Plant Cell Reports
Plant Cell Reports (1995) 14:393 397
9 Springer-Verlag1995
Efficient callus formation and plant regeneration from leaves of oats (Arena sativa L.) Hancai Chen, Guanju Xu, David C. Loschke, Luba Tomaska, and Barry G. Rolfe Plant Microbe Interactions Group, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, ACT 0200 Australia Received 1 July 1994/Revised version received 11 August 1994 - Communicated by I.K. Vasil
Abstract. An efficient plant regeneration system from leaf-derived callus in 6 genotypes of Arena sativa L. has been established. Regenerable callus was induced in the basal 1-2 m m region of 2 to 5 day old seedlings. Plants were regenerated from the regenerable callus and grown to maturity. The frequency of regenerable callus formation and plant regeneration was correlated with the position, developmental stage and genotype of the explant. The regeneration capacity of the first one mm of the leaf basal region from three day old seedlings was comparable to that of immature embryos. Furthermore, the leaf regenerable calli were subcultured for 8 months without loss of their regeneration capabilities.
Introduction Recent advances in plant tissue culture and transformation technologies have made it possible to genetically modify the most recalcitrant of crops such as cereals (Vasil 1994). The biolistic procedure has emerged as a simple and promising alternative for cereal transformation. The advantage of this method is that it can directly transfer DNA into intact plant cells without the constraints of protoplast culture and A g r o b a c t e r i u m host-specificity (Christou 1992). Recently, transgenic oat plants have been obtained following microprojectile bombardment with DNA-coated tungsten particles of either oat suspension culture cells or immature-embryo-derived calli ( S o m e r s e t al. 1992). However, establishment of regenerable suspension cultures of oats is difficult and genotype dependent (Bregitzer et al. 1991; Somerset al. 1992). Immature embryos are also not a convenient source of material for transformation studies because they require mature plants for their production, require specialized growth environments and may be restricted to a short season. Therefore, it is still of importance to develop a reliable alternative regeneration system in oats. Callus cultures of oats can be initiated from seeds or mature embryos, or from the roots of germinating Correspondence to: H. Chen
seedlings and hypocotyls or mesocotyls of germinating mature embryos. However, the regeneration capacities of these callus cultures are much lower (Carter et al. 1967; Cummings et al. 1976; Cure and Mott 1978; Heyser and Nabors 1982; Lorz ct al. 1976) than that of the callus derived from immature embryos (Cummings et al. 1976; Rines and McCoy 1981). Therefore, these oat callus cultures, although convenient to isolate, may be less attractive to be used as target tissue in transformation experiments. Regenerable callus formation and plant regeneration from leaf tissues has been reported in a number of cereals, but not yet for oats (Becher et al. 1992; Wernicke and Brettell 1980; Wernicke and Milkovits 1984; Zamora and Scott 1983). The ready availability of oat seedlings makes them a suitable source of explants for tissue culture if a high regeneration frequency can be achieved. We describe a high frequency regeneration system from leaf tissues of various cultivars of oats which also may have general application for oat transformation via the biolistic method.
Materials and Methods MS medium containing MS salts (Murashige and Skoog 1962) with 150 mg/1 asparagine, 0.5 rag/1 thiamine-HCl, 20 g/1 sucrose, 100 mg/l m-inositol, 7 mg/l glycine, 2.0 mg/1 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma) and 0.7% Bacto-agar (Difco), pH 5.8 was used for routine callus induction, growth and maintainance. N6 medium containing N6 salts (Chu et al. 1975) with 146 rag/1 glutamine, 30 g/1 sucrose, 100 mg/1 m-inositol, 200 rag/1 casein hydrolysate, 2 mg/1 kinetin, 2 mg/l 1naphthaleneacetic acid (NAA) and 0.7% agar, pH 5.8 was used for shoot regeneration. MS medium containing 0.3 mg/1 kinetin and 0.7% agar, plt 5.8 was used for root regeneration. Tissue culture media.
