reversion of limonium hybrid ‘misty blue’ inflorescence development and its...

Reversion of Limonium hybrid `Misty Blue'

inflorescence development and its

applicability in micropropagation

Nopmanee Topoonyanonta,b, Rungsima Ampawanc,Pierre C. Debergha,*

aDepartment of Plant Production ± Horticulture, University Gent,

Coupure links 653, 9000 Gent, BelgiumbFaculty of Science, Maejo University, Sansai, 50290 Chiangmai, Thailand

cOffice 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 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

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).

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

1. Introduction

Hybrids between Limonium latifolium and L. bellidifolium, are well accepted inthe flower market for their attractive colours and long life, both fresh and dried.Different selections `Misty Blue', `Misty White' and `Misty Pink' have beenrecently introduced. Conventional propagation by means of root cuttings takes 6±8 months and gives usually only 20±30% success and the offspring areheterogeneous. Therefore, efforts have been directed towards micropropagationtechniques.

The switch from vegetative to floral morphology is a process by which a singlevegetative shoot apex is transformed, depending on the species, into aninflorescence that contains one, several or many individual flowers. Subsequentlythe flowers often arise directly from the inflorescence meristem without apreceding vegetative phase (Steeves and Sussex, 1972). This flowering processcan be subdivided into four different stages: induction, evocation, initiation andflower morphogenesis (McDaniel, 1994).

However, on some occasions, inflorescence meristems of a number of plantspecies can be forced to return transiently or permanently to vegetativefunctioning by some physiological phenomenon (Bernier, 1992). The mostremarkable example is the production of so-called `vegetative inflorescences' inwhich the shoot takes the branching pattern of an inflorescence but withoutforming any flowers. Bracts may take the size and shape of normal leaves,inflorescence internodes may have an abnormal length, branches or shoots (ofinflorescence or vegetative nature or a mixture of both) may substitute for flowersand inflorescence phyllotaxis may be altered. Such a return is usually called a`reversion'. However, flower reversion is not frequently reported, maybe partlybecause plants usually grow under conditions that eventually result in normalflower development. It is often a sporadic and unpredictable situation and itsoccurrence and part reversion, is sometimes ignored or treated as an aberration ora teratoma (Battey and Lyndon, 1990).

Reversion may or may not involve the terminal flower, depending on whetheror not it occurs before this flower is formed. At the level of the individualmeristem, 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 ofcontamination of the explants. Consequently, other explants, distant from theshoot tip, were considered. Successful shoot formation by reversion ofinflorescence 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).

284 N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299

Preliminary studies on plantlet induction from inflorescence explants of threespecies of statice namely L. bellidifolium, L. gmellinii and L. latifolium, showedsatisfactory results with a lower contamination percentage compared to shoot tipexplants.

The following experiments have been conducted to assess the influence of thedevelopmental stage and the lateral branch position on the reversion ofinflorescence explants of Limonium hybrid `Misty Blue' in vitro and itssuitability 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 � 38Cand 11±13 h day-length in Thailand. They were transplanted into 6 cm diameterround 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 woodenboxes, 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 wereinvestigated until they reached 50% anthesis. Five distinct developmental stageswere used for further experimentation (Table 1): stage 1, elongated inflorescencestem; stage 2, main axis was elongated with first-order branches emerging; stage3, the first- and second-order branches were completely elongated; stage 4, calyxformation was complete at the terminal end of second-order branches; and stage5, corolla formation complete.

2.2. In vitro reversion of inflorescence explants from different developmentalstages and positions

For each developmental stage (Table 1), except stage 1, explants taken from themother plant were divided into two sub-categories depending on their branchposition: main axis or lateral branch. Five inflorescences were used asreplications. Every inflorescence node of the main axis, and the first- andsecond-order branches were numbered according to their position.

