Duval, Yves

Horizon / Pleins textes

INSTITUT DE RECHERCHES 
POUR 
LES HUILES ET OLEAGINEUX 
, 
MISSION TO THE SOCFINDO 
VEGETATIVE PROPAGATION LABORATORY " ,; 
. _ .  - - . -  -&b?*-a.- 
i DOC. 
-_._--  *.-: 184J ..j 
Y. DUVAL Mgrch 1984 
*  
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11, SQUARE PÉTRARQUE, 75116 PARIS 
SUMMARY AND CONCLUSION 
1- STATE OF THE PROGRAMME 
The programme initially planned (culture of 2 trees 
per month) is being carried out normally. At the end of February 
11 trees have been put to culture. Calluses from the 2 trees 
(SOC OOA and SOC 4.6) have been placed in embryogenesis cycle 
and 3 other clones (SOC 4.6, SOC 3.1 and SOC 3.2) show calluses 
which Will be isolated in March 1984. 
Several improvements have been proposed concerning 
cultures and disinfection and modifications of the culture media. 
It has been agreed that they Will be applied as soon as March 
samplings. 
It is recommended to send to IRHO every two months 
a record of the cultures in order to follow the programme's pro- 
gress. 
II- EQUIPMENT OF THE LABORATORY 
Well equipped and operational, the Bangun Bandar labo- 
ratory does not show any important problem. However three points 
must still be improved : 
l- The pH of the water supplied to the purification 
system is too basic and provokes a quick degradation of the fil- 
,_ tering membrane. Considering its high cost, we advise to contact 
MILLIPORE in Jakarta in order to equip the laboratory with a 
water softener. 74 
2- The air-conditioning system of the lighted culture 
room is not good. Two improvements cari be brought : 
- place the regulation sensor on the shelves and 
not in the air-ducts, 
- replace the one-speed engine of the air-condi- 
tioning system by a multi-speed engine which would allow to adjust 
the air flow to requirements. In the meantime the two fans (normal 
+ spare one) must be used simultaneously during the lighting 
phase. 
3- The horizontal steriliser does not work and the 
intervention of a LEQUEUX's agent has been asked and planned 
from April 6th. 
1- NOVEMBER 1983 CULTURES - CALLOGENESIS 
The 5 trees put to culture in November have given 
average results for callus production. Practically no neofor- 
mation has appeared on 2 clones (< 1 % at 12 weeks) ; the three 
other trees have given more encouraging results (table 1). 
We Will simply recall that the media have been prepared 
with BDH agar, different from the advised SYGMA agar, which geli- 
fication is not good at the pH used and at a sterilisation tem- 
perature of 120" C. Explants of trees sampled before 1984 have 
been transferred after 6 weeks culture on a new and more solid 
medium (Mi 034 - BDH agar sterilisation t" 115" C). This has 
involved no noticeable browning or contamination which proves 
the quality of the laboratory and team. Also the parafilm was 
only available during January and most of the callogenesis stage 
has been done without parafilm (which has not increased the con- 
tamination rate). Al1 these points, as well as yield, have been 
summed up in table 2. After these precisions, several facts are 
noteworthy : 
I.l- Callogenesis time 
The observation of the callogenesis on SOC OOA shows 
a clear evolution between the 12th and 15th week. 
SOC OOA - 8th week : 0 % 
10th II : 1.7 % 
12th II : 5.9 % 
15th II : 15.3 % 
The unexpected evolution of yield between TO + 12 weeks 
and TO -t- 15 weeks cari be explained by the subculture at TO + 
12 weeks, the calluses of clones SOC 03.1, SOC 03.3 Will be iso- 
lated at TO + 15 weeks. For the trees not transferred at TO + 
6 weeks and cultured on a medium prepared with agar of the requi- 
red quality, the calluses Will continue to be isolated at TO + 
12 weeks. 
1.2- Height of sampling 
The detailcd observation of callogenesis shows that 
the first 5 levels (30 to 37.5 cm from the apex) are lower yiel- 
ding than the 5 levels immediately below. The results given by 
the first ten cultures Will confirm this point. Up to now a11 
samplings have been done between 30 cm and 67.5 cm from the apex 
and we propose to increase the size of the cylinder in order 
to know the callogenesis aptitude at the 67.5-75 cm level on 
the trees of Bangun Bandar (annex 1). This is justified by the 
fact that the sampling standards have been defined in the La Mé 
laboratory on older trees (20 years to 9-12 years) living in a 
very different ecology, and whose apex has perhaps a different 
development. 
