Influence of 2,4-D and BAP on callus growth and the subsequent regeneration of somatic embryos in long-term cultures of Pelargonium x domesticum cv. Madame Layal

Klaus-Thomas Haensch
Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V.
Department Plant Propagation
Kühnhäuser Str. 101, D-99189 Erfurt-Kühnhausen
Germany
Tel: 49 0 36 201 785 224
Fax: 49 0 36 201 785 250
E-mail: haensch@erfurt.igzev.de

Web page: http://www.igzev.de

Financial support: This investigation was undertaken with the support of the Ministries of Agriculture of the Federal Republic of Germany and the States of Brandenburg and Thüringen.

Keywords: auxin, cytokinin, pelargonium, propagation, somatic embryogenesis.

Pelargoniums are very popular ornamental plants, and their mass propagation is performed using cuttings or seeds, which is very expensive. Somatic embryogenesis has the potential for establishing more efficient propagation systems. This kind of propagation allows the regeneration of embryos from somatic cells of plants with the advantage that such embryos have the same genetic characteristics as the donor plant. Moreover, it is expected that this method is more efficient than other procedures because a lot of structures with a shoot and a root meristem could be produced in a very small volume at very high propagation rates. Possibilities for somatic embryogenesis in pelargoniums are restricted at present to the production of embryos on primary explants. In other species, for example carrots, it is possible to propagate undifferentiated embryogenic callus on a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D)  on which the development of embryos is suppressed by this growth regulator. Afterwards, the differentiation of embryos can be initiated by transferring the callus to a medium lacking this growth regulator. Until now, there has been no procedure available in pelargoniums that allows the continuous propagation of undifferentiated embryogenic material followed by the regeneration of somatic embryos. Therefore, the objective of this study was to examine to what extent such a procedure would be possible in this species. For this purpose, we investigated the connection between different amounts of the growth regulators 2,4-D and 6-benzylaminopurine (BAP) in the medium and the resultant growth and regeneration responses.

The experiment was performed using petioles of the cultivar Madame Layal (Pelargonium x domesticum). The petioles were cultivated in vitro on a medium with 2,4-D and BAP at concentrations between 0 and 18 µmol l^-1 according to the experimental scheme (Figure 4). The primary culture was followed by five or seven subcultures on the same medium (Figure 1). After this time, the material in each treatment was cultivated on the same medium without growth regulators for a period of up to 44 weeks. For growth characterization, the fresh mass of the cultures was measured after the primary culture and after each of the first five subcultures on the growth regulator-containing medium. The kind of regeneration was determined after primary culture and after different periods of cultivation on the growth regulator-free medium. The regeneration of somatic embryos on media with 2,4-D and BAP was examined histologically.

The results show that the cultures produced different amounts of fresh mass dependent on the growth regulator combination and subculture (Figure 2). The region with the highest fresh mass after primary culture is with 6-8 µmol l^-1 2,4-D in combination with 6-18 µmol l^-1 BAP. In the following five subcultures, a shift in the growth maximum towards lower 2,4-D concentrations could be observed. For BAP, such a shift in the growth maximum is less clear. Considering the complete experiment, the maximal growth decreases from the primary culture to the following subcultures.

Most growth regulator combinations have already given rise to the formation of globular embryos during primary culture (Figure 3a and Figure 4). The histological examinations revealed that the globular stage somatic embryos are new individuals (Figure 3b), which develop to bipolar structures showing clearly well developed shoot and root meristems (Figure 3c and Figure 3d). These examinations confirm a previous histological report about the regeneration of somatic embryos in this cultivar. Combinations without 2,4-D and those without BAP showed no somatic embryogenesis. Almost all treatments with both BAP and 2,4-D gave rise to somatic embryogenesis (Figure 4).

A check after the fifth subculture on growth regulator-containing medium on the ability to regenerate somatic embryos on growth regulator-free medium showed that there were a few embryos in some combinations. A check after the seventh subculture on growth regulator-containing medium demonstrated that there were no somatic embryos on growth regulator-free medium, even 26 weeks after the transfer. Therefore, most of this material was discarded after this stage. Only calluses of some treatments were transferred again to fresh growth regulator-free medium. With all of these maintained combinations, somatic embryos with the same appearance as in the primary culture (Figure 3a) were found after 18 weeks. Their number varied between 1 and 22.

This long-term study demonstrates for the first time that it is possible to propagate embryogenic cultures in pelargoniums, and to subsequently initiate the differentiation of embryos using the cultivar Madame Layal (Pelargonium x domesticum). Propagation of callus was only possible with combinations of 2,4-D and BAP, which gave rise to embryos from the primary culture stage on.However, the propagation of cells, as well as the differentiation of embryos, was inhibited for a long time by a continuous application of these growth regulators. For this reason, a long period on medium lacking growth regulators was necessary before the differentiation of embryos occurred again. This knowledge could contribute to improving the propagation of embryogenic cultures and the subsequent regeneration of embryos in pelargoniums. One approach for such improvements might be, for example, the elimination of growth regulators after a certain time by binding them to active charcoal or washing them away by frequently changing the medium. Other approaches might be the repeated switching between development and disorganization of somatic embryos or the substitution of growth regulators during the induction of somatic embryogenesis by alternative means such as salt stress.