Effect of genotype on micropropagation and post-propagation growth of 35 commercial clones of Castanea sp
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- MATERIAL AND METHODS Chestnut clones
- “In vitro” propagation and postpropagation
- Multiplication Stage Heller (1953) modified (Vieitez et al., 1986)
- Elongation stage Rooting stage
- Nursery
- Height 7 Height Increment 1-7 Stem Form 4
- Establishment stage Elongation stage Multiplication stage
- Parameter Trait GD-Hm GD-MS(½N) Hm- MS(½N) r xy
- Plantation Nursery
| Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 313
Effect of genotype on micropropagation and post-propagation growth of 35 commercial clones of Castanea sp.
M. E. Miranda- Fontaíña and J. Fernández-López
Centro de Investigacions Forestais de Lourizán. Departamento de Producción Forestal. Xunta de Galicia. Apartado 127. 36080. Pontevedra. Spain. e-mail: memiranda.cifal@siam-cma.org Keywords: Chestnut, in vitro, clonal heritability, genetic correlations, plantation, genotype-environment interaction. Abstract
chestnut, the genotype-environment interaction during “in vitro” multiplication and the genetic correlations between “in vitro” and “ex vitro” traits. High genetic variability and high clonal heritabilities were obtained for all studied traits. Mean values of all traits increase from the “in vitro” establishment to “in vitro” multiplication stage. Low phenotypic and genetic correlations were obtained between “in vitro” establishment and multiplication traits, only the variable number of shoots per explant had significative and moderated genetic correlations between these stages. High genetic correlations between Hm-MS(½N) media were obtained during “in vitro” multiplication and this result indicates that both media induced a similar guideline for the group of clones. Low values of genetic correlation of the same trait in different culture media indicate that the origin of the genotype- environment interaction. No significative correlations were obtained between “in vitro” and growth in plantation traits. Contrary there were significative correlations between growth and stem form in nursery and height and stem form in plantations, it is possible to predict the growth in plantations from nursery behaviour. INTRODUCTION 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 micropropagation. Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 314
MATERIAL AND METHODS Chestnut clones Thirty five chestnut hybrid clones between the European chestnut (Castanea sativa Mill.) with the Asiatic chestnut (C. crenata Sieb. et Zucc. or C. mollissima Blume) (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). Culture media for “in vitro” Establishment, Multiplication and Elongation stages 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.
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 obtain clusters of shoots. Cultures were incubated in a growth chamber at 25ºC and 16 hours of photoperiod, under cool white fluorescent lamps (Sylvania gro-lux 40W) with a Photon-flux density 50 µmol.m -2 .s -1 .
Shoots with at least 3-cm long of all clones were ex vitro rooted. For rooting, the 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.
1.- Establishment and Multiplication stages: The variables were the same studied by Miranda-Fontaíña and Fernández-López (2001): Number of Shoots per Explant (NSH), Length of the Tallest Shoot (LS), Number of one-centimetre Segments (NS) per explant, Apical Necrosis (AN) in the shoot, Percentage of Responsive Explants (%RE) and Multiplication Coefficient (MC). 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
Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 315
favorable results during rooting stage, Miranda-Fontaíña and Fernández-López, 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 ).
4.- Plantation: Height and stem form were evaluated: height in centimetres after four and 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.
Establishment traits were measured from the first to the fifth subculture. The multiplication traits were measured after the fifth subculture.
establishment and multiplication tests. For “in vitro” elongation only basal explants were used to obtain clusters of
shoots. 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 ijk
= µ + C i +
S j(i)
+ ε k(ij) (model 1),
where: C: Clone (i=54), S: Subculture (k=3) within the clone, ε : Residual effect. Clonal heritabilities (H 2 c
2 C = σ 2 c
/ (σ
2 e /RS+ σ 2 S(C)
/R + σ
2 C ), where σ 2 C , σ 2 S(C)
, σ 2 e are variances due to clones, subcultures in clone and due to 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 ijkl
= µ + C i + M
j +
C*M ij + S k(ij) +
ε l(ijk),
(model 2) where M, C*M and S are culture medium, interaction 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 ijkl =
µ + C
i +
ε i(j) (model 3) .
