Plant Cell, Tissue and Organ Culture 63: 67–72, 2000. © 2001 Kluwer Academic Publishers.

Printed in the Netherlands.

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Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis
Park So Young, H.N. Murthy & Paek Kee Yoeup∗
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-762, Korea (∗ requests for offprints; E-mail: paekky@chucc.chungbuk.ac.kr)
Received 31 May 2000; accepted in revised form 7 November 2000

Key words: bioreactor, liquid cultures, micropropagation, orchid

Abstract Protocorm-like bodies (PLBs) formed on leaf segments in vitro were used as explants for bioreactor cultures. Continuous immersion cultures (air lift column and air lift-balloon bioreactor), and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections. A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. About 18,000 PLBs were harvested from 20 g of inoculum (∼1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Aeration in a bioreactor at 0.5 or 2.0 volume of air per volume of medium min−1 (vvm) yielded similar levels of biomass production. PLBs grown in bioreactors were cultured on solid Murashige and Skoog, Vacin and Went, Knudson C, Lindemann and Hyponex media. Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors, rate of proliferation, and regeneration. Abbreviations: BA – N6 -benzyladenine; NAA – α-naphthaleneacetic acid; MS – Murashige and Skoog medium; VW – Vacin and Went medium; KC – Knudson C medium; LM – Lindemann medium; PLBs – Protocorm-like bodies; vvm – volume air/volume medium min−1 Introduction Phalaenopsis is an important ornamental orchid, but is difficult to propagate vegetatively. Propagation is generally by in vitro seed germination. However, seedlings obtained through this method are not uniform and there will be segregation of flower color. Therefore, Phalaenopsis’ growers prefer tissue-cultured plants. Induction of PLBs from leaf segments is a popular method but this yields low number of PLBs and the time taken for PLB multiplication is long when compared to seed culture. Recently, cells, somatic embryos or organogenic propagules like bulblets, corms, microtubers or shoots have been cultured in liquid suspension in bioreactors (Stuart et al., 1987; Akita and Takayama, 1988; Preil and Beck, 1991; Takahashi et al., 1992; Contliffe et al., 1993; Gupta et al., 1993; Shigeoko et al., 1994; Seon et al., 2000). The use of bioreactors for micropropagation will help in scaling-up of production and decreases the cost of production (Preil, 1991; Vasil, 1994; Leathers et al., 1995; Son et al., 1999). Large scale production of embryos in bioreactors was achieved in alfalfa, birch, carrot, coffee, sandalwood, spruce, sweet potato (see Leathers et al., 1995). Similarly, organogenic propagules were cultured in bioreactors for mass propagation in lily, potato, Stevia sps. (Akita and Takayama, 1988; Takahashi et al., 1992; Shigeoko et al., 1994; Son et al., 2000). There are few reports on propagation of orchids in bioreactors. Aitken-Christie et al. (1995) have suggested the possibility of mass production of protocorms in bioreactors. We have initiated various experiments for mass multiplication of Phalaenopsis hybrids using bioreactor systems. In this paper we present a successful method for mass cultivation of PLBs of Phalaenopsis in bioreactors. We have also

68 established a convenient protocol for conversion of PLBs into plantlets. inoculum (∼1000 PLB sections) were inoculated into the medium. For the temporary immersion bioreactors, PLB sections were placed on a plastic net (sieve) installed in the vessel. The specially designed feeding line was equipped to change or supply of medium. The system was programmed to immerse the PLB sections in medium for 5 min followed by without medium 2 h by the timer and solenoid valve. In continuous immersion culture, PLB sections were submerged in liquid medium during the whole period of culture. The medium was aerated by membrane filtered air through sinter disc at the base of bubble or column airlift bioreactors. The rate of airflow was measured by a flow meter (Dwyer In., USA). 0.5 and 2.0 vvm (volume air/volume medium/min) were tested on the growth of biomass. In addition, an aseptic sampling device, an air vent apparatus and an air flow meter was attached to each bioreactor. All the bioreactor cultures were kept at 25±2 ◦ C and illuminated continuously by cool white fluorescent lamps, 15–20 µmol m−1 s−1 at the outer jacket of the vessel. All the bioreactor cultures were maintained up to 8 weeks and after 8 weeks each PLB clusters were used to estimate proliferation rate (number of PLBs developed on each protocorm section or explant) and fresh weight of total biomass was calculated. Plantlet regeneration from PLBs PLBs which were harvested from bioreactor cultures were inoculated individually on MS, VW (Vacin and Went, 1949), KC (Knudson, 1946), LM (Lindemann et al., 1970) and Hyponex (6.5N – 4.5P – 19K 1 g l−1 + 20N – 20P – 20K 1 g l−1 ) media. All the media were supplemented with 45 g l−1 sucrose, pH was adjusted 5.7±0.1 and the media were solidified with 7 g l−1 agar. 30 ml of medium was taken in 115 mm×50 mm polystyrene boxes (Tong Yang Moolsan Co., Seoul, Korea) and was autoclaved at 110 k Pa for 20 min at 120 ◦ C. The cultures were incubated at 25±2 ◦ C and 16-h photoperiod under 60 µmol m−2 s−1 . The plantlets were subcultured at intervals of 4 weeks to fresh medium containing 0.5% activated charcoal (Sigma). Experimental design, data analysis and transplantation of plantlets Each experiment had six replicates and was repeated twice. The average was calculated and compared using Duncan’s multiple range test. Mean values with different letter differ significantly at p < 0.05. The plantlets (3–4 cm height) were transferred to pots

