Plant Cell Reports
Plant Cell Reports (1995) 15:254-258
9 Springer-Verlag1995
Transformation of peas (Pisum sativum L.) using immature cotyledons Jan E. Grant, Pauline A. Cooper, Alastair E. McAra, and Tonya J. Frew New Zealand Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch,New Zealand Received 31 January 1995/Revisedversion received 17 May 1995 - Communicatedby E.D. Earle
Abstract. A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 rag/1 phosphinothricin. The nptlI and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the "Buster' (='Basta') leaf-paint test and the phosphinothricin acetyl transferase enzyme assay. Keywords. Pisum sativum, transformation, regeneration, phosphinothricin, Agrobacterium tumefaciens Abbreviations. BA: 6-benzylaminopurine;
Introduction Grain legumes have generally been difficult to regenerate and transform. To harness the potential of biotechnology, efficient and reliable transformation systems are necessary. Of the major grain legume crops - soybean, chickpeas, peas, cowpea, peanut, common bean, faba bean and lentils confirmed transgenic plants have been produced in all except faba bean, lentils and cowpea (reviewed by Christou, 1994). The only reports using Agrobacterium-mediated transformation were for soybean, chickpeas and peas. For soybean, the method was not reproducible, and for chickpeas, inheritance of the Iransgenes was not demonstrated. For peas, there are three published reports of Agrobacteriummediated transformation of peas where mature flowering transgenic plants have been produced (Davies et al. 1993; Puonti-Kaerlas et al. 1990; Schroeder et al. 1993). These three reports used Agrobacterium tumefaciens with different Correspondence to: J.E. Grant
explants and culture regimes to achieve transformation. Puonti-Kaerlas et al. (1990) used cultivars Stivo and Puget and recovered transgenic plants after co-cultivation of explants from shoot cultures and epicotyls, and a lengthy callus phase. Hygromycin as the selection agent allowed the recovery of transgenic plants whereas kanamycin did not. The process took 15 months from explant to seed-bearing primary regenerant. The primary regenerants that produced viable seed were tetraploid (Puonti-Kaerlas et al. 1992), probably due to the long callus phase required in this procedure. Schroeder et al. (1993) co-cultivated longitudinal slices of immature embryos of cultivars Greenfeast and Rondo and used phosphinothricin as the selection agent. This method took approximately 9 months from explant to seed-beating primary regenerant. Between 1.5 and 2.5% of starting explants gave rise to transformed plants. These authors considered explant source, bacterial strain, choice of selectable marker and the presence of hormones during cocultivation to be important factors for consistent production of transgenic pea plants. Davies et al. (1993) obtained transgenic plants by injecting A. tumefaciens into the cotyledonary node of cultivar Puget. After co-cultivation, explants were grown on a medium containing kanamycin as the selection agent. Approximately 1.4% of injected seed gave rise to transgenic plants. The time taken from inoculation to transfer to soil was about 4 months. Another 2-3 months would be required to obtain mature seed from such plants. This method has been unreliable for consistent production of transformed plants (Davies, pers. comm.). For the benefits of biotechnology to be realised, a transformation system should be widely applicable - i.e., genotype independent, efficient, reproducible and fast (Jacobsen 1993). Of the three protocols published for peas thus far, the one developed by Schroeder et al. (1993) is promising.
