Somatic embryogenesis in
Narcissus pseudonarcissus
cvs. Golden
Harvest and St. Keverne
Darren O. Sage *, James Lynn, Neil Hammatt
Horticulture Research International,Wellesbourne,Warwick CV35 9EF,UK
Received 14 May 1999; received in revised form 20 September 1999; accepted 20 September 1999
Abstract
Somatic embryos (SEs) have been produced from bulb and shoot culture leaf explants ofNarcissus pseudonarcissuscvs. Golden Harvest and St. Keverne. Initial experiments with cv. Golden Harvest resulted in SEs from leaf lamina, leaf base, bulb scale and scape (flower stem) explants. Embryogenesis was induced on media with a range of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP) concentrations. There were significantly more SEs with media containing 5mM 2,4-D and 0.5mM or
5mM BAP than any other growth regulator combination. Scape explants produced more early SEs than the other explant types,
and when orientated with their basipetal surface away from the medium, they produced significantly more advanced SEs than those with this surface in contact with the medium. Leaf explants from shoot cultures of cv. Golden Harvest produced SEs on medium with BAP combined with 2,4-D or 1-naphthaleneacetic acid (NAA), but 4-amino-3, 5, 6-trichloro-2-pyridinecarboxylic acid (picloram) was ineffective. SEs converted to plantlets efficiently following a 4°C treatment in addition to 4.9 mM
indole-3-butyric acid (IBA). These plantlets readily transferred to ex vitro conditions. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Bulb; Clone; Daffodil; Vegetative propagation
www.elsevier.com/locate/plantsci
1. Introduction
Narcissus growers face a number of problems including pests, diseases and the slow rate ofNar -cissus sexual and vegetative propagation. It has been estimated that it takes 25 – 30 years to pro-duce enough bulbs to complete variety trials and to bulk-up stock for commercial release [1]. There have been attempts to speed up vegetative propa-gation by chipping and twin scaling [2], and by micropropagation, e.g. [3 – 7]. However, existing procedures are either unable to produce the re-quired number of propagules or are not
economi-cally viable for the rapid development of new commercial varieties [4,8]. Somatic embryogenesis in liquid media could provide a means for rapid, automated plant production via bioreactors at re-duced cost.
Conventional control of pests and diseases, pri-marily via agrochemicals, can be a substantial ongoing expense. Introgression of genes for pest and disease resistance should improve control measures. There is some resistance within Narcis-sus cultivars, e.g. St. Keverne is resistant to basal and neck rot, but the long juvenile period of 3 – 8 years [9 – 12] slows the transfer of this resistance into other cultivars by conventional breeding. Transformation could provide a more rapid means to move desirable genes betweenNarcissusspecies/ cultivars, and allow introduction of genes from other genera. Potentially, somatic embryogenesis * Corresponding author. Tel.: +44-1789-470382; fax: +
44-1789-470552.
E-mail address:[email protected] (D.O. Sage)
could provide the foundation of a transformation protocol for Narcissus.
The research described here defines conditions for induction of somatic embryogenesis inNarcis -sus pseudonarcis-sus cvs. Golden Harvest and St. Keverne, and for conversion of somatic embryos into plantlets and subsequent transfer to soil.
2. Materials and methods
2.1. Media preparation
Unless otherwise stated, the following condi-tions applied: all media in somatic embryogenesis and rooting experiments comprised MS (Mu-rashige and Skoog) medium [13] (Imperial labora-tories, Andover, UK), modified with 100 mM
FeNa ethylenediaminetetraacetic acid (EDTA) and 30 mM ZnSO4.7H2O instead of the Na2
-EDTA, FeSO4.7H2O and ZnSO4.4H2O, and
with-out edamin. This, in addition to 87.6 mM sucrose and 0.6% (w/v) agar (both from Merck, UK), pH 5.65 (prior to autoclaving for 5 min at 121°C), constituted ‘basal MS medium’. Where required, media were supplemented with one or more of the following growth regulators: 4-amino-3,5,trichloro-2-pyridinecarboxylic acid (picloram), 6-benzylaminopurine (BAP), 2,4-dichloropheno-xyacetic acid (2,4-D), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA) (all from Sigma). Where used, these were added prior to adjusting pH and autoclaving. Media were dispensed into either 30 ml volume Coulter counter pots (CCPs; consisting of polystyrene body and polyethylene cap with 10 ml medium per CCP), or 90 mm diameter polypropylene Petri dishes (10 mm deep, triple-vented or 20 mm deep, hexi-vented both with 25 ml per dish) sealed with Nescofilm. The latter are referred to as ‘standard’ and ‘deep’ Petri dishes, respectively. All culture vessels were from Greiner Labortechnik, Gloucester, UK.