Materials and methods Plant material A Phalaenopsis hybrid that bears pink striped flowers was used in this work. Initially, flower stalk sections containing nodal buds (2 cm long) were sterilized in 0.2% mercuric chloride for 10 min and washed thoroughly in sterilized distilled water. They were cultured on Murashige and Skoog (1962) solid medium supplemented with sucrose (45 g l−1 ) and BA (3 mg l−1 ). The leaves emerging from nodes were used for PLB induction. The young leaves were cut into 5×10 mm sections and cultured on MS medium containing 45 g l−1 sucrose and BA (15 mg l−1 )+NAA (1 mg l−1 ). PLBs developed on leaf sections after 8 weeks of culture. These PLBs were cut transversely and sections bearing apical meristems (about 2 mm in size) were used as explants for PLB proliferation study. Agitated flask cultures Erlenmeyer flasks (250 ml) each containing modified Hyponex medium (Kano, 1965; 6.5N – 4.5P – 19K 1 g l−1 + 20N – 20P – 20K 1 g l−1 + 1% potato homogenate) were agitated on a horizontal gyrotory shaker at 100 revolutions per min. To verify the optimum medium volume needed for growth of PLBs, 0.5 g inoculum (protocorm sections) were cultured in various volumes of medium (25, 50, 100 ml). For the analysis of optimum inoculum density on PLB proliferation, 0.5, 1.0 or 2.0 g of inoculum was cultured in 50 ml of medium. The cultures were incubated under white fluorescent light of 60 µmol m−2 s−1 intensity and at 25±2 ◦ C for the period of 8 weeks. Bioreactor cultures Two types of culture system, temporary and continuous immersion cultures were used to select a suitable method for PLB proliferation. An air lift column type (3 l capacity); and an air lift balloon type (5 l capacity) were used as continuous cultures and temporary immersion type with or without activated charcoal filter attached were used as temporary cultures. In all four cases, 2 l modified Hyponex medium (Kano, 1965; 6.5N – 4.5P – 19K 1 g l−1 +20N – 20P – 20K 1 g l−1 +1% potato homogenate) was taken and 20 g of

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Table 1. The effect of medium volume in Erlenmeyer flasks on the PLB proliferation after 8 weeks of culture with 0.5 g inoculum (∼25 PLB sections) Medium volume (ml) 25 50 100 Number of PLBs/protocorm section

5.8bc 7.2b 10.2a

Mean separation within column by DMRT, p < 0.05 (12 samples for each treatment) Table 2. The effect of inoculum density in Erlenmeyer flasks on the PLB proliferation after 8 weeks of cuture, 50 ml and 1000 ml in flasks and bioreactors respectively Culture types Inoculum density (g) 0.5 (∼25 PLB sections) 1.0 (∼50 PLB sections) 2.0 (∼100 PLB sections) 5.0 (∼250 PLB sections) 10.0 (∼500 PLB sections) Number of PLBs/ protocorm section 9.2a 7.0b 6.5b 8.2b 12.6a

Flask

Bioreactor (air-lift ballon)

Mean separation within column by DMRT, p<0.05 (12 samples for each treatment).

containing peatmoss and perlite (1:1) and kept in greenhouse conditions under high humidity of 60– 70% and day/night temperature of 25/15 ◦ C and 16-h photoperiod of 500 µmol m−2 s−1 . The plants were gradually acclimatized to greenhouse conditions.

Results and discussion Effect of culture conditions on PLB proliferation The interaction between inoculum density and volume of the medium was studied in two experiments in Erlenmeyer flasks. The medium volume had a distinct effect on PLB proliferation (Table 1). In flasks inoculated with 0.5 g of inoculum, ca. 10 PLBs were produced in 100 ml. The PLB production was lowest in 25 ml of medium. In flasks containing 50 ml of medium, 0.5 g of inoculum was superior to 1.0 g or 2.0 g inoculum. In a bioreactor, 10 g of inoculum was superior to 5 g inoculum (Table 2). Similarly, Lilien-Kipnis et al. (1990) and Ilan et al. (1995) have correlated the inoculum density with growth values of tissues in liquid cultures of Brodiaea.

