Plant Cell Reports
P l a n t Cell R e p o r t s (1993) 1 2 : 1 8 5 - 1 8 8
9 Springer-Verlag 1993
Cambial tissue culture and subsequent shoot regeneration from mature black locust (Robiniapseudoacacia L.) Kyung-Hwan Han 1, Daniel E. Keathley 2, and Milton P. Gordon 1 t D e p a r t m e n t o f Biochemistry, SJ-70, U n i v e r s i t y o f W a s h i n g t o n , Seattle, WA 98195, U S A
2 Department of Forestry, Michigan State University, East Lansing, MI 48824, USA Received September 6, 1992/Revised version received November 25, 1992 - Communicated by J. M. Widholm
Abstract. Callus initiation and subsequent shoot regeneration were obtained from cambial tissue of mature black locust trees. Shoot regeneration was highly tree specific, and was only achieved when calli derived from a particular genotype (tree #1) were transferred to medium containing 6benzylaminopurine. The studies described here Show that the immediate use of a mature genotype as starting material for tissue cultural manipulation is possible by using cambial explants with the proper in vitro conditions. We also found that stems could be stored at 4~ for 8 months prior to culture initiation, with no decline in the viability of the cultures. Tissue sources can thus be continuously available.
The use of cambial tissues provides an advantage over other tissues from mature trees due to its year-round availability. Additionally, cambial explants offer the possibility of immediate gene transfer in mature genotypes without waiting through an initial culture induction period. For cambial tissues to be successfully incorporated into transformation systems, the ability to regenerate 'whole plants is critical. Our preliminary regeneration studies from seedling-derived callus tissues of black locust showed that initiation and maintenance of green callus tissues was required. In this paper, we report procedures for callus initiation and culture from four mature black locust trees and subsequent shoot regeneration from the callus.
Abbreviations: BAP = 6-benzylaminopurine; NAA --1-naphthyleneacetic acid; IBA = indolebutyric acid.
Materials and Methods Eaplantpreparation. Branches (0.5-1.0 cm in diameter) were removed from
Introduction Genetic manipulation of plants at the cellular and molecular levels for use in plant improvement programs requires the ability to regenerate whole plants from unorganized tissues. In black locust (Robiniapseudoacacia L.), successful shoot regeneration has been reported from seedling-derived callus (Han and Keathley 1989) and callus derived from shoot cultures of mature trees (Han et al. 1990). In vitro micropropagation of black locust has been reported from auxiliary buds (Chalupa 1983; Davis and Keatlfley 1987; Barghchi 1987) and from leaf disks (Davis and Keathley 1985). Along with these, isolation and culture of protoplasts (Han mad Keathley 1988) and regeneration of black locust via somatic embryogenesis (Merkle and Wiecko 1989) have extended current in vitro methods for the species. Davis and Keathley (1989) were able to transform black locust with several strains of Agrobacterium tumefaciens and detect stably incorporated T-DNA sequences in the plant genome. Furthermore, we are able to regenerate lransgenic black locust using Ri plasmid-mediated Iransfonnation (H~a et al. 1992). Correspondence to: K . - H . H a n
4 mature trees (23-26 years old; Davis and Keathley 1987) in March and stored at 4"C until use. The branches were cut into 3-4cm pieces, washed with detergent (Liqui-nox) and placed under running water for 1 hr. Following a wash with tap water, the materials were immersed in 70% ethanol for 1-2 min and soaked in a 50% Clorox solution for 10-15 min followed by 5 washes with sterile distilled water. The bark layers external to the vascular cambium were discarded and cambial explants 0.2-0.5 m m thick and 3x4 m m in size were excised using a scalpel.
Explant culture. Cambial tissue explants were placed in petri dishes (15x100 mm) containing 25 ml of MS (Murashige and Skoog 1962) basal medium supplemented with various growth regulators, 0.8% Sigma-agar, 2% sucrose, and adjusted to pH 5.8 and then autoclaved for 15-20 min at 120~ The number of explants started and growth regulator levels are given in the table for each experiment. Cultures were grown in a growth chamber at 25~ with an 18 hr light/6 hr dark lighting regime using fluorescent lamps (50 uE m-2 s1). Callus was maintained by subculturing at 4-week intervals. Callus growth was scored by measuring the diameter of the callus.
Slwot regeneration and rooting. Twenty ealli per treatment were transferred to MS medium containing various concentrations (10, 25, and 50 uM) of BAP alone or BAP combined with 10 uM NAA. The number of shoots regenerated and callus growth were scored at 13 weeks. Shoots were excised from the callus tissue and transferred individually to 10 ml of shoot multiplication medium (MS + 0.5 uM BAP) in 25x200 m m test tubes. The resulting shoots were transferred to rooting medium (1/10 strength MS supplemented with 1
186 uM IBA and no sucrose), and rooted plantlets were then potted in a container covered with plastic wrap. The potting medium was peat moss, perlite and vermiculite (1:1:1). Plants were watered with Peter's 20:20:20 (lg/l). Statistical analysis. The data for callus initiation and growth were analyzed separately for each treatment using a general linear model analysis of variance/ covariance (GLM-ANOVA; Hintze 1987). The data for each treatment were separated by Duncan's New Multiple-Range Test (Steele and Torrie 1980) where the GLM-ANOVA indicated significance. To perform pairwise comparison between treatments for shoot regeneration, 2x2 contingency tables were constructed (Steele and Torrie 1980).
