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Somaclonal variation in plants regenerated from callus culture of hybrid aspen (Populus alba L. x P. grandidentata Michx.)
Plant Science, 90 (1993) 89-94 Elsevier Scientific Publishers Ireland Ltd.
89
Somaclonal variation in plants regenerated from callus culture of hybrid aspen (Populus alba L. x P. grandidentata Michx.) S u n g H o Son a'b, H e u n g K y u M o o n b a n d R i c h a r d B. Hall a aDepartment of Forestry, Iowa State University, Ames, IA 50010 (USA) and bLab of Biotechnology, Institute of Forest Genetics, Forestry Administration, P.O. Box 24, Suwon, 441-350 (Republic of Korea) (Received July 9th, 1992; revision received January 19th, 1993; accepted January 25th, 1993)
Calli-clones of hybrid poplar are known to exhibit high levels of somaclonal variation. This study was undertaken to understand the causes of this variation. Multiple-shoot regeneration from callus cultures was obtained using woody plant medium (WPM) supplemented with 20/~M zeatin. Of the 400 calli-clones maintained in vitro for 6 weeks on WPM without plant growth regulators (PGR) 75.5% appeared to be growing normally, while the rest were putative albino (7.5%), slow-growing (8.25%), and rapid-growing (16.3%) variants. For the 200 calli-elones grown in the greenhouse for 6 months after transplanting, 4% were slow-growing, 6% were rapidgrowing and 10% had aberrant leaf morphology. Chromosome studies using in vitro calli-clones showed a broad range of variation from haploidy to tetraploidy. Protein studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong relationship between the staining intensity of specific bands and calli-clone growth patterns. Finally, the albino phenotype was associated with the loss of at least two proteins.
Key words: poplar; tissue culture; putative albino; calli-clones; phenotypic variation; chromosome number; growth related protein
Introduction
Among Populus species, hybrid aspen (Populus alba L. x P. grandidentata Michx.) is the most productive. It has been bred in Canada, Hungary, and the U.S., producing progeny of outstanding quality, with an above-average growth pattern, compared with that of the parental lines [1,21. This hybrid aspen also occurs naturally, and was first discovered in southeastern Iowa in 1947 [21. It is considered a potentially valuable tree species for energy plantations in central Iowa due to its rapid growth rate. The biomass of this species can be used to supply commercial energy needs by direct combustion or conversion into liquid fuels. It has been suggested that this hybrid can also be used for conservation reserve plantings [1] and sawlog rotations [3]. Correspondence to: Richard B. Hall, Department of Forestry, Iowa State University, Ames, IA 50010, USA.
Vegetative macropropagation of this aspen is accomplished by using either stem cuttings [4,5] or the root sprouting method [1]. As with other poplars, these methods routinely produce several thousand propagules every growing season. However, large-scale clonal propagation of this hybrid aspen by conventional methods is still difficult. To overcome this obstacle, extensive in vitro studies have been conducted on this genotype of hybrid aspen [6-11]. Two of the more useful micropropagation methods include cell- and callus-based regeneration systems [8,12]. Although the latter systems show great potential for large-scale micropropagation of Populus, previous reports revealed relatively high genotypic and phenotypic variation among the calli-clones [13,14]. This suggests that plants obtained by callus culture systems should be examined at in vitro or ex vitro levels to verify the genetic uniformity before commercial-scale micropropagation is attempted. In addition, the potential enrichment
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
90 of genetic diversity by in vitro-generated mutations and/or the amplication of pre-existing genetic heterogeneity in somatic cells will influence the success of tree breeding and improvement programs. The purpose of present study was to qualitatively characterize the extent of somaclonal variation observed in ramets derived from callus via in vitro shoot induction. The results of this study could have implications for future field studies, both applied and basic. Materials and Methods
Callus culture, regeneration, and transplanting in the greenhouse Callus was induced on stem cambial tissue from 1-year-old greenhouse-grown stock plants. Procedures for explant surface sterilization, in vitro establishment of callus culture, callus proliferation and isolation of organogenic callus have been described previously [8,12]. Multiple shoot regeneration from callus culture was obtained using WPM [16] supplemented with 20/~M zeatin. Each shoot excised from callus tissue was cultured on shoot elongation and root induction media, PGR-free WPM. Two-thirds of the shoots with well-expanded leaves and actively growing root systems were maintained in vitro while the other one-third of the regenerants (200 plants) were transplanted in the greenhouse, following previously described hardening protocols [7,8]. The media used in all culture steps contained 3% (w/v) sucrose and was adjusted to pH 5.7 before addition of 0.5% (w/v) Difco bacto agar and autoclaving at 121°C for 15 min. Cultures were maintained in plastic Petri dishes (10 x 1.5 cm; Fisher Sci. Co., Canada) sealed with Nesco film (Bando Chemical Ind., Ltd., Kobe, Japan). Cultures were maintained at 25 4- 2°C with 60 4- 10% relative humidity, under a 16-h photoperiod per day (except for the callus induction and proliferation steps, which were done in continuous darkness), with a photosynthetically active photon flux rate of 40-60/~E m -2 s -~ from cool white fluorescent lamps.
