Turkey ranks the third in the production of chestnuts in the world having an important place both in domestic and global markets. However, the chestnut production and the number of trees have been diminishing in recent years. Therefore, in vitro propagation of the chestnut, in addition to the classical propagation techniques, should be applied. Especially the propogation of the early maturing cultivars and production of the quality chestnuts will provide a better income to the producer. Here, somatic embryo production and regeneration from the immature cotyledons of the early maturing cultivars of the European chestnut ( Castanea sativa Mill), Haciibis and Karamehmet, were studied using the somatic embryogenesis, one of the in vitro propagation techniques. To induce the somatic embryogenesis, 168 different combinations were applied to both cultivars. The somatic embryogenesis rate in Haciibis cultivar, in which the interactions were observed among the applications, was found to be 9.9% while it was 11.1% for the Karamehmet cultivar. Dessication, cold treatment, gibberellic acid (GA 3 ) and benzyladenine (BA) + naphthaleneacetic acid (NAA) applications were performed on the regeneration of the somatic embryos, and 40% conversion to plant was obtained with desiccation together with BA + NAA supplementation to the medium.
The genus Castanea (chestnut) belongs to the Fagaceae family with its 13 different species, and it is found in the natural flora of the northern hemisphere. Among the chestnut species, the origin of European chestnut (Castanea sativa Mill.) is Anatolia, and it also spreads throughout the Southern Europe, Northern Africa, and Caucasia [
According to the FAO 2013 statistics, Turkey ranks the third in the world with 60.019 tonnes of chestnut pro- duction after China (925 thousand tonnes) and South Korea (70 thousand tonnes), and has a share of 5.2% in the global production (2 million tonnes) [
The early maturing chestnut cultivars Hacıibiş and Karamehmet are grown in the Marmara Region in Turkey [
Somatic embryogenesis from the zygote-originated tissues is an important potential for the clonal propagation of the hybrids obtained via hybridization breeding [
The early maturing cultivars of European chestnut (C. sativa Mill) Hacıibiş and Karamehmet were used as plant material in this research. The trees grew in the chestnut collection parcel in Atatürk Central Horticultural Research Institute, Yalova, Turkey and in the gardens of two manufacturers located at the skirts of the Uludağ Mountain (Bursa-Turkey).
For the first step of somatic embryogenesis,explants were obtained from the cotyledons of immature seeds because of their high levels of auxin and cytokinin levels. For 3 yr, burs (fruiting heads) were manually harvested 7 wk (50 d) after the female flowers were in full bloom, between the end of July and the beginning of August (with slight seasonal variation), and were divided into their fruits [
Immature fruits were separated from the bur and suspended for 15 min in 20% (v/v) commercial sodium hypochlorite solution containing a few drops of Tween 20. This was followed by five rinses with sterile distilled water (dH2O) for 5 min (with agitation). The immature fruits were opened in a laminar flow cabinet and cotyledonary explants, about 0.5 cm2 in size, were excised using a scalpel.
In order to induce initial stage somatic embryos: 1) Murashige and Skoog medium (MS) [
The rate of somatic embryogenesis (%) was calculated dividing the number of total cotyledon explants generated somatic embryos to the number of total cotyledon explants planted onto the initiation media, and the resulting number was multiplied with 100.
The average number of somatic embryos (number/cotyledon explant) was calculated dividing the number of embryos (at the stage of cotyledon) to the number of total explants.
The cotyledon explants were placed horizontally on the media under aseptic conditions. Each of the Petri dish contained five explants, and was cultured for a period of 4 wk in a growth chamber at 25˚C ± 1˚C in the dark. In order to induce growth of somatic embryos, after 4-wk culture to initiate somatic embryogenesis, cotyledon explants were sub-cultured on the same media without PGRs, AgNO3 or L-glutamine, for a total of four times at 4-wk intervals onto the basal media with the same supplementation.
