Abstract: Somatic embryogenesis in sweet corn has been reported by a number of workers. However, the knowledge maintaining storage life, vigor and viability of these somatic embryos are limited. A model system of synchronous somatic embryos production combined with encapsulation to synthetic seed was studied in sweet corn (Zea mays var. saccharata). In this study immature zygotic embryo cultured on N6 medium, contained 2, 4-D 2 mg L-1 and sucrose 60 g L-1 form the embryogenic callus. Higher 2, 4-D levels did not show increasing in inducing embryogenic callus. If the concentration of 2, 4-D decreased globular-stage, somatic zygote form the roots. Somatic embryo develop without surrounding nutritive tissues and protective seed coat has been devoted to causing somatic embryos to functionally mimic embryo, then was encapsulated by 3% (w/v) sodium alginate with 4-6 mm in diameter. It was found that when synthetic seed were treated with 60 g L-1 sucrose and stored at 15±2 degree Celsius for 2 weeks, the survival rate of synthetic seed were 44%, after 8 days of germination test, it was found that there were 91% of which were normal seedling and 9% were abnormal seedling. This result indicated that there is a possibility in sweet corn synthetic seed production. Anyhow, more research for better technique are further required.
INTRODUCTION
Plant regeneration via somatic embryos start with one or only a few cells, this type of regeneration is important for plant multiplication, mass production and plant biotechnology such as clonal propagation and especially genetic transformation (Gordon-Kamm et al., 1990). Synthetic seed technology is an exciting and rapidly-growing research area in plant cell and tissue culture (Fujii et al., 1987; Datta et al., 1999). Even though this technique is still at a non-commercial phase because of several constraints that limit its practical use (Kozai et al., 1991; Carlson and Hartle, 1995), synthetic seeds offer the potential advantages of being genetically identical materials, easy to handle and transport and which display an increased efficiency for in vitro propagation in terms of space, time, labor and overall costs. The usefulness of encapsulation of in vitro-cultured explants has also been demonstrated in other fields of application, such as cryopreservation of plant genetic resources (Ishikawa et al., 1997; Bouafia et al., 1996; Mix-Wagner, 1999). The concept of synthetic seeds having first been publicly discussed by Murashige and Skoog (1962) and initially, the first synthetic seeds were simply somatic embryos produced from tissue cultured vegetative cells that had the particular advantage of enabling rapid clonal multiplication. Presently, the development of synthetic seed technology as expanded to the artificial encapsulation of somatic embryos, shoot, cell aggregates, or any other tissue that can be used for sowing and that retains this potential after storage (Capuano et al., 1998).
The encapsulation materials and methods for the production of synthetic seeds have seldom varied since the concept was introduced by Murashige and Skoog (1962). A series of studies dealing with the encapsulation of somatic embryos in conventional calcium alginate solid beads have been published in the past two decades (Redenbaugh et al., 1987; Sheeba and Padmaja, 1999). In these cases, the embryos protruded or were located close to the surface, so that complete protection was ensured by used of calcium alginate beads. These beads offer complete protection and allow for continued development of the explant within the beads (Patena et al., 2002).
Little attention has been directed towards encapsulation in calcium alginate hallow beads and storage of the encapsulated sweet corn propagules. Furthermore, storage of synthetic seeds using an alginate-encapsulation protocol has been attempted in only a few species, with minimal success (Redenbaugh et al., 1986, 1987; Fujii et al., 1987). In this study, results of the optimized production, storage and regeneration of sweet corn somatic embryos encapsulated in calcium alginate under various storage conditions and the re-growth under in vitro condition are presented.
