Plant Cell Reports (1985) 4:126-128
PlantCell Reports © Springer-Verlag 1985
Studies on shoot regeneration of lupins (Lupinusspp.) Christine Sator Institut for Pflanzenbau und Pflanzenzt~chtung, Bundesforschungsanstalt for Landwirtschaft Braunschweig-V61kenrode (FAL),
Bundesallee 50, D-3300 Braunschweig, FRG Received January 3, 1985 / Revised version received March 18, 1985 - Communicated by J. Potrykus
Abstract Tests have been made to regenerate plants from different lupin species using leaf, leaf petlole and hypocotyl tissue of t. polyphyllus, L. hartwegii, L. angustiFolius and L. luteus. Callus induction rates have been high with all plant parts from all tested lupin species. Regeneration success has been rather limited. One plant could be regenerated from hypoeotyl material of L. polyphyllus. Only shoots appeared when using leaf explants from L. luteus or L. hartwegii as well as from leaf petiole tissue of L. hartwegii. With L. angustifolius one regeneration of unknown character occurred.
Introduction Many plant species have been tested in respect to their regeneration capability but only few of them are belonging to the Family of Leguminosae (PierJk 1979, Vasil 1980 and Evans et el. 1983). N vertheless, success in regenerating plants has been reported for some legumes like e. g. Arachis hypogea (Bajaj et el. 1981), Lathyrus sativus (Gharyal et al. I"983 ), Lotus corniculatus (Niizeeki and Grant 1971), Medioago saliva (Saunders et at. 1972), Onobrychis vieiaefolia (Areioni eL at. 1983), Phaseolus vulgaris (Crocomo et al. 1976), Stylosanthes guyanensis (Meijer et at. 1982), Psophocarpus tetragonolobus (Bottino et el. 1979), Trifolium resupinatum (Oelck et al. 1983) and Vigna aconitifolia (Krishnamurthy et al, 1984). Within the genus Lupinus rooting of L. albus shoot tips has been achieved by Bail (1946), and Lee (1955) succeeded in rooting or shoot tips of L. hartwegii. Up to date no Further literature seems to be concerned with "in vitro" propagation or lupins. Because of the great importance of "in vitro" techniques for lupin breeders, some experiments have been done in this direction (see also Sator et el. 1982, Sator 1983, Sator 1984, Sator 1985).
Material and Methods Studies have been conducted using tissue of leaf petiole, leaves, hypocoLyl and roots of Lupinus polyphyllus, L. luteus cv. 'Palra', L. hartwegii and t. angustifolius ev. 'Kubesa'. Additionally, the rooting capability or shoot tips from L. polyphyltus, L. luteus and L. angustifolius has been examined. Plants or the above mentioned species have been grown aseptically after sterilising the seeds 20 - 30 min
in 5 % Domestos solution Followed by a short incubation in 96 % ethanol. Afterwards the seeds have been washed with autoclaved water three times under sterile conditions and scratched seeds remained in water For several hours to swell. The seed coats have been removed from the swollen seeds before the embryos have been placed on culture medium. Two to eiqht weeks old p l a n t s have been cut i n t o pieces which were placed on c a l l u s i n d u c t i o n medium. After callus induction under dark conditions at 24 °C the plant material has been transferred to a differentiation medium and placed in chambers having a constant temperature of 24 °C with ~tlumination For 12 hours or 200/15 ° C day/night temr;rature with a 16 h daylength. Culture medium t o grow p l a n l s aseptically has been prepared from Lap water adding 6 g o f a g a r / l . For %esLing the r o o t i n g c a p a b i l i t y o f shoot t i p s Lhe same medium w i t h 20 g r e f i n e d s u g a r / l has been used, For callus induction several media have been tested, the composition of which varied in respect to hormon~ combination and concentration as well as in vitamin content and addition of caseinhydrolysate, as shown in table ]. Table i: Media composition of callus induction media (Basic medium according to Nurashige and Skoog 1962) Organics mg/l
II
I2
IAA ~A 2,4 D KIN NAA vitamins glycin pyridoxine HC1 nicotinic acid thiamine HCl caseinhydrolysate
1 1 i
. 2 2 N&S . . . . .
