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Non Structural Carbohydrate Variability in Hevea Clones: Conference Paper
Non Structural Carbohydrate Variability in Hevea Clones: Conference Paper
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Abstract
The aim of smdy was m s e s . d clonal variabiliiy of starch und wgar sol&Ee (SS) in
lmnk of rubber [we. The two experimertls were ser up, [he first fidd compared 25
clones icsied itt arr interndona1 clone wchclngr program which is now 30 years old. A
stcondfield clone trial iw used for 15 years trees, with 18 clones. The results showed
that nn average starch irr ~ v w d71 tngmJgsDM and SS uvood21 mgcLJgSDDCI recorded 25
clo~resexchange trial at 30 years were similar to the average obtained the same .Fensun
i~ the previuiw rrnr dy on' standard clone RRIM600 age 1I years, stnrch 76 mgGLLdgSDM
a~zd SS 16 Howver, nrean conrents SS bark 37 ~ n g ~ ~ d and g s ~ ~ ,
particulariy starch in bark 25 rngcLdgso~ were higher in the 30 years tree shmr the
previous study SLY22 ingcdgsDM and starch J I mgcLJgsDM. The 15 years trial wiih 2 8
clufies, mean co~lrentin rvood were a bit higher fur borh starch 94 mgctdgsDMand SS
28 mgGLd,.TnMthan the previous sittdy. bark SS 22 r n g c t d g s ~w m similar as
above but starch 41 mgct JgSDM was much higher //tun RHM 600 age I I yeors. Stcvch
attd SS in wood seems not much variable with age, However, carbohydrate cmtenfs in
bark vaned widdy. ?"he main source of variation may be the sfatus of the bark
Nevertheless, there wcu d rugutive corie~utionbeween cumulative yield and srarch
content in the bark Carbohydrate contents in clones hod a high variability ond
heritability. Som interesting ire~lds linking cuti tents in the diflerent fonns of
cnrbohydm fe to clonal origin or fo a well known rnetubolic characten'sfic were oafso
detected.
1. Introduction
Sucrose content within Iaticiferous vessels is often a major factor limiting production (Jacob
ef nl., 1998; Gohct, 1996;Goher et al., 1998): intensive exploitation induces a shortage of
intra-laticiferous sucrose, particularly when production is stimulated by the application of
ethylene gerlerntors. Qn the o ~ e hand,
r carbohydrate demand created by regeneration of latex
194 International Rubber Conference 2007, S j en] Rean. Cambodia
did not deplete wood reserves, but resultsu : i ~increase of such reserves (Silpi er al.. 2007,
Cbanturna, 2007). Therefore, carbohydrate reserves are considered a competing sink, not just
a mere buffer.
Sampling was performed during January 2006: i.e. at the end of the vegetative season in this
area. This is a period when carbohydrate reserves are the highest (Silpi et al. 2007, Chantuma
2007). In the international exchange clone trial, 3 trees were sampled in each cione and cores
were taken at 20, 100 and 300 crn from ground and 1 sample on taproot, Jn collection clone
trial for disease resistance, only one core was sampled at 50 cm from ground on 2 trees per
clone. Sarnples consisted in 0.5 cm diameter, 5 cm long cores, including 1 cm of bark and 4
cm of wood. They were made with a wood auger. Wood m d bark were separated. After each
corz was sampled, it was soaked immediately in liquid nitrogen and was kept i11 cryo-tube , .
immersed in liquid nitrogen until transfer to the laboratory and stored at -80 'C, before freeze-
drj0ing using a -50°C freeze-dryer (Telstar Cryodos, Spain). Thereafter, the samples were
blended usirlg ball-blender hlM20Q (Retsch, Germany), ball d-iarneter7 nlm. Storage after this
step until ex traction and chemical analysis was at -80 OC.
3. Chemical Analysis
international Rubber C o n f e r e n c e 2 0 0 7 , a r n b o d i a 195
Powder was re-dried in the oven for 2 hours at 6S°C. Soluble sugars were extract& from 20
mg samples with 80% ethanol during 30 min at 80°C, then centrifuged. This step was
repeated twice, first with 80% ethanol and then with SO% ethanol and dl the supernatants
were pooled. The sediment, which contained starch, was filled with 80% ethanol and kepi at -
80 OC until analysis. The supernatant was filtered in crushed glass mini columns added with a
mixture of polyvinyl pol ypyrrolidone and activated charcoal to eliminate pigments and
plyphenols. Ethanol was evaporated using a vacuum dryer (Maxi Dry plus', Hem,
Denmark). Soluble sugars (SS) and smch were quantifted by enzymatic analysis. Sucrose
was transformed into glucose and fructose by invertase (P-fmctofuranosidase). The glucose
and fructose were quantified using hexakinase, glucose-6-phosphate-dehydmgenas and
phosphoglucose isomerase followed by spectro-photometry of resulting NADPH at 340 m.
