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本頁は“日本作物学会関東支部報,16:62-63,2001”に掲載したものです.図表一部省略.

ソルガムの葯及び小花由来カルスへのパーティクルガンによる直接遺伝子導入

Anas1)・高溝正2)・吉田智彦1)
(1)宇都宮大学・2)草地畜産研究所)

Direct Gene Transfer to Anther and Floret -Derived Callus of Sorghum by Particle Bombardment
Anas1), Tadashi Takamizo2) and Tomohiko Yoshida1)
( 1)Utsunomiya University; 2)NILGS )

The preliminary microprojectile bombardment experiment was conducted to identify (1) the possibility and effect of plasmid DNA gene constructs on transient GUS expression in sorghum anther and floret-derived callus of Sorghum; (2) the effect of tissue culture condition.

Materials and Methods
Plant Materials. Callus was induced from 4 sorghum
genotypes: SPA2, C9H11/C9H13, C9H11/SPA2, C9H13/SPA2, C8/H2 and seed-derived callus of tall fescue was used as a control. SPA2 is Al - tolerant genotypes (Anas and Yoshida, 2000. PPS 3:246 - 252).
Media. Murashige and Skoog medium supplemented with 2.0 mg L-1 kinetin, 1.0 mg L-1 IAA and 2.5 mg L-1 2,4-D was used for callus induction. Selective medium was supplemented with 100 mg/l hygromycin. Plasmid DNA, microprojectile bombardment, GUS assay and selection of hygromycin resistant-calli. Three plasmid DNAs, namely, pAct1-D, pWI-GUS, pEX7113 each encoding b-glucuronidase (GUS) as a reporter gene were used. The plasmid pAcH1 carrying the selectable marker gene for hygromycin resistance (hpt) was used for stable transformation experiment. Anther and floret-derived callus of sorghum and mature seed-derived tall fescue callus 2-9 wk after induction were transferred on to fresh medium and placed in the center of a petri dish one day prior to microprojectile bombardment. All anther-derived callus were cut in to small pieces (0.5 - 0.8 cm in diameter) before bombardment. More than 30 anther calli were placed in a petri dish. Floret-derived calli were not cut before bombardment and bombarded in a little big size (0.7 - 1.5 cm in diameter). PDS-1000/He Particle Delivery System was used. For routine bombardment, the distance from rupture dish to microcarrier, from microcarrier to stopping screen aperture, and from stopping screen aperture to target tissue was 2.5, 1.0 and 9.5 cm, respectively. Bombardment was conducted twice per petri dish with 1100 psi bombardment pressure. GUS activity was assayed histochemically 3 d after bombardment. Bombarded calli were incubated in phosphate buffer (150 mM, pH 7.0) containing x-Gluc, Potassium Ferricyanide, Potassium Ferrocyanide, and Trition X-100 at 370C.Callus was transferred to selective medium 1 wk after bombardment for selection of hygromycin resistant-calli.

Results and Discussion
The overall percentage of anther and floret-derived callus induction varied among genotypes. The percentage of callus induction from anther ranged from 10% to 35% and callus induction from floret ranged from 68% to 80%. GUS activity was observed both in sorghum and tall fescue control callus (Table 1). Presence of any blue spots in a callus indicated transient expression of the uidA (GUS) gene activity (Fig.1). However, number of blue spots was not so high comparing with the tall fescue control callus. No GUS activities were found for anther callus. These results showed that sorghum calli were very sensitive in sub-culture. GUS activity was observed in all plates of floret calli 2-3 wk after induction. Only two plates of floret callus 6 and 7 wk after induction showed blue spots. These results suggest that young, vigorously growing fresh callus is more suitable for direct gene transfer. The plasmid pWI-GUS gave the highest on transient GUS expression in floret callus of sorghum. Eleven blue spots were observed in callus over genotype bombarded with pWI-GUS plasmid (Table 2). The selection of hygromycin - resistant calli is now going on.

Table 1. Effect of genotypes, callus age and plasmid DNA on the number 
of blue spots observed on anther and floret- derived callus bombarded 
with 1100 psi 
-----------------------------------------------------------
Geno-	 Week after Kind of     Number of    Plasmid   Number of 	
type	 planting	     callus	   plasmid	                    blue spots
-----------------------------------------------------------
SPA2	   6 wk	Floret callus	7	pAct1-D	 5	
	   6 wk	Floret callus	8	pAct1-D	 0	
	   6 wk	Floret callus	9	pWI-GUS	 0	
	   3 wk	Floret callus	15	pEX7113	 2	
C9H11/ICR2 2 wk	Floret callus	11	pEX7113	 2	
	   7 wk	Floret callus	6	pWI-GUS	 3	
	   7 wk	Floret callus	4	pWI-GUS	 0	
C9H13/ICR2 2 wk	Floret callus	5	pWI-GUS	 8	
C9H11/C9H13 3 wk	Floret callus	10	pEX7113	 4	
	  4 wk	Anther callus	45	pAct1-D	 0	
	  9 wk	Anther callus	45	pWI-GUS	 0	
	  9 wk	Anther callus	35	pAct1-D	 0	
	  9 wk	Anther callus	30	pAcH1	 -	
	  9 wk	Anther callus	30	pAcH1	 -	
	  9 wk	Anther callus	32	pAcH1	 -	
C8/H2	  7 wk	Floret callus	7	pEX7113	 0	
	  7 wk	Floret callus	6	pEX7113	 0	
	  7 wk	Floret callus	8	pAcH1	 -	
	  7 wk	Floret callus	6	pAcH1	 -	
Tall fescue  1 day after  -	              -	pAct1-D	59	
(control)     sub-culture
-----------------------------------------------------

Table 2. Effect of various plasmid on transient GUS expression of floret 
callus of sorghum over genotypes.
---------------------------------------------
Plasmid	                  pAct1-D  pWI-GUS  pEX7113	
---------------------------------------------
Number of blue spots	5	11	8	
---------------------------------------------

Fig. 1. The blue spots in embryogenic calli of sorghum bombarded with  pWI-GUS plasmid.(略)
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