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

doi:10.3969/j.issn.1000-2561.2021.09.004

Abstract

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

CLC Number:

S794.1

FAN Jingzhang, $\boxed{\hbox{HUA Yuwei}}$ , FAN Yueting, XIN Shichao, DAI Xuemei, HUANG Tiandai, HUANG Huasun, LI Ji. Parameter Optimization and Accessory Improvement of GDS-80 Handheld Gene Gun for Rubber Tree Genetic Transformation[J]. Chinese Journal of Tropical Crops, 2021, 42(9): 2458-2467.

Figures/Tables 12

Fig. 1 Sketch of pJIT163-hGFP (Wang)

Fig. 2 Improvements to bombardment accessories A: Front view of the filter screen; B: Top view of the filter screen; C: Not covered with a filter screen, the blue circle is the bombardment range; D: Cover with filter screen; E: Bombardment range was cut; F: The filter screen is stuck in the hollow of the petri dish, and it is placed on the test tube rack for bombardment.

Fig. 3 Experimental results of target spacers at different distances A: 3 cm; B: 6 cm; C: 9 cm.

Fig. 4 Experimental results under different bombardment pressures A: 20 psi; B: 30 psi; C: 40 psi; D: 50 psi; E: 60 psi.

Fig. 5 Effect of needle control valve on coomassie brilliant blue bombardment A: 1 circle; B: 2 circles; C: 3 circles; D: 4 circles.

Fig. 6 Experimental results of different sample volumes A: 5 µL; B: 10 µL; C: 15 µL; D: 20 µL.

Tab. 1 Effect of different bombardment pressures on the transformation efficiency of embryos

轰击压力 Bombardment pressure/psi	目标间隔盘距离 Target spacer distance/cm	荧光数 Fluorescence numbers
30	6	33.00±31.31^c
40	6	168.80±98.89^b
50	6	166.20±38.35^b
60	6	329.80±88.87^a
70	6	75.60±24.20^c

Tab. 2 Experimental program and results after bombardment equipment improvement

材料 Material	目标间隔距离 Target spacer distance/cm	轰击压力 Bombardment pressure/psi	推荐圈数 Recommended number of laps	改进方式 Improvement mode	荧光数 Fluorescence numbers
胚	3	60	3	遮盖	574.00±121.54^b
	3	60	3	不遮盖	368.00±102.23^c
	6	60	3	遮盖	263.40±20.21^c
		60	3	不遮盖	76.60±19.33^d
		60	3	底部漏气	834.40±147.77^a
愈伤	3	50	4	遮盖	147.40±17.34^b
愈伤	6	50	4	遮盖	635.00±143.62^a

Fig. 7 Bombardment effect of embryos and fragile calli A: 60 psi, 6 cm, 3 circles, no cover; B: 60 psi, 3 cm, 3 circles, cover; C: 60 psi, 6 cm, 3 circles, hollow petri dish; D: 50 psi, 3 cm, 4 circles, cover; E: 50 psi, 6 cm, 4 circles, cover.

Tab. 3 Difference of green fluorescent spots between counting by naked eyes and Image J

处理 Treatments	统计方式 Statistical methods	荧光数 Fluorescence numbers	误差 Deviation	误差百分比 Deviation percentage/%	时长 Time/min
60 psi,3 cm,3圈,不遮盖	肉眼	93	4	4.30	1.0
60 psi,3 cm,3圈,不遮盖	Image J	89	4	4.30	0.5
60 psi,3 cm,3圈,遮盖	肉眼	144	-4	2.78	2.0
60 psi,3 cm,3圈,遮盖	Image J	148	-4	2.78	0.5
50 psi,3 cm,4圈,遮盖	肉眼	224	22	9.82	3.0
50 psi,3 cm,4圈,遮盖	Image J	202	22	9.82	0.5

Fig. 8 Converted count image

Tab. 4 Comparison of transformation efficiency and work efficiency between handheld gene gun GDS-80 and desktop gene gun PDS-1000/He

仪器 Apparatus	打枪次数 Shot	荧光数 Fluorescence number	荧光数均值±标准差 Fluorescence numbers mean±SD	用时 Time/min	用时均值±标准差 Time mean±SD/min
手持基因枪	1	853	744.67±107.02^a	1.5	1.77±0.25^b
	2	639		2.0
	3	742		1.8
台式基因枪	1	401	691.67±372.81^a	13.5	14.17±0.76^a
	2	1112		14.0
	3	562		15.0

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