INDUCTION OF SOMATIC EMBRYOS IN PEA, PISUM SATIVUM L.

PNL Volume 17

1985

RESEARCH REPORTS

Kyse1y, W.

University of Bonn

Federal Republic of Germany

Successful application of tissue culture technology to the improvement

of crop plants depends upon the ability to regenerate plants in vitro.

Plant regeneration, however, has been quite difficult with seed legumes

as peas and beans (Glycine, Phaseolus, Vicia). In pea, an attempt was

made to overcome the regeneration problem by screening several genotypes

for their in vitro behavior in order to select embryogenic cell lines.

Ten genotypes of pea were tested, including the variety 'Dippes

Gelbe Viktoria' (DGV), five X-ray and neutron-induced mutants of the

variety DGV (A89C, 251A, 37A, 39, and 176A), three recombinants from

crosses with the fasciated mutant 489C and DGV (R 1115, R 15±1, R 20±1

—the last two are named according to the number ol their sterile

nodes), and a line of Pisum arvense.

For callus formation leaf explants (6 mm in diameter) of 13-day-old

pea seedlings were transferred to a modified MS-medium (MS-salts, 2 mg/1

thiamine HC1 and 250 mg/1 inositol) supplemented with 3% sucrose and

0.8% agar. The callus medium with 0.06 mg/1 picloram (4-amino-3,5,6-

-trichloropicolinic acid) in combination with 0.1 mg/1 BA (benzyl-

adenine) was found to be optimal. The pH of the media was adjusted to

5.8. Callus formation was performed at 27C in the dark. For suspension

cultures, the same modified MS-medium was used.

The genotypes showed different genetic behavior, not only for

callus growth (Fig. 1) but also for callus morphology. The recombinants

R 1115, R 15±1, and R 20±1 as well as the accession of P. arvense

developed a compact and nodular embryogenic callus whereas the other

genotypes produced only a compact callus which however exhibited nodular

structures upon transfer to liquid MS-medium. The callus cultures of

genotype 37A segregated into a slow growing, yellowish, soft, and com-

pact callus and into a fast growing, yellowish-white, compact callus.

In suspension cultures, some of the nodular callus structures de-

veloped into embryos (Fig. 2, 3). When torpedo-shaped embryos were

transferred to hormone-free solid MS-medium, some of them showed root

formation, but no shoot morphogenesis has yet occurred. In a limited

number of cases, an additional root was formed in the apparent shoot

apical region after 2-5 days. Growing the embryos in liquid MS- media

with added soluble starch, casein hydrolysate, activated charcoal, or

culturing with varying combinations of BA alone or in combination with

IBA (indole-3-butyric acid) or PAA (pheny1-acetIc acid) did not result

in shoot morphogenesis.



	PNL Volume 17 1985	RESEARCH REPORTS 39



	Fig. 1. Mean fresh weight of leaf-derived callus after a culture period of 5 and 10 weeks. Bars give ± S.E. of 9-12 replicates.



	Fig. 2. Embryogenic callus with Fig. 3. Single embryo (vertical embryoids and embryos in bar : 1 mm). suspension (vertical bar : 1 cm).