Chromosome counting Root tips (2-3 cm in size) were collected from in vitro grown shoot cultures of calli-clones which were maintained for four weeks on PGR-free WPM. Root tips were pretreated with 0.002 M 8hydroxyquinoline solution for 12 h, soaked in a fixative (alcohol/glacial acetic acid, 3:1, v/v) for 1-3 h in the dark at 25°C, hydrolyzed in 2 N HC1 for 20 min at 55-58°C and stained in CarbolFuchsin for 2-5 h. Chromosome numbers were calculated from 113 independent observations made under a microscope at 1000 x, 1250 x and 1500 x. Prote& analysis Leaf proteins were extracted from in vitro- (6 weeks of culture on PGR-ffee WPM) and greenhouse-grown (6-month-old) calli-clones. To minimize the appearance of nascent proteins synthesized during different developmental stages and/or by stress, the same age and size of leaves from each of 70 plants were used immediately after collection. For sample preparation, a known volume of leaf sample was ground in liquid nitrogen with 1 x SDS gel-loading buffer (sample: buffer, 1:3, v/v) and heated at 100°C for 3-5 min. The debris was removed by micro-centrifugation before 30 #g of total protein was loaded in each lane of the gel (15% resolving and 5% stacking gel) and subjected to electrophoresis at 72 mA for 8 h (until the bromophenol blue marker reached the bottom 10 cm from the origin). After soaking in the fixative (methanol/glacial acetic acid/water, 1:2:7, by vol.) for a few min, the gels were stained with Coomassie Brilliant Blue. Preparation of solutions used for SDS-PAGE in this study followed the protocol described in the manual by Sambrook et al. [17]. Rainbow TM protein molecular weight markers (RPN 755 & 756, Amersham, Corp., Arlington Hts, IL) were also included for size determination. Results and Discussion
Phenotypic variation was observed among the calli-clones maintained in vitro and under greenhouse conditions. Large differences in pigment accumulating ability and growth pattern var-
91
iation were observed among 400 calli-clones grown in vitro. When callus-derived shoots were subcultured and maintained for 6 weeks on shoot elongation and rooting media, 6 putative albinos with healthy root systems and 24 putative albinos which did not produce root systems were observed. The putative albino plants could be divided into three classes. These are plants with: (1) nearly white leaves on the top and one or two pale-green leaves on the bottom, (2) white leaves with palegreen margins and midvein and (3) pale-green and yellowish leaves. Putative albino plants were extracted using a previously described method [14] and indicated loss of pigments. Similar results have been reported for both chimeric [9,14] and naturally occurring albino aspen [181.
