Plant Cell Reports
Plant Cell Reports (1986) 5:464-467
© Springer-Verlag1986
High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.) Eun-Woon Noh and Subhash C. Minocha Department of Botany and Plant Pathology, University of New Hampshire, Durham, NH 03824, USA Received August 25, 1986 / Revised version received October 19, 1986 - Communicated by A. R. Gould
ABS~R~
Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/l benzyladenine (BA) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/l BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3-4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/l BA for shoot regeneration. Almost 100% of the clumps formed shoots on W P M when subcultureddirectly from mB5 with anaverage number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or W P M with 0.2 mg/l indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2-4 weeks before normal growth in the greenhouse.
AEB~mr~TICRS
BA: 6-Benzyl-adenine, IBA: indole-3-butyric acid, 2,4-D: 2,4-dichlorophenoxyacetic acid, mB5 medium: modified B5 medium, WPM: Woody Plant medium.
Shoot differentiation from callus is a crucial s t e p for s o m a t i c cell f u s i o n and g e n e t i c transformation of plants by foreign DNA. It has, however, been very difficult to regenerate shoots from callus of woody plants. This has made many woody plants unsuitable for application of recombinant DNA technology. Among the woody plants, various species of the genus Populus have been studied extensively for their capacity to regenerate in i__nnvitro cultures (P. tremula: Winton 1968, 1970, 1971; P__L.tremuloides: Wolter 1968; P. tremula. P__L.euramericana: Chalupa, 1974; P__~.trgmula and P. alba _x glandulosa: Christie 1978; P__~.alba _x glandulosa and P__~.euramericana: Kim et al 1982; P__~. tremula, P_~.tremuloides, and their hybrid: Ahuja, 1983 a,b, 1984, 1986, Ahuja and Muhs 1982; P__~.
Offprint requests to: S. C. Minocha
tremula and P. tremuloides: FrShlich and Weisgerber 1985; P_~.albax P__~.grandidentata: Chun et al 1986; P__~. tremuloides: Wann and Einspar 1986). Whereas adventitious shoots have been successfully regenerated from stem and leaf segments or axillary buds of poplars, shoot regeneration from callus has been very difficult. Wolter (1968) and Winton (1968, 1970) reported limited success with shoot regeneration from callus of aspen. Douglas (1982), using suspension culture techniques, reported good shoot regeneration from internode-derivedcallus of hybrid poplar (Populus hybrid TT32). We report here a reproducible high frequency shoot regeneration method from callus and suspension cultures of quaking aspen (Populus tremuloides). ~
AND M~n~3uS
Callus Induction Full sib seeds (XT-19-84) of Populus tremuloides were obtained from the Institute of Paper Chemistry, Appleton, WI and stored at 4°C. Seeds were surface sterilized with 0.2% HgCI 2 solution for 2 minutes, washed with sterile distilled water and placed on the MS (Murashige and Skoog 1962) medium containing 0.2 mg/l BA. Germinated shoots were subcultured onto the same medium for multiple shoot production. Twenty to forty shoots were regenerated from each shoot segment in 25 days on this medium (data not shown). These plantlets were excised again and transferred to mB5 (Gamborg et al 1968) with 0.2 mg/l IBA for rooting. Leaves were excised only from rooted shoots (about 4 cm tall) and placed on the roB5 media containing 0.5 or 5.0 mg/l 2,4-D alone and 0.i mg/l BA combined with 0.5, 5.0, and i0 mg/l 2,4-D. Shoot Reaeneration Callus clumps grew to a diameter of 3-5 m m in 20 days. Callus clumps were subcultured directly onto W P M (Lloyd and McCown 1981) with 0.5 mg/l BA for shoot induction. For suspension culture, approximately 500 mg of callus clumps grown on solidified mB5 medium with 0.i mg/l BA plus 0.5 mg/l 2,4-D were passed through a 1 mm sieve and grown in 50 ml of liquid mB5 medium with 0.I mg/l BA and 0.5 mg/l 2,4-D in 125 ml Erlenmyer flask. The flasks were kept on gyratory shakers at 150 rpm. The cell
465 I~KLT
clumps grew to 3-4 mm in diameter after 3 weeks in suspension culture. Tney were then transferred onto solidified W P M with 0.05, 0.5 and 5.0 mg/l BA in petri dishes or in 125 ml Erlenmeyer flasks for shoot inductioru
Initiation and growth of callus: Calli were formed easily on mB5 medium. The frequency, growth and color of calli varied with the concentration of hormones (Table i). Cultures on medium lacking BA or having high concentration of 2,4-D resulted in calli with red pigmentation. The c o m b i n a t i o n of 0.I m g / l BA and 0.5 mg/l 2,4-D produced the best quality of callus. These calli were relatively compact and when subcultured onto W P M for shoot induction, turned to a yellowish-red color. In suspension culture, calli grew rapidly and reached a 3-4 mm diameter within 2 weeks. These calli were dark green and compact. When subcultured to W P M with 0.5 mg/l BA for shoot induction, however, the calli developed red pigmentation and many soft fiber-like surface cells.
