Journal of Plant Biotechnology 2015; 42(3): 223-227
Published online September 30, 2015
https://doi.org/10.5010/JPB.2015.42.3.223
© The Korean Society of Plant Biotechnology
Correspondence to : Y. W. Kim e-mail: bravekim@korea.kr
Embryogenic callus (EC) was created from mature embryos of
Keywords Embryogenic callus, Japanese larch, Somatic embryos, Zygotic mature embryos
The genus
The mature embryos of
After the mature embryos were extracted from the female megagametophytes, they were bisected transversally, and placed on EC medium. The ovuliferous scales were discarded, and the mature zygotic embryos were cultured on three EC initiation media such as modified LP (Quoirin and Lepoivre 1977), LM (Litvay et al. 1985), and SH (Schenk and Hildebrandt 1972). Each medium was composed of full-strength salts and vitamines supplemented with 1,000 mg/L casein hydrolysate, 3.4 mM glutamine, 29.2 mM sucrose, and 0.2% (v/v) gelrite (Sigma) as a gelling agent. L-Glutamine was sterilized with a disposable filter unit (0.2 μm pore size) and then added to cooled medium after autoclaving. The medium was supplemented with one of the three types of auxins; 2,4-D, Picloram or pCPA, at the concentrations ranging from 1.0 to 5.0 mg/L, and one cytokinin (BA) first at the concentrations of 1.0 mg/L (see Table 1 and 2). Two different culture methods were employed, the first was culturing the mature embryos in the 25
Table 1 Effect of media and plant growth regulators on embryogenic tissue initiation grown in the light from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiation (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloramb | pCPAc | BAP | ||
LMe | 1.0 | 1.0 | 16.0±1.9cdefgd | ||
2.0 | 1.0 | 28.0±3.7abcde | |||
5.0 | 1.0 | 12.5±3.1efg | |||
1.0 | 1.0 | 20.9±3.3bcdefg | |||
2.0 | 1.0 | 28.0±3.8abcde | |||
5.0 | 1.0 | 12.0±2.2fg | |||
1.0 | 1.0 | 18.0±3.9bcdefg | |||
2.0 | 1.0 | 20.0±3.9bcdefg | |||
5.0 | 1.0 | 8.0±1.0g | |||
LPf | 1.0 | 1.0 | 10.1±0.1fg | ||
2.0 | 1.0 | 17.0±1.7bcdefg | |||
5.0 | 1.0 | 8.5±1.9g | |||
1.0 | 1.0 | 24.7±2.3bcdef | |||
2.0 | 1.0 | 24.4±4.3bcdef | |||
5.0 | 1.0 | 15.4±2.1defg | |||
1.0 | 1.0 | 13.5±2.8efg | |||
2.0 | 1.0 | 12.7±3.6efg | |||
5.0 | 1.0 | 17.2±3.0bcdefg | |||
SHg | 1.0 | 1.0 | 32.5±2.7ab | ||
2.0 | 1.0 | 16.0±8.0cdefg | |||
5.0 | 1.0 | 17.8±2.9bcdefg | |||
1.0 | 1.0 | 24.8±3.8bcdef | |||
2.0 | 1.0 | 31.4±2.9abc | |||
5.0 | 1.0 | 20.6±2.6bcdefg | |||
1.0 | 1.0 | 29.6±10.4abcd | |||
2.0 | 1.0 | 41.7±13.0a | |||
5.0 | 1.0 | 11.3±4.5fg |
aThe mature embryos were cultured in the light (25μEm-2s-1) for one week followed by darkness for 7 weeks.
