Summary. Callus cultures were initiated from in vitro grown leaf, stem and root segments of Lonicera japonica "Hall's Prolific', on a medium containing 10.7.
Plant Cell Reports
Plant Cell Reports (1993) 13:91-94
9 Springer-Verlag1993
Plant regeneration from aged-callus of the woody ornamental species Lonicera japonica cv. "Hall's Prolific" D. Georges 1, J.-C. C h e n i e u x 2, and S.J. Ochatt 1 1 I.N.R.A. Station d'Am61ioration des Esp6ces Fruiti6res et Omementales, F-49071 Beaucouz6-Angers, France 2 E.A. 1370 DRED, Plant Biotechnology Laboratory, Frangois Rabelais University, F-37200 "tours, France Received 14 June 1993/Revised version received 4 August 1993 - Communicated by A.M. Boudet
Summary. Callus cultures were initiated from in vitro grown leaf, stem and root segments o f L o n i c e r a japonica "Hall's Prolific', on a medium containing 10.7 p M ~-naphthtylacetic acid and 2.7 p M benzyladenine, while media with 2,4-dichlorophenoxyacetic acid led to a rapid necrosis of explants. Shoot regeneration from true-callus (i.e. without any part of the original explant) was achieved for the three different source ti~ues within 12 weeks. The highest rate of regeneration was obtained by using benzyladenine (4.4 to 44.4 p M ) as the sole hormone in the medium. The regenerated shoots were readily elongated and rooted on the same medium as used for multiplication, and plantlets were subsequently transferred to greenhouse conditions, where nearly 100% of them were successfully acclimatized. This is the first example of plant regeneration from aged (> 6 month-old) true-callus of a woody ornamental species.
Key words. Lonicera sp. ; woody ornamentals ; trne-callus ; plant regeneration. Abbreviations. 2,4-D : 2,4-dichlorophenoxyacetic acid ; 2iP : 2-isopentenyladenine ; BA : benzyladenine ; KIN : 6furfurylaminopurine = kinetin ; LS : Linsmaier and Skoog ; N A A ct-naphthylacetic acid ; TDZ : N-phenyl-N'-l,2,3thidiazol-5-ylurea (thidiazuron) ; WPM : woody plant medium ; Z : zeatin ; GA3 : gibberellic acid.
Introduction The genus Lonicera is divided in two groups : climbing and shrubby species. The climbing honeysuckles are characterized by their ornamental qualities, particularly with respect to the colour, size and fragrance of flowers (Wright 1983), but these plants need a support to grow. Shrubby species have less ornamental value than climbing ones, but their growth habit leads to the formation of a bush more valuable for the small european garden. Regeneration o f shoots from callus is a critical requirement for the successful exploitation of in vitro selection, somaclonal variation and genetic engineering techniques as applied to the genetic manipulation o f plants (Puite 1992). In spite o f the obvious advantages that could derive from the use of such biotechnological methods as a novel tool for their breeding, w o o d y ornamental species have seldom been studied in this Correspondence to: D. Georges
respect. This is particularly true for Lonicera species with just three articles on micropropagation of different honeysuckles (Boonour and Wainwright 1988 ; Laine et al. 1990 ; Suzuki et al. 1986) and, for the shrubby species L. nitida 'MaigrUn', two reports on direct regeneration o f adventitious buds from leaf explants (Cambec~des et al. 1991 and 1992), and one on the establishment of a protoplast-to-plant system (Ochatt 1991). Against this background, the aim o f this study was to develop a strategy for the obtention o f highly regenerating true-callus (i.e. without any part of the original explant) for the climbing honeysuckle, L. japonica "Hall's Prolific", a species never studied in vitro before.