Shoots formed on W P M with 0.5 mg/l BA were excised and subcultured on either WPM or mB5 with 0.2 mg/l IBA for rooting. Rooted plants were transplanted to pots containing vermiculite, perlite and sand (I:I:I) and kept under intermittent mist for 2-4 weeks before normal growth in the green house. Culture Conditions and Media Throughout the experiment, cultures were kept under constant light (20 pEm-2sec -I for callus induction and shoot regeneration and 50 ~Em-2sec-I for root induction) at 25+2°C. All m e d i a w e r e adjusted to pH 5.6 prior to autoclaving at 121°C and 15 ibs psi for 25 rain. The mB5 medium used for callus induction and liquid culture contained the following in 1 liter: 150 mg KCI, 2500 mg KNO 3, 250 mg MgSO4.7H20, 150 mg CaCI2.2H20, 300 mg NaH2PO4.H20, 150 mg (NH4)2SO4, 27.8 mg FeSO4.7H20, 37.3 mg Na 2I~3TA, and micronutrients descrlbed in Gamborg et al (1968). The medium also contained i00 mg myoinositol, 1 mg thiamine-H~l, 1 mg pyridoxine-HCl, 1 mg nicotinic acid, 7 g Phytagar (deleted in liquid medium) and 30 g sucrose. Woody Plant Medium of Lloyd and M c C o w n (1981) was used for shoot regeneration.
Shoot Reqeneration: Most of the shoots were regenerated from the surface of the callus, whereas some came from embedded portions of the callus. When newly induced callus was directly subcultured to shoot inducing medium (WPM), most of the calli regenerated shoots within 30 days. The frequency of shoot regeneration on WPM containing 0.5mg/l BA was quite high and the total number of shoots produced was greatly affected by the hormonal constituents of the callus induction media (Table 2). The presence of BA in the callus inducing medium greatly
Table I. Callus induction from leaves of Populus tremuloides on modified B5 medium. Culture period was 30days.
BA (mg/l)
2,4-D (mg/l)
No. of explants
No. of explants forming callus
Relative growth
Color
0.0
0.5
30
8
+
red
0.0
5.0
40
16
++
red
0.i
0.5
30
27
+++
green
0.1
5.0
28
20
++
green
0.1
10.0
22
6
++
r ed-green
Table 2. The effect of hormonal constituents of callus inducing medium (roB5) on shoot regenration when subcultured to WPM with 0.5 mg/l B ~ Culture period was 30 days.
