Abstract

Under identical culture conditions, only 8 out

of 12 species and subspecies of the genus

Daucus proved

capable of somatic embryogenesis. Random amplified



polymorphic DNA analysis indicated a polymorphism



between the genomes of individual species that were capable



of embryogenesis and those that were not. Two



specific bands (1.1 kbp, 0.68 kbp) were detected only in



the genomes of individuals with the capacity for embryogenesis.



These were cloned and sequenced, and the homology



of the nucleotide sequences of the various species



was detected: this ranged from 74% to 92% for the



larger sequence and from 92% to 97% for the smaller



one. These DNA sequences would appear to be useful as



a marker of the capacity for somatic embryogenesis in



the genus

Daucus.

Keywords

Daucus · DNA marker · Random amplified

polymorphic DNA · Somatic embryogenesis



Abbreviations

IAA: Indole-3-acetic acid ·

PCR:

Polymerase chain reaction · RAPD: Random

amplified polymorphic DNA ·



2D-PAGE:

Two-dimensional polyacrylamide gel

electrophoresis



Introduction



Although there is a continuous steady increase in the



number of species of higher plants for which protocols to



induce somatic embryogenesis have been reported, little



is understood about this developmental process. Genetic



factors related to the recalcitrance to somatic embryogenesis



have been discussed for one or the other species and



even for varieties of one species (Vasil 1984), but no conclusive



evidence for a direct genetic correlation at the



molecular level has yet been reported. In order to gain a



better understanding of the significance of genetic factors



for detecting possible markers for somatic embryogenesis,



we conducted studies at the DNA level under identical



experimental conditions on selected regenerative and



non-regenerative

Daucus species and subspecies.

In the investigation reported here we initially tested



the embryogenic capacity of 12

Daucus species or subspecies;

of these, 8 were successfully induced to undergo



somatic embryogenesis through the use of cultured petiole



explants. It appears that a finely tuned developmental



program is initiated in these explants when they are



placed into a medium containing an auxin. Following extensive



biochemical and histological investigations,



which also included a study of transgenic plant material



containing a mannopine synthase promoter/glucuronidase



reporter gene to observe auxin distribution within



cultured explants (Li and Neumann 1985; Neumann and



Grieb 1992; Neumann 1995, 2000; Grieb et al. 1997;



Imani 1999; Imani et al. 1999), we propose that this developmental



program is as follows.



Upon the initiation of embryogenesis, the endogenous



auxin is more or less evenly distributed among the cells



of the explanted petiole sections. After 3–4 days of culture



in an IAA-supplemented nutrient medium, a large



increase in cytoplasmic volume occurs near the vascular



bundles in cells that were previously vacuolated; this is



followed by the initiation of cell division. After 7 days of



culture, an accumulation of IAA can be observed in cells



near the vascular bundles, leaving the other tissue almost



free of IAA. Concurrently, the initiation of root primor-



Communicated by K.K. Kamo



J. Imani · L. Tran Thi · B. Arnholdt-Schmitt · C. Lein



K.-H. Neumann (

✉)

Institute of Plant Nutrition, Division of Plant Tissue Culture,



Justus Liebig University, Heinrich-Buff-Ring 26-32,



35392 Giessen, Germany



e-mail: Karl-Hermann.Neumann@ernaehrung.uni-giessen.de



Tel.: +49-641-9939190, Fax: +49-641-9939199



G. Langen



Institute for Phytopathology, and Applied Zoology,



Justus Liebig University, Heinrich-Buff-Ring 26-32,



35392 Giessen, Germany



S. Roy · A. Kumar



Biotechnology Laboratory, Department of Botany,



University of Rajasthan, Jaipur 302004, India



Plant Cell Rep (2001) 20:537–541



DOI 10.1007/s002990100363



GENETICS AND GENOMICS



J. Imani · L. Tran Thi · G. Langen



B. Arnholdt-Schmitt · S. Roy · C. Lein · A. Kumar



K.-H. Neumann



Somatic embryogenesis and DNA organization of genomes



from selected

Daucus species

Received: 17 November 2000 / Accepted: 22 May 2001 / Published online: 5 July 2001



© Springer-Verlag 2001