Plant Cell Reports
Plant Cell Reports (1987) 6:431-434
© Springer-Verlag 1987
Differential responses to in vitro bud culture in mature Robiniapseudoacacia L. (black locust)* J. M. Davis and D. E. Keathley Department of Forestry, Michigan State University, East Lansing, MI 48824, USA Received July 10, 1987 / Revised version received October 5, 1987 - Communicated by J. M. Widholm
ABSTRACT
Buds excised from the stems of five dormant, mature (20- to 30-year old) black locust (Robinia pseudoacacia L. ) trees were placed on MS basal medium with various levels of 6-benzylaminopurine. In all treatments, bud explants from two of the trees produced shoots which could be subcultured. Whole plants were obtained from cultures of these two trees. Explants from two other trees became vitrified or produced callus, respectively, when cultured on medium containing between 0.032 and 1.0 ~M 6-benzylaminopurine; suhculturable shoots were only obtained when the buds from these trees were cultured on medium containing 3.2 ~M 6-benzylaminopurine. No shoot cultures which could be subcultured were obtained from the fifth tree used in these experiments. The whole plants produced in these experiments were transferred to a greenhouse, and were phenotypically normal five months after culture initiation (three months after transfer to the greenhouse). AB~TIONS: BAP, 6-benzylaminopurine; IBA, indole3-butyric acid INTRODUCTION Black locust (Robinia pseudoaoacia L. ) is a papilionaceous legt~e which is native to the southeastern United States, but has a worldwide naturalized range (Harlow et al., 1979). A method for propagating shoots of black locust (Chalupa, 1983) has been reported which used shoot nodal segments as the primary explant. Another report (Brown and Sommer, 1982) indicated that whole black locust plantlets were regenerated from seedling-derived callus tissue. Shoots have also been regenerated from leaf disks using the primary leaves of seedlings as explant source (Davis and Keathley, 1985). A simple in vitro method was sought which could be used to propagate mature, as well as juvenile, black locust trees. Shoot meristem culture was the selected strategy, because in vitro-induced genetic variation is probably minimized by the use of shoot meristem cultures (Scowcroft, 1985). The goal was to produce clonal propagules without causing appreciable damage to the donor tree.
Offprint requests to: D. Keathley *Michigan Ag. Exp. Station Journal Article No. 12351
The initial barrier to accomplishing this goal was the initiation of aseptic cultures. Surface sterilization of stems which possessed dormant lateral buds was only successful with one of five trees which were tested (Davis and Keathley, 1987). To ciretmlvent this problem, dormant winter buds from stems of five different 20- to 30-year old black locust trees were excised, and placed directly onto culture medit~. Culture contamination was substantially reduced in the experiments described in this paper, permitting the systematic study of in vitro shoot cultures from mature explants of this species. MATERIALS AND
Five black locust trees were selected from four different locations (two trees, #2 and #3, were from the same uneven-aged stand) in southern Michigan, to minimize the possibility that the chosen trees were closely related. Age was determined for each tree by counting annual rings on a core sample taken at breast height with an increment borer. Stems which represented the 1986 growing season were taken from the upper branches of each tree on March 9 (Experiment 1) and March 25 (Experiment 2), 1987. After removal from each tree, the cut ends of the stems were placed: in double-distilled water, and the stems were transported to the lab. To reduce stem surface contaminants, stems were cut into 20- to 30-cm segments, placed in a 500 ml graduated cylinder, and immersed in a sterilizing solution of 2.5% NaOCI with I0 drops/l Tween-20 for 20 minutes, followed by three separate one-vollm~ rinses with sterile double-distilled water. The stem segments were allowed to stand in a fourth rinse for 5 min, after which the water was decanted. The stem segments were then removed singly from the cylinder and placed on sterile filter paper for excision of lateral buds. The dormant buds of black locust lack true bud scales, are 1-2 mm in length, and lie sunken beneath three apparent "plates" of the leaf scar (Harlow et al., 1979). The basal attachment of each plate was cut, and the plates were removed to expose the buds. The buds were then excised (Fig. ] ) and placed vertically in 100x15 mm petri dishes which contained 25 mi of MS basal medium (Murashige and Skoog, 1962) with 1.2% agar (w/v) and various levels of BAP (Table 2). The range of BAP concentrations was selected on the basis of preliminary studies and the results presented in Davis and Keathley (1987). Four buds were
