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Somatic Embryogenesis Induction in Delonix regia (Boger.) Raf (Royal Poinciana)

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Somatic Embryogenesis Induction in Delonix regia (Boger.) Raf (Royal Poinciana)
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Myers, Alba
Vendrame, Wagner ( Mentor )
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Gainesville, Fla.
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University of Florida
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Somatic Embryogenesis Induction in Delonix regia (Boger.) Raf (Royal Poinciana)

Alba Myers


ABSTRACT


Induction of somatic embryogenesis in Delonix regia has been successfully achieved in cultures initiated

from immature seeds. Two induction culture media were evaluated, combined with three levels of 2,4-D and

three levels of 6 BA. Three different explants were evaluated, consisting of: 1) nicked immature zygotic embryos;

2) immature whole seeds; and 3) immature seeds cut in half. Cultures were successfully established with a

low contamination level of 3%. Callus and proembryogenic mass (PEM) formation were observed for all

treatments, media, and explant types. Somatic embryos developed on the surface of PEMs and no

significant differences existed for the type of medium used, explant type, and the plant growth

regulator combinations and levels tested. Future perspectives include the regeneration of plantlets from

somatic embryos and the use of somatic embryogenesis as a technique for rapid multiplication of royal

Poinciana trees.



INTRODUCTION


Delonix regia (Boger.) Raf. (royal poinciana, flamboyant) is an ornamental flowering tree related to the mimosa

tree family (Leguminosae, subfamily Caesalpinioideae), native and endemic to Madagascar. Royal poincianas

can reach up to 40 feet tall, usually with a canopy wider than its height (Turner, 1999). This species is

considered one of the five most beautiful flowering trees in the world, blooming between spring and early

summer. The flower is very showy and its color varies from orange to orange-red, including yellow and a rare

white (Stebbins, 1999).



In The United States, specifically in South Florida, the ornamental and landscape characteristics of poincianas

add commercial value for nursery production. Poinciana trees grow best from seed and flowering can be

achieved within five to seven years (Stebbins, 1999). However, propagation of poincianas by seeds has

several limitations and rooting of cuttings is not an efficient alternative. The seeds hard coating imply in

reduced germination percentage (Duarte, 1974), the seed position in the pod influences germination and quality

of seed product (Srimathi et al., 1992) and unfavorable changes in climate during flowering and pollination

periods result in low seed production (Gutteridge and Stur, 1994).







Tissue culture techniques have been applied to a number of woody ornamental species with success, allowing the

in vitro propagation of species that are difficult to propagate by conventional methods. Somatic embryogenesis

has been a tissue culture technique of particular interest for woody species. In poincianas, young

meristematic tissues, such as immature embryos and developing leaves have been used as explants to

induce somatic embryogenesis (Rahman and Hossain, 1992; Lakshmanan and Taji, 1999). However, the success

has been limited and no further attempts have been reported to date.



The objective of this work is to develop an improved protocol for inducing somatic embryogenesis in royal poinciana.



MATERIALS AND METHODS


Plant material selection and sterilization. Immature seedpods of poinciana were collected during mid-June

2003 from a tree located in Miami, Florida (Figure 1). Seedpods were cut in sections and pre-washed with soap

and water to remove external dirt and contaminants. The sterilization protocol consisted of placing the pre-

washed seedpods into a sterile beaker containing a solution of 50% (v/v) Clorox. A surfactant was added for

better sterilization coverage, consisting of 16 drops of Tween 20. Seedpods were kept under agitation for 20

minutes, followed by threel5-minute rinses using autoclaved distilled water. The procedure was performed in

sterile conditions inside a laminar flow hood (Figure 2).


Figure 1. A. Royal poinciana tree. B. Detail of Royal poinciana flowers.




























Figure 2. Sterilization setup. A. Immature seedpods drying after pre-sterilization wash. B.