1
2
Four distinctive growth patterns were observed in the green-colored shoots grown in vitro and derived from calli-clones. By grouping calli-clones of similar growth patterns, we observed: (1) 33 slowgrowing plants which usually lost their root induction ability, (2) 302 normal growth pattern plants which produced actively growing root systems, (3) 42 with rapid growth and healthy root systems and (4) 23 with rapid growth but without root induction. Six months after transplantation of 200 greenhouse-grown calli-clones, variation in growth pattern and leaf morphology were apparent. Most of the plants observed had normal growth but a few (8 plants) displayed below-average growth rates and 12 plants exhibited very rapid growth. This
,q
b 4
F
6
"
8
9
Fig. 1. Foliar variation of calli-clone-derived plants of hybrid aspen (Populus alba L. x P. grandidentata Michx. 'Crandon') 6 months after transplanting in the greenhouse. (1-1) Cordate leaf, (1-2) normal poplar leaf, (1-3) ovate leaf, (1-4) to (1-5) unbalanced growth from leaf mid vein, (1-6) pseudomorphic growth, (1-7) split-leaf, (l-g) to (1-9) split and stacked leaf. Bar = 2.5 cm.
92
level of variability was similar to previously reported for somaclonal variation in studies with poplar [13]. Leaf morphological variation was also apparent ( - 10%), with most of the unusually shaped leaves concentrated at the bottom one-third of the stem. The variations in leaf morphology could be grouped as (1) intact leaf with different morphology such as cordate or ovate, (2) leaves of unbalanced growth from the mid vein, (3) leaves of pseudomorphic growth, (4) split and unbalanced leaves and (5) split and stacked leaves. Leaf morphological variations observed from greenhousegrown calli-clones are shown in Fig. 1. Asymmetric leaves from anther culture-derived aneuploid poplar have been previously reported [19]. Chromosome counts from root tips of calliclones grown in vitro are summarized in Table I. Among the 150 randomly selected root tips from different calli-clones, 113 cells were in metaphase. Compared with axillary bud-derived root tips,
Table I. V a r i a t i o n in c h r o m o s o m e n u m b e r o f root tip cells derived from in v i t r o - g r o w n calli-clones o f h y b r i d aspen (Populus alba L. x P. grandidentata Michx. ' C r a n d o n ' ) . Ploidy/chromosome. no.
= = = =
I1 13 16 19
No. of observed cells a
Frequency of occurrence
1 1 1 14
0.88 0.88 0.88 12.39
(%)
Haploid or less
n n n n
Aneuploid b
2n 2n 2n 2n 2n 2n 2n
23 24 26 27 29 30 33
1 1 1 2 1 1 1
0.88 0.88 0.88 1.77 0.88 0.88 0.88
Diploid
2n = 38
66
58.40
Tetraploid
4n = 76
22
19.46
= = = = = = =
a Total of 113 cells were observed from 150 root tips. b C h r o m o s o m e n u m b e r s between 19 and 38 were g r o u p e d as aneuploid.
which contained only diploid cells, calli-clones had highly variable ploidy levels; high frequencies of haploid, aneuploid and tetraploids were observed (Fig. 2). Although we observed some aneuploid (chromosome number range from 2n = 23 to 33) and haploid chromosome numbers, it is doubtful that the haploid state can be maintained, because in previous studies field-grown haploid plants doubled their chromosome numbers within 3 years [20-22]. However, it is not clear whether chromosomal number variations among our calli-clones is responsible for the growth pattern variation observed. To characterize somaclonal variants, various types of biotechnological approaches have been used. Isozyme analysis has frequently been used to verify the correlation between observed differences and somaclonal variation [14,23]. Southern hybridization [24] and protein electrophoresis techniques (zymogram, electrofocussing) have been applied to detect somaclonal variants of potato at the molecular level and to verify the phenotypic variations of protoclones [25]. Coleman and Ernst [26] have also reported high levels of a 32-kDa protein related to callus growth and shoot regeneration capacity. Specific proteins related to callus growth, regeneration potential and embryogenic determined characteristics have also been reported in rice and sorghum [27,28]. In the present study, protein bands of in vitro leaf sources showed less variation in pattern and weakly stained band intensities as compared with those of ex vitro leaf materials. Albino plants were missing two bands at 32 and 67.5 kDa. Axillary bud-derived plant leaf protein showed the same banding pattern as that of the normal and rapidgrowing plants, whereas slow-growing plants were missing a band between 69 and 97.4 kDa. Among the calli-clones, differences in the staining intensity of at least 3 bands were exactly matched by their growth habits (data not shown). The observed variation in phenotype, chromosome number and protein content may be due to the high levels of PGR in the culture media and/or heterogeneity in the competent cell population. However, to increase genetic diversity of given genotype via the callus culture system, more detailed experiments and field studies are needed with
93
Fig. 2. Chromosomal variation in root tip cells derived from in vitro calli-clones of hybrid aspen (Populus alba L. x P. grandidentata Michx. 'Crandon'). (2-1) to (2-2) cells with haploid n = 16 ( !000 × ) and n = 19 (1000 × ); (2-3) to (2-4) aneuploid 2n = 24 (1250 x ) and 2n = 27 (1500x); (2-5) diploid 2n = 38 (1250x); (2-6) tetraploid 4n = 76 (1000x).
