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Protein variation and efficient in vitro culture of scale segments from Hyacinthus orientalis L. cv. Carnegie
Scientia Horticulturae 92 (2002) 367±374
Short communication
Protein variation and ef®cient in vitro culture of scale segments from Hyacinthus orientalis L. cv. Carnegie Young-Byung Yia,*, Kyeoung-Soon Leea, Chung-Han Chungb a
Faculty of Life Science and Resources, Department of Horticultural Science, Dong-A University, 840 Ha-Dan-Dong, Sa-Ha-Gu, Pusan 604-714, South Korea b Faculty of Life Science and Resources, Division of Biotechnology, Dong-A University, 840 Ha-Dan-Dong, Sa-Ha-Gu, Pusan 604-714, South Korea Accepted 21 May 2001
Abstract SDS±PAGE analysis of scale extracts of Hyacinthus orientalis L. cv. Carnegie exhibited various polypeptide bands in electrophoresis gels. As the culture period of the hyacinth bulb scales increased, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was that a very strong 29 kD polypeptide band was observed during the early stages of scale culture, but after 8 weeks of culture, the polypeptide band was nearly undetectable in the gel, indicating that this polypeptide may be one of a family of major storage proteins in the hyacinth scales. Treatment with IBA at concentrations of 1.5 and 3.0 mg/l showed higher regeneration and growth of the bulblets formed from the scale segments of bulbs than those treated with IAA at the same concentrations. No recognizable difference was observed between cultures on either perlite or agar media. The normal (base-down) orientation of the explants had greater in¯uence on the root growth than their inverted (base-up) orientation. The above data suggested that a two-step culture system (®rst cultured in the agar medium, then in the perlite medium) may be more useful for ef®cient in vitro culture of this hyacinth cultivar. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Hyacinthus orientalis L.; IBA; IAA; Explant orientation; Perlite
1. Introduction Hyacinths (Hyacinthus orientalis L.) have been widely cultivated for several hundred years as a horticulturally important bulbous plant. Most hyacinth cultivars can grow in a * Corresponding author. Tel.: 82-51-200-7596/7522; fax: 82-51-200-7505. E-mail address: [email protected] (Y.-B. Yi).
0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 9 7 - 7
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Y.-B. Yi et al. / Scientia Horticulturae 92 (2002) 367±374
wide range of environmental conditions. Although hyacinth has the cultural advantages of tolerating various environmental conditions, the natural reproduction rates of their bulblets are very slow, and the parent bulbs form very few bulblets. To achieve effective propagation of hyacinth bulblets, in vitro culture techniques were introduced and, as a result, various in vitro culture systems were established. According to a recent review (Paek and Thorpe, 1990), the in vitro multiplication of hyacinth bulblets is greatly in¯uenced by some factors such as plant growth regulators, culturing temperatures, culture media, conditions of dark and light, explant sources, etc. For promotion of hyacinth bulblet formation, IAA and IBA were generally used, although their ef®ciency is dependent on such factors as their concentrations and explant sources (Paek and Thorpe, 1990). It is known that physiological changes occur during morphogenesis of explants of ¯ower bulbs. In particular, some metabolic activities during morphogenesis of explants have been related to their protein pro®les analyzed by SDS±PAGE (De Hertogh and Le Nard, 1993). However, until now there has been no report on protein changes occurring during morphogenesis of hyacinth explants. Our experiment was divided into two parts. One was to investigate protein variation during morphogenesis of the scale segments of the cultivar Carnegie. The other was to study the effects of three factors on bulblet regeneration and growth. These factors are treatments with IAA and IBA, the orientation of explants (Pierik and Ruibing, 1973; Paek and Thorpe, 1990; Yi et al., 1996), and the use of perlite as an alternative medium to agar. Although agar is most effective for the support of hyacinth explants, when their regenerated bulblets or plantlets are transferred to other culturing materials such as potting or soils, some dif®culties with handling are frequently encountered (Chalupa, 1974; Whitehead and Giles, 1976, 1977; Cassells, 1979; Rugini and Verma, 1983; Hutchinson, 1984). 