In regeneration experiments, 30 g/L sucrose and 6 g/L agar were added to DKW or half-strength MS basal salts (1/2 MS). The somatic embryos were used at the stage of the cotyledon derived from the cotyledon explants in the regeneration experiments. The somatic embryos (five pieces/Petri dish) were placed in a vertical position so that the root tip of the root/epicotyl axis was in contact with the media. All the cultures were incubated for a period of 4 wk 25˚C ± 1˚C temperature, 16 h light (35 μmol∙m−2∙s−1) and 8 h dark for regeneration. Three different trials were conducted for regeneration of the somatic embryos.
For the desiccation procedure, a Nalgene® desiccator (1.5 L capacity) was sterilized by autoclaving with 40 ml saturated MgCl2∙6H2O solution and then, in a laminar air flow cabinet, 20 somatic embryos at the cotyledon stage were placed in Petri dishes without media (four pieces per plate), and were placed in the desiccator. The desiccator was wrapped with stretch film and kept in the growth chamber at 25˚C ± 1˚C in the dark for 4 d. After treatment with or without desiccation, somatic embryos were cultured on DKW basal media containing 0, 1, 3, 5, 7, or 9 mg/L GA3.
These treatment conditions were performed according to Corredoira et al. [
In the second trial, the aim was to determine whether the somatic embryos respond better when they were pre-treated with desiccation, cold (cold treatment was conducted at 4˚C, in the dark for 8 wk), or GA3 (3 mg/L for 10d), before being cultured on DKW with BA + NAA (0.1 mg/L each). The effect of the same media with or without 438 g/L L-glutamine were tested [
In this study, all trials were established according to Coincidence Pattern Experimental Design (Minitab Inc. 814-238-3280 WS112102553). The effects of five different applications (basal media × PGR combinations × organic matters × AgNO3 × solidifying substances) were investigated on the somatic embryogenesis of two early cultivars Hacıibiş and Karamehmet. The experiments were repeated five times in each case, with five explants per treatment. All trials were repeated throughout two years.
The trials of somatic embryo regeneration were evaluated by analyzing the interactions among “cultivars × desiccation × GA3” in the first trial, “cultivars × BA applications” in the second trial, and “cultivars × pre-application combination × BA + NAA” in the third trial. The experiments were repeated five times in each case, with five explants per treatment. Each experiment was repeated at least twice.
Data obtained from somatic embryogenesis and regeneration experiments were calculated as the average of repeats using the analysis of variance (ANOVA) in Minitab Package Program, and controlled with F test (P ≤ 0.05, 0.01, and 0.001). Significant differences were determined depending on 5% error limit with the Duncan test, and the differences were indicated with letters. In statistical analyses, transformed angle values were utilized for the percentage data.
The effects of basal media, combinations of plant growth regulators (PGRs), organic substance, AgNO3, and solidfying substance on somatic embryo formation as well as the impacts of desiccation, cold treatment, and GA3 were shown in
A significant interaction among the basal media, combinations of PGRs, organic substance, AgNO3, and solidfying substance could not be observed in Hacıibiş chestnut cultivar. However, the interactions among PGR combination × organic substance × solidifying substance were found to be significant (P = 0.045). In the content of PGR combination × organic substance × solidifying substance interaction in this cultivar, the PGR com- bination number 3 [KIN (2 mg/l) + TDZ (0.1 mg/l) + IBA (0.01 mg/l)] × L-glutamin × gelrite showed the highest somatic embryogenesis rate with 9.9% (