the p r o p a g u l e s a n d their p r o g e n y . Nevertheless, o u r results suggest t h a t selecting r a p i d g r o w i n g a n d t e t r a p l o i d p l a n t s f r o m calli-clones m a y be a useful tool for tree i m p r o v e m e n t p r o g r a m s .
2 3
Acknowledgement T h e a u t h o r s .wish to t h a n k Drs. R . C . Schultz, E.S. W u r t e l e a n d R. M e i l a n for their helpful advice a n d Mr. J.W. E o m for his t e c h n i c a l assistance.
4
References 5 1 R.B. Hall, J.P. Colletti, R.C. Schultz, R.R. Faltonson, S.H. Kolison Jr., R.D. Hanna, T.D. Hillson and J.W.
6
Morrison, Commercial-scale vegetative propagation of aspen, Proc. Aspen Symposium, July 25-27, Duluth, MN, 1989, pp. 211-219. E.L. Little, K.A. Brinkman Jr. and A.L. McComb, Two natural Iowa hybrid poplars. For. Sci., 3 (1957) 253-262. R.B. Hall, G.D. Hilton and C.A. Maynard, Construction lumber from hybrid aspen plantations in the central states. J. For., 80 (1982) 291-294. R.R. Faltonson, D. Thompson and J.C. Gordon, Propagation of poplar clones for controlled-environmental studies, in: Methods of Rapid Early Selection of Poplar Clones for Maximum Yield Potential: A Manual of Procedures. North Central Forest Experiment Station, USDA For. Ser. Gen. Tech. Rep. NC-81, 1983, pp. 1-11. H.T. Hartmann and D.E. Kester, Plant propagation: principles and practices, 4th edn., Prentice-Hall, Englewood Cliffs, NJ, 1983, pp. 727. Y.W. Chun and R.B. Hall, Survival and early growth of
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7
8
9
l0
11
12
13
14
15 16
17
Populus alba x P. grandidentata in vitro cultured plantlets in soil. J. Kor. For. Soc., 66 (1984) 1-7. S.H. Son and R.B. Hall, Multiple shoot regeneration from root organ cultures of Populus alba x P. grandidentata. Plant Cell Tiss. Org. Cult., 20 (1990) 53-57. S.H. Son and R.B. Hall, Plant regeneration capacity of callus derived from leaf, stem, and root segments of Populus alba L. x P. grandidentata Michx: Plant Cell Rep., 9 (1990) 344-347. S.H. Son, Y.W. Chun and R.B. Hall, Cold storage of in vitro cultures of hybrid poplar shoots (Populus alba L. x P. grandidentata Michx.). Plant Cell Tiss. Org. Cult., 27 (1991) 161-168. S.H. Son and R.B. Hall, Multiple shoot regeneration capacity from ex vitro and in vitro derived stem node cultures of hybrid aspen (Populus alba L. x P. grandidentata Michx.). Plant Cell Rep., (1993) in press. J.C. Sellmer, B.H. McCown and B.E. Haissig, Shoot culture dynamics of six Populus clones. Tree Physiol., 5 (1989) 219-227. Y.G. Park and S.H. Son, Regeneration from plantlets from cell suspension derived callus of Populus alba. Plant Cell Rep., 7 (1988) 567-570. D.T. Lester and J.G. Berbee, Within-clonal variation among black poplar trees derived from callus culture. For. Sci., 23 (1977) 122-132. E.W. Noh and S.C. Minocha, Pigment and isozyme variation in aspen shoots regenerated from callus culture. Plant Cell Tiss. Org. Cult., 23 (1990) 39-44. L.L. Winton, Plantlet formation from aspen tissue culture. Science, 160 (1968) 1234-1235. G.B. Lloyd and B.H. McCown, Commercially feasible micropropagation of mountain laurel (Kalmia latifolia) by use of shoot tip culture. Prop. Int. Plant Proc. Soc., 30 (1981) 421-427. J. Sambrook, E.F. Fritch and T. Maniatis, in: Molecular
18 19
20
21 22 23
24
25