2. Materials and methods 2.1. Extraction of protein Scale segments of the `Carnegie' bulbs were pulverized in liquid nitrogen with a mortar and pestle and then 0.2 g of the pulverized material was homogenized in an extraction buffer (800 ml) containing 1 mM EDTA, 5 mM dithiothreitol, 2% Tirton X-100 (pH 7.2). The homogenate was then centrifuged at 10 000g for 40 min at 4 8C and the supernatants used for protein analysis by SDS±PAGE. For SDS±PAGE analysis, after preparation of a 5% stacking and 12.5% separating gel, about 20 mg of the total protein extracts were loaded on the gel and electrophoresis was run at 200 V using the Bio-Rad mini-gel system according to the method of Laemmli (1970). After electrophoresis, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 and destained with a mixture of 7% acetic acid and 10% methanol. 2.2. Plant materials and treatments Bulbs of H. orientalis L. cv. Carnegie, whose diameters were 5.6±6.0 cm, were obtained from a commercial seed company (ZaboPlant) and maintained at 20 8C after their
Y.-B. Yi et al. / Scientia Horticulturae 92 (2002) 367±374
369
purchase. When shoots developed to approximately 1.0 cm, the bulbs were ®rst immersed in a diluted (2500) solution of PHYSAN 20 for 20 min, and then dried on a clean bench for approximately 16 h. Afterwards, the bulbs were cut longitudinally into eight pieces and their outermost covers removed, 48 scale segments of approximately equal size 1:0 1:7 cm per bulb were prepared by removing the layered scales from the bulb pieces. Thereafter, the segments were sonicated at 47 kHz for 10 min, then soaked in 70% ethanol for 20 sec followed by short washings in sterile-distilled water. Finally, they were sterilized using a solution of 8% calcium hypochlorite for 15 min with intermittent shakings and then washed three times with sterile-distilled water. The modi®ed Heller's (1953) medium (lacking FeCl3) containing NaFe±EDTA 12.5 mg/l, glycine 1.0 mg/l, nicotinic acid 0.25 mg/l, pyridoxine 0.25 mg/l, thiamineHCl 0.05 mg/l, myo-inositol 50 mg/l and 20 g/l sucrose was used. Four auxin treatments IAA 1.5 mg/l, IAA 3.0 mg/l, IBA 1.5 mg/l or IBA 3.0 mg/l were individually supplemented to the above medium. Before Bacto agar 6 g/l was added to the above medium, its pH was adjusted to 5.4 and then it was melted. Approximately, 13 ml of the melted agar medium was poured into a 22 160 mm culture tubes and autoclaved at 121 8C for 20 min (Yi et al., 1996). To examine the ef®ciency of explant orientation, the sterile scale segments were prepared as mentioned above and placed into the above media to a depth of 3 mm and oriented base-down (normal) or base-up (inverted). To analyze the usefulness of perlite as an alternative to agar, about 13 g of the aseptic perlite saturated with the sterile media containing the auxins mentioned above were packed into the culture tubes. Then the vigorous bulblets whose diameter reached about 5.0 mm were selected after 20 weeks of culture and transferred to the culture tubes packed with the saturated perlite or solidi®ed with agar. Throughout these experiments, the cultural conditions were maintained in a room at a temperature of 23 2 C under cool-white ¯uorescent lamps (Toshiba FL40 SW) of 30 50 mmol m 2 s 1 for 16/8 h periods of light/dark per 24 h. The bulblet regeneration and root formation percentages were calculated after 20 weeks of culturing the scale segments using the following formulae: bulblet regeneration % number of bulblets regenerated=total number of segments used 100, and the root formation % number of bulblets rooted=total number of the regenerated bulblets used 100. 3. Results and discussion 3.1. Protein variation of `Carnegie' bulb scales Total protein was extracted from the scale segments of `Carnegie' bulbs and analyzed by SDS±PAGE. Fig. 1 shows that various banding patterns of some polypeptides were clearly observed. The most outstanding feature was the presence of a polypeptide band of 29 kD. This polypeptide appeared most strongly in the lane of zero week culture, and after 4 weeks of culture its intensity began to decrease and ®nally disappeared. Based on the change in its band intensity, this polypeptide seemed to be one of a family of major
370
Y.-B. Yi et al. / Scientia Horticulturae 92 (2002) 367±374
Fig. 1. SDS±PAGE analysis of protein changes in bulb scale segments of H. orientalis L. cv. Carnegie cultured from 4 to 20 weeks. The electrophoresis was performed on a 5% stacking and 12.5% separating gel in a Tris± glycine buffer system. M and kD indicate size marker and kilodalton, respectively.