432 cultured in each dish, and five dishes were used for each hormone treatment (20 buds/tree/phytohormone level ). Three days after excision from the tree, and every 4 to 7 days thereafter, buds were transferred to I0 ml of fresh medium in 25x150 mm culture tubes. Transfers were relatively frequent, because results from preliminary experiments using stem cuttings indicated that mature black locust tissues secrete significant amounts of inhibitory metabelites into the culture medium. Necrotic tissue, if present, was trimmed from the base of each explant at the time of transfer. Twenty-three days after excision from the tree, data were collected on the number of shoots per aseptically cultured bud, and the lengt/~ of the longest shoot from each of six randomly chosen explants in each treatment (Table 2). The number of cultures which had been lost to contamination was also counted at that time (Table i). Individual shoots were then excised from the primary bud explants and transferred to fresh medit~ containing 0.32 ~M BAP for elongation (this BAP concentration was chosen based on preliminary experiments). The shoots were maintained on this medium for a total of 24 days, with one transfer to fresh mediom after 12 days. Shoots which had attained a length of 1.0 cm after 24 days were transferred to rooting medium containing 5 ml of 0.1 strength MS basal medium with no sucrose, 1.0% (w/v) agar, and I. 0 pM IBA. All other shoots were maintained on elongation meditm~. For all experiments, cultures were placed in a growth chamber maintained at 26-27~C, with an 18~h light / 6 h dark lighting regime (75 ~ o l / s / m z PAR).
these bud explants (Fig. 2), since black locustlbuds are often superposed (Harlow et al., 1979). Observation of the explants during culture growth indicated that bud explants from trees #I, #2, and #4 were producing multiple shoots on 0.1 and 0.32 BAP, but that higher BAP levels should be tested in an attempt to increase the response of trees #3 and #5. Experiment 2 was then performed. The higher levels of BAP did not increase the number of shoots produced by the explants of trees #3 and #5 (Table 2), but the viability of the shoots from tree #5 increased (Table 3). Buds from tree #1 produced the longest shoots, trees #3 and #5 the shortest, and the other trees were intermediate. The s~me hierarchy was apparent when values for mean shoot number per explant were compared. Although the bud cultures from tree #2 produced many shoots, most of these did not continue to elongate in later stages of the experiment due to vitrification. Analysis of the shoot length data using analysis of variance techniques showed that both the cytokinin level and the explant source (tree) had a significant effect on shoot elongation (F-test, alpha = 0.01) in Experiment I. Only differences due to the explant source were significant in Experiment 2 (F-test, alpha = 0 . 0 1 ). The 0.32 )~M BAP treatment was included in Experiment 2 to test the repeatability of the results obtained in Experiment I. Both the mean nt~ber of shoots produced and the average length of the longest shoot per bud (Table 2) were compared across the two 0.32 pM BAP treatments for each tree. None of the averages for shoot number or shoot length differed significantly when tested pair-wise across the two experiments (t-test, alpha = 0.05).
RESULTS The percentage of the explants which had visible fungal or bacterial contamination varied among the trees which were tested (Table I). Results from Experiments 1 and 2 were similar. Values ranged from 1.7% (tree #3) to 38.3% (tree #4), and trees #I, #3, and #5 had significantly lower levels of contamination than trees #2 and #4.
Table 1. Tree ages (years), the number and mean number of contaminated explants per treatment, and the total percentage of contaminated explants for each black locust tree sampled (data from Experiment 2).
Tree
Age
1 2 3 4 5
21 24 30 20 27
Contaminated Explants BAP level (pM) 0.32 1.0 3.2 Ib 7 0 9 3
3 5 1 9 5
Mean number of Contaminated Explants Total (~)a
0 5 0 5 1
1.3a 5.7c 0.3a 7.7c 3.0ab
6.7 28.3 1.7 38.3 15.0
aMeans which have different letters are significantly different at alpha = 0.05, using Duncan's New Multiple Range test.
