Abstract: This study describes the investigation on the synthetic seed technology in two varieties (Loda and China) of eggplant with a particular focus on the carbon source in seed bead. To assess exact type and concentration of carbon source, three types of carbon sources (viz., sucrose, mannitol and sorbitol) and their different concentrations were used in alginate seed bead to achieve the optimum germination of synthetic seed on MS0. Among the different carbon sources, sucrose showed better performance for both varieties when nodal segment with bud was used as explants. But when somatic embryos were used as explants sucrose + sorbitol (1:1) was found to be more efficient for both varieties in germination of synthetic seeds. On the other hand, among the different concentrations 1% sucrose gave good result for the germination of synthetic seed made of nodal segment. In case of somatic embryo 3% sucrose showed best performance for both varieties.
INTRODUCTION
Numerous techniques are now available for rapid and large-scale commercial multiplication of elite and desirable plant species in vitro. Somatic embryogenesis can provide a more efficient method for mass propagation. In order to make the system economically viable many workers have encapsulated the somatic embryo (Radenbaugh et al., 1986). In some instances, instead of embryos, axillary buds also have been encapsulated and germinated (Machii, 1992; Kinoshita and Satto, 1990). Encapsulation of embryos to produce synthetic seeds could serve as a substitute for the seeds produced in vivo. Eggplant is only propagated by seeds without having opportunity of asexual propagation. Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to response well in plant tissue culture, notably plant regeneration, has allowed the application of Biotechnology, particularly the exploitation of somaclonal variation, haploidization, somatic hybridization and gene transfer.
Synthetic seed technology offers many useful advantages towards commercial application of these biotechnological approaches. The resultant plant population from the synthetic seed will be uniform and the direct delivery of somatic embryos will save many subcultures to obtain plantlets from regenerated embryos. The encapsulated embryos could also be packed with pesticides, fertilizers, nitrogen fixing bacteria (Redenbaugh et al., 1986; Ohishi et al., 1995). Concentration and source of carbon in the bead medium has been found to affect photosynthetic potentiality of plant growing from synthetic seed. But Hyndman et al. (1982) and Landford and Wainwright (1988) reported that high concentration of sucrose cause to reduce the photosynthetic activity and germination rate may be decreased.
In this present study an attempt was made to assess the influence of carbon source in seed bead medium on synthetic seed germination and in vitro shoot development.
MATERIALS AND METHODS
The experiment was conducted in the Institute of Biological sciences, Rajshahi University, Bangladesh in 2006.
Induction of Explant
In this experiment, two varieties of Solanum melongena viz. Loda
and China were used and the seeds of these two varieties were cultured on MS0
(Murashig and Skoog without hormone) medium for seedlings and seedlings were
cultured on nutrient medium. Nodal segment with axillary bud formed within 30
days of culture which was used as explants following the methods of Maruyama
(1996). On the other hand, the cotyledon pair of seedlings was cultured as explant
on callus inducing medium (2.0 mg L-1 NAA + 0.05 mg L-1
BAP) for callus induction. Best calli derived from cotyledonary explants of
the two varieties were subcultured separately on MS medium having 2 mg L-1
NAA with 0.05 mg L-1 BAP. Somatic embryos, formed and matured within
7-28 days, were used as explant for making synthetic seed (Saiprasad, 2001).
Carbon sources used were: Sucrose, Sorbitol and Mannitol.
Preparation of Solutions for Encapsulation
Encapsulation of synthetic seed was done according to the method reported
by Kinoshita and Saito (1990). Two hundred milliliters MS media was prepared
with 6 g sugar (sucrose, sorbitol and mannitol) supplemented with 0.5 mg L-1
BAP + 0.1 mg L-1 GA3 for nodal segment but in case somatic
embryo the medium was supplemented with 1.0 mg L-1 BAP + 0.1 mg L-1
GA3. Twenty milliliters of the above mentioned solution was taken
and 0.8 g of sodium alginate was added and taken in a small beaker (50 mL beaker).
With a small piece of glass rod efforts were made to mix the alginate in solution.
Alginate was partially dissolved and it was then kept aside. During autoclaving
alginate was completely dissolved. Fifty milliliters of the above mentioned
solution (200 mL MS) was taken in a small beaker and 0.7 g (70 mg) CaCl2
was added to it and dissolved. Out of 200 and 70 mL (50+20) was used during
the preparation of alginate and CaCl2 solutions and rest 130 ml was
remained as reserved. After autoclaving it was used during washing of the encapsulated
beads. All prepared solutions were autoclaved at 15-lb/sq inch’s pressure
and at 120-121°C temperatures for 21 min for sterilization.
Encapsulation of Explants
The nodal segments with active buds and somatic embryos were placed to the
beaker containing alginate solution. The explants were dipped in alginate solution
and taken by a forcep and placed to the beaker of CaCl2. During picking
up the explants, the forceps, also took some addition alginate solution together
with explants.. The rolling explants with the liquid of alginate
were dropped in to the CaCl2 solution. The explants were kept inside
the alginate solution for about 30 min. After 30 min each explant became a hardball
encoated by alginate.
Culture of Synthetic Seed
The encapsulated explants or synthetic seeds were washed with 130 mL MS
liquid medium (above mentioned). After washing, synthetic seeds were cultured
on MS0 medium under in vitro condition in growth chamber where photoperiod
was maintained generally 16 h light 8 h dark.
RESULTS AND DISCUSSION
In order to understand the regeneration potentiality of different carbon source in seed bead medium two types of explants were studied for eggplant germination. In this experiment, somatic embryo and nodal segment with axillary buds were used as explants for artificial seed production and data were taken for germination percentage, days to germination and shoot length. The results are given in Table 1 and 2.
| Table 1: | Effect of different types of carbon sources on synthetic seed formation and germination |
| Table 2: | Effect of different levels of sucrose (carbon source) on synthetic seed formation and germination |
In the present investigation, three types of carbon sources viz., sucrose, sorbitol, mannitol and their combination in the ratio 1:1 were used in alginate bead (Table 1). Best result was found when sucrose was used alone using nodal segment and in this case 85% seeds of Loda variety were germinated within 4-7 days of culture and 82% seeds of China were germinated within 5-7 days of culture. But highest shoot length (3.7±0.6) was observed when sorbitol was used instead of sucrose. Among all the carbon sources used in the experiment, sucrose and its combination gave better result in artificial seed germination.
In case of somatic embryo, best result for synthetic seed germination was obtained when sucrose + sorbitol (1:1) was used as carbon source (Fig. 1C and D) and in this case 70% seeds of Loda variety were germinated within 5-7 days of culture and 54% seeds of China variety were germinated within 15-20 days of culture. Second highest result was obtained when sucrose was used alone and in this case, 67% seeds of Loda variety and 50% seeds of China variety were germinated.
As sucrose and its combination gave better results then it was necessary to understand the right concentration of sucrose, best effective in artificial seed germination. For this purpose different concentrations of sucrose were used in seed bead medium using the cultivars of eggplant and their performance was presented in Table 2. It was evident from this experiment that among different sucrose levels, treated in MