Plant Cell Reports
Plant Cell Reports (1991) 9:549-554
9 Springer-Verlag1991
Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro John E. Hachey, Kiran K. Sharma, and Maurice M. Moloney Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4 Received July 24, 1990/Revisedversion received October 5, 1990 Communicated by F. Constabel
SUMMARY A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as explants and a combination of BA and NAA in the regeneration media, up to 70% of explants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL -1 and NAA concentration of lmgL -I. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the explants after the initiation of meristematic activity in the cells about 100~m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium - mediated transformation protocols involving cotyledonary petioles. Key Words 9 Brassica campestris ; cotyledon culture ; histology; organogenesis; regeneration ; tissue culture INTRODUCTION Brassica campestris is cultivated widely both as a vegetable and oilseed crop. In Canada, for example, Brassica campestris comprises 40 - 50% of total Canola production with the remainder contributed by B. napus. Similarly, on the Indian subcontinent B. campestris accounts for a large proportion of vegetable oil production. In addition to its cultivation for edible oil, some cultivars of B. campestris are grown for their
Offprint requests to: M. M_ Moloney
high erucic acid oil. This oil has industrial applications in plastics, lubricants, lacquers and detergents (Lennox,1984). B. campestris has consistently proven to be one of the most recalcitrant members of the Brassicaceae in tissue culture. This is evident from studies on shoot regeneration from callus (Dietert, 1982; Murata and Orton, 1987), leaf discs (Dunwell, 1981) or cotyledons (Jain et al., 1988; Narasimhulu and Chopra, 1988) and from isolated protoplasts (Glimelius, 1984) or anther culture (Keller and Armstrong, 1979). In spite of these problems there is great interest in the genetic manipulation of Brassica campestris for the production of transgenic plants Which could be used for crop improvement, as sources of protoplasts with selectable markers for fusion experiments, and as a diploid model for studies on such phenomena as self incompatibility. We have recently shown that the major limitation in Agrobacterium - mediated transformation of Brassica n a p u s was the shoot regeneration potential of accessible cells within the explant (Moloney et al., 1989). A shoot regeneration system was developed that had high morphogenic potential from cells in the cut surface of an explant. These cells, at the base of cotyledonary petioles, were susceptible to Agrobacterium transformation and yielded large numbers of regenerating transgenic plants, This system was adapted from the regeneration protocol originally developed for Brassicajuncea (Sharma et al., 1990a, b; Sharma and ]3hojwani, 1990). We have attempted to extend the applicability of this system for the regeneration of Brassica campestris . Initial experiments using the same media and conditions as reported in Moloney et al., (1989) produced very few regenerants and thus limited the potential of this system for the production of transgenic plants from this species. We, therefore, investigated modifications of the cotyledonary petiole regeneration system and have optimized this system for regeneration from a number ofBrassica campestris genotypes. Histological studies
550 were carried out to confirm the origin and ontogeny of multiple shoot formation in this system. MATERIALS AND METHODS Plant materials Brassica campestris cultivars used in this study were the Canola types Tobin, Colt, Parkland, Horizon, Echo and a high erucic acid type, R-500 (supplied by Agriculture Canada, Saskatoon, Saskatchewan). Seeds were sterilized in a solution of 20% commercial bleach with 1-2 drops of TWEEN for 10 minutes, followed by 3 rinses in sterile distilled water. The seeds were placed on germination medium comprising Murashige and Skoog (1962) salts and vitamins (MS), 3% sucrose and 0.7% phytagar at a density of 20 seeds per plate and maintained at 24 ~ C in a 16h light/8h dark photoperiod at a light intensity of 60-80REm-2s -1 (GRO-LUX | Sylvania). Shoot regeneration Cotyledons were excised from 4-day old seedlings (or from 4, 5, and 6-day old seedlings in one experiment) so that they included -~2mm of petiole at the base. The petioles were embedded into the regeneration media tested here to a depth of ~-2mm. The culture vessels used were 100x15 mm sterile disposable petri dishes for germination and regeneration and Magenta| jars for