Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue3-4.March2000:
Scientia Horticulturae 83 (2000) 283±299
Reversion of Limonium hybrid `Misty Blue'
inflorescence development and its
applicability in micropropagation
Nopmanee Topoonyanonta,b, Rungsima Ampawanc,
Pierre C. Debergha,*
a
Department 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 inflorescences of Limonium `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: Limonium sp.; Micropropagation
* Corresponding author. Tel.: +32-9-264-70-71; fax: +32-9-264-62-25.
E-mail address: [email protected] (Pierre C. Debergh).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 8 - 3
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1. Introduction
Hybrids between Limonium latifolium and L. 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 prefloral 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,
1991), Gerbera jamesonii (Topoonyanont and Dillen, 1988), and Lycopersicon
esculentum Mill. (Compton and Veilleux, 1991).
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
285
Preliminary studies on plantlet induction from inflorescence explants of three
species of statice namely L. bellidifolium, L. gmellinii and L. 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 vitro Limonium `Misty Blue' plants were grown in a greenhouse at 25 38C
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 100 60 30 cm3 wooden
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 : K 15 : 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,
286
Developmental stages
Inflorescence length (cm)
Number of days after
bolting
Number of nodes on
main axis of inflorescence
Cumulated number of
nodes in lateral branches
per inflorescence
Inflorescence morphology
1
2
3
4
5
3.8 0.5
4 1.3
22.9 0.7
13 1.5
56.7 1.2
33 2.4
101.4 3.6
41 2.1
125.8 5.7
79 4.2
5 2.0
11 3.0
15 3.0
22 2.0
26 3.0
15±20
90±100
>1300
>1500
Main axis is
elongated, with >10
visible nodes, and
first-order branches
emerging from axils
of main axis
First- and second-order
branches completely
elongated; at the terminal
part of all branches,
flowers could be observed
as green clusters
At the terminal end of
second-order branches,
calyx formation is
completed, showing
blue colour
Corolla formation is
completed, showing
white inflorescence
with 50% anthesis
Inflorescence with
5 visible nodes on
main axis, covered
by large bracts
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
Table 1
Some characteristics of Limonium inflorescences at five different developmental stages
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287
0.5 mg lÿ1 nicotinic acid, 100 mg lÿ1 inositol, 30 g lÿ1 sucrose, 35 mg lÿ1
NaFeEDTA and 8 g lÿ1 BDH agar. 2 mg lÿ1 KIN 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 23 28C under a 16 h daylength with a photosynthetic photon flux density (PPFD) of 43 mmol mÿ2 sÿ1
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 floralshoot 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 nondeveloped 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).
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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 10 mm 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.
Histological examination showed that, in general, each node of a greenhouse
inflorescence had an inflorescence bud, no matter the order of the branches
(Fig. 2). The main axis nodes had a central bud which developed into an
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
289
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 of Limonium `Misty Blue' inflorescence in stage 2
of development after 6 weeks culturing in vitro: (fb) first-order and (sb) second-order branches.
Bar 150 m.
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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
Fig. 3. Diagram showing the in vitro behaviour of four different explant types from inflorescences
in stages 1±5 of Limonium `Misty Blue' after 1 month in culture: (a) stage 1, (b) stage 2, (c) stage 3,
(d) stage 4 and (e) stage 5. (*) no development; (&) vegetative shoot; (~) mixed shoots; (#) floral
shoot.
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
291
Fig. 3. (Continued ).
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.
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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
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293
Fig. 3. (Continued ).
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
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Fig. 4. Reversion of inflorescence meristem to vegetative meristem after 6 weeks cultured in vitro.
Bar 150 m.
Fig. 5. Main floral bud emerged as floral shoot (a) while side bud changed to be a vegetative bud
(b) after 6 weeks cultured in vitro. (fs): floral shoot, (vs): vegetative shoot. Bar 100 m.