storage proteins that are common in storage or reproductive organs of plants (De Hertogh and Le Nard, 1993; MuÈntz, 1998). Another feature was that some other larger polypeptides with much weaker band intensities (Fig. 1) appeared after 4 weeks of culture (arrow to the right of about 53± 55 kD) and disappeared after 8 weeks of culture (arrow to the right of about 32±34 kD). Although no direct evidence for protein variation in other hyacinth cultivars is available, it was assumed that these polypeptides might be metabolic proteins involved in bulb formation or growth (Paek and Thorpe, 1990; De Hertogh and Le Nard, 1993). 3.2. Effects of auxin treatment and explant orientation on bulblet regeneration and root formation Fig. 2 shows that the highest percent bulblet regeneration of cultivar Carnegie occurred at a concentration of 1.5 mg/l IBA with the normal orientation of the explants. In contrast, the lowest was observed with 3.0 mg/l IBA in the inverted orientation of the explants. However, with the exception of 3.0 mg/l IAA, relatively higher bulblet regeneration was found in the lots placed in the normal orientation of the explants (Fig. 2). These results indicate that explant orientation might in¯uence bulblet regeneration in cultivar Carnegie. Fig. 3 exhibits the effects of auxins, IAA and IBA, and explant orientation on root formation of cultivar Carnegie. The greatest percent of root formation was found with 3.0 mg/l IBA in the inverted orientation of the explants, while the lowest was with 1.5 mg/l IAA in the inverted orientation of the explants (Fig. 3). These results also demonstrate that higher root formation occurred with treatment of IBA rather than IAA (Fig. 3). Accordingly, it was concluded that IBA promoted root formation more than IAA, while explant orientations may be less involved in root formation. Some workers have reported that most hyacinths do not require growth regulators for bulblet regeneration (Hussey, 1975a,b; Yi et al., 1996), others reported that addition of IAA or IBA (Chung et al., 1981; Lee, 1998) promotes their regeneration. Accordingly, their reports indirectly support our results although the materials and methods used were different.
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371
Fig. 2. Comparison of bulblet regeneration from bulb scale segments of H. orientalis L. cv. Carnegie by treatments with IAA and IBA at concentrations of 1.5 and 3.0 mg/l and by explant orientation. The number of bulblets was counted after 20 weeks of culturing the scale segments and the values are the means of five replications (20 explants per representation).
3.3. Effects of auxins and explant orientation on bulblets and root growth The growth status of bulblets and roots formed from in vitro culture of the scale segments of cultivar Carnegie was analyzed by measuring the height and diameter of the bulblets,
Fig. 3. Comparison of the root formation from bulb scale segments of H. orientalis L. cv. Carnegie by treatments of IAA and IBA at concentrations of 1.5 and 3.0 mg/l and by explant orientation. The number of roots was counted after 20 weeks of culturing the scale segments and the values are the means of five replications (20 explants per representation).
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Table 1 Effect of explant orientation and auxins on bulblet and root growth formed from bulb scale segments of H. orientalis L. cv. Carnegie cultured for 20 weeksa Growth regulators (mg/l)
Bulblet Height (cm) b
IAA 1.5 IAA 3.0 IBA 1.5 IBA 3.0
Root
c
Diameter (cm)
Length (cm)
Number per explant
Down
Up
Down
Up
Down
Up
Down
Up
0:9 0:1 0:7 0:1 0:8 0:1 0:8 0:1
0:7 0:1 0:7 0:1 0:8 0:1 0:7 0:1
0:5 0:1 0:4 0:1 0:4 0:0 0:4 0:1
0:4 0:1 0:4 0:1 0:5 0:1 0:4 0:0
0:2 0:0 0:2 0:0 0:4 0:1 0:4 0:1
0:2 0:0 0:2 0:0 0:2 0:0 0:3 0:0
1:1 0:1 1:0 0:1 1:0 0:1 1:0 0:1
1:1 0:1 1:0 0:0 1:1 0:2 1:3 0:1
a
The values were obtained from 40 explants and expressed with mean S.E. The bases of explants were placed in the medium in downward orientation. c The bases of explants were placed in the medium in upward orientation. b