For starch analysis, aftcr the ethanol was evaporated, the sediment was hydrolysed with
NaOH 0.02N for 1.5h at 90°C,then with a-amyloglucosidase for I h at 50°C and then
glucose was quantified as described above. The results were expressed as mg glucose
equivalent per gram of sst~ctura~ dry matter (m&&p,DM, mg C~IUw,.i'g mcniral D~).Sum ~f
starch and soluble sugar represented the total non-structural carbohydrate (TNC)(Bothringer,
1984).
4. Statistical: PPocedure
The carbohydrate reserves results were processed in the principle component amly sis (PCA)
and varcomp procedure. SAS version 8.0 (96250628) was used in both cases.
Main' features of girth, y ietd and carbohydrate contenrs are shown in table 1. BPM1 had the
fighest girth (higher than 80 cm), whereas KRS21 had the lowest (below 70 cm). BPM24 had
the highest yield (gltlt higher than 50) and BPM3 was particularly low (10 dtlt).
RRIM722 had the highest non-structural carbohydrate content in the trunk, far ahead of the
second clone. R R M 7 12, in the wood as weU as in the bark. This was the result of very high
SS content. RIiIM712 had the highest starch content in wood. The lowest contenis in TNC
were exhibited by BPM22 and BPM26 in the wood and by RRIClOl and PR302 in the bark.
Well known clones such as GT1, RRIC 100, RRIM600, and RRIM703 are medium.
In average, the percentage of starch within total wmd carbohydrate content is 77 5%.
RRIM722 has the lowest percentage (60 8)due to its high SS content. Starch percentage
within thc bark is only 40 8. Again RRIM122 has one of the lowest percentage (28 %), as
well as other clones such as KRSZI and BPM24, whereas it is particularly high. for MlM725
(76 %), the only Wickham x Amazonian clone of this trial, which had the lowest SS content
in bark. SS in wood had the highest CV (coefficient of variation), whereas girth had the
Iowest.
I96 --
International Rubber Conference 2007, Siem Reap, Cambodia
Figure 1 shows that distribution of starch in wood was almost nonnal, whereas in bark, almost
half. the clones (1 1 in 25) had a starch content within the lowest class (less than 20
mgGLu/gsDM). ~ d SS,
r it was the contrary. distribution was almost normal in bark, but in wood
content ranged 25 I I I ~ for~ 10~clones / 25.
~ in ~ SS ~ ~ in both bark and wood in
~ content
RRIM722 was outstnnding.
T h e correlation matrix (table 2, df = 23, statistical significance threshold for a = 0.05 = 0.396)
shows that girth is positively correlated with latex yield per me (@&I (r = 0.43). Girth
correlates negatively with starch in wood (S'tarch~,r = - 0.28).
h most cases all the carbohydrate content variables are positively currelated berween each
ather (r varies from 0.28 to 0.82). They probably act together for generating the global
variation.
198 Internat iond Rubber Conference
-2007, Siem Reap, Cambodia
Solrible siignrs in woad (SSW), Girth, S&rcb in wood (SmrckW), Total mn stmctrard
carbohydrates in wood (TNCW). Soluble sugars in bark (SSB), Starch in bark (SlorchB)and
To#a1 non structl~ralcarbohydrates in bark (TArCB), Latex yield in gram per free per tapping
(G-T-T) -
The principal component analysis (Table 3) shows that the 3 fmt principal components make
85 9% (53 % + 18 8 + 13 5%) of the total variance displayed by the variables (girth, yield, and
cafbohydrate contents). There is a very clear distinction between principal cornponcnt I that is
closely related with carbohydrate content variables, and principal component 2, that is closely
related with girth and yield (Figure 2 and 3). Therefore, we can assume that 2 groups of
variable wif I determine the global variation, one group madc of the 4 (or 6) variables related
with carbohydrates, and one group made of girth and production. The 6 first components
cover 100 % of the variance, which is due to the redundancy of TNCW and TNCB.