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28
cloning a laboratory manual. Cold Spring Harbor Laboratory Press, 1989. K.A. Green, J.C. Zasada and K. Van Cleve, An albino aspen sucker. For. Sci., 17 (1971) 272. M.U. Stoehr and L. Zsuffa, Induction of haploids in Populus maximowiczii via embryogenic callus. Plant Cell Tiss. Org. Cult., 23 (1990) 49-58. J.H. Kim, H.K. Moon and J.l. Park, Haploid plantlet induction through anther culture of Populus maximowiczii. Res. Rep. Inst. Gen. Korea, 22 (1986) 116-121. C.C. Wang, Z.C. Chu and C.S. Sun, The induction of Populus pollen plants. Acta Bot. Sin., 17 (1975) 56-59. X.Y. Zhu, R.L. Wang and Y. Liang, Induction of poplar pollen plantlets. Sci. Silvae Sin., 16 (1990) 190-197. R. Allicchio, C. Antonioli, L. Graziani, R. Roncarati and C. Vannini, Isozyme variation in leaf-callus regenerated plants of Solanum tuberosurn. Plant Sci., 53 (1987) 81-86. E. Southern, Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98 (1975) 503. K. Sree Ramula, P. Dijkhuis, G.M.M. Bredemeijer, H.C.J. Burg, S. Roest, G.S. Bahelmann, C.H. Hanisch Ten Cate and L. Ennik, Protoclonal variation in a butch commercial cultivar of potato (Solanum tuberosum L. cv. Bintje). Experimentia Suppl., 45 (1983) 148-149. G.D. Coleman and S.G. Ernst, Protein differences among Populus deltoides internodal stem explants determined for shoot regeneration or callus growth. Plant Sci., 75 (1991) 83-92. L. Chen and D.S. Luthe, Analysis of proteins from embryogenic and non embryogenic rice (Oryza sativa L.) calli. Plant Sci., 48 (1987) 181-188. C.A. Wozniak and J.E. Partridge, Analysis of growth in sorghum callus cultures and association with a 27-kDa peptide. Plant Sci., 57 (1988) 235-246.
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Somaclonal variation in plants regenerated from callus culture of hybrid aspen (Populus alba L. x P. grandidentata Michx.) S u n g H o Son a'b, H e u n g K y u M o o n b a n d R i c h a r d B. Hall a aDepartment of Forestry, Iowa State University, Ames, IA 50010 (USA) and bLab of Biotechnology, Institute of Forest Genetics, Forestry Administration, P.O. Box 24, Suwon, 441-350 (Republic of Korea) (Received July 9th, 1992; revision received January 19th, 1993; accepted January 25th, 1993)
Calli-clones of hybrid poplar are known to exhibit high levels of somaclonal variation. This study was undertaken to understand the causes of this variation. Multiple-shoot regeneration from callus cultures was obtained using woody plant medium (WPM) supplemented with 20/~M zeatin. Of the 400 calli-clones maintained in vitro for 6 weeks on WPM without plant growth regulators (PGR) 75.5% appeared to be growing normally, while the rest were putative albino (7.5%), slow-growing (8.25%), and rapid-growing (16.3%) variants. For the 200 calli-elones grown in the greenhouse for 6 months after transplanting, 4% were slow-growing, 6% were rapidgrowing and 10% had aberrant leaf morphology. Chromosome studies using in vitro calli-clones showed a broad range of variation from haploidy to tetraploidy. Protein studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong relationship between the staining intensity of specific bands and calli-clone growth patterns. Finally, the albino phenotype was associated with the loss of at least two proteins.