Callus inducing medium (mg/l)
No. of callus pieces
No. of pieces forming shoots
No. of shoots produced
0.5
2,4-D
3
2
5
5.0
2,4-D
4
2
2
0.5
2,4-D
+
0.i
BA
9
9
47
5.0
2,4-D
+
0.1
BA
8
5
49
10.0
2,4-D
+
0.1
BA
6
4
36
466 enhanced the number of shoots produced on the shoot inducing medium. One hundred percent of the calli produced on 0.5 mg/1 2,4-D and 0.1 mg/l BA regenerated shoots. Subculturing from liquid roB5 containing 0.i mg/l BA plus 0.5 mg/l 2,4-D onto WPM with 0.5 rag/1 caused shoot regeneration in approximately 50% of the callus pieces within 5 weeks . Other concentrations (0, 0.05 and 5.0 rag/l) of BA tested showed no shoot regeneration on WPM during this period (Table 3). On long term cultures, however, most of the remaining
Douglas (1982) reported that maximum shoot/bud regeneration occurred at 1.0 mg/l BA on MS medium (up to 33% with an average number of ii shoots/buds per callus) and at 1.0 mg/l BA + 1.0 mg/l NAA (80% shoot regeneration with an average number of 5 shoots/buds per callus). However, concentrations of 0.05 - 0.15 mg/l (Winton 1970) and 0.i0 - 0.3 mg/l (Wolter 1968) of BA have also been reported as optimum. In our experiments, no shoot regeneration occurred on 0.05 mg/l BA within 30 days. We also tried to regenerate shoots from callus on W P M or Nitsch medium with various concentrations of auxin
Table 3. The effect of BA concentration on shoot regeneration from callus of aspen on WPM. The callus was transferred from liquid roB5 medium containing 0.1 mg/l BA + 0.5 mg/l 2,4-D. Culture period was 35 days. The data are combined from 3 separate experiment~ Conc. of BA (rag/l)
No. of callus pieces
No. of pieces forming shoots
No. of shoots produced
0.0
13
0
0
0.05
15
0*
0
0.5
82
5.0
10
42 0*
248 0
* Two calli formed shoots (one each) within 3 months. ** Four calli formed shoots (1-4 shoots) within 2 months. calli also produced shoots. Many roots were observed from callus growing on the medium containing 0 and 0.05 mg/l BA, probably the result of high concentration of 2,4-D carried over from the previous culture medium. Compact andgreenish-white undifferentiated cells grew on the surface of the callus on WPM containing 5.0 mg/l BA. When these undifferentiated calli were transferred onto the same medium with 0.5 mg/l BA, they began to regenerate shoots within 2 weeks. Root Induction and Transplant t__qGreenhouse Shoots regenerated from callus were excised and placed on solidified WPM or mB5 with 0.2 mg/l IBA for rooting. Nearly 100% of the shoots produced roots within 10 days. Rooted plants were transplanted to sterilized soil in 2-6" diameter pots. The plants had to be acclimatized under intermittent mist for 2-4 weeks or covered with transparent polyethylene bag~ before resuming normal growth in the green house.
DISC~RSIGN
Wolter (1968) reported that aspen (Populus tremuloides) callus showed shoot regeneration when left on the same medium for several months. In earlier experiments, we had observed that some calli eventually regenerated shoots or roots if maintained for long periods of time without subculture. Ageing and changing of hormonal balance in the tissue with time might be responsible for such regeneration. However, in many cases, regeneration was sporadic and required long periods of time (2-4 months). In the current experiments, shoot regeneration occurred within 16-30 days in culture.
(2,4-D) in combination with 0.5 mg/l BA which were unsuccessful. The fact that callus frequently produced roots on media with a low concentration of BA (0.05 mg/l) or without BA and lacking auxin indicates that the calli contained substantial amounts of auxin carried over from previous media (roB5 with 0.i mg/l BA and 0.5 mg/l 2,4-D). Exogenous auxin was found to be unnecessary in shoot inducing medium. Wolter (1968) had earlier suggested that the degree of aeration might be a controlling factor for shoot regeneration on the underside of callus embedded in agar. His attempts to explore this possibility using liquid culture, however, proved unsuccessful. Douglas (1982) reported shoot regeneration from suspension cultured cell colonies of hybrid poplar with MS medium. In our studies, however, MS medium was unsuitable for suspension cultur~ On the other hand, in roB5 medium containing 0.I mg/l BA and 0.5 mg/l 2,4-D, we observed many nodular structures on the surface of sieved cell clumps within 2 weeks of suspension culture. Examining the tissue after sectioning showed that there was no organized shoot tip-like structure on the surface. When transferred to shoot inducing media, 30-60 shoot tips were observed on the surfaces of many clumps. Up to 26 shoot tips from callus clumps (3-5 mm in diameter) developed into plantlets, with most clumps producing less than 20 shoots because of dedifferentiation of many shoot tips. Attempts to induce shoots from clumps in liquid W P M with 0.i or 0.5mg/l BA were unsuccessful. The color and texture of the callus proved interesting. When newly induced, most of calli were green and relatively compact on mB5 with 0.1 mg/l BA plus 0.5 or 5.0 mg/l 2,4-D. These calli turned yellowish-red (sometimes light brown) on W P M with
