Reversion of
Limonium
hybrid `Misty Blue'
inflorescence development and its
applicability in micropropagation
Nopmanee Topoonyanont
a,b, Rungsima Ampawan
c,
Pierre C. Debergh
a,* aDepartment of Plant Production ± Horticulture, University Gent, Coupure links 653, 9000 Gent, Belgium
b
Faculty of Science, Maejo University, Sansai, 50290 Chiangmai, Thailand
c
Office of Agricultural Research and Extension, Maejo University, Sansai, 50290 Chiangmai, Thailand
Accepted 22 May 1999
Abstract
Five different developmental stages and lateral branch positions of Limonium hybrid `Misty Blue' inflorescences cultured in vitro were investigated for floral reversion. The results indicated that there was a gradient of expression along the inflorescence. Explants from the proximal ends, either from the main axis or the lateral branches of inflorescences in whatever stage of development, tended to exhibit vegetative traits, while the terminal ends continued to form floral organs. Reversion percentages and multiplication rates of shoot production in vitro were examined for three generations. Explants taken from the main axis of stage 1 yielded approx. 30% of nodes that produced vegetative shoots but decreased to less than 10% from the more advanced stages. Explants taken from lateral branches produced many floral shoots, especially from the advanced developmental stages 3±5. Vegetative shoots, harvested from nodal explants on the main axis produced between 2.5 to 4 newly developed vegetative shoots in the first subculture and continued to multiply 3±4 new shoots in the second and third subcultures. It was recommended that inflore-scences ofLimonium`Misty Blue' at stages 4 and 5, main axis as well as lateral branches, should be used as initial explants for micropropagation.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Limoniumsp.; Micropropagation
Scientia Horticulturae 83 (2000) 283±299
* Corresponding author. Tel.: +32-9-264-70-71; fax: +32-9-264-62-25.
E-mail address:[email protected] (Pierre C. Debergh).
1. Introduction
Hybrids betweenLimonium latifoliumandL. bellidifolium, are well accepted in the flower market for their attractive colours and long life, both fresh and dried. Different selections `Misty Blue', `Misty White' and `Misty Pink' have been recently introduced. Conventional propagation by means of root cuttings takes 6± 8 months and gives usually only 20±30% success and the offspring are heterogeneous. Therefore, efforts have been directed towards micropropagation techniques.
The switch from vegetative to floral morphology is a process by which a single vegetative shoot apex is transformed, depending on the species, into an inflorescence that contains one, several or many individual flowers. Subsequently the flowers often arise directly from the inflorescence meristem without a preceding vegetative phase (Steeves and Sussex, 1972). This flowering process can be subdivided into four different stages: induction, evocation, initiation and flower morphogenesis (McDaniel, 1994).
However, on some occasions, inflorescence meristems of a number of plant species can be forced to return transiently or permanently to vegetative functioning by some physiological phenomenon (Bernier, 1992). The most remarkable example is the production of so-called `vegetative inflorescences' in which the shoot takes the branching pattern of an inflorescence but without forming any flowers. Bracts may take the size and shape of normal leaves, inflorescence internodes may have an abnormal length, branches or shoots (of inflorescence or vegetative nature or a mixture of both) may substitute for flowers and inflorescence phyllotaxis may be altered. Such a return is usually called a `reversion'. However, flower reversion is not frequently reported, maybe partly because plants usually grow under conditions that eventually result in normal flower development. It is often a sporadic and unpredictable situation and its occurrence and part reversion, is sometimes ignored or treated as an aberration or a teratoma (Battey and Lyndon, 1990).
Reversion may or may not involve the terminal flower, depending on whether or not it occurs before this flower is formed. At the level of the individual meristem, reversion that occurs very early, is indistinguishable from those pre-floral changes in morphology that may result from partial induction (Greyson, 1994).
Generally, shoot tip culture of rosette plants is risky due to the high degree of contamination of the explants. Consequently, other explants, distant from the shoot tip, were considered. Successful shoot formation by reversion of inflorescence explants has been reported in several crops, such as Allium cepa
L. (Dunstan and Short, 1979), Cymbidium goeringii (Shimasaki and Uemoto,
Preliminary studies on plantlet induction from inflorescence explants of three species of statice namelyL. bellidifolium, L. gmelliniiandL. latifolium, showed satisfactory results with a lower contamination percentage compared to shoot tip explants.