255 In this paper we report the production of transgenic pea plants from immature cotyledons. We have introduced a herbicide resistance gene and an antibiotic resistance gene into four cultivars of pea. Two of the cultivars were fieldgrown process peas (Bolero, Trounce) and two cultivars were glasshouse-grown white field peas (Huka and Bohatyr). This system is consistent and efficient with respect to rate of transformation, time taken to obtain mature transformed plants and number of cultivars successfully transformed. Materials and Methods Bacterial strain. The binary vector pLN27 contained the bar gene fused to the cauliflower mosaic virus 35S promoter with 5'7' terminator. The vector was based around pGA643 (An et al. 1988) and was constructed by Kevin Davies (Levin Research Centre, Crop & Food Research, Levin, New Zealand) who ligated the bar gene fragment into the HindlII site. This plasmid was transferred into A. tumefaciens strain AGLI (Lazo etal. 1991). Agrobacterium tumefaciens strain AGL1 with the plasmid pLN27 was grown overnight in LB containing 200/zM acetosyringone. Plant Material and Transformation Procedure. Pea cultivars Bolero (Asgrow) and Trounce (Goulden and Scott 1993) were grown in the field, Huka (Crampton 1975) and Bohatyr (Selgen Ltd) were grown in the glasshouse. Immature pods were harvested at the "eating pea' stage, where the seed had obtained maximum size but had not started to dry. Pods were surface sterilized for 20 mins in 0.75% (w/v) sodium hypochlorite containing 2 drops/1 Tween 80 as the wetting agent. Seeds were removed from the pods and cut in half from the seed attachment scar. Half the seed, distal to the embryonic axis, was discarded. The testa was removed from the remaining half and the embryonic axis (including the shoot/root axes and the cotyledonary node), was cut out adjacent to the cotyledonary stalk (Fig. 1) and discarded, thus separating the cotyledons. The remaining cotyledon segments were immersed for one hour in an overnight culture of A. tumefaciens. Cotyledons were plated on B5 medium (Gamhorg et al. 1968) containing 1.3 mg/l BA, 30 g/1 sucrose, 8 g/1 agar (Difco), pH 5.5 and 19.6 mg/l acetosyringone (Colby et aL 1991) and were placed so the cotyledonary stalk was perpendicular to the medium. After six days cocultivation, they were washed three times with sterile water and rinsed once in 400 mg/l timentin (Beecham Research Laboratories). Cotyledons were blotted and transferred at two-weekly intervals to B5 medium with 1.3 mg/l BA, 30 g/l sucrose, 8 g/1 agar, pH 5.8, 10 mg/l phosphinothricin (a gift from Hcechst Ltd, NZ) and 150 mg/l timentin. After 3 to 4 transfers, the cotyledon tissue was cut away from the growing callus and shoots. When shoots reached more than 10 mm they were excised and transferred to root initiation medium (B5 medium with 1 mg/l indole-3-butyric acid, 30 g/l sucrose, 8 g/l agar, 150 mg/1 timentin, pH 5.8). After 7 days shoots were transferred to B5 medium, with no plant growth regulators, 150 mg/1 timentin and 10 rag/1 phosphinothricin, for reselection and root elongation. Where axillary shoots elongated at the base of the main stem, they were cut off and placed on rooting medium. In this way some multiplication occurred. Shoots approximately 4 cm in height with roots actively growing on selection medium were transferred to soil and grown in the glasshouse (16 hr 18-23~ day/8 hr 12-16~ night). Analyses for Transformation. For bar gene expression the leaf paint test and phosphinothricin acetyltransferase (PAT) enzyme assays were conducted and followed the method of Schroeder et al. (1993). The levels of herbicide ('Buster' = "Basta'; active ingredient 200 g/1 glufosinate-ammonium) used for the leaf paint test on plants in the glasshouse were equivalent to 3 l/ha at approximately 4 weeks old and 5 I/ha approximately three weeks later. DNA isolation and Southern analysis followed the method of Timmerman
et al. (1993) and used PCR-amplified coding regions as probes, to confirm the presence of the bar and nptlI genes. Genomic DNA from the peas was cut with EcoRl which, for pLN27, cuts once adjacent to the left border. Cytology. Chromosome counts were made from the root tips of one Huka and one Trounce primary regenerant. Root tips were harvested from pots in the glasshouse, fixed in 6:3:1 chloroform:ethanol:acetic acid for 1 hour and stained with Fenlgen stain according to the method of Grant et al. (1984).
