Plant Cell Reports
Plant Cell Reports (I 985) 4:267-268
© Springer-Verlag 1985
The effect of anther orientation on the production of microspore-derived embryoids and plants of Hordeum vulgare cv. Sabarlis C. P. Hunter Shell Research Ltd., Sittingbourne Research Centre, Sittingbourne, Kent, ME9 8AG, UK Received August 13, 1985 - Communicated by J. Potrykus
Abstract The orientation of barley anthers on culture medium had a marked effect on their response. Embryoids developed only in the upper lobes of anthers cultured on edge, i.e. with a single lobe in contact with the culture medium. Anthers cultured flat, i.e. with both lobes in contact with the medium, did not respond. Careful orientation of anthers at the start of incubation resulted in a mean yield of 20 green plants per i00 anthers cultured. Introduction Techniques for the regeneration of plants from microspores of barley via anther culture are well established in the literature (for review see Dunwell, in press). However, current methods are very inefficient when compared with the alternative bulbosum method of producing doubled haploids (Huang et al. 1984). The efficiency of anther culture needs to be improved substantially before the potential of the technique as a breeding tool can be properly evaluated.
and/or embryoids were counted (= number of responding anthers). The cultures were then transferred to continuous light. Any callus or embryoid larger than 2nml, that was observed during the next 4 weeks was transferred to regeneration medium (Foroughi-Wehr et al. 1976) solidified with 0.5% Sea Plaque agarose. The numbers of green and albino plants that developed from these were recorded. Results and Discussion Embryoid development was only observed in the upper lobes of anthers placed on edge, i.e. with a single lobe in contact with the culture medium (Figure i). No development was seen in the lower lobe. When anthers were lying flat, i.e. with both lobes in contact with the medium, the microspores were generally shed onto the medium and did not develop further (Figure 2).
A research programme on direct embryogenesis from barley anther culture has been undertaken in this laboratory (Lyne et al, in press). During the course of this work it was observed that orientation of the anthers on the culture medium affected their response. The results of experiments designed to quantify this effect are reported here. Materials
and Methods
Seed of Barley (Hordeum vulgare L.) variety Sabarlis, was kindly donated by Dr. N.Sunderland, John Innes Institute, Norwich, U.K. Plants were grown in controlled environment cabinets (12°C; 350~ E.mf 2 s ~ at top of pot; 16h day; 60-80% RH). Spikes containing anthers at the mid-uninucleate stage were selected and pretreated for 28 days at 4°C as described by Huang and Sunderland (1982). At the end of the pretreatment microspore viability was assessed by acetocarmine staining and spikes were discarded if the viability was less than 40%. Anthers were cultured in 5 cm Petri dishes containing i0 ml of the callus induction medium of Foroughi-Wehr et al. (1976) solidified with 0.5% Sea Plaque agarose. Cultures were incubated in the dark at 25°C. After 28 days, the anthers bearing callus
Figure
i.
Embryoid development only observed in the upper lobes of anthers placed on edge, i.e. with a single lobe in contact with the culture medium.
268 Tab le i.
Effect of orientation on anther response and plant regeneration. Experiment On edge
Flat
On edge
Flat
Number of anthers
99
252
553
230
Anthers responding
60
1
480
O
Plants regenerated green
19
O
160
O
23
0
408
O
32
0
33
38
O
85
Plants regenerated per iOO anthers responding green - albino Anthers lying flat, i.e. with both lobes in contact with the medium generally shed microspores onto the medium. These did not develop further.
Table 1 shows results from two experiments carried out to examine the relationship between anther orientation and plant regeneration. In the first experiment anther orientation was random, while in the second experiment anthers were placed on edge. As some anthers placed on edge fell into the flat position, orientation was checked after 28 days incubation. The proportions of anthers remaining on edge were 28% and 71% respectively. The data show clearly that little or no response was obtained from anthers lying flat on the medium. However, a high proportion of anthers lying on edge responded, producing green and albino plants. Mean yields of 5 and 20 green plants per iOO anthers cultured were obtained in experiments 1 and 2 respectively. Most of this difference is attributable to the higher proportion of anthers remaining on edge, and therefore capable of response, in the second experiment. Anthers that did respond produced plants at a similar frequency in both experiments. This emphasises the importance of maximising the numbers of anthers responding when seeking to improve the overall yields from anther culture. Effects of anther orientation on response in culture have been reported previously for Datura (Sopory and Maheshwari, 1976) and Nicotiana (Misoo et al. 1981). Anthers of Datura responded better cultured flat than on edge, in contrast to our results with barley. The experiments with tobacco indicated that anthers responded best when cultured vertically, with one end partially embedded in the medium. Thus it appears that orientation is an important factor in determining the efficiency of plant regeneration from cultured anthers, although the preferred orientation varies with the species.
