Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms

Abstract

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.

Inoculation Methods to Determine Resistance of Phalaenopsis amabilis (L.) Regeneratded from Irradiated Protocorms to Dickeya dadantii

“…Experiment 1 was conducted at the Laboratory of Tissue Culture I and Plant Molecular Biology I, Faculty of Agriculture, IPB University, Bogor, Indonesia. The protocol to obtain isolation of D. dadantii has been described by Putri et al (2021).…”

“…Inoculum Concentration of D. dadantii A bacterium isolated from an infected leaf of Phalaenopsis amabilis was confirmed as a soft rot pathogen by the Koch Postulate method (Sudarsono et al, 2018;Sanjaya et al, 2021;Putri et al, 2021). A single colony from the selected strain was grown into liquid medium (Lactose Broth/LB) of as much as 15 ml, shaken for 21hour at 100 ppm, and harvested by centrifugation at 8000 rpm for 6 min until the final volume was OD 600 =0.2 (Putri et al, 2021). The leaf disk assay evaluation method was carried out following Sudarsono et al (2018).…”

“…Breeders design a mutant superior using various techniques like somaclonal variations (Ferreira et al, 2020), mutation induction (Jeong et al, 2017), and transgenic plants (Chen et al, 2019). Mutation induction through physical and chemical mutagens is widely used in Phalaenopsis amabilis (Raihanun, 2017;Mohammadi et al, 2020;Putri et al, 2021;Azis et al, 2021). Gamma irradiation is one of the physical mutagens widely used to improve genetic diversity, such as resistance to soft rot disease.…”

Relationship of Resistance-Related Enzyme Activity and Salicylic Acid Content in Phalaenopsis Species with Different Levels of Resistance to Dickeya dadantii

“…Plant inoculation with D. dadantii D. dadantii isolate was obtained from Plant Molecular Biology Laboratory (PMB Lab), Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University. The isolate was previously confirmed as pathogenic to Phalaenopsis (Lubis et al 2020;Sanjaya et al 2020;Putri et al 2021). Isolate preparation was made, according to Sanjaya et al (2020).…”

“…The pellet was diluted with sterilized water to the concentration OD600 = 0.2. The inoculation was performed using detached leaves following Putri et al (2021) protocol. The leaf of each plant was cut into 2 cm long parts.…”