2.2. Culture conditions
Unless otherwise stated, the following condi-tions applied: light-grown cultures were main-tained under Philips colour TLD 36W/84 fluorescent tubes, providing an irradiance of 60 – 70 mM m−2s−1, in a growth room at 24.6°C with
a 16 h daily photoperiod; dark cultures were incu-bated at the same temperature.
2.3. Bulb preparation
All bulb explants were derived from terminal bulb units. These were dissected from sexually mature bulbs and surface-sterilised in a 10% (v/v) aqueous solution of ‘Domestos’ commercial bleach solution (Lever Bros., London), giving at least a 0.5% (v/v) final concentration of NaOCl, for 15 min on a rotary shaker. These explants were then rinsed at least six times with sterile water deionised by reverse osmosis.
2.4. Bulb explants for somatic embryogenesis experiments
All explants were taken from immediately above the basal plate (Fig. 1A). Bulb scale explants (four per bulblet) and leaf base and lamina explants (three each per bulblet) were cut transversely into 10 mm lengths; scapes (flower stems) were cut transversely into two to four (depending on exper-iment) slices, 1 mm in thickness. Leaf and scale explants were cultured with their abaxial surfaces in contact with the medium and scape explants with their basipetal cut-surfaces in contact with the medium. Explants were either cultured singly in CCPs or in Petri dishes at a rate of three or four explants per dish (depending on experiment).
2.5. Establishment and maintenance of c6. Golden
Har6est shoot cultures
Bulbs were sterilised for 30 min. Leaf bases and scapes were cut 10 mm above the basal plate and were left attached to a 1.5 mm length of basal plate. Scape explants were divided longitudinally into thirds while leaf bases were left entire.
Each bulb yielded three of each explant type, which were cultured vertically in individual CCPs with the basal plate end away from the medium. This medium was described by Seabrook et al. [14]. They were subcultured onto fresh medium after 3 weeks. After 4 weeks, shoot clusters arising from the basal plate region, were divided into single shoots and subcultured at 4 – 8 week inter-vals onto a similar medium but with less NAA (0.6
mM) and BAP (17.8 mM). At each subculture,
basal plate tissue and cut longitudinally into pieces approximately 5 – 10 mm in diameter. Large shoots (\3 mm diameter, measured immediately above the basal plate tissue) were isolated and split longi-tudinally through the meristem into 2 – 4 pieces of approximately equal size before being transferred individually to fresh medium. Twelve months after culture initiation, one vigorous shoot line was retained to supply leaf explants.
2.6. Preparation of leaf explants from shoot cultures
The first 5 mm of leaves above the basal plate tissue was excised from 2-week-old shoot cul-tures. These were only taken from complete shoots 6 – 15 mm in height and were cultured with their abaxial surfaces in contact with media.
Fig. 1. (A) Dissection of explants fromNarcissusterminal bulb units, bar=10 mm. (B) Development of early somatic embryos (E. SEs) from a scape-derived culture ofNarcissuscv. Golden Harvest after 4 weeks on standard induction medium and 2 weeks on standard regeneration medium, bar=10 mm. (C) Development of early (E.SEs) and an advanced (A.SE) somatic embryos after culture of a scape explant for 4 weeks on standard induction medium and 10 weeks on standard regeneration medium, bar=10 mm. (D) Advanced somatic embryo formation of cv. St. Keverne (c is cotyledon) from scape tissues cultured for 4 weeks on standard induction medium and 10 weeks on standard regeneration medium, bar=10 mm. (E) Longitudinal section of a zygotic embryo (c is cotyledon, a is aperture, sm is shoot meristem), bar=5 mm. (F) Longitudinal section of a somatic embryo (c is cotyledon, a is aperture, sm is shoot meristem), bar=5 mm. (G) A plantlet of cv. St. Keverne produced after an advanced SE had been cultured for 20 weeks at 24°C and 8 weeks at 4°C on basal MS medium with 4.9 mM IBA (p is plumule, c is
2.7. Comparison of somatic and zygotic embryos
Somatic and zygotic embryos from cv. St. Keverne, at different developmental stages, were cut in half longitudinally to reveal their internal structure. Zygotic embryos were obtained from seeds derived from cv. St. Keverne flowers polli-nated with a mixture of cv. St. Keverne and cv. Golden Harvest pollen. Seeds were collected at time of shedding, stored at room temperature, and a sample cut open periodically to study embryo development.