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
295
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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Percentages of four different types of development
No
development
Vegetative
shoots
Mixed
shoots
Floral
shoots
30.3d
21.4e
16.2ef
10.2f
39.1c
28.8a
10.6c
16.1b
6.8cd
8.8c
25.7b
32.0ab
25.8b
33.9a
32.6ab
15.2ef
36.0d
41.9cd
49.1b
19.5e
90.0a
48.6b
21.6e
12.8f
0.0e
1.9e
2.9de
3.0de
0.0c
2.8c
6.0c
6.0c
10.0f
46.7bc
69.5a
70.2a
a
The 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%).
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
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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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Origin of the inoculum
Number of newly formed
vegetative shoots
Number of nodes in a developing
inflorescence
Vegetative Mixed
shoots
shoots
Base
Mixed
shoots
Floral
shoots
Base
4.0 0.2b
3.1 0.5
3.5 0.9
3.1 0.6
2.3 0.3
1.5 0.2
1.3 0.3
1.3 0.1
2.0 0.5
1.0 0.1
3.6 0.5
3.5 0.6
4.4 0.5
4.0 0.8
3.0 1.0
1.0 0.2
1.0 0.1
1.3 0.2
1.2 0.1
2.3 1.0
3.3 1.0
1.5 0.3
2.2 1.1
1.9 0.2
1.5 0.3
1.6 0.4
3.5 0.3
2.2 0.2
1.9 0.2
1.5 0.3
±c
1.0 0.1
1.9 0.3
2.0 0.3
±
1.1 0.1
1.3 0.3
1.0 0.2
±
2.1 0.2
2.6 0.2
2.8 0.2
±
1.3 0.3
1.0 0.2
1.3 0.1
±
1.3 0.1
1.3 0.2
1.2 0.1
±
1.7 0.1
1.9 0.2
1.6 0.1
a
Data are scored as number of vegetative shoots or as number of nodes in a developing
inflorescence.
b
Mean S.E.
c
No data.
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
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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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
Type of inoculum taken from the first subculture
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Vegetative shoots
Vegetative shoots
from ``base''
Floral shoots
Nosc 2
Nos 2 Nos 3
Nos 2
Nos 3
100.0d
53.8c
±
11.3a
51.1c
±
63.6c
±
80.0d
25.0b
Nos 3
d
100.0b
100.0b
46.2a
100.0b
100.0b
95.6b
100.0b
98.8b
99.0b
98.9b
57.1b
75.0c
±
63.6b
75.0c
±
±
±
100.0a
99.0a
±
57.1a
100.0b
92.2b
±
99.0b
97.7b
59.8a
±
96.6c
55.0b
27.9a
±
100.0a
100.0a
100.0a
±
25.6ab
32.7b
21.8ab
±
12.5a
33.3bc
95.9e
a
Data are scored after 30 days in each subculture, as in Table 3.
Means followed by the same letter are not significantly different (LSD 95%).
c
Number of subcultures.
d
No data.
b
Table 5
Inocula taken from mother plants at five developmental stages (1±5) and from different branch
positions (main or lateral)a
Origin
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
a
Multiplication rate
stage
stage
stage
stage
stage
branches
branches
branches
branches
Vegetative shoots
from ``base''
Floral shoots
Nosb 2
Nos 2
Nos 2
Nos 3
4.3 1.6
4.5 1.0
3.1 0.7
3.8 0.9
3.3 1.0
1
2
3
4
5
stage
stage
stage
stage
Vegetative shoots
2
3
4
5
±
4.4 0.7
3.7 0.8
2.7 0.3
Nos 3
c
Nos 3
d
4.3 0.4
3.0 0.5
3.1 0.3
3.3 0.2
3.3 0.4
2.6 0.1
1.6 0.1
±
3.6 0.1
3.8 0.3
±
±
±
1.9 0.1
2.9 0.2
1.0 0.2
1.0 0.2
±
1.2 0.3
1.6 0.3
±
1.3 0.5
±
1.4 0.2
1.2 0.2
±
4.4 0.7
3.2 0.8
3.2 0.2
±
3.1 0.5
3.1 0.7
2.0 0.0
±
1.6 0.2
3.1 0.8
3.0 0.4
±
0.7 0.1
1.4 0.1
1.2 0.0
±
1.1 0.2
0.7 0.0
0.8 0.0
Multiplication rate of three different explant types in second and third subcultures. Data scored
after 30 days.
b
Number of subcultures.
c
Mean S.E.
d
No data.