Donor plants. Seeds of 6 different Avena sativa L. cultivars
were used. Four (Coolabah, Cooba, Blackbutt, and Mortlock) are Australian cultivars (Cleanseeds Pty Ltd, Bungendore, NSW, Australia) and two (Victorgrain and HVR) are North American Cultivars (Professor H. H. Lukel USDA Department of Plant Pathology, University of Florida, Florida, USA). The de-husked seeds were surface sterilised in 75% ethanol for
394 Mean first leaf length 10
I st leaf
30
60
(ram) 80
N
\
5-
2ud leaf i
Apical -mefistem~
2 days
3 days
4 days
5 days
Plant regeneration. Calli, five weeks or older, were transferred to a shoot regeneration medium and were incubated at 25~ with low intensity light (10 btE m -2 sec - l ) for 16 h day in a controlled growth cabinet. Three to four days later, the light intensity was increased to 190 g E m -2 sec -1. The callus cultures were subcultured on the shoot regeneration medium at two week intervals. During a period of one to three weeks, shoots about 3 mm long were dissected from calli and grown on the same medium in 100 x 20 mm Petri dishes. Shoots about 15 mm long were transferred to a tissue culture jar (70 x 75 mm) containing 30 ml root regeneration medium. The plantlets were transferred to fresh root regeneration medium in the tissue culture jars at two week intervals. Whole plants with a shoot length of between 7 and 10 cm and with well developed roots were transplanted to soil in pots and grown in a greenhouse (22~ for 10 h day and 15~ for 14 h night). A plant nutrient solution containing 0.18% Thrive (Arthur Yates & Co. Pty. Ltd. Australia) was applied once a week.
Fig. 1. Diagram illustrating leaf growth of oat seedlings for the first five days. The coleoptile-free leaves from each seedling were cut into 1 mm transverse section, starting from the leaf base. The first segment consists of the leaf base and the apical meristem. The subsequent segments contain either the first leaf tissue or the first and second leaf tissue.
2 rain, followed by two washes with sterile distilled water, and then in a 5% sodium hypochlorite solution for 10 min followed by five washes with sterile distilled water. The sterilised seeds were germinated on solid MS medium in tissue culture jars (70 x 75 ram) at 27~ in the dark for one day. The germinated seeds were grown in a 25~ incubator in the dark or in a growth cabinet at 25~ with 16 h day illumination (light intensity, 190 g E m -2 sec-1). Immature embryos were dissected from immature seeds approximately 10-12 days after pollination and sterilised using the same procedure as the mature seed sterilisation. Embryos were 1 to 3 mm long at this stage.
Explant preparation. Immature embryos were immediately excised from the sterilized de-husked seed and transferred onto solid callus-inducing medium with the embryo axis oriented down. Leaf base explants were prepared from 2 to 5 day old seedlings grown in the dark or with illumination. Leaves of the seedlings were very young without a complete leaf structure during this growth period. The length of the first leaf was between 10 mm and 80 ram, and the second leaf was up to 4 mm long during this growth period (Fig. 1). The leaves were separated from coleoptiles and sequentially cut into 1 mm transverse sections, starting from the original leaf base (Fig. 1) using a scalpel in a clear sterile Petri dish with a sheet of millimetre paper underneath to allow accurate sizing during dissection. The first segment refers to the basal segment consisting of the leaf base and the shoot meristem.
Callus initiation and subculture. The explants from the same position were placed onto a Petri dish (90 x 12 ram) containing 20 ml MS2D medium for callus induction. The explants were incubated at 25~ in the dark. After 14 days, callus was dissected from the leaf tissue and transferred to fresh MS2D medium. Callus cultures were subcultured on MS2D medium at 25~ in the dark every two to three weeks.
Results L e a f segments isolated from seedlings grown for 2 to 5 days in the dark were subjected to an initial screening for callus formation. Callus was induced from cultured leaf base segments on MS m e d i u m containing 2 rag/1 2,4-D. No organised callus tissue was formed in the absence o f 2,4-D. L e a f segments Of seedlings grown in the dark or in the light had a similar response for callus induction. The frequency of callus formation in leaf explants was strongly dependent on the position in the seedling and the age o f the seedling (Table 1). First, callus was induced only from the basal s e g m e n t s o f leaves. S e c o n d l y , the frequency of callus induction of the first segment from the leaf base was higher than that o f the subsequent segments from the leaf base. Thirdly, the basal region o f leaves with callus-forming ability in the younger seedlings was broader than that in the older seedlings. The highest response (100%) was observed in the one m m leaf basal region (the first segments) of 2-3 day old seedlings (Table 1). Callus tissue first became visible on the leaf explants within a week on callus medium. The initial callus tissue formed was soft and translucent. S o m e c o m p a c t and opaque callus d e v e l o p e d within soft callus masses one week later. The c o m p a c t callus tissues were increased c o n s i d e r a b l y f o l l o w i n g two w e e k s i n c u b a t i o n o f the cultures on callus medium (Fig. 2a). F o r 4 to 5 week old calli, o r g a n i z e d somatic e m b r y o - l i k e structures were observed on the compact callus (Fig. 2b) but not on the soft, translucent callus. The four to five week old calli were transferred to a shooting m e d i u m . Green spots appeared on the compact callus tissue 2 to 3 days later. In contrast, the soft, translucent callus tissues were unable to form green spots. Shoots were formed on the green spotforming calli 4 to 5 days later (Fig. 2c). Callus without green spots did not regenerate shoots. Shoots about 3 m m long were excised from the callus and transferred to fresh shooting medium. Seven days later, when most of
395 Table 1. Frequency of callus formation and plant regeneration from leaf segments of 2 to 5 day old seedlings and immature embryos of Avena sativa L. (cv. Coolabah) No. of Plantlets regeneratedb Source of explanta
No. of calli formed (%)
Immature embryo
No. of regenerable calli (%)
total
averagec
46
(92)
44
(87)
281
6.4
50 30 3 0
(100) (60) (6)
23 5 0
(46) (10)
74 15
3.2 3.0
50 26 2 0
(100) (52) (4)
43 9 0
(86) (18)
292 28
6.8 3.1
43 5 0
(86) (10)
28 0
(56)
64
2.3
45 3 0
(90) (6)
24 0
(48)
31
1.3
Two day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment 4th leaf segment
Three day seedling lst leaf segment 2nd leaf segment 3rd leaf segment 4th leaf segment
Four day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment
Five day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment
aFifty explants were tested in each treatment. bThe number of plantlets was counted for up to 6 weeks after the transfer of the five week old callus to the shooting medium. CAverage number of plantlets regenerated from each regenerable callus.