Results
Transformation and characterization of primary transgenic plants The immature cotyledons formed callus on and around the cut surface of the cotyledon attachment stalk (Fig. 2a). This callus then regenerated into multiple shoots (Fig. 2b). Two types of shoots developed on the selection medium - shoots that were green and actively growing and shoots that bleached and died. Healthy-looking shoots were placed on a phosphinothricin-free medium for root initiation. When these shoots with root initials were placed on selection medium, the roots of putative, transformed shoots elongated rapidly and continued to grow. The roots of non-transformed shoots grew slowly for 2 days, stopped elongating and gradually turned brown. All of the primary transformants except Bolero 64, Trounce 105 and Trounce 1054, were multiplied in culture by nodal cuttings, to give five plantlets each that could be transferred to the glasshouse. The first transformed shoots were transferred to the glasshouse after 4 months in culture. Transformed plants (Table 1, Fig. 3) were recovered from all four cultivars tested - Bolero, Trounce, Bohatyr and Huka. Table 1. Numbers of pea cotyledon explants used in transformation experiments Number cocultivated
Number Lost1
Number remaining
Transformed plants
Bolero
150
100
50
3
Trounce
225
184
41
3
Huka
100
0
100
2
Bohatyr
135
0
135
1
I cotyledons lost due to media contamination 3 months after co-cultivation
Transformation of pea plants was confirmed by Southern analysis probing with both the bar gene (Fig. 4, lanes 1-7; Table 2) and the nptll gene (data not shown). As the EcoR1 restriction enzyme cuts the pLN27 plasmid once, one Bolero regenerant, 64, was shown to have at least 3 copies of the gene, and Bohatyr regenerant, 10, had at least 4 copies. The other primary regenerants appeared to have one copy (Fig. 4). Trounce 70, Bolero 59 and Huka 12 lacked the npt II
256 gene. Expression of the phosphinothricin-resistance gene was determined by the PAT enzyme assay and the leaf paint test. The results of the leaf paint test at 5 1/ha and the PAT enzyme assay corresponded. Plants that did not express the phosphinothricin-resistance gene in the leaf tissue but were shown to have the gene by Southern analysis were also recovered (Trounce 105; Table 2). For the five micropropagated Huka 11 plants Huka 11/1 expressed phosphinothricin resistance but Huka 11/2, 11/3, 11/4 and 11/5 did not. Table 2. Analysis of the primary transformants for presence and expression of the bar gene. (ND = not determined).
Cultivar/ Transformant
Southern analysis
PAT assay
leaf paint (5 1/ha)
105 70 1054
+ +
+ +
+ +
11 12
+ +
+ +
+ +
Bohatyr I0
+
ND
+
Bolero 59 64 500
+ + +
+ + ND
+ + +
Trounce
Huka
Chromosome counts in the two primary transformants tested showed a diploid (2n = 14) chromosome complement. Individual pods from the primary regenerants were harvested separately and grown to form the next progeny (R1) generation. Analyses of the progeny
Progeny from primary transgenic peas were tested for presence of the bar gene by Southern analyses and for PAT expression by the PAT enzyme assay (Fig. 5) and the leaf paint test. Table 3. Inheritance of the bar gene in progeny of 4 of the primary transformants
Cultivar/ Transforrnant
positive Southern
negative Southern
30
12
11 12
50 33
18 8
Bolero 64
11
0
Trounce 105 Huka
Inheritance of the bar gene in the progeny of four primary transformants is shown in Table 3. Progeny from primary transformants Trounce 105, Huka 11 and Huka 12 inheritance showed the Mendelian single gene pattern for presence: absence of 3:1. For Bolero 64, which has at least 3 copies of the gene, all the progeny were positive although only a small number were available for testing. In general for the R1 progeny, the peas with a positive Southem showed a positive leaf paint and PAT assay results. However, progeny from non-expressing primary transformants did not express the gene. The primary transformants recovered in this system and their progeny showed a wide range in their resistance to phosphinothricin as measured by the leaf paint test and the PAT assay. From any given transformant that expressed the gene, the first generation progeny containing the gene showed variable resistance - from those plants that gave a resistant leaf test at the equivalent of 10 1/ha "Buster', to those that showed susceptibility at 3 1/ha "Buster' equivalent. The possibility that the primary transformants were chimeras was assessed in the progeny. Trounce and Huka progeny in forty individual multi-seeded pods were scored for the presence of the inserted gene by Southern analysis. In only one of these pods was the gene absent from all of the progeny. This pod was a two-seeded Huka pod.