For the Hacıibiş cultivar,when the interaction group of “PGR combination × AgNO3 × solidifying substance” is considered (P = 0.001), among the basal media solidified with gelrite, the PGR combination number 3 provided 9.2% embryogenesis rate with AgNO3, and 8.6% without AgNO3, which are the highest rates. The embryogenesis rate of PGR combination number 2 without AgNO3 (7.7%), and the PGR combination number 1 with AgNO3 (7.1%) were found to be statistically significant. Moreover, in the media solidified with agar, the PGR combination numbers 2 and 3 provided significant results with AgNO3 (4.9%) and without AgNO3 (5.8%), respectively. In this context, low rates (0.3% - 3.1%) were obtained for the PGR combination numbers 4, 5, and 6 containing 2,4-D as auxin, and PGR combination number 7 containing NAA, with two solidifiers and AgNO3 dose (
PGR | Organic substance | ||||
---|---|---|---|---|---|
L-glutamine | Casein hydrolyzate | L-glutamine | Casein hydrolyzate | ||
Agar | Gelrite | ||||
1 | BA + KIN + IBA | 3.2 (4.08)** defghi* | 2.3 (2.86) fghi | 2.7 (3.11) fghi | 8.0 (8.42) abc |
2 | BA + TDZ + IBA | 3.0 (4.25) cdefgh | 4.1 (5.06) bcdef | 6.1 (7.09) abcd | 5.0 (5.71) bcdefg |
3 | KIN + TDZ + IBA | 6.0 (7.26) abcde | 3.0 (3.55) efghı | 9.9 (9.85) a | 7.2 (8.61) ab |
4 | BA + KIN + 2,4-D | 0.7 (0.89) hi | 0.3 (0.44) i | 1.4 (1.75) ghi | 1.4 (1.70) hi |
5 | BA + TDZ + 2,4-D | 2.2 (3.02) fghi | 0.7 (0.89) hi | 1.7 (1.50) hi | 2.0 (2.66) fghi |
6 | KIN + TDZ + 2,4-D | 1.0 (1.10) hi | 1.4 (1.77) fghi | 3.3 (3.71) efghi | 0.7 (0.89) hi |
7 | BA + NAA | 2.2 (3.10) fghi | 2.2 (2.87) fi | 1.5 (1.54) hi | 0.7 (0.89) hi |
BA (1 mg/L), KIN (2 mg/L), IBA (0.01 mg/L), TDZ (0.1 mg/L), 2,4-D (0.02 mg/L), NAA (1 mg/L). *Different letters indicate significant differences between the treatments (P = 0.045). **The numbers in parenthesis represents the angle value of percentages.
PGR | AgNO3 | ||||
---|---|---|---|---|---|
0.0 mg/L | 1.7 mg/L | 0.0 mg/L | 1.7 mg/L | ||
Agar | Gelrite | ||||
1 | BA + KIN + IBA | 4.1 (5.45) bcdef* | 1.4 (1.55) fgh | 2.3 (3.10) defgh | 7.1 (8.11) abc |
2 | BA + TDZ + IBA | 3.5 (4.34) cdefg | 4.9 (6.27) abcde | 7.7 (8.56) ab | 3.7 (4.77) bcdefg |
3 | KIN + TDZ + IBA | 5.8 (6.80) abcd | 3.2 (4.11) defgh | 8.6 (9.61) a | 9.2 (10.17) a |
4 | BA + KIN + 2,4-D | 0.3 (0.45) h | 0.7 (0.89) gh | 1.3 (1.77) fgh | 1.3 (1.77) fgh |
5 | BA + TDZ + 2,4-D | 2.1 (2.56) efgh | 1.0 (1.33) gh | 1.9 (1.99) efgh | 2.0 (2.04) fgh |
6 | KIN + TDZ + 2,4-D | 1.3 (1.54) fgh | 1.0 (1.33) gh | 2.0 (2.22) efgh | 2.0 (1.99) efgh |
7 | BA + NAA | 1.2 (1.77) fgh | 3.1 (4.15) defgh | 1.7 (1.98) fgh | 0.3 (0.44) h |
BA (1 mg/L), KIN (2 mg/L), IBA (0.01 mg/L), TDZ (0.1 mg/L), 2,4-D (0.02 mg/L), NAA (1 mg/L) *Different letters indicate significant differences between the treatments (P = 0.001). **The numbers in parenthesis represents the angle value of percentages.
In the Karamehmet cultivar of the chestnut, only the interaction between “basal medium × PGR combination” was found to be significant in terms of the somatic embryogenesis rate and the number of the somatic embryos (P = 0.000) (
Although 7 different PGR combinations were used for the Karamehmet cultivar, any somatic embryogenesis was induced by the WPM basal medium because of its lower nitrogen content with respect to DKW and MS media. No positive result was obtained also with MS medium PGR combination numbers 3, 4, 5, 6, and 7, as well as DKW medium PGR combination number 7.