rooting. Petri dishes were sealed with 2 layers of Parafilm| Regeneration media comprised MS salts and vitamins, 2% sucrose, and various concentrations of anaphthalene.acetic acid (NAA) and N6-benzyladenine (BA). BA at 2mgL-1 (8.8p2vl) was used in conjunction with NAA at 0, 0.5, 1 and 2mgL-1 (0, 2.7, 5.4, and I0.8pA4 respectively). Also, NAA at lmg-lL was used with 0, 1, 2, and 4mgL-1 BA. Explants (10 per plate) were kept at 24 ~ C in a 16h light/Sh dark photoperiod at a low light intensity (30-40 gEm'2s'l). After 3 weeks, shoots and roots arising from the explants were counted. Percentage regeneration per plate (number of explants regenerating/total number of explants) was averaged for 6-7 plates for each treatment. A score of 'one' was given to an explant even when multiple regenerants were produced. Rooting and acclimatisation was performed on 2 week old explants containing regenerating shoots by placing them on germination media (see above) under low light intensity. Two weeks later the regenerated shoots with accompanying root mass were transferred to potting mix (Peat Moss 9 Vermiculite : Terra-Green| 2:1:1) supplemented with Osmocote| granular fertilizer and placed in a misting chamber (average relative humidity of ~-60%). Histological studies To study the ontogeny of adventitious shoot bud differentiation in culture , petioles from cotyledons at day 0 and those Cultured on shoot regeneration medium (MS + 2% sucrose, 2mg/L BA, lmg/L NAA and 0.7% phytagar) were fixed after 2, 5, 7, and 11 days and
processed for glycol methacrylate (GMA) sectioning. After fixing the material in a freshly prepared, chilled solution of 2% glutaraldehyde and 2% formaldehyde in 0.05M phosphate buffer at pH 6.8, the material was dehydrated in methyl cellosolve followed by two changes of absolute alcohol (O'Brien and McCully, 1981). The specimens were then infiltrated with the LKB infillration solution containing GMA. After the tissue had been infiltrated it was embedded and polymerized in LKB embedding solution. Sections 3gin thick were obtained on a Reichert - Jung rotary microtome using dry glass knives. All sections were stained with periodic acid Schiff's reaction (PAS) for total insoluble polysaccharides and counterstained with amido black 10B for proteins (Yeung, 1984). RESULTS Factors affecting regeneration efficiency Media composition Initial experiments on regeneration from B. campestris cotyledon explants were aimed at reducing the necrosis of explants. This was approximately 90% after 3 weeks using the protocol we previously developed for B. napus regeneration (Moloney et al. ,1989). Upon reducing the light intensity from 60-801xEm-2s"1 to 3040 laEm-2s-1 explant survival increased considerably and callus formation was favoured, but regeneration was still a rare event. Lowering the BA concentration from 4mg]L to 2mg/L increased regeneration to 5%. The most critical factor for enhancement of shoot regeneration was the inclusion of NAA in the medium. With NAA, regeneration frequency increased to an optimum of ~70% at lmgL-1 NAA and 2mgL-1 BA. 80
8n.. 60 O O -r" 03 40
03 Iz ,< ..I o.
20
x
u.I
0
0.5
1
2
C O N C E N T R A T I O N OF N A A (mg/L)
Figure 1. Regeneration of shoots (Ill) and roots (El) from cotyledon explants of B. campestris cv. Tobin on 2mg/L BA and various concentrationsof NAA. Values are average +s.c. Data consists of 5 replicates, each with 10 cotyledons.
551 Figure 1 shows the dose - response relationship of these explants to increasing NAA concentrations. Inthe presence of NAA some root formation was also promoted, but this did not adversely affect the appearance of shoot buds. The amount of BA in the medium was also varied to establish the concentration of this growth regulator required for maximum shoot regeneration (Figure 2). It was found that BA at a concentration of 2mgL-1 was optimum for shoot regeneration. While 4mgL-1 BA was also effective for shoot regeneration, the shoots arising on this medium were of a lesser quality often being more vitreous than those arising from explants on 2mgL-1 BA. It was also difficult to obtain shoots with well defined apical dominance at the higher BA concentration. We decided to determine whether the auxin - cytokinin ratio was the major determinant for shoot regeneration or whether the absolute concentrations of BA and NAA were important. When we reduced the concentrations of NAA and BA while maintaining the same molar ratio (1 : 1.63~NAA : BA) we found a six-fold reduction in regeneration frequency after a five-fold reduction in absolute concentrations of these growth substances. o')
i--
80
o o
n.' 03
i.-
o o -I-
60
09
3:: I-,.