Discussion We have developed a reliable transformation system for peas using immature cotyledons as the explant source. The transferred genes were shown to be stably inherited through the first sexual generation of transformed plants. As in previous reports (Puonti-Kaerlas et al. 1990; Schroeder et al. 1993), the choice of selectable marker was found to b e important for recovering transformed plants. Puonti-Kaerlas et al. (1990) successfully used a gene for hygromycin resistance and Schroeder et al. (1993) successfully used a phosphinothricin resistance gene. Both groups found the nptlI gene to be ineffective. We have used the npt II gene in conjunction with the nos promoter and found this to be ineffective in our system for selection of transformed plants (unpublished results). Davies et al. (1993) used a kanamycin resistance gene fused to a double enhancer CaMV 35S promoter to transform peas by their injection method. Experiments are underway to test a construct that has a CaMV 35S promoter fused to a kanamycin resistance gene. The immature pea cotyledons form callus at the cotyledon attachment scar and from this callus shoots develop. We have observed the development of embryo-like structures with intact root and shoot axes on this callus. It seems likely that in this regeneration system we get a mixture of organogenesis and embryogenesis. Investigation into the nature of regeneration is forming a separate study. It is possible that chimeric plants could be obtained
257 using immature cotyledons as the explant source. So far we have no evidence for this as only one of 40 multi-seeded pods contained progeny seed that did not have the gene. This pod was two-seeded and it could be expected that, by chance alone, both the progeny would not have the gene. This method for producing Ixansformed peas is reliable and efficient. It has a short time for production of transgenic plants and appears to be cultivar independent. All four cultivars tested produced morphologically normal, fertile, transformed plants. Further studies are underway to improve the efficiency of transformation and to test a wider range of cultivars. While transformation systems can be continually improved and "fine-tuned', our transformation system allows genes of economic importance and genes that contribute to basic research to be put into a range of pea cultivars. We are currently introducing genes with the potential to confer virus resistance, into a wide range of pea cultivars. Acknowledgements. We would like to thank Ms J. Reader for care of the plants in the glasshouse, Mr R. Lamberts and Ms J. Smith for photography and illustrations, Drs G. Timmerman-Vaughan, M. Christey, C. Eady and Ms T. Williams for helpful comments on early versions of the manuscript.
Figure 3 Transformed plants of Huka and Trounce flowering and setting seed in the glasshouse.
cotyledo.nry node.
cotFledol I
Figure 1 Diagrammatic representation of a pea seed showing where the cotyledon is cut. The explant is the piece of cotyledon between the cuts. Figure 4 Southern analysis for the bar gene of primary transformants: Bolero 64 (lane 1), Huka 11/1 (lane 2), Huka 11/3 (lane 3), Huka 12 (lane 4) Trounce 70/2 (lane 5), Trounce 105/! (lane 6), Trounce 105/3 (lane 7), progeny of Huka 11/1 (lanes 8-18), and a non-transformed pea - cultivar Huka (lane 19) and plasmid pLN27 (lane 20, cut with EcoR1).
Figure 2a Three week old cotyledon explants forming callus from the cotyledonary stalk; Figure 2b Multiple shoots that have formed from the callused embryo attachment stalk. The remaining cotyledon pieces have been removed.
Figure 5 PAT enzyme assay for progeny of ltuka 11/1 (lanes 1-11), a nontransformed negative control (lane 12), and the substrate mix without plant extract (lane 13). The top line is the non-incorperated acetyl CoA and the bottom line is the incorporated phosphinothricin product.
