Screening of variation through callus formation and potential plant growth promoting endophytic bacteria for biohardening of tissue culture plantlet’s Etlingera elatior [EMBARGOED]

Abstract

Torch ginger (Etlingera elatior) is a promising horticultural plant with various purposes such as medicinal, antibacterial agent, culinary, ornamental, and floral arrangement. The extravagant and showy inflorescence has made E. elatior suitable to be promoted as cut flower and floral arrangements in many countries such as Australia, Brazil, and Hong Kong. The increasing demand has led to the varietal improvements of E. elatior via tissue culture technology. In this project, white friable calluses were produced through indirect regeneration from the innermost part of inflorescence and screened for their genetic variation using Inter-simple sequence repeat (ISSR) marker. Special attention is needed to estimate the molecular genetic variation between the mother plant and the calluses. Furthermore, plant growth promoting endophytic (PGPE) bacteria isolated from E. elatior were also investigated in this project for biohardening purpose. PGPE bacteria were believed to improve adaptation of plantlets for transplanting during in vitro culture and acclimatization phase. The results showed that the Murashige and Skoog (MS) medium supplemented with glucose and 1.5 mg/L Benzylaminopurine (BAP) and 3 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) produced significantly higher percentage of callus induction (50%) after 20 weeks of culture. Next, the calluses were transferred into shoot induction media with different concentrations of BAP, Naphthaleneacetic acid (NAA) and Thidiazuron (TDZ). The calluses from shoot induction media were evaluated for their genetic variations by seven primers of ISSR. A total of 72 bands were generated of which 51 were polymorphic with mean percentage of polymorphic bands was 72%. Next, six endophytic bacteria were successfully isolated from E. elatior and screened for their PGPE behaviour. This study found that most of the endophytic bacterial exhibit a positive result for phosphate solubilization, Indole-3-acetic acid (IAA) production, and nitrogen fixation. A3 is a high solubilizer bacteria with PSI value 3.13±0.03. Another promising potential of phosphate solubilizing bacteria was shown by both B2 and A4 with PSI value 2.97±0.17 and 2.56±0.05 respectively. All PGPE bacteria also show positive results for IAA production while five of six of them were found to fix nitrogen. Three of six endophytes with the best result of PGPE properties were molecularly identified. PGPE bacteria A3 belongs to the species Enterobacter cloacae strain UIA3, while A4 is Bacillus velezensis strain UIA4 and B2 belongs to Bacillus siamensis strain UIA5, as per the 16S rRNA sequence analysis. In brief, these results showed that the explant used was affected by various concentrations of auxin and cytokinin for callus induction. Additionally, ISSR analysis revealed the occurrence of genetic variations during callus induction process. Then, PGPE bacteria obtained in this study can be utilised to promote plant growth and increase E. elatior productivity in future.