]Figure 2. Position of factors according to the two first axes of the PCA, explaining 72% of
the variance
P C P r h 2 5 d c r l e s c n m 3-2
-----------------
I BM1
-
-- -- - - - - - - - - - --
FIFPC110
m3M A A mMm1
4 8
RHC 101 r -26
A m.171~
- - - - - - - -- - - - - - - -
rPAW -
A mrn
-
-
r n o
-..- - --.. ,. ,
Figure 3. Position of the different clones according to the two frrst axes of the PCB
200 International Rubber Conference 2007. S iem Reap, Cambodia
1-2. Focus on the 4 carbohvdrate content variables (StarchW, SSW, St&B. SSB)
StarchW h t i 3
' hsW SSB TNCW TNCB
Clone 165.56 0.03
. 0.02 192.91 388.19 3 14.20
Rep 0.45 0.00 0.00 0.17 0.49 2.77
Height 143.62 0.01 0.00 17.85 179.64 71.09
Error 126.14 0.01 0.00 16.27 132.22 62.09
Total 435.77 0.06 0.03 227.20 700.54 450.15
Cto?e+Error 291.70 0.04 0.03 209.18 520.41 37629
hS 0.57 0,68 0.87 0.92 0.75 0.83
%Clone 38 47 80 85 55 70
%Rep 0 3 0 0 0 1
%Height 33 27 7 8 26 16
%Error 29 23 13 7 19 14
Tranfon~data By logaritkm starch in bark (LrfB) Qncd Tranfonn data by IoganShrn soluble
ntgm in wood fLssW)
The four variables tent to be positively correlated two by two, but not always sigmfmntly
(Table 7). Starch in the wood was correlated with starch in the bark, but not with soiuble
sugars in the wood and in tlle bark.'~tarchin the bark was dso correlated with soluble sugars
in the wood. Soluble sugars in the wood and in the bark were correlated between each other.
But sol ubIe sugars in the bark had low correlation coefficients with starch ( w d and bark).
As variability due to the outstanding SS content in RRIM722 could hide trends among other
clones. we processed the analysis without this cIonc (Table 8 and 9).
The PCA of these 4 variables shows that the 3 first components make 94. % of the variation
(62 % + 220 % + 12 %I. The first component exhibited the variation due to the joined effects
of the 4 variables. The second component indicatd a divergence between StarchW and SSB.
The third component indicated a divergence between StarchW and StarchB (Figure 4 and 5).
202 International Rubber Conference 2007. Siem Reap. Cambodia
.-.
rn a
- - - - - - .. - .. - - - a4- - - - - - . - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -*=
- - -.
I --
. - .- .
,I . , - .. . - - - - - . - - - - - - - - - - - . . . - - .
4
.-
sb-
- .- - - - - .
-0.2
.-
0.1
------- - - - -
. . . . . .-
4
P
-42 - - - - - - - - - - - -
II
- - -
a1
. .
.
0.2
- - - .
. . .. . -
03
. . .
0.4
- - - - - - - - - - - .- - - .. - - - .
ASSW
. . . . . . -- nm
A
05
-ow
Figure 4. Position of factors according to the two first axes of the PCA without RRTM722
Figure 5. Position of the 24 clones (without RRlM722) according to the two first axes of rhe
PCA
PB235 and PB217 had .the highest girth, whereas KRS156 and RRLM7 12 have the lowest.
PB260 has the hiehest starch content in wood. whereas PI3217 has the lowes! {Table 10).
hternational Rubber Conference 2007, Siem Reap, Cambodia
- 203
StarchW / Starcm ratio is 1 far PI3217, whereas it's higher than 2 for most the other clones.
In bark, GTI had the lowest starch content, whereas PB235 had the highest. RREM703 had by
far the highest SS in w d . h bark, PR261 had the lowest SS, KRS 156 having the highest
SS. TNC in bark had ~e lowest CV (coefficient of variation), whereas SS in wood had the
highest.
Soluble sugars in wood {SSW), Starch in wood (StarchW), Total non srructuru€
carbohydrates in wood (TNCW),Soluble sugars in bark (SSB), Srarch in bark (StarchB) and
Totad non stnrctural curbohyiIralt.s in bcr rk (TiZJCB).
Dak grey: highest vdue among clones. Pale grey: lowest value among clones.
204 International Rubber Conference 2007, Siem Reap. Cambodia
Figure 6. Distribution of Starch in wood (A) and in bark (C), and soluble sugars in wood (B)
and in bark (D)
R*
0.98
1 Cv-m%
5.0
Clone
***
'*
Rep
StarchB 0.98
.. 3.5 *** Ns
LssW 0.9 2. I *** Ns
SsB 0.99 6.8 *** Ms
TncW 0.99 . 4.2 4** Ns
-
0.9s 3.5 *+* Ns
Clonal effect was very significant for all variables, Heritability was very high for all variables
too, higher than in the 25 clones experiment because them was no height factor (Table f 1 and
12).
Table f 3. CorreIation 111atrixfor experiment 2 ,
-
Girth
-
Girth
1.00
-
StarchW
-0.20
-
StarchW -0.20 3 -00 0.21 0.04 0.33
SSW 0.19 0.21 1.00 -0.14 -0.05
StarchB -0.01. 0.04 -0.14 1.Oo 0.2 1
SS B 4.62 0.33 -0.05 - 0.21 1.00
Girth was negatively correlated witb SS content in bark (Table 13). Other currelations were
not high. There was notably no c or relation ktween contents in bark and in wood for either
starch or SS,contrary to what was obtained in the 30 yem old trees.