258 References
An G, Ebert PR, Mitra A, Ha SB (1988) In: Gelvin SB, Schilperoot RA, Verma DPS (eds) Plant Molecular Biology Manual, Kluwer Academic Publishers, Dordreeht pp. A3/1-A3/19 Christou P (1994) Euphytica 74:165-185 Colby SM, Juncosa AM, Meredith CP (1991) J Am Soc Hort Sei 116(2):356-361 Crampton NJ (1975) NZ J of Agric 131:11-183 Davies DR, Hamilton J, Mullineaux P (1993) Plant Cell Rep 12:180-183 Gamborg OL, Miller RA, Ojima K (1968) Exp Cell Res 50:151-158 Goulden DS, Scott RE (1993) NZ J of Crop and Hort Sei 21:265-266 Grant JE, Brown AI-ID,Grace JP (1984) Aust J Bot 32:665-677 Jacobsen H-J (1993) Grain Legumes 2:12-13 Lazo GR, Stein PA, Ludwig RA (1991) Bio/Technology9:963-967 Puonti-Kaerlas J, Eriksson T, EngstrOmP (1990) Theor Appl Genet 80:246252 Puonti-Kaerlas J, Eriksson T, Engstr{JmP (1992) Theor Appl Genet 84:443450 Schroeder HE, Schotz All, Wardley-Richardson T, SpencerD, Higgins TJV (1993) Plant Physiol 101:751-757 Timmerman GM, Frew TJ, Miller AL, Weeden NF, Jermyn WA (1993) Theor Appl Genet 85:609-615
Plant Cell Reports (1995) 15:254-258
9 Springer-Verlag1995
Transformation of peas (Pisum sativum L.) using immature cotyledons Jan E. Grant, Pauline A. Cooper, Alastair E. McAra, and Tonya J. Frew New Zealand Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch,New Zealand Received 31 January 1995/Revisedversion received 17 May 1995 - Communicatedby E.D. Earle
Abstract. A reliable Agrobacterium tumefaciens-mediated transformation method has been developed for peas (Pisum sativum) using immature cotyledons as the explant source. Transgenic plants were recovered from the four cultivars tested: Bolero, Trounce, Bohatyr and Huka. The method takes approximately 7 months from explant to seed-bearing primary regenerant. The binary vector used carried genes for kanamycin and phosphinothricin resistance. Transformed pea plants were selected on 10 rag/1 phosphinothricin. The nptlI and bar genes were shown to be stably inherited through the first sexual generation of transformed plants. Expression of the phosphinothricin-resistance gene in the transformed plants was demonstrated using the "Buster' (='Basta') leaf-paint test and the phosphinothricin acetyl transferase enzyme assay. Keywords. Pisum sativum, transformation, regeneration, phosphinothricin, Agrobacterium tumefaciens Abbreviations. BA: 6-benzylaminopurine;
Introduction Grain legumes have generally been difficult to regenerate and transform. To harness the potential of biotechnology, efficient and reliable transformation systems are necessary. Of the major grain legume crops - soybean, chickpeas, peas, cowpea, peanut, common bean, faba bean and lentils confirmed transgenic plants have been produced in all except faba bean, lentils and cowpea (reviewed by Christou, 1994). The only reports using Agrobacterium-mediated transformation were for soybean, chickpeas and peas. For soybean, the method was not reproducible, and for chickpeas, inheritance of the Iransgenes was not demonstrated. For peas, there are three published reports of Agrobacteriummediated transformation of peas where mature flowering transgenic plants have been produced (Davies et al. 1993; Puonti-Kaerlas et al. 1990; Schroeder et al. 1993). These three reports used Agrobacterium tumefaciens with different Correspondence to: J.E. Grant
explants and culture regimes to achieve transformation. Puonti-Kaerlas et al. (1990) used cultivars Stivo and Puget and recovered transgenic plants after co-cultivation of explants from shoot cultures and epicotyls, and a lengthy callus phase. Hygromycin as the selection agent allowed the recovery of transgenic plants whereas kanamycin did not. The process took 15 months from explant to seed-bearing primary regenerant. The primary regenerants that produced viable seed were tetraploid (Puonti-Kaerlas et al. 1992), probably due to the long callus phase required in this procedure. Schroeder et al. (1993) co-cultivated longitudinal slices of immature embryos of cultivars Greenfeast and Rondo and used phosphinothricin as the selection agent. This method took approximately 9 months from explant to seed-beating primary regenerant. Between 1.5 and 2.5% of starting explants gave rise to transformed plants. These authors considered explant source, bacterial strain, choice of selectable marker and the presence of hormones during cocultivation to be important factors for consistent production of transgenic pea plants. Davies et al. (1993) obtained transgenic plants by injecting A. tumefaciens into the cotyledonary node of cultivar Puget. After co-cultivation, explants were grown on a medium containing kanamycin as the selection agent. Approximately 1.4% of injected seed gave rise to transgenic plants. The time taken from inoculation to transfer to soil was about 4 months. Another 2-3 months would be required to obtain mature seed from such plants. This method has been unreliable for consistent production of transformed plants (Davies, pers. comm.). For the benefits of biotechnology to be realised, a transformation system should be widely applicable - i.e., genotype independent, efficient, reproducible and fast (Jacobsen 1993). Of the three protocols published for peas thus far, the one developed by Schroeder et al. (1993) is promising.