Table 2. Frequency of callus formation and plant regeneration from the first leaf segment of 3 day old seedlings in five genotypes of Avena sativa L. No. of plantlets regenerateda Genotype
Cooba Blackbutt Mortlock Victorgrain HVR
No. of explants tested
No. of calli formed (%)
50 50 50 50 50
50 (100) 46 (92) 50 (100) 47 (94) 45 (90)
No. of regenerable calli (%) 42 40 36 31 26
(84) (87) (72) (66) (58)
total 143 132 97 87 39
averageb
-
3.4 3.3 2.7 2.8 1.5
aThe number of plantlets was counted for up to 6 weeks after the transfer of the five week old Callus to the shooting medium. bAverage number of plantlets regenerated per each regenerable callus.
396 Immature embryos of cultivar Coolabah were also used for callus induction and plant regeneration, using the same procedure. The callus induced from the immature embryos (data not shown) was similar to the leaf callus (Fig. 2). The frequency of callus initiation, regenerable callus formation and plant regeneration of the immature embryos was similar to that of the first leaf segment of 3 day old seedlings (Table 1). The first leaf segment of 3 day old seedlings of the other five oat cultivars (Cooba, Blackbutt, Mortlock, Victorgrain and HVR) were also used for callus induction and plant regeneration (Table 2). Depending upon the cultivar investigated, the frequency of callus induction was between 80% and 100%. About 48% to 86% of the leaf basal explants were able to form regenerable callus tissues, and an average of 1.5 to 3.4 plants were regenerated from each regenerable callus (Table 2). The regenerable callus from the leaf explants was subcultured at three week intervals, and has maintained its capacity to form plants for more than eight months without any significant loss of regeneration potential.
Discussion
Fig.2. Callus induction and plant regeneration from leaf segments of Avena sativa L. a Three week old callus consisting of opaque and compact callus, and soft and translucent callus obtained from leaf segment, b somatic embryo-like structure (arrow) on compact callus, c Shoot (arrow) regenerated from compact callus, d, e Whole and mature plants obtained from leaf callus.
the shoots had grown 15 to 20 mm long, they were transferred to a rooting medium. Roots developed within 5 days. The plants with shoots between 7 to 10 cm long and well developed roots (Fig. 2d) were transplanted to pots containing soil. Whole plants were able to grow in soil to normal mature plants with the abilities of tillering, flowering and grain formation (Fig. 2e). Total time taken from culture initiation to the transplanting of regenerated plants was approximately 10 weeks. The callus induced from different leaf explants exhibited quite different regeneration capacity (Table 1). The callus from the first leaf segment of 2 to 5 day old seedlings had a relatively high regeneration capacity. In contrast, the callus derived from the second leaf segment of 2 to 3 day old seedlings had a low regeneration capacity. The callus induced from the second leaf segment of 4 to 5 day old seedlings or the third leaf segment of 2 day old seedlings was unable to regenerate. The callus induced from the first leaf segment of the 3 day old seedling had the highest regeneration capacity (86%) and averaged 6.8 plants regenerated from each regenerable callus. The regeneration capacity of the callus induced from the first leaf segment of 2 day or 4 to 5 day old seedlings was much reduced in comparison to that of the first leaf segment of 3 day old seedlings (Table 1).