The following experiments have been conducted to assess the influence of the developmental stage and the lateral branch position on the reversion of
inflorescence explants of Limonium hybrid `Misty Blue' in vitro and its
suitability as initial explants for micropropagation.
2. Materials and methods
2.1. Limonium inflorescence development
In vitroLimonium`Misty Blue' plants were grown in a greenhouse at 2538C and 11±13 h day-length in Thailand. They were transplanted into 6 cm diameter round plastic pots with a sterilised soil : burned-rice-husk : sand (1 : 1 : 1 v/v) mixture. After 2 months, they were transplanted into 1006030 cm3wooden boxes, containing 25 l of a soil : peanut shell : burned-rice-husk : compost (3 : 1 : 1 : 0.5 v/v) mixture. Slow release fertiliser (N : P : K15 : 15 : 15, 25 g/ plant) was added. Growth and development of the inflorescences were investigated until they reached 50% anthesis. Five distinct developmental stages were used for further experimentation (Table 1): stage 1, elongated inflorescence stem; stage 2, main axis was elongated with first-order branches emerging; stage 3, the first- and second-order branches were completely elongated; stage 4, calyx formation was complete at the terminal end of second-order branches; and stage 5, corolla formation complete.
2.2. In vitro reversion of inflorescence explants from different developmental stages and positions
For each developmental stage (Table 1), except stage 1, explants taken from the mother plant were divided into two sub-categories depending on their branch position: main axis or lateral branch. Five inflorescences were used as replications. Every inflorescence node of the main axis, and the first- and second-order branches were numbered according to their position.
Individually marked branches were surface sterilised with 0.1% HgCl2 for
10 min, followed by three rinses with sterile distilled water, cut into single nodes (approx. 2 cm long), except the terminal end of each branch and used as initial explants. They were cultured individually in tubes (; 24 mm, height 150 mm, closed with Cap Uts) containing 10 ml Murashige and Skoog (1962) basal medium supplemented with 1 mg lÿ1 thiamine-HCl, 0.5 mg lÿ1 pyridoxine,
Table 1
Some characteristics ofLimoniuminflorescences at five different developmental stages Developmental stages
5 visible nodes on main axis, covered
0.5 mg lÿ1 nicotinic acid, 100 mg lÿ1 inositol, 30 g lÿ1 sucrose, 35 mg lÿ1 NaFeEDTA and 8 g lÿ1BDH agar. 2 mg lÿ1KIN and 2 mg lÿ1(3-indolyl) acetic acid (IAA) were added as growth regulators, based on preliminary experiments (N. Topoonyanont, unpublished). The pH was adjusted to 5.8 prior to autoclaving at 1218C for 20 min. The cultures were incubated at 2328C under a 16 h day-length with a photosynthetic photon flux density (PPFD) of 43mmol mÿ2sÿ1
provided by fluorescent tubes (OSRAM 31).
After 1 month in vitro, four different types of inocula origins taken from tissue cultures were considered, depending on the morphology of the organs which arose from the axil of each node explant: vegetative shoot, floral shoot, mixed shoot (vegetative and floral) or none. Vegetative shoots were characterised by the development of a leaf rosette, while floral shoots elongated and formed 1±4 nodes. A nodal explant can produce one or more vegetative shoots. From a floral-shoot development, many single nodes can be used as inoculum for subculture. The mixed development yielded both vegetative shoots and floral single nodes as shown in Fig. 1. Moreover, from every explant, the tissues around the axil from where both vegetative shoots and floral shoots developed, were also used as another source of inocula during the first subculture. We called this type of inoculum `base' with a size of approximately 0.3±0.5 cm3. In case of non-developed explants, if they looked green and healthy their `bases' were used as initial explants as well.
The explant type and the way they developed were positioned in diagrams to examine the distribution and degree of reversion of inflorescence buds along the branches.