and the length and number of roots per explant to investigate the effects of the auxins and explant orientation on their growth. As shown in Table 1, the greatest height and diameter of the bulblets were seen with 1.5 mg/l IAA used in the normal orientation of the explants. In contrast, the longest roots were observed at both IBA concentrations inoculated in the normal orientations of the explants. In general, however, the effects of growth regulator and of explant orientation were small. It has generally been known that because the bulblet formation from hyacinth scales is initiated at their base due to their basipetal polarity (Pierik and Woets, 1971), the normal orientation may be more effective for bulblet formation and root growth than the inverted orientation. Nonetheless, several workers reported that when the explants were placed in the inverted orientation, their bulblet regeneration and growth were considerably enhanced (Hackett, 1969; Pierik and Ruibing, 1973; Pierik and Post, 1975). However, our results do not agree with these reports. 3.4. Comparison of the effects of perlite and agar on the growth of bulblets and roots To compare the effects of perlite and agar on the growth of bulblets and roots, wellgrown bulblets (about 0.6±0.8 cm high and 0.3±0.5 wide) with roots (about 0.2±0.4 cm long) cultured for 20 weeks were selected and transferred to either the agar-solidi®ed medium (agar medium) or the medium containing perlite saturated with the liquid medium. Because IBA had been more effective for bulblet regeneration and growth than IAA, the bulblets formed only in the media containing 1.5 and 3.0 mg/l IBA were transferred to the above media supplemented with the same IBA concentrations (Table 2). After 36 weeks of culture, their growth was scored. Table 2 shows that bulblet heights slightly increased, while no change was found in their diameters. After transfer, the bulblets formed in the perlite medium were slightly taller than in the agar medium (Table 2), suggesting that perlite can be used as an alternative to agar. Secondly, there was no effect of treatment on root length after transfer, although there was a slight effect of explant orientation. The greatest increase in root number per explant was seen with 3.0 mg/l IBA placed in the normal orientation, while the most severe decreases were
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373
Table 2 Comparison between the effects of agar and perlite media on the growth of bulblets and roots subcultured for 16 weeksa Support
Growth Bulblet regulator Height (cm) (mg/l) b
Down
Root
c
Up
Diameter (cm)
Length (cm)
Number per explant
Down
Down
Down
Up
Up
Up
Agar
IBA 1.5 IBA 3.0
0:9 0:1 0:9 0:1 0:4 0:1 0:4 0:1 0:2 0:0 0:2 0:0 1:0 0:0 0:4 0:1 0:9 0:1 0:9 0:1 0:4 0:1 0:4 0:0 0:2 0:0 0:3 0:0 1:2 0:1 0:4 0:0
Perlite
IBA 1.5 IBA 3.0
1:0 0:1 1:0 0:1 0:5 0:1 0:6 0:1 0:3 0:0 0:2 0:0 1:0 0:0 0:6 0:1 1:1 0:1 0:9 0:1 0:5 0:1 0:4 0:0 0:2 0:0 0:3 0:0 2:0 0:3 0:4 0:0
a
The values were obtained from 20 bulblets and expressed with mean S.E. The bases of explants were placed in the medium in downward orientation. c The bases of explants were placed in the medium in upward orientation. b
found in the inverted orientation. This ®nding also suggests that the use of perlite could be recommended for support of hyacinth explants in their in vitro cultures. Until now agar has generally been used as a material for supporting explants in in vitro cultures (Paek and Thorpe, 1990). However, when the explants are transferred for subculture or the plantlets are transplanted to soil, their roots are frequently damaged as the agar attached to their roots are removed, sometimes resulting in the complete failure of growth. Accordingly, perlite may be more ef®cient than agar because perlite is convenient for handling the explants or the plantlets formed in in vitro cultures, although our results showed that there was no difference between the effects of perlite and agar media on the bulblet and root growth. 3.5. Conclusion The protein pro®le showed that various polypeptide bands were present in the gel during early culture periods of the hyacinth scales. As their cultures continued, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was the presence of a strong polypeptide band with 29 kD. After 8 weeks of culture, this polypeptide band almost disappeared, suggesting that this polypeptide may belong to a family of major storage proteins of the hyacinth scales. From the results of the in vitro culture experiment, it could be cautiously concluded that a two-step culture system may be more useful for ef®cient in vitro bulblet formation and root growth. In the ®rst step of the culture, the explants are cultured in the agar medium supplemented with 1.5±3.0 mg/l IBA in base-down (normal) orientation. Then in the second step, the bulblets with roots are cultured in the perlite medium with the same concentrations of IBA. Acknowledgements This study was supported by a research grant from Dong-A University.
