PlantCell Reports
Plant Cell Reports (1995) 14:560-565
9 Springer-Verlag1995
Organogenesis in Taxus brevifolia tissue cultures Paula P. Chee Discovery Research, Unit 7242, The Upjohn Company, 301 Henrietta St. Kalamazoo, MI 49007, USA Received 14 July 1994/Revised version received 14 December 1994 - Communicated by J.M. Widholm
Summary. A method is described for multiple shoot and plantlet formation from zygotic embryos of Taxus brevifolia. Adventitious bud primordia were best induced by culturing zygotic embryos on 89 medium supplemented with i0 HM BA for 14 days. Further vegetative buds were produced following subculture to half-strength McCown's basal salt medium containing 1.0% activated charcoal. Individual adventitious shoots were excised and approximately 5% of these formed roots. Rooting frequency was increased to 58% by a single treatment with ABT rooting powder. Vigorous growing Taxus brevifolia plants were established after transfer to plant growth medium. Key words:
BA, N6-benzyladenine
Introduction Taxus brevifolia L. is a slow growing evergreen species occurring primarily in the ancient forest of the United States Pacific Northwest. Interest in the culture of this and other Taxus species has intensified with the discovery that taxol, an anticancer drug, is found in various parts of the tree (Vidensek et al., 1990). The total synthesis of taxol has recently been reported (Holton et al., 1994; Nicolaou et al., 1994); however, because of the complexity and numerous steps involved in the synthesis, largescale production may not yet be feasible. Because T. b r e v i f o l i a grows very slowly and has lengthy seed dormancy requirements (1.5 to 2 years), the supply of taxol is limited (Steinfeld, 1992). To ensure the long term survival of T. b r e v i f o l i a as a species, it is necessary to explore
alternative approaches for propagation of T. b r e v i f o l i a and the production of taxol. Flores and Sgrignoli (1991), and Chee (1994) reported embryo culture methods that could overcome the dormancy requirement of T. brevifolia. Eccher (1988) and Chee et al. (1994) reported stem cutting methods that could increase the number of available T. b r e v e f o l i a trees. Fett-Neto et al. (1992) and Wickremesinhe et al. (1994) reported callus culture and suspension culture methods, respectively, that could increase the production of taxol. Additional improvements in taxol production may be achievable through genetic engineering; however, an effective plant regeneration system is needed to facilitate genetic modification of T a x u s b r e v i f o l i a . As yet no reports have been made describing the regeneration of T a x u s spp. plants or any other member of the non-cone-bearing Taxaceae family. However, regeneration has been achieved in different families of Coniferophyta, which possess well-developed seedbearing cones, such as P i n a c e a e (Attree et al., 1990; Becwar et al.,, 1989; Lu et al., 1991) and Taxodiaceae (Bourgkard and Favre, 1988). The objectives of the present study were to investigate conditions for i n v i t r o organogenesis of T a x u s b r e v i f o l i a , and to develop a regeneration procedure that could be useful for mass production of T a x u s b r e v i f o l i a in vitro and which may serve as an entry point for genetic engineering. Materials and Methods Plant material and embryo isolation. Seeds of T. brevifolia, stage III (Chee, 1994), were
purchased from Special Trees, Corvallis, OR. Seeds were surface-sterilized for i0 min in
561 concentrated HCI, and then rinsed 5 times in sterile, distilled water. After sterilization, seeds were soaked in sterile, distilled water for 24 h. Embryos were excised aseptically from surrounding gametophytic tissue under a binocular microscope using fine forceps and a scalpel.