Fig. 1. Diagrammatic representation of mixed shoots (vegetative and floral) proliferating at the axil of the initial explant 30 days after inoculation: (a) vegetative shoot; (b) floral shoot with 3±5 nodes; (c) the base (the tissues around the axil, approx. 0.5 cm3).
2.3. Application of inflorescence reversion for Limonium micropropagation
The aforementioned types of inocula were evaluated for another two generations. Percentages of reacting inocula and the type of development were examined and recorded in every generation at one month intervals.
The anatomical characteristics of reverted inflorescences from all five different developmental stages and two branch positions (first- and second-order) were investigated histologically following the procedure of Johansen (1940). They were fixed in FAA (5% formalin, 5% glacial acetic acid and 90% ethyl alcohol) and dehydrated with a tertiary butyl alcohol series (TBA). Then they were
embedded in paraffin, sectioned at 10mm and stained with Delafield's
hematoxylin.
The experiment was designed as a factorial in completely randomised design. The factors were five developmental stages of the inflorescence and two branching positions. Results were analysed by ANOVA.
3. Results
3.1. Inflorescence development
During the vegetative stage, Limonium `Misty Blue' grew as a rosette with many leaves arranged spirally with very short internodes. The transition from the vegetative to the reproductive phase became apparent with the development of the rapidly bolting inflorescence meristem, covered by modified leaf primordia, called bracts. Several stages could be distinguished during the development of an inflorescence (Table 1). First, the inflorescence meristem started to elongate and had large bracts (stage 1). After about 15 days the first-order branches developed acropetally from the axils of the main axis (stage 2). While there was a gradual acropetal development of first-order branches, the main axis continued to elongate, and gradually second-order branches were initiated on the first-order branches (stage 3). They followed the same pattern of the first-order branches. At the terminal end of second-order branches, 10±12 spikes differentiated in a centrifugal order; subsequently spikelets differentiated into spikes in centrifugal order too. The number of spikelets per spike was reduced towards the distal end of the branches. Flower development started with calyx formation (stage 4) and later on each spikelet produced a white corolla starting at the outer side (stage 5). General information on the different developmental stages of the Limonium
`Misty Blue' inflorescence is presented in Table 1.
inflorescence branch, but moreover, two axillary buds were observed, which could eventually develop into second-order branches. The degree of development of these buds depended on the position of the node. Buds from the main axis grew faster and were more developed than those from first- or second-order nodes, as shown in Fig. 2.
Fig. 2. Longitudinal section of main axis node ofLimonium`Misty Blue' inflorescence in stage 2 of development after 6 weeks culturing in vitro: (fb) first-order and (sb) second-order branches. Bar150m.
3.2. In vitro reversion of inflorescence explants in different developmental stages and positions
Diagrams of the in vitro behaviour during initiation of the cultures of the explants from each position of the five different developmental stages (Table 1) are illustrated in Fig. 3(a±e). The results indicate there is a base-to-apex gradient of reversion along the inflorescence for the main axis as well as for the lateral branches. Explants from the proximal end of the main axis of each stage tended to exhibit vegetative traits while the terminal ends continued to form floral organs. This gradient was repeated in the first- and second-order branches, but the specific early stage 3 (Fig. 3c) seems to have a lower degree of reversion
than the later stages 4 (Fig. 3d) and 5 (Fig. 3e). In stages 4 and 5, mixed shoots were obtained from proximal end explants and floral shoot from the distal ones.
It was clear from the histological study that the shoots emerged directly from axillary buds, without callus intervention. Inflorescence meristems of the main axis could revert to a vegetative meristem (a meristematic dome with leaf primordia, Fig. 4), or floral and vegetative shoots could be obtained from the same node, as shown in Fig. 5a and b.
Fig. 3. (Continued).
3.3. Use of inflorescence reversion for Limonium micropropagation
3.3.1. Initiating the cultures
The developmental stage of the mother plant and the position of the explant in the inflorescence influenced the development in vitro. Stage 1 explants taken from the main axis yielded approx. 30% of nodes that produced vegetative shoots; this decreased to less than 10% for the more advanced stages (Table 2). On the
contrary, stage 1 explants yielded only 15% of nodes producing floral shoots, and this increased to almost 50% for stage 4 explants, to decrease significantly again for stage 5. Each nodal explant could produce different shoots, which were either of the same developmental type or from a different type (mixed shoots).