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References Cassells, A.C., 1979. The effect of 2,3,5-tri-iodobenzoic acid on caulogenesis in callus cultures of tomato and Pelargonium. Physiol. Plant. 46, 159±164. Chalupa, V., 1974. Control of root and shoot formation and production of trees from poplar callus. Biol. Plant. 16, 316±320. Chung, J.D., Chun, C.K., Lee, E.M., Paek, K.Y., 1981. In vitro propagation of Hyacinthus orientalis L. II. Effect of auxin on callus formation and bulblet differentiation from bulb scale segments. J. Kor. Plant Tissue Cult. 8 (1), 45±48. De Hertogh, A.A., Le Nard, M., 1993. Physiological and biochemical aspects of flower bulbs. In: De Hertogh, A.A., Le Nard, M. (Eds.), The Physiology of Flower Bulbs. Elsevier, Amsterdam, pp. 53±69. Hackett, W.P., 1969. Aseptic multiplications of lily bulblets from bulb scales. Proc. Int. Plant. Prop. Soc. 19, 105±108. Heller, R., 1953. Recherche sur la nutrition minerale des tissus vegetaux cultiveÂs ``in vitro''. Ann. Sci. Nat. Biol. Veg. 14, 1±123. Hussey, G., 1975a. Propagation of hyacinths by tissue culture. Sci. Hort. 3, 21±28. Hussey, G., 1975b. Totipotency in tissue explants and callus of some members of Liliaceae, Iridaceae, and Amaryllidaceae. J. Exp. Bot. 26 (91), 253±262. Hutchinson, J.F., 1984. Factors affecting shoot proliferation and root initiation in organ cultures of the apple `Northern Spy'. Sci. Hort. 22, 347±358. Laemmli, U.K., 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Lee, K.S., 1998. Establishment of plantlet production system by in vitro culture in Hyacinthus orientalis L. Ph.D. Thesis. Dong-A University, Pusan, South Korea. MuÈntz, K., 1998. Deposition of storage proteins. Plant Mol. Biol. 38, 77±99. Paek, K.Y., Thorpe, T.A., 1990. Hyacinth. In: Ammirato, P.V., Evans, D.A., Sharp, W.R., Bajaj, Y.P.S. (Eds.), Handbook of Plant Cell Culture, Vol. 5. McGraw-Hill, New York, pp. 479±508. Pierik, R.L.M., Post, A.J.M., 1975. Rapid vegetative propagation of Hyacinthus orientalis L. in vitro. Sci. Hort. 3, 293±297. Pierik, R.L.M., Ruibing, M.A., 1973. Regeneration of bulblets on bulb scale segments of hyacinth in vitro. Neth. J. Agric. Sci. 21, 129±138. Pierik, R.L.M., Woets, J., 1971. Regeneration of isolated bulb scale segments of hyacinth. Acta Hort. 23, 423± 428. Rugini, E., Verma, D.C., 1983. Micropropagation and difficult to propagate Almond (Prunus amygdalus Batch) cultivar. Plant Sci. Lett. 28, 273±281. Whitehead, H.C.M., Giles, K.L., 1976. Rapid propagation of poplars by tissue culture methods. Comb. Proc. Int. Plant Prop. Soc. 26, 340±343. Whitehead, H.C.M., Giles, K.L., 1977. Rapid propagation of poplars by tissue culture methods. N.Z. J. For. Sci. 7, 40±43. Yi, Y.B., Park, J.K., Lee, K.S., 1996. Effect of medium on regeneration and growth of bulblets from the culture of bulb scale segments of Hyacinthus orientalis L. in vitro. J. Kor. Soc. Hort. Sci. 37 (1), 146±151.
Short communication
Protein variation and ef®cient in vitro culture of scale segments from Hyacinthus orientalis L. cv. Carnegie Young-Byung Yia,*, Kyeoung-Soon Leea, Chung-Han Chungb a
Faculty of Life Science and Resources, Department of Horticultural Science, Dong-A University, 840 Ha-Dan-Dong, Sa-Ha-Gu, Pusan 604-714, South Korea b Faculty of Life Science and Resources, Division of Biotechnology, Dong-A University, 840 Ha-Dan-Dong, Sa-Ha-Gu, Pusan 604-714, South Korea Accepted 21 May 2001
Abstract SDS±PAGE analysis of scale extracts of Hyacinthus orientalis L. cv. Carnegie exhibited various polypeptide bands in electrophoresis gels. As the culture period of the hyacinth bulb scales increased, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was that a very strong 29 kD polypeptide band was observed during the early stages of scale culture, but after 8 weeks of culture, the polypeptide band was nearly undetectable in the gel, indicating that this polypeptide may be one of a family of major storage proteins in the hyacinth scales. Treatment with IBA at concentrations of 1.5 and 3.0 mg/l showed higher regeneration and growth of the bulblets formed from the scale segments of bulbs than those treated with IAA at the same concentrations. No recognizable difference was observed between cultures on either perlite or agar media. The normal (base-down) orientation of the explants had greater in¯uence on the root growth than their inverted (base-up) orientation. The above data suggested that a two-step culture system (®rst cultured in the agar medium, then in the perlite medium) may be more useful for ef®cient in vitro culture of this hyacinth cultivar. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Hyacinthus orientalis L.; IBA; IAA; Explant orientation; Perlite
1. Introduction Hyacinths (Hyacinthus orientalis L.) have been widely cultivated for several hundred years as a horticulturally important bulbous plant. Most hyacinth cultivars can grow in a * Corresponding author. Tel.: 82-51-200-7596/7522; fax: 82-51-200-7505. E-mail address: [email protected] (Y.-B. Yi).