In this study, leaf base segments of oat seedlings have been successfully used as explants for regenerable callus induction and subsequent regeneration of fertile oat plants. The opaque, compact callus derived from oat leaf tissue is similar to the callus obtained from the immature embryos of oats (Rines and McCoy 1981). The frequency of regenerable callus formation and plant regeneration of the leaf segments is comparable to that of the immature embryos. Therefore, with the protocol developed in this study, regenerable oat cell cultures with high regeneration potential could be easily established without the limitation of donor explant source. Our results confirm the existence of a gradient of response of callus formation from the base to the apex in leaves of cereal species which include barley (Becher et al. 1992), rice (Wernicke et al. 1981), sorghum (Wernicke and Brettell 1980) and wheat (Wernicke and Milkovits 1984; Zamora and Scott 1983). In sorghum, wheat, Lolium multiflorum and Pennisetum purpureum, the abilities of callus formation and plant regeneration of leaf tissue was correlated not only with leaf explant position but also with developmental stage of the explants (Haydu and Vasil 1981; Wernicke and Brettell 1982; Wernicke and Milkovits 1984, Joarder et al. 1986; Rajasekaran et al. 1987a, 1987b). Such correlation also has been observed in this study. In oats, the optimum response was shown by the first one mm long leaf basal region (including the first basal leaf segment and the shoot meristem) of three day old seedlings. However, the same region of seedlings grown for 2 days or more than 3 days had a dramatic reduction of their regeneration capability, although they did not change in callus forming abilities. These results indicate that the developmental stage of the leaf explants plays a key role in the persistence of totipotency activity of the leaf callus in oats, as well as in other cereals (Wernicke and Brettell 1982; Wernicke and Milkovits
397 1984) and grasses (Joarder et al. 1986; Rajasekaran et al. 1987a, 1987b). Thus, seedling age must be considered to be a crucial factor for in vitro regeneration from leaf explants in oats. In oats, the establishment of regenerable cell cultures has often encountered the problem of cultivar-dependence. Cummings et al. (1976) reported that with immature embryos obtained from 25 oat genotypes as explants for starting callus cultures, two out of the 25 genotypes failed to initiate callus tissue and 9 out of the other 23 genotypes, although able to form callus, had no regeneration ability. Subsequently, Rines and McCoy (1981) reported that the frequency of regenerable callus formation with immature embryos obtained from 23 oat cultivars was between 5% and 75% of the number of embryos tested. Nineteen out of the 23 cultivars showed a frequency below 20%. In this study, six oat genotypes were investigated for plant regeneration from leaf base segments. Although all of these genotypes showed 100% or almost 100% frequency of callus formation, variation between genotypes was observed for the frequencies of regenerable callus formation and plant regeneration. However, the frequency of regenerable callus formation obtained from leaf base segments was considerably higher than immature embryos (Cummings et al. 1976; Rines and McCoy 1981). This indicates that leaf explants are less genotype dependent and are suitable donor material for the establishment of regenerable callus cultures of oats. Regenerable callus derived from immature embryos has been shown to be a suitable target tissue for the transformation of plants in rice, oats and wheat (Li et al. 1993; Somerset al. 1992; Vasil et al. 1992) by the biolistic method. In our future studies, we will focus on oat transformation using the regenerable leaf callus as the target for DNA delivery via the biolistic method. Acknowledgments. We thank Richard I. S. Brettell for critical reading of the manuscript. This study was supported by ANUTECH Pry. Ltd. and City Bank Ltd. through a syndicatedR & D program.