467 0.5mg/l BA and produced shoots. Although Nitsch medium with thesame combination and concentration of growth regulators produced vigorous callus growth, most calli were soft and could not regenerate shoots when subcultured onto shoot inducing medium (WPM). Clumps passed through sieve and grown in suspension culture were much more compact anddarker gree~ Many new cells with red pigmentation grew on the surface of the clumps following transfer onto WPM for shoot induction. Although aspen is known to be difficult to root from woody cuttings (Ahuja 1983a,b, 1986), plantlets regenerated from callus were easily rooted on WPM, mB5 or Nitsch medium with 0.2 mg/l IBA. Ease of rooting was possibly due to juvenility. When the rooted plants were transplanted directly to the green
house, many were infected by fungi. Planting the rooted aspen in boxes containing sterilized soil protected by polyethylene film and leaving these boxes in the culture room for two weeks prior to transplanting to the green house yielded almost 100% survival. There was no striking morphological variation among plants regenerated from callus. However, isoenzyme studies showed that there were some variation in iso-citric dehydrogenase (IDH) and Shikimic dehydrogenase (SDH). We are now trying to induce callus from these variants and to regenerate plants again. Change of isoenzyme pattern from these variants will be examined in details to establish the nature of this somaclonal variatio~
Scientific Contribution No. 1455 from New Hampshire Agricultural Experiment Station.
Ahuja MR (1983a) Silvae Genet. 32: 131-135. Ahuja MR (1983b) Silvae Genet. 32: 225-227.
Gamborg OL, Miller A, Ojima K (1968) Exp. Cell Res. 50: 151-158.
Ahuja MR (1984) Silvae Genet. 33: 174-176.
Kim JH, Shim SY, Noh EW, Park JI (1982) Res. Rep. Inst. For. Gen. Korea. 18: 80-85.
Ahuja MR (1986) In: tissue culture in forestry. J.M. Bonga and D . J . Durzan (eds.) Martinus-Nijhoff, Amsterdam. In Press.
Lloyd G, McCown B (1981) In: Comb Proc Intern Plant Prop Soc. 30:421-427
Ahuja MR, Muhs H-J (1982) In: Proc. 5th Intern.Cong. Plant Tissue and Cell Culture, Tokyo. pp 177-178. Chalupa V (1974) Biol. Plant. 16: 316-320. Christie CB (1978) Comb. Proc. Intern Plant Prop Soc. 28: 255-260. Chun YW, Hall R, Stephens L (1986) Plant Cell Tissue Organ Culture. 5: 179-185. Douglas G (1982) In: Proc. 5th Intl. Cong. Plant Tissue and Cell Culture, Tokyo. pp 605-606. Fr~hlich HJ, Weisgerber H (1985) Silvae Genet. 34: 132-137.
Murashige T, Skoog F (1962) Physiol. Plant. 15: 473492 Wann SR, Einspar DW (1986) Silvae Genet. 35:19-24 Winton LL (1968) Science 160:1234-1235 Winton LL (1970) Amer. J. Bot. 57(8): 904-909 Winton
LL (1971) Forest Sci. 17:348-350
Wolter
KE (1968) Nature 219:509-510
Plant Cell Reports (1986) 5:464-467
© Springer-Verlag1986
High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.) Eun-Woon Noh and Subhash C. Minocha Department of Botany and Plant Pathology, University of New Hampshire, Durham, NH 03824, USA Received August 25, 1986 / Revised version received October 19, 1986 - Communicated by A. R. Gould
ABS~R~
Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/l benzyladenine (BA) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/l BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3-4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/l BA for shoot regeneration. Almost 100% of the clumps formed shoots on W P M when subcultureddirectly from mB5 with anaverage number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or W P M with 0.2 mg/l indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2-4 weeks before normal growth in the greenhouse.
AEB~mr~TICRS
BA: 6-Benzyl-adenine, IBA: indole-3-butyric acid, 2,4-D: 2,4-dichlorophenoxyacetic acid, mB5 medium: modified B5 medium, WPM: Woody Plant medium.