Key words: poplar; tissue culture; putative albino; calli-clones; phenotypic variation; chromosome number; growth related protein
Introduction
Among Populus species, hybrid aspen (Populus alba L. x P. grandidentata Michx.) is the most productive. It has been bred in Canada, Hungary, and the U.S., producing progeny of outstanding quality, with an above-average growth pattern, compared with that of the parental lines [1,21. This hybrid aspen also occurs naturally, and was first discovered in southeastern Iowa in 1947 [21. It is considered a potentially valuable tree species for energy plantations in central Iowa due to its rapid growth rate. The biomass of this species can be used to supply commercial energy needs by direct combustion or conversion into liquid fuels. It has been suggested that this hybrid can also be used for conservation reserve plantings [1] and sawlog rotations [3]. Correspondence to: Richard B. Hall, Department of Forestry, Iowa State University, Ames, IA 50010, USA.
Vegetative macropropagation of this aspen is accomplished by using either stem cuttings [4,5] or the root sprouting method [1]. As with other poplars, these methods routinely produce several thousand propagules every growing season. However, large-scale clonal propagation of this hybrid aspen by conventional methods is still difficult. To overcome this obstacle, extensive in vitro studies have been conducted on this genotype of hybrid aspen [6-11]. Two of the more useful micropropagation methods include cell- and callus-based regeneration systems [8,12]. Although the latter systems show great potential for large-scale micropropagation of Populus, previous reports revealed relatively high genotypic and phenotypic variation among the calli-clones [13,14]. This suggests that plants obtained by callus culture systems should be examined at in vitro or ex vitro levels to verify the genetic uniformity before commercial-scale micropropagation is attempted. In addition, the potential enrichment
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
90 of genetic diversity by in vitro-generated mutations and/or the amplication of pre-existing genetic heterogeneity in somatic cells will influence the success of tree breeding and improvement programs. The purpose of present study was to qualitatively characterize the extent of somaclonal variation observed in ramets derived from callus via in vitro shoot induction. The results of this study could have implications for future field studies, both applied and basic. Materials and Methods
Callus culture, regeneration, and transplanting in the greenhouse Callus was induced on stem cambial tissue from 1-year-old greenhouse-grown stock plants. Procedures for explant surface sterilization, in vitro establishment of callus culture, callus proliferation and isolation of organogenic callus have been described previously [8,12]. Multiple shoot regeneration from callus culture was obtained using WPM [16] supplemented with 20/~M zeatin. Each shoot excised from callus tissue was cultured on shoot elongation and root induction media, PGR-free WPM. Two-thirds of the shoots with well-expanded leaves and actively growing root systems were maintained in vitro while the other one-third of the regenerants (200 plants) were transplanted in the greenhouse, following previously described hardening protocols [7,8]. The media used in all culture steps contained 3% (w/v) sucrose and was adjusted to pH 5.7 before addition of 0.5% (w/v) Difco bacto agar and autoclaving at 121°C for 15 min. Cultures were maintained in plastic Petri dishes (10 x 1.5 cm; Fisher Sci. Co., Canada) sealed with Nesco film (Bando Chemical Ind., Ltd., Kobe, Japan). Cultures were maintained at 25 4- 2°C with 60 4- 10% relative humidity, under a 16-h photoperiod per day (except for the callus induction and proliferation steps, which were done in continuous darkness), with a photosynthetically active photon flux rate of 40-60/~E m -2 s -~ from cool white fluorescent lamps.