2.8. Somatic embryos from bulb explants of c6.
Golden Har6est
Bulb scale, leaf base, leaf lamina and scape explants were incubated on the basal MS medium with the addition of 0, 0.5, 5 or 50 mM 2,4-D in
combination with either 0, 0.5 or 5 mM BAP.
Explants were placed singly into CCPs, with 20 per growth regulator combination, and treatments randomised in blocks before culturing in the dark. Cultures were recorded 11 weeks after initiation.
2.9. Effect of induction time on somatic embryo production by scape explants
Bulbs were obtained from Dr Gordon Hanks (HRI, Kirton, UK) after they had been harvested and hot water treated (3 h at 44°C) in August. Bulbs were then stored in nets, in the dark, at 20°C (from 28/8/97). For each of four replicate experiments established on 3/12/97, 5/2/98, 30/3/ 98 and 3/6/98, four scape explants were excised from each surface-sterilised terminal bulb unit (cvs. Golden Harvest and St. Keverne). These were placed onto standard induction medium (basal MS medium supplemented with 5mM 2,4-D
and 5mM BAP) in standard Petri dishes with their
morphological bases in contact with the medium. The 0 week control treatments were immediately placed onto standard regeneration medium (as the standard induction medium but without 2,4-D). These were plated so that each dish (one replicate) had four explants, one from each of the four positions up the scape, and from four different bulbs. There were five replicate dishes per treat-ment. Treatments comprised dark culture of scape explants on standard induction medium for 0, 2, 4, 6 and 14 weeks followed by transfer to standard
regeneration medium. The treatments were ran-domised in blocks. The 0 and 14 week treatments were subcultured every 4 weeks to standard regen-eration medium only and standard induction medium only, respectively. The 2, 4 and 6 week treatments were not subcultured onto fresh induc-tion medium prior to transfer to regenerainduc-tion medium. After transfer to regeneration medium, cultures were transferred to fresh regeneration medium every 4 weeks. At their first subculture and thereafter, explants were plated into deep Petri dishes. Cultures were recorded 14 weeks after initiation.
2.10. Effects of scape explant orientation on somatic embryo production in c6s. Golden Har6est
and St. Ke6erne
From each surface-sterilised terminal bulb unit, two transverse scape slices were placed on stan-dard induction medium either with their morpho-logical base, apex or side in contact with the medium. Three explants were placed in each stan-dard Petri dish (one replicate), with position 1 as morphological base down, position 2 on their side and 3 with the morphological base up. Explants from the first bulb were placed in positions 1 and 2 of the first plate, those from the second bulb placed in position 3 of this plate and position 1 of the second plate and so on in succession. Explants from the two cultivars were kept in separate dishes, and replicates were randomised in blocks. After 5 and 9 weeks, cultures were subcultured to standard regeneration medium in deep Petri dishes. Cultures were recorded 15 weeks after initiation.
2.11. Comparison of the effects of 2,4-D, NAA and picloram on induction of somatic embryos from shoot culture leaf explants of c6. Golden
Har6est
Leaf explants were placed onto basal MS medium supplemented with 5 mM BAP and auxin
concentrations of either 0, 0.5, 5.0 or 10.0 mM of
either 2,4-D, NAA or picloram.
D.O.Sage et al./Plant Science150 (2000) 209 – 216
Fig. 2. Early somatic embryos from bulb explants on media containing 5mM 2,4-D and different concentrations of BAP
(9standard errors).
2.12. Con6ersion of somatic embryos of c6. St.
Ke6erne and transfer to soil
Scape explants were cultured for 4 weeks on induction medium, in standard petri dishes, after which they were transferred to standard regenera-tion medium, in deep petri dishes, and subcultured at 4 week intervals. After 14 weeks of culture, advanced SEs were harvested and cultured on half-concentration basal MS, but with 87 mM sucrose, 0.6% (w/v) agar, either with or without 4.9 mM IBA, in CCPs. Cultures were placed in a
dark incubator at 24°C. Approximately half of the cultures from each treatment were moved to a dark incubator (4°C) after 10 weeks and the rest after 20 weeks. Only cultures that had produced both a root and a shoot were recorded 28 – 30 weeks after culture initiation. After recording in August 1998 (late Summer), plantlets were trans-ferred to a sterilised peat-based growing medium (Levington M2 compost, Littleton-Badsey Grow-ers, Worcestershire, UK) and placed in a cooled incubator (1792°C; 16 h photoperiod of 10 – 20
mM m−2 s−1irradiance from 8 W Osram type 33
fluorescent tubes).