Shoot differentiation from callus is a crucial s t e p for s o m a t i c cell f u s i o n and g e n e t i c transformation of plants by foreign DNA. It has, however, been very difficult to regenerate shoots from callus of woody plants. This has made many woody plants unsuitable for application of recombinant DNA technology. Among the woody plants, various species of the genus Populus have been studied extensively for their capacity to regenerate in i__nnvitro cultures (P. tremula: Winton 1968, 1970, 1971; P__L.tremuloides: Wolter 1968; P. tremula. P__L.euramericana: Chalupa, 1974; P__~.trgmula and P. alba _x glandulosa: Christie 1978; P__~.alba _x glandulosa and P__~.euramericana: Kim et al 1982; P__~. tremula, P_~.tremuloides, and their hybrid: Ahuja, 1983 a,b, 1984, 1986, Ahuja and Muhs 1982; P__~.
Offprint requests to: S. C. Minocha
tremula and P. tremuloides: FrShlich and Weisgerber 1985; P_~.albax P__~.grandidentata: Chun et al 1986; P__~. tremuloides: Wann and Einspar 1986). Whereas adventitious shoots have been successfully regenerated from stem and leaf segments or axillary buds of poplars, shoot regeneration from callus has been very difficult. Wolter (1968) and Winton (1968, 1970) reported limited success with shoot regeneration from callus of aspen. Douglas (1982), using suspension culture techniques, reported good shoot regeneration from internode-derivedcallus of hybrid poplar (Populus hybrid TT32). We report here a reproducible high frequency shoot regeneration method from callus and suspension cultures of quaking aspen (Populus tremuloides). ~
AND M~n~3uS
Callus Induction Full sib seeds (XT-19-84) of Populus tremuloides were obtained from the Institute of Paper Chemistry, Appleton, WI and stored at 4°C. Seeds were surface sterilized with 0.2% HgCI 2 solution for 2 minutes, washed with sterile distilled water and placed on the MS (Murashige and Skoog 1962) medium containing 0.2 mg/l BA. Germinated shoots were subcultured onto the same medium for multiple shoot production. Twenty to forty shoots were regenerated from each shoot segment in 25 days on this medium (data not shown). These plantlets were excised again and transferred to mB5 (Gamborg et al 1968) with 0.2 mg/l IBA for rooting. Leaves were excised only from rooted shoots (about 4 cm tall) and placed on the roB5 media containing 0.5 or 5.0 mg/l 2,4-D alone and 0.i mg/l BA combined with 0.5, 5.0, and i0 mg/l 2,4-D. Shoot Reaeneration Callus clumps grew to a diameter of 3-5 m m in 20 days. Callus clumps were subcultured directly onto W P M (Lloyd and McCown 1981) with 0.5 mg/l BA for shoot induction. For suspension culture, approximately 500 mg of callus clumps grown on solidified mB5 medium with 0.i mg/l BA plus 0.5 mg/l 2,4-D were passed through a 1 mm sieve and grown in 50 ml of liquid mB5 medium with 0.I mg/l BA and 0.5 mg/l 2,4-D in 125 ml Erlenmyer flask. The flasks were kept on gyratory shakers at 150 rpm. The cell
465 I~KLT
clumps grew to 3-4 mm in diameter after 3 weeks in suspension culture. Tney were then transferred onto solidified W P M with 0.05, 0.5 and 5.0 mg/l BA in petri dishes or in 125 ml Erlenmeyer flasks for shoot inductioru
Initiation and growth of callus: Calli were formed easily on mB5 medium. The frequency, growth and color of calli varied with the concentration of hormones (Table i). Cultures on medium lacking BA or having high concentration of 2,4-D resulted in calli with red pigmentation. The c o m b i n a t i o n of 0.I m g / l BA and 0.5 mg/l 2,4-D produced the best quality of callus. These calli were relatively compact and when subcultured onto W P M for shoot induction, turned to a yellowish-red color. In suspension culture, calli grew rapidly and reached a 3-4 mm diameter within 2 weeks. These calli were dark green and compact. When subcultured to W P M with 0.5 mg/l BA for shoot induction, however, the calli developed red pigmentation and many soft fiber-like surface cells.
