily monitored with a data logger model Li-1000 (LI-COR, Lincoln, Nebr.). Plant height, number of leaves, leaf area, total fresh weight, chlorophyll content and CO 2 assimilation rate were investigated after 4 weeks of culture. Leaf area and chlorophyll content were measured with a leaf area meter (Skye Co, UK) and a chlorophyll meter (SPAD-502, Minolta, Japan). Sucrose concentrations Plantlets were grown at three levels of sucrose (0, 15,and30mgl 1 ) for 4 weeks. Culture conditions were maintained the same as those of the previous experiment except increased air exchanges (4.4 h 1 ) and CO 2 supply (1000 µmol mol 1 ). Investigation of growth characteristics was also conducted the same as that of the previous experiment. CO 2 concentrations of inside and outside of the culture vessel were measured with a gas chromatograph (HP 6890, Hewlett Packard, Wilmington, DE). Extraction of total sugar and reducing sugar was carried out by a modified method of Martin et al. (2000). Leaves (0.5 g) and roots (0.5 g) were ground in a mortar with liquid nitrogen, to which 1 ml of 80% ethanol was added then filtered with a filter paper (0.45 µm, Whatman). The filtrates were recovered and the residues were washed again with 70% ethanol, filtered and both filtrates were mixed to which distilled water (3 ml) was added. The extract was centrifuged at a g for 15 min and 1 ml supernatant was taken. Dinitrosalicylic acid (3 ml) was added to the supernatant and the mixture was heated in boiling water for 5 min then cooled. Evaluation of the reducing sugar was done by the method of Somogyi (1952) and Nelson (1944). 0.2 ml of Nelson reagent plus 2.4 ml of distilled water were added to the mixture and the

3 221 mixture was shaken to eliminate CO 2. Absorbance was measured at 550 nm and D-glucose was used as a standard. To evaluate total sugar, method of Dubois et al. (1956) was employed. 0.5 ml of distilled water and 0.5 ml of 5% phenol were added to 0.1 ml of dry ethanolic extract. After shaking, 2.5 ml of concentrated H 2 SO 4 was added then the mixture was left for 10 min and absorbance was read at 470 nm. D-glucose was used as astandard. The method of Takahashi et al. (1995) was used for extraction of starch. The residue obtained after ethanol extraction was resuspended with 0.1 M sodium acetate buffer (ph 4.8) and boiled for 20 min. The gelatinized starch was digested with amyloglucosidase (Sigma, St. Louis, MO) for 4 h at 37 C, then boiled again to stop the enzymatic reaction. After cooling, the mixture was centrifuged and the level of glucose in the supernatant was determined by the method of Somogyi (1952) and Nelson (1944). Evaluation of starch was done by converting glucose to starch equivalents using a factor of 0.9 (Pucher et al., 1948). The net photosynthetic rates, Pn (µg plantlet 1 h 1 ) of the plantlets were estimated by the following equation: Pn = k F (C in C out )/n where k is a conversion factor of CO 2 from volume to weight ( gcm 3 at 25 C), F is the flow rate of the gas across the culture vessel (cm 3 h 1 ), C in and C out are CO 2 concentration at the gas inlet and outlet of the culture vessel (mg l 1 ) respectively, and n is the number of plantlets cultured in the culture vessel (Fujiwara et al., 1987). PPF and differences between photoperiod and dark period temperature (DIF) PPF was varied at 70, 140, and 210 µmol m 2 s 1. DIF was varied at 18/26 C ( 8 DIF), 25/25 C (0 DIF), and 25/18 C (+8 DIF) (photoperiod/dark period). Cultures were maintained for 4 weeks under the same environmental conditions as those in the previous experiment, after which measurements were done on plantlet growth and sugar determination. Statistical analysis The experiments were carried out in duplicate; there were 5 single-plantlet replications within each treatment and data were tested using the Statistical Analysis System (SAS Institute, Cary, NC). Results and discussion Effect of the number of air exchanges of the culture vessel After 4 weeks of culture, 4.4 h 1 of air exchanges resulted in the greatest plantlet growth (Table 1). More than three-fold leaf area was obtained at 4.4 h 1 of air exchanges (40.3 mm 2 ) compared to control (0 h 1 ) (12.1 mm 2 ). Shoot weight total fresh weight, and chlorophyll content were also greatest at 4.4 h 1 of air exchanges. Increasing number of air exchanges improved plantlet growth except number of leaves. On the other hand, no air exchanges resulted in retarded growth, showing lowest increases in plant height, leaf area, shoot weight and total fresh weight. Likewise, chlorophyll content of the leaves under control was only half of that under 4.4 h 1 of air exchanges. Inhibition of Magnolia plantlet growth under no air exchanges was caused by increase in C 2 H 4 concentration and toxic compounds inside the culture vessel (De Proft et al., 1985). Fujiwara and Kozai (1995) suggested increasing the number of air exchanges of culture vessels by using a gas permeable filter or by forced ventilation systems, resulting in significant increases in growth of in vitro plantlets. There are other reports that confirmed the distinctive effect of increased numberofairexchangesofculturevesselsongrowthof in vitro strawberry and Brassica campestris plantlets (Kozai and Sekimoto, 1988; Kozai et al., 1991). Effect of sucrose concentration on in vitro growth and ex vitro survival Sucrose concentration affected shoot and root growth but not survival rate during acclimatization (Table 2). A high sucrose concentration (30 g l 1 ) increased root weight, while decreasing shoot growth. In contrast, plantlet growth on the medium containing no sucrose and containing 15 g l 1 sucrose was great. No differences in plant height, number of leaves, leaf area, shoot weight, total fresh weight, and total dry weight were obtained between the two treatments (0gl 1 sucrose and containing 15 g l 1 sucrose) (Table 2). The reason for significant plantlet growth without sucrose supply can be explained with the culture environments during the experiment; increased number of air exchanges, CO 2 supply and high PPF. It has already been acknowledged that improvement of culture environments in micropropagation resulted in photoautotrophic growth of in vitro plantlets. That is, in vitro plantlets photosynthesize and do not require

