- Email: [email protected]
Bulblet-productivity of explants from scales, leaves, stems and teplas of Lilium rubellum Baker
Scientia Horticulturae, 22 (1984) 391-394 Elsevier Science PUblishers B.V., Amsterdam -Printed in The Netherlands
391
BULBLET-PRODUCTIVITY OF EXPLANTS FROM SCALES, LEAVES, STEMS AND TEPALS OF LILIUM RUBELLUM BAKER
YOSHIJI NIIMI Faculty of Agriculture, Laboratory of Horticultural Science, Niigaia Uniuersity, Niigata 950·21 (Japan)
(Accepted for publication 25 August 1983)
ABSTRACT Niimi, Y., 1984. Bulblet-productivity of explants from scales, leaves, stems and tepals of Lilium rubellum Baker. Scieniia Horiic., 22: 391-394. The number of bulblets per cultured explant was greatest in the explants of leaves, i.e. 2.2 bulblets, followed in decreasing order by scale-, stem- and tepal-axplants. Bulblets formed in the explants of stems were heaviest, 135 mg on average. The explants ofleaves were excellent for regenerating many bulblets per explant, while those of stems produced the heaviest bulblets. Keywords: in vitro; Lilium rubellum Baker.
INTRODUCTION
In vitro propagation of Lilium rubellum Baker has been performed by culturing explants of leaves (Niimi and Onozawa, 1979), scales, stems, tepals and other organs (Niimi and Watanabe, 1982). Some of these cultures had almost the same bulblet productivity as the explants of scales. Considering that scales from bulbs grown in soil were very often contaminated and thus difficult to culture in vitro (Niimi and Onozawa, 1977; Niimi and Watanabe, 1982), it was considered that explants excised from organs above the soil might be more suitable sources for producing new bulblets in vitro than those from scales. On the other hand, scaly segments excised from parent plants have commonly been cultured because scales seemed to be the most productive (Stimart and Ascher, 1978; Takayama and Misawa, 1979). The present study was made to compare explants of leaves, stems and tepals with those of scales in producing new bulblets in vitro in terms of number and/or weight.
0304-4238/84/$03.00
© 1984 Elsevier Science Publishers B.V.
392 MATERIALS AND METHODS
The procedures for sterilizing materials, dissecting explants, and the composition of the culture medium have been previously described in detail. Explants were excised from portions of each organ at the optimal time of excision (Niimi and Onozawa, 1979; Niimi and Watanabe, 1982).
Plant materials. - Nine parent plants of Lilium rubellum Baker, cultivated in an experimental field under the same conditions as described in a previous paper (Niimi and Watanabe, 1982), were harvested at the end of April 1982, during the 10-20 days after sprouting. Fifteen unexpended young leaves, one flower bud, the 7 em terminal part of a stem, and one mother bulb were separated from each parent plant and washed thoroughly with tap water. Culture conditions. - A medium consisting of Murashige and Skoog salt (1962), myo-inositol, vitamins, glycine, 50 g 1-1 sucrose, 7 g 1- 1 Difco Bacto agar, 1mg r ' o-naphthaleneacetic acid (NAA), and 0.1 mg r 1 6-benzylaminopurine (BA) was used (Niimi and Onozawa, 1979). About 20 ml of the dissolved medium was poured into each 50-ml Erlenmeyer flask, and these were then capped with aliminum foil and autoclaved for 10 .min at 121°C, under a pressure of 1.2 kg cm". Flasks with explants were kept in the dark at 24 ± 1°C. Disinfection of organs. - Leaves, stems and flower buds were surface-sterilized by swabbing them with cotton wool containing 70% ethanol, followed by immersing the organs for 5 min in a 10% solution of a commercial bleach containing 6% sodium hypochloride in water. The organs were then rinsed 3 times in sterile distilled water. After removal of a few outer scales, bulbs were immersed in a 0.1% solution of HgClz for 30 min and disinfected using the methods described for the other organs, except that the bulbs were immersed in the solution of sodium hypochloride for 20 min. Excision and culturing of explants. - Three 5-mm transverse strips of a leaf, one 5-mm transverse strip of a tepal and a scale section of about 1 X 1 em were dissected from the proximal portion of each organ, and each was then placed with the abaxial side down on the basal medium in each flask. Ten 5-mm explants were dissected from the terminal portion of each stem, and each was laid on the basal medium in each flask. Determining number and weight of developed bulblets. - 120 days after culture, bulblets were dissected from each of the explants. Their fresh weight was determined after removal of the roots. The results were subjected to statistical analysis at the 5% level following Duncan's multiple range test (Duncan, 1955).