IAA BA 2,4 KIN NAA M&S
= = D = = =
-
M&S . . . . .
. . . . .
13 .
14
. . 2 1,5 0,2 2 M&S M&S
. . . . .
indolylaeetic acid benzyladenine = 2,4 diehlorphenoxyacetic acid kinetine naphtylaeetic acid according to Murashige and Skoog 1962
15
2 1,25 2 i0 5 5 2g
127 Altogether, 95 different hormon combinations and concentrations have been tested during regeneration experiments. All the media have been adjusted to pH 5.8 with NaOH/HCI before being autoclaved at 120 °C/ 1.1 bar for 15 min.
shoot or root formation. It did not develop further except showing branching. Root formation only could be achieved with I. polyphyllus on hormon free medium. Because of the difficulties in rooting the regenerated shoots of I. hartwegii and L. luteus, the root forming capability has been tested with shoot tips of seedlings from I. polyphyllus, L. luteus and L. angustifolius. L. hartwegii has not been tested because of the known rooting capability of this lupin species from literature (Lee 1955). L. polyphyllus shoot tips could all be rooted (i0 from lO), L. angustiFolius shoot tips could be rooted to 70 Z (7 from i0) (Fig. 3) as well as 60 Z (3 from 5) of the I. luteus shoot tips.
Results and Discussion Even though callus induction rates have been high for all plant parts on all tested media (table 2), regeneration could only be achieved after callus induction on medium I3. Callus induction period has been two to three weeks. After this shoot regeneration showed up within four weeks from leaf tissue of L. hartwegii, within six weeks using hypoeotyl tissue of L. polyphyllus and within ten weeks using leaf tissue of L. luteus. Details are given in table 3.
No precise explanation can be given for the bad rooting capability of isolated apices after shoot regeneration from different plant parts in contrast to the relatively good rooting capability of isolated apices from normally grown seedlings. The regenerated shoots of L. hartwegii even continued to multiply
Table 2: Callus induction of different plant parts from several lupin species on different culture media (absolute figures)* Medium
Species L. angustifolius
L. luteus 1
2
1
3
2
L. polyphyllus 1
3
2
I. hartwegii 3
1
2
I1
40/37
40/40
40/39
40/40
40/40
40/36
40/40
-
-
40/38
40/40
60/50
12
40/35
40/35
40/40
40/40
40/40
40/36
40/24
40/40
40/37
40/39
40/40
50/49
I3
320/313
310/288
160/150
130/]30
70/70
90/90
120/90
100/46
]]0/87
180/]75
250/122
260/176
I4
40/39
40/40
40/40
40/40
40/40
40/40
40/32
40/40
40/33
40/35
40/32
60/58
15
40/36
40/40
40/38
40/13
40/36
-
-
40/39
40/34
40/18
* The first figure indicates the number of cultivated plant parts, the second Figure gives the number of plant parts showing callus 1 = leaf, 2 = hypocotyl,
3 = root
Table 3: Shoot regeneration of Lupinus species from different plant parts (results after elimination of infected cultures) SpeciesJcv.
plant part
differentiation
L. polyphyllus
hypocotyl
1 plant (from 6 pieces)
D1
L. hartwegii
leaf
2 bulks of shoals (from 19 pieces
D2
leaf petiole
1 bulk of shoots (from 5 pieces)
D2
L. luteus
'Palfa'
L. angustifolius
leaf
'Kubesa'
leaf
medium
i shoot (from 15 pieces)
D1
2 shoots (from 15 pieces)
D2
] unknown differn.