The impact of gibberellic acid (GA3) and desiccation applications on root, shoot, and plantlet regeneration from somatic embryos was examined in Hacıibiş and Karamehmet early cultivars. The interactions among “cultivar ×
PGR Combinations | Somatic embryogenesis (%) | Somatic embryos (embryos/explants) | ||||
---|---|---|---|---|---|---|
DKW | ||||||
1 | BA + KIN + IBA | 11.1 (12.08)** | a* | 0.46 ± 0.07 | a | |
2 | BA + TDZ + IBA | 9.2 (10.26) | ab | 0.41 ± 0.07 | ab | |
3 | KIN + TDZ + IBA | 8.0 (9.11) | bc | 0.33 ± 0.06 | b | |
4 | BA + KIN + 2,4-D | 3.0 (4.12) | def | 0.14 ± 0.04 | cdefg | |
5 | BA + TDZ + 2,4-D | 7.0 (7.41) | cd | 0.22 ± 0.05 | cd | |
6 | KIN + TDZ + 2,4-D | 4.0 (4.79) | def | 0.16 ± 0.05 | cdef | |
7 | BA + NAA | 2.5 (3.28) | efg | 0.13 ± 0.04 | defgh | |
MS | ||||||
1 | BA + KIN + IBA | 5.0 (5.95) | de | 0.21 ± 0.04 | cde | |
2 | BA + TDZ + IBA | 7.1 (7.56) | cd | 0.24 ± 0.05 | c | |
3 | KIN + TDZ + IBA | 2.2 (2.99) | efg | 0.12 ± 0.04 | defgh | |
4 | BA + KIN + 2,4-D | 1.6 (1.63) | fg | 0.07 ± 0.03 | efgh | |
5 | BA + TDZ + 2,4-D | 0.7 (0.96) | g | 0.04 ± 0.02 | gh | |
6 | KIN + TDZ + 2,4-D | 1.2 (1.55) | fg | 0.05 ± 0.03 | fgh | |
7 | BA + NAA | 3.0 (3.50) | efg | 0.12 ± 0.04 | efgh | |
WPM | ||||||
1 | BA + KIN + IBA | 0.0 (0.00) | g | 0.0 ± 0.0 | h | |
2 | BA + TDZ + IBA | 0.0 (0.00) | g | 0.0 ± 0.0 | h | |
3 | KIN + TDZ + IBA | 1.2 (1.36) | fg | 0.05 ± 0.02 | fgh | |
4 | BA + KIN + 2,4-D | 0.0 (0.00) | g | 0.0 ± 0.0 | h | |
5 | BA + TDZ + 2,4-D | 0.0 (0.00) | g | 0.0 ± 0.0 | h | |
6 | KIN + TDZ + 2,4-D | 0.0 (0.00) | g | 0.0 ± 0.0 | h | |
7 | BA + NAA | 0.0 (0.00) | g | 0.0 ± 0.0 | h |
BA (1 mg/L), KIN (2 mg/L), IBA (0.01 mg/L), TDZ (0.1 mg/L), 2,4-D (0.02 mg/L), NAA (1 mg/L). *Values followed by different letters indicate significant differences between the treatments (P = 0.000). **The numbers in parenthesis represents the angle value of percentages.
desiccation × GA3 dosage”, “cultivar × desiccation”, “cultivar × GA3 dosage”, and “desiccation × GA3 dosage”, and the differences among the cultivar, desiccation, and GA3 dosage were also studied. However, no statistically significant interactions were observed (
The supplementation of BA in the media did not significantly affect the regeneration rates of the somatic embryos of Hacıibiş and Karamehmet early cultivars after the desiccation and cold treatments (cultivar × BA) (P = 0.982). Furthermore, the differences among the cultivars were also not significant (P = 0.787). On the other hand, the differences between the control and the BA, BA + NAA, and BA + IBA treatments were found to be statistically significant (P = 0.000). All PGRs were used at a concentration of 0.1 mg/L. The impacts of the applications on root, shoot, and plantlet regeneration of the cultivars are presented in
Origin | Degree of freedom | P value | ||
---|---|---|---|---|
Root | Shoot | Plant reg. | ||
Cultivar | 1 | 0.728 | 1.00 | 0.681 |
Desiccation | 1 | 0.215 | 0.220 | 1.00 |
GA3 dosages | 5 | 0.645 | 0.681 | 0.681 |
Cultivar × desiccation | 1 | 0.728 | 1.00 | 1.00 |
Cultivar × GA3 dosages | 5 | 0.404 | 0.325 | 0.325 |
Desiccation × GA3 dosages | 5 | 0.645 | 0.681 | 0.681 |
Cultivar x desiccation × GA3 dosages | 5 | 0.404 | 0.325 | 0.325 |
*P < 0.05, **P < 0.01, ***P < 0.001.