Table 2. Shoot regeneration from cambial callus of mature black locust
after culture on regeneration medium for 13 weeks. Tree Medium No. of Explants % Explants Producing Shoots # NAA BAP (uM) #1 #2 #1 #2 0
10
0 0 10 10 10
25 50 10 25 50
20 18 20 20 20 20
12 18 12 18 18 18
0.0 27.8 0.0 0.0 0.0 0.0
8.3 33.3 16.7 0.0 0.0 0.0
2
all treatments
120
102
0.0
0.0
3
all treatments
120
108
0.0
0.0
4
all treatments
120
108
0.0
0.0
1
Result and Discussion
B a s e d on previous w o r k (Han and Keathley 1989; Han et at. 1990), 5 u M B A P and 10 u M N A A were selected as growth regulators for this study. T h e frequency o f callus induction from mature cambial tissues (90-100 %) was similar to that of juvenile tissues, indicating that unlike organogenesis (Han and Keathley 1989; H a n et al. 1990), cellular growth in vitro is not constrained by explant age. Tree-specific responses in cambial caUus growth were observed for both freshly collected explants and explants f r o m branches that h a d been stored for eight months (Table I). Analysis of variance showed that individual trees differed significantly for callus diameter growth (F-test; alpha=0.01).
(1) Experiment 1: Regeneration from fresh cambial tissues. Experiment 2: Regeneration from eambial tissues stored for 8 months. Table 3. XZanalysisby 2-way contingency table from regeneration data.
Comparison
% Shoot Regeneration
BAP concentration (uM) 10vs25 3.1 / 30.6 10vs50 3.1 / 6.3 25 vs 50 30.6 / 6.3
Xz 8.8** 0.4 6.5*
Table 1. The effects of fresh or stored (8 months) explant sources on
cambium callus growth after 4 weeks of culture initiation. Trees 1
(i) (2)
Explants
No. Explants
Avg. Diam. (mm)
Fresh Stored
P l a n t Cell R e p o r t s (1993) 1 2 : 1 8 5 - 1 8 8
9 Springer-Verlag 1993
Cambial tissue culture and subsequent shoot regeneration from mature black locust (Robiniapseudoacacia L.) Kyung-Hwan Han 1, Daniel E. Keathley 2, and Milton P. Gordon 1 t D e p a r t m e n t o f Biochemistry, SJ-70, U n i v e r s i t y o f W a s h i n g t o n , Seattle, WA 98195, U S A
2 Department of Forestry, Michigan State University, East Lansing, MI 48824, USA Received September 6, 1992/Revised version received November 25, 1992 - Communicated by J. M. Widholm
Abstract. Callus initiation and subsequent shoot regeneration were obtained from cambial tissue of mature black locust trees. Shoot regeneration was highly tree specific, and was only achieved when calli derived from a particular genotype (tree #1) were transferred to medium containing 6benzylaminopurine. The studies described here Show that the immediate use of a mature genotype as starting material for tissue cultural manipulation is possible by using cambial explants with the proper in vitro conditions. We also found that stems could be stored at 4~ for 8 months prior to culture initiation, with no decline in the viability of the cultures. Tissue sources can thus be continuously available.
The use of cambial tissues provides an advantage over other tissues from mature trees due to its year-round availability. Additionally, cambial explants offer the possibility of immediate gene transfer in mature genotypes without waiting through an initial culture induction period. For cambial tissues to be successfully incorporated into transformation systems, the ability to regenerate 'whole plants is critical. Our preliminary regeneration studies from seedling-derived callus tissues of black locust showed that initiation and maintenance of green callus tissues was required. In this paper, we report procedures for callus initiation and culture from four mature black locust trees and subsequent shoot regeneration from the callus.
Abbreviations: BAP = 6-benzylaminopurine; NAA --1-naphthyleneacetic acid; IBA = indolebutyric acid.