References Becher T, Haberland G, Koop H (1992) Plant Cell Rep. 11:39-43 Bregitzer P, Bushnell WR, Rines HW, Somers DA (1991) Plant Cell Rep. 10:234-246 Carter O, Yamada Y, Takahashi E (1967) Nature 214: 1029-1030 Christou P (1992) Plant J. 2:275-281 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Sci. Sin. 18:659-668 Cummings DP, Green CE, Stuthman DD (1976) Crop Science 16:465-470 Cure WW, Mott RL (1978) Physiol. Plant. 42:91-96 Ilaydu Z, Vasil IK (1981) Theor. Appl. Genet. 59: 269273 Ileyser JW, Nabors MW (1982) Z. Pflanzenphysiol. 107: 153-160
Joarder OI, Joarder NII, Dale PJ (1986) Theor. Appl. Genet. 73:286-291 Li L, Qu R, Kochko A, Fauquet C, Beachy RN (1993) Plant Cell Rep. 12:250-255 Lorz Il, Harms CT, Potrykus I (1976) Z. Pflanzenzuchtg. 77:257-259 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Rajasekaran K, Hein MB, Davis GC, Carnes MG, Vasil IK (1987a) J. Plant Physiol. 130:13-25 Rajasekaran K, Hein MB, Vasil IK (1987b) Plant Physiol. 84:47-51 Rines IlW, McCoy TJ (198 I) Crop Science 21: 837-842 Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR (1992) Bio/Tech. 10:1589-1594 Vasil IK (1994) Plant Mol. Biol. 25:926-937 Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Bio/Tech. 10:667-674 Wernicke W, Brettell R (1980) Nature 287:138-139 Wernicke W, Brettell R (1982) Protoplasma 111:19-27 Wernicke W, Brettel] R, Wakizuka T, Potrykus I (1981) Z. Pflanzenphysiol. 103:361-365 Wernicke W, Milkovits L (1984) J. Plant Physiol. 115: 49-58 Zamora AB Scott KJ (1983) Plant Sci. Lett. 29i 183-189
Plant Cell Reports (1995) 14:393 397
9 Springer-Verlag1995
Efficient callus formation and plant regeneration from leaves of oats (Arena sativa L.) Hancai Chen, Guanju Xu, David C. Loschke, Luba Tomaska, and Barry G. Rolfe Plant Microbe Interactions Group, Research School of Biological Sciences, Institute of Advanced Studies, Australian National University, Canberra, ACT 0200 Australia Received 1 July 1994/Revised version received 11 August 1994 - Communicated by I.K. Vasil
Abstract. An efficient plant regeneration system from leaf-derived callus in 6 genotypes of Arena sativa L. has been established. Regenerable callus was induced in the basal 1-2 m m region of 2 to 5 day old seedlings. Plants were regenerated from the regenerable callus and grown to maturity. The frequency of regenerable callus formation and plant regeneration was correlated with the position, developmental stage and genotype of the explant. The regeneration capacity of the first one mm of the leaf basal region from three day old seedlings was comparable to that of immature embryos. Furthermore, the leaf regenerable calli were subcultured for 8 months without loss of their regeneration capabilities.
Introduction Recent advances in plant tissue culture and transformation technologies have made it possible to genetically modify the most recalcitrant of crops such as cereals (Vasil 1994). The biolistic procedure has emerged as a simple and promising alternative for cereal transformation. The advantage of this method is that it can directly transfer DNA into intact plant cells without the constraints of protoplast culture and A g r o b a c t e r i u m host-specificity (Christou 1992). Recently, transgenic oat plants have been obtained following microprojectile bombardment with DNA-coated tungsten particles of either oat suspension culture cells or immature-embryo-derived calli ( S o m e r s e t al. 1992). However, establishment of regenerable suspension cultures of oats is difficult and genotype dependent (Bregitzer et al. 1991; Somerset al. 1992). Immature embryos are also not a convenient source of material for transformation studies because they require mature plants for their production, require specialized growth environments and may be restricted to a short season. Therefore, it is still of importance to develop a reliable alternative regeneration system in oats. Callus cultures of oats can be initiated from seeds or mature embryos, or from the roots of germinating Correspondence to: H. Chen
seedlings and hypocotyls or mesocotyls of germinating mature embryos. However, the regeneration capacities of these callus cultures are much lower (Carter et al. 1967; Cummings et al. 1976; Cure and Mott 1978; Heyser and Nabors 1982; Lorz ct al. 1976) than that of the callus derived from immature embryos (Cummings et al. 1976; Rines and McCoy 1981). Therefore, these oat callus cultures, although convenient to isolate, may be less attractive to be used as target tissue in transformation experiments. Regenerable callus formation and plant regeneration from leaf tissues has been reported in a number of cereals, but not yet for oats (Becher et al. 1992; Wernicke and Brettell 1980; Wernicke and Milkovits 1984; Zamora and Scott 1983). The ready availability of oat seedlings makes them a suitable source of explants for tissue culture if a high regeneration frequency can be achieved. We describe a high frequency regeneration system from leaf tissues of various cultivars of oats which also may have general application for oat transformation via the biolistic method.
Materials and Methods MS medium containing MS salts (Murashige and Skoog 1962) with 150 mg/1 asparagine, 0.5 rag/1 thiamine-HCl, 20 g/1 sucrose, 100 mg/l m-inositol, 7 mg/l glycine, 2.0 mg/1 2,4-D (2,4-dichlorophenoxyacetic acid, Sigma) and 0.7% Bacto-agar (Difco), pH 5.8 was used for routine callus induction, growth and maintainance. N6 medium containing N6 salts (Chu et al. 1975) with 146 rag/1 glutamine, 30 g/1 sucrose, 100 mg/1 m-inositol, 200 rag/1 casein hydrolysate, 2 mg/1 kinetin, 2 mg/l 1naphthaleneacetic acid (NAA) and 0.7% agar, pH 5.8 was used for shoot regeneration. MS medium containing 0.3 mg/1 kinetin and 0.7% agar, plt 5.8 was used for root regeneration. Tissue culture media.