0304-4238/02/$ ± see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 1 ) 0 0 2 9 7 - 7
368
Y.-B. Yi et al. / Scientia Horticulturae 92 (2002) 367±374
wide range of environmental conditions. Although hyacinth has the cultural advantages of tolerating various environmental conditions, the natural reproduction rates of their bulblets are very slow, and the parent bulbs form very few bulblets. To achieve effective propagation of hyacinth bulblets, in vitro culture techniques were introduced and, as a result, various in vitro culture systems were established. According to a recent review (Paek and Thorpe, 1990), the in vitro multiplication of hyacinth bulblets is greatly in¯uenced by some factors such as plant growth regulators, culturing temperatures, culture media, conditions of dark and light, explant sources, etc. For promotion of hyacinth bulblet formation, IAA and IBA were generally used, although their ef®ciency is dependent on such factors as their concentrations and explant sources (Paek and Thorpe, 1990). It is known that physiological changes occur during morphogenesis of explants of ¯ower bulbs. In particular, some metabolic activities during morphogenesis of explants have been related to their protein pro®les analyzed by SDS±PAGE (De Hertogh and Le Nard, 1993). However, until now there has been no report on protein changes occurring during morphogenesis of hyacinth explants. Our experiment was divided into two parts. One was to investigate protein variation during morphogenesis of the scale segments of the cultivar Carnegie. The other was to study the effects of three factors on bulblet regeneration and growth. These factors are treatments with IAA and IBA, the orientation of explants (Pierik and Ruibing, 1973; Paek and Thorpe, 1990; Yi et al., 1996), and the use of perlite as an alternative medium to agar. Although agar is most effective for the support of hyacinth explants, when their regenerated bulblets or plantlets are transferred to other culturing materials such as potting or soils, some dif®culties with handling are frequently encountered (Chalupa, 1974; Whitehead and Giles, 1976, 1977; Cassells, 1979; Rugini and Verma, 1983; Hutchinson, 1984). 2. Materials and methods 2.1. Extraction of protein Scale segments of the `Carnegie' bulbs were pulverized in liquid nitrogen with a mortar and pestle and then 0.2 g of the pulverized material was homogenized in an extraction buffer (800 ml) containing 1 mM EDTA, 5 mM dithiothreitol, 2% Tirton X-100 (pH 7.2). The homogenate was then centrifuged at 10 000g for 40 min at 4 8C and the supernatants used for protein analysis by SDS±PAGE. For SDS±PAGE analysis, after preparation of a 5% stacking and 12.5% separating gel, about 20 mg of the total protein extracts were loaded on the gel and electrophoresis was run at 200 V using the Bio-Rad mini-gel system according to the method of Laemmli (1970). After electrophoresis, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 and destained with a mixture of 7% acetic acid and 10% methanol. 2.2. Plant materials and treatments Bulbs of H. orientalis L. cv. Carnegie, whose diameters were 5.6±6.0 cm, were obtained from a commercial seed company (ZaboPlant) and maintained at 20 8C after their
Y.-B. Yi et al. / Scientia Horticulturae 92 (2002) 367±374
369
purchase. When shoots developed to approximately 1.0 cm, the bulbs were ®rst immersed in a diluted (2500) solution of PHYSAN 20 for 20 min, and then dried on a clean bench for approximately 16 h. Afterwards, the bulbs were cut longitudinally into eight pieces and their outermost covers removed, 48 scale segments of approximately equal size 1:0 1:7 cm per bulb were prepared by removing the layered scales from the bulb pieces. Thereafter, the segments were sonicated at 47 kHz for 10 min, then soaked in 70% ethanol for 20 sec followed by short washings in sterile-distilled water. Finally, they were sterilized using a solution of 8% calcium hypochlorite for 15 min with intermittent shakings and then washed three times with sterile-distilled water. The modi®ed Heller's (1953) medium (lacking FeCl3) containing NaFe±EDTA 12.5 mg/l, glycine 1.0 mg/l, nicotinic acid 0.25 mg/l, pyridoxine 0.25 mg/l, thiamineHCl 0.05 mg/l, myo-inositol 50 mg/l and 20 g/l sucrose was used. Four auxin treatments IAA 1.5 mg/l, IAA 3.0 mg/l, IBA 1.5 mg/l or IBA 3.0 mg/l were individually supplemented to the above medium. Before Bacto agar 6 g/l was added to the above medium, its pH was adjusted to 5.4 and then it was melted. Approximately, 13 ml of the melted agar medium was poured into a 22 160 mm culture tubes and autoclaved at 121 8C for 20 min (Yi et al., 1996). To examine the ef®ciency of explant orientation, the sterile scale segments were prepared as mentioned above and placed into the above media to a depth of 3 mm and oriented base-down (normal) or base-up (inverted). To analyze the usefulness of perlite as an alternative to agar, about 13 g of the aseptic perlite saturated with the sterile media containing the auxins mentioned above were packed into the culture tubes. Then the vigorous bulblets whose diameter reached about 5.0 mm were selected after 20 weeks of culture and transferred to the culture tubes packed with the saturated perlite or solidi®ed with agar. Throughout these experiments, the cultural conditions were maintained in a room at a temperature of 23 2 C under cool-white ¯uorescent lamps (Toshiba FL40 SW) of 30 50 mmol m 2 s 1 for 16/8 h periods of light/dark per 24 h. The bulblet regeneration and root formation percentages were calculated after 20 weeks of culturing the scale segments using the following formulae: bulblet regeneration % number of bulblets regenerated=total number of segments used 100, and the root formation % number of bulblets rooted=total number of the regenerated bulblets used 100. 3. Results and discussion 3.1. Protein variation of `Carnegie' bulb scales Total protein was extracted from the scale segments of `Carnegie' bulbs and analyzed by SDS±PAGE. Fig. 1 shows that various banding patterns of some polypeptides were clearly observed. The most outstanding feature was the presence of a polypeptide band of 29 kD. This polypeptide appeared most strongly in the lane of zero week culture, and after 4 weeks of culture its intensity began to decrease and ®nally disappeared. Based on the change in its band intensity, this polypeptide seemed to be one of a family of major
370
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Fig. 1. SDS±PAGE analysis of protein changes in bulb scale segments of H. orientalis L. cv. Carnegie cultured from 4 to 20 weeks. The electrophoresis was performed on a 5% stacking and 12.5% separating gel in a Tris± glycine buffer system. M and kD indicate size marker and kilodalton, respectively.
