The use of mini and twin plants for the genetic analysis of symbiotic variation of Pisum sativum L. towards Rhizobium leguminosarum strains

For the study of symbiotic nitrogen fixation, small-seeded legumes like clover and alfalfa can be grown under aseptic conditions within test tubes enclosed with cotton plugs. However, such manipulations are not feasible with the large-seeded legumes like peas, bean and soybean, even by using large tubes of up to 30 cm in length. Lie et al. (2) reported that mini plants can be obtained by removing the cotyledons at an early stage. Using this method plantlets of 2-5 cm height were obtained, which grow well both in nutrient solution and in solid substrate (Perlite), and complete their normal life cycle by forming flowers and pods with seeds.

Root nodule formation and nitrogen fixation proceeded normally, when inoculated with the specific Rhizobium strain. In agreement with intact plants, combined nitrogen in the root medium was found to inhibit root nodule formation. When growing under controlled climatic conditions, it was found that the mini plants were quite sensitive to high light intensities, and the light intensity should be reduced to ca. 25% of that normally used for intact plants. This effect was also found in experiments with detached primary leaves from leguminous plants (Phaseolus vulgaris) which were induced to form roots and root nodules (1).

In this paper a simple method will be reported to obtain two identical individuals (twins) from each plant. This method is very helpful in the genetic analysis of plant crossings (F₂ generation and backcrosses), because it is now possible to expose the two identical individuals to two different Rhizobium strains.

Pea seeds were surface-sterilized using 5% hydrogen peroxide and germinated on 1% agar in Petri dishes at 20°C. To obtain mini plants the cotyledons were aseptically removed, after 2-3 days, when the roots were about 2-3 cm long. The seedlings were then either wrapped in cotton plugs and transferred to tubes containing sterile N-free nutrient solution or directly sown in pots containing sterile Perlite with the same nutrient solution. To obtain twins, the seed coat of the surface-sterilized seeds was removed after one day germination, and the exposed embryo was cut into two halves using a sharp razor. Each halved embryo, still attached to one cotyledon, was further cultivated in the Petri dish and then transferred to pots containing sterile N-free nutrient solution.

Data from Table 1 show that the results obtained with mini plants are in a good agreement with our results obtained earlier with intact pea plants. An analysis was made of a crossing between pea cv. Rondo and cv. Afghanistan. Again similar results were obtained with mini plants and with intact plants. A single gene in pea cv. Afghanistan was found to determine the resistance to European Rhizobium strains.

Table 1. Growth and nitrogen fixation of mini plants of pea cv. Rondo in association with R. leguminosarum strains.

An example of an analysis using twin plants is given in Table 2. A cross was made between pea lines Afghanistan and L110. The Afghanistan pea was known to be resistant to European Rhizobium strains, only forming nodules with Rhizobium strains from the Middle East (e.g. strain Tom). In contrast, L110, when growing in nutrient solution, nodulated well with European Rhizobium strains, but nodulation with Rhizobium strain Tom was strongly delayed, and more than 50% of the plants were non-nodulating (3). In this experiment 47 seedlings were used, but only 41 twins, 82 individuals, were available for the analysis.

Table 2. Segregation of twin individuals of a F₂ generation of a crossing between pea lines Afghanistan and L110, exposed to Rhizobium strains PRE or Tom

1 - no nodulation, + nodulation

These results show that the resistance in L110 to Rhizobium strain Tom is controlled by a single host gene which segregated independently from the gene in Afghanistan controlling resistance to European Rhizobium strains.