Table 2. Average nt~nber of shoots black locust bud explant culture, and the average est shoot per explant in Experiments 1 and 2.
Tree
produced by each during in vitro length of the longeach treatment for
BAP level (~M) Experiment 1 Experiment2 0.032 0.1 0.32 0.32 1.0 3.2
Average Shoots per Explant a
1 2 3 4 5
0.8 0.7 0.2 0.8 0.0
1.8 1.5 0.6 1.6 0.4
2.6 1.5 0.5 1.7 0.6
1.7 1.2 0.7 1.7 0.6
2.4 1.3 0.6 1.8 0.9
2.1 1.7 0.5 1.6 0.9
Average Shoot Length b (mm)
1 2 3 4 5
2.2 0.3 0.7 1.0 0.0
4.0 2.3 1.2 2.5 0.8
6.5 2.3 1.2 3.0 0.8
6.2 2.3 1.3 3.3 1.0
4.3 1.5 0.5 3.7 0.8
4.0 2.5 0.8 3.5 1.2
aAverages were calculated from the number of visible shoot meristems 23 days after culture initiation. Only aseptic cultures were scored.
hTwenty observations per datum.
bAverages were calculated from the length of the longest shoot in each of six randomly chosen culture tubes.
The mean number of shoot meristems per cultured bud, and the average length of the longest shoot per bud explant, are shown in Table 2. More than one center of shoot proliferation was present on many of
The treatment that produced the most nonvitrified shoots longer than 1.0 cm for each tree is listed in Table 3. The response of buds from trees #I and #4 was uniform across BAP levels, as is indi-
433 eated by the relatively low percentage of the total shoots that were produced in the best treatment. In contrast, almost all of the shoots which were prod u c e d b y buds from trees #2 and #5 came from a single treatment (3.2 ~tM RAP). In the treatments with'lower BAP, extensive callus growth was observed on the buds from tree #5. Shoots from the buds of tree #2 appeared to be vitrified after culture at these lower BAP levels, while shoots from tree #3 appeared vitrified in all treatments.
Table
Tree
3. The treatment which produced the highest percentage of non-vitrified shoots !l.0 cm in length, the number of shoots
Plant Cell Reports (1987) 6:431-434
© Springer-Verlag 1987
Differential responses to in vitro bud culture in mature Robiniapseudoacacia L. (black locust)* J. M. Davis and D. E. Keathley Department of Forestry, Michigan State University, East Lansing, MI 48824, USA Received July 10, 1987 / Revised version received October 5, 1987 - Communicated by J. M. Widholm
ABSTRACT
Buds excised from the stems of five dormant, mature (20- to 30-year old) black locust (Robinia pseudoacacia L. ) trees were placed on MS basal medium with various levels of 6-benzylaminopurine. In all treatments, bud explants from two of the trees produced shoots which could be subcultured. Whole plants were obtained from cultures of these two trees. Explants from two other trees became vitrified or produced callus, respectively, when cultured on medium containing between 0.032 and 1.0 ~M 6-benzylaminopurine; suhculturable shoots were only obtained when the buds from these trees were cultured on medium containing 3.2 ~M 6-benzylaminopurine. No shoot cultures which could be subcultured were obtained from the fifth tree used in these experiments. The whole plants produced in these experiments were transferred to a greenhouse, and were phenotypically normal five months after culture initiation (three months after transfer to the greenhouse). AB~TIONS: BAP, 6-benzylaminopurine; IBA, indole3-butyric acid INTRODUCTION Black locust (Robinia pseudoaoacia L. ) is a papilionaceous legt~e which is native to the southeastern United States, but has a worldwide naturalized range (Harlow et al., 1979). A method for propagating shoots of black locust (Chalupa, 1983) has been reported which used shoot nodal segments as the primary explant. Another report (Brown and Sommer, 1982) indicated that whole black locust plantlets were regenerated from seedling-derived callus tissue. Shoots have also been regenerated from leaf disks using the primary leaves of seedlings as explant source (Davis and Keathley, 1985). A simple in vitro method was sought which could be used to propagate mature, as well as juvenile, black locust trees. Shoot meristem culture was the selected strategy, because in vitro-induced genetic variation is probably minimized by the use of shoot meristem cultures (Scowcroft, 1985). The goal was to produce clonal propagules without causing appreciable damage to the donor tree.
