Soft-rot disease (SRD) in Phalaenopsis, caused Dickeya dadantii, has resulted in significant losses in the orchid sector in Indonesia. This study aimed to evaluate the inoculation method of Dickeya dadantii and identify the resistance response of individual regenerated plantlets of Phal. amabilis from irradiated protocorm. A detached leaf assay was used to evaluate the inoculation method and resistance response of SRD. Based on the results of this study, Dickeya dadantii bacteria could only infect the leaves through wounding tissue. The density of bacteria that could infect leaf tissue was OD600 = 0.2. All dilution factors tested caused soft rot symptoms in P. amabilis. On the other hand, Vanilla planifolia only showed symptoms at a dilution factor of 10-0. Four accessions of regenerated plantlets from irradiated protocorms were resistant to SRD. They were from irradiation 5 Gy (IP 05 Gy-23, IP 05 Gy-31, and IP 05 Gy-33) and one accession from the control treatment or without irradiation (IP 0 Gy -1). These results showed that 5 Gy irradiation increased plant resistance to SRD in Phalaenopsis. A dose of 5 Gy can potentially produce mutant lines resistant to SRD in Phalaenopsis or other plants, too.

Abstract. Putri HA, Purwito A, Sudarsono, Sukma D. 2021. Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms. Biodiversitas 22: 1077-1090. Phalaenopsis amabilis (L.) Blume is a prominent donor for the white petal and sepal trait in Phalaenopsis breeding. However, it has an undesirable character, such as susceptible to soft-rot disease. Therefore, developing soft-rot resistance mutants through gamma irradiation could be explored. This study aimed to evaluate the variability of plantlets regenerated from irradiated and non-irradiated protocorms using morphology, stomatal size and molecular markers and to test responses of the plantlets against soft-rot disease. The plantlets were regenerated from irradiated (5, 10, 15 or 20 Gy) and non-irradiated protocorms. The results showed that P. amabilis plantlet variants were successfully identified based on their leaf morphology and stomatal size variations. A few plantlets have low stomatal densities, large stomatal size, and high chloroplast numbers, which indicated they were polyploids. Leaf disc assay for soft-rot disease response grouped most of the plantlets into very susceptible or susceptible. Moreover, four soft-rot resistant plantlets regenerated from irradiated and non-irradiated protocorms were successfully identified. The resistant plantlets were identified after three consecutive periods of inoculations with pathogens causing soft-rot disease. The evaluation also confirmed nucleotide variation in the Pto gene isolated from different levels of plantlet variant resistance responses.

Protocorm multiplication through the regeneration of protocorm-like bodies (PLBs) is used to obtain clonal progenies of hybrid orchids. The aim of the present study was to identify effective media for commercial orchid propagation through PLB proliferation. We evaluated the effects of basal media and either coconut water or chitosan supplementation on the growth of PLBs and the proliferation of Phalaenopsis hybrid PLBs, using two experiments, with random complete block designs (RBCD). The first experiment evaluated the progeny of white (V3) x pink standard hybrid (KHM0421, population 1) and their reciprocal hybridization (population 2) and different medium compositions. The hybrid seeds germinated and developed into protocorms on a half-strength Murashige and Skoog (1/2 MS) medium supplemented with 15% coconut water (CW). The protocorms were then transferred to four different media: 1) 1/2 MS; 2) 1/2 MS + 15% CW; 3) 2 g L-1 complete fertilizer (CF); and 4) CF + 15% CW. Experiment two evaluated the effects of chitosan on the growth and proliferation of protocorms from population 1, using four different media: 1) 1/2 MS; 2) 2 g L-1 CF; 3) 1/2 MS + 5 ppm chitosan; and 4) CF + 5 ppm chitosan, supplemented with 15% CW. The first experiment showed that the protocorm from population 1 generated more PLBs (5.4 PLBs in 16 weeks) than that of population 2 (2.2 PLBs in 16 weeks). The best media for protocorm proliferation and plantlet regeneration was CF and CF + 15% CW. The results of the second experiment revealed CF + 15% CW was the best medium for protocorm multiplication and plantlet regenerations. Chitosan tended to have a negative effect on protocorm growth and did not increase protocorm multiplication or plantlet regeneration. The use of CF medium could benefit orchid propagation, as it was cheaper than the MS medium.