4 222 Table 1. Effect of number of air exchanges on plant height, number of leaves, leaf area, total fresh weight and chlorophyll content of Rehmannia glutinosa plantlets after 4 weeks of culture No. of air Plant height No. leaves Leaf area Total fresh Chlorophyll exchange per (cm) (cm 2 plantlet 1 ) weight content hour (mg plantlet 1 ) (mgcm 2 ) 0 3.7b z 12.2 a 12.1 e c 2.90 d a 7.6 b 14.5 d c 3.38 c a 7.2 bc 18.3 c b 3.97 b a 7.0 bc 20.7 b b 4.10 b a 6.6 c 40.3 a a 4.51 a Table 2. Effect of sucrose concentration on growth of Rehmannia glutinosa after 4 weeks of culture Sucrose Plant No. of Leaf area Total fresh Total dry Chlorophyll Ex vitro conc. height leaves (cm 2 plantlet 1 ) weight weight content Survival (mg l 1 ) (cm) (mg plantlet 1 ) (mg plantlet 1 ) (mg plantlet 1 ) (%) a Z 8.2 a 83.5 a ab c 4.18 ab ab 8.6 a 75.5 a a b 4.53 a b 9.0 a 53.1 b b a 3.75 b 97 sucrose as a carbon source (Kozai and Iwanami, 1988; Desjardins, 1995; Roche et al., 1996). Total sugar, reducing sugar, and starch contents of the plantlets were also significantly affected by sucrose concentration in the culture medium. Increasing sucrose concentration resulted in high contents of total sugar, reducing sugar and starch of the plantlets (Table 3). The reason for greatest root and total dry weight at sucrose 30 g l 1 sucrose (Table 2) explains this result. Particularly, root weight at 30 g l 1 sucrose was almost double that at 0 g l 1 sucrose but shoot weight at 30 g l 1 sucrose was lower than that at 0 mg l 1 sucrose (Table 2). This result indicates that high contents of carbohydrates are accumulated in roots rather than other organs (Reuther et al., 1992). Sucrose concentration also affected net photosynthetic rate (NPR) of the plantlets, resulting in higher NPRin0gl 1 sucrose as compared to 15 g l 1 and30gl 1 sucrose (Figure 1). Studies have proved that high concentrations of sucrose in the culture medium prevented photosynthesis of in vitro plantlets and lowering sucrose concentration under improved culture environment increased CO 2 assimilation rate (Nakayama et al., 1991). This result indicated that sucrose is not necessarily required for the growth of in vitro plantlets under the culture conditions of high PPF, increased number of air exchanges, and CO 2 enrichment as has already been suggested (Kozai, 1991). The effect of DIF and PPF on growth of the plantlets Analysis of variance indicated that DIF and PPF affected plantlet growth, respectively. Plantlet growth increased as PPF and DIF levels increased. Particularly, increasing PPF level had a more distinctive effect on plantlet growth than increasing DIF level (Table 4). Negative DIF ( 8 DIF) significantly inhibited plantlet growth, resulting in the lowest plant height, number of leaves, leaf area, total fresh weight and total dry weight. This result is in agreement with the result of reduced growth of rosebay rhododendron seedlings under negative DIF (Starrett et al., 1993). Kozai et al. (1995) reported that 0 or negative DIF remarkably suppressed stem length of in vitro potato plantlets but not leaf area and number of leaves. It has already been acknowledged that the effect of DIF on plant growth was dependent on the responses of plants to photoperiod (Myster and Moe, 1995). The interaction of DIF PPF was also significant. The effect of PPF on plantlet growth was significant when combined with positive and zero DIF but became less effective under negative DIF. The reason for decreased effect of PPF under