Shoots formed on W P M with 0.5 mg/l BA were excised and subcultured on either WPM or mB5 with 0.2 mg/l IBA for rooting. Rooted plants were transplanted to pots containing vermiculite, perlite and sand (I:I:I) and kept under intermittent mist for 2-4 weeks before normal growth in the green house. Culture Conditions and Media Throughout the experiment, cultures were kept under constant light (20 pEm-2sec -I for callus induction and shoot regeneration and 50 ~Em-2sec-I for root induction) at 25+2°C. All m e d i a w e r e adjusted to pH 5.6 prior to autoclaving at 121°C and 15 ibs psi for 25 rain. The mB5 medium used for callus induction and liquid culture contained the following in 1 liter: 150 mg KCI, 2500 mg KNO 3, 250 mg MgSO4.7H20, 150 mg CaCI2.2H20, 300 mg NaH2PO4.H20, 150 mg (NH4)2SO4, 27.8 mg FeSO4.7H20, 37.3 mg Na 2I~3TA, and micronutrients descrlbed in Gamborg et al (1968). The medium also contained i00 mg myoinositol, 1 mg thiamine-H~l, 1 mg pyridoxine-HCl, 1 mg nicotinic acid, 7 g Phytagar (deleted in liquid medium) and 30 g sucrose. Woody Plant Medium of Lloyd and M c C o w n (1981) was used for shoot regeneration.
Shoot Reqeneration: Most of the shoots were regenerated from the surface of the callus, whereas some came from embedded portions of the callus. When newly induced callus was directly subcultured to shoot inducing medium (WPM), most of the calli regenerated shoots within 30 days. The frequency of shoot regeneration on WPM containing 0.5mg/l BA was quite high and the total number of shoots produced was greatly affected by the hormonal constituents of the callus induction media (Table 2). The presence of BA in the callus inducing medium greatly
Table I. Callus induction from leaves of Populus tremuloides on modified B5 medium. Culture period was 30days.
BA (mg/l)
2,4-D (mg/l)
No. of explants
No. of explants forming callus
Relative growth
Color
0.0
0.5
30
8
+
red
0.0
5.0
40
16
++
red
0.i
0.5
30
27
+++
green
0.1
5.0
28
20
++
green
0.1
10.0
22
6
++
r ed-green
Table 2. The effect of hormonal constituents of callus inducing medium (roB5) on shoot regenration when subcultured to WPM with 0.5 mg/l B ~ Culture period was 30 days.
Callus inducing medium (mg/l)
No. of callus pieces
No. of pieces forming shoots
No. of shoots produced
0.5
2,4-D
3
2
5
5.0
2,4-D
4
2
2
0.5
2,4-D
+
0.i
BA
9
9
47
5.0
2,4-D
+
0.1
BA
8
5
49
10.0
2,4-D
+
0.1
BA
6
4
36
466 enhanced the number of shoots produced on the shoot inducing medium. One hundred percent of the calli produced on 0.5 mg/1 2,4-D and 0.1 mg/l BA regenerated shoots. Subculturing from liquid roB5 containing 0.i mg/l BA plus 0.5 mg/l 2,4-D onto WPM with 0.5 rag/1 caused shoot regeneration in approximately 50% of the callus pieces within 5 weeks . Other concentrations (0, 0.05 and 5.0 rag/l) of BA tested showed no shoot regeneration on WPM during this period (Table 3). On long term cultures, however, most of the remaining
Douglas (1982) reported that maximum shoot/bud regeneration occurred at 1.0 mg/l BA on MS medium (up to 33% with an average number of ii shoots/buds per callus) and at 1.0 mg/l BA + 1.0 mg/l NAA (80% shoot regeneration with an average number of 5 shoots/buds per callus). However, concentrations of 0.05 - 0.15 mg/l (Winton 1970) and 0.i0 - 0.3 mg/l (Wolter 1968) of BA have also been reported as optimum. In our experiments, no shoot regeneration occurred on 0.05 mg/l BA within 30 days. We also tried to regenerate shoots from callus on W P M or Nitsch medium with various concentrations of auxin
Table 3. The effect of BA concentration on shoot regeneration from callus of aspen on WPM. The callus was transferred from liquid roB5 medium containing 0.1 mg/l BA + 0.5 mg/l 2,4-D. Culture period was 35 days. The data are combined from 3 separate experiment~ Conc. of BA (rag/l)
No. of callus pieces
No. of pieces forming shoots
No. of shoots produced
0.0
13
0
0
0.05
15
0*
0
0.5
82
5.0
10
42 0*
248 0
* Two calli formed shoots (one each) within 3 months. ** Four calli formed shoots (1-4 shoots) within 2 months. calli also produced shoots. Many roots were observed from callus growing on the medium containing 0 and 0.05 mg/l BA, probably the result of high concentration of 2,4-D carried over from the previous culture medium. Compact andgreenish-white undifferentiated cells grew on the surface of the callus on WPM containing 5.0 mg/l BA. When these undifferentiated calli were transferred onto the same medium with 0.5 mg/l BA, they began to regenerate shoots within 2 weeks. Root Induction and Transplant t__qGreenhouse Shoots regenerated from callus were excised and placed on solidified WPM or mB5 with 0.2 mg/l IBA for rooting. Nearly 100% of the shoots produced roots within 10 days. Rooted plants were transplanted to sterilized soil in 2-6" diameter pots. The plants had to be acclimatized under intermittent mist for 2-4 weeks or covered with transparent polyethylene bag~ before resuming normal growth in the green house.