b4-amino-3,5,6-trichloropicolinic acid
cpara-chlorophenoxyacetic acid
dMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 3.32
Table 2 Effect of media and plant growth regulators on embryogenic tissue initiation grown in the dark from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiationb (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloram | pCPA | BAP | ||
LM | 1.0 | 1.0 | 51.2±8.1abcdef | ||
2.0 | 1.0 | 42.0±2.0bcdefgh | |||
5.0 | 1.0 | 30.0±5.8ghijklm | |||
1.0 | 1.0 | 56.4±4.9abcd | |||
2.0 | 1.0 | 56.0±9.5abcd | |||
5.0 | 1.0 | 18.8±2.4ijklm | |||
1.0 | 1.0 | 62.8±5.4a | |||
2.0 | 1.0 | 41.2±7.1bcdefgh | |||
5.0 | 1.0 | 54.0±5.7abcde | |||
LP | 1.0 | 1.0 | 60.0±2.9ab | ||
2.0 | 1.0 | 37.8±4.5cdefghi | |||
5.0 | 1.0 | 14.0±0.7lm | |||
1.0 | 1.0 | 62.8±10.2a | |||
2.0 | 1.0 | 32.1±5.0fghijkl | |||
5.0 | 1.0 | 16.0±4.6klm | |||
1.0 | 1.0 | 14.0±3.3lm | |||
2.0 | 1.0 | 16.0±4.6klm | |||
5.0 | 1.0 | 10.4±3.2m | |||
SH | 1.0 | 1.0 | 44.7±7.2abcdefgh | ||
2.0 | 1.0 | 35.4±4.1efghijk | |||
5.0 | 1.0 | 28.0±8.5hijklm | |||
1.0 | 1.0 | 55.0±9.8abcdefg | |||
2.0 | 1.0 | 4.7±2.2abcdefgh | |||
5.0 | 1.0 | 17.4±2.0jklm | |||
1.0 | 1.0 | 57.1±9.2abc | |||
2.0 | 1.0 | 42.0±9.9bcdefgh | |||
5.0 | 1.0 | 36.2±1.9defghij |
aThe mature embryos were cultured in the dark throughout the 8 week culture.
bMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 7.71.
The effect of various concentrations (14.6, 29.2, 58.4, 87.6, 116.8, 146 mM) of sucrose on EC initiation was tested. Totally 60 embryos were cultured on each of the 6 different sucrose concentrations with three replicates. Ten mature embryos were placed on each petri-dish (87×15 mm) with medium. The cultures were kept in darkness at 25±1°C for 8 weeks. The frequency of EC initiation was recorded after the initial 8 weeks of culture.
For the investigation of somatic embryogenesis by scanning electron microscope, several pieces of embryogenic or non-embryogenic callus were collected after 8 weeks in culture. No dehydration or fixation treatment was done on the sample. Observations and photographs were made on a ABT-55 scanning electron microscope (Toshiba, Japan).
Analyses of variance were performed for frequency of embryogenic tissue initiation and somatic embryo maturation. Statistically significant mean differences were determined with Duncan’s mutiple range test.
When the mature embryos were placed on EC initiation medium within 1 to 2 days in culture, the explants developed red color in the hypocotyl and radicle parts (Fig. 1a). After the 5 days, the embryos became gradually swollen, and started EC initiation from the hypocotyle zone (Fig. 1b). At this time, the induced EC showed the typical features that appeared only in EC such as white to translucent, and mucilaginous texture (Fig. 1c) as has been reported (Liao and Amerson 1995). When examined by SEM, the EC appeared to have long filamentous shape with embryonal head as well as suspensor (Fig. 2b). On the other hand, non-embryogenic callus (NEC) with isodiametric small cells could also be observed (Fig. 2a). After 7-9 days in culture, the early-staged somatic embryos having both putative embryonal head and suspensor were induced from the cultured EC. After 2 weeks, the distinctive proembryos having both embryonal head and suspensor could also be observed (Fig. 1d). The effect of the three basal media on the EC initiation from mature embryos was compared. Regardless of light conditions, the best results were obtained from LM medium (45.8%), followed by SH medium (39.5%), and LP medium (29.2%) (Table 1 and 2). Since the LM salt formulation appeared to be the most satisfactory for EC initiation, it was selected thereafter as a basal induction medium in the immature zygotic embryos. The LM medium has been reported to be effective in other conifer species (von Aderkas and Bonga 1987; Beardmore and Charest 1995). Keeping the culture in the dark condition throughout the experiment (38.2%) seemed to give better results than exposing them to 16 h light (25
The embryogenic callus (EC) initiation in
Scanning electron micrographs of non-embryogenic or EC callus in
As for the effective sucrose concentration on initiation of EC, 29.2 mM sucrose (38.6%) gave the best results (Fig. 3). Studying with the megagametophytes of
Effect of sucrose concentrations on the initiation of EC from mature zygotic embryos in
The low number of viable embryos of
Journal of Plant Biotechnology 2015; 42(3): 223-227
Published online September 30, 2015 https://doi.org/10.5010/JPB.2015.42.3.223
Copyright © The Korean Society of Plant Biotechnology.