Immature seeds are located in the upper part of the seedpod (arrows). C. Location of immature

seeds inside the pods (arrows). D. Pods sections after sterilization, ready for dissection inside

the laminar flow hood.



Culture medium preparation. Two different media were used; Medium A, a modified induction medium

(Merkle and Sommer, 1986); and Medium B, a modified Woody Plant Medium (Lloyd and McCown,

1980). Modifications for both culture media consisted of additions of plant growth regulators in combination

at different concentrations (treatments), as described below. Culture media were autoclaved and dispensed

into plastic disposable Petri dishes.



Treatments. Treatments consisted of different combined levels of 2,4-D (an auxin) and 6 BA (a cytokinin) for

each culture medium. Auxins and cytokinins have been used to successfully induce somatic embryogenesis in

several woody species. The combinations and levels of 2,4-D and 6 BA were used as follows:



- Dl: 1 mg/I of 2,4-D + 0.125 mg/I of 6 BA

- D2: 2 mg/I of 2,4-D + 0.250 mg/I of 6 BA

- D3: 4 mg/I of 2,4-D + 0.500 mg/I of 6 BA


Culture initiation and somatic embryogenesis induction. Sterilized seedpods were dissected and the

seeds removed under aseptic conditions. In order to evaluate the potential of different seed explants for

inducing somatic embryogenesis, seeds were either used intact or dissected with a scalpel and the immature

zygotic embryo removed (Figure 3). Three different explant types were used, as follows:



1. Seeds were dissected and the immature zygotic embryos (El) were removed and isolated, nicked with a scalpel and

placed in each culture medium for each of the treatments (Figure 3A).




2. Entire seeds (E2) containing the immature zygotic embryos were placed in the culture medium for each of the

treatments (Figure 3B).

3. Entire seeds were cut in half (E3) and halves were placed with the cut side down in the culture medium for each

of the treatments (Figure 3C).



Between 2-4 explants were placed per Petri dish. Cultures were kept in the dark at 25 � 2...C. The total number

of explants obtained was 136 immature zygotic embryos (El), 156 entire seeds (E2), and 142 half seeds

(E3). Explants were measured, varying from 1mm in length for zygotic embryos to 10mm in length for

immature seeds (Figure 3).














Figure 3. Explant types used in the experiment. A. Zygotic immature embryo. Bar = 1 mm. B.

Immature whole seed. Note the location of the immature zygotic embryo inside the seed (arrow). Bar

= 10 mm. C. Immature seed cut in half. Bar = 10 mm.



Experimental Design and Statistical Analysis. From 20 seedpods collected from the source tree, the number

of immature seeds that were extracted from each pod ranged from 15 to 24. For each culture medium a total of

60 Petri dishes were used, consisting of 20 Petri dishes replicationss) per treatment (Table 1). A total of

186 explants were randomly distributed among the different treatments. The Analysis of Variance (ANOVA)

procedure (SAS Institute, 1989) was used to evaluate the effects of explant type, treatment, culture medium,

and the interaction among them on the induction of embryogenic cultures.




Table 1

Experimental design showing the number of replications (number of Petri dishes) per

treatment (D1, D2, D3) for each culture medium used

Number of Replications

Treatment 2, 4-D Level 6 BA Level Medium A Medium B

D1 1 mg/l 0.125 mg/l 20 20

D2 2 mg/l 0.250 mg/l 20 20

D3 4 mg/l 0.500 mg/l 20 20


RESULTS AND DISCUSSION








Embryogenic Callus Formation. After two weeks in culture, callus formation was observed for all explant types

in every medium and for all treatments (Figure 4). Contamination was detected in some cultures, although

the overall level of contamination was under 3%. Contaminated cultures were promptly discarded.

















Figure 4. Embryogenic callus formation. A. Embryogenic callus formation in immature zygotic

embryos (arrows). Bar = 2 mm. B. Immature embryo showing callus formation at the tip (arrow).