Donor plants. Seeds of 6 different Avena sativa L. cultivars
were used. Four (Coolabah, Cooba, Blackbutt, and Mortlock) are Australian cultivars (Cleanseeds Pty Ltd, Bungendore, NSW, Australia) and two (Victorgrain and HVR) are North American Cultivars (Professor H. H. Lukel USDA Department of Plant Pathology, University of Florida, Florida, USA). The de-husked seeds were surface sterilised in 75% ethanol for
394 Mean first leaf length 10
I st leaf
30
60
(ram) 80
N
\
5-
2ud leaf i
Apical -mefistem~
2 days
3 days
4 days
5 days
Plant regeneration. Calli, five weeks or older, were transferred to a shoot regeneration medium and were incubated at 25~ with low intensity light (10 btE m -2 sec - l ) for 16 h day in a controlled growth cabinet. Three to four days later, the light intensity was increased to 190 g E m -2 sec -1. The callus cultures were subcultured on the shoot regeneration medium at two week intervals. During a period of one to three weeks, shoots about 3 mm long were dissected from calli and grown on the same medium in 100 x 20 mm Petri dishes. Shoots about 15 mm long were transferred to a tissue culture jar (70 x 75 mm) containing 30 ml root regeneration medium. The plantlets were transferred to fresh root regeneration medium in the tissue culture jars at two week intervals. Whole plants with a shoot length of between 7 and 10 cm and with well developed roots were transplanted to soil in pots and grown in a greenhouse (22~ for 10 h day and 15~ for 14 h night). A plant nutrient solution containing 0.18% Thrive (Arthur Yates & Co. Pty. Ltd. Australia) was applied once a week.
Fig. 1. Diagram illustrating leaf growth of oat seedlings for the first five days. The coleoptile-free leaves from each seedling were cut into 1 mm transverse section, starting from the leaf base. The first segment consists of the leaf base and the apical meristem. The subsequent segments contain either the first leaf tissue or the first and second leaf tissue.
2 rain, followed by two washes with sterile distilled water, and then in a 5% sodium hypochlorite solution for 10 min followed by five washes with sterile distilled water. The sterilised seeds were germinated on solid MS medium in tissue culture jars (70 x 75 ram) at 27~ in the dark for one day. The germinated seeds were grown in a 25~ incubator in the dark or in a growth cabinet at 25~ with 16 h day illumination (light intensity, 190 g E m -2 sec-1). Immature embryos were dissected from immature seeds approximately 10-12 days after pollination and sterilised using the same procedure as the mature seed sterilisation. Embryos were 1 to 3 mm long at this stage.
Explant preparation. Immature embryos were immediately excised from the sterilized de-husked seed and transferred onto solid callus-inducing medium with the embryo axis oriented down. Leaf base explants were prepared from 2 to 5 day old seedlings grown in the dark or with illumination. Leaves of the seedlings were very young without a complete leaf structure during this growth period. The length of the first leaf was between 10 mm and 80 ram, and the second leaf was up to 4 mm long during this growth period (Fig. 1). The leaves were separated from coleoptiles and sequentially cut into 1 mm transverse sections, starting from the original leaf base (Fig. 1) using a scalpel in a clear sterile Petri dish with a sheet of millimetre paper underneath to allow accurate sizing during dissection. The first segment refers to the basal segment consisting of the leaf base and the shoot meristem.
Callus initiation and subculture. The explants from the same position were placed onto a Petri dish (90 x 12 ram) containing 20 ml MS2D medium for callus induction. The explants were incubated at 25~ in the dark. After 14 days, callus was dissected from the leaf tissue and transferred to fresh MS2D medium. Callus cultures were subcultured on MS2D medium at 25~ in the dark every two to three weeks.