MATERIALS AND METHODS
The experiment was conducted at Department of Agricultural Technology, Faculty of Technology, Maha Sarakham University and Department of Agronomy, Faculty of Agriculture, Chiang Mai University, Thailand in 2007-2009. Sweet corn variety of FAH 01 somatic embryos were cultured from immature zygotic embryos 11 days after pollinated. Callus initiation with sterilized zygotic embryos was cultured on agar-solidified N6 medium, containing with different levels of sucrose (30 and 60 g L-1) and 2, 4-D (2, 3 and 4 mg L-1). In all experiments culture temperature was 25±2°C in the dark. Callus maintenance was incubated under temperature 25±2°C in the dark. Sweet corn somatic embryos were developed by transfer friable type II callus aggregates on regenerate medium N6 contained with 1 mg L-1 NAA and incubated under temperature 25±2°C in the dark for 2 weeks. Then, embryogenic callus were transferred for 2 weeks on plant growth regulator- free MS medium and cultured under temperature 25±2°C in the light condition. The in vitro plantlets were mutiplied in liquid medium culture; one cycle lasted 3-4 weeks. The single cell of somatic embryo raised on MS liquid medium at 25±2°C in the 14 h photoperiod condition. The single cell sweet corn somatic embryos of in vitro-propagated were dissected and trimmed as described by Schäfer-Menuhr et al. (1996) and pre-cultured on liquid MS medium. Under sterile conditions, the precultured somatic embryos (3-4 mm in length) were then mixed with 10 mL of 100 mmole calcium chloride solution. Using a hypodermic syringe the suspension was dropped in 500 mL 3% sodium alginate solution. After 20 min of polymerization, the alginate hollow beads were collected on a sterile sieve and washed with 500 mL of sterile water. After that, the experiment was conducted in factorial in complete randomized design with 4 replications, while synthetic seeds were stored under various conditions; 15±2°C and 25±2°C, various dessicated levels; 20, 40 and 60% by silica gel method and then storage for 2 weeks. After synthetic seed germinated, the percentage of sweet corn synthetic seed germination, normal seedling and abnormal seedling was observed. The analysis of variance was performed for data analysis and differentiated with Last Significant Different (LSD) test at p<0.05 using the software SX release 8.0 (Analytical software, Tallahassee, USA).
RESULTS AND DISCUSSIONS
The sweet corn synthetic seeds capsules were consisted of the somatic embryos
in a liquid core of nutrients surrounded by a calcium alginate. The nutrients
were added in the capsule core during incubated capsules in the MS liquid medium.
Of the 400 measured capsules, a mean were between 0.54-0.68 cm diameters. The
encapsulated somatic embryos that were not stored were 100% regenerated on MS
solid medium. It was indicated that trimming and encapsulating had no effect
on viability of somatic embryos and they could maintained their meristematic
characteristics. Emergence occurred when the elongating shoot tips with new
leaves burst through the calcium-alginate capsule walls. The first shoot tip
emergence was observed at 7 days and the last emergence at 10 days after transferred
to the regenerated MS solid medium. 10 days after regenerated, the seedling
developed in 1-2 cm high plantlets. The synthetic seeds after storage under
15±2°C and 25±2°C condition were germinated 43 and 55%
and emergence in 9 and 8 days, respectively (Fig. 1). For
the seedling characteristics, the results was indicated that the seedling of
sweet corn synthetic were normal seedling for 90 and 95%, abnormal seeding 10
and 5% (Fig. 2). Aggrey et al. (2003) reported regeneration
of 80% of potato synthetic seeds on MS solid medium, while attained 60% regeneration
of banana encapsuled. The somatic embryos were inoculated with various concentration
of sucrose (0, 30 and 60 g L-1), that affected on synthetic seeds
germinated to 43, 57 and 46% and plantlets were regenerated in 7-9 days (Fig.
3).
| Fig. 1: | The effect of storage temperature (15 and 25°C) on sweet corn synthetic seed germination percentage and date of germination |
| Fig. 2: | The seedling characteristics of sweet corn synthetic seed after germinated under various storage temperatures (15 and 25°C) |
| Fig. 3: | The effect of various sucrose concentration (0, 30 and 60 g L-1) on sweet corn synthetic seed germination percentage and date of germination |
| Fig. 4: | The seedling characteristics of sweet corn synthetic seed after germinated under various sucrose concentrations (0, 30 and 60OC) |
Moreover, the result was showed 89-94% of normal seedlings and 5-10% of abnormal seedlings (Fig. 4).Synthetic seeds were decreased their germination to 60, 44 and 13% and normal seedling characteristics were also reduced 92, 89 and 81%, the abnormal seedling 7, 11 and 19% and wide range of germination date when desicated at level 20, 40 and 60%, respectively (Table 1, Fig. 5A-D).