D2
Shoot regeneration has been achieved with either M&S basic medium plus 2 mg BA and 2 mg IAA/I (D1) or with M&S basic medium plus 2 mg BA and 0,2 mg IAA/I (D2). For root regeneration a hormon free medium with M&S mineral salts of half the concentration reported by Murashige and Skoog (1962) has been used as well as a medium with only IAA in various concentrations up to 5 mg/l. Regeneration of shoots achieved with L. hartwegii leaf material started in one case as single shoot as shown in Figure 1. After this shoot had been separated From the old leaf tissue and transferred to fresh medium it started to multiply at the basis of the separated shoot. In the second case direct multiple shoot formation occurred at the original leaf tissue as it also appeared when using petiole tissue (fig. 2). The regeneration which has been achieved with L. angustifolius leaf material could not be identified as either
(from 3 pieces
after transfer to rooting medium. Shoots of L. ]uteus died after several transfers on rooting medium but before that new shoots started to develop at the basis of the isolated regenerated shoots, as can be seen in figure 4.
128 References Arcioni I e t al. (1983) Tissue culture and plant regeneration in Onobrychis viciaefolia Stop. Z. Pflanzenz. 90:192-197 Bajaj YPS et al. (1981) Regeneration of plants from seedling explants and callus of Arachis hypogea L. Ind. J. Exp. Biol. 19:1026-1029 Ball E (1946) Development in sterile culture of stemtips and subjacent regions of Tropaedum majus L. and Lupinus albus L. American J. of Bot. 33: 301318 Bottino PJ et al. (1979) Tissue culture and organogenesis in the winged bean. Can. J. Bot. 57: 1773-1776 Crocomo OJ et al. (1976) Plantlet morphogenesis and the control of callu~ growth of P~a~eolus vuigaris in tissue culture, Arquivos de Biologiae Technologia 18:25-31
Martinus Nijhoff Publishers, The Hague Boston London astor C (1984) Chances of Crop Improvement in Lupins using Tissue Culture Techniques In: 3rd International Lupln Conference, Proceedings, La Rochelle, France pp 183-190 Saint C (1985) Regeneration yon Lupinenpflanzen aus Antheren. Landbauforschung V61kenrode 35/1, im Druck Sator C (1985) Regeneration yon Lupinenpflanzen aus Embryonen,Landbauforschung V61kenrode 35/1, im Druck astor C (1985) Induktion einer "in vitro-VieifachsproBbiJdung" an Samen yon Lupinen.Landbauforschung V61kenrode 35/1, im Druck Saunders JW et al. (1972) Production of alfalfa plants from callus tissue. Crop Sci. 12:804-808 Vasil IK (1980) International Review of Cytology Suppl IIA and Perspectives in Plant Cell and Tissue Culture, Academic Press
Evans DA et al. (1983) Applications of tissue culture technology to development of improved crop varieties In: Biotech 83 Proceedings of the international conference on the commercial application and implications of biotechnology, Online Publications, Northwood Mills Gharyal PK et al. (1983) Genetic and physiological influences as differentiation in tissue culture of a legume, tsthyrus sativus. Theor. Appl. Geneti 66: 123-126 Krishnamurthy KV et al. (1984) Studies on a drought resistant legume: The moth bean, Vigna aconitifolia I Protoplast culture and organogenesis. Plant Cell Rep. 3 : 3 0 Lee AE (1955) Growth in culture of excised portions of lupin embryos. Bot. Gaz. 116:359-364 Meijer EGM (1982) High frequency plant regeneration from hypocotyl derived tissue cultures of the tropical pasture legume Stylosanthes humiliso Phys. Plant 56:381-385 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497 Niizeeki M e t al. (1971) Callus plantlet formation and ploidy from cultured anthers of Lotus and Nicotisna. Can. J. Bot. 49:2041-2051 Oelck MM et al. (1983) Genotypic differences in some legume species affecting the redifferentiation ability from callus to plants. Z. Pflanzenz. 91: 312-321 Pierik RLM (1979) In vitro culture of higher plants. Wageningen, The Netherlands Sator C et al. (1982) Antherenkulturversuche mit Lupinus polyphyllus, tandbauforschung V~lkenrode 32: 37-42 Sator C (1983) In vitro breeding of lupins In: Perspectives for peas and lupins as protein crops.