Individually marked branches were surface sterilised with 0.1% HgCl2 for10 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 initialexplants. They were cultured individually in tubes (; 24 mm, height 150 mm,closed with Cap Uts) containing 10 ml Murashige and Skoog (1962) basalmedium supplemented with 1 mg lÿ1 thiamine-HCl, 0.5 mg lÿ1 pyridoxine,

N. Topoonyanont et al. / Scientia Horticulturae 83 (2000) 283±299 285

Table 1

Some characteristics of Limonium inflorescences at five different developmental stages

Developmental stages

1 2 3 4 5

Inflorescence length (cm) 3.8 � 0.5 22.9 � 0.7 56.7 � 1.2 101.4 � 3.6 125.8 � 5.7

Number of days after

bolting

4 � 1.3 13 � 1.5 33 � 2.4 41 � 2.1 79 � 4.2

Number of nodes on

main axis of inflorescence

5 � 2.0 11 � 3.0 15 � 3.0 22 � 2.0 26 � 3.0

Cumulated number of

nodes in lateral branches

per inflorescence

15±20 90±100 >1300 >1500

Inflorescence morphology Inflorescence with

�5 visible nodes on

main axis, covered

by large bracts

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

28

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283±299

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) aceticacid (IAA) were added as growth regulators, based on preliminary experiments(N. Topoonyanont, unpublished). The pH was adjusted to 5.8 prior to autoclavingat 1218C for 20 min. The cultures were incubated at 23 � 28C under a 16 h day-length 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 whicharose from the axil of each node explant: vegetative shoot, floral shoot, mixedshoot (vegetative and floral) or none. Vegetative shoots were characterised by thedevelopment of a leaf rosette, while floral shoots elongated and formed 1±4nodes. 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 asshown in Fig. 1. Moreover, from every explant, the tissues around the axil fromwhere both vegetative shoots and floral shoots developed, were also used asanother source of inocula during the first subculture. We called this type ofinoculum `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 asinitial explants as well.

The explant type and the way they developed were positioned in diagrams toexamine the distribution and degree of reversion of inflorescence buds along thebranches.

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 twogenerations. Percentages of reacting inocula and the type of development wereexamined and recorded in every generation at one month intervals.

The anatomical characteristics of reverted inflorescences from all five differentdevelopmental stages and two branch positions (first- and second-order) wereinvestigated histologically following the procedure of Johansen (1940). Theywere fixed in FAA (5% formalin, 5% glacial acetic acid and 90% ethyl alcohol)and dehydrated with a tertiary butyl alcohol series (TBA). Then they wereembedded in paraffin, sectioned at 10 mm and stained with Delafield'shematoxylin.

The experiment was designed as a factorial in completely randomised design.The factors were five developmental stages of the inflorescence and twobranching positions. Results were analysed by ANOVA.

3. Results

3.1. Inflorescence development

During the vegetative stage, Limonium `Misty Blue' grew as a rosette withmany leaves arranged spirally with very short internodes. The transition from thevegetative to the reproductive phase became apparent with the development of therapidly bolting inflorescence meristem, covered by modified leaf primordia,called bracts. Several stages could be distinguished during the development of aninflorescence (Table 1). First, the inflorescence meristem started to elongate andhad large bracts (stage 1). After about 15 days the first-order branches developedacropetally from the axils of the main axis (stage 2). While there was a gradualacropetal development of first-order branches, the main axis continued toelongate, and gradually second-order branches were initiated on the first-orderbranches (stage 3). They followed the same pattern of the first-order branches. Atthe terminal end of second-order branches, 10±12 spikes differentiated in acentrifugal order; subsequently spikelets differentiated into spikes in centrifugalorder too. The number of spikelets per spike was reduced towards the distal endof the branches. Flower development started with calyx formation (stage 4) andlater 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 greenhouseinflorescence 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


inflorescence branch, but moreover, two axillary buds were observed, whichcould eventually develop into second-order branches. The degree of developmentof these buds depended on the position of the node. Buds from the main axis grewfaster and were more developed than those from first- or second-order nodes, asshown 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.


3.2. In vitro reversion of inflorescence explants in different developmental stagesand positions

Diagrams of the in vitro behaviour during initiation of the cultures of theexplants 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 gradientof reversion along the inflorescence for the main axis as well as for the lateralbranches. Explants from the proximal end of the main axis of each stage tendedto exhibit vegetative traits while the terminal ends continued to form floralorgans. 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.


than the later stages 4 (Fig. 3d) and 5 (Fig. 3e). In stages 4 and 5, mixedshoots were obtained from proximal end explants and floral shoot from thedistal ones.