40
u) I-.-
~"
Plant Cell Reports (1991) 9:549-554
9 Springer-Verlag1991
Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro John E. Hachey, Kiran K. Sharma, and Maurice M. Moloney Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4 Received July 24, 1990/Revisedversion received October 5, 1990 Communicated by F. Constabel
SUMMARY A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as explants and a combination of BA and NAA in the regeneration media, up to 70% of explants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL -1 and NAA concentration of lmgL -I. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the explants after the initiation of meristematic activity in the cells about 100~m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium - mediated transformation protocols involving cotyledonary petioles. Key Words 9 Brassica campestris ; cotyledon culture ; histology; organogenesis; regeneration ; tissue culture INTRODUCTION Brassica campestris is cultivated widely both as a vegetable and oilseed crop. In Canada, for example, Brassica campestris comprises 40 - 50% of total Canola production with the remainder contributed by B. napus. Similarly, on the Indian subcontinent B. campestris accounts for a large proportion of vegetable oil production. In addition to its cultivation for edible oil, some cultivars of B. campestris are grown for their
Offprint requests to: M. M_ Moloney
high erucic acid oil. This oil has industrial applications in plastics, lubricants, lacquers and detergents (Lennox,1984). B. campestris has consistently proven to be one of the most recalcitrant members of the Brassicaceae in tissue culture. This is evident from studies on shoot regeneration from callus (Dietert, 1982; Murata and Orton, 1987), leaf discs (Dunwell, 1981) or cotyledons (Jain et al., 1988; Narasimhulu and Chopra, 1988) and from isolated protoplasts (Glimelius, 1984) or anther culture (Keller and Armstrong, 1979). In spite of these problems there is great interest in the genetic manipulation of Brassica campestris for the production of transgenic plants Which could be used for crop improvement, as sources of protoplasts with selectable markers for fusion experiments, and as a diploid model for studies on such phenomena as self incompatibility. We have recently shown that the major limitation in Agrobacterium - mediated transformation of Brassica n a p u s was the shoot regeneration potential of accessible cells within the explant (Moloney et al., 1989). A shoot regeneration system was developed that had high morphogenic potential from cells in the cut surface of an explant. These cells, at the base of cotyledonary petioles, were susceptible to Agrobacterium transformation and yielded large numbers of regenerating transgenic plants, This system was adapted from the regeneration protocol originally developed for Brassicajuncea (Sharma et al., 1990a, b; Sharma and ]3hojwani, 1990). We have attempted to extend the applicability of this system for the regeneration of Brassica campestris . Initial experiments using the same media and conditions as reported in Moloney et al., (1989) produced very few regenerants and thus limited the potential of this system for the production of transgenic plants from this species. We, therefore, investigated modifications of the cotyledonary petiole regeneration system and have optimized this system for regeneration from a number ofBrassica campestris genotypes. Histological studies
550 were carried out to confirm the origin and ontogeny of multiple shoot formation in this system. MATERIALS AND METHODS Plant materials Brassica campestris cultivars used in this study were the Canola types Tobin, Colt, Parkland, Horizon, Echo and a high erucic acid type, R-500 (supplied by Agriculture Canada, Saskatoon, Saskatchewan). Seeds were sterilized in a solution of 20% commercial bleach with 1-2 drops of TWEEN for 10 minutes, followed by 3 rinses in sterile distilled water. The seeds were placed on germination medium comprising Murashige and Skoog (1962) salts and vitamins (MS), 3% sucrose and 0.7% phytagar at a density of 20 seeds per plate and maintained at 24 ~ C in a 16h light/8h dark photoperiod at a light intensity of 60-80REm-2s -1 (GRO-LUX | Sylvania). Shoot regeneration Cotyledons were excised from 4-day old seedlings (or from 4, 5, and 6-day old seedlings in one experiment) so that they included -~2mm of petiole at the base. The petioles were embedded into the regeneration media tested here to a depth of ~-2mm. The culture vessels used were 100x15 mm sterile disposable petri dishes for germination and regeneration and Magenta| jars for rooting. Petri