A PROPOSED ASSAY FOR DETECTION OF AUXIN-SENSITIVE MUTANTS OF PISUM USING INTACT SEEDLINGS

PNL Volume 14

1982

RESEARCH REPORTS 19

A PROPOSED ASSAY FOR DETECTION OF AUXIN-SENSITIVE MUTANTS OF PISUM USING

INTACT SEEDLINGS

Ingensiep, H. W. Institute of Genetics, University of Bonn

Federal Republic of Germany

Many of the morphological differences among mutants or recombinants

may result from the action of genes that control plant growth regulators

such as auxins. Experiments show that exogeneously applied auxins are

able to induce several morphological changes in plant form and size. In

such experiments different morphogenetic effects often are caused by

different auxins applied under equimolar conditions, and for Pisum

sativum these different effects could be correlated with differences in

metabolic availability and translocation of the applied auxins (1).

These studies led us to propose a simple test system to screen for

mutants which affect auxin metabolism, using intact pea seedlings.

Experimental background: Our previous experiments showed that nor-

mal pea seedlings exhibit a characteristic morphogenetic effect when

auxins 2,4-D, IAA, and NAA were applied via the root system (1).

Seedlings treated with 2,4-D were strongly inhibited in root and shoot

development, while IAA- and NAA-treated seedlings were not. This could

be correlated with higher metabolic availability of the IAA and NAA

molecules by different mechanisms: IAA is mainly decarboxylated or

conjugated to aspartic acid; NAA is mainly conjugated; and 2,4-D is

neither decarboxylated nor conjugated to any extent. In the case of IAA

and NAA, these mechanisms prevent the morphogenetic alteration of the

seedling by inactivating many of the free molecules. Hence, these

auxins are not able to reach the vascular tissue in the root in high

amounts and are not translocated into the shoot to cause morphogenetic

aberrations there. On the other hand, 2,4-D does reach the vascular

tissue and is actively translocated in lethal amounts into the shoot.

PNL Volume 14

1982

RESEARCH REPORTS

This experimental background was used for the following two-step

screening system for auxin-sensitive pea mutants. It is supposed that

the roots act as selective "filters" for the auxins and the shoots as

visible indicators of auxin action in the plants. If the shoot of a

given plant differs morphologically from the initial line, this may be a

hint that the plant exhibits genetically controlled changes in auxin me-

tabolism. For instance, if IAA-treated pea seedlings show strong

inhibition compared to the control, this may be due to the lower inac-

tivation capacity of this mutant. On the other hand, plants may occur

that show normal shoot development after being treated with 2,4-D. This

could be due to metabolic changes affecting the free molecule to greater

extent and resulting in resistance. Between these extreme reactions

other seedlings may occur which show auxin-sensitivity. These mor-

phological deviations could be observed in the first step, where the

initial line and mutants are treated with the three auxins as described.

In a second step, suspicious plants could be analyzed by physiological

methods to characterize quantitative changes in auxin metabolism, for

instance by chromatographical analysis of labelled auxins as described

(1). It must, of course, be recognized that other factors may be in-

volved in the observed changes.

Nevertheless this assay could be of help in detecting auxin-

sensitive pea mutants in those cases where auxin-dependent variations

are suspected. The advantage is that the procedure is simple, inexpen-

sive, and rapid (possible effects are detected within two weeks with a

few seeds). The more involved in vitro culture methods could be applied

after prescreening intact pea seedlings with this system.

1. Ingensiep, H. W. , ejt_ ai- 1981. PNL 13:21-23.