Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of spine gourd (Momordica dioica Roxb. ex Willd)

Abstract

Spine gourd (Momordica dioica Roxb. ex Willd) is a medicinally and economically important plant and also used as vegetable. In this study, we established an Agrobacterium tumefaciens-mediated transformation procedure for M. dioica. Leaf explants were incubated with A. tumefaciens strain LBA4404 containing a binary vector pBAL2 carrying the reporter gene β-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Following co-cultivation, leaf explants were cultured on Murashige and Skoog (MS) + Gamborg et al., (B 5) medium supplemented with 6.6 µM 2,4dichlorophenoxyacetic acid (2,4-D) combined with 3.3 µM 6-benzylaminopurine (BAP) containing 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Kanamycin-resistant calluses were induced from the leaf explants after three weeks. Shoot regeneration was achieved after transferring the calluses onto fresh selection medium 8.8 µM BAP and 2.2 µM 2,4-D. Transgenic shoots were excised from callus and elongated in MS medium fortified with 3.0 µM gibberellic acid (GA 3), 100 mg L-1 kanamycin and 300 mg L-1 carbenicillin. Finally, the shoots were rooted on MS basal medium supplemented with 3.0 µM indole 3-butyric acid (IBA) and 100 mg L-1 kanamycin. High transformation frequency was achieved by using three-day-old precultured leaf explants. Furthermore, the presence of acetosyringone (200 µM), infection of explants for 30 min and three days of cultivation proved to be critical factors for greatly improving the transformation efficiency. Incorporation and expression of the transgenes confirmed by polymerase chain reaction (PCR), Southern blot analysis, reverse transcription (RT)-PCR and GUS histochemical assay. Using this protocol, transgenic M. dioica plants can be obtained in approximately three months with a high transformation frequency of 9%.

Molecular confirmation of sex in regenerated plantlets of spine gourd (Momordica dioica Roxb. Ex. wild) by using RAPD markers

“…Similar observation were made in leaf explants (Devendra et al, 2009;Thiruvengadam & Chung, 2011;Mustafa et al, 2012), petiole explants of M. dioica (Thiruvengadam et al, 2012) leaf explants of Melothria maderaspatana (Baskaran et al, 2009), nodal explants of M. charantia (Al Munsur et al, 2009). …”

Induction of hairy roots by Agrobacterium rhizogenes -mediated transformation of spine gourd ( Momordica dioica Roxb. ex. willd) for the assessment of phenolic compounds and biological activities

“…It is commonly known as spine gourd or small bitter gourd or teasle gourd is an annual or perennial dioecious climber. It has native of tropical regions on Africa, South America and Asia with extensive distribution in Bangladesh, China, India, Japan, and Pakistan (Thiruvengadam and Chung, 2011;Talukdar and Hossain, 2014). Spine gourd contains significant amount of alkaloids, steroids, triterpenoids, glycosides, saponins, carotenoids, polyphenols, vitamins, and other health promoting phytochemicals, these may be helpful used for antioxidant, diabetes, cancer, neurodegenerative diseases, asthma, leprosy, hepatoprotective, analgesic, postcoital anti-fertility, nematocidal, jaundice, bleeding piles, anti-allergic, anti-malarial, anti-feedant, anti-bacterial, anti-fungal and anti-viral activities Talukdar and Hossain, 2014).…”

Momordica dioica Roxb (Spine Gourd)- An underutilized vegetable and medicinal plant in Sri Lanka

“…Seeds have dormancy for 5-6 months. Dipping the seeds in water for a day at the 30 0 C showed better germination (Thiruvengadam et al, 2011). The cutting of terminal vine with 2-3 nodes is the best portion for easily rooting (Ponnusamy et al, 2019).…”

“…MSHP +Ads+ IBA +Agar are the best combination treatment combination for root development (Deokar et al, 2003). Other than conventional breeding methods, direct organogenesis is the novel method for the propagation of diocia (Thiruvengadam and Chung, 2011).…”