393 RESULTS
Of the cultured explants, 74% developed bulblets. The ability to regenerate bulblets was greatest in explants of stems, of which 89% developed bulblets (Table I), but there was no statistical differences among explants of stems, leaves and scales. Table I also indicates significant differences in the numbers of bulblets per cultured explant, and the greatest bulblet formation occurred in the explants of leaves. This was also true for the numbers of bulblets per regenerated explant. Therefore, the explants of leaves appeared to be the best source for producing the greatest number of bulblets. The explants of stems produced, on average, by far the heaviest bulblets (Table I). Almost 50% of the bulblets formed in the explants of stems weighed more than 100 mg. In contrast, over 70% of bulblets regenerated from the explants of both leaves and scales weighed less than 50 mg. In conclusion, the explants of leaves were the best for regenerating bulblets per explant, while those of stems were the best for developing heavy bulblets. TABLE I Comparison of the regenerative ability of explants excised from different organs. Means in rows not followed by the same letter are significantly different at the 5% level. Absence of a letter shows that statistical analysis was not carried out Explants from: Scale
Leaf
Stem
Tepa!
Number of explants 405 90 54 180 Explants forming bulblets (%) Boa 89a 55 b 72 a Numbers of bulblets per cultured explant 1.9 a 2.2 b 1.5 c o.a d Numbers of bulblets per regenerated explant 1.7 1.4 2.4 3.1 Mean fresh weight of bulblet (mg) 24a 52a 40 a 135 b DISCUSSION
The results support the previous surmise that in L. rubellum Baker, the explants of leaves could be an excellent source for producing new bulblets in vitro, although leaf propagation of monocotyledonous plants is generally regarded as difficult in several species of Liliaceae, Iridaceae and Amaryllidaceae (Hussey, 1975). Takayama and Misawa (1979) reported that in L. speciosum also , segments of leaves excised just before anthesis were a poor source for the production of bulblets, as only lout of 150 segments sur-
394
vived. On the other hand, segments excised from young leaves of L. longiflorum (Stenberg et al., 1977), L. auratum, L. japonicum, L. speciosum X L. auratum, and a few other species of lily plants (Niimi and Onozawa, 1979) developed bulblets. These inconsistent results on the regenerative ability of leaf explants cannot be explained satisfactorily from the present results. More work will be needed to answer the inconsistent results. The present study shows not only that explants excised from the terminal portion of stems can easily regenerate bulblets, as reported for L. longiflorurn (Sheridan, 1968) and tulip (Wright and Alderson, 1980) but also that these excised explants can produce the heaviest bulblets in a short period of time. Furthermore, use of the terminal portions of a stem with young leaves has the advantage of not disturbing the parent bulbs, as well as providing explants without serious fungal pathogens. ACKNOWLEDGEMENTS
The author is grateful to Mr. Y. Kato and Mr. K. Sotome for their cooperation in this study and to Mr. D.H. Goldstein of the Indiana University for correcting the English text.
REFERENCES Duncan, D.B., 1955 . Multiple range and multiple F tests. Biometrics, 11: 1-42. Hussey, G., 1975. Totipotency in tissue explants and callus of some members of the Lilia· ceae, Iridaceae, and Amaryllidaceae. J. Exp. Bot., 26 : 253-262. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-479. Niimi, Y. and Onozawa, T. , 1977. Vegetative propagation of L. rubel/urn Baker; corriparlson of bulblet formation between scaling and segment cultured in vitro. Abstracts Fall Meeting Jpn. Soc. Hortie. ScL, pp. 348-349 (in Japanese). Niirni, Y. and Onozawa, T., 1979. In vitro bulblet formation from leaf segments of lilies, especially Lilium rubellurn Baker. Scientia Hortic., 11: 379-389. Niimi, Y. and Watanabe, H., 198Z. In vitro propagation of Liliurn rubellum Baker with special reference to bulblet formation from stem segments. Jpn. Soc. Hortic. Sci., 51: 344-349 (in Japanese, with English summary). Sheridan, W.F., 1968. Tissue culture of the monocot Lilium. Planta, 82: 189-192. Stenberg, N.E., Chen, C.H . and Ross, J.G., 1977. Regeneration of plantlets from leaf cultures of Lilium longiflorurn Thunb. Proc, South Dakota Acad. Sci., 56: 152-158. Stimart, D.P. and Ascher, P.D., 1978. Tissue culture of bulb scale sections for asexual propagation of Lilium longiflorum Thunb. J. Am. Soc . Hortic. ScL, 103: 182-184. Takayama. S. and Misawa, M., 1979. Differentiation in Lilium bulb-scales grown in vitro. Effect of various cultural conditions. Physio!. Plant. , 46: 184-190. Wright, N.A. and Alderson, P.G., 1980 . The growth of tulip tissue in vitro. Acta Hortie., 109 : 263-270 .