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 B
= r xy /(h Cx
h Cy ), where r xy is the phenotypic correlation between genetic group means in environments x and y, and h Cx
and h Cy
are square–roots of heritabilities of the genetic 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.
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
Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 316
the percentage of responsive explants increased 11% during multiplication stage, also 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 factors and for the interaction between clone and culture media, as was obtained in 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 (Falconer, 1989). Correlations between establishment and multiplication stages: Low phenotypic and genetic correlations were obtained between “in vitro” establishment and multiplication traits Only the variable number of shoots per explant had moderated genetic correlations. Interaction genotype-environment during multiplication stage: the estimates of 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.
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 elongation stage.
possible to predict the growth in nursery or plantations from in vitro traits. Correlations between nursery and plantation traits: Table 7 shows that there were 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.
Burdon, R.D. 1977. Genetic correlation as a concept for studying genotype-environment interaction in forest tree breeding. Silvae Genet. 26:168-175. Chauvin, J.E. and Salesses, G. 1988. Advances in chestnut micropropagation (Castanea sp.). Acta Horticulturae. No 227, 340-345; International Symposium on vegetative propagation of woody species, Pisa, Italy, 3-5 Se. 1987.
Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 317
Dickerson, G.E. 1962. Implications of genetic environment interaction in animal breeding. Anim. Prod. 4: 47-64. Falconer, D. S. 1989. Introduction to quantitative genetics. Longman, Essex. Fernández- López , J. 1996. Variabilidad isoenzimática, morfológica y selección clonal en de Castanea sativa Miller Castanea crenata Sieb. et Zucc., Castanea mollissima e híbridos interespecíficos. Tesis doctoral, Univ. Polit. de Madrid. Gresshof PM, Doy CH. 1972. Development and differentiation of haploid Lycopersicon
Heller, R. 1953. Recherches sur la nutrition minérale des tissus végétaux cultivés in vitro. Ann. Sci. Nat. Bot. Biol., 14: 473-497. Ivkovich, M. 1996. Genetic variation of wood properties in Balsam Poplar (Populus balsamifera L.). Silvae genetica 45, (2-3): 119-124. Johnson, G.R. 1997. Site-to-site correlations and their implications on breeding zone size and optimum number of progeny test sites for coastal Douglas-fir. Silvae genetica, 46 (5): 280-285. Johnson, G.R. and Burdon, R.D. 1990. Family-site interaction in Pinus radiata: implications for Progeny Testing strategy and regionalised breeding in New Zeland. Silvae Genetica, 39 (2) : 55-62. Miranda, M.E. and Fernández, J. 1990. Estudio de las condiciones para el enraizamiento de brotes de castaño híbridos producidos in vitro. II. Congreso Forestal Nacional, Porto, Portugal. Vol I : 433-436. Miranda, M.E. and Fernández, J. 1992. The micropropagation of chestnut tree: “in vivo” establishment and post-propagation growth. Mass production technology for genetically improved fast growing forest tree species. Symposium Bordeaux, France, Tome I: 421-426. Miranda-Fontaíña, M. E. and Fernández-López, J. 2001. Genotypic and environmental variation of Castanea crenata x C. sativa clones in aptitude to micropropagation. Silvae Genetica. 50 (3 - 4) pp. 153 - 162. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Peng-xin Lu, T.L. White, D.A. Huber. 1999. Estimating type B Genetic Correlations with unbalanced data and heterogeneous variances for half-sib experiment. Forest Science 45 (4), 562-572. Pswarayi, I.Z., Barnes, R.D., Birks and Kanowski, j. 1997. Genotype-Environment Interaction in a population of Pinus elliotti Engelm. Var. elliotti. Silvae Genetica, 46 (1): 35-40. Sánchez, M.C. and Vieitez, A.M.. 1991. In vitro morphogenetic competence of basal sprouts and crown branches of mature chestnut. Tree Physiology. 8: 59-70. Urquijo, P. 1956. La regeneración del castaño. Bol. Pat. Veg. Ent. Agr. XXII, 217-232. Vieitez, E. 1960. Obtención de castaños resistentes a la enfermedad de la tinta. Centro Regional de Enseñanzas y experiencias Forestales de Lourizán, Pontevedra. Vieitez, A.M., Ballester, A., Vieitez, M.L. and Vieitez, E. 1983. In vitro plantlet regeneration of mature chestnut. J. Hortic. Sci., 58: 457-463. Vieitez, A.M., Vieitez, M.L. and Vieitez, E. 1986. Chestnut (Castanea spp.) In: Y.P.S. Bajaj (Editor), Biotechnology in Agriculture and Forestry. Vol. I: Trees I. Springer-Verlag, Berlin, pp. 393-414. Yamada, Y. 1962. Genotype by environment interaction and genetic correlation of the same trait under different environments. Japan. J. Genet., 37, 498-509.
Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 318
Table 1. The mean squares, significance levels of the analyses of variance, clonal heritabilities and mean values with standard deviation (SD) for 4 variables of establishment stage.
Source of variation
df
Number of shoots per explant
Length of the tallest shoot
df
% of Apical Necrosis Responsive Explants (%)
Clone
Subculture(clone) Error
34
70 1.395
12.05 *** 2.49*** 0.98
14.41 *** 2.76*** 1.06
34
70
0.04*
0.02 0.05**
0.02 H 2 c 0.79 0.81
0.47 0.50 Mean
± SD
2.40 ± 0.64
2.12 ± 0.69 7.49
± 16.55
85.51 ± 16.67 Hc 2 : 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. Multiplication Stage Heller (1953) modified (Vieitez et al., 1986) Source of variation
df Number shoots per explant Length of the tallest shoot
Number one-cm
segments
df Apical Necrosis Responsive Explants (%) Multiplication Coefficient
Clone Subculture(clone) Error
34
70 1.995
13.47*** 3.01*** 1.22
25.75*** 3.93*** 1.24
111.55*** 14.08*** 3.63
34
70
0.06***
0.01 78.80***
17.61
6.18***
0.71 H 2 c 0.77 0.84 0.87
0.71 0.77 0.88 Mean ±
2.77
± 0.56
2.79 ± 0.74 4.11 ± 1.71 19.57
± 17.49 96.14 ± 6.13
4.00 ± 1.58 Gresshoff and Doy (1972)
Clone Subculture(clone) Error
34
70 1.995
25.35*** 6.39*** 1.59
43.19*** 4.76*** 1.64
129.09*** 18.89*** 4.10
34
70
0.02***
0.01 103.96***
26.42 7.36***
0.89 H 2 c 0.74 0.89 0.85
0.60 0.74 0.88 Mean ±
3.01
± 0.79
2.80 ± 0.93 4.11 ± 1.65 6.61
± 8.47
94.38 ± 7.19 3.94 ± 1.73 Murashige and Skoog (1962) (½ N) (Vieitez et al., 1986)
Clone Subculture(clone) Error
34
70 1.995
21.97*** 2.80*** 1.34
73.51*** 11.44*** 2.39
185.05*** 21.56*** 4.61
34
70
0.04***
0.01 118.27***
5.71 10.33***
1.01 H 2 c 0.87
0.84 0.87
0.68 0.95 0.90 Mean ±
2.53
± 0.67
3.23 ± 1.25 4.38 ± 1.93 12.95
± 11.42 95.71 ± 6.52
4.25 ± 2.01 Hc 2 : 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.
Rooting stage Source of variation
df Number of shoots >3cm Length of shoot >3cm Diameter of shoot >3cm
df
Percentage of rooting
Number of roots per rooted shoot
Clone
Error
26 1.124
14.47*** 1.25
25.19*** 3.09
0.71*** 0.09
27
1.813
5.84*** 0.79
61.89*** 3.78 H 2 c
0.91 0.87 0,86
0.81 0.92 Mean
± SD
2.03 ± 0.64 5.20 ± 0.85 cm 1.30 ± 0.18 mm 90.31±15.60 8.61±4.75 Hc 2 : clonal heritability; ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001 Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 319
Table 4. Mean squares, significance levels of analyses of variance, clonal heritabilities, clonal means and Standard Deviation (SD) for variables of nursery and plantation.