255 In this paper we report the production of transgenic pea plants from immature cotyledons. We have introduced a herbicide resistance gene and an antibiotic resistance gene into four cultivars of pea. Two of the cultivars were fieldgrown process peas (Bolero, Trounce) and two cultivars were glasshouse-grown white field peas (Huka and Bohatyr). This system is consistent and efficient with respect to rate of transformation, time taken to obtain mature transformed plants and number of cultivars successfully transformed. Materials and Methods Bacterial strain. The binary vector pLN27 contained the bar gene fused to the cauliflower mosaic virus 35S promoter with 5'7' terminator. The vector was based around pGA643 (An et al. 1988) and was constructed by Kevin Davies (Levin Research Centre, Crop & Food Research, Levin, New Zealand) who ligated the bar gene fragment into the HindlII site. This plasmid was transferred into A. tumefaciens strain AGLI (Lazo etal. 1991). Agrobacterium tumefaciens strain AGL1 with the plasmid pLN27 was grown overnight in LB containing 200/zM acetosyringone. Plant Material and Transformation Procedure. Pea cultivars Bolero (Asgrow) and Trounce (Goulden and Scott 1993) were grown in the field, Huka (Crampton 1975) and Bohatyr (Selgen Ltd) were grown in the glasshouse. Immature pods were harvested at the "eating pea' stage, where the seed had obtained maximum size but had not started to dry. Pods were surface sterilized for 20 mins in 0.75% (w/v) sodium hypochlorite containing 2 drops/1 Tween 80 as the wetting agent. Seeds were removed from the pods and cut in half from the seed attachment scar. Half the seed, distal to the embryonic axis, was discarded. The testa was removed from the remaining half and the embryonic axis (including the shoot/root axes and the cotyledonary node), was cut out adjacent to the cotyledonary stalk (Fig. 1) and discarded, thus separating the cotyledons. The remaining cotyledon segments were immersed for one hour in an overnight culture of A. tumefaciens. Cotyledons were plated on B5 medium (Gamhorg et al. 1968) containing 1.3 mg/l BA, 30 g/1 sucrose, 8 g/1 agar (Difco), pH 5.5 and 19.6 mg/l acetosyringone (Colby et aL 1991) and were placed so the cotyledonary stalk was perpendicular to the medium. After six days cocultivation, they were washed three times with sterile water and rinsed once in 400 mg/l timentin (Beecham Research Laboratories). Cotyledons were blotted and transferred at two-weekly intervals to B5 medium with 1.3 mg/l BA, 30 g/l sucrose, 8 g/1 agar, pH 5.8, 10 mg/l phosphinothricin (a gift from Hcechst Ltd, NZ) and 150 mg/l timentin. After 3 to 4 transfers, the cotyledon tissue was cut away from the growing callus and shoots. When shoots reached more than 10 mm they were excised and transferred to root initiation medium (B5 medium with 1 mg/l indole-3-butyric acid, 30 g/l sucrose, 8 g/l agar, 150 mg/1 timentin, pH 5.8). After 7 days shoots were transferred to B5 medium, with no plant growth regulators, 150 mg/1 timentin and 10 rag/1 phosphinothricin, for reselection and root elongation. Where axillary shoots elongated at the base of the main stem, they were cut off and placed on rooting medium. In this way some multiplication occurred. Shoots approximately 4 cm in height with roots actively growing on selection medium were transferred to soil and grown in the glasshouse (16 hr 18-23~ day/8 hr 12-16~ night). Analyses for Transformation. For bar gene expression the leaf paint test and phosphinothricin acetyltransferase (PAT) enzyme assays were conducted and followed the method of Schroeder et al. (1993). The levels of herbicide ('Buster' = "Basta'; active ingredient 200 g/1 glufosinate-ammonium) used for the leaf paint test on plants in the glasshouse were equivalent to 3 l/ha at approximately 4 weeks old and 5 I/ha approximately three weeks later. DNA isolation and Southern analysis followed the method of Timmerman
et al. (1993) and used PCR-amplified coding regions as probes, to confirm the presence of the bar and nptlI genes. Genomic DNA from the peas was cut with EcoRl which, for pLN27, cuts once adjacent to the left border. Cytology. Chromosome counts were made from the root tips of one Huka and one Trounce primary regenerant. Root tips were harvested from pots in the glasshouse, fixed in 6:3:1 chloroform:ethanol:acetic acid for 1 hour and stained with Fenlgen stain according to the method of Grant et al. (1984).