storage proteins that are common in storage or reproductive organs of plants (De Hertogh and Le Nard, 1993; MuÈntz, 1998). Another feature was that some other larger polypeptides with much weaker band intensities (Fig. 1) appeared after 4 weeks of culture (arrow to the right of about 53± 55 kD) and disappeared after 8 weeks of culture (arrow to the right of about 32±34 kD). Although no direct evidence for protein variation in other hyacinth cultivars is available, it was assumed that these polypeptides might be metabolic proteins involved in bulb formation or growth (Paek and Thorpe, 1990; De Hertogh and Le Nard, 1993). 3.2. Effects of auxin treatment and explant orientation on bulblet regeneration and root formation Fig. 2 shows that the highest percent bulblet regeneration of cultivar Carnegie occurred at a concentration of 1.5 mg/l IBA with the normal orientation of the explants. In contrast, the lowest was observed with 3.0 mg/l IBA in the inverted orientation of the explants. However, with the exception of 3.0 mg/l IAA, relatively higher bulblet regeneration was found in the lots placed in the normal orientation of the explants (Fig. 2). These results indicate that explant orientation might in¯uence bulblet regeneration in cultivar Carnegie. Fig. 3 exhibits the effects of auxins, IAA and IBA, and explant orientation on root formation of cultivar Carnegie. The greatest percent of root formation was found with 3.0 mg/l IBA in the inverted orientation of the explants, while the lowest was with 1.5 mg/l IAA in the inverted orientation of the explants (Fig. 3). These results also demonstrate that higher root formation occurred with treatment of IBA rather than IAA (Fig. 3). Accordingly, it was concluded that IBA promoted root formation more than IAA, while explant orientations may be less involved in root formation. Some workers have reported that most hyacinths do not require growth regulators for bulblet regeneration (Hussey, 1975a,b; Yi et al., 1996), others reported that addition of IAA or IBA (Chung et al., 1981; Lee, 1998) promotes their regeneration. Accordingly, their reports indirectly support our results although the materials and methods used were different.
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Fig. 2. Comparison of bulblet regeneration from bulb scale segments of H. orientalis L. cv. Carnegie by treatments with IAA and IBA at concentrations of 1.5 and 3.0 mg/l and by explant orientation. The number of bulblets was counted after 20 weeks of culturing the scale segments and the values are the means of five replications (20 explants per representation).
3.3. Effects of auxins and explant orientation on bulblets and root growth The growth status of bulblets and roots formed from in vitro culture of the scale segments of cultivar Carnegie was analyzed by measuring the height and diameter of the bulblets,
Fig. 3. Comparison of the root formation from bulb scale segments of H. orientalis L. cv. Carnegie by treatments of IAA and IBA at concentrations of 1.5 and 3.0 mg/l and by explant orientation. The number of roots was counted after 20 weeks of culturing the scale segments and the values are the means of five replications (20 explants per representation).