As concerns the behaviour of explants from the level 
comprised between 30 to 37.5 cm from the apex, two hypotheses 
cari be retained : 
- The tissues are too Young and are not apt to callo- 
genesis. 
- The tissues are apt to callogeensis but do not resist 
to the hypochlorite treatment. 
The observation of the explants leads us to favour 
the second hypothesis and a particular study Will be carried 
out (cf 1.3). 
If levels 67.5-75 cm present a good callogenesis, 
sampling cari be done 5 cm farther in order not to use blocks 
between 30 and 37.5 cm for culture since they do not present 
an interesting yield. 
1.3- Hypochlorite treatment 
The hypochlorite used for the cultures is of industrial 
type and we have as yet no technical specification about it. 
In these conditions it is possible that the solutions prepared 
at 45 g/l are too concentrated and damage the explants (mainly 
the Young ones). This seems to be confirmed by the state of the 
explants in culture and could explain the relative weakness of 
the callogenesis yield. 
In order to solve this problem we advise : 
BDH. 
l- TO get laboratory calcium hypochlorite at 35 % Cl- 
2- TO carry out the tria1 proposed in annex 2 on a 
tree in order to use the available calcium hypochlorite at a 
lower and less damaging concentration until the laboratory product 
is delivered. 
1.4- Emergence of roots 
About 40 % of the neoformations present unfavourable 
structures of root type. The apparition of such structures should 
decrease with the use of a new callogenesis medium tested in 
the La Me Laboratory, the Mi 113 medium. 
The composition of this medium has been communicated 
to Mr KUSNADI who Will use it for a11 cultures from now on. 
1.5- Conclusions 
Excepting failures recorded on clones SOC 01.2 which 
remain unexplained, we cari consider that the first callogenesis 
results are encouraging since these works have been done on the 
starting of the laboratory. The use of a more homogeneous (quali- 
ty of the agar) and slightly modified medium (Mi 1131, a gentler 
hypochlorite treatment and a somewhat more adapted sampling level 
would allow to increase noticeably the yield of this stage of 
process. 
II- INVENTORY OF THE CULTURES - FEBRUARY 1984 
CLONE 
soc 3.1 
2.3 
3.3 
4.3 
2.7 
3.4 
2.2 
CLONE 
SOC OOA 
SOC 4.6 
CLONE 
BC 068 
II.l- Callogenesis stage 
NUMBER DATE OF CULTURE 
1 592 24.11.83 
1 946 12.12.83 
1 999 19.12.83 
1 969 13.01.84 
1 936 31.01.84 
1 994 11.02.84 
2 000 23.02.84 
11.2- Embryogenesis stage 
NUMBER DATE OF ISOLATION 
16 24.02.84 
32 25.02.84 
11.3- Embryoids proliferation stage 
DATE OF ISOLATION 
8.03.84 
26.03.84 
12.03.84 
6.04.84 
23.04.84 
5.05.84 
17.05.84 
NEXT SUBCULTURE 
20.04.84 
21.04.84 
NUMBER NEXT SUBCULTURE 
83 9.04.84 
IV- TECHNICAL PROBLEMS 
The laboratory has been working for 4 months quite 
satisfactorily. However the following points are still to be 
solved : 
IV.l- Steriliser 
Thanks to the demand to Ets LEQUEUX a more complete 
handbook has been sent to the laboratory, but this is not a 
maintenance handbook as required. The inflatable joint has been 
changed on the advice of LEQUEUX, but the sterilization cycle 
is not satisfactory. The pressure within the chamber does not 
stabilize during the sterilization phase and it seems from the 
contacts with LEQUEUX that the thermometer does not work. A tech- 
nician Will bring the necessary parts to the laboratory and solve 
this problem SO that the sterilizer cari function at last. 
IV.2- Milli Ro 60 
It has started to function at the beginning of November 
1983 the Milli Ro 60 cartridge containing the filtering membrane 
has been changed twice in 4 months, and should have lasted 1 
year on average according to MILLIPORE. It seems, after the 
results demanded by Mr KUSNADI that the water supplied to the 
laboratory is too basic (pH 8) and calcium rich, considering 
the builder's specifications. 
The cost of the Milli Ro 60 cartridge being very high, 
it is urgent to provide a water softener in order to supply the 
Milli Ro with a water of pH around 4.5 to 7.5. We advise to 
ask the characteristics of this apparatus to MILLIPORE Jakarta. 