Yong-Wook Kim
1Department of Forest Genetics, Korea Forest Research Institute, Suwon, 441-847, Korea
Correspondence to: Y. W. Kim e-mail: bravekim@korea.kr
Embryogenic callus (EC) was created from mature embryos of
Keywords: Embryogenic callus, Japanese larch, Somatic embryos, Zygotic mature embryos
The genus
The mature embryos of
After the mature embryos were extracted from the female megagametophytes, they were bisected transversally, and placed on EC medium. The ovuliferous scales were discarded, and the mature zygotic embryos were cultured on three EC initiation media such as modified LP (Quoirin and Lepoivre 1977), LM (Litvay et al. 1985), and SH (Schenk and Hildebrandt 1972). Each medium was composed of full-strength salts and vitamines supplemented with 1,000 mg/L casein hydrolysate, 3.4 mM glutamine, 29.2 mM sucrose, and 0.2% (v/v) gelrite (Sigma) as a gelling agent. L-Glutamine was sterilized with a disposable filter unit (0.2 μm pore size) and then added to cooled medium after autoclaving. The medium was supplemented with one of the three types of auxins; 2,4-D, Picloram or pCPA, at the concentrations ranging from 1.0 to 5.0 mg/L, and one cytokinin (BA) first at the concentrations of 1.0 mg/L (see Table 1 and 2). Two different culture methods were employed, the first was culturing the mature embryos in the 25
Table 1 . Effect of media and plant growth regulators on embryogenic tissue initiation grown in the light from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiation (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloramb | pCPAc | BAP | ||
LMe | 1.0 | 1.0 | 16.0±1.9cdefgd | ||
2.0 | 1.0 | 28.0±3.7abcde | |||
5.0 | 1.0 | 12.5±3.1efg | |||
1.0 | 1.0 | 20.9±3.3bcdefg | |||
2.0 | 1.0 | 28.0±3.8abcde | |||
5.0 | 1.0 | 12.0±2.2fg | |||
1.0 | 1.0 | 18.0±3.9bcdefg | |||
2.0 | 1.0 | 20.0±3.9bcdefg | |||
5.0 | 1.0 | 8.0±1.0g | |||
LPf | 1.0 | 1.0 | 10.1±0.1fg | ||
2.0 | 1.0 | 17.0±1.7bcdefg | |||
5.0 | 1.0 | 8.5±1.9g | |||
1.0 | 1.0 | 24.7±2.3bcdef | |||
2.0 | 1.0 | 24.4±4.3bcdef | |||
5.0 | 1.0 | 15.4±2.1defg | |||
1.0 | 1.0 | 13.5±2.8efg | |||
2.0 | 1.0 | 12.7±3.6efg | |||
5.0 | 1.0 | 17.2±3.0bcdefg | |||
SHg | 1.0 | 1.0 | 32.5±2.7ab | ||
2.0 | 1.0 | 16.0±8.0cdefg | |||
5.0 | 1.0 | 17.8±2.9bcdefg | |||
1.0 | 1.0 | 24.8±3.8bcdef | |||
2.0 | 1.0 | 31.4±2.9abc | |||
5.0 | 1.0 | 20.6±2.6bcdefg | |||
1.0 | 1.0 | 29.6±10.4abcd | |||
2.0 | 1.0 | 41.7±13.0a | |||
5.0 | 1.0 | 11.3±4.5fg |
aThe mature embryos were cultured in the light (25μEm-2s-1) for one week followed by darkness for 7 weeks.