Explants taken from lateral branches hardly produced any vegetative shoots, but many developed floral shoots, especially from the advanced developmental
Fig. 3. (Continued).
Fig. 4. Reversion of inflorescence meristem to vegetative meristem after 6 weeks cultured in vitro. Bar150m.
stages 3±5 of the mother plants. These explants produced elongated floral shoots with 3±4 nodes. These were used in subsequent subcultures.
It was obvious that shoots regenerated from both positions (main axis or lateral branches) showed different quality. Shoots derived from the explants on the main axis were larger, with 6±7 leaves, and of a better visual quality; those from lateral branches were smaller with 2±3 leaves. However, the disadvantages of main axis explants are the high degree of bacterial contamination (Pseudomonas sp.) and the difficulty to root them (data not presented); this is not the case for material derived from lateral branches.
3.3.2. First subculture
Vegetative shoots, harvested from nodal explants on the main axis, produced between 2.5 and 4 newly developed vegetative shoots (Table 3). When these vegetative shoots originated from what was originally a mixed shoot, the propagation ratio was almost limited to 1 or 2. Interesting results were obtained when the tissues around the axil of the primary explant were recultured, indeed each ``base'' yielded approximately four new shoots, notwithstanding the developmental stage of the mother plant from which they originated.
For vegetative shoots harvested from side branches the shoot production was much reduced (Table 3).
Vegetative shoots never formed floral shoots. The other types of inocula could develop floral shoots again, and be used as a further source of inocula in the
Table 2
Development (%) of explants taken from mother plants at five developmental stages (1±5) and from different branch positions (main or lateral)a,b
Developmental inflorescence stage and branch position
Percentages of four different types of development
No
Main axis stage 1 30.3d 28.8a 25.7b 15.2ef
Main axis stage 2 21.4e 10.6c 32.0ab 36.0d
Main axis stage 3 16.2ef 16.1b 25.8b 41.9cd
Main axis stage 4 10.2f 6.8cd 33.9a 49.1b
Main axis stage 5 39.1c 8.8c 32.6ab 19.5e
Lateral branches stage 2 90.0a 0.0e 0.0c 10.0f Lateral branches stage 3 48.6b 1.9e 2.8c 46.7bc Lateral branches stage 4 21.6e 2.9de 6.0c 69.5a Lateral branches stage 5 12.8f 3.0de 6.0c 70.2a
aThe explants either do not develop, or they form a vegetative or floral shoot or a mixture of both
30 days after initiation.
b
Within columns means followed by the same letter are not significantly different (LSD 95%).
successive subcultures, and are therefore interesting, as each floral shoot produced 2±4 nodes.
3.3.3. Second and third subcultures
All types of development obtained from the first subculture (vegetative shoots and nodes from floral shoots) were used as inocula for the second and third subcultures. Almost every vegetative shoot which originated from the main axis continued to multiply (Table 4) by producing 3±4 new shoots at the base (Table 5). There was only one exception, which we cannot explain for stage 3 main axis inocula. The results were not so clear-cut for inocula from lateral branches, although good results were obtained, approaching 100% or at least 60%. Vegetative shoots which originated from the base in the first subculture were rather unpredictable in percentage of inocula which produced vegetative shoots again in the second subculture, but all of them yielded almost 100% in the third subculture, notwithstanding whether they originated from the main axis or from lateral branches.
