J Plant Biotechnol 2017; 44(3): 343-348
Published online September 30, 2017
https://doi.org/10.5010/JPB.2017.44.3.343
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: philiprobin81@gmail.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords Protocorm, Orchids, KC, 1/2 MS, VW media, NAA, BAP, IAA and IBA
Orchids are outstanding in many ways, like diverse shapes, colors and its medicinal values. They are marketed both as plants and as cut flowers and their production has been increased in recent years due to demand in the floriculture field (Tokuhara and Mii 1993, 2001; Chang and Chang 2000).
Hard and intelligent decisions are needed to save the valuable taxa from the vastly changing landscape. As part of an
Purpose of this article is to report our success in aseptic seed germination of
The mature undehisced capsules of
Initially it was washed with mild detergent and then soaked in 15% (v/v) NaOCl augmented with Tween-80 for 15 min. Capsules were again washed with fungicide (Bavistin) for 10 min. and subsequently rinsed five times with sterile distilled water. The surface sterilized capsules were longitudinally dissected and around 100±10 seeds were transferred into a 200 ml sterile glass jar containing 50ml of 0.8% (w/v) agar-solidified KC, 1/2 MS and VW basal media. Effect of Coconut water (10% v/v) and Peptone (0.2% w/v) were studied in modified KC, 1/2 MS and VW media in which both were supplemented separately. Exact number of seeds was counted under a microscope after the successful transfer.
Plant cultures were maintained at 25°C±2°C for 12 h photoperiod provided by White fluorescent lights, M/s:Philipsof 3000 lux intensity. Percentage of germination was calculated using the following formula:
X = Number of seeds inoculated
Y = Number of seeds germinated
The KC was used for proliferation of the protocorm based on the obtained result (Table 1). In KC media 2% of sucrose served as carbon source and additionally two different plant growth regulators (PGRs) namely BAP and NAA were supplemented to each type of medium in different combinations as mentioned in the Table 2. The pH of the media was adjusted to 5.3, followed by an autoclave at 121°Cfor 15 min under 15 lbs pressure. These cultures were maintained under a 16 h- photoperiod at 25±2°CThe experiment was performed in triplicate and average number of shoots per protocorm was determined after four weeks.
Table 1 Effect of different media on the germination and protocorm development in
Media employed for seed germination | % of germination | Time taken for initiation of germination (days) | Shoot growth (%) |
---|---|---|---|
1/2 MS – B | 50 | 85 | 10 |
1/2 MS – CW | 30 | 80 | - |
1/2 MS – P | 20 | 90 | - |
KC – CW | 80 | 70 | 50 |
KC – P | 70 | 75 | 40 |
VW – B | - | - | - |
VW – CW | - | - | - |
VW – P | - | - | - |
B-Basal medium, CW-Coconut Water (10% v/v), P- Peptone (0.2%, w/v)
Table 2 Effect on BAP and NAA on the development of protocorm into microshoots and regeneration on KC medium
Hormone concentration (mg/l) | Mean number of shoots (M±SD) | Mean length (cm) of shoots (M±SD) | Mean number of roots (M±SD) | |
---|---|---|---|---|
BAP | NAA | |||
0.5 | - | 1.3±1.34 lm | 0.72±0.21 kl | 0 |
1.0 | - | 2.0±1.34 i | 0.54±0.37 mn | 0 |
1.5 | - | 1.4±1.01 k | 0.57±0.39 m | 1.3±0.64 de |
2.0 | - | 1.9±1.3 ij | 0.80±0.30 h | 1.4±0.48 cd |
0.5 | 0.5 | 2.7±1.41 gh | 0.73±0.34 ij | 0 |
1.0 | 0.5 | 2.8±1.46 fg | 1.32±0.14 de | 0 |
1.5 | 0.5 | 4.8±1.46 b | 1.46±0.28 c | 1.6±0.19 c |
2.0 | 0.5 | 3.2±2.00 ef | 1.06±0.55 g | 1.4±0.80 cd |
0.5 | 1.0 | 4.5±1.24 bc | 1.70±0.15 ab | 2.5±1.02 b |
1.5 | 1.0 | 3.8±0.97 d | 1.21±0.31 f | 1.6±0.66 c |
2.0 | 1.0 | 3.4±1.42 de | 1.40±0.26 cd | 0 |
For the induction of root, the regenerated multiple shoots of
Table 3 Effect of IAA and IBA on root induction in KC medium after 25th day of inoculation
Medium+ Hormone (mg/l) | Concentration (mg/l) | Mean root number (M±SD) | Mean root (cm) length (M±SD) |
---|---|---|---|
IAA | 0.5 | 1.7±0.90 cd | 0.3 ±0.35 hi |
1.0 | 2.5±0.80 c | 0.58±0.45 ef | |
1.5 | 0.60±0.38 cd | ||
2.0 | 1.4±0.91 de | ||
2.5 | 1.2±0.60 ef | 0.41±0.33 gh | |
IBA | 0.5 | 2.0±0.77 c | 0.51±0.31 fg |
1.0 | |||
1.5 | 1.4±0.8 de | 0.68±0.21 e | |
2.0 | 0.6±0.66 g | 0.26±0.16 jk | |
2.5 | 0.3±0.64 gh | 0.16±0.16 bc |
Well-developed rooted shoots were taken out from the culture vessels and washed under running tap water then the plantlets were washed with fungicide solution. Individual regenerates were placed onto net pots containing equal proportion of charcoal (0.5 ~ 1.0 cm each), brick-gravels and coconut husk as supporting materials. The plantlets were sprayed with balanced fertilizers (1 g/l 17N-17P-17k) twice weekly. The transplanted plants were finally kept in the shade house for further acclimatization.