5 223 Table 3. Content of reducing sugar, total sugar and starch in leaves and roots of in vitro Rhemannia glutinosa grown under different sucrose concentrations Sucrose Reducing sugar Total sugar Starch content concentration (mg FW g 1 ) (mg FWg 1 ) (mgfw g 1 ) (mg l 1 ) Leaf Root Leaf Root Leaf Root c Z 2.08 c c 9.94 c c c b b b b b b a a a a a a Figure 1. Difference in growth of Rehmannia glutinosa grown at different culture conditions. (A) The number of air exchanges; (B) Sucrose concentrations; (C) DIFs;(D) PPFs;(E) Transplants of Rehmannia glutinosa after acclimatization in a green house. negative DIF was thought to be the low day temperature (18 C) that suppressed shoot growth. Among the treatments, positive DIF (+8 DIF) and a high PPF (210 µmol m 2 s 1 ) resulted in the greatest increases in plant height (9.3 cm), leaf area (7.65 cm 2 ), fresh weight (5.67 g plantlet 1 ), and dry weight (0.47 g plantlet 1 ) (Table 4). These results indicate that the effect of DIF was enhanced when other environmental factors were combined, which has already been suggested from other experiments (Moe and Heins, 1990; Erwin et al., 1991; Kaczperski et al., 1991). Conclusion The results of our experiment suggest that high rates of in vitro growth and acclimatization of Rhemannia glutinosa plantlets could be obtained by optimizing the culture conditions. Increased number of air exchanges (4.4 h 1 ) resulted in a significant shoot and root growth compared to no air exchanges. There was no need to maintain high sucrose concentration in the culture medium under proper culture conditions (increased number of air exchanges, CO 2 supply, positive DIF and high PPF), showing greater plantlet growth

6 224 Table 4. Plant height, number of leaves and leaf area of Rehmannia glutinosa plantlets grown at different DIF and PPF levels after 4 weeks of culture Treatment Plant height No. leaves Leaf area DIF (A) PPF (B) (cm) (cm 2 plantlet 1 ) (Day/night, C) (µmol m 2 s 1 ) 18/ c z 10.5 bc 36.7 e c 10.0 bc 38.9 de c 9.7 c 51.3 c 25/ bc 11.2 abc 44.5 d abc 12.5 a 61.6 b a 11.5 ab 55.9 bc 26/ ab 11.2 abc 39.7 de ab 11.0 abc 57.9 bc a 11.2 abc 76.5 a Significance Temperature (A) PPFD (B) Ns Interaction (A B) Ns Ns Ns, : Nonsignificant or significant at p 0.05 or 0.01, respectively. Table 5. Shoot weight, root weight and total fresh and dry weight of Rehmannia glutinosa plantlets grown at different DIFs and PPFs after 4 weeks of culture Treatment Mean shoot Mean root Total fresh Total dry DIF (A) PPF (B) weight weight weight weight (Day/night, C) (µmol m 2 s 1 ) (mg plantlet 1 ) (mg plantlet 1 ) (mg plantlet 1 ) (mg plantlet 1 ) 18/ c cd f fg (+8 DIF) c c ef def c ab bc bc 25/ c d f efg (0 DIF) b cd de cde b b b b 26/ c d f g ( 8 DIF) b c cd cd a a a a Significance Temperature (A) PPFD (B) Interaction (A B) Ns, : Nonsignificant or significant at p 0.05 or 0.01, respectively. at sucrose concentrations of 0 g l 1 and/or 15 g l 1 compared to 30 g l 1. The present results could allow productionof Rhemannia glutinosa plantlets with time and cost reduction by solving problems of low rates of in vitro growth and acclimatization.

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