It was clear from the histological study that the shoots emerged directly fromaxillary buds, without callus intervention. Inflorescence meristems of the mainaxis could revert to a vegetative meristem (a meristematic dome with leafprimordia, Fig. 4), or floral and vegetative shoots could be obtained from thesame 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 inthe inflorescence influenced the development in vitro. Stage 1 explants takenfrom 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, andthis increased to almost 50% for stage 4 explants, to decrease significantly againfor stage 5. Each nodal explant could produce different shoots, which were eitherof 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. 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.


stages 3±5 of the mother plants. These explants produced elongated floral shootswith 3±4 nodes. These were used in subsequent subcultures.

It was obvious that shoots regenerated from both positions (main axis or lateralbranches) showed different quality. Shoots derived from the explants on the mainaxis were larger, with 6±7 leaves, and of a better visual quality; those from lateralbranches were smaller with 2±3 leaves. However, the disadvantages of main axisexplants are the high degree of bacterial contamination (Pseudomonas sp.) andthe difficulty to root them (data not presented); this is not the case for materialderived from lateral branches.

3.3.2. First subculture

Vegetative shoots, harvested from nodal explants on the main axis, producedbetween 2.5 and 4 newly developed vegetative shoots (Table 3). When thesevegetative shoots originated from what was originally a mixed shoot, thepropagation ratio was almost limited to 1 or 2. Interesting results were obtainedwhen the tissues around the axil of the primary explant were recultured, indeedeach `̀ base'' yielded approximately four new shoots, notwithstanding thedevelopmental stage of the mother plant from which they originated.

For vegetative shoots harvested from side branches the shoot production wasmuch reduced (Table 3).

Vegetative shoots never formed floral shoots. The other types of inocula coulddevelop 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

development

Vegetative

shoots

Mixed

shoots

Floral

shoots

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.bWithin columns means followed by the same letter are not significantly different (LSD 95%).


successive subcultures, and are therefore interesting, as each floral shootproduced 2±4 nodes.

3.3.3. Second and third subcultures

All types of development obtained from the first subculture (vegetative shootsand nodes from floral shoots) were used as inocula for the second and thirdsubcultures. Almost every vegetative shoot which originated from the main axiscontinued 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 3main axis inocula. The results were not so clear-cut for inocula from lateralbranches, although good results were obtained, approaching 100% or at least 60%.

Vegetative shoots which originated from the base in the first subculture wererather unpredictable in percentage of inocula which produced vegetative shootsagain in the second subculture, but all of them yielded almost 100% in the thirdsubculture, notwithstanding whether they originated from the main axis or fromlateral branches.

Nodes originating from floral shoots during initiation were rather unreliable inthe following subcultures. However, it was obvious that the younger stage ofdevelopment 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

Vegetative

shoots

Mixed

shoots

Base Mixed

shoots

Floral

shoots

Base

Main axis stage 1 4.0 � 0.2b 1.5 � 0.2 3.6 � 0.5 1.0 � 0.2 3.3 � 1.0 1.6 � 0.4

Main axis stage 2 3.1 � 0.5 1.3 � 0.3 3.5 � 0.6 1.0 � 0.1 1.5 � 0.3 3.5 � 0.3

Main axis stage 3 3.5 � 0.9 1.3 � 0.1 4.4 � 0.5 1.3 � 0.2 2.2 � 1.1 2.2 � 0.2

Main axis stage 4 3.1 � 0.6 2.0 � 0.5 4.0 � 0.8 1.2 � 0.1 1.9 � 0.2 1.9 � 0.2

Main axis stage 5 2.3 � 0.3 1.0 � 0.1 3.0 � 1.0 2.3 � 1.0 1.5 � 0.3 1.5 � 0.3

Lateral branches stage 2 ±c ± ± ± ± ±

Lateral branches stage 3 1.0 � 0.1 1.1 � 0.1 2.1 � 0.2 1.3 � 0.3 1.3 � 0.1 1.7 � 0.1



aData are scored as number of vegetative shoots or as number of nodes in a developing

inflorescence.bMean � S.E.cNo data.