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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 development 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.
Nodes from lateral branches can be used as initial explants because they can
produce both vegetative and floral shoots; although the multiplication rate was
low (approx. 1 vegetative shoot per explant). However, lateral branches are a huge
source of initial explants; more than 1000 nodes could be obtained from one
inflorescence in stages 4 and 5 (Table 1). Moreover, they are also a source of
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
299
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.
References
Battey, N.H., Lyndon, R.F., 1990. Reversion of flowering. Bot. Rev. 56(2), 12±189.
Bernier, G., 1992. Attempts to bridge the molecular genetics and physiology of inflorescence and
flower morphogenesis are an exciting experience. Flowering Newslett. 14, 34±40.
Compton, M.E., Veilleux, R.E., 1991. Shoot, root and flower morphogenesis of tomato
inflorescence explants. Plant Cell, Tissue Organ Cult. 24, 223±231.
Debergh, P.C., Maene, L.J., 1981. A scheme for commercial propagation of ornamental plants by
tissue culture. Sci. Hortic. 14, 335±345.
Dunstan, D.I., Short, K.C., 1979. Shoot production from the flower head of Allium cepa L.. Sci.
Hortic. 10, 345±356.
Ferguson, C.J., Huber, S.C., Hong, P.H., Singer, S.R., 1991. Determination for inflorescence
development is a stable state, separable from determination for flower development in Pisum
sativum L. buds. Planta 185, 518±522.
Greyson, R.I., 1994. The Development of Flowers. Oxford University Press, New York, Oxford.
Johansen, D.A., 1940. Plant Microtechnique. McGraw Hill, New York.
Larkin, J.C., Felsheim, R., Das, A., 1990. Floral determination in the terminal bud of the short-day
plant Pharbitis nil. Dev. Biol. 137, 434±443.
McDaniel, C.N., 1994. Photoperiodic induction, evocation and floral initiation. In: Greyson, R.I.
(Ed.), The Development of Flowers. Oxford University Press, NewYork, Oxford.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol. Plant. 15, 473±497.
Shimasaki, K., Uemoto, S., 1991. Rhizome induction and plantlet regeneration of Cymbidium
goeringii from flower bud cultures in vitro. Plant Cell, Tissue Organ Cult. 25, 49±52.
Singer, S.R., McDaniel, C.N., 1986. Floral determination in the terminal and axillary buds of
Nicotiana tabacum L.. Dev. Biol. 118, 587±592.
Steeves, T.A., Sussex, I.M., 1972. Patterns in Plant Development. Prentic-Hall, Englewood Cliffs,
NJ, pp.152±169.
Topoonyanont, N., Dillen, W., 1988. Capitulum explant as a start for micropropagation of Gerbera:
culture technique and applicability. Med. Fac. Landbouww. RijksUniv. Gent 53(1), 169±173.
Reversion of Limonium hybrid `Misty Blue'
inflorescence development and its
applicability in micropropagation
Nopmanee Topoonyanonta,b, Rungsima Ampawanc,
Pierre C. Debergha,*
a
Department 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 inflorescences of Limonium `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: Limonium sp.; Micropropagation
* Corresponding author. Tel.: +32-9-264-70-71; fax: +32-9-264-62-25.
E-mail address: [email protected] (Pierre C. Debergh).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 8 - 3
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1. Introduction
Hybrids between Limonium latifolium and L. 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 prefloral 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,
1991), Gerbera jamesonii (Topoonyanont and Dillen, 1988), and Lycopersicon
esculentum Mill. (Compton and Veilleux, 1991).