Data were recorded on the basis of different parameters subjected to analysis of variance (ANOVA) and mean values of treatments were compared by least significant difference (LSD).
Values are Mean ±SD (n = 10) of two independent experiments. Mean values followed by the same letter in a column are not significantly different as indicated by Duncan’s multiple range test (P = 0.05). Values within a column having the same alphabet are not statistically significant and sharing at least one letter are no significantly different at p < 0.05 level according to Duncan’s multiple range test.
The first sign of germination on the 30thday on KC basal medium was that the embryo turned into spherical form and was enclosed in dark yellow color. After a few days some adventive tissues appeared on their tips and the embryo ruptured at one of the poles and after a few days it showed 4-5fold increases in size with abundant chloroplasts and starch cells. The embryos by the 50thday exhibited a prominent zone of pro-meristematic cells and developed a pair of leaf primordial (
Protocorm like bodies formation on KC- b medium
Regeneration of plantlets and aerial root formation on ½ MS medium supplemented with 1.0 mg/l of BAP and 1.0 mg/l of NAA
Different stages of the prtocorm like bodies (PLbs)
The seedlings before processed for hardening
Potted plant of
The well-developed protocorm like bodies (Plbs) were inoculated with KC media augmented with BAP (1.0 mg/l) and NAA (1.0 mg/l) which yields High frequency of plant regeneration (Table 2). One of the primordial developed more rapidly than the other to produce an unequal pair of first embryogenic photosynthetic leaves. Simultaneously with the development of embryonic leaves at the proximal end of the embryo the marginal cells at the distal end of the embryo started giving rise to tubular and unicellular rhizoids.
The seeds are thin and transparent. The cells of seed coats are varied in size and shape. Such variations that were observed in the present study are in agreement with the observations noted by the previous workers (Clifford & Smith 1969; Ekanthappa, 1981). It has been earlier established that the nature of seed coat is of great taxonomic value within the Orchidaceae (Stoutamire, 1963, Kalimuthu
Thus the early concept that the orchid seeds are sterile and or at least incapable of germination is no more valid (Arditti, 1967). That the germination of seeds of epiphytic orchids posed no problem has been pointed out by various workers (Stoutamire, 1964a; Stoutamire 1964b; Warcap 1971; McIntyre et al., 1972; McIntyre et al., 1974). However, they have indicated that difficulty was encountered in the germination of terrestrial orchid seeds in contrast to those of epiphytic taxa.
The well-developed shoots were removed from the culture tubes and transferred to the rooting media. About 90% of the shoots rooted well after transfer to the KC medium supplemented with 0.2 mg/l IBA.
In the present investigation the developed plantlets were transferred into KC medium supplemented with IAA and IBA separately which was more essential for regeneration of roots. The similar results were obtained in
The present investigation was mainly aimed at understanding the mode of seed germination and organogenesis of the developing seedling. Although different workers have suggested several nutrient media for orchids, three well-known media were tried in the present study to prove into the germination response of seeds selected species. While Knudson C medium promoted the germination of
Authors are grateful to Dr. P. Ponmurugan, Professor and Head, Department of Biotechnology, K. S. R College of Technology, Tiruchengode for his encouragement and Dr. K. S. Rangasamy, Chairman, K. S. R. Group of Institutions for his support.