Results
Transformation and characterization of primary transgenic plants The immature cotyledons formed callus on and around the cut surface of the cotyledon attachment stalk (Fig. 2a). This callus then regenerated into multiple shoots (Fig. 2b). Two types of shoots developed on the selection medium - shoots that were green and actively growing and shoots that bleached and died. Healthy-looking shoots were placed on a phosphinothricin-free medium for root initiation. When these shoots with root initials were placed on selection medium, the roots of putative, transformed shoots elongated rapidly and continued to grow. The roots of non-transformed shoots grew slowly for 2 days, stopped elongating and gradually turned brown. All of the primary transformants except Bolero 64, Trounce 105 and Trounce 1054, were multiplied in culture by nodal cuttings, to give five plantlets each that could be transferred to the glasshouse. The first transformed shoots were transferred to the glasshouse after 4 months in culture. Transformed plants (Table 1, Fig. 3) were recovered from all four cultivars tested - Bolero, Trounce, Bohatyr and Huka. Table 1. Numbers of pea cotyledon explants used in transformation experiments Number cocultivated
Number Lost1
Number remaining
Transformed plants
Bolero
150
100
50
3
Trounce
225
184
41
3
Huka
100
0
100
2
Bohatyr
135
0
135
1
I cotyledons lost due to media contamination 3 months after co-cultivation
Transformation of pea plants was confirmed by Southern analysis probing with both the bar gene (Fig. 4, lanes 1-7; Table 2) and the nptll gene (data not shown). As the EcoR1 restriction enzyme cuts the pLN27 plasmid once, one Bolero regenerant, 64, was shown to have at least 3 copies of the gene, and Bohatyr regenerant, 10, had at least 4 copies. The other primary regenerants appeared to have one copy (Fig. 4). Trounce 70, Bolero 59 and Huka 12 lacked the npt II
256 gene. Expression of the phosphinothricin-resistance gene was determined by the PAT enzyme assay and the leaf paint test. The results of the leaf paint test at 5 1/ha and the PAT enzyme assay corresponded. Plants that did not express the phosphinothricin-resistance gene in the leaf tissue but were shown to have the gene by Southern analysis were also recovered (Trounce 105; Table 2). For the five micropropagated Huka 11 plants Huka 11/1 expressed phosphinothricin resistance but Huka 11/2, 11/3, 11/4 and 11/5 did not. Table 2. Analysis of the primary transformants for presence and expression of the bar gene. (ND = not determined).
Cultivar/ Transformant
Southern analysis
PAT assay
leaf paint (5 1/ha)
105 70 1054
+ +
+ +
+ +
11 12
+ +
+ +
+ +
Bohatyr I0
+
ND
+
Bolero 59 64 500
+ + +
+ + ND
+ + +
Trounce
Huka
Chromosome counts in the two primary transformants tested showed a diploid (2n = 14) chromosome complement. Individual pods from the primary regenerants were harvested separately and grown to form the next progeny (R1) generation. Analyses of the progeny
Progeny from primary transgenic peas were tested for presence of the bar gene by Southern analyses and for PAT expression by the PAT enzyme assay (Fig. 5) and the leaf paint test. Table 3. Inheritance of the bar gene in progeny of 4 of the primary transformants
Cultivar/ Transforrnant
positive Southern
negative Southern
30
12
11 12
50 33
18 8
Bolero 64
11
0
Trounce 105 Huka
Inheritance of the bar gene in the progeny of four primary transformants is shown in Table 3. Progeny from primary transformants Trounce 105, Huka 11 and Huka 12 inheritance showed the Mendelian single gene pattern for presence: absence of 3:1. For Bolero 64, which has at least 3 copies of the gene, all the progeny were positive although only a small number were available for testing. In general for the R1 progeny, the peas with a positive Southem showed a positive leaf paint and PAT assay results. However, progeny from non-expressing primary transformants did not express the gene. The primary transformants recovered in this system and their progeny showed a wide range in their resistance to phosphinothricin as measured by the leaf paint test and the PAT assay. From any given transformant that expressed the gene, the first generation progeny containing the gene showed variable resistance - from those plants that gave a resistant leaf test at the equivalent of 10 1/ha "Buster', to those that showed susceptibility at 3 1/ha "Buster' equivalent. The possibility that the primary transformants were chimeras was assessed in the progeny. Trounce and Huka progeny in forty individual multi-seeded pods were scored for the presence of the inserted gene by Southern analysis. In only one of these pods was the gene absent from all of the progeny. This pod was a two-seeded Huka pod.