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

Nosc � 2 Nos � 3 Nos � 2 Nos � 3 Nos � 2 Nos � 3

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.bMeans followed by the same letter are not significantly different (LSD 95%).cNumber of subcultures.dNo 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

Vegetative shoots Vegetative shoots

from `̀ base''

Floral shoots

Nosb � 2 Nos � 3 Nos � 2 Nos � 3 Nos � 2 Nos � 3

Main axis stage 1 4.3 � 1.6c 4.3 � 0.4 2.6 � 0.1 ±d 1.0 � 0.2 ±

Main axis stage 2 4.5 � 1.0 3.0 � 0.5 1.6 � 0.1 ± 1.0 � 0.2 1.3 � 0.5

Main axis stage 3 3.1 � 0.7 3.1 � 0.3 ± ± ± ±

Main axis stage 4 3.8 � 0.9 3.3 � 0.2 3.6 � 0.1 1.9 � 0.1 1.2 � 0.3 1.4 � 0.2

Main axis stage 5 3.3 � 1.0 3.3 � 0.4 3.8 � 0.3 2.9 � 0.2 1.6 � 0.3 1.2 � 0.2

Lateral branches stage 2 ± ± ± ± ± ±




aMultiplication rate of three different explant types in second and third subcultures. Data scored

after 30 days.bNumber of subcultures.cMean � S.E.dNo data.


second subculture, but the remaining results are rather confusing and no real trendcan be deduced from them.

Limonium `Misty Blue' taken from in vitro have been grown on the greenhousefor 6 months. All plants behaved normally and no phenotypic variation wasobserved.

4. Discussion

Inflorescence development phases of Limonium hybrid `Misty Blue' can beseparated into two major transition phases: the transition from rosette to earlyinflorescence when the vegetative meristem begins to bolt (stage 1), and later onthe inflorescence develops (stages 2 and 3) and flowers (stages 4 and 5).

The results of the reversion of inflorescence nodes in vitro showed that therewas a gradient along the axis. The reversion could be partial or complete,depending on the developmental stage of the inflorescence and the position of thenodes. In the early stages of development, a high percentage of reversion wasobtained and it decreased in more advanced stages. On the contrary, the develop-ment of floral shoots increased as the explants were taken from more developedinflorescences, except for stage 5. In this stage flowers were fully developed andshowed 50% anthesis, floral shoot formation on the main axis explants decreasedagain. This is probably due to the fact that the inflorescences reached a point ofno return in their development. This suggests that the determination toinflorescence development in Limonium is separated from determination toflower development, as is the case in Pisum sativum (Ferguson et al., 1991). Inother crops such as Nicotiana tabacum (Singer and McDaniel, 1986) andPharbitis nil (Larkin et al., 1990) they were reported to be non-separable steps.

Inflorescence reversion in Limonium `Misty Blue' can be made use of inmicropropagation. The developmental stage of an inflorescence and the branchposition can affect the percentage and rate of shoot formation. Main axis explantsof stages 4 and 5 plants are the best explant source for gaining more and vigorousvegetative shoots, and these shoots can be used as inocula in subsequentgenerations. However, main axis explants are only recommended when thegrowing conditions in the greenhouse are well controlled during mother plantpreparation (stage 0, Debergh and Maene, 1981) and they should not betransplanted to the greenhouse immediately after initiation, because of rootingdifficulties.

Nodes from lateral branches can be used as initial explants because they canproduce both vegetative and floral shoots; although the multiplication rate waslow (approx. 1 vegetative shoot per explant). However, lateral branches are a hugesource of initial explants; more than 1000 nodes could be obtained from oneinflorescence in stages 4 and 5 (Table 1). Moreover, they are also a source of


newly formed base inocula and floral nodes as well. These newly formed floralnodes provide new shoots in the second subculture (2 months after initiation).Although the rate of formation of new shoots from lateral branches was low (oneshoot per node), the multiplication rate increased during the third subculture (3±4fold every 6 weeks). Moreover, shoots regenerated from lateral branches easilyformed roots (data not presented).

Based on our experiments, we recommend that nodes from inflorescences ofLimonium `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.


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