Results L e a f segments isolated from seedlings grown for 2 to 5 days in the dark were subjected to an initial screening for callus formation. Callus was induced from cultured leaf base segments on MS m e d i u m containing 2 rag/1 2,4-D. No organised callus tissue was formed in the absence o f 2,4-D. L e a f segments Of seedlings grown in the dark or in the light had a similar response for callus induction. The frequency of callus formation in leaf explants was strongly dependent on the position in the seedling and the age o f the seedling (Table 1). First, callus was induced only from the basal s e g m e n t s o f leaves. S e c o n d l y , the frequency of callus induction of the first segment from the leaf base was higher than that o f the subsequent segments from the leaf base. Thirdly, the basal region o f leaves with callus-forming ability in the younger seedlings was broader than that in the older seedlings. The highest response (100%) was observed in the one m m leaf basal region (the first segments) of 2-3 day old seedlings (Table 1). Callus tissue first became visible on the leaf explants within a week on callus medium. The initial callus tissue formed was soft and translucent. S o m e c o m p a c t and opaque callus d e v e l o p e d within soft callus masses one week later. The c o m p a c t callus tissues were increased c o n s i d e r a b l y f o l l o w i n g two w e e k s i n c u b a t i o n o f the cultures on callus medium (Fig. 2a). F o r 4 to 5 week old calli, o r g a n i z e d somatic e m b r y o - l i k e structures were observed on the compact callus (Fig. 2b) but not on the soft, translucent callus. The four to five week old calli were transferred to a shooting m e d i u m . Green spots appeared on the compact callus tissue 2 to 3 days later. In contrast, the soft, translucent callus tissues were unable to form green spots. Shoots were formed on the green spotforming calli 4 to 5 days later (Fig. 2c). Callus without green spots did not regenerate shoots. Shoots about 3 m m long were excised from the callus and transferred to fresh shooting medium. Seven days later, when most of
395 Table 1. Frequency of callus formation and plant regeneration from leaf segments of 2 to 5 day old seedlings and immature embryos of Avena sativa L. (cv. Coolabah) No. of Plantlets regeneratedb Source of explanta
No. of calli formed (%)
Immature embryo
No. of regenerable calli (%)
total
averagec
46
(92)
44
(87)
281
6.4
50 30 3 0
(100) (60) (6)
23 5 0
(46) (10)
74 15
3.2 3.0
50 26 2 0
(100) (52) (4)
43 9 0
(86) (18)
292 28
6.8 3.1
43 5 0
(86) (10)
28 0
(56)
64
2.3
45 3 0
(90) (6)
24 0
(48)
31
1.3
Two day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment 4th leaf segment
Three day seedling lst leaf segment 2nd leaf segment 3rd leaf segment 4th leaf segment
Four day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment
Five day seedling 1st leaf segment 2nd leaf segment 3rd leaf segment
aFifty explants were tested in each treatment. bThe number of plantlets was counted for up to 6 weeks after the transfer of the five week old callus to the shooting medium. CAverage number of plantlets regenerated from each regenerable callus.
Table 2. Frequency of callus formation and plant regeneration from the first leaf segment of 3 day old seedlings in five genotypes of Avena sativa L. No. of plantlets regenerateda Genotype
Cooba Blackbutt Mortlock Victorgrain HVR
No. of explants tested
No. of calli formed (%)
50 50 50 50 50
50 (100) 46 (92) 50 (100) 47 (94) 45 (90)
No. of regenerable calli (%) 42 40 36 31 26
(84) (87) (72) (66) (58)
total 143 132 97 87 39
averageb
-
3.4 3.3 2.7 2.8 1.5
aThe number of plantlets was counted for up to 6 weeks after the transfer of the five week old Callus to the shooting medium. bAverage number of plantlets regenerated per each regenerable callus.
396 Immature embryos of cultivar Coolabah were also used for callus induction and plant regeneration, using the same procedure. The callus induced from the immature embryos (data not shown) was similar to the leaf callus (Fig. 2). The frequency of callus initiation, regenerable callus formation and plant regeneration of the immature embryos was similar to that of the first leaf segment of 3 day old seedlings (Table 1). The first leaf segment of 3 day old seedlings of the other five oat cultivars (Cooba, Blackbutt, Mortlock, Victorgrain and HVR) were also used for callus induction and plant regeneration (Table 2). Depending upon the cultivar investigated, the frequency of callus induction was between 80% and 100%. About 48% to 86% of the leaf basal explants were able to form regenerable callus tissues, and an average of 1.5 to 3.4 plants were regenerated from each regenerable callus (Table 2). The regenerable callus from the leaf explants was subcultured at three week intervals, and has maintained its capacity to form plants for more than eight months without any significant loss of regeneration potential.