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
285
Preliminary studies on plantlet induction from inflorescence explants of three
species of statice namely L. bellidifolium, L. gmellinii and L. 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 vitro Limonium `Misty Blue' plants were grown in a greenhouse at 25 38C
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 100 60 30 cm3 wooden
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 : K 15 : 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,
286
Developmental stages
Inflorescence length (cm)
Number of days after
bolting
Number of nodes on
main axis of inflorescence
Cumulated number of
nodes in lateral branches
per inflorescence
Inflorescence morphology
1
2
3
4
5
3.8 0.5
4 1.3
22.9 0.7
13 1.5
56.7 1.2
33 2.4
101.4 3.6
41 2.1
125.8 5.7
79 4.2
5 2.0
11 3.0
15 3.0
22 2.0
26 3.0
15±20
90±100
>1300
>1500
Main axis is
elongated, with >10
visible nodes, and
first-order branches
emerging from axils
of main axis
First- and second-order
branches completely
elongated; at the terminal
part of all branches,
flowers could be observed
as green clusters
At the terminal end of
second-order branches,
calyx formation is
completed, showing
blue colour
Corolla formation is
completed, showing
white inflorescence
with 50% anthesis
Inflorescence with
5 visible nodes on
main axis, covered
by large bracts
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
Table 1
Some characteristics of Limonium inflorescences at five different developmental stages
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287
0.5 mg lÿ1 nicotinic acid, 100 mg lÿ1 inositol, 30 g lÿ1 sucrose, 35 mg lÿ1
NaFeEDTA and 8 g lÿ1 BDH agar. 2 mg lÿ1 KIN 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 23 28C under a 16 h daylength with a photosynthetic photon flux density (PPFD) of 43 mmol mÿ2 sÿ1
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 floralshoot 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 nondeveloped 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).
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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 10 mm 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.
Histological examination showed that, in general, each node of a greenhouse
inflorescence had an inflorescence bud, no matter the order of the branches
(Fig. 2). The main axis nodes had a central bud which developed into an
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
289
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 of Limonium `Misty Blue' inflorescence in stage 2
of development after 6 weeks culturing in vitro: (fb) first-order and (sb) second-order branches.
Bar 150 m.
290
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
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
Fig. 3. Diagram showing the in vitro behaviour of four different explant types from inflorescences
in stages 1±5 of Limonium `Misty Blue' after 1 month in culture: (a) stage 1, (b) stage 2, (c) stage 3,
(d) stage 4 and (e) stage 5. (*) no development; (&) vegetative shoot; (~) mixed shoots; (#) floral
shoot.
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
291
Fig. 3. (Continued ).
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.
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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
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
293
Fig. 3. (Continued ).
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
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Fig. 4. Reversion of inflorescence meristem to vegetative meristem after 6 weeks cultured in vitro.
Bar 150 m.
Fig. 5. Main floral bud emerged as floral shoot (a) while side bud changed to be a vegetative bud
(b) after 6 weeks cultured in vitro. (fs): floral shoot, (vs): vegetative shoot. Bar 100 m.
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
295
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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Percentages of four different types of development
No
development
Vegetative
shoots
Mixed
shoots
Floral
shoots
30.3d
21.4e
16.2ef
10.2f
39.1c
28.8a
10.6c
16.1b
6.8cd
8.8c
25.7b
32.0ab
25.8b
33.9a
32.6ab
15.2ef
36.0d
41.9cd
49.1b
19.5e
90.0a
48.6b
21.6e
12.8f
0.0e
1.9e
2.9de
3.0de
0.0c
2.8c
6.0c
6.0c
10.0f
46.7bc
69.5a
70.2a
a
The 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%).