391
BULBLET-PRODUCTIVITY OF EXPLANTS FROM SCALES, LEAVES, STEMS AND TEPALS OF LILIUM RUBELLUM BAKER
YOSHIJI NIIMI Faculty of Agriculture, Laboratory of Horticultural Science, Niigaia Uniuersity, Niigata 950·21 (Japan)
(Accepted for publication 25 August 1983)
ABSTRACT Niimi, Y., 1984. Bulblet-productivity of explants from scales, leaves, stems and tepals of Lilium rubellum Baker. Scieniia Horiic., 22: 391-394. The number of bulblets per cultured explant was greatest in the explants of leaves, i.e. 2.2 bulblets, followed in decreasing order by scale-, stem- and tepal-axplants. Bulblets formed in the explants of stems were heaviest, 135 mg on average. The explants ofleaves were excellent for regenerating many bulblets per explant, while those of stems produced the heaviest bulblets. Keywords: in vitro; Lilium rubellum Baker.
INTRODUCTION
In vitro propagation of Lilium rubellum Baker has been performed by culturing explants of leaves (Niimi and Onozawa, 1979), scales, stems, tepals and other organs (Niimi and Watanabe, 1982). Some of these cultures had almost the same bulblet productivity as the explants of scales. Considering that scales from bulbs grown in soil were very often contaminated and thus difficult to culture in vitro (Niimi and Onozawa, 1977; Niimi and Watanabe, 1982), it was considered that explants excised from organs above the soil might be more suitable sources for producing new bulblets in vitro than those from scales. On the other hand, scaly segments excised from parent plants have commonly been cultured because scales seemed to be the most productive (Stimart and Ascher, 1978; Takayama and Misawa, 1979). The present study was made to compare explants of leaves, stems and tepals with those of scales in producing new bulblets in vitro in terms of number and/or weight.
0304-4238/84/$03.00
© 1984 Elsevier Science Publishers B.V.
392 MATERIALS AND METHODS
The procedures for sterilizing materials, dissecting explants, and the composition of the culture medium have been previously described in detail. Explants were excised from portions of each organ at the optimal time of excision (Niimi and Onozawa, 1979; Niimi and Watanabe, 1982).
Plant materials. - Nine parent plants of Lilium rubellum Baker, cultivated in an experimental field under the same conditions as described in a previous paper (Niimi and Watanabe, 1982), were harvested at the end of April 1982, during the 10-20 days after sprouting. Fifteen unexpended young leaves, one flower bud, the 7 em terminal part of a stem, and one mother bulb were separated from each parent plant and washed thoroughly with tap water. Culture conditions. - A medium consisting of Murashige and Skoog salt (1962), myo-inositol, vitamins, glycine, 50 g 1-1 sucrose, 7 g 1- 1 Difco Bacto agar, 1mg r ' o-naphthaleneacetic acid (NAA), and 0.1 mg r 1 6-benzylaminopurine (BA) was used (Niimi and Onozawa, 1979). About 20 ml of the dissolved medium was poured into each 50-ml Erlenmeyer flask, and these were then capped with aliminum foil and autoclaved for 10 .min at 121°C, under a pressure of 1.2 kg cm". Flasks with explants were kept in the dark at 24 ± 1°C. Disinfection of organs. - Leaves, stems and flower buds were surface-sterilized by swabbing them with cotton wool containing 70% ethanol, followed by immersing the organs for 5 min in a 10% solution of a commercial bleach containing 6% sodium hypochloride in water. The organs were then rinsed 3 times in sterile distilled water. After removal of a few outer scales, bulbs were immersed in a 0.1% solution of HgClz for 30 min and disinfected using the methods described for the other organs, except that the bulbs were immersed in the solution of sodium hypochloride for 20 min. Excision and culturing of explants. - Three 5-mm transverse strips of a leaf, one 5-mm transverse strip of a tepal and a scale section of about 1 X 1 em were dissected from the proximal portion of each organ, and each was then placed with the abaxial side down on the basal medium in each flask. Ten 5-mm explants were dissected from the terminal portion of each stem, and each was laid on the basal medium in each flask. Determining number and weight of developed bulblets. - 120 days after culture, bulblets were dissected from each of the explants. Their fresh weight was determined after removal of the roots. The results were subjected to statistical analysis at the 5% level following Duncan's multiple range test (Duncan, 1955).
