Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro
Acta Hort. 693, ISHS 2005
M. E. Miranda- Fontaíña and J. Fernández-López
Centro de Investigacions Forestais de Lourizán. Departamento de Producción Forestal. Xunta de Galicia.
This work studies the effect of genotype on micropropagation of 35 clones of
Euroasiatic hybrid clones are used for plantations in Atlantic areas of Spain
affected by Phytophthora sp.. Micropropagation is used for the vegetative multiplication
of chestnut clones resistant to Phytophthora sp. and selected by growing and crown form.
The genotype determines the aptitude to micropropagation in chestnut (Chauvin and
Salesses, 1988; Miranda-Fontaíña and Fernández-López, 2001; Sánchez and Vieitez,
1991; Vietitez et al, 1986). The study of the genetics of metric characters can to predict
the performance of clones during micropropagation and in their post-propagation growth.
Thus type B genetic correlation is defined as the genetic correlation of the same trait
measured in different environments (Dickerson, 1962; Yamada, 1962; Burdon, 1977).
Also type B genetic correlations have been used as quantitative measures of genotype-by-
environment interactions (Burdon, 1977; Johnson and Burdon, 1990; Pswarayi et al.,
1997) and for predicting genetic responses for indirect selection (Johnson, 1997; Peng-
xin et al.,1999).
The objectives of this work were to study 1) genetic variability in each stage of
micropropagation, assessed as the variability among clones and the clonal heritability; 2)
the influence of the culture media on multiplication rates and the genotype-environment
interaction during the multiplication stage; 3) the genetic correlations in traits of “in vitro”
and “ex vitro” stages and so to predict of the performance of clones during
Thirty five chestnut hybrid clones between the European chestnut (Castanea
(Urquijo, P. 1956; Vieitez, E. 1960), selected in field trials by growth and crown form
(Fernández-López, 1996) were used for this study. The genealogical origin of this clones
was determined using diagnostic isozyme loci of chestnut (Fernández-López, 1996).
Heller (1953) medium modified by Vieitez (Vieitez et al., 1983, 1986) (Hm) was
used during “in vitro” establishment and multiplication stages, Murashige and Skoog
(1962) with half-strength of nitrates (MS ½N) (Vieitez et al., 1983, 1986) was used
during multiplication and elongation stage, Gresshoff and Doy (1972) (GD) was used
during multiplication stage. All media supplemented with 30 g/l sucrose, agar 6 g/l,
Vitamins, Micronutrients and Fe-EDTA of Murashige and Skoog (1962) and 0.2 mg/l of
Benzylaminopurine, pH 5.6.
“In vitro” propagation and postpropagation
These clones were “in vitro” established and multiplied as described in Miranda-
Fontaíña and Fernandez-López (2001). “In vitro” cultures were initiated in spring from
field-grown young stump shoots from trees more than thirty years old. Multiplication was
developed by axillary shoots production.
For elongation stage basal explants were used to
hours of photoperiod, under cool white fluorescent lamps (Sylvania gro-lux 40W) with a
Photon-flux density 50 µmol.m
lower leaves were removed from the microcuttings and the bases of elongated-excised
shoots were dipped for two minutes in Captan® and then into a solution of AIB (1 g/l),
and placed into a moistly and sterilized substrate of perlite and composted pine bark
(mixe 2:1) in a polystyrene trays (Miranda and Fernández, 1990), immediately watered
and covered with a 3 mm polycarbonate sheet to maintain moisture, and kept in growth
chamber under the same conditions as for the foregoing phases. After four week this step
was evaluated. After eight weeks in growth chamber the plants were acclimatized in a
microtunnel with a fog system in a greenhouse with controlled temperature of 24±3ºC
(Miranda and Fernández, 1992). The plants in the polystyrene tray were fertilized during
the development in greenhouse, with the Murashige and Skoog salt solution (1962) with
half-strength of nitrates. In spring the plants were established in nursery for bare root
cultivation. After one year in nursery the plants obtained by micropropagation were
planted in the field with the objective of clonal selection.
Variables : variables recorded in each stage were:
Miranda-Fontaíña and Fernández-López (2001): Number of Shoots per Explant (NSH),
2.- Elongation stage: Number of Shoots with at least 3 cm per basal explant (NS>3cm),
that can be used as microcutting for rooting (this is the minimum length to obtain
unpublished data) and Length (LS>3cm) and Diameter of shoot with at least 3 cm.
3.- Nursery: Height (Height 1) and Stem form after one year (Stem form 1) (the stem
quality is rated among 1-10,
more strength stem form is corresponded with higher values
seven years (Height 4, 7), stem form after four years (Stem form 4) and also height
increment between the first and the seventh year (Height Inc 1-7)were also calculated to
minimize the influence of different height among plants in the moment of plantation.
Number of subcultures
(or blocks): three.
The Number of replications in establishment and multiplication subcultures were twenty.
Establishment traits were measured from the first to the fifth subculture. The
multiplication traits were measured after the fifth subculture.