Offprint requests to: D. Keathley *Michigan Ag. Exp. Station Journal Article No. 12351
The initial barrier to accomplishing this goal was the initiation of aseptic cultures. Surface sterilization of stems which possessed dormant lateral buds was only successful with one of five trees which were tested (Davis and Keathley, 1987). To ciretmlvent this problem, dormant winter buds from stems of five different 20- to 30-year old black locust trees were excised, and placed directly onto culture medit~. Culture contamination was substantially reduced in the experiments described in this paper, permitting the systematic study of in vitro shoot cultures from mature explants of this species. MATERIALS AND
Five black locust trees were selected from four different locations (two trees, #2 and #3, were from the same uneven-aged stand) in southern Michigan, to minimize the possibility that the chosen trees were closely related. Age was determined for each tree by counting annual rings on a core sample taken at breast height with an increment borer. Stems which represented the 1986 growing season were taken from the upper branches of each tree on March 9 (Experiment 1) and March 25 (Experiment 2), 1987. After removal from each tree, the cut ends of the stems were placed: in double-distilled water, and the stems were transported to the lab. To reduce stem surface contaminants, stems were cut into 20- to 30-cm segments, placed in a 500 ml graduated cylinder, and immersed in a sterilizing solution of 2.5% NaOCI with I0 drops/l Tween-20 for 20 minutes, followed by three separate one-vollm~ rinses with sterile double-distilled water. The stem segments were allowed to stand in a fourth rinse for 5 min, after which the water was decanted. The stem segments were then removed singly from the cylinder and placed on sterile filter paper for excision of lateral buds. The dormant buds of black locust lack true bud scales, are 1-2 mm in length, and lie sunken beneath three apparent "plates" of the leaf scar (Harlow et al., 1979). The basal attachment of each plate was cut, and the plates were removed to expose the buds. The buds were then excised (Fig. ] ) and placed vertically in 100x15 mm petri dishes which contained 25 mi of MS basal medium (Murashige and Skoog, 1962) with 1.2% agar (w/v) and various levels of BAP (Table 2). The range of BAP concentrations was selected on the basis of preliminary studies and the results presented in Davis and Keathley (1987). Four buds were
432 cultured in each dish, and five dishes were used for each hormone treatment (20 buds/tree/phytohormone level ). Three days after excision from the tree, and every 4 to 7 days thereafter, buds were transferred to I0 ml of fresh medium in 25x150 mm culture tubes. Transfers were relatively frequent, because results from preliminary experiments using stem cuttings indicated that mature black locust tissues secrete significant amounts of inhibitory metabelites into the culture medium. Necrotic tissue, if present, was trimmed from the base of each explant at the time of transfer. Twenty-three days after excision from the tree, data were collected on the number of shoots per aseptically cultured bud, and the lengt/~ of the longest shoot from each of six randomly chosen explants in each treatment (Table 2). The number of cultures which had been lost to contamination was also counted at that time (Table i). Individual shoots were then excised from the primary bud explants and transferred to fresh medit~ containing 0.32 ~M BAP for elongation (this BAP concentration was chosen based on preliminary experiments). The shoots were maintained on this medium for a total of 24 days, with one transfer to fresh mediom after 12 days. Shoots which had attained a length of 1.0 cm after 24 days were transferred to rooting medium containing 5 ml of 0.1 strength MS basal medium with no sucrose, 1.0% (w/v) agar, and I. 0 pM IBA. All other shoots were maintained on elongation meditm~. For all experiments, cultures were placed in a growth chamber maintained at 26-27~C, with an 18~h light / 6 h dark lighting regime (75 ~ o l / s / m z PAR).