Eigenvalaes (corn)
Component Eigenvdue Difference Proportion
1 1.84 -- 0.60 0.37
2 1.24 0.26 0.25
3 0.98 0.35 0.20
4 8.63 0.30 0.13 0.94
Two first axes e~pIained62 % of the variance (Table 14). Axis 1 showed mainly the
divergence between girth and SS bark, whereas axes 2 showed mainly the dnergence betuleen
SS in wood aid btarch in bark (Figure 7 and 8).
206 International Rubber Conference 2007, Skm Reap, Cambodia
"-.
L"-- -
Figure 8. Location of clones according to the two first axes of PCA
. - Reap, Cambodia
hternariand Kubber Conferen= 2007, Siem - 207
4, Discussion
As conlparison to mean contents obtained the s m e season in an extensive study on stnndard
clone RRIM600, I t years old crees, by Chantuma (2007).An average value recorded 25
clones exchange trial at 30 years werc similar into wood for both starch and SS wood. In bark
however, mean contents SS. and particularly swch were higher in tbe 30 yews taees 25
clones than the 11 years RRIM600. In the 15 years old trial wilh 18 clones. mean cantent in
wood were a bit higher for both starch md SS than RRIEA600 in the extensive trial. h bark,
SS content was similar as abvve but: starch content was much higher. A directed cornparisfin
of contents in RRM600 clone only showed the same trend. Starch in bark was lower in the
extensive trial 1 I years old, medium at 30 years and higher at 15 yeas. 5s content in bark
was lower at 15 years, medium at 11 years and much higher at 30 yews. Thus, contents in
wood scem not much variable with age and location. contents in bark varied widely. The
main source of variation may be the stams of the back. In the extensive study at 11 y m , most
samples consisted h virgin hark, whereas in Ihc 30 years old experiment all hunk samples but
except the one at 300 cm high were made of m e w e d bark. Similarly, irl h e 15 years old &ial,
the only one sample was taken in the renewed bark too, However, at 30 years there was no
difference in either starch or SS content between bark sampled at 300 cm. which was virgin
bark, and bark sampled at 100 cm,which was renewed bark (Table 5).
Carbohydrate variables were correlatsd together in the 30 years experiment, but not so much
in the 15 years old experiment. In the Iater, paticlrlarly there wac nu correlation betweet~
content in bark arid in wood. We know that much the resemes are located in the wood (SiIpi
ct ~2.2007,Chimtuma, 20071, but as sanlpling waQd creates more damage io the tree, a g d
cone]ation between wood and bark content would d1ow to sample batk only. Tfiis seems not
appropriate according to the present results.
Clonal effect on all carbohydrate variables was highly significant a d heritability was high.
This i s a very interesting result showing that it is possible to select clones on the basis of their
carbohydrate content. Among clones, some remarkable features were shown. The very high
SS content recorded in RRM722 raises questi~nsas it shows that some dorm can hhave in
drastically different way fmm others according to carbohydrates accumulation. Zt was
measured in both bark and wood, where starch content was medium. Thus this high SS
content was not a recuft of higher s t a h iuvbilization, but of hig-her total carbohydrate
accumulation. Yield of RRIM722 was medium-low (25 &t/t) and girth was high (79 cm). It
would be interesting to assess if clones with the same pace.nts (RR1M60q x TK14) have the
same pattern. Other remarkable patterns were observed. PB217 is known to have a peculiar
latex metabolism, as it is able to maintain a high level of sucrose in latex despite high latex
yield. This make this clone particularly adapted to ethylene stimulation (Gohet et d,1996).
Our study dernonslrated that this clone had the lowest starch content in wood among 18
clones, with the lowest StarchW / StarchB ratio too. This may indicate a higher allwatior~of
cubohydrate in the vicinity of the laticiftrous tissues. Clones well known for their active
latex metabolism such ~ F SPB'ZGD, PB235 or BPM24 had on the contrary high starch cunkent in
wood. The only clone with an Amazonian parent (RRIM 725) had the lowest SS conten! in
bark and by far the highest percentage of starch in TNC in the bark. It wvuld be ~f interest to
assess if such a character is linked to Amazonian (wild) genotypes as compared to Wickham
gmotypcs.
Ludovici, K.H., H.L.Allen, T.3. Albaugh and P.M. Dougherty. 2002. The Influence of
Nutrient and Wata Availability on Carbohydrate Storage in LobloIly Pine. For. Ecol.
and Manage. 159:261-270.
SiIpi U.,A. bcointe, P.Kasemsap, S. Thanisawanymgkura, P, Chantuma. E.Gohet, N.
Musigamart, A. Clement, T.Ameglio and P.Thaler. 2007. Carbohydrate Reserves as a
Campeting Sink:Evidence from Tapping Rubber Trees. Tree Physiol. 27,881-889.