Discussion We have developed a reliable transformation system for peas using immature cotyledons as the explant source. The transferred genes were shown to be stably inherited through the first sexual generation of transformed plants. As in previous reports (Puonti-Kaerlas et al. 1990; Schroeder et al. 1993), the choice of selectable marker was found to b e important for recovering transformed plants. Puonti-Kaerlas et al. (1990) successfully used a gene for hygromycin resistance and Schroeder et al. (1993) successfully used a phosphinothricin resistance gene. Both groups found the nptlI gene to be ineffective. We have used the npt II gene in conjunction with the nos promoter and found this to be ineffective in our system for selection of transformed plants (unpublished results). Davies et al. (1993) used a kanamycin resistance gene fused to a double enhancer CaMV 35S promoter to transform peas by their injection method. Experiments are underway to test a construct that has a CaMV 35S promoter fused to a kanamycin resistance gene. The immature pea cotyledons form callus at the cotyledon attachment scar and from this callus shoots develop. We have observed the development of embryo-like structures with intact root and shoot axes on this callus. It seems likely that in this regeneration system we get a mixture of organogenesis and embryogenesis. Investigation into the nature of regeneration is forming a separate study. It is possible that chimeric plants could be obtained
257 using immature cotyledons as the explant source. So far we have no evidence for this as only one of 40 multi-seeded pods contained progeny seed that did not have the gene. This pod was two-seeded and it could be expected that, by chance alone, both the progeny would not have the gene. This method for producing Ixansformed peas is reliable and efficient. It has a short time for production of transgenic plants and appears to be cultivar independent. All four cultivars tested produced morphologically normal, fertile, transformed plants. Further studies are underway to improve the efficiency of transformation and to test a wider range of cultivars. While transformation systems can be continually improved and "fine-tuned', our transformation system allows genes of economic importance and genes that contribute to basic research to be put into a range of pea cultivars. We are currently introducing genes with the potential to confer virus resistance, into a wide range of pea cultivars. Acknowledgements. We would like to thank Ms J. Reader for care of the plants in the glasshouse, Mr R. Lamberts and Ms J. Smith for photography and illustrations, Drs G. Timmerman-Vaughan, M. Christey, C. Eady and Ms T. Williams for helpful comments on early versions of the manuscript.
Figure 3 Transformed plants of Huka and Trounce flowering and setting seed in the glasshouse.
cotyledo.nry node.
cotFledol I
Figure 1 Diagrammatic representation of a pea seed showing where the cotyledon is cut. The explant is the piece of cotyledon between the cuts. Figure 4 Southern analysis for the bar gene of primary transformants: Bolero 64 (lane 1), Huka 11/1 (lane 2), Huka 11/3 (lane 3), Huka 12 (lane 4) Trounce 70/2 (lane 5), Trounce 105/! (lane 6), Trounce 105/3 (lane 7), progeny of Huka 11/1 (lanes 8-18), and a non-transformed pea - cultivar Huka (lane 19) and plasmid pLN27 (lane 20, cut with EcoR1).
Figure 2a Three week old cotyledon explants forming callus from the cotyledonary stalk; Figure 2b Multiple shoots that have formed from the callused embryo attachment stalk. The remaining cotyledon pieces have been removed.
Figure 5 PAT enzyme assay for progeny of ltuka 11/1 (lanes 1-11), a nontransformed negative control (lane 12), and the substrate mix without plant extract (lane 13). The top line is the non-incorperated acetyl CoA and the bottom line is the incorporated phosphinothricin product.
258 References
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