IV.3- Air-conditioning in the lighted room 
There has been no change since the november 1983 visit 
and it is impossible to obtain 27" C constant on 24 hours with 
3 fluorescent tubes per shelf. We observe a difference of about 
3" C between lighted and dark phase. Since the lighted room 
has been in use since the end of February , we advise : 
- to use only 2 fluorescent tubes to ensure lighting, 
- to contact CARRIER in order to modify the positions 
of the sensor, placing it on a shelf instead of within the air 
duct. This should allow to get the actual temperature at the 
shelf level. If this is not enough, a more powerful multi-speed 
engine must be installed in order to modulate the cold air flow 
according to the light or obscurity phase. In the meantime, we 
suggest to use the two fan coils simultaneously. 
IV.4- Quality of the media 
The use of SYGMA agar has solved the problems met with 
Oxoid agar. Observations have shown that the sterilisation above 
115" C at the pH used prevents the gel from solidifying normally. 
At lower temperatures, the gel presents better qualities. However 
in order to prevent any problem, it is recommended to use only 
SYGMA agar. 
ANNEX 1 
PROTOCOLE 
Aim 
Method 
Culture 
: Observing the callogenesis on the area between 
30 cm and 75 cm below the apex. 
: Each leaf is tut in 30 blocks of 1.5 cm length. 
The levels are numbered 1 to 30 and they a11 Will 
be treated until rinsing, G solution included. 
Levels 5, 10, 15, 20 and 25 Will not be used 
(25 blocks are cultured according to the usual 
process). 
: Each block is disinfected in a Ca hypochlorite 
solution (concentration defined for the triai). 
They are rinsed in a glucose (20 g/l) solution 
then transferred directly to Mi 113 medium (20 
explants per block). 
Observations : According to the usual process at TO + 8, 10, 12 
weeks. Results are expressed in frequency of callo- 
genesis for each block, each level (4 leaves simul- 
taneously) and regrouped for 4 levels, according 
to the usual way. 
This protocole must be used on 4 trees. 
ANNEX II 
PROTOCOLE 
Aim 
Method 
Results 
: Disinfection of the blocks - Concentration of 
hypochlorite 
: A tree is sampled according to the usual process 
(sec instructions for culture in November 1983 
report). 
- 1/3 of the blocks is disinfected with a 
hypochlorite solution at 22.5 g/l, 
- 1/3 of the blocks is disinfected with a 
' hypochlorite solution at 34 g/l, 
- 1/3 of the blocks is disinfected with a 
hypochlorite solution at 45 g/l. 
The blocks are transferred at TO + 20 mn in G 
solution then in Mi 032. Then they are kept at 
27" C for 8 days. 
: At TO + 8 days the presence of contaminations in 
the flasks indicates the quality of the disin- 
fection. Results are given by the number of 
contaminated flasks to the number of disinfected 
flasks for each concentration of hypochlorite. 
The lowest concentration giving a good result Will 
be used for the next cultures. 
Level 
(distance from SOC OA SOC 04.6 soc 03.1 TOTAL 
the apex - cm) 
1.5 3/191 4/383 27/328 34/902 
(30 to 37.5) (1.51 %) (1.01 %) (8.23 %) (3.76 %) 
6.10 19/199 58/387 65/393 142/972 
(37.5 to 45 ) (9.5 %) (14.98 %) (16.53 8) (14.50 %) 
11.15 10/186 40/391 39/397 89/974 
(45 to 52.5) (5.3 %) (10.2 %) (9.82 %) (9.13 %) 
16.20 49/393 31/398 80/79 1 
(52.5 to 60 1 (12.46 %) (7.78 %) (10 %) 
20.25 
(60 to 67.5) 
TOTAL 32/576 161/1554 162/1516 345/3646 
(5.56 %) (9.72 %) j (10.7 %) (9.46 %) 
Table 1 : Callogenesis yield according to the distance from the 
apex at TO + 12 weeks. 
. . 
CLONE Date of Date of Date of Total yield % 5 best Isolation of callusei culture transfert11 parafilming 12 weeks consecutive 
levels Date Number 
SOC OOA 9.11.83 24.12.83 25.01.84 5.93 % 10.5 % 24.2.84 16 
soc 01.1 15.11.83 29.12.83 11.01.84 0.14 % 
soc 01.2 17.11.83 30.12.83 17.02.84 0.63 % 
SOC 04.6 22.11.83 31.12.83 9.02.84 9.72 % 14.98 % 25.2.84 32 
soc 03.1 24.11.83 3.01.84 3.01.84 10.7 % 16.5 % not isolated 
Table 2 : Sequence of the operations on the trees put to culture in November 1983 
(on poor quality agar). 
(1) on SYGMA agar. 


Mission to the Socfindo Vegetative Propagation Laboratory

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