After 2 weeks, plants were transferred to an unheated polyethylene tunnel where they were maintained over winter. In April 1998 these were planted outside.
2.13. Statistical analyses
Statistical analyses were applied using either a generalised linear or a log-linear model [15] with the appropriate links, errors and aggregation parameters being applied where necessary. All analyses were carried out with Genstat 5 software [16].
Figs. 2 – 5 illustrate predicted means from the modelled data.
Fig. 3. Effect of induction time and bulb storage length on somatic embryo yield from scape explants (9standard er-rors).
Fig. 4. Effect of scape morphological base orientation on the yield of advanced somatic embryos (9standard errors).
Fig. 5. Effect of auxins on yield of advanced somatic embryos from shoot culture leaf explants (9standard errors). weeks after culture initiation. Twelve weeks after
3. Results
3.1. Somatic embryos from bulb explants of c6.
Golden Har6est
After 5 – 6 weeks, organised, semi-translucent to opaque structures were produced (Fig. 1B) which resembled early somatic embryos (hereafter re-ferred to as early SEs). The majority of the cul-tures began to develop fine trichome-like hairs after 4 weeks, which obscured some of the cul-tures, particularly the developing SEs. Conse-quently, only cultures with visible early SEs could be recorded 11 weeks after culture initiation. There were no SEs produced without 2,4-D and only a minimal response with 0.5 and 50 mM 2,4-D (data not shown). With 5 mM 2,4-D
and no BAP only one explant produced SEs. The only data analysed were for 5 mM 2,4-D
and 0.5 or 5 mM BAP (Fig. 2). BAP
concen-trations tested had no effect (P=0.05), but there were highly significant differences between explant types (PB0.001). Scape explants pro-duced most early SEs and scale explants the least.
In some cultures, after 4 – 8 weeks, early SEs developed into structures resembling more ad-vanced somatic embryos (Fig. 1C and D). The most advanced SEs were white/grey in colour, and were usually associated with a single structure similar to a Narcissus zygotic embryo cotyledon, either partially ensheathing, attached basally to, or slightly distant from the SE (Fig. 1D). In some cultures, advanced SEs did not appear to develop from early SEs but emerged from below the cut surface of the explant.
3.2. Somatic embryo identity
The study of the internal organisation of so-matic and zygotic embryos revealed similar struc-tures. Both were bipolar structures with a shoot and root pole. Zygotic embryos had a single cotyledon and an aperture towards the root pole containing the shoot meristem (Fig. 1E). SEs nearly always contained a shoot meristem inside an aperture (Fig. 1F), and a single cotyledon-like structure. From this evidence we suggest that the structures produced were indeed somatic em-bryos.
3.3. Effect of induction time and bulb storage on somatic embryo production by scape explants
The two cultivars behaved differently, with dif-ferences in terms of both time on induction medium and storage period prior to experimental set up (Fig. 3). Following the shortest storage period, cv. St. Keverne gave a significantly higher (PB0.001) response than cv. Golden Harvest with four weeks induction. However, as the time in storage increased, the overall response from cv. Golden Harvest did not change (P=0.05), but that for St. Keverne fell significantly (PB0.001). Thus, by the last date, cv. Golden Harvest had a much higher overall response than cv. St. Keverne (PB0.001).
3.4. Effect of scape explant orientation on somatic embryo production in c6s. Golden
Har6est and St. Ke6erne
The orientation of the explant significantly (PB
0.001) affected the numbers of SEs produced per explant (Fig. 4). The morphological base down orientation produced significantly (PB0.001) more SEs than the other orientations. The differ-ence between morphological base up and side ori-entation was just significant (PB0.05). The base up and side orientations also showed a cultivar effect, with cv. St. Keverne producing more SEs (PB0.001) than Golden Harvest.
3.5. Comparison of 2,4-D, NAA and picloram on induction of somatic embryos from shoot culture leaf explants
SEs were produced with all treatments (Fig. 5), but NAA and 2,4-D promoted SE production while picloram inhibited it (PB0.05). With 2,4-D at 5 mM and 10 mM there were significantly more
SEs produced than at 0.5 mM (PB0.05).
How-ever, with NAA there were no significant differ-ences between the treatments over the range 0.5 – 10 mM (P=0.05).