Experiment 2.
Orientation after 28 days culture
- albino
Figure 2.
i.
Experiment
I.
Anthers from 5 spikes, random orientation.
Experiment
2.
Anthers from 12 spikes, on edge orientation.
Acknowledgements This paper will form part of a thesis to be submitted for a postgraduate degree to the University of London, School of Agriculture, Wye College, Kent. I am indebted to Dr. Rob L.Lyne for his guidance and encouragement throughout the duration of this work and Drs. Sinclair H.Mantell and Robin S.Nelson for their advice and stimulating discussions. I also thank Mr. C.Nicholson for his expert photography. Finally, I express my gratitude to Shell Research Limited for sponsorship of this work. References Dunwell JM (In press). In: Alderson PG, Withers LA (eds) Plant Tissue Culture and its Agricultural Application, Butterworths, London. Foroughi-Wehr B, Mix G, Gaul H, Wilson HM (1976). Z.Pflanzenzuchtg 77: 198-204. Huang B, Sunderland N (1982).
Annal.Bot.
49: 77-88.
Huang B, Dunwell JM, Powell W, Hayter AM, Wood W (1984). Z.Pflanzenz~chtg 92: 22-29. Lyne RL, Bennett R, Hunter CP (In press). In: Alderson PG, Withers LA (eds) Plant Tissue Culture and its Agricultural Application, Butterworths, London. Misoo S, Yokota F, Matsubayashi M (1981). Rep. Soc. Crop Sci. Breed., Kinki 26: 44-48. Sopory SK, Maheshwari 27 : 49-57.
SC (1976).
J. Expt.
Bot.
Plant Cell Reports (I 985) 4:267-268
© Springer-Verlag 1985
The effect of anther orientation on the production of microspore-derived embryoids and plants of Hordeum vulgare cv. Sabarlis C. P. Hunter Shell Research Ltd., Sittingbourne Research Centre, Sittingbourne, Kent, ME9 8AG, UK Received August 13, 1985 - Communicated by J. Potrykus
Abstract The orientation of barley anthers on culture medium had a marked effect on their response. Embryoids developed only in the upper lobes of anthers cultured on edge, i.e. with a single lobe in contact with the culture medium. Anthers cultured flat, i.e. with both lobes in contact with the medium, did not respond. Careful orientation of anthers at the start of incubation resulted in a mean yield of 20 green plants per i00 anthers cultured. Introduction Techniques for the regeneration of plants from microspores of barley via anther culture are well established in the literature (for review see Dunwell, in press). However, current methods are very inefficient when compared with the alternative bulbosum method of producing doubled haploids (Huang et al. 1984). The efficiency of anther culture needs to be improved substantially before the potential of the technique as a breeding tool can be properly evaluated.
and/or embryoids were counted (= number of responding anthers). The cultures were then transferred to continuous light. Any callus or embryoid larger than 2nml, that was observed during the next 4 weeks was transferred to regeneration medium (Foroughi-Wehr et al. 1976) solidified with 0.5% Sea Plaque agarose. The numbers of green and albino plants that developed from these were recorded. Results and Discussion Embryoid development was only observed in the upper lobes of anthers placed on edge, i.e. with a single lobe in contact with the culture medium (Figure i). No development was seen in the lower lobe. When anthers were lying flat, i.e. with both lobes in contact with the medium, the microspores were generally shed onto the medium and did not develop further (Figure 2).
