This study was conducted to establish callus cultures from leaf, stem and root explants of
Physalis minima using different combinations of 2,4-D and kinetin. Callus growth and anti-cancer
compound, physalin B production were further enhanced by optimising the cell explants and media
compositions such as basal media, salts concentration, carbon sources and plant growth regulators.
The results indicated that callus cultures derived from leaf, stem and root explants were best initiated
using a combination of 9.0 µM 2,4-D and 4.5 µM kinetin. Callus growth and synthesis of physalin B
were peaked at the late exponential growth phase over 25 d of culture. Callus growth did not vary
between explants, but physalin B was observed higher in leaf (0.78 mg g-1 dry wt.), followed by root
(0.71 mg g-1 dry wt.) and stem (0.64 mg g-1 dry wt.). MS basal medium was found superior to B5, SH
and WH basal media in supporting growth and physalin B production. Further tests on the media
compositions obtained a half strength of MS salts (½MS), 2.5% (w/v) sucrose and 9.0:4.5 µM of 2,4-
D:kinetin combination, which were the preferred salts strength, carbon sources and plant growth
regulators for optimum growth (0.23 g dry wt.) and physalin B production (1.75 mg g-1 dry wt.) of
callus cultures derived from leaf.
The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms.
Three different morphological callus types, identified as type A, B and C, and tips of in vitro inflorescences were used as target tissues for genetic transformation. Five different DNA plasmids carrying a synthetic green fluorescent protein (gfp) gene driven by different promoters, CaMV 35S, HBT, and Ubi1 were tested for the genetic transformation of Dendrobium Sonia 17. 35S-sgfp-TYG-nos (p35S) with the CaMV 35S promoter showed the highest GFP transient expression rate, while the HBT and Ubi1 promoters showed a relatively lower expression rate in all of the target tissues tested. The highest number of GFP-expressing cells was observed on day 2 post-bombardment, and the number declined gradually over the course of the next 2 weeks. Type A and B callus were found to be the best potential target tissues for genetic transformation.
The effects of medium strategy, number of impellers, aeration mode, and mode of operation on Morinda elliptica cell suspension cultures in a stirred-tank bioreactor are described. A lower number of impellers and continuous aeration contributed toward high cell growth rate, whereas a higher number of impellers reduced cell growth rate, although not anthraquinone yield. The semicontinuous mode could indirectly imitate the larger scale version of production medium strategy and improved anthraquinone production even with 0. 012% (v/v) antifoam addition. Production medium promoted both growth (maximum dry cell weight of 24.6 g/L) and anthraquinone formation (maximum content of 19.5 mg/g of dry cell weight), without any necessity for antifoam addition. Cultures in production medium or with higher growth rate and anthraquinone production were less acidic than cultures in growth medium or with lower growth rate and anthraquinone production. Using the best operating variables, growth of M. elliptica cells (24.6 g/L) and anthraquinone yield (0.25 g/L) were 45% and 140%, respectively, lower than those using a shake flask culture after 12 days of cultivation.