Note the desiccation and distortion of the seed at the tip. Bar = 9 mm. C. Dissected seed producing

large amounts of callus around both halves (arrows). Bar = 13 mm.



For El explants, callus formed around the immature zygotic embryos causing increase in size and volume

(Figure 4A) for all treatments tested. Callus characteristics consisted of a disorganized cell growth around

the explant, showing round to elongated cells of translucent color. For E2 explants, desiccation and distortion at

the tip of the immature seed was observed (Figure 4B). Callus production was observed only at the base of

the immature seeds, although for all treatments. E3 explants showed large amounts of callus around the two

halves placed in culture, varying froml0mm to 13mm in length (Figure 4C).



Callus formation is likely to have resulted from auxin and cytokinin action in the culture medium. Auxins induce

the formation of embryogenic cells and promote repetitive cell division, while cytokinins are required to

induce embryogenesis in several dicotyledonous species (Gray, 2000). In this experiment, we used three

different explants to evaluate their potential for the induction of embryogenic callus. Seeds split in half (E3) showed

a noticeable larger volume of callus formation, although no volume measurements were evaluated. Nicking

the immature zygotic embryo with a scalpel (El) also showed to be an effective method to induce callus

formation, while the use of an entire seed (E2) showed a limited amount of callus produced. All explants showed

to be successful in producing callus.



Proembryogenic Masses (PEMs) Formation. Five weeks after cultures were initiated and callus growth

was observed, some PEMs started to form (Figure 5). Proembryogenic masses are groups of cells showing

organized growth, with small, densely cytoplasmic (embryogenic) cells of isodiametric shape (Gray, 2000).

The responses to PEM formation were similar for both media and treatment levels for all explants used.









































Figure 5. Proembryogenic mass (PEM) formation in the different explants used. A. PEM formation

in nicked immature zygotic embryos (El). Bar = 5 mm. B. Same photo showing a close up of PEMs

(10X). Bar = 50 mm. C. PEMs forming at the base of immature whole seeds (E2). Bar = 11 mm. D.

Same photo showing close up of PEMs (10X). Bar = 110 mm. E. PEM formation in split seeds (E3). Bar

= 15 mm. F. Close up of PEMs shown in D (10X). Bar = 150 mm.



Immature zygotic embryos (El) showed prolific PEM formation. The size of explants increased an average of 3.0

mm in size. PEMs distinguished themselves from callus by showing particular characteristics for embryogenic

cells (Figures 5A,B), as described by Gray (2000). Immature seeds (E2) developed PEMs on top of callus tissue

and an average of 2.0 mm increase in size was observed (Figures 5C,D). Immature seeds split in halves (E3)

were almost completely covered with callus and PEMs. The increase in size averaged 2.0 mm. (Figures 5E,

F). Overall, PEM formation for all explants indicates an excellent potential for inducing somatic embryogenesis. As

a matter of fact, somatic embryos can arise from different cell and tissue types, directly on the explant

tissue, without the formation of callus, or indirectly, via formation of embryogenic callus (Gray, 2000).



Somatic Embryo Formation. Four weeks after PEMS were observed, somatic embryos started to form.

Three distinct stages of somatic embryos could be clearly observed consisting of:


a. globular stage, where embryos appear as well-formed round structures (Figure 6A).






b. torpedo stage, showing an elongated axle (Figure 6B).

c. cotyledonary stage, where cotyledons are clearly visible (Figure 6B).


Figure 6. Somatic embryo induction in Delonix regia explants. A. Globular stage somatic

embryos (arrows). Bar = 150 mm. B. Torpedo stage and cotyledonary stage somatic embryos

(arrows). Bar = 150 mm.



This process happened independently of the explant type. The process of somatic embryo development was

observed to be asynchronous, where somatic embryos can be observed at different stages of development within

the same treatment and replication unit. However, this is a common characteristic of somatic embryogenesis.