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Table 1 Effect of explant orientation and auxins on bulblet and root growth formed from bulb scale segments of H. orientalis L. cv. Carnegie cultured for 20 weeksa Growth regulators (mg/l)
Bulblet Height (cm) b
IAA 1.5 IAA 3.0 IBA 1.5 IBA 3.0
Root
c
Diameter (cm)
Length (cm)
Number per explant
Down
Up
Down
Up
Down
Up
Down
Up
0:9 0:1 0:7 0:1 0:8 0:1 0:8 0:1
0:7 0:1 0:7 0:1 0:8 0:1 0:7 0:1
0:5 0:1 0:4 0:1 0:4 0:0 0:4 0:1
0:4 0:1 0:4 0:1 0:5 0:1 0:4 0:0
0:2 0:0 0:2 0:0 0:4 0:1 0:4 0:1
0:2 0:0 0:2 0:0 0:2 0:0 0:3 0:0
1:1 0:1 1:0 0:1 1:0 0:1 1:0 0:1
1:1 0:1 1:0 0:0 1:1 0:2 1:3 0:1
a
The values were obtained from 40 explants and expressed with mean S.E. The bases of explants were placed in the medium in downward orientation. c The bases of explants were placed in the medium in upward orientation. b
and the length and number of roots per explant to investigate the effects of the auxins and explant orientation on their growth. As shown in Table 1, the greatest height and diameter of the bulblets were seen with 1.5 mg/l IAA used in the normal orientation of the explants. In contrast, the longest roots were observed at both IBA concentrations inoculated in the normal orientations of the explants. In general, however, the effects of growth regulator and of explant orientation were small. It has generally been known that because the bulblet formation from hyacinth scales is initiated at their base due to their basipetal polarity (Pierik and Woets, 1971), the normal orientation may be more effective for bulblet formation and root growth than the inverted orientation. Nonetheless, several workers reported that when the explants were placed in the inverted orientation, their bulblet regeneration and growth were considerably enhanced (Hackett, 1969; Pierik and Ruibing, 1973; Pierik and Post, 1975). However, our results do not agree with these reports. 3.4. Comparison of the effects of perlite and agar on the growth of bulblets and roots To compare the effects of perlite and agar on the growth of bulblets and roots, wellgrown bulblets (about 0.6±0.8 cm high and 0.3±0.5 wide) with roots (about 0.2±0.4 cm long) cultured for 20 weeks were selected and transferred to either the agar-solidi®ed medium (agar medium) or the medium containing perlite saturated with the liquid medium. Because IBA had been more effective for bulblet regeneration and growth than IAA, the bulblets formed only in the media containing 1.5 and 3.0 mg/l IBA were transferred to the above media supplemented with the same IBA concentrations (Table 2). After 36 weeks of culture, their growth was scored. Table 2 shows that bulblet heights slightly increased, while no change was found in their diameters. After transfer, the bulblets formed in the perlite medium were slightly taller than in the agar medium (Table 2), suggesting that perlite can be used as an alternative to agar. Secondly, there was no effect of treatment on root length after transfer, although there was a slight effect of explant orientation. The greatest increase in root number per explant was seen with 3.0 mg/l IBA placed in the normal orientation, while the most severe decreases were
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373
Table 2 Comparison between the effects of agar and perlite media on the growth of bulblets and roots subcultured for 16 weeksa Support
Growth Bulblet regulator Height (cm) (mg/l) b
Down
Root
c
Up
Diameter (cm)
Length (cm)
Number per explant
Down
Down
Down
Up
Up
Up
Agar
IBA 1.5 IBA 3.0
0:9 0:1 0:9 0:1 0:4 0:1 0:4 0:1 0:2 0:0 0:2 0:0 1:0 0:0 0:4 0:1 0:9 0:1 0:9 0:1 0:4 0:1 0:4 0:0 0:2 0:0 0:3 0:0 1:2 0:1 0:4 0:0
Perlite
IBA 1.5 IBA 3.0
1:0 0:1 1:0 0:1 0:5 0:1 0:6 0:1 0:3 0:0 0:2 0:0 1:0 0:0 0:6 0:1 1:1 0:1 0:9 0:1 0:5 0:1 0:4 0:0 0:2 0:0 0:3 0:0 2:0 0:3 0:4 0:0
a
The values were obtained from 20 bulblets and expressed with mean S.E. The bases of explants were placed in the medium in downward orientation. c The bases of explants were placed in the medium in upward orientation. b
found in the inverted orientation. This ®nding also suggests that the use of perlite could be recommended for support of hyacinth explants in their in vitro cultures. Until now agar has generally been used as a material for supporting explants in in vitro cultures (Paek and Thorpe, 1990). However, when the explants are transferred for subculture or the plantlets are transplanted to soil, their roots are frequently damaged as the agar attached to their roots are removed, sometimes resulting in the complete failure of growth. Accordingly, perlite may be more ef®cient than agar because perlite is convenient for handling the explants or the plantlets formed in in vitro cultures, although our results showed that there was no difference between the effects of perlite and agar media on the bulblet and root growth. 3.5. Conclusion The protein pro®le showed that various polypeptide bands were present in the gel during early culture periods of the hyacinth scales. As their cultures continued, some polypeptide bands disappeared and some new bands appeared. The most outstanding feature was the presence of a strong polypeptide band with 29 kD. After 8 weeks of culture, this polypeptide band almost disappeared, suggesting that this polypeptide may belong to a family of major storage proteins of the hyacinth scales. From the results of the in vitro culture experiment, it could be cautiously concluded that a two-step culture system may be more useful for ef®cient in vitro bulblet formation and root growth. In the ®rst step of the culture, the explants are cultured in the agar medium supplemented with 1.5±3.0 mg/l IBA in base-down (normal) orientation. Then in the second step, the bulblets with roots are cultured in the perlite medium with the same concentrations of IBA. Acknowledgements This study was supported by a research grant from Dong-A University.
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