Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea

Table 1 Success rate of sym10 complementation in pea mutants P5 and P56

Pea line	Treatment *	Number of plants	Number of plants with nodulated roots	Complementation efficiency
P5 (sym10)	Water	36	10	0.28
P5 (sym10)	IAA	21	4	0.19
P5 (sym10)	Roots excised	22	12	0.55
P56 (sym10)	Water	20	4	0.20
P56 (sym10)	IAA	14	5	0.36
P56 (sym10)	Roots excised	14	10	0.71
Latest round of transformation
P5 (sym10)	Roots excised	22	16	0.73
P56 (sym10)	Roots excised	10	7	0.70

The excised shoots of P5 and P56 plants were exposed to Agrobacterium rhizogenes AR1193 carrying the SYM10 gene. Complementation efficiency represents the ratio of transformed plants with at least one root bearing nodules. With each passing round of transformation, adjustments were made to fine-tune the protocol. Thus the latest round is distinguished from the other trials to illustrate the outcome of the optimized protocol. * The different treatments were performed after the Agrobacterium inoculation in order to promote the formation of roots. The treatment where plants received only water until roots developed were considered as control. The plants undergoing the IAA treatment were given 10 μM IAA for 48 hours after transformation. The plants undergoing the root excision treatment were given water until roots developed; all roots protruding around the callus were excised before the callused epicotyls were transferred to pots.

ISSN: 1746-4811