Bud induction, culture media, and conditions. For assaying bud induction, the embryos were cultured on hormone-containing medium for 7, 14 or 21 days. Depending on seed availability, twenty embryos were cultured in a 100 x 20-mm petri dish containing 25 ml of culture medium. Occasionally, some replicate dishes contained either more or fewer embryos. Frequencies of adventitious bud formation from each treatment were compared after a 6-week incubation period. All cultures were maintained at a 16-h photoperiod (80 pmol'~2's -l) at 26~ Unless otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8% agar (Sigma). The pH of all media was adjusted to 5.8 prior to autoclaving at 121~ for 20 minutes. Initially, the effect of five basal salt formulations on bud induction was assayed. These media were B5(Gamborg et al., 1968), DBM2(Gresshoff and Doy, 1972), MCM(Lloyd and McCown, 1981), QP(Quoirin and LePoivre, 1977), and SH(Schenk and Hildebrandt, 1972). Each formulation was supplemented with SH vitamins(Schenk and Hildebrandt, 1972), and referred as BSSH, DBM2SH, MCMSH, QPSH, SHSH. These basal salt formulations were supplemented with I0 ~M BA. The three best basal salt formulations were then tested at ~ salt dilutions. Various dilutions of B5SH in combination with i0 ~M BA were used to test effects of B5SH concentrations on frequency of bud induction. Full-strength B5SH with various concentrations of BA (7, i0, 15 and 20 b~4), kinetin (2.5, 5, I0 ~M), or thidiazuron (0.05, 0.i, 0.2 ~M) were used to test effects of growth regulators on bud induction. Half-strength B5SH with varying concentrations (i0, 20, 30, 40, 50 g/liter) of sucrose were used to test effects of sucrose on bud induction. Bud development and shoot elongation. Embryos were standardized on half-strength B5SH and embryos bearing adventitious buds were transferred to growth-regulator-free halfstrength MCM medium supplemented with 1% activated charcoal. After about one month, the cultured tissue was transferred to fresh medium of the same composition. The effects of two types of activated charcoal (acid washed, Sigma C4386; neutralized, Sigma C3790) on elongation of shoots at different concentrations (0%, 0.1%, 0.5%, 1%) were tested. In addition, the effect of gelling agents at various concentrations (0.6% agar, 0.8% agar, 0.5% agar + 0.2% gelrite, 0.2% gelrite) was also tested. Cultures were transferred to fresh medium every three to four weeks.
Statistical Analyses. If not otherwise stated all experiments were repeated at least twice. The total number of embryos cultured in each treatment is reported in the Tables. For each treatment examined, the data were entered into a contingency table. The response or non-response of different treatment groups was compared using the statistical procedure CATMOD(SAS Institute, 1989). Results Bud
and Discussion
induction
from
whole
embryos.
Primary explants were intact white mature zygotic embryos approximately 2 mm long. The selection of BA for use in organogenesis was based upon experimental evidence found for the Pinaceae family (von Arnold, 1982). During the first three days of culture in the presence of BA, the embryos elongated slightly. After about seven days in culture, further elongation of the embryos ceased. While swelling of the explants was observed, the surface of these organs was smooth. After approximately 14-21 days of culture, the explants continued to swell, and their upper surfaces developed nodular structures. These nodular structures further developed into bud primordialike structures upon transfer to growth-regulator-free medium (Fig. I).
Fig.1. Adventitious buds induced on zygotic embryos of T. brevifolia cultured on h B5SH with i0 btM BA for 14 days. Bar = 5 mm.
Rooting Rooting under sterile condition. After about two months, when the stems of adventitious shoots had reached a length of about 5 mm, the shoots were dissected from explants and transferred individually to growth-regulator-free-halfstrength MCM medium supplemented with 1.0% activated charcoal. Root formation was scored after four weeks. Rooting under non-sterile condition. Adventitious shoots were excised from the tissue and treated with ABT rooting powder (ABT Research Center, Beijing, China). Subsequently, rooted shoots were transferred to 2 inch-pots of plant growth medium [vermiculite and Perlite (i:i), J. Mollenma Co., Grand Rapids, MI]. During the first month ex vitro, the transplants were handmisted three times daily.