these bud explants (Fig. 2), since black locustlbuds are often superposed (Harlow et al., 1979). Observation of the explants during culture growth indicated that bud explants from trees #I, #2, and #4 were producing multiple shoots on 0.1 and 0.32 BAP, but that higher BAP levels should be tested in an attempt to increase the response of trees #3 and #5. Experiment 2 was then performed. The higher levels of BAP did not increase the number of shoots produced by the explants of trees #3 and #5 (Table 2), but the viability of the shoots from tree #5 increased (Table 3). Buds from tree #1 produced the longest shoots, trees #3 and #5 the shortest, and the other trees were intermediate. The s~me hierarchy was apparent when values for mean shoot number per explant were compared. Although the bud cultures from tree #2 produced many shoots, most of these did not continue to elongate in later stages of the experiment due to vitrification. Analysis of the shoot length data using analysis of variance techniques showed that both the cytokinin level and the explant source (tree) had a significant effect on shoot elongation (F-test, alpha = 0.01) in Experiment I. Only differences due to the explant source were significant in Experiment 2 (F-test, alpha = 0 . 0 1 ). The 0.32 )~M BAP treatment was included in Experiment 2 to test the repeatability of the results obtained in Experiment I. Both the mean nt~ber of shoots produced and the average length of the longest shoot per bud (Table 2) were compared across the two 0.32 pM BAP treatments for each tree. None of the averages for shoot number or shoot length differed significantly when tested pair-wise across the two experiments (t-test, alpha = 0.05).
RESULTS The percentage of the explants which had visible fungal or bacterial contamination varied among the trees which were tested (Table I). Results from Experiments 1 and 2 were similar. Values ranged from 1.7% (tree #3) to 38.3% (tree #4), and trees #I, #3, and #5 had significantly lower levels of contamination than trees #2 and #4.
Table 1. Tree ages (years), the number and mean number of contaminated explants per treatment, and the total percentage of contaminated explants for each black locust tree sampled (data from Experiment 2).
Tree
Age
1 2 3 4 5
21 24 30 20 27
Contaminated Explants BAP level (pM) 0.32 1.0 3.2 Ib 7 0 9 3
3 5 1 9 5
Mean number of Contaminated Explants Total (~)a
0 5 0 5 1
1.3a 5.7c 0.3a 7.7c 3.0ab
6.7 28.3 1.7 38.3 15.0
aMeans which have different letters are significantly different at alpha = 0.05, using Duncan's New Multiple Range test.
Table 2. Average nt~nber of shoots black locust bud explant culture, and the average est shoot per explant in Experiments 1 and 2.
Tree
produced by each during in vitro length of the longeach treatment for
BAP level (~M) Experiment 1 Experiment2 0.032 0.1 0.32 0.32 1.0 3.2
Average Shoots per Explant a
1 2 3 4 5
0.8 0.7 0.2 0.8 0.0
1.8 1.5 0.6 1.6 0.4
2.6 1.5 0.5 1.7 0.6
1.7 1.2 0.7 1.7 0.6
2.4 1.3 0.6 1.8 0.9
2.1 1.7 0.5 1.6 0.9
Average Shoot Length b (mm)
1 2 3 4 5
2.2 0.3 0.7 1.0 0.0
4.0 2.3 1.2 2.5 0.8
6.5 2.3 1.2 3.0 0.8
6.2 2.3 1.3 3.3 1.0
4.3 1.5 0.5 3.7 0.8
4.0 2.5 0.8 3.5 1.2
aAverages were calculated from the number of visible shoot meristems 23 days after culture initiation. Only aseptic cultures were scored.
hTwenty observations per datum.
bAverages were calculated from the length of the longest shoot in each of six randomly chosen culture tubes.
The mean number of shoot meristems per cultured bud, and the average length of the longest shoot per bud explant, are shown in Table 2. More than one center of shoot proliferation was present on many of
The treatment that produced the most nonvitrified shoots longer than 1.0 cm for each tree is listed in Table 3. The response of buds from trees #I and #4 was uniform across BAP levels, as is indi-
433 eated by the relatively low percentage of the total shoots that were produced in the best treatment. In contrast, almost all of the shoots which were prod u c e d b y buds from trees #2 and #5 came from a single treatment (3.2 ~tM RAP). In the treatments with'lower BAP, extensive callus growth was observed on the buds from tree #5. Shoots from the buds of tree #2 appeared to be vitrified after culture at these lower BAP levels, while shoots from tree #3 appeared vitrified in all treatments.
Table
Tree
3. The treatment which produced the highest percentage of non-vitrified shoots !l.0 cm in length, the number of shoots