Materials and methods Plant material and establishment of callus cultures. L. japonica "Hall's Prolific" plants were micropropagated on WPM (Lloyd and MeCown 1981) hormone-free medium with 3 g/l charcoal (pH 5.7), with cultures kept at 25~ under a 16 h light photoperiod of 54 Ixmol. s -1. m -2 (cool white fluorescent tubes), and subcultured every 2 months. Callus cultures were initiated from the leaves, stems and roots, harvested from rooted in vitro plants (6-8 cm tall). For leaf explants, the petiole was removed and the lamina wounded with the scalpel, perpendicularly to the central vein. Stem internodes (1 cm long), and root portions (of 1 cm and taken from the middle part) were also employed. Explants were placed onto 10 ml of LS (Linsmaier and Skoog 1965) medium with 5.4, 10.7, 21.5 or 43 pM NAA, or 0.45, 4.5 or 9 pM 2,4-D and 2.7 pM BA. Cultures were maintained either in darkness or under a 16 h l i g h t photoperiod of 14 ~tmol. s-1. m-2 (cool white fluorescent tubes). Each treatment was repeated twice, including at least 25 explants (5/dish) subcultured every 4 weeks, and observations on induction of callogenesis were made after the first 4 weeks. Caulogenesis and plant regeneration. For all source explants (leaves, stems and roots), 0.5 to 1 cm in diameter portions (4-5/dish) of well-established callus, subcultured for at least 6 months on I ~ medium with 10.7/~M NAA and 2.7 /~M BA were used and estimation of the RC (regenerating callus) was performed after 6 months on the regenerating medium. Experiments were carried out under the same conditions as for callogenesis, except that the mineral concentration of the medium was reduced by half
92
(i.e. medium LS/2). Various hormones were tested, including the cytokinins BA (from 4.4 to 44.4 pM), 2iP, KIN, Z or TDZ (at 4.5 pM each), and with 10 to 45 callus pieces per medium per explant source. In addition, the effect of the callus age on the regeneration competence was studied with leaf-callus after 2, 6 or 12 passages on callusing medium (i.e. 75, 182 and 365 day-old, respectively), and subcultured for 6 months on induction media supplemented with 4.4, 13.3, 22.2 or 44.4/aM of BA. Each experiment included 10 to 20 callus portions per medium per age. Other factors were also examined for their influence on regeneration, using 10 to 20 leaf-derived callus (aged 182 days) per treatment, placed on a medium with 13.3 pM BA. These included the use of the multiplication medium (WPM with 3 g/l charcoal) instead of LS/2 medium, the addition of 2.9/AM GA 3 to facilitate the elongation of regenerated shoots, the exposure to a higher temperature (30~ dark culture throughout or only during the first month of culture, and a hormonal induction treatment with 13.3/~M BA limited to the first month or to the first two months followed by culture on the hormonefree multiplication medium. Regenerated stems were transferred onto the multiplication nicdiuin Ir-.:-_ O l [~t I O.l.l ~. .d t l O l [ l and t- -o- 'v: - u a g , ~ik ^tt~, plttttu~,tS _,._,,,, voualncu -,,,,; aa were acclimatized in pots containing peat and pedite (t: 1), under an intermittent mist for 2 weeks, before normal growth in the greenhouse.
and was not greatly affected by the explant origin (e.g. 90 • 10 days for leaves, 90 • 21 for stems and 75 • 10 for roots). Likewise, the callus source tissue did not greatly affect its competence for caulogenesis (Fig. lb). However, leaf-derived callus seemed to be the most responsive (Fig. lc), with an overall percentage o f regeneration of 44% (61 of 137), compared with 35% (23 o f 66) and 36% (31 o f 86) for callus derived from stems and roots, respectively. In addition, all BA concentrations considered, leaf tissues were the m o s t reliable source, with callus of this origin showing the smallest standard deviation in terms of the percentage of regenerating callus. Table 1. Effect of the explant source and cytokinin concentration on shoot regeneration from 6 month-old callus of L. japonica
LS/2
explant + source(a) BA (~uM) L 4.4 S R
TOR(b) (days) 75 90 90
RC(C) overall (%) RC/medium (%) 48 27 36 • 8.8 33
L
13.3
Results
Initiation and growth of callus Leaves, stems and roots efficiently callused on media containing NAA, while 2,4-D led to a rapid browning of the explants. Even at a low concentration (0.45/aM), 2,4-D gave callus that rapidly browned during the first subcultures. Of the 3 explant sources tested, the leaves were the most responsive, with 100% o f explants giving callus. Morphology o f callus from the 3 different sources was quite similar and the texture o f all callus was hard. Darkness was not beneficial for callus initiation, as tissues maintained in darkness were brownish and typically of a very slow growth rate. All the callus obtained was compact; only under dark conditions small friable portions were sometimes noticed, but without any further growth. No shoot bud regeneration was observed during callus initiation, but roots appeared at low frequency on NAA-based media, mostly for leaf explants and in the dark. The medium containing 10.7/~M NAA and 2.7/~M BA (under photoperiodic illumination) gave the best results, and was therefore retained for callus maintenance. Upon subeulturing on this medium, the callus seemed to enter a redifferentiation state showing a more globular texture (Fig. la) and a slower growth rate; it also became very heterogenous with brown, white and green parts close to each other.