J Plant Biotechnol 2017; 44(3): 343-348
Published online September 30, 2017 https://doi.org/10.5010/JPB.2017.44.3.343
Copyright © The Korean Society of Plant Biotechnology.
Philip Robinson J.
Department of Biotechnology, K. S. Rangasamy College of Technology, Tiruchengode 637 215, Tamil Nadu, India,
Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli -620020, Tamil Nadu, India
Correspondence to: e-mail: philiprobin81@gmail.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Protocorm, Orchids, KC, 1/2 MS, VW media, NAA, BAP, IAA and IBA
Orchids are outstanding in many ways, like diverse shapes, colors and its medicinal values. They are marketed both as plants and as cut flowers and their production has been increased in recent years due to demand in the floriculture field (Tokuhara and Mii 1993, 2001; Chang and Chang 2000).
Hard and intelligent decisions are needed to save the valuable taxa from the vastly changing landscape. As part of an
Purpose of this article is to report our success in aseptic seed germination of
The mature undehisced capsules of
Initially it was washed with mild detergent and then soaked in 15% (v/v) NaOCl augmented with Tween-80 for 15 min. Capsules were again washed with fungicide (Bavistin) for 10 min. and subsequently rinsed five times with sterile distilled water. The surface sterilized capsules were longitudinally dissected and around 100±10 seeds were transferred into a 200 ml sterile glass jar containing 50ml of 0.8% (w/v) agar-solidified KC, 1/2 MS and VW basal media. Effect of Coconut water (10% v/v) and Peptone (0.2% w/v) were studied in modified KC, 1/2 MS and VW media in which both were supplemented separately. Exact number of seeds was counted under a microscope after the successful transfer.
Plant cultures were maintained at 25°C±2°C for 12 h photoperiod provided by White fluorescent lights, M/s:Philipsof 3000 lux intensity. Percentage of germination was calculated using the following formula:
X = Number of seeds inoculated
Y = Number of seeds germinated
The KC was used for proliferation of the protocorm based on the obtained result (Table 1). In KC media 2% of sucrose served as carbon source and additionally two different plant growth regulators (PGRs) namely BAP and NAA were supplemented to each type of medium in different combinations as mentioned in the Table 2. The pH of the media was adjusted to 5.3, followed by an autoclave at 121°Cfor 15 min under 15 lbs pressure. These cultures were maintained under a 16 h- photoperiod at 25±2°CThe experiment was performed in triplicate and average number of shoots per protocorm was determined after four weeks.
Table 1 . Effect of different media on the germination and protocorm development in
Media employed for seed germination | % of germination | Time taken for initiation of germination (days) | Shoot growth (%) |
---|---|---|---|
1/2 MS – B | 50 | 85 | 10 |
1/2 MS – CW | 30 | 80 | - |
1/2 MS – P | 20 | 90 | - |
KC – CW | 80 | 70 | 50 |
KC – P | 70 | 75 | 40 |
VW – B | - | - | - |
VW – CW | - | - | - |
VW – P | - | - | - |
B-Basal medium, CW-Coconut Water (10% v/v), P- Peptone (0.2%, w/v).
Table 2 . Effect on BAP and NAA on the development of protocorm into microshoots and regeneration on KC medium.
Hormone concentration (mg/l) | Mean number of shoots (M±SD) | Mean length (cm) of shoots (M±SD) | Mean number of roots (M±SD) | |
---|---|---|---|---|
BAP | NAA | |||
0.5 | - | 1.3±1.34 lm | 0.72±0.21 kl | 0 |
1.0 | - | 2.0±1.34 i | 0.54±0.37 mn | 0 |
1.5 | - | 1.4±1.01 k | 0.57±0.39 m | 1.3±0.64 de |
2.0 | - | 1.9±1.3 ij | 0.80±0.30 h | 1.4±0.48 cd |
0.5 | 0.5 | 2.7±1.41 gh | 0.73±0.34 ij | 0 |
1.0 | 0.5 | 2.8±1.46 fg | 1.32±0.14 de | 0 |
1.5 | 0.5 | 4.8±1.46 b | 1.46±0.28 c | 1.6±0.19 c |
2.0 | 0.5 | 3.2±2.00 ef | 1.06±0.55 g | 1.4±0.80 cd |
0.5 | 1.0 | 4.5±1.24 bc | 1.70±0.15 ab | 2.5±1.02 b |
1.5 | 1.0 | 3.8±0.97 d | 1.21±0.31 f | 1.6±0.66 c |
2.0 | 1.0 | 3.4±1.42 de | 1.40±0.26 cd | 0 |
For the induction of root, the regenerated multiple shoots of
Table 3 . Effect of IAA and IBA on root induction in KC medium after 25th day of inoculation.