393 RESULTS
Of the cultured explants, 74% developed bulblets. The ability to regenerate bulblets was greatest in explants of stems, of which 89% developed bulblets (Table I), but there was no statistical differences among explants of stems, leaves and scales. Table I also indicates significant differences in the numbers of bulblets per cultured explant, and the greatest bulblet formation occurred in the explants of leaves. This was also true for the numbers of bulblets per regenerated explant. Therefore, the explants of leaves appeared to be the best source for producing the greatest number of bulblets. The explants of stems produced, on average, by far the heaviest bulblets (Table I). Almost 50% of the bulblets formed in the explants of stems weighed more than 100 mg. In contrast, over 70% of bulblets regenerated from the explants of both leaves and scales weighed less than 50 mg. In conclusion, the explants of leaves were the best for regenerating bulblets per explant, while those of stems were the best for developing heavy bulblets. TABLE I Comparison of the regenerative ability of explants excised from different organs. Means in rows not followed by the same letter are significantly different at the 5% level. Absence of a letter shows that statistical analysis was not carried out Explants from: Scale
Leaf
Stem
Tepa!
Number of explants 405 90 54 180 Explants forming bulblets (%) Boa 89a 55 b 72 a Numbers of bulblets per cultured explant 1.9 a 2.2 b 1.5 c o.a d Numbers of bulblets per regenerated explant 1.7 1.4 2.4 3.1 Mean fresh weight of bulblet (mg) 24a 52a 40 a 135 b DISCUSSION
The results support the previous surmise that in L. rubellum Baker, the explants of leaves could be an excellent source for producing new bulblets in vitro, although leaf propagation of monocotyledonous plants is generally regarded as difficult in several species of Liliaceae, Iridaceae and Amaryllidaceae (Hussey, 1975). Takayama and Misawa (1979) reported that in L. speciosum also , segments of leaves excised just before anthesis were a poor source for the production of bulblets, as only lout of 150 segments sur-
394
vived. On the other hand, segments excised from young leaves of L. longiflorum (Stenberg et al., 1977), L. auratum, L. japonicum, L. speciosum X L. auratum, and a few other species of lily plants (Niimi and Onozawa, 1979) developed bulblets. These inconsistent results on the regenerative ability of leaf explants cannot be explained satisfactorily from the present results. More work will be needed to answer the inconsistent results. The present study shows not only that explants excised from the terminal portion of stems can easily regenerate bulblets, as reported for L. longiflorurn (Sheridan, 1968) and tulip (Wright and Alderson, 1980) but also that these excised explants can produce the heaviest bulblets in a short period of time. Furthermore, use of the terminal portions of a stem with young leaves has the advantage of not disturbing the parent bulbs, as well as providing explants without serious fungal pathogens. ACKNOWLEDGEMENTS
The author is grateful to Mr. Y. Kato and Mr. K. Sotome for their cooperation in this study and to Mr. D.H. Goldstein of the Indiana University for correcting the English text.
REFERENCES Duncan, D.B., 1955 . Multiple range and multiple F tests. Biometrics, 11: 1-42. Hussey, G., 1975. Totipotency in tissue explants and callus of some members of the Lilia· ceae, Iridaceae, and Amaryllidaceae. J. Exp. Bot., 26 : 253-262. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-479. Niimi, Y. and Onozawa, T. , 1977. Vegetative propagation of L. rubel/urn Baker; corriparlson of bulblet formation between scaling and segment cultured in vitro. Abstracts Fall Meeting Jpn. Soc. Hortie. ScL, pp. 348-349 (in Japanese). Niirni, Y. and Onozawa, T., 1979. In vitro bulblet formation from leaf segments of lilies, especially Lilium rubellurn Baker. Scientia Hortic., 11: 379-389. Niimi, Y. and Watanabe, H., 198Z. In vitro propagation of Liliurn rubellum Baker with special reference to bulblet formation from stem segments. Jpn. Soc. Hortic. Sci., 51: 344-349 (in Japanese, with English summary). Sheridan, W.F., 1968. Tissue culture of the monocot Lilium. Planta, 82: 189-192. Stenberg, N.E., Chen, C.H . and Ross, J.G., 1977. Regeneration of plantlets from leaf cultures of Lilium longiflorurn Thunb. Proc, South Dakota Acad. Sci., 56: 152-158. Stimart, D.P. and Ascher, P.D., 1978. Tissue culture of bulb scale sections for asexual propagation of Lilium longiflorum Thunb. J. Am. Soc . Hortic. ScL, 103: 182-184. Takayama. S. and Misawa, M., 1979. Differentiation in Lilium bulb-scales grown in vitro. Effect of various cultural conditions. Physio!. Plant. , 46: 184-190. Wright, N.A. and Alderson, P.G., 1980 . The growth of tulip tissue in vitro. Acta Hortie., 109 : 263-270 .