Plant regeneration from true-callus Of the cytokinins assessed, BA gave the best caulogenic responses, with about 40% of callus regenerating shoots with either concentration o f BA tested. Strikingly though, the overall percentage of regenerating callus per medium showed little difference depending on the BA concentration used (Table 1). Replacing BA by Z resulted in a reduced caulogenesis (25 • 7.5% for the various source tissues), while the use of 2iP, K I N or T D Z did not permit shoot regeneration at all. The mean time to the onset o f shoot bud regeneration from 6 month-old callus o f L. japonica was typically 3 months
S 90 25 34 • 13.2 R 60 25 L 105 42 22.2 S ! 20 30 38 • 5.9 R 75 43 L 90 41 44.4 S 60 67 48 • 13.3 R 75 37 a, type of explant : L=leaf ; S~tem ; R=root b, TOR : time to the onset of shoot regeneration c, RC : regenerating callus after 6 months on inducing medium. On these grounds, in a separate series o f experiments aimed at assessing the effects o f callus age on regeneration competence, only leaf-derived callus was used. In general, the younger the callus, the higher its ability to undergo caulogenesis and the sooner was the first regeneration event recorded (Table 2). This trend was particularly noticeable for those tissues grown on the medium with the highest BA concentration tested (i.e. 44.4/AM) where, for one year-old callas, the average time to the onset o f shoot bud regeneration nearly trebled while the percentage of regenerating callus was just under 20% of that observed for 75 day-old callus. Table 2. Effect of callus age on competence for regeneration from leaf-derived callus callus age (days) 75 75 75 75 182 182 182 182 365 365 365 365 (b,c : see table
BA (/AM)
TOR(b) (days)
4.4 13.3 22.2 44.4 4.4 13.3 22.2 44.4 4.4 13.3 22.2 44.4 1)
60 60 60 60 60 90 120 90 30 60 90 150
RC(C) (%) 100 50 87 75 62 35 50 70 38 33 47 13
overall RC/age (%) 78 • 18.4
54 • 13.2
33 • 12.5
93 Another group of experiments examined the effects of various other factors on the organogenic ability of callus. Thus, the use of the multiplication medium (hormone-free WPM) supplemented with 13.3/~M BA, as well as the addition of GA 3 to LS/2 medium supplemented with 13.3/~M BA were detrimental for the induction of organogenesis, with only 24% and 16% of calluses regenerating, respectively. Continuous darkness or even culture in the dark during the first month prevented shoot bud regeneration. Indeed, a photoperiodic illumination of 14/~mol. s -1. m -2 was needed throughout for a successful induction of shoots on callus of L. japonica. Of the two temperatures used, 240C proved better for shoot regeneration, while 300C induced the regeneration of roots at a high level (90% of the callus) and was associated with a browning of the cultured tissues. Experiments on the induction of organogenesis, with an exposure to hormones for 1 or 2 months only, showed that induction is completed after the first 60 days on inducing medium.
Noteworthy was that, sporadically and irrespective of the callus explant origin and medium, more or less welldeveloped somatic embryos were also obtained, their germination occurred and the recovery of plants was possible. Although no clear inducing factor could be distinguished, somatic embryos appeared mostly on caulogenic callus. The buds regenerated on callus could be isolated or grouped in an actively regenerating portion of the callus. They could arise from green parts of callus or from parts showing a brown surface. Elongation of the shoots was better on media with 4.4/~M BA as compared with 44.4 /,M BA. When shoots were long enough (1 cm), they were excised from the callus and transferred directly to the multiplication medium. Alternatively, the whole callus, with shoots still attached, was transferred to this medium to obtain elongation before excision of the stems. After two months, all elongated shoots were rooted (Fig. ld) and the regenerated plants w e r e transferred to the greenhouse where they were acclimatized with a success rate of nearly 100% (Fig. ie).
Fig, 1. Shoot bud regeneration and plant recovery from true-callus .of Lonicera japonica cv. "Hall's Prolific" : a) Typical 182 day-old leaf callus on medium containing 10.7/~M NAA and 2.'7 pM BA, Bar = 0.5 cm. b) A shoot bud regenerating from a stem callus (112 days on inducing medium), Bar = 0.1 cm. c) Highly regenerating leaf calluses, Bar = 1 cm. d) A rooted regenerated shoot (2 months on the multiplication medium), Bar = 1 cm. e) Sample of regenerated plants after 5 months in vivo.