Fig. i: RegeneraLed shooL from a leaf of L. i,artwegii Fig. 2: Starting multiple shoot formation from a - petiole piece of L. hartwegii Fig. 3: Rooted shoot tip of L. angustifolius Fig. 4: Regenerated shoot from L. luteus leaf starting ' multiplication
PlantCell Reports © Springer-Verlag 1985
Studies on shoot regeneration of lupins (Lupinusspp.) Christine Sator Institut for Pflanzenbau und Pflanzenzt~chtung, Bundesforschungsanstalt for Landwirtschaft Braunschweig-V61kenrode (FAL),
Bundesallee 50, D-3300 Braunschweig, FRG Received January 3, 1985 / Revised version received March 18, 1985 - Communicated by J. Potrykus
Abstract Tests have been made to regenerate plants from different lupin species using leaf, leaf petlole and hypocotyl tissue of t. polyphyllus, L. hartwegii, L. angustiFolius and L. luteus. Callus induction rates have been high with all plant parts from all tested lupin species. Regeneration success has been rather limited. One plant could be regenerated from hypoeotyl material of L. polyphyllus. Only shoots appeared when using leaf explants from L. luteus or L. hartwegii as well as from leaf petiole tissue of L. hartwegii. With L. angustifolius one regeneration of unknown character occurred.
Introduction Many plant species have been tested in respect to their regeneration capability but only few of them are belonging to the Family of Leguminosae (PierJk 1979, Vasil 1980 and Evans et el. 1983). N vertheless, success in regenerating plants has been reported for some legumes like e. g. Arachis hypogea (Bajaj et el. 1981), Lathyrus sativus (Gharyal et al. I"983 ), Lotus corniculatus (Niizeeki and Grant 1971), Medioago saliva (Saunders et at. 1972), Onobrychis vieiaefolia (Areioni eL at. 1983), Phaseolus vulgaris (Crocomo et al. 1976), Stylosanthes guyanensis (Meijer et at. 1982), Psophocarpus tetragonolobus (Bottino et el. 1979), Trifolium resupinatum (Oelck et al. 1983) and Vigna aconitifolia (Krishnamurthy et al, 1984). Within the genus Lupinus rooting of L. albus shoot tips has been achieved by Bail (1946), and Lee (1955) succeeded in rooting or shoot tips of L. hartwegii. Up to date no Further literature seems to be concerned with "in vitro" propagation or lupins. Because of the great importance of "in vitro" techniques for lupin breeders, some experiments have been done in this direction (see also Sator et el. 1982, Sator 1983, Sator 1984, Sator 1985).
Material and Methods Studies have been conducted using tissue of leaf petiole, leaves, hypocoLyl and roots of Lupinus polyphyllus, L. luteus cv. 'Palra', L. hartwegii and t. angustifolius ev. 'Kubesa'. Additionally, the rooting capability or shoot tips from L. polyphyltus, L. luteus and L. angustifolius has been examined. Plants or the above mentioned species have been grown aseptically after sterilising the seeds 20 - 30 min
in 5 % Domestos solution Followed by a short incubation in 96 % ethanol. Afterwards the seeds have been washed with autoclaved water three times under sterile conditions and scratched seeds remained in water For several hours to swell. The seed coats have been removed from the swollen seeds before the embryos have been placed on culture medium. Two to eiqht weeks old p l a n t s have been cut i n t o pieces which were placed on c a l l u s i n d u c t i o n medium. After callus induction under dark conditions at 24 °C the plant material has been transferred to a differentiation medium and placed in chambers having a constant temperature of 24 °C with ~tlumination For 12 hours or 200/15 ° C day/night temr;rature with a 16 h daylength. Culture medium t o grow p l a n l s aseptically has been prepared from Lap water adding 6 g o f a g a r / l . For %esLing the r o o t i n g c a p a b i l i t y o f shoot t i p s Lhe same medium w i t h 20 g r e f i n e d s u g a r / l has been used, For callus induction several media have been tested, the composition of which varied in respect to hormon~ combination and concentration as well as in vitamin content and addition of caseinhydrolysate, as shown in table ]. Table i: Media composition of callus induction media (Basic medium according to Nurashige and Skoog 1962) Organics mg/l
II
I2
IAA ~A 2,4 D KIN NAA vitamins glycin pyridoxine HC1 nicotinic acid thiamine HCl caseinhydrolysate
1 1 i
. 2 2 N&S . . . . .