The lack of external nutrients support necessary for synthetic seed germinated growth and developed and did not regenerate when transferred onto medium (Armstrong and Green, 1985). Chand and Singh (2004) showed that the uncapsulated axillary buds of six mulberry species failed to develop shoots after 7 days after storage at 4°C. It was also observed that the hollow beads capsules progressively become drier and shrunk in size during storage, probably due to decreasing humidity within storage condition. It is also further thought that the decline in the regeneration frequency observed among the stored encapsulated propagules may be due to the inhibited respiration of plant tissue by the alginate and accumulated of metabolic wastes in the alginate capsules during the long storage (Redenbaugh et al., 1987). The result indicated that at present the maximum storability of the synthetic seeds is 2 weeks. A 20% desicated were 46% regenerated is achieved and shown significantly on normal seedling characteristic, but it was shown that, synthetic seeds viability reduced when stored time and desicated level was increased (Table 2).
Other studies have shown lower regeneration levels under various storage conditions,
Ghosh and Sen (1994) acheived a maximum regeneration of only 34% after 90 days
storage of asparagus encapsulated somatic embryos. Datta et al. (1999)
found that artificial seeds of an endangered orchid stored at 4°C for 120
days shown no reduction in viability, whereas non-encapsulated protocorn-like
bodies showed no viability after 30 days at 4°C. Unlike zygotic embryos,
synthetic seeds do not accumulate storage reserves, do not develop tolerance
to drying, can be stored for a limited time and simply regenerate into plantlets,
developing by emerging through the capsule, a process that can be slowed down
by lowering the temperature (Duncan et al., 1985). The high viability
of sweet corn synthetic seeds stored at 15±2°C indicated that they
can be useful alternatives to be handled and transported cheaply like seeds
due to their small size. Synthetic seeds can also allow breeders to store unique
gene combinations that cannot be maintained by conventional seed production
due to genetic recombination (Emons and Kieft, 1991). The usefulness of synthetic
seeds was storage of vegetative propagules, especially for ex situ conservation
germplasm as an alternative for field or greenhouse nursery is brought into
focus in this paper, which was congruence with Fransz and Schel (1991), their
high frequently regeneration, viability and re-growth imply that they can be
very useful to sweet corn seed producers Fransz and Schel (1994).
| Fig. 5: | Sweet corn synthetic seeds; (A) Sweet corn somatic embryos were encapsulated with calcium-alginate polymerized beads, (B) Germinated of sweet corn synthetic seeds, (C) Seedling characteristic of sweet corn synthetic seed; N: Normal seedling, AB: Abnormal seedling and (D) Plantlet was regenerated from sweet corn synthetic seeds |
| Table 1: | The effect of desiccation levels on synthetic seeds viability |
| The different letters indicate the statistically significant difference by LSD at 5% level. ns: Not significant | |
| Table 2: | The effect of storage time and desiccated level interaction on sweet corn synthetic seed storability and viability |
| The different letters indicate the statistically significant difference by LSD at 5% level | |
This study was successful in developing and optimizing a procedure of the production and storage of sweet corn synthetic seeds by encapsulating somatic embryos in calcium alginate hollow beads and retrieving plantlets, as an alternative sweet corn propagation practice. However, a detailed study towards the development of a cost-effective delivery protocol for synthetic seeds in the field is under way. In order to confirm their possible economic use in agricultural system and whether they could be handled as traditional plant seeds or seedling transport as the natural true seeds.
ACKNOWLEDGMENTS
This research is an activity of the programme Subject-related partnership between the University of Göttingen (Germany) and Faculty of Agriculture, Department of Agronomy, Chiang Mai University (Thailand) in the area of Academic Co-operation in Teaching and Research and acknowledged to Department of Agricultural Technology, Faculty of Technology, Maha Sarakham University, Thailand.