Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2021, Vol. 42 ›› Issue (9): 2458-2467.DOI: 10.3969/j.issn.1000-2561.2021.09.004

• Omics & Biotechnology • Previous Articles     Next Articles

Parameter Optimization and Accessory Improvement of GDS-80 Handheld Gene Gun for Rubber Tree Genetic Transformation

FAN Jingzhang1, $\boxed{\hbox{HUA Yuwei}}$ 2, FAN Yueting3, XIN Shichao2, DAI Xuemei2, HUANG Tiandai2,*(), HUANG Huasun2,*(), LI Ji2   

  1. 1. College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China
    2. Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory of Biology and Genetic Resources of Rubber Tree, Ministry of Agriculture & Rural Affairs, Haikou, Hainan 571101, China
    3. Xixian Senior Middle School Affiliated to Central China Normal University, Xixian, Henan 464300, China
  • Received:2021-01-12 Revised:2021-02-24 Online:2021-09-25 Published:2021-11-01
  • Contact: HUANG Tiandai,HUANG Huasun

Abstract:

GDS-80 handheld gene gun is a gene delivery system launched by Wealtec Company in 2009, which is easy to operate and efficient. All the published researches on the transformation of Hevea brasiliensis by gene gun are based on desktop gene gun. In this paper, Coomassie Brilliant Blue was firstly used to bombard filter paper to screen out obviously not suitable parameters, and then the green fluorescent protein (GFP) gene was used as the reporter gene to bombard the somatic embryos and callus of H. brasiliensis, finally the green fluorescence was observed by fluorescence microscope to explore the suitable bombardment parameters for H. brasiliensis. The difference in fluorescent spot numbers calculated by Image J software and naked-eye and the difference between GDS-80 handheld gene gun and PDS-1000/He desktop gene gun was compared. The results showed that it was difficult to obtain good transformation efficiency by using original accessories for somatic embryos and callus of H. brasiliensis. In this paper, a filter screen with a diameter of 3cm (same as the bombardment range), the height of 2.5 cm and pore diameter of 60 mesh was designed. At the same time, a petri dish containing the experimental materials was improved. A circle with a diameter of 3cm was cut out in the center of the petri dish by a blade. The filter screen designed above was clamped on the circle with forwarding direction, and the embryos were put into the filter screen. When bombarding, the petri dish was put on the test tube rack, and the pressure would decompose from the mesh and the cavity at the bottom, and the micro bullet would completely bombard the embryos. The optimal parameters for embryos were as follows: bombardment pressure 60 psi, needle-shaped regulating valve 3 circles, and target spacing plate 6 cm. When the callus was bombarded, the material was placed in the center of a traditional petri dish and covered by the designed filter screen reversely. The optimal bombardment parameters for callus were as follows: bombardment pressure 50 psi, needle-shaped regulating valve 4 circles, and target spacing disc 6 cm. In this paper, Image J software was used to count fluorescent spots. The accuracy was equivalent to that counted by naked eyes, but it saved time. The transformation efficiency of the GDS-80 handheld gene gun was similar to that of the PDS-1000 / He desktop gene gun, but the GDS-80 handheld gene gun was 12 minutes faster than PDS-1000 / He desktop gene gun for one shot. The research results would provide an efficient gene gun transformation system for H. brasiliensis genetic transformation and a efficient software to count fluorescent spots.

Key words: Hevea brasiliensis, GDS-80, gene gun, GFP gene, Image J

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