IAA BA 2,4 KIN NAA M&S
= = D = = =
-
M&S . . . . .
. . . . .
13 .
14
. . 2 1,5 0,2 2 M&S M&S
. . . . .
indolylaeetic acid benzyladenine = 2,4 diehlorphenoxyacetic acid kinetine naphtylaeetic acid according to Murashige and Skoog 1962
15
2 1,25 2 i0 5 5 2g
127 Altogether, 95 different hormon combinations and concentrations have been tested during regeneration experiments. All the media have been adjusted to pH 5.8 with NaOH/HCI before being autoclaved at 120 °C/ 1.1 bar for 15 min.
shoot or root formation. It did not develop further except showing branching. Root formation only could be achieved with I. polyphyllus on hormon free medium. Because of the difficulties in rooting the regenerated shoots of I. hartwegii and L. luteus, the root forming capability has been tested with shoot tips of seedlings from I. polyphyllus, L. luteus and L. angustifolius. L. hartwegii has not been tested because of the known rooting capability of this lupin species from literature (Lee 1955). L. polyphyllus shoot tips could all be rooted (i0 from lO), L. angustiFolius shoot tips could be rooted to 70 Z (7 from i0) (Fig. 3) as well as 60 Z (3 from 5) of the I. luteus shoot tips.
Results and Discussion Even though callus induction rates have been high for all plant parts on all tested media (table 2), regeneration could only be achieved after callus induction on medium I3. Callus induction period has been two to three weeks. After this shoot regeneration showed up within four weeks from leaf tissue of L. hartwegii, within six weeks using hypoeotyl tissue of L. polyphyllus and within ten weeks using leaf tissue of L. luteus. Details are given in table 3.
No precise explanation can be given for the bad rooting capability of isolated apices after shoot regeneration from different plant parts in contrast to the relatively good rooting capability of isolated apices from normally grown seedlings. The regenerated shoots of L. hartwegii even continued to multiply
Table 2: Callus induction of different plant parts from several lupin species on different culture media (absolute figures)* Medium
Species L. angustifolius
L. luteus 1
2
1
3
2
L. polyphyllus 1
3
2
I. hartwegii 3
1
2
I1
40/37
40/40
40/39
40/40
40/40
40/36
40/40
-
-
40/38
40/40
60/50
12
40/35
40/35
40/40
40/40
40/40
40/36
40/24
40/40
40/37
40/39
40/40
50/49
I3
320/313
310/288
160/150
130/]30
70/70
90/90
120/90
100/46
]]0/87
180/]75
250/122
260/176
I4
40/39
40/40
40/40
40/40
40/40
40/40
40/32
40/40
40/33
40/35
40/32
60/58
15
40/36
40/40
40/38
40/13
40/36
-
-
40/39
40/34
40/18
* The first figure indicates the number of cultivated plant parts, the second Figure gives the number of plant parts showing callus 1 = leaf, 2 = hypocotyl,
3 = root
Table 3: Shoot regeneration of Lupinus species from different plant parts (results after elimination of infected cultures) SpeciesJcv.
plant part
differentiation
L. polyphyllus
hypocotyl
1 plant (from 6 pieces)
D1
L. hartwegii
leaf
2 bulks of shoals (from 19 pieces
D2
leaf petiole
1 bulk of shoots (from 5 pieces)
D2
L. luteus
'Palfa'
L. angustifolius
leaf
'Kubesa'
leaf
medium
i shoot (from 15 pieces)
D1
2 shoots (from 15 pieces)
D2
] unknown differn.
D2
Shoot regeneration has been achieved with either M&S basic medium plus 2 mg BA and 2 mg IAA/I (D1) or with M&S basic medium plus 2 mg BA and 0,2 mg IAA/I (D2). For root regeneration a hormon free medium with M&S mineral salts of half the concentration reported by Murashige and Skoog (1962) has been used as well as a medium with only IAA in various concentrations up to 5 mg/l. Regeneration of shoots achieved with L. hartwegii leaf material started in one case as single shoot as shown in Figure 1. After this shoot had been separated From the old leaf tissue and transferred to fresh medium it started to multiply at the basis of the separated shoot. In the second case direct multiple shoot formation occurred at the original leaf tissue as it also appeared when using petiole tissue (fig. 2). The regeneration which has been achieved with L. angustifolius leaf material could not be identified as either
(from 3 pieces
after transfer to rooting medium. Shoots of L. ]uteus died after several transfers on rooting medium but before that new shoots started to develop at the basis of the isolated regenerated shoots, as can be seen in figure 4.