dishes were sealed with 2 layers of Parafilm| Regeneration media comprised MS salts and vitamins, 2% sucrose, and various concentrations of anaphthalene.acetic acid (NAA) and N6-benzyladenine (BA). BA at 2mgL-1 (8.8p2vl) was used in conjunction with NAA at 0, 0.5, 1 and 2mgL-1 (0, 2.7, 5.4, and I0.8pA4 respectively). Also, NAA at lmg-lL was used with 0, 1, 2, and 4mgL-1 BA. Explants (10 per plate) were kept at 24 ~ C in a 16h light/Sh dark photoperiod at a low light intensity (30-40 gEm'2s'l). After 3 weeks, shoots and roots arising from the explants were counted. Percentage regeneration per plate (number of explants regenerating/total number of explants) was averaged for 6-7 plates for each treatment. A score of 'one' was given to an explant even when multiple regenerants were produced. Rooting and acclimatisation was performed on 2 week old explants containing regenerating shoots by placing them on germination media (see above) under low light intensity. Two weeks later the regenerated shoots with accompanying root mass were transferred to potting mix (Peat Moss 9 Vermiculite : Terra-Green| 2:1:1) supplemented with Osmocote| granular fertilizer and placed in a misting chamber (average relative humidity of ~-60%). Histological studies To study the ontogeny of adventitious shoot bud differentiation in culture , petioles from cotyledons at day 0 and those Cultured on shoot regeneration medium (MS + 2% sucrose, 2mg/L BA, lmg/L NAA and 0.7% phytagar) were fixed after 2, 5, 7, and 11 days and
processed for glycol methacrylate (GMA) sectioning. After fixing the material in a freshly prepared, chilled solution of 2% glutaraldehyde and 2% formaldehyde in 0.05M phosphate buffer at pH 6.8, the material was dehydrated in methyl cellosolve followed by two changes of absolute alcohol (O'Brien and McCully, 1981). The specimens were then infiltrated with the LKB infillration solution containing GMA. After the tissue had been infiltrated it was embedded and polymerized in LKB embedding solution. Sections 3gin thick were obtained on a Reichert - Jung rotary microtome using dry glass knives. All sections were stained with periodic acid Schiff's reaction (PAS) for total insoluble polysaccharides and counterstained with amido black 10B for proteins (Yeung, 1984). RESULTS Factors affecting regeneration efficiency Media composition Initial experiments on regeneration from B. campestris cotyledon explants were aimed at reducing the necrosis of explants. This was approximately 90% after 3 weeks using the protocol we previously developed for B. napus regeneration (Moloney et al. ,1989). Upon reducing the light intensity from 60-801xEm-2s"1 to 3040 laEm-2s-1 explant survival increased considerably and callus formation was favoured, but regeneration was still a rare event. Lowering the BA concentration from 4mg]L to 2mg/L increased regeneration to 5%. The most critical factor for enhancement of shoot regeneration was the inclusion of NAA in the medium. With NAA, regeneration frequency increased to an optimum of ~70% at lmgL-1 NAA and 2mgL-1 BA. 80
8n.. 60 O O -r" 03 40
03 Iz ,< ..I o.
20
x
u.I
0
0.5
1
2
C O N C E N T R A T I O N OF N A A (mg/L)
Figure 1. Regeneration of shoots (Ill) and roots (El) from cotyledon explants of B. campestris cv. Tobin on 2mg/L BA and various concentrationsof NAA. Values are average +s.c. Data consists of 5 replicates, each with 10 cotyledons.
551 Figure 1 shows the dose - response relationship of these explants to increasing NAA concentrations. Inthe presence of NAA some root formation was also promoted, but this did not adversely affect the appearance of shoot buds. The amount of BA in the medium was also varied to establish the concentration of this growth regulator required for maximum shoot regeneration (Figure 2). It was found that BA at a concentration of 2mgL-1 was optimum for shoot regeneration. While 4mgL-1 BA was also effective for shoot regeneration, the shoots arising on this medium were of a lesser quality often being more vitreous than those arising from explants on 2mgL-1 BA. It was also difficult to obtain shoots with well defined apical dominance at the higher BA concentration. We decided to determine whether the auxin - cytokinin ratio was the major determinant for shoot regeneration or whether the absolute concentrations of BA and NAA were important. When we reduced the concentrations of NAA and BA while maintaining the same molar ratio (1 : 1.63~NAA : BA) we found a six-fold reduction in regeneration frequency after a five-fold reduction in absolute concentrations of these growth substances. o')
i--
80
o o
n.' 03
i.-
o o -I-
60
09
3:: I-,.
40
u) I-.-
~"
