Dally, A.M.

Second, Gérard

Zhang, S.H.

Horizon / Pleins textes

- - . ,  
144 
about 4-5 cm and be a smeary band near the dye front. 
An alternative to the phenol extraction for DNA purification, is centrifuga- 
tion on a CsCl gradient. The digested lysate may be diluted to the desired volume 
(determined by the size of the ultracentrifuge tube) and made 1.7 g/ml with CsC1. 
The solution is centrifuged as 50,000 rpm in the VTiS0, and then fractionated by 
dripping from the bottom. The nucleic acid-containing fractions are analyzed by 
electrophoresis, and the desired fractions are dialyzed and nucleic acids collected 
Rice Genetics Newsletter Vol. 5 
. 
ethanol precipitation. 
HB (homogenization buffer). 0.4 M mannitol, 1 mM EDTA, 0.05% cysteine, 
and 10 mM TES-Na+, pH 7.2. Autoclave and store at 4°C. Supplement with 
0.1% BSA and 0.03% beta-mercaptoethanol before use. Add BSA to the 
surface and allow to dissolve, do not stir. 
Sucrose.,solutions are percent (w/v) prepared in 1.0 mM TES-Na+ pH 7.2, 10 
mM EDTA and autoclaved. 
50 mM Tris-HCl,-lO mM EDTA (autoclave). 
Solutions : 
. 59. Non-aqueous method for purification of cytoplasmic DNA from individual 
rice plants for RFLP analysis 
._ _-_ 
Shang-Hong ZHANG, Andreas M. DALLY andiGerard SECOND-' 
Centre Emberger, CNRS, B. P. 5051, 34033 Montpellier Cedex, France 
L--- ~- __i 
A non-aqueous method for the purification of chloroplast DNA (ctDNA) 
was developed and successfully applied to wheat (Bowman and Dyer 1982). 
However, in our experience, it can hardly be applied directly to rice. Also, there 
is no report on the isolation of mitochondrial DNA (mtDNA) from dried plants, 
which has some advantages: easier sampling, possibility of mailing and long term 
conservation, etc. 
Several important improvements have been made in the method of purifica- 
tion of ctDNA of rice in non-aqueous media (Dally 1988). Among them are 1) 
the less dangerous 1, 2,4-trichlorobenzene (CB) and decahydronaphthalene (HN) 
are used instead of carbon tetrachloride and n-hexane in the isolation of chloro- 
plasts, allowing mechanical blending in addition, and 2) to prevent nuclear 
contamination, 4% Triton X-100 is used for the first lysis. At a low temprerature, 
this detergent destroys preferentially the organellar membranes, so that most of 
cytoplasmic DNA may be recovered in the supernatant while (the contaminant) 
nuclear DNA can be precipitated. 
A 4 : 1 (v : v) mixture of CB and HN is used, with a protective layer of 1 : 3 
mixture overlaid. After centrifugation, a band containing chloroplasts is collected 
at the 4 : 111 : 3 interface. Eventually, there are mitochondria in this collection, 
but they are in negligible quantities compared with chloroplasts. In our trials, 
I 
i 
Research Notes 
a kb M A A' B B' C C' D D' E E' F F' G G' H H' 
48 
19 
15 
l 
10.6 
8.9 
7.3 
5.6 
4.36 
3.99 
3.61 
2.93 
2.32 
1.81 
1.41 
1.25 
x 
155 i- 
b kb M A A ' B B ' C C ' O D ' E  E ' F  F ' G G ' H H '  
Fig. 1. a) Rice cytoplasmic DNA digested by EmRI (A-D) and AvaI (E-H). 
b) The same pattern revealed by Southern blot hybridization with a cytochrome 
oxidase II (COX II) probe f h m  aheat. 
In both a) and b), M is a size marlier (Raoul from Appligene). The samples used are: 
O. rufipogon (OR42) in A and E, O. oficinalis (W1198)in B and F, O. sativa (VZOA, male 
sterile) in C and G, and O. Nfipogon (W1187) inD and H. In each sample, lanes A, B. 
etc. are from the upper band (lighter band), and lanes A', B', etc. are from the inferior 