Materials and Methods Eaplantpreparation. Branches (0.5-1.0 cm in diameter) were removed from
Introduction Genetic manipulation of plants at the cellular and molecular levels for use in plant improvement programs requires the ability to regenerate whole plants from unorganized tissues. In black locust (Robiniapseudoacacia L.), successful shoot regeneration has been reported from seedling-derived callus (Han and Keathley 1989) and callus derived from shoot cultures of mature trees (Han et al. 1990). In vitro micropropagation of black locust has been reported from auxiliary buds (Chalupa 1983; Davis and Keatlfley 1987; Barghchi 1987) and from leaf disks (Davis and Keathley 1985). Along with these, isolation and culture of protoplasts (Han mad Keathley 1988) and regeneration of black locust via somatic embryogenesis (Merkle and Wiecko 1989) have extended current in vitro methods for the species. Davis and Keathley (1989) were able to transform black locust with several strains of Agrobacterium tumefaciens and detect stably incorporated T-DNA sequences in the plant genome. Furthermore, we are able to regenerate lransgenic black locust using Ri plasmid-mediated Iransfonnation (H~a et al. 1992). Correspondence to: K . - H . H a n
4 mature trees (23-26 years old; Davis and Keathley 1987) in March and stored at 4"C until use. The branches were cut into 3-4cm pieces, washed with detergent (Liqui-nox) and placed under running water for 1 hr. Following a wash with tap water, the materials were immersed in 70% ethanol for 1-2 min and soaked in a 50% Clorox solution for 10-15 min followed by 5 washes with sterile distilled water. The bark layers external to the vascular cambium were discarded and cambial explants 0.2-0.5 m m thick and 3x4 m m in size were excised using a scalpel.
Explant culture. Cambial tissue explants were placed in petri dishes (15x100 mm) containing 25 ml of MS (Murashige and Skoog 1962) basal medium supplemented with various growth regulators, 0.8% Sigma-agar, 2% sucrose, and adjusted to pH 5.8 and then autoclaved for 15-20 min at 120~ The number of explants started and growth regulator levels are given in the table for each experiment. Cultures were grown in a growth chamber at 25~ with an 18 hr light/6 hr dark lighting regime using fluorescent lamps (50 uE m-2 s1). Callus was maintained by subculturing at 4-week intervals. Callus growth was scored by measuring the diameter of the callus.
Slwot regeneration and rooting. Twenty ealli per treatment were transferred to MS medium containing various concentrations (10, 25, and 50 uM) of BAP alone or BAP combined with 10 uM NAA. The number of shoots regenerated and callus growth were scored at 13 weeks. Shoots were excised from the callus tissue and transferred individually to 10 ml of shoot multiplication medium (MS + 0.5 uM BAP) in 25x200 m m test tubes. The resulting shoots were transferred to rooting medium (1/10 strength MS supplemented with 1
186 uM IBA and no sucrose), and rooted plantlets were then potted in a container covered with plastic wrap. The potting medium was peat moss, perlite and vermiculite (1:1:1). Plants were watered with Peter's 20:20:20 (lg/l). Statistical analysis. The data for callus initiation and growth were analyzed separately for each treatment using a general linear model analysis of variance/ covariance (GLM-ANOVA; Hintze 1987). The data for each treatment were separated by Duncan's New Multiple-Range Test (Steele and Torrie 1980) where the GLM-ANOVA indicated significance. To perform pairwise comparison between treatments for shoot regeneration, 2x2 contingency tables were constructed (Steele and Torrie 1980).
Table 2. Shoot regeneration from cambial callus of mature black locust
after culture on regeneration medium for 13 weeks. Tree Medium No. of Explants % Explants Producing Shoots # NAA BAP (uM) #1 #2 #1 #2 0
10
0 0 10 10 10
25 50 10 25 50
20 18 20 20 20 20
12 18 12 18 18 18
0.0 27.8 0.0 0.0 0.0 0.0
8.3 33.3 16.7 0.0 0.0 0.0
2
all treatments
120
102
0.0
0.0
3
all treatments
120
108
0.0
0.0
4
all treatments
120
108
0.0
0.0
1
Result and Discussion
B a s e d on previous w o r k (Han and Keathley 1989; Han et at. 1990), 5 u M B A P and 10 u M N A A were selected as growth regulators for this study. T h e frequency o f callus induction from mature cambial tissues (90-100 %) was similar to that of juvenile tissues, indicating that unlike organogenesis (Han and Keathley 1989; H a n et al. 1990), cellular growth in vitro is not constrained by explant age. Tree-specific responses in cambial caUus growth were observed for both freshly collected explants and explants f r o m branches that h a d been stored for eight months (Table I). Analysis of variance showed that individual trees differed significantly for callus diameter growth (F-test; alpha=0.01).
(1) Experiment 1: Regeneration from fresh cambial tissues. Experiment 2: Regeneration from eambial tissues stored for 8 months. Table 3. XZanalysisby 2-way contingency table from regeneration data.
Comparison
% Shoot Regeneration
BAP concentration (uM) 10vs25 3.1 / 30.6 10vs50 3.1 / 6.3 25 vs 50 30.6 / 6.3
Xz 8.8** 0.4 6.5*
Table 1. The effects of fresh or stored (8 months) explant sources on
cambium callus growth after 4 weeks of culture initiation. Trees 1
(i) (2)
Explants
No. Explants
Avg. Diam. (mm)
Fresh Stored