Nodes originating from floral shoots during initiation were rather unreliable in the following subcultures. However, it was obvious that the younger stage of development of the mother plant gave a better yield of vegetative shoots in the
Table 3
Inocula yielded from the initial cultures (Table 2) were subcultured and their development was observed after 30 daysa
Explant type in stage 1 Origin of the inoculum
Number of newly formed vegetative shoots
Number of nodes in a developing inflorescence
Data are scored as number of vegetative shoots or as number of nodes in a developing inflorescence.
b
MeanS.E.
c
Table 4
Development (%) of inocula taken from the organ developing in the first subculture (Table 3) and subcultured two more times (second and third subcultures)a,b
Explant type in initiation stage
Type of inoculum taken from the first subculture
Vegetative shoots Vegetative shoots from ``base''
Floral shoots
Nosc2 Nos3 Nos2 Nos3 Nos2 Nos3
Main axis stage 1 100.0b 95.6b 57.1b ±d 100.0d ± Main axis stage 2 100.0b 100.0b 75.0c ± 53.8c 63.6c
Main axis stage 3 46.2a 98.8b ± ± ± ±
Main axis stage 4 100.0b 99.0b 63.6b 100.0a 11.3a 80.0d Main axis stage 5 100.0b 98.9b 75.0c 99.0a 51.1c 25.0b
Lateral branches stage 2 ± ± ± ± ± ±
Lateral branches stage 3 57.1a 99.0b 96.6c 100.0a 25.6ab 12.5a Lateral branches stage 4 100.0b 97.7b 55.0b 100.0a 32.7b 33.3bc Lateral branches stage 5 92.2b 59.8a 27.9a 100.0a 21.8ab 95.9e
aData are scored after 30 days in each subculture, as in Table 3. b
Means followed by the same letter are not significantly different (LSD 95%).
cNumber of subcultures. d
No data.
Table 5
Inocula taken from mother plants at five developmental stages (1±5) and from different branch positions (main or lateral)a
Origin Multiplication rate
Multiplication rate of three different explant types in second and third subcultures. Data scored after 30 days.
bNumber of subcultures. c
MeanS.E.
dNo data.
second subculture, but the remaining results are rather confusing and no real trend can be deduced from them.
Limonium`Misty Blue' taken from in vitro have been grown on the greenhouse for 6 months. All plants behaved normally and no phenotypic variation was observed.
4. Discussion
Inflorescence development phases of Limonium hybrid `Misty Blue' can be separated into two major transition phases: the transition from rosette to early inflorescence when the vegetative meristem begins to bolt (stage 1), and later on the inflorescence develops (stages 2 and 3) and flowers (stages 4 and 5).
The results of the reversion of inflorescence nodes in vitro showed that there was a gradient along the axis. The reversion could be partial or complete, depending on the developmental stage of the inflorescence and the position of the nodes. In the early stages of development, a high percentage of reversion was obtained and it decreased in more advanced stages. On the contrary, the develop-ment of floral shoots increased as the explants were taken from more developed inflorescences, except for stage 5. In this stage flowers were fully developed and showed 50% anthesis, floral shoot formation on the main axis explants decreased again. This is probably due to the fact that the inflorescences reached a point of no return in their development. This suggests that the determination to
inflorescence development in Limonium is separated from determination to
flower development, as is the case in Pisum sativum(Ferguson et al., 1991). In
other crops such as Nicotiana tabacum (Singer and McDaniel, 1986) and
Pharbitis nil(Larkin et al., 1990) they were reported to be non-separable steps.
Inflorescence reversion in Limonium `Misty Blue' can be made use of in
micropropagation. The developmental stage of an inflorescence and the branch position can affect the percentage and rate of shoot formation. Main axis explants of stages 4 and 5 plants are the best explant source for gaining more and vigorous vegetative shoots, and these shoots can be used as inocula in subsequent generations. However, main axis explants are only recommended when the growing conditions in the greenhouse are well controlled during mother plant preparation (stage 0, Debergh and Maene, 1981) and they should not be transplanted to the greenhouse immediately after initiation, because of rooting difficulties.
newly formed base inocula and floral nodes as well. These newly formed floral nodes provide new shoots in the second subculture (2 months after initiation). Although the rate of formation of new shoots from lateral branches was low (one shoot per node), the multiplication rate increased during the third subculture (3±4 fold every 6 weeks). Moreover, shoots regenerated from lateral branches easily formed roots (data not presented).
Based on our experiments, we recommend that nodes from inflorescences of
Limonium`Misty Blue' in stages 4 and 5, main axis as well as lateral branches, should be used as initial explants for micropropagation.
Acknowledgements
The authors thank the Thai Government for financial support.
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