Size and characteristics of the explants: the first apical centimeter was used to perform
establishment and multiplication tests. For “in vitro” elongation only basal explants were
used to obtain clusters of
The influence of genotype was estimated by ANOVA (generated using SAS (SAS
Institute, Cary NC)), with clone as main factor and with three subcultures: X
where: C: Clone (i=54), S: Subculture (k=3) within the clone,
Clonal heritabilities (H
) were calculated: h
), where σ
error variance. R and S are replicates and subcultures. The second model of analyses of
variance was applied to study the interaction genotype-environment: X
between clone and culture medium and subculture in clone and culture medium
interaction. The third model of analyses of variance was applied to traits of elongation,
rooting, growth in nursery and plantation stages: X
i(j) (model 3)
Phenotypic and Genotypic correlations: Pearson phenotypic correlation coefficients were
determined to study strength of relation among traits (PROC CORR).
Type B Genotypic correlations between traits were estimated between pairs of
traits to determine: 1) correlations of the same trait in different stages, to estimate the
possibility of predicting genetic responses for traits among different stages of
propagation; 2) correlations of the same trait in different environments (culture media)
inside the “in vitro” multiplication stage, as quantitative measures of genotype-by-
environment (GxE) interactions.
Genetic correlations have been estimated using the method of Burdon (1977): r
), where r
is the phenotypic correlation between genetic group means in
environments x and y, and h
group means in environments (or steps) x and y, respectively. Genetic correlation ranged
from –1 to 1, a positive correlation means that two traits are usually associated with each
other, a negative correlation means that one trait tends to go down as the other goes up as
a response of genetic manipulation.
RESULTS AND DISCUSSION
Significative differences among clones were obtained for all variables (Tables 1 to
4), which was already mentioned for chestnut by Miranda-Fontaíña and Fernández-López
(2001) for the “in vitro” multiplication stage. Mean values of all traits increased from
establishment (first five subcultures) to multiplication stage (after the fifth subculture), so
Sanchez and Vieitez (1991) obtained for five clones of chestnut that the multiplication
coefficient increased among the sixth and the twelfth subculture.
The analysis of variance, in multiplication stage, is highly significant for main
Miranda-Fontaíña and Fernández-López (2001). Mean values of traits in the
multiplication stage varied with the different culture media (Table 2).
Clonal heritabilities are very high for all studied traits. Heritability of clonal means
is a parameter used to measure the degree of genetic determination of clonal behavior
and genetic correlations were obtained between “in vitro” establishment and
multiplication traits Only the variable number of shoots per explant had moderated
genetic correlations between culture media for the same “in vitro” multiplication trait
(Table 6) shows that a high genetic correlations between Hm-MS(½N) media and this
indicate that the two media gave similar rankings of clones is their response to culture
media, even though there are different clonal mean values. Low values of genetic
correlation of the same trait in different media indicate that the origin of the genotype-
environment interaction and every of the medium have different guideline for the group
of clones. Miranda-Fontaíña and Fernández-López (2001) obtained for chestnut clones
very high values of phenotypic correlations between micropopagation traits and so, inside
a concrete stage of “in vitro” propagation, it is possible to select simultaneously pairs of
growth traits and at the same time to improve against the production of apical necrosis.
Correlations between multiplication and elongation stages: The number of shoots
with more than 3 centimetres per explant developed during the elongation stage was
significantly correlated with the variables of “in vitro” multiplication stage (Table 5);
therefore the clones with higher multiplication rates produced longer shoots during
Correlations between in vitro traits and ex vitro growth (nursery and plantation
growth): No significant correlations were obtained between these stages, thus it is not
possible to predict the growth in nursery or plantations from in vitro traits.
moderate correlations between growth at the end of the first year in nursery with height
and stem form in plantations. Very high correlations between nursery stem form and
growth and stem form in plantations were obtained. Highly positive phenotypic,
environmental and genetic correlations among growth characteristics have been
mentioned by Ivkovich (1996) in plantations of Populus. So it seems that it is possible to
predict the growth in plantations from nursery performance.
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heritabilities and mean values with standard deviation (SD) for 4 variables of
: clonal heritability. ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 2. The mean squares, significance levels of the analyses of variance, clonal
heritabilities and mean values with standard deviation (SD) for variables of multiplication
stage on three culture media.
: clonal heritability; ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 3. The mean squares, significance levels of the analyses of variance, clonal
heritabilities and mean values with Standard deviation (SD) for variables of elongation
and rooting stage.
per rooted shoot
clonal means and Standard Deviation (SD) for variables of nursery and plantation.
ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 5. Phenotypic correlation (r
) and genetic correlations (r
) between traits for
multiplication-elongation (both data groups in Murashige and Skoog (½N) medium).
ns= P>0,05; * = P>0,01; ** = P<0,01; *** = P<0,001
) and genotypic correlations (r
) between environments (culture
media) for different traits.
ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 7. Phenotypic correlation (r
) and genotypic correlations (r
between traits for
Stem Form 4
Stem Form 1
ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001