Summary
An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- 2,4-D:
-
2,4-Dichlorophenoxyacetic acid
- BA:
-
6-ben-zyladenine
- hpt :
-
hygromycin phosphotransferase gene
- IAA:
-
indole acetic acid, kin, kinetin
- NAA:
-
α-naphtalene acetic acid
- picloram:
-
4-amino-3,5,6-trichloropicolinic acid
References
Ahmed R, Gupta SD, Ghosh PD (1987) The cytological status of plants regenerated from shoot-meristem culture of Pisum sativum L. Plant Breed 98:306–311
Bedbrook J (1981) A plant nuclear DNA preparation procedure. Plant Mol Biol Newsl 2:24
Christou P, Swain WF, Yang NS, McCabe DE (1989) Inheritance and expression of foreign genes in transgenic soybean plants. Proc Natl Acad Sci USA 86:7500–7504
D'Amato F (1977) Cytogenetics of differentiation in tissue and cell cultures. In: Reinert J, Bajaj YPS (eds) Applied and fundamental aspects of plant cell, tissue, and organ culture. Springer, Berlin Heidelberg New York, pp 343–357
De KK, Roy SC (1985) Morphogenetic investigation on pea under in vitro conditions. Bull Torrey Bot Club 112:363–367
De Kathen A, Jacobsen H-J (1990) Agrobacterium tumefaciens-mediated transformation of Pisum sativum L. using binary and cointegrate vectors. Plant Cell Rep 9:276–279
Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J (1985) Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acid Res 13:4774–4778
Evans DA (1986) Case histories of genetic variability in vitro: tomato. In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol 3. Academic Press, New York, pp 419–448
Fry J, Barnason A, Horsch RB (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321–325
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158
Gheysen G, Dhaese P, Van Montagu M, Schell J (1985) DNA flux across genetic barriers: the crown gall phenomenon. In: Hohn B, Dennis ES (eds) Genetic flux inplants. Springer, Wien, pp 11–47
Gould AR (1984) Control of the cell cycle in cultured plant cells. CRC Crit Rev Plant Sci 1:315–344
Hinchee MAW, Connor-Ward DV, Newell CA, McDonnell RE, Sato SJ, Gasser CS, Fischoff DA, Re DB, Fraley RT, Horsch RB (1988) Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer. Bio/Technology 6:915–922
Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inzé D, Engler G, Villaroel R, Van Montagu M, Schell J (1980) The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230
Karp A (1986) Chromosome variation in plants regenerated from protoplasts and cultured plant tissues. In: Semal J (ed) Somaclonal variations and crop improvement. Martinus Nijhoff Publ. Dortrecht, pp 28–34
Kublakova M, Tejklova E, Griga M (1988) Some factors affecting root formation on in vitro-regenerated pea shoots. Biol Plant 30:179–184
Kysely W, Myers JR, Lazzari PA, Collins GB, Jacobsen H-J (1987) Plant regeneration via somatic embryogenesis in pea (Pisum sativum L.). Plant Cell Rep 6:305–308
Lulsdorf MM, Rempel H, Jackson JA, Baliski DS, Hobbs SLA (1991) Optimizing the production of transformed pea (Pisum sativum L.) callus using disarmed Agrobacterium tumefaciens strains. Plant Cell Rep 9:479–483
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y.
Manners JM (1988) Transgenic plants of the tropical pasture legume Stylosanthes humilis. Plant Sci 55:61–68
Matthysse AG, Torrey JG (1967) Nutritional requirements for polyploid mitoses in cultured pea root segments. Physiol Plant 20:661–672
McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 5:81–84
Miller HJ (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Natali L, Cavallini A (1987a) Nuclear cytology of callus and plantlets regenerated from pea (Pisum sativum L.) meristems. Protoplasma 141:121–125
Natali L, Cavallini A (1987b) Regeneration of pea (Pisum sativum L.) plantlets by in vitro culture of somatic embryos. Plant Breed 99:172–176
Pickardt T, Meixner M, Schade V, Schieder O (1991) Transformation of Vicia narbonensis via Agrobacterium-mediated gene transfer. Plant Cell Rep 9:535–538
Pijnacker LP, Walch K, Ferwerda MA (1986) Behaviour of chromosomes in potato leaf tissue cultured in vitro as studied by BrdC-Giemsa labelling. Theor Appl Genet 72:833–839
Puonti-Kaerlas J, Stabel P, Eriksson T (1989) Transformation of pea (Pisum sativum L.) by Agrobacterium tumefaciens. Plant Cell Rep 8:321–324
Puonti-Kaerlas J, Eriksson T, Engström P (1990) Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens-mediated gene transfer. Theor Appl Genet 80:246–252
Rubluo A, Kartha KK, Mroginski LA, Dyck J (1984) Plant regeneration from pea leaflets cultured in vitro and stability of regenerants. J Plant Physiol 117:119–130
Shahin E, Simpson R (1986) Gene transfer system for potato. Hortic Sci 21:1199–1201
Sree Ramulu K (1986) Case histories of genetic variability in vitro: potato. In: Vasil IK (ed) Cell culture and somatic cell genetics of plants, vol 3. Academic Press, New York, pp 449–473
Van den Elzen P, Townsend J, Lee KY, Bedbrook J (1985) A chimeric hygromycin resistance gene as a selectable marker in plant cells. Plant Mol Biol 5:299–302
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–414
Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48
Weising K, Schell J, Kahl G (1988) Foreign genes in plants: transfer, structure, expression, and applications. Annu Rev Genet 22:421–477
Zambryski P (1988) Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet 22:1–30
Author information
Authors and Affiliations
Additional information
Communicated by I. Potrykus
Rights and permissions
About this article
Cite this article
Puonti-Kaerlas, J., Eriksson, T. & Engström, P. Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants. Theoret. Appl. Genetics 84, 443–450 (1992). https://doi.org/10.1007/BF00229505
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00229505