DISC~RSIGN
Wolter (1968) reported that aspen (Populus tremuloides) callus showed shoot regeneration when left on the same medium for several months. In earlier experiments, we had observed that some calli eventually regenerated shoots or roots if maintained for long periods of time without subculture. Ageing and changing of hormonal balance in the tissue with time might be responsible for such regeneration. However, in many cases, regeneration was sporadic and required long periods of time (2-4 months). In the current experiments, shoot regeneration occurred within 16-30 days in culture.
(2,4-D) in combination with 0.5 mg/l BA which were unsuccessful. The fact that callus frequently produced roots on media with a low concentration of BA (0.05 mg/l) or without BA and lacking auxin indicates that the calli contained substantial amounts of auxin carried over from previous media (roB5 with 0.i mg/l BA and 0.5 mg/l 2,4-D). Exogenous auxin was found to be unnecessary in shoot inducing medium. Wolter (1968) had earlier suggested that the degree of aeration might be a controlling factor for shoot regeneration on the underside of callus embedded in agar. His attempts to explore this possibility using liquid culture, however, proved unsuccessful. Douglas (1982) reported shoot regeneration from suspension cultured cell colonies of hybrid poplar with MS medium. In our studies, however, MS medium was unsuitable for suspension cultur~ On the other hand, in roB5 medium containing 0.I mg/l BA and 0.5 mg/l 2,4-D, we observed many nodular structures on the surface of sieved cell clumps within 2 weeks of suspension culture. Examining the tissue after sectioning showed that there was no organized shoot tip-like structure on the surface. When transferred to shoot inducing media, 30-60 shoot tips were observed on the surfaces of many clumps. Up to 26 shoot tips from callus clumps (3-5 mm in diameter) developed into plantlets, with most clumps producing less than 20 shoots because of dedifferentiation of many shoot tips. Attempts to induce shoots from clumps in liquid W P M with 0.i or 0.5mg/l BA were unsuccessful. The color and texture of the callus proved interesting. When newly induced, most of calli were green and relatively compact on mB5 with 0.1 mg/l BA plus 0.5 or 5.0 mg/l 2,4-D. These calli turned yellowish-red (sometimes light brown) on W P M with
467 0.5mg/l BA and produced shoots. Although Nitsch medium with thesame combination and concentration of growth regulators produced vigorous callus growth, most calli were soft and could not regenerate shoots when subcultured onto shoot inducing medium (WPM). Clumps passed through sieve and grown in suspension culture were much more compact anddarker gree~ Many new cells with red pigmentation grew on the surface of the clumps following transfer onto WPM for shoot induction. Although aspen is known to be difficult to root from woody cuttings (Ahuja 1983a,b, 1986), plantlets regenerated from callus were easily rooted on WPM, mB5 or Nitsch medium with 0.2 mg/l IBA. Ease of rooting was possibly due to juvenility. When the rooted plants were transplanted directly to the green
house, many were infected by fungi. Planting the rooted aspen in boxes containing sterilized soil protected by polyethylene film and leaving these boxes in the culture room for two weeks prior to transplanting to the green house yielded almost 100% survival. There was no striking morphological variation among plants regenerated from callus. However, isoenzyme studies showed that there were some variation in iso-citric dehydrogenase (IDH) and Shikimic dehydrogenase (SDH). We are now trying to induce callus from these variants and to regenerate plants again. Change of isoenzyme pattern from these variants will be examined in details to establish the nature of this somaclonal variatio~
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