94 Discussion The auxin NAA induced callogenesis on L. japonica and allowed to maintain it throughout a large number of subcultures, in contrast to other w o o d y plants (Greinwald and Czygan 1991 ; Noh and M i n o c h a 1986), where 2,4-D was used for callogenesis. Comparing our results with those of Warrag et al. (1991) indicated that, for Lonicera japonicaexplants, as little as 0.45 p M 2,4-D led to ultimate necrosis, whereas, for Eucalyptus grandis explants, more than 27.1 p M were required for this. The rapid necrotic effect of 2,4-D on Lonicera explants might be due to the herbicide action of this partially non-metabolizable molecule, whose biochemical derivatives accumulated in the tissues, and coupled with the natural high level of endogenous auxin in this species with strong apical dominance. Dark conditions also had a negative effect on induction and growth of callus, contrasting data for other genera (Blumenfeld and Gazit 1971 ; King and Morehart 1987 ; Subbaiah and Minocha 1990), where caiiogenesis was better in darkness. A true-callus of L. japonica was a patchwork of undifferentiated parts and redifferentiated parts (globular structures); also for colour, the same callus exhibited differences with brown, yellow, white and green parts close to each other. These heterogeneities were probably linked to the use of NAA (10.7 pM) and BA (2.7 pM), that allowed good induction and growth of callus of the three types of explants but also, allowed a certain degree of redifferentiation (i.e. roots on some calluses). In fact, the conditions for induction of viable callogenesis were very close to those for rhizogenesis on callus from this species. This was in agreement with the general diagram drawn by Gaspar (1988), and is in accordance with the role of the exogenous auxirdcytokinin balance as first described by Skoog and Miller (1957). With respect to the regenerating capacity of callus, results were better with leaf-callus compared with the other two types of explant origins tested (stems and roots). Few reports, and none for Lonicera species, have compared results on regeneration from true-callus initiated from leaves, stems and roots of woody plants. For Populus, Son and Hall (1990) also concluded that leaf-derived callus had the greatest capacity for shoot formation in comparison with stem and root-callus. Leafcallus was thus preferred for our further experiments on regeneration ability. The callus age was critical for regeneration competence, as already shown for other species. For example, for Chamaecytisus (Greinwald and Czygan 1991), the younger was the callus, the higher was the regeneration capacity. Conversely, different factors interacted in our system : a relatively slow growth rate and the tendency to redifferentiation along subcultures led us to use callus as aged as possible to try to obtain somaclonal variation in cultures, whilst retaining a correct capacity to regenerate shoots. Six month-old callus was chosen as a compromise. When working with leaves of L. nitida, TDZ was essential for successful shoot bud regeneration (Cambec~des 1991), while true-callus (of protoplast origin) of this same genotype underwent caulogenesis only on a medium containing BA and/or Z (Ochatt 1991). This is in line with our results for L. japonica, where only BA and Z gave consistent, frequent regeneration from true-callus. Thus, Z (4.5 pM) gave significantly fewer regeneration events than BA at all
concentrations tested, whereas BA allowed the induction of similar rates of shoot formation at concentrations of 4.4 to 44.4 pM. Only the further development, and particularly the elongation, of induced buds was affected by high concentrations of BA. This indifference to the cytokinin concentration for percentage of regeneration might be ascribed either to a high endogenous level of auxin in tissues (that might then require a large amount of cytokinins to establish a hormonal balance favourable to bud induction), or to a limited amount of cytokinin receptors, so that the cytokinins in excess would not be used by cells. In this context, either (or both) of these suggestions could explain the long delay before the onset of shoot bud regeneration from callus of L. japonica, where 90 days (3 months) were necessary in most of the experiments. After well-formed buds appeared on callus, their further development into whole rooted plants was not critical, contrasting with results for most woody species (N~meth 1986), but in line with the ease for rooting of regenerated shoots of other honeysuckles (Ochatt 1991 ;Cambec&iesetal. 1991). The availability of this novel methodology for the regeneration from true-callus of Lonicera japonica cv. "Hall's Prolific" paves the way for the induction of somaclonal variation with a view to producing ornamental novelties within this species and could also be a suitable regeneration system to produce transgenic plants. Regenerated plants are now under examination to assess any potential somaclonal variants thus produced.
Acknowledgements.
DG held a fellowship from the "D~partement du Maine-et-Loire (France)."
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