Chromosome counting Root tips (2-3 cm in size) were collected from in vitro grown shoot cultures of calli-clones which were maintained for four weeks on PGR-free WPM. Root tips were pretreated with 0.002 M 8hydroxyquinoline solution for 12 h, soaked in a fixative (alcohol/glacial acetic acid, 3:1, v/v) for 1-3 h in the dark at 25°C, hydrolyzed in 2 N HC1 for 20 min at 55-58°C and stained in CarbolFuchsin for 2-5 h. Chromosome numbers were calculated from 113 independent observations made under a microscope at 1000 x, 1250 x and 1500 x. Prote& analysis Leaf proteins were extracted from in vitro- (6 weeks of culture on PGR-ffee WPM) and greenhouse-grown (6-month-old) calli-clones. To minimize the appearance of nascent proteins synthesized during different developmental stages and/or by stress, the same age and size of leaves from each of 70 plants were used immediately after collection. For sample preparation, a known volume of leaf sample was ground in liquid nitrogen with 1 x SDS gel-loading buffer (sample: buffer, 1:3, v/v) and heated at 100°C for 3-5 min. The debris was removed by micro-centrifugation before 30 #g of total protein was loaded in each lane of the gel (15% resolving and 5% stacking gel) and subjected to electrophoresis at 72 mA for 8 h (until the bromophenol blue marker reached the bottom 10 cm from the origin). After soaking in the fixative (methanol/glacial acetic acid/water, 1:2:7, by vol.) for a few min, the gels were stained with Coomassie Brilliant Blue. Preparation of solutions used for SDS-PAGE in this study followed the protocol described in the manual by Sambrook et al. [17]. Rainbow TM protein molecular weight markers (RPN 755 & 756, Amersham, Corp., Arlington Hts, IL) were also included for size determination. Results and Discussion
Phenotypic variation was observed among the calli-clones maintained in vitro and under greenhouse conditions. Large differences in pigment accumulating ability and growth pattern var-
91
iation were observed among 400 calli-clones grown in vitro. When callus-derived shoots were subcultured and maintained for 6 weeks on shoot elongation and rooting media, 6 putative albinos with healthy root systems and 24 putative albinos which did not produce root systems were observed. The putative albino plants could be divided into three classes. These are plants with: (1) nearly white leaves on the top and one or two pale-green leaves on the bottom, (2) white leaves with palegreen margins and midvein and (3) pale-green and yellowish leaves. Putative albino plants were extracted using a previously described method [14] and indicated loss of pigments. Similar results have been reported for both chimeric [9,14] and naturally occurring albino aspen [181.
1
2
Four distinctive growth patterns were observed in the green-colored shoots grown in vitro and derived from calli-clones. By grouping calli-clones of similar growth patterns, we observed: (1) 33 slowgrowing plants which usually lost their root induction ability, (2) 302 normal growth pattern plants which produced actively growing root systems, (3) 42 with rapid growth and healthy root systems and (4) 23 with rapid growth but without root induction. Six months after transplantation of 200 greenhouse-grown calli-clones, variation in growth pattern and leaf morphology were apparent. Most of the plants observed had normal growth but a few (8 plants) displayed below-average growth rates and 12 plants exhibited very rapid growth. This
,q
b 4
F
6
"
8
9
Fig. 1. Foliar variation of calli-clone-derived plants of hybrid aspen (Populus alba L. x P. grandidentata Michx. 'Crandon') 6 months after transplanting in the greenhouse. (1-1) Cordate leaf, (1-2) normal poplar leaf, (1-3) ovate leaf, (1-4) to (1-5) unbalanced growth from leaf mid vein, (1-6) pseudomorphic growth, (1-7) split-leaf, (l-g) to (1-9) split and stacked leaf. Bar = 2.5 cm.
92
level of variability was similar to previously reported for somaclonal variation in studies with poplar [13]. Leaf morphological variation was also apparent ( - 10%), with most of the unusually shaped leaves concentrated at the bottom one-third of the stem. The variations in leaf morphology could be grouped as (1) intact leaf with different morphology such as cordate or ovate, (2) leaves of unbalanced growth from the mid vein, (3) leaves of pseudomorphic growth, (4) split and unbalanced leaves and (5) split and stacked leaves. Leaf morphological variations observed from greenhousegrown calli-clones are shown in Fig. 1. Asymmetric leaves from anther culture-derived aneuploid poplar have been previously reported [19]. Chromosome counts from root tips of calliclones grown in vitro are summarized in Table I. Among the 150 randomly selected root tips from different calli-clones, 113 cells were in metaphase. Compared with axillary bud-derived root tips,
Table I. V a r i a t i o n in c h r o m o s o m e n u m b e r o f root tip cells derived from in v i t r o - g r o w n calli-clones o f h y b r i d aspen (Populus alba L. x P. grandidentata Michx. ' C r a n d o n ' ) . Ploidy/chromosome. no.