Induction of embryogenic cultures showed no significant differences for the means for the explant type (P =

0.7109), treatment (P = 0.9567) and medium (P = 0.8742) tested (Table 2). The interaction among the

variables also showed no significant differences (P = 1.000) at a = 0.05.




Table 2

Number of Petri Dishes (mean �SD) Showing Somatic Embryo Induction after Five Weeks in

Culture Media. Numbers are shown for each explant type and treatment used

Explant Type Treatment Medium A* Medium B*

D1 11.5 � 2.12a 10.5 � 6.36a
Immature Zygotic Embryos
D2 11.5�3.54a 11.5 � 3.54a
(El)
D3 11.5�0.71a 11.0 � 4.24a



D1 13.5 � 6.36a 12.5 � 6.36a
Immature Whole Seeds
D2 13.0 � 2.83a 12.5 � 6.36a
(E2)
D3 13.0 � 7.07a 13.5 � 7.78a


D1 10.0 � 4.24a 13.0 � 2.83a
Immature Half Seeds
D2 14.0 � 2.83a 12.0 � 4.24a
(E3)
D3 13.0 � 5.66a 12.0 � 8.49a


* Values followed by the same letter within columns are not significantly different at a � = 0.05.





In this experiment we demonstrated the feasibility of inducing somatic embryogenesis in royal poinciana.

Although some contamination was initially detected, the level was very low and it did not affect the experiment.

No additional contamination was detected along the course of the experiment. Both culture media showed to

be similar and effective in inducing somatic embryogenesis. No significant differences were observed among

the different treatments regarding the induction of callus, PEMs, or somatic embryos. Likewise, all explant types

were able to produce callus, PEMS and somatic embryos.



Cultures have been routinely maintained and future perspectives include the optimization of the culture

system aiming synchronous and increased production of somatic embryos, and ultimately the regeneration of

royal poinciana plantlets. Somatic embryogenesis can be a feasible system for the rapid multiplication of

royal Poinciana trees.






ACKNOWLEDGEMENTS



I would like to thank The University of Florida, Dr. Wagner Vendrame and Ian Maguire for assisting and teaching

me the steps of tissue culture and somatic embryogenesis during my internship.






REFERENCES



1. Cutteridge, R.C. and W.C. Stur. 1994. Seed production of forage tree legumes. In: Cutteridge, R.C. and Shelton, H.

M. (ed.). Forage tree legumes in tropical agriculture. 168-174.

2. Duarte, 0. 1974. Improving Royal Poinciana seed germination. Plant Propagator. 20(1): 15-16.

3. Gray, D.J. 2000. Nonzygotic embryogenesis. In: Trigiano, R.N. and D.J.Gray (eds.). Plant tissue culture concepts

and laboratory exercises. CRC Press, Boca Raton, FL. 175-189.

4. Lakshmanan, P. and A. Taji. 2000. Somatic embryogenesis in leguminous plants. Plant Biology, 2: 136-148.

5. Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use

of shoot-tip culture. Proc. Int. Plant Propag. Soc., 30: 421-427.

6. Merkle, S.A. and H.E. Sommer. 1986. Somatic embryogenesis in tissue cultures of Liriodendron tulipifera. Can. J.

For. Res., 16: 420-422.

7. Rahman, S.M. and M. Hossain. 1992. Micropropagation in Delonix regia. Pakistan Journal of Botany, 24(1): 60-63.

8. SAS Institute. 1989. SAS/STAT user's guide, 4th ed., version 6. SAS Institute, Cary, North Carolina.

9. Stebbins, M.K. 1999. Flowering Trees of Florida. Pineapple Press, Sarasota , Florida.

10. Srimathi, P., Swaminathan, C., Sivagnanam, K. and C. Surendran. 1992. Seed attributes in relation to their






position in the pod and its influence on seedling establishment of four ornamental tree species. Journal of

Tropical Forest Science, 4(3): 245-248.

11. Turner Jr., R.G. 1999. Botanica. Barnes and Noble, Hong Kong, China.


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