Transformasi kedelai dengan gen proteinase inhibitor II melalui Agrobacterium dan penembakan partikel

Abstract

The objectives of these research were (1) to obtain the best protocols of soybean transformation using Agrobacterhm-mediated bransformation and Particle Bombardment, (2) to transfer and regenerate soybean explants transformed with the proteinase inhibitor I1 gene, and to obtain soybean plants that contained the proteinme inhibitor II gene and resistant to pod barer. Research was included three experiments i.e 1) Soybean transformation using gus gen, 2) Soybean trmsfarmation using proteinase inhibitor (pin) II gene, and 3) Evaluation of soybean transformant plants. Two soybean cdtivars (Wilis and Tidar) were used in this experiments. Both cdtivars were chosen because it shown good response on early in vitro plant regeneration study and very popular to the farmers, especially cv. Wilis. On the first activity, soybean explants were inoculated with A. tumefaciens strain EHA 105 which is contained plasmid pCambia 1301 with p s gene in T-DNA region. The treatments were included an optical density (0.5; 1 and 1.5), inoculation time (60 and 90 minutes), co-cultivation time (3 and 5 days) and type of explants (young embryo and cotyledon). Result indicated that the best protocol for inoculation was using young cotyledon as explants with 1 x 10' celYrnl of Agrobacterium for 90 minutes inoculation and 5 days co-cdtivation. While on the particle bombardment method, young embryos and cotyledons were bombarded with pRQ6 plasmid. Treatment were included Helium gas pressure (1 1 00 and 1 300 psi), shooting distance (5 and 7 cm) and number of bombardment (once and twice). The best treatment was using young cotyledon as explant, bombarded once or twice with gold particles at 1 100 psi of the Helium gas pressure and 5 crn of shooting distance. On the second activity, 1539 young cotyledon explants from Wilis and 984 explants from Tidar were inoculated with A. tumefaciens which is contained proteinase inhibitor II gene on plasmids pGApin LI construct. Result from plant regeneration and selection of tsansformant explants on media with 200 mgA Kariamycine (spesific for nptII gene selection) indicated that c.v Wilis were better than Tidar, because its produced more plantlets/plants 8 plants (coded: A W I -AW8), while Tidar produced only one plant (coded: AT& On the Particle Bombardment experiments, 1274 young cotyledon explants of Wilis and 1 763 explants of Tidar were bombarded using gold particle coated with pTWa plasmids (contained pinn and bar genes). Result of plant regeneration and selection of transformant explants on media with 3 mg~l Basta (spesific for bar gene selection) indicated that cv. Tidar produced three plants (TPI-TP3) while cv.Wi1is produced only 2 plants (WPI and WP2). On the third activity, transformant plants were evaluated through molecular and bioassay analysis. Molecular analysis of nine soybean plants obtained from Agrobacterium-mediated transformation (8 Wilis and 1 Tidar) using Polymerase Chain Reaction (PCR) technique indicated that only event ATI (Tidar) was positive to pin I1 gene test, while 8 plants from event AW (Wilis) were negative. PCR test of five soybean plants obtained from bombardments experiments (2 Wilis and 3 Tidar ) indicated that no plants were positive to pin 11 gene, except WP2 (Wilis). Seeds fiom these positive plants were collected for further evaluation. Furthermore, PCR test of 50 WPtRl plants and 40 AT1 R1 plants indicated that there were several of Rl plants still positive on PCR test. Jt indicated that the proteinase inhibitor II gene was transmitted from parent plants (h)to the progeny @I). There were three DNA samples of WPzRl(No. 44,45,46) and six DNA samples of ATIRl(No. 6, 9, 11,20,22,25) were positive to pin I1 gene. Those RI plants were also challenged to pod borer (Etielfa zinckenella, Tr.) larvae in the Biosafety Containment. Observation on pod and seed damages percentage after harvesting indicated that WP2 progeny were categoried into rather susceptible to pod borer (pod damages = 45.4%), while AT1 progeny were susceptible to pod borer @od damages = 58.8%). WP2aS and W2-46 were the best event of WP2 progeny (categoried into rather resistant to pod borer with 20°/0 pod damages) and ATI-11 was the best event of ATl progeny (categoried into rather susceptible to pod born with 30% pod damages). those piants were also positive on PCR test.