Nursery
Source of variation df Height 1 Stem Form 1 df Height 4 Height 7 Height Increment 1-7 Stem Form 4
Clone Error
12 26
2.218.27*** 230.92
4.70*** 0.17
23 38
6.064.61*** 1.255.30
15.197*** 3.765.87
11.305*** 3.929.67
5.51*** 0.45 H 2 c
0.89 0.96
0.79 0.75 0.65 0.91 Mean
± SD
75.03
± 19.84
8.55±1.24
239.16 ± 46.71 402.46 ± 78.65
317.64 ± 96.46 7.31 ± 1.41 Hc 2 : clonal heritability; ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 5. Phenotypic correlation (r xy ) and genetic correlations (r gxy ) between traits for different stages (multiplication-establishment (both in Heller mod medium) and multiplication-elongation (both data groups in Murashige and Skoog (½N) medium).
NSH
LS AN
%RE NS>3cm
LS>3cm NSH
r xy
r gxy
0.38* 0.49 0.21ns 0.26 0.06ns 0.10 0.40* 0.65 0.35ns 0.42 0.11ns 0.13 LS
r xy
r gxy
0.01ns 0.01 0.31ns 0.37 -0.10ns -0.16 0.21 0.32 0.59** 0.67 0.21ns 0.24 NS
r xy
r gxy
- - -
- 0.55** 0.62 0.15ns 0.17 AN
r xy
r gxy
0.24ns 0.32 0.10ns 0.13 0.10ns 0.17 0.24ns 0.40 -0.32ns 0.40 -0.47* -0.60 %RE
r xy
r gxy
0.02ns 0.02 -0.04ns -0.05 -0.12ns -0.20 0.15ns 0.24 0.46* 0.55 0.32ns 0.39 MC
r xy
r gxy
- - -
. 0.56** 0.62 0.16ns 0.18 ns= P>0,05; * = P>0,01; ** = P<0,01; *** = P<0,001 Proc. IIIrd Intl. Chestnut Congress Eds.: C.G. Abreu, E. Rosa & A.A. Monteiro Acta Hort. 693, ISHS 2005 320
Table 6. Phenotypic (r xy ) and genotypic correlations (r gxy ) between environments (culture media) for different traits.
GD-Hm GD-MS(½N) Hm- MS(½N) r xy r gxy
NSH
LS NS
AN %RE
MC
NSH LS NS
AN %RE
MC 0.46** 0.55*** 0.57*** 0.54*** 0.19ns 0.55***
0.61 0.63 0.68
0.82 0.25
0.62 0.56*** 0.63*** 0.68*** 0.42* 0.28ns 0.66***
0.69 0.84 0.79
0.65 0.39
0.74 0.71*** 0.64*** 0.74*** 0.42* 0.42* 0.74***
0.86 0.76 0.85
0.60 0.49
0.83 ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001
Table 7. Phenotypic correlation (r xy ) and genotypic correlations (r gxy) between traits for nursery and plantation stage.
Plantation Nursery
Height 4 Height 7 Height
Increment 1-7 Stem Form 4 Height 1
0.28ns 0.33 0.34* 0.41 0.32* 0.42 0.48** 0.53 Stem Form 1 r xy r gxy 0.89*** 1.00 0.89*** 1.00 0.80* 1.02 0.93*** 1.00 ns= P>0.05; * = P>0.01; ** = P<0.01; *** = P<0.001 Kataloq: wp-content -> files wp-content -> Klinik protokol Az ərbaycan Respublikas wp-content -> Soalan: Kawasan Perdagangan Bebas asean (afta) yang dipersetujui oleh pertubuhan asean menjanjikan pelbagai kebaikan untuk kawasan asean. Namun, pelaksanaan perjanjian ekonomi juga mendatangkan pelbagai implikasi. Bincangan wp-content -> Issn 0143-6597 print/issn 1360-2241 online/03/020235-19 wp-content -> Вместе к высотам стоматологии files -> Fármacos Boletín electrónico latinoamericano para fomentar el acceso y el uso adecuado de medicamentos Yüklə 156,01 Kb. Dostları ilə paylaş:
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