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
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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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Origin of the inoculum
Number of newly formed
vegetative shoots
Number of nodes in a developing
inflorescence
Vegetative Mixed
shoots
shoots
Base
Mixed
shoots
Floral
shoots
Base
4.0 0.2b
3.1 0.5
3.5 0.9
3.1 0.6
2.3 0.3
1.5 0.2
1.3 0.3
1.3 0.1
2.0 0.5
1.0 0.1
3.6 0.5
3.5 0.6
4.4 0.5
4.0 0.8
3.0 1.0
1.0 0.2
1.0 0.1
1.3 0.2
1.2 0.1
2.3 1.0
3.3 1.0
1.5 0.3
2.2 1.1
1.9 0.2
1.5 0.3
1.6 0.4
3.5 0.3
2.2 0.2
1.9 0.2
1.5 0.3
±c
1.0 0.1
1.9 0.3
2.0 0.3
±
1.1 0.1
1.3 0.3
1.0 0.2
±
2.1 0.2
2.6 0.2
2.8 0.2
±
1.3 0.3
1.0 0.2
1.3 0.1
±
1.3 0.1
1.3 0.2
1.2 0.1
±
1.7 0.1
1.9 0.2
1.6 0.1
a
Data are scored as number of vegetative shoots or as number of nodes in a developing
inflorescence.
b
Mean S.E.
c
No data.
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
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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
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
stage
stage
stage
stage
stage
branches
branches
branches
branches
Type of inoculum taken from the first subculture
1
2
3
4
5
stage
stage
stage
stage
2
3
4
5
Vegetative shoots
Vegetative shoots
from ``base''
Floral shoots
Nosc 2
Nos 2 Nos 3
Nos 2
Nos 3
100.0d
53.8c
±
11.3a
51.1c
±
63.6c
±
80.0d
25.0b
Nos 3
d
100.0b
100.0b
46.2a
100.0b
100.0b
95.6b
100.0b
98.8b
99.0b
98.9b
57.1b
75.0c
±
63.6b
75.0c
±
±
±
100.0a
99.0a
±
57.1a
100.0b
92.2b
±
99.0b
97.7b
59.8a
±
96.6c
55.0b
27.9a
±
100.0a
100.0a
100.0a
±
25.6ab
32.7b
21.8ab
±
12.5a
33.3bc
95.9e
a
Data are scored after 30 days in each subculture, as in Table 3.
Means followed by the same letter are not significantly different (LSD 95%).
c
Number of subcultures.
d
No data.
b
Table 5
Inocula taken from mother plants at five developmental stages (1±5) and from different branch
positions (main or lateral)a
Origin
Main
Main
Main
Main
Main
axis
axis
axis
axis
axis
Lateral
Lateral
Lateral
Lateral
a
Multiplication rate
stage
stage
stage
stage
stage
branches
branches
branches
branches
Vegetative shoots
from ``base''
Floral shoots
Nosb 2
Nos 2
Nos 2
Nos 3
4.3 1.6
4.5 1.0
3.1 0.7
3.8 0.9
3.3 1.0
1
2
3
4
5
stage
stage
stage
stage
Vegetative shoots
2
3
4
5
±
4.4 0.7
3.7 0.8
2.7 0.3
Nos 3
c
Nos 3
d
4.3 0.4
3.0 0.5
3.1 0.3
3.3 0.2
3.3 0.4
2.6 0.1
1.6 0.1
±
3.6 0.1
3.8 0.3
±
±
±
1.9 0.1
2.9 0.2
1.0 0.2
1.0 0.2
±
1.2 0.3
1.6 0.3
±
1.3 0.5
±
1.4 0.2
1.2 0.2
±
4.4 0.7
3.2 0.8
3.2 0.2
±
3.1 0.5
3.1 0.7
2.0 0.0
±
1.6 0.2
3.1 0.8
3.0 0.4
±
0.7 0.1
1.4 0.1
1.2 0.0
±
1.1 0.2
0.7 0.0
0.8 0.0
Multiplication rate of three different explant types in second and third subcultures. Data scored
after 30 days.
b
Number of subcultures.
c
Mean S.E.
d
No data.
298
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
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 development 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.
Nodes from lateral branches can be used as initial explants because they can
produce both vegetative and floral shoots; although the multiplication rate was
low (approx. 1 vegetative shoot per explant). However, lateral branches are a huge
source of initial explants; more than 1000 nodes could be obtained from one
inflorescence in stages 4 and 5 (Table 1). Moreover, they are also a source of
N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299
299
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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