GA3 (mg/L) | Root reg. (%) | Shoot reg. (%) | Plant reg. (%) | Root reg. (%) | Shoot reg. (%) | Plant reg. (%) |
---|---|---|---|---|---|---|
Hacıibiş | Karamehmet | |||||
Control | ||||||
0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
3 | 20.0 | 20.0 | 20.0 | 0.0 | 0.0 | 0.0 |
5 | 0.0 | 0.0 | 0.0 | 40.0 | 20.0 | 20.0 |
7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
Desiccation | ||||||
0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
1 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
3 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
5 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
7 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
9 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
The highest rate of the root formation (22.0%) was obtained using media supplemented with either BA + NAA or BA + NAA. There was no response (i.e., 0.0% root regeneration) from all other treatments tested (
In terms of the effects of BA + NAA application in combination with desiccation, cold, or GA3 pre-treatments, on root, shoot, and plantlet regeneration of the somatic embryos of both chestnut cultivars, only pre-application combinations followed by the BA + NAA treatment (average of the cultivar responses) were statistically significant (P = 0.050). No difference was observed between “cultivar × pre-application combinations × BA + NAA” (P = 0.986). Only somatic embryos treated with BA + NAA regenerated into plantlets (
The aim of “earliness” in agriculture is to obtain high yield with quality product in the proper time period. In addition to the classical production techniques, in vitro propagation of the European chestnut (C. sativa Mill), a valuable tree in agriculture and forestry, has an important potential. Development of a standard protocol both in
Application | Root reg. (%) | Shoot reg. (%) | Plant reg. (%) | |||
---|---|---|---|---|---|---|
Control | 0.0 | b* | 0.0 | b | 0.0 | b |
BA | 0.0 | b | 8.0 | b | 0.0 | b |
BA + NAA | 22.0 | a | 24.0 | a | 22.0 | a |
BA + IBA | 0.0 | b | 0.0 | b | 0.0 | b |
BA (0.1 mg/L), IBA (0.1 mg/L), NAA (0.1 mg/L). *Different letters indicate significant differences between treatments.
Application | Root reg. (%) | Shoot reg. (%) | Plant reg. (%) | Plantlet/ Emb.** | Root reg. (%) | Shoot reg. (%) | Plant reg. (%) | Plantlet/ Emb. ** | |
---|---|---|---|---|---|---|---|---|---|
Control BA (0.0 mg/L) + NAA (0.0 mg/L) | BA (0.1 mg/L) + NAA (0.1 mg/L) | ||||||||
Desicc. Cold GA3 | (-) (-) (-) | 0.0 c* | 0.0 c | 0.0 c | 0/45 | 20.0 b | 20.0 b | 20.0 b | 9/45 |
Desicc. Cold GA3 | (+) (-) (-) | 0.0 c | 0.0 c | 0.0 c | 0/45 | 40.0 a | 40.0 a | 40.0 a | 18/45 |
Desicc. Cold GA3 | (+) (+) (-) | 0.0 c | 0.0 c | 0.0 c | 0/45 | 40.0 a | 40.0 a | 40.0 a | 18/45 |
Desicc. Cold GA3 | (+) (-) (+) | 0.0 c | 0.0 c | 0.0 c | 0/45 | 28.8 a | 28.8 a | 28.8 a | 13/45 |
*Different letters indicate significant differences between treatments (P = 0.000). **The number of the plantlet/embryo.
classical and in vitro production, and emphasizing its results with repetitive and diverse trials has an importance in terms of time and economy. The present study was conducted for the optimization of an in vitro propagation protocol for the two early maturing cultivars of European chestnut, Hacıibiş and Karamehmet.