Medium+ Hormone (mg/l) | Concentration (mg/l) | Mean root number (M±SD) | Mean root (cm) length (M±SD) |
---|---|---|---|
IAA | 0.5 | 1.7±0.90 cd | 0.3 ±0.35 hi |
1.0 | 2.5±0.80 c | 0.58±0.45 ef | |
1.5 | 0.60±0.38 cd | ||
2.0 | 1.4±0.91 de | ||
2.5 | 1.2±0.60 ef | 0.41±0.33 gh | |
IBA | 0.5 | 2.0±0.77 c | 0.51±0.31 fg |
1.0 | |||
1.5 | 1.4±0.8 de | 0.68±0.21 e | |
2.0 | 0.6±0.66 g | 0.26±0.16 jk | |
2.5 | 0.3±0.64 gh | 0.16±0.16 bc |
Well-developed rooted shoots were taken out from the culture vessels and washed under running tap water then the plantlets were washed with fungicide solution. Individual regenerates were placed onto net pots containing equal proportion of charcoal (0.5 ~ 1.0 cm each), brick-gravels and coconut husk as supporting materials. The plantlets were sprayed with balanced fertilizers (1 g/l 17N-17P-17k) twice weekly. The transplanted plants were finally kept in the shade house for further acclimatization.
Data were recorded on the basis of different parameters subjected to analysis of variance (ANOVA) and mean values of treatments were compared by least significant difference (LSD).
Values are Mean ±SD (n = 10) of two independent experiments. Mean values followed by the same letter in a column are not significantly different as indicated by Duncan’s multiple range test (P = 0.05). Values within a column having the same alphabet are not statistically significant and sharing at least one letter are no significantly different at p < 0.05 level according to Duncan’s multiple range test.
The first sign of germination on the 30thday on KC basal medium was that the embryo turned into spherical form and was enclosed in dark yellow color. After a few days some adventive tissues appeared on their tips and the embryo ruptured at one of the poles and after a few days it showed 4-5fold increases in size with abundant chloroplasts and starch cells. The embryos by the 50thday exhibited a prominent zone of pro-meristematic cells and developed a pair of leaf primordial (
Protocorm like bodies formation on KC- b medium
Regeneration of plantlets and aerial root formation on ½ MS medium supplemented with 1.0 mg/l of BAP and 1.0 mg/l of NAA
Different stages of the prtocorm like bodies (PLbs)
The seedlings before processed for hardening
Potted plant of
The well-developed protocorm like bodies (Plbs) were inoculated with KC media augmented with BAP (1.0 mg/l) and NAA (1.0 mg/l) which yields High frequency of plant regeneration (Table 2). One of the primordial developed more rapidly than the other to produce an unequal pair of first embryogenic photosynthetic leaves. Simultaneously with the development of embryonic leaves at the proximal end of the embryo the marginal cells at the distal end of the embryo started giving rise to tubular and unicellular rhizoids.
The seeds are thin and transparent. The cells of seed coats are varied in size and shape. Such variations that were observed in the present study are in agreement with the observations noted by the previous workers (Clifford & Smith 1969; Ekanthappa, 1981). It has been earlier established that the nature of seed coat is of great taxonomic value within the Orchidaceae (Stoutamire, 1963, Kalimuthu
Thus the early concept that the orchid seeds are sterile and or at least incapable of germination is no more valid (Arditti, 1967). That the germination of seeds of epiphytic orchids posed no problem has been pointed out by various workers (Stoutamire, 1964a; Stoutamire 1964b; Warcap 1971; McIntyre et al., 1972; McIntyre et al., 1974). However, they have indicated that difficulty was encountered in the germination of terrestrial orchid seeds in contrast to those of epiphytic taxa.
The well-developed shoots were removed from the culture tubes and transferred to the rooting media. About 90% of the shoots rooted well after transfer to the KC medium supplemented with 0.2 mg/l IBA.