Discussion
Fig.2. Callus induction and plant regeneration from leaf segments of Avena sativa L. a Three week old callus consisting of opaque and compact callus, and soft and translucent callus obtained from leaf segment, b somatic embryo-like structure (arrow) on compact callus, c Shoot (arrow) regenerated from compact callus, d, e Whole and mature plants obtained from leaf callus.
the shoots had grown 15 to 20 mm long, they were transferred to a rooting medium. Roots developed within 5 days. The plants with shoots between 7 to 10 cm long and well developed roots (Fig. 2d) were transplanted to pots containing soil. Whole plants were able to grow in soil to normal mature plants with the abilities of tillering, flowering and grain formation (Fig. 2e). Total time taken from culture initiation to the transplanting of regenerated plants was approximately 10 weeks. The callus induced from different leaf explants exhibited quite different regeneration capacity (Table 1). The callus from the first leaf segment of 2 to 5 day old seedlings had a relatively high regeneration capacity. In contrast, the callus derived from the second leaf segment of 2 to 3 day old seedlings had a low regeneration capacity. The callus induced from the second leaf segment of 4 to 5 day old seedlings or the third leaf segment of 2 day old seedlings was unable to regenerate. The callus induced from the first leaf segment of the 3 day old seedling had the highest regeneration capacity (86%) and averaged 6.8 plants regenerated from each regenerable callus. The regeneration capacity of the callus induced from the first leaf segment of 2 day or 4 to 5 day old seedlings was much reduced in comparison to that of the first leaf segment of 3 day old seedlings (Table 1).
In this study, leaf base segments of oat seedlings have been successfully used as explants for regenerable callus induction and subsequent regeneration of fertile oat plants. The opaque, compact callus derived from oat leaf tissue is similar to the callus obtained from the immature embryos of oats (Rines and McCoy 1981). The frequency of regenerable callus formation and plant regeneration of the leaf segments is comparable to that of the immature embryos. Therefore, with the protocol developed in this study, regenerable oat cell cultures with high regeneration potential could be easily established without the limitation of donor explant source. Our results confirm the existence of a gradient of response of callus formation from the base to the apex in leaves of cereal species which include barley (Becher et al. 1992), rice (Wernicke et al. 1981), sorghum (Wernicke and Brettell 1980) and wheat (Wernicke and Milkovits 1984; Zamora and Scott 1983). In sorghum, wheat, Lolium multiflorum and Pennisetum purpureum, the abilities of callus formation and plant regeneration of leaf tissue was correlated not only with leaf explant position but also with developmental stage of the explants (Haydu and Vasil 1981; Wernicke and Brettell 1982; Wernicke and Milkovits 1984, Joarder et al. 1986; Rajasekaran et al. 1987a, 1987b). Such correlation also has been observed in this study. In oats, the optimum response was shown by the first one mm long leaf basal region (including the first basal leaf segment and the shoot meristem) of three day old seedlings. However, the same region of seedlings grown for 2 days or more than 3 days had a dramatic reduction of their regeneration capability, although they did not change in callus forming abilities. These results indicate that the developmental stage of the leaf explants plays a key role in the persistence of totipotency activity of the leaf callus in oats, as well as in other cereals (Wernicke and Brettell 1982; Wernicke and Milkovits
397 1984) and grasses (Joarder et al. 1986; Rajasekaran et al. 1987a, 1987b). Thus, seedling age must be considered to be a crucial factor for in vitro regeneration from leaf explants in oats. In oats, the establishment of regenerable cell cultures has often encountered the problem of cultivar-dependence. Cummings et al. (1976) reported that with immature embryos obtained from 25 oat genotypes as explants for starting callus cultures, two out of the 25 genotypes failed to initiate callus tissue and 9 out of the other 23 genotypes, although able to form callus, had no regeneration ability. Subsequently, Rines and McCoy (1981) reported that the frequency of regenerable callus formation with immature embryos obtained from 23 oat cultivars was between 5% and 75% of the number of embryos tested. Nineteen out of the 23 cultivars showed a frequency below 20%. In this study, six oat genotypes were investigated for plant regeneration from leaf base segments. Although all of these genotypes showed 100% or almost 100% frequency of callus formation, variation between genotypes was observed for the frequencies of regenerable callus formation and plant regeneration. However, the frequency of regenerable callus formation obtained from leaf base segments was considerably higher than immature embryos (Cummings et al. 1976; Rines and McCoy 1981). This indicates that leaf explants are less genotype dependent and are suitable donor material for the establishment of regenerable callus cultures of oats. Regenerable callus derived from immature embryos has been shown to be a suitable target tissue for the transformation of plants in rice, oats and wheat (Li et al. 1993; Somerset al. 1992; Vasil et al. 1992) by the biolistic method. In our future studies, we will focus on oat transformation using the regenerable leaf callus as the target for DNA delivery via the biolistic method. Acknowledgments. We thank Richard I. S. Brettell for critical reading of the manuscript. This study was supported by ANUTECH Pry. Ltd. and City Bank Ltd. through a syndicatedR & D program.
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