band (denser band) in the prepration. In b). arrows on the left indicate the major hy- 
bridization fragments which should correspond to mtDNA. Note that the inferior band 
gives a better yield, especially when the DNA-quantities are small (D versus D'; H versus 
H'). The origin of other fragments r e v d e d  by the probe are uncertain, perhaps they are 
nuclear DNA, but are not ctDNA. In B. chet wo additional fragments which do not exist 
in other lanes are perhaps due to au  incomplete digestion. 
- \  <% 
1>6 Rice Genetics Newsletter Vol. 5 
the results of Southern blot hybridization with a wheat mtDNA probe were ir- 
regular with this preparation. 
Contrary to the situation in aqueous solution, we found that the niitochon- 
dria are a little denser than the chloroplasts in the mixture of organic solvents 
used. Thus, for the enrichment of mitochondria, various combinations of solvent 
mixtures such as 1 :O (pure CB) with 4: 1, 9: 1 with 4: 1, 8:  1 with 6 :  1 were 
tried, but the following procedure gave the best results, using a 8 : 1 mixture of 
the same solvents (d= 1.38) for blending and intercalating a layer of 4 : 1 mixture 
(d= 1.34) in between the 8 : 1 and 1 : 3 layers. 
Two bands appear after centrifugation at the interface between 4 : 1 and 1 : 3 
(upper band), and a t  the interface between 8 : 1 and 4 : 1. Though the contamina- 
tion ,of chloroplasts is inevitable (Fig. la), the inferior band is enriched with 
mitochondria as can be judged .from. Southern blot hybridization. with a aon- 
radioactively labeled .wheat mtDNA' cytochrome oxidase' II probe (Fig. lb). In .' 
addition, .from the DNA abtained from the inferior band, traces of restriction. 
fragment pattern of -mtDNÄ can in some cases be seen directly along the ctDNA 
pattern in the ethidium bromide stained electrophoresis gel (Fig. la). 
So the upper band recovered by the centrifugation may be used for the pre- 
paration of cytoplasmic DNA, in which mtDNA concentration is sufficiently high 
to be easily revealed by Southern blot hybridization. 
References 
Bowman, C. M. and T. A. Dyer, 1982. Purification and analysis of DNA from wheat chloroplasts 
isolated in nonaqueous media. Analytical Biochemisstry 122: 108-1 18. 
Dally, A. M., 1988. Polymorphisme des longueurs des fragments de restriction de I'ADN chloro- 
plastique dans la section Eu-Oryza du genre Oryza (riz) et implications phylogénétiques. 
Thèse de Doctorat. U.S.T.L., Montpellier. 160p. 
60. Enhancement of division frequency of protoplasts and plant regeneration in 
rice 
Bao-Lin SUN, Xiang-Hui LI, Yong-Ru SUN and Shi-Min ZHAO 
Institute of Genetics, Academia Sinica, Beijing 100012, China 
Calli were initiated from young inflorescences of Oryza sativa L. on MS 
medium supplemented with 2 mg/l 2,4-D, 3% (w/v) sucrose and 1% (w/v) agar 
adjusted to pH 5.6-5.8. The calli of 1-1.5 g were introduced into a flask contain- 
ing 30 ml of AA liquid medium. The suspension cell line was established after 
being cultured for 7-8 weeks on a gyratory shaker at 100 rpm. 
Protoplasts were isolated from established cell line at 3-4 days after sub- 
culture. Protoplasts were cultured at densities between 2--5X lO'/ml in KM8p 
media solidified with 0.8% (w/v) agarose. KM8p liquid medium was added when 
the agarose drops were solidified. The percentage of the protoplasts having divided 


Non-aqueous method for purification of cytoplasmic DNA from individual rice plants for RFLP analysis

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