In the literature, varying results have been reported for the somatic embryogenesis and regeneration in chestnut [
The transformation of somatic embryos into plants for chestnut genotypes is a limiting step in all in vitro culture systems [
In this report, 22% - 24% plantlets were obtained via PGR application, which increased to 28.0% - 40.0% when using pre-applications such as desiccation, cold and GA3 (
Desiccation treatment together with BA + NAA supplementation to the medium was shown to be optimum condition for the somatic embryo formation and regeneration of the chestnut early cultivars Hacıibiş and Karamehmet. The in vitro somatic embryogenesis is an important technique for propagation of the early cultivars and development of novel genotypes.
This study was supported by the Ankara University Institute of Biotechnology.
Mehmet Sezgin,Hatice Dumanoğlu, (2016) In Vitro Propagation Potential via Somatic Embryogenesis of the Two Maturing Early Cultivars of European Chestnut (Castanea sativa Mill.). American Journal of Plant Sciences,07,1001-1012. doi: 10.4236/ajps.2016.77095
(A) MACRO ELEMENTS | g/mol | MS Medium | DKW Medium | WPM Medium |
---|---|---|---|---|
mg/L | mg/L | mg/L | ||
Ammonium nitrate (NH4NO3) | 80.04 | 1650 | 1416 | 400 |
Potassium nitrate (KNO3) | 101.11 | 1900 | - | - |
Calcium chloride dihydrate (CaCl2∙2H2O) | 147.02 | 440 | 149 | 96 |
Calcium nitrate tetrahydrate (Ca(NO3)2∙4H2O) | 236.15 | - | 1968 | - |
Zinc nitrate hexahydrate (Zn(NO3)2∙6H2O) | 297.48 | - | 26.68 | - |
Potassium sulfate (K2SO4) | 174.27 | - | 1559 | - |
Potassium dihydrogen phosphate (KH2PO4) | 136.09 | 170 | 265 | - |
Magnesium sulfate heptahydrate (MgSO4∙7H2O) | 246.48 | 370 | 740 | 370 |
(B) FERROUS | ||||
Sodium-ferrous EDTA (C10H12N2FeNa) | 367.1 | 36.7 | - | - |
Sodium EDTA (Na2EDTA) | 372.24 | - | 45.4 | 37.3 |
Ferrous sulfate heptahydrate (FeSO4∙7H2O) | 278.02 | - | 33.8 | 27.8 |
(C) MICRO ELEMENTS | ||||
Manganese sulfate monohydrate (MnSO4∙H2O) | 169.02 | 16 | 33.4 | 22.3 |
Zinc sulfate heptahydrate (ZnSO4∙7H2O) | 287.54 | 8.6 | - | 8.6 |
Nickel sulfate hexahydrate (NiSO4∙6H2O) | 262.90 | - | 0.05 | - |
Boric acid (H3BO3) | 61.83 | 6.2 | 4.8 | 6.2 |
Potassium iodide (KI) | 166.01 | 0.83 | - | - |
Potassium dihydrogen phosphate (KH2PO4) | 136.09 | - | 265.0 | - |
Sodium molybdate dihydrate (Na2MoO4∙2H2O) | 241.95 | 0.25 | 0.39 | 0.25 |
Copper sulfate pentahydrate (CuSO4∙5H2O) | 249.68 | 0.025 | 0.25 | 0.25 |
Cobalt chloride hexahydrate (CoCl2∙6H2O) | 237.9 | 0.025 | - | - |
(D) VITAMINS | ||||
Myo-Inositol (C6H12O6) | 180.16 | 100 | 100 | 100 |
Glycine (C2H5NO2) | 75.07 | 2 | 2 | 2 |
Thiamin chloride hydrochloride (C12H8Cl2N4OsxH2O) | 337.27 | 0.1 | 2 | 1 |
Nicotinic acid (C6H5NO2) | 123.11 | 0.5 | 1 | 0.5 |
Pridoksol hydrochloride (C8H12ClNO3) | 205.64 | 0.5 | - | 0.5 |