I. Effects of of basal medium
type and concentration on bud induction
Many reports have described the importance of the type and concentration of the basal medium on bud induction. Bornman (1983) c o m p a r e d four different basal media and found that MCM was most effective for inducing morphogenesis of Norway spruce cotyledons. Patel and Thorpe (1986) evaluated three basal media and observed that AE medium (von Arnold and Eriksson, 1981) induced the greatest
562 number of pitch pine buds per embryo. However, Lu et al. (1991) c o m p a r e d five d i f f e r e n t basal m e d i a and found that ~MCMSH was most e f f e c t i v e for i n d u c i n g buds from isolated embryos of red spruce. Chalupa (1977) r e p o r t e d that vegetative buds of P i c e a abies grew better on half-strength MS medium. Chee (1994) reported that zygotic embryos e x c i s e d from T a x u s spp. showed their highest frequency of radicle emergence and s e e d l i n g d e v e l o p m e n t on growth-regulator-free B5 basal salt medium. Thus, the effects of several basal media were tested. During the first three weeks, bud formation from i s o l a t e d embryos was compared on B5SH, DBM2SH, MCMSH, QPSH and SHSH media (Table I) . Bud formation was s i g n i f i c a n t l y h i g h e r on day 14 than on day 7 (Z2=30.8, p
Plant Cell Reports (1995) 14:560-565
9 Springer-Verlag1995
Organogenesis in Taxus brevifolia tissue cultures Paula P. Chee Discovery Research, Unit 7242, The Upjohn Company, 301 Henrietta St. Kalamazoo, MI 49007, USA Received 14 July 1994/Revised version received 14 December 1994 - Communicated by J.M. Widholm
Summary. A method is described for multiple shoot and plantlet formation from zygotic embryos of Taxus brevifolia. Adventitious bud primordia were best induced by culturing zygotic embryos on 89 medium supplemented with i0 HM BA for 14 days. Further vegetative buds were produced following subculture to half-strength McCown's basal salt medium containing 1.0% activated charcoal. Individual adventitious shoots were excised and approximately 5% of these formed roots. Rooting frequency was increased to 58% by a single treatment with ABT rooting powder. Vigorous growing Taxus brevifolia plants were established after transfer to plant growth medium. Key words:
BA, N6-benzyladenine
Introduction Taxus brevifolia L. is a slow growing evergreen species occurring primarily in the ancient forest of the United States Pacific Northwest. Interest in the culture of this and other Taxus species has intensified with the discovery that taxol, an anticancer drug, is found in various parts of the tree (Vidensek et al., 1990). The total synthesis of taxol has recently been reported (Holton et al., 1994; Nicolaou et al., 1994); however, because of the complexity and numerous steps involved in the synthesis, largescale production may not yet be feasible. Because T. b r e v i f o l i a grows very slowly and has lengthy seed dormancy requirements (1.5 to 2 years), the supply of taxol is limited (Steinfeld, 1992). To ensure the long term survival of T. b r e v i f o l i a as a species, it is necessary to explore
alternative approaches for propagation of T. b r e v i f o l i a and the production of taxol. Flores and Sgrignoli (1991), and Chee (1994) reported embryo culture methods that could overcome the dormancy requirement of T. brevifolia. Eccher (1988) and Chee et al. (1994) reported stem cutting methods that could increase the number of available T. b r e v e f o l i a trees. Fett-Neto et al. (1992) and Wickremesinhe et al. (1994) reported callus culture and suspension culture methods, respectively, that could increase the production of taxol. Additional improvements in taxol production may be achievable through genetic engineering; however, an effective plant regeneration system is needed to facilitate genetic modification of T a x u s b r e v i f o l i a . As yet no reports have been made describing the regeneration of T a x u s spp. plants or any other member of the non-cone-bearing Taxaceae family. However, regeneration has been achieved in different families of Coniferophyta, which possess well-developed seedbearing cones, such as P i n a c e a e (Attree et al., 1990; Becwar et al.,, 1989; Lu et al., 1991) and Taxodiaceae (Bourgkard and Favre, 1988). The objectives of the present study were to investigate conditions for i n v i t r o organogenesis of T a x u s b r e v i f o l i a , and to develop a regeneration procedure that could be useful for mass production of T a x u s b r e v i f o l i a in vitro and which may serve as an entry point for genetic engineering. Materials and Methods Plant material and embryo isolation. Seeds of T. brevifolia, stage III (Chee, 1994), were
purchased from Special Trees, Corvallis, OR. Seeds were surface-sterilized for i0 min in
561 concentrated HCI, and then rinsed 5 times in sterile, distilled water. After sterilization, seeds were soaked in sterile, distilled water for 24 h. Embryos were excised aseptically from surrounding gametophytic tissue under a binocular microscope using fine forceps and a scalpel.