A research programme on direct embryogenesis from barley anther culture has been undertaken in this laboratory (Lyne et al, in press). During the course of this work it was observed that orientation of the anthers on the culture medium affected their response. The results of experiments designed to quantify this effect are reported here. Materials
and Methods
Seed of Barley (Hordeum vulgare L.) variety Sabarlis, was kindly donated by Dr. N.Sunderland, John Innes Institute, Norwich, U.K. Plants were grown in controlled environment cabinets (12°C; 350~ E.mf 2 s ~ at top of pot; 16h day; 60-80% RH). Spikes containing anthers at the mid-uninucleate stage were selected and pretreated for 28 days at 4°C as described by Huang and Sunderland (1982). At the end of the pretreatment microspore viability was assessed by acetocarmine staining and spikes were discarded if the viability was less than 40%. Anthers were cultured in 5 cm Petri dishes containing i0 ml of the callus induction medium of Foroughi-Wehr et al. (1976) solidified with 0.5% Sea Plaque agarose. Cultures were incubated in the dark at 25°C. After 28 days, the anthers bearing callus
Figure
i.
Embryoid development only observed in the upper lobes of anthers placed on edge, i.e. with a single lobe in contact with the culture medium.
268 Tab le i.
Effect of orientation on anther response and plant regeneration. Experiment On edge
Flat
On edge
Flat
Number of anthers
99
252
553
230
Anthers responding
60
1
480
O
Plants regenerated green
19
O
160
O
23
0
408
O
32
0
33
38
O
85
Plants regenerated per iOO anthers responding green - albino Anthers lying flat, i.e. with both lobes in contact with the medium generally shed microspores onto the medium. These did not develop further.
Table 1 shows results from two experiments carried out to examine the relationship between anther orientation and plant regeneration. In the first experiment anther orientation was random, while in the second experiment anthers were placed on edge. As some anthers placed on edge fell into the flat position, orientation was checked after 28 days incubation. The proportions of anthers remaining on edge were 28% and 71% respectively. The data show clearly that little or no response was obtained from anthers lying flat on the medium. However, a high proportion of anthers lying on edge responded, producing green and albino plants. Mean yields of 5 and 20 green plants per iOO anthers cultured were obtained in experiments 1 and 2 respectively. Most of this difference is attributable to the higher proportion of anthers remaining on edge, and therefore capable of response, in the second experiment. Anthers that did respond produced plants at a similar frequency in both experiments. This emphasises the importance of maximising the numbers of anthers responding when seeking to improve the overall yields from anther culture. Effects of anther orientation on response in culture have been reported previously for Datura (Sopory and Maheshwari, 1976) and Nicotiana (Misoo et al. 1981). Anthers of Datura responded better cultured flat than on edge, in contrast to our results with barley. The experiments with tobacco indicated that anthers responded best when cultured vertically, with one end partially embedded in the medium. Thus it appears that orientation is an important factor in determining the efficiency of plant regeneration from cultured anthers, although the preferred orientation varies with the species.
Experiment 2.
Orientation after 28 days culture
- albino
Figure 2.
i.
Experiment
I.
Anthers from 5 spikes, random orientation.
Experiment
2.
Anthers from 12 spikes, on edge orientation.
Acknowledgements This paper will form part of a thesis to be submitted for a postgraduate degree to the University of London, School of Agriculture, Wye College, Kent. I am indebted to Dr. Rob L.Lyne for his guidance and encouragement throughout the duration of this work and Drs. Sinclair H.Mantell and Robin S.Nelson for their advice and stimulating discussions. I also thank Mr. C.Nicholson for his expert photography. Finally, I express my gratitude to Shell Research Limited for sponsorship of this work. References Dunwell JM (In press). In: Alderson PG, Withers LA (eds) Plant Tissue Culture and its Agricultural Application, Butterworths, London. Foroughi-Wehr B, Mix G, Gaul H, Wilson HM (1976). Z.Pflanzenzuchtg 77: 198-204. Huang B, Sunderland N (1982).
Annal.Bot.
49: 77-88.
Huang B, Dunwell JM, Powell W, Hayter AM, Wood W (1984). Z.Pflanzenz~chtg 92: 22-29. Lyne RL, Bennett R, Hunter CP (In press). In: Alderson PG, Withers LA (eds) Plant Tissue Culture and its Agricultural Application, Butterworths, London. Misoo S, Yokota F, Matsubayashi M (1981). Rep. Soc. Crop Sci. Breed., Kinki 26: 44-48. Sopory SK, Maheshwari 27 : 49-57.
SC (1976).
J. Expt.
Bot.