= = = =
I1 13 16 19
No. of observed cells a
Frequency of occurrence
1 1 1 14
0.88 0.88 0.88 12.39
(%)
Haploid or less
n n n n
Aneuploid b
2n 2n 2n 2n 2n 2n 2n
23 24 26 27 29 30 33
1 1 1 2 1 1 1
0.88 0.88 0.88 1.77 0.88 0.88 0.88
Diploid
2n = 38
66
58.40
Tetraploid
4n = 76
22
19.46
= = = = = = =
a Total of 113 cells were observed from 150 root tips. b C h r o m o s o m e n u m b e r s between 19 and 38 were g r o u p e d as aneuploid.
which contained only diploid cells, calli-clones had highly variable ploidy levels; high frequencies of haploid, aneuploid and tetraploids were observed (Fig. 2). Although we observed some aneuploid (chromosome number range from 2n = 23 to 33) and haploid chromosome numbers, it is doubtful that the haploid state can be maintained, because in previous studies field-grown haploid plants doubled their chromosome numbers within 3 years [20-22]. However, it is not clear whether chromosomal number variations among our calli-clones is responsible for the growth pattern variation observed. To characterize somaclonal variants, various types of biotechnological approaches have been used. Isozyme analysis has frequently been used to verify the correlation between observed differences and somaclonal variation [14,23]. Southern hybridization [24] and protein electrophoresis techniques (zymogram, electrofocussing) have been applied to detect somaclonal variants of potato at the molecular level and to verify the phenotypic variations of protoclones [25]. Coleman and Ernst [26] have also reported high levels of a 32-kDa protein related to callus growth and shoot regeneration capacity. Specific proteins related to callus growth, regeneration potential and embryogenic determined characteristics have also been reported in rice and sorghum [27,28]. In the present study, protein bands of in vitro leaf sources showed less variation in pattern and weakly stained band intensities as compared with those of ex vitro leaf materials. Albino plants were missing two bands at 32 and 67.5 kDa. Axillary bud-derived plant leaf protein showed the same banding pattern as that of the normal and rapidgrowing plants, whereas slow-growing plants were missing a band between 69 and 97.4 kDa. Among the calli-clones, differences in the staining intensity of at least 3 bands were exactly matched by their growth habits (data not shown). The observed variation in phenotype, chromosome number and protein content may be due to the high levels of PGR in the culture media and/or heterogeneity in the competent cell population. However, to increase genetic diversity of given genotype via the callus culture system, more detailed experiments and field studies are needed with
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Fig. 2. Chromosomal variation in root tip cells derived from in vitro calli-clones of hybrid aspen (Populus alba L. x P. grandidentata Michx. 'Crandon'). (2-1) to (2-2) cells with haploid n = 16 ( !000 × ) and n = 19 (1000 × ); (2-3) to (2-4) aneuploid 2n = 24 (1250 x ) and 2n = 27 (1500x); (2-5) diploid 2n = 38 (1250x); (2-6) tetraploid 4n = 76 (1000x).
the p r o p a g u l e s a n d their p r o g e n y . Nevertheless, o u r results suggest t h a t selecting r a p i d g r o w i n g a n d t e t r a p l o i d p l a n t s f r o m calli-clones m a y be a useful tool for tree i m p r o v e m e n t p r o g r a m s .
2 3
Acknowledgement T h e a u t h o r s .wish to t h a n k Drs. R . C . Schultz, E.S. W u r t e l e a n d R. M e i l a n for their helpful advice a n d Mr. J.W. E o m for his t e c h n i c a l assistance.
4
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