Efficient genetic transformation of Momordica charantia L. by microprojectile bombardment

Abstract

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6 µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene () and neomycin phosphotransferase II gene ( II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2 ± 1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of L. were determined as follows: 650 psi helium pressure and 6 cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50 mg l kanamycin) round. Finally, the transformants produced were subjected to histochemical assay, and integration of the transgenes ( and II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L

“…The longer the bombardment distance, the slower the speed of the gold particles and the more discrete their distribution. In the present experiment, with increase in the bombardment distance, the expression of GUS in safflower calli decreased, which is consistent with reports by Narra et al [ 33 ]. Increasing the number of bombardments increases the probability of plasmids entering the explant; however, there were no differences in GUS expression among safflower calli bombarded for different durations.…”

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L. (safflower)

“…The longer the bombardment distance, the slower the speed of gold particals and the more discrete the distribution. Therefore, this experiment found that with the increase of the bombardment distance, the expression of GUS in sa ower callus decreased, which was consistent with the study by Narra M et al [33]. Increasing the times of bombardment can increase the probability of plasmids entering the explant, but there is no difference in the expression of GUS in the sa ower callus bombarded with different times in this experiment.…”

Rapid and Efficient Optimization Method for a Genetic Transformation System of Medicinal Plants Erigeron breviscapus

“…It is widely used and efficient in DNA transfer in mammalian cells, microbes, and monocots [ 29 ]. So far, particle bombardment has been used in many medicinal plants, i.e., Catharanthus roseus [ 30 ], Hypericum perforatum [ 31 ], Centella asiatica [ 32 ], Tripterygium wilfordii [ 33 ], Momordica charantia [ 34 ], Scoparia dulcis [ 35 ], etc. In this study, a simple and effective biolistic transformation method was established in E. breviscapus with a transformation efficiency of 36.7%.…”