Bud induction, culture media, and conditions. For assaying bud induction, the embryos were cultured on hormone-containing medium for 7, 14 or 21 days. Depending on seed availability, twenty embryos were cultured in a 100 x 20-mm petri dish containing 25 ml of culture medium. Occasionally, some replicate dishes contained either more or fewer embryos. Frequencies of adventitious bud formation from each treatment were compared after a 6-week incubation period. All cultures were maintained at a 16-h photoperiod (80 pmol'~2's -l) at 26~ Unless otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8% agar (Sigma). The pH of all media was adjusted to 5.8 prior to autoclaving at 121~ for 20 minutes. Initially, the effect of five basal salt formulations on bud induction was assayed. These media were B5(Gamborg et al., 1968), DBM2(Gresshoff and Doy, 1972), MCM(Lloyd and McCown, 1981), QP(Quoirin and LePoivre, 1977), and SH(Schenk and Hildebrandt, 1972). Each formulation was supplemented with SH vitamins(Schenk and Hildebrandt, 1972), and referred as BSSH, DBM2SH, MCMSH, QPSH, SHSH. These basal salt formulations were supplemented with I0 ~M BA. The three best basal salt formulations were then tested at ~ salt dilutions. Various dilutions of B5SH in combination with i0 ~M BA were used to test effects of B5SH concentrations on frequency of bud induction. Full-strength B5SH with various concentrations of BA (7, i0, 15 and 20 b~4), kinetin (2.5, 5, I0 ~M), or thidiazuron (0.05, 0.i, 0.2 ~M) were used to test effects of growth regulators on bud induction. Half-strength B5SH with varying concentrations (i0, 20, 30, 40, 50 g/liter) of sucrose were used to test effects of sucrose on bud induction. Bud development and shoot elongation. Embryos were standardized on half-strength B5SH and embryos bearing adventitious buds were transferred to growth-regulator-free halfstrength MCM medium supplemented with 1% activated charcoal. After about one month, the cultured tissue was transferred to fresh medium of the same composition. The effects of two types of activated charcoal (acid washed, Sigma C4386; neutralized, Sigma C3790) on elongation of shoots at different concentrations (0%, 0.1%, 0.5%, 1%) were tested. In addition, the effect of gelling agents at various concentrations (0.6% agar, 0.8% agar, 0.5% agar + 0.2% gelrite, 0.2% gelrite) was also tested. Cultures were transferred to fresh medium every three to four weeks.
Statistical Analyses. If not otherwise stated all experiments were repeated at least twice. The total number of embryos cultured in each treatment is reported in the Tables. For each treatment examined, the data were entered into a contingency table. The response or non-response of different treatment groups was compared using the statistical procedure CATMOD(SAS Institute, 1989). Results Bud
and Discussion
induction
from
whole
embryos.
Primary explants were intact white mature zygotic embryos approximately 2 mm long. The selection of BA for use in organogenesis was based upon experimental evidence found for the Pinaceae family (von Arnold, 1982). During the first three days of culture in the presence of BA, the embryos elongated slightly. After about seven days in culture, further elongation of the embryos ceased. While swelling of the explants was observed, the surface of these organs was smooth. After approximately 14-21 days of culture, the explants continued to swell, and their upper surfaces developed nodular structures. These nodular structures further developed into bud primordialike structures upon transfer to growth-regulator-free medium (Fig. I).
Fig.1. Adventitious buds induced on zygotic embryos of T. brevifolia cultured on h B5SH with i0 btM BA for 14 days. Bar = 5 mm.
Rooting Rooting under sterile condition. After about two months, when the stems of adventitious shoots had reached a length of about 5 mm, the shoots were dissected from explants and transferred individually to growth-regulator-free-halfstrength MCM medium supplemented with 1.0% activated charcoal. Root formation was scored after four weeks. Rooting under non-sterile condition. Adventitious shoots were excised from the tissue and treated with ABT rooting powder (ABT Research Center, Beijing, China). Subsequently, rooted shoots were transferred to 2 inch-pots of plant growth medium [vermiculite and Perlite (i:i), J. Mollenma Co., Grand Rapids, MI]. During the first month ex vitro, the transplants were handmisted three times daily.
I. Effects of of basal medium
type and concentration on bud induction
Many reports have described the importance of the type and concentration of the basal medium on bud induction. Bornman (1983) c o m p a r e d four different basal media and found that MCM was most effective for inducing morphogenesis of Norway spruce cotyledons. Patel and Thorpe (1986) evaluated three basal media and observed that AE medium (von Arnold and Eriksson, 1981) induced the greatest
562 number of pitch pine buds per embryo. However, Lu et al. (1991) c o m p a r e d five d i f f e r e n t basal m e d i a and found that ~MCMSH was most e f f e c t i v e for i n d u c i n g buds from isolated embryos of red spruce. Chalupa (1977) r e p o r t e d that vegetative buds of P i c e a abies grew better on half-strength MS medium. Chee (1994) reported that zygotic embryos e x c i s e d from T a x u s spp. showed their highest frequency of radicle emergence and s e e d l i n g d e v e l o p m e n t on growth-regulator-free B5 basal salt medium. Thus, the effects of several basal media were tested. During the first three weeks, bud formation from i s o l a t e d embryos was compared on B5SH, DBM2SH, MCMSH, QPSH and SHSH media (Table I) . Bud formation was s i g n i f i c a n t l y h i g h e r on day 14 than on day 7 (Z2=30.8, p