b4-amino-3,5,6-trichloropicolinic acid
cpara-chlorophenoxyacetic acid
dMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 3.32
Table 2 . Effect of media and plant growth regulators on embryogenic tissue initiation grown in the dark from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiationb (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloram | pCPA | BAP | ||
LM | 1.0 | 1.0 | 51.2±8.1abcdef | ||
2.0 | 1.0 | 42.0±2.0bcdefgh | |||
5.0 | 1.0 | 30.0±5.8ghijklm | |||
1.0 | 1.0 | 56.4±4.9abcd | |||
2.0 | 1.0 | 56.0±9.5abcd | |||
5.0 | 1.0 | 18.8±2.4ijklm | |||
1.0 | 1.0 | 62.8±5.4a | |||
2.0 | 1.0 | 41.2±7.1bcdefgh | |||
5.0 | 1.0 | 54.0±5.7abcde | |||
LP | 1.0 | 1.0 | 60.0±2.9ab | ||
2.0 | 1.0 | 37.8±4.5cdefghi | |||
5.0 | 1.0 | 14.0±0.7lm | |||
1.0 | 1.0 | 62.8±10.2a | |||
2.0 | 1.0 | 32.1±5.0fghijkl | |||
5.0 | 1.0 | 16.0±4.6klm | |||
1.0 | 1.0 | 14.0±3.3lm | |||
2.0 | 1.0 | 16.0±4.6klm | |||
5.0 | 1.0 | 10.4±3.2m | |||
SH | 1.0 | 1.0 | 44.7±7.2abcdefgh | ||
2.0 | 1.0 | 35.4±4.1efghijk | |||
5.0 | 1.0 | 28.0±8.5hijklm | |||
1.0 | 1.0 | 55.0±9.8abcdefg | |||
2.0 | 1.0 | 4.7±2.2abcdefgh | |||
5.0 | 1.0 | 17.4±2.0jklm | |||
1.0 | 1.0 | 57.1±9.2abc | |||
2.0 | 1.0 | 42.0±9.9bcdefgh | |||
5.0 | 1.0 | 36.2±1.9defghij |
aThe mature embryos were cultured in the dark throughout the 8 week culture.
bMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 7.71.
The effect of various concentrations (14.6, 29.2, 58.4, 87.6, 116.8, 146 mM) of sucrose on EC initiation was tested. Totally 60 embryos were cultured on each of the 6 different sucrose concentrations with three replicates. Ten mature embryos were placed on each petri-dish (87×15 mm) with medium. The cultures were kept in darkness at 25±1°C for 8 weeks. The frequency of EC initiation was recorded after the initial 8 weeks of culture.
For the investigation of somatic embryogenesis by scanning electron microscope, several pieces of embryogenic or non-embryogenic callus were collected after 8 weeks in culture. No dehydration or fixation treatment was done on the sample. Observations and photographs were made on a ABT-55 scanning electron microscope (Toshiba, Japan).
Analyses of variance were performed for frequency of embryogenic tissue initiation and somatic embryo maturation. Statistically significant mean differences were determined with Duncan’s mutiple range test.
When the mature embryos were placed on EC initiation medium within 1 to 2 days in culture, the explants developed red color in the hypocotyl and radicle parts (Fig. 1a). After the 5 days, the embryos became gradually swollen, and started EC initiation from the hypocotyle zone (Fig. 1b). At this time, the induced EC showed the typical features that appeared only in EC such as white to translucent, and mucilaginous texture (Fig. 1c) as has been reported (Liao and Amerson 1995). When examined by SEM, the EC appeared to have long filamentous shape with embryonal head as well as suspensor (Fig. 2b). On the other hand, non-embryogenic callus (NEC) with isodiametric small cells could also be observed (Fig. 2a). After 7-9 days in culture, the early-staged somatic embryos having both putative embryonal head and suspensor were induced from the cultured EC. After 2 weeks, the distinctive proembryos having both embryonal head and suspensor could also be observed (Fig. 1d). The effect of the three basal media on the EC initiation from mature embryos was compared. Regardless of light conditions, the best results were obtained from LM medium (45.8%), followed by SH medium (39.5%), and LP medium (29.2%) (Table 1 and 2). Since the LM salt formulation appeared to be the most satisfactory for EC initiation, it was selected thereafter as a basal induction medium in the immature zygotic embryos. The LM medium has been reported to be effective in other conifer species (von Aderkas and Bonga 1987; Beardmore and Charest 1995). Keeping the culture in the dark condition throughout the experiment (38.2%) seemed to give better results than exposing them to 16 h light (25