In the present investigation the developed plantlets were transferred into KC medium supplemented with IAA and IBA separately which was more essential for regeneration of roots. The similar results were obtained in
The present investigation was mainly aimed at understanding the mode of seed germination and organogenesis of the developing seedling. Although different workers have suggested several nutrient media for orchids, three well-known media were tried in the present study to prove into the germination response of seeds selected species. While Knudson C medium promoted the germination of
Authors are grateful to Dr. P. Ponmurugan, Professor and Head, Department of Biotechnology, K. S. R College of Technology, Tiruchengode for his encouragement and Dr. K. S. Rangasamy, Chairman, K. S. R. Group of Institutions for his support.
Protocorm like bodies formation on KC- b medium
Regeneration of plantlets and aerial root formation on ½ MS medium supplemented with 1.0 mg/l of BAP and 1.0 mg/l of NAA
Different stages of the prtocorm like bodies (PLbs)
The seedlings before processed for hardening
Potted plant of
Table 1 . Effect of different media on the germination and protocorm development in
Media employed for seed germination | % of germination | Time taken for initiation of germination (days) | Shoot growth (%) |
---|---|---|---|
1/2 MS – B | 50 | 85 | 10 |
1/2 MS – CW | 30 | 80 | - |
1/2 MS – P | 20 | 90 | - |
KC – CW | 80 | 70 | 50 |
KC – P | 70 | 75 | 40 |
VW – B | - | - | - |
VW – CW | - | - | - |
VW – P | - | - | - |
B-Basal medium, CW-Coconut Water (10% v/v), P- Peptone (0.2%, w/v).
Table 2 . Effect on BAP and NAA on the development of protocorm into microshoots and regeneration on KC medium.
Hormone concentration (mg/l) | Mean number of shoots (M±SD) | Mean length (cm) of shoots (M±SD) | Mean number of roots (M±SD) | |
---|---|---|---|---|
BAP | NAA | |||
0.5 | - | 1.3±1.34 lm | 0.72±0.21 kl | 0 |
1.0 | - | 2.0±1.34 i | 0.54±0.37 mn | 0 |
1.5 | - | 1.4±1.01 k | 0.57±0.39 m | 1.3±0.64 de |
2.0 | - | 1.9±1.3 ij | 0.80±0.30 h | 1.4±0.48 cd |
0.5 | 0.5 | 2.7±1.41 gh | 0.73±0.34 ij | 0 |
1.0 | 0.5 | 2.8±1.46 fg | 1.32±0.14 de | 0 |
1.5 | 0.5 | 4.8±1.46 b | 1.46±0.28 c | 1.6±0.19 c |
2.0 | 0.5 | 3.2±2.00 ef | 1.06±0.55 g | 1.4±0.80 cd |
0.5 | 1.0 | 4.5±1.24 bc | 1.70±0.15 ab | 2.5±1.02 b |
1.5 | 1.0 | 3.8±0.97 d | 1.21±0.31 f | 1.6±0.66 c |
2.0 | 1.0 | 3.4±1.42 de | 1.40±0.26 cd | 0 |
Table 3 . Effect of IAA and IBA on root induction in KC medium after 25th day of inoculation.
Medium+ Hormone (mg/l) | Concentration (mg/l) | Mean root number (M±SD) | Mean root (cm) length (M±SD) |
---|---|---|---|
IAA | 0.5 | 1.7±0.90 cd | 0.3 ±0.35 hi |
1.0 | 2.5±0.80 c | 0.58±0.45 ef | |
1.5 | 0.60±0.38 cd | ||
2.0 | 1.4±0.91 de | ||
2.5 | 1.2±0.60 ef | 0.41±0.33 gh | |
IBA | 0.5 | 2.0±0.77 c | 0.51±0.31 fg |
1.0 | |||
1.5 | 1.4±0.8 de | 0.68±0.21 e | |
2.0 | 0.6±0.66 g | 0.26±0.16 jk | |
2.5 | 0.3±0.64 gh | 0.16±0.16 bc |
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Journal of
Plant BiotechnologyProtocorm like bodies formation on KC- b medium
|@|~(^,^)~|@|Regeneration of plantlets and aerial root formation on ½ MS medium supplemented with 1.0 mg/l of BAP and 1.0 mg/l of NAA
|@|~(^,^)~|@|Different stages of the prtocorm like bodies (PLbs)
|@|~(^,^)~|@|The seedlings before processed for hardening
|@|~(^,^)~|@|Potted plant of