128 References Arcioni I e t al. (1983) Tissue culture and plant regeneration in Onobrychis viciaefolia Stop. Z. Pflanzenz. 90:192-197 Bajaj YPS et al. (1981) Regeneration of plants from seedling explants and callus of Arachis hypogea L. Ind. J. Exp. Biol. 19:1026-1029 Ball E (1946) Development in sterile culture of stemtips and subjacent regions of Tropaedum majus L. and Lupinus albus L. American J. of Bot. 33: 301318 Bottino PJ et al. (1979) Tissue culture and organogenesis in the winged bean. Can. J. Bot. 57: 1773-1776 Crocomo OJ et al. (1976) Plantlet morphogenesis and the control of callu~ growth of P~a~eolus vuigaris in tissue culture, Arquivos de Biologiae Technologia 18:25-31
Martinus Nijhoff Publishers, The Hague Boston London astor C (1984) Chances of Crop Improvement in Lupins using Tissue Culture Techniques In: 3rd International Lupln Conference, Proceedings, La Rochelle, France pp 183-190 Saint C (1985) Regeneration yon Lupinenpflanzen aus Antheren. Landbauforschung V61kenrode 35/1, im Druck Sator C (1985) Regeneration yon Lupinenpflanzen aus Embryonen,Landbauforschung V61kenrode 35/1, im Druck astor C (1985) Induktion einer "in vitro-VieifachsproBbiJdung" an Samen yon Lupinen.Landbauforschung V61kenrode 35/1, im Druck Saunders JW et al. (1972) Production of alfalfa plants from callus tissue. Crop Sci. 12:804-808 Vasil IK (1980) International Review of Cytology Suppl IIA and Perspectives in Plant Cell and Tissue Culture, Academic Press
Evans DA et al. (1983) Applications of tissue culture technology to development of improved crop varieties In: Biotech 83 Proceedings of the international conference on the commercial application and implications of biotechnology, Online Publications, Northwood Mills Gharyal PK et al. (1983) Genetic and physiological influences as differentiation in tissue culture of a legume, tsthyrus sativus. Theor. Appl. Geneti 66: 123-126 Krishnamurthy KV et al. (1984) Studies on a drought resistant legume: The moth bean, Vigna aconitifolia I Protoplast culture and organogenesis. Plant Cell Rep. 3 : 3 0 Lee AE (1955) Growth in culture of excised portions of lupin embryos. Bot. Gaz. 116:359-364 Meijer EGM (1982) High frequency plant regeneration from hypocotyl derived tissue cultures of the tropical pasture legume Stylosanthes humiliso Phys. Plant 56:381-385 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497 Niizeeki M e t al. (1971) Callus plantlet formation and ploidy from cultured anthers of Lotus and Nicotisna. Can. J. Bot. 49:2041-2051 Oelck MM et al. (1983) Genotypic differences in some legume species affecting the redifferentiation ability from callus to plants. Z. Pflanzenz. 91: 312-321 Pierik RLM (1979) In vitro culture of higher plants. Wageningen, The Netherlands Sator C et al. (1982) Antherenkulturversuche mit Lupinus polyphyllus, tandbauforschung V~lkenrode 32: 37-42 Sator C (1983) In vitro breeding of lupins In: Perspectives for peas and lupins as protein crops.
Fig. i: RegeneraLed shooL from a leaf of L. i,artwegii Fig. 2: Starting multiple shoot formation from a - petiole piece of L. hartwegii Fig. 3: Rooted shoot tip of L. angustifolius Fig. 4: Regenerated shoot from L. luteus leaf starting ' multiplication