Two kinds of bioreactor systems were studied for multiplication of PLBs continuous immersion system (air-lift type) and temporally immersion system. The results are presented in Table 3. All types of bioreactor system were suitable for PLB proliferation in liquid medium. However, temporary immersion bioreactor with activated charcoal filter attached was most suitable (Figure 1A). Protocorm sections produced 17 PLBs and even biomass of PLB was highest compared to other bioreactor systems. We have harvested ∼18,000 PLBs from each batch of culture (Figure 1B). Thus PLB culture in bioreactors will reduce laborintensive manipulations required for media replenishment and propagule transfer, and facilitates scaling up of PLB production. Similarly, liquid bioreactor culture systems were used to facilitate scaling up of vegetative propagules by Ziv (1989, 1992b, 1999), Ilan et al. (1995) and Watad et al. (1999). A serious problem in liquid cultures is high frequency of hyperhydrated plants when organogenic propagules like bulblets, corms, microtubers and shoots were used as explants (Vanderschaege and Debergh, 1988; Ziv, 1992a). Various growth retardants were used in the medium to overcome shoot growth

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Table 3. Types of bioreactors for PLB proliferation and the extent of proliferation, after 8 weeks of culture Biorector system used Mean number of PLBs per protocorm section 10.8ab 6.1b 9.0b Biomass of PLBs (fresh weight – g l−1 ) 94.6 93.9 87.9

Air lift balloon type Air lift column type Temporary immersion type Temporary immersion type with charcoal filter attached

17.0a

138.9

Mean separation within the column by DMRT, p<0.05 (12 samples for each treatment).

Figure 1. Multiplication of PLBs of Phalaenopsis using bioreactor system and plant regeneration. (A) Multiplication of PLBs in temporary immersion bioreactor system (arrows indicated proliferating PLBs and charcoal filter attached to temporary immersion bioreactor system). (B) Biomass of PLBs harvested from temporary immersion bioreactor system. (C) Plantlet regeneration from PLBs on Hyponex medium. (D) Acclimatized plantlets.

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Table 4. Effect of MS, Hyponex, VW, KC and LM media on plantlet regeneration from PLB sections, after 8 weeks of culture Medium % of regeneration % of rooting Fresh weight of plantlets (mg/plantlet)

Murashige and Skoog Hyponex Vacin and Went Knudson C Lindemann

22.6b 83.0a 45.1b 25.0b 23.9b

83.3a 83.0a 61.0a 47.0ab 14.0c

73.1b 207.4a 98.3b 104.3b 127.7b

Mean separation within column by DMRT, p<0.05 (12 samples for each treatment).

and prevent leaf expansion, and to achieve shoot proliferation (Ziv and Hadar, 1991; Ziv, 1992a, b). In our experiments, we used protocorms as explants to achieve only PLB multiplication (not for plantlet regeneration) in liquid bioreactor cultures. Subsequently, the PLBs were used for plantlet production through conventional micropropagation system (solid medium cultures) and thus we have not faced the problems of hyperhydricity of plantlets. Aeration rate is one of the factors that may affect the growth of propagules/explants in liquid cultures. The effects of two aeration levels were tested during PLB proliferation. A low aeration rate (0.5 volumes of air per volume of medium min−1 (vvm)) was compared with a high rate of aeration (2.0 vvm). An identical growth was observed for both aeration rates, indicating that in the range of tested speed, aeration had no effect on biomass growth. Similar response was observed by Ilan et al. (1995) during propagation of Brodiaea in liquid cultures. A common problem encountered during the in vitro culture of Phalaenopsis tissues and organs was the high level of phenolics released by tissues, which is toxic for the growth of the tissues in culture. Researchers (see Arditti and Ernst, 1993), tested many antioxidants like polyphenoloxidase inhibitors, polyvinylpyrrolidone, and activated charcoal to overcome phenolic compound accumulation. In one of the liquid bioreactors of this study, phenolics were removed by filtering the medium through charcoal. This strongly enhanced PLB growth. Regeneration of PLBs into plantlets For conversion of PLBs of Phalaenopsis into plantlets, systematic efforts have not been done in the past. Many scientists have not described the proced-

ure of PLB conversion into plantlets when they have developed PLB regeneration methods from various organs and tissues. Griesbach (1983) used Murashige and Skoog medium for production of plantlets from PLBs, whereas Lin (1986) used modified KC medium supplemented with benzylaminopurine (1 mg l−1 ) for conversion of PLBs into plantlets. In present study, we have used MS, KC, VW, LM and Hyponex media for the regeneration of plantlets from PLBs. Hyponex medium is suitable for plantlet regeneration from PLBs (Figure 1C) and on this medium 83% of PLBs regenerated into plantlets in 8 weeks, and percentage of rooting and fresh weight of the plantlets was also very high (Table 4). During regeneration of plantlets frequent subculturing of plantlets to the medium containing 0.5% activated charcoal favors the growth of the plantlets. The plantlets of size ∼3–4 cm height were successfully transplanted to community pots containing peatmoss and perlite (1:1) (Figure 1D). This is the first report of multiplication of PLBs of orchid species using bioreactor system. This procedure can be conveniently applied for mass multiplication of other commercial orchid species with little modifications. This system/methodology will reduce the labor and space, cost of micropropagation, and also overcomes the problems of hyperhydricity since protocorms are used for multiplication in liquid medium and subsequently plantlets are regenerated on solid medium.

Acknowledgements We are thankful to the Korean Science and Engineering Foundation, for funding to Research Center for the Development of Advanced Horticultural Technology

72 at Chungbuk National University, Cheongju, 361-763, Korea. We are grateful to Professor J. Miller for the correction of English in the manuscript.
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