The embryogenic callus (EC) initiation in
Scanning electron micrographs of non-embryogenic or EC callus in
As for the effective sucrose concentration on initiation of EC, 29.2 mM sucrose (38.6%) gave the best results (Fig. 3). Studying with the megagametophytes of
Effect of sucrose concentrations on the initiation of EC from mature zygotic embryos in
The low number of viable embryos of
The embryogenic callus (EC) initiation in
Scanning electron micrographs of non-embryogenic or EC callus in
Effect of sucrose concentrations on the initiation of EC from mature zygotic embryos in
Table 1 . Effect of media and plant growth regulators on embryogenic tissue initiation grown in the light from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiation (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloramb | pCPAc | BAP | ||
LMe | 1.0 | 1.0 | 16.0±1.9cdefgd | ||
2.0 | 1.0 | 28.0±3.7abcde | |||
5.0 | 1.0 | 12.5±3.1efg | |||
1.0 | 1.0 | 20.9±3.3bcdefg | |||
2.0 | 1.0 | 28.0±3.8abcde | |||
5.0 | 1.0 | 12.0±2.2fg | |||
1.0 | 1.0 | 18.0±3.9bcdefg | |||
2.0 | 1.0 | 20.0±3.9bcdefg | |||
5.0 | 1.0 | 8.0±1.0g | |||
LPf | 1.0 | 1.0 | 10.1±0.1fg | ||
2.0 | 1.0 | 17.0±1.7bcdefg | |||
5.0 | 1.0 | 8.5±1.9g | |||
1.0 | 1.0 | 24.7±2.3bcdef | |||
2.0 | 1.0 | 24.4±4.3bcdef | |||
5.0 | 1.0 | 15.4±2.1defg | |||
1.0 | 1.0 | 13.5±2.8efg | |||
2.0 | 1.0 | 12.7±3.6efg | |||
5.0 | 1.0 | 17.2±3.0bcdefg | |||
SHg | 1.0 | 1.0 | 32.5±2.7ab | ||
2.0 | 1.0 | 16.0±8.0cdefg | |||
5.0 | 1.0 | 17.8±2.9bcdefg | |||
1.0 | 1.0 | 24.8±3.8bcdef | |||
2.0 | 1.0 | 31.4±2.9abc | |||
5.0 | 1.0 | 20.6±2.6bcdefg | |||
1.0 | 1.0 | 29.6±10.4abcd | |||
2.0 | 1.0 | 41.7±13.0a | |||
5.0 | 1.0 | 11.3±4.5fg |
aThe mature embryos were cultured in the light (25μEm-2s-1) for one week followed by darkness for 7 weeks.
b4-amino-3,5,6-trichloropicolinic acid
cpara-chlorophenoxyacetic acid
dMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 3.32
Table 2 . Effect of media and plant growth regulators on embryogenic tissue initiation grown in the dark from mature zygotic embryos of
Medium | Plant growth regulators (mg/L) | ET initiationb (%±S.E.) | |||
---|---|---|---|---|---|
2,4-D | Picloram | pCPA | BAP | ||
LM | 1.0 | 1.0 | 51.2±8.1abcdef | ||
2.0 | 1.0 | 42.0±2.0bcdefgh | |||
5.0 | 1.0 | 30.0±5.8ghijklm | |||
1.0 | 1.0 | 56.4±4.9abcd | |||
2.0 | 1.0 | 56.0±9.5abcd | |||
5.0 | 1.0 | 18.8±2.4ijklm | |||
1.0 | 1.0 | 62.8±5.4a | |||
2.0 | 1.0 | 41.2±7.1bcdefgh | |||
5.0 | 1.0 | 54.0±5.7abcde | |||
LP | 1.0 | 1.0 | 60.0±2.9ab | ||
2.0 | 1.0 | 37.8±4.5cdefghi | |||
5.0 | 1.0 | 14.0±0.7lm | |||
1.0 | 1.0 | 62.8±10.2a | |||
2.0 | 1.0 | 32.1±5.0fghijkl | |||
5.0 | 1.0 | 16.0±4.6klm | |||
1.0 | 1.0 | 14.0±3.3lm | |||
2.0 | 1.0 | 16.0±4.6klm | |||
5.0 | 1.0 | 10.4±3.2m | |||
SH | 1.0 | 1.0 | 44.7±7.2abcdefgh | ||
2.0 | 1.0 | 35.4±4.1efghijk | |||
5.0 | 1.0 | 28.0±8.5hijklm | |||
1.0 | 1.0 | 55.0±9.8abcdefg | |||
2.0 | 1.0 | 4.7±2.2abcdefgh | |||
5.0 | 1.0 | 17.4±2.0jklm | |||
1.0 | 1.0 | 57.1±9.2abc | |||
2.0 | 1.0 | 42.0±9.9bcdefgh | |||
5.0 | 1.0 | 36.2±1.9defghij |
aThe mature embryos were cultured in the dark throughout the 8 week culture.
bMeans with the same letter are not significantly different as determined by analysis of variance with Duncan’s mutiple range test. Pr>F: 0.0001, F value: 7.71.
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Plant BiotechnologyThe embryogenic callus (EC) initiation in
Scanning electron micrographs of non-embryogenic or EC callus in
Effect of sucrose concentrations on the initiation of EC from mature zygotic embryos in