3.6. Con6ersion of somatic embryos into plantlets
and transfer to soil
with no significant (P=0.05) differences between cultures kept previously for either 10 weeks or 20 weeks at 24°C. Inclusion of 4.9 mM IBA further
increased (PB0.001) the number of cold-treated SEs that developed into whole plantlets. Out of 24 plantlets, 23 were successfully acclimatised to a peat-based compost. These underwent further leaf elongation and plants moved into a polyethylene tunnel in August 1998 still had green leaves the following July.
4. Discussion
This paper describes a novel method for produc-ing plants of N. pseudonarcissus cvs. Golden Har-vest and St. Keverne using somatic embryogenesis. The hypothesis was that, SEs usually developed from globular structures into cotyledon-like struc-tures containing an aperture, within which the shoot and root meristem were housed (Fig. 1B, C and F). These SEs frequently developed further, so that a bulbil was produced from the meristems which emerged through the aperture (Fig. 1D). However, variations of these structures and devel-opmental pathways were observed. Thus, we de-cided to describe all bipolar structures, with both shoot and root poles, as somatic embryos. Work is underway to characterise these structures and de-velopmental pathways further.
Scape explants of cv. Golden Harvest produced most SEs, and subsequently similar explants of cv. St. Keverne also produced such structures. Effi-cient somatic embryogenesis from similar explants has also been reported recently in Tulipa[17]. The orientation of explants in the two species was important in optimising SE yields. With scapes of both Narcissus cvs., a base up orientation pro-duced most SEs. This was also observed in some cultivars of Tulipa, while in others a morphologi-cal base down orientation slowed embryo develop-ment [18].
Relatively high levels of plant growth regulators are generally inhibitory to further SE development [19]. In Tulipa, prolonged exposure to induction conditions slowed subsequent embryo develop-ment once removed from the inductive environ-ment [18]. In the current study, differences were found between the two Narcissus cvs. in their response to varying induction time and length of bulb storage. This suggests that these factors may
need to be optimised for each new genotype. The embryogenic response of cv. St. Keverne scape explants deteriorated rapidly during storage. How-ever, it was promising to find SEs forming from leaf explants of shoot cultures. Thus, where sea-sonality or numbers of bulbs limits availability of bulb explants, the establishment of an initial shoot culture could provide a year-round supply of explants.
Different auxins vary in their ability to induce somatic embryogenesis. Thus, in Freesia hybrida, over two concentrations, picloram induced be-tween three and twelve fold more SEs than 2,4-D [20]. In some Lilium-hybrids, at the same concen-tration, 2,4-D was able to induce somatic embryo-genesis while picloram was not [21]. Our work has shown that, in conjunction with BAP, NAA and 2,4-D did induce reasonable yields of SEs, while picloram produced fewer SEs under the conditions tested.
Evidence suggested the need for chilling of SEs to convert them into plantlets. This was signifi-cantly improved by including IBA in the medium. In the native habitat of wild Narcissus, predomi-nantly Mediterranean regions [22], seeds are shed in early summer and then have a period of warm weather before germinating in the cooler condi-tions of winter/early spring. The SEs produced during this research may be mimicking the be-haviour of Narcissus zygotic embryos by convert-ing durconvert-ing a 4°C treatment after a number of weeks at 24°C.
It is promising that Narcissus plantlets estab-lished in soil easily, with little requirement for a costly and time-consuming humidity-controlled weaning phase. Furthermore, plantlets transferred into a polyethylene tunnel in August 1998 contin-ued to grow throughout autumn, winter, spring and into summer 1999. The increased length of their first growing season, compared to plants grown from seeds in situ, may result in larger bulbs and thus an overall reduction in time to their first flowering. This could be important to Narcissus breeders in generating sexually mature plants sooner for crossing.
To our knowledge this is the first report on somatic embryogenesis from tissues of sexually mature Narcissus plants.
The results presented here are being used as a foundation for research into the rapid, economical, production of new and improvedNarcissusvarieties in bioreactors. Our experiments are now concentrat-ing on the production and regeneration of embryo-genic callus, as a prelude to proliferating material in liquid culture. In addition, research is focusing on developing a transformation system for Narcissus, as a possible route to cultivar improvement.
Acknowledgements
The Ministry of Agriculture, Fisheries and Food funded this research. We are grateful to Professor Malcolm Bennett, Dr Brian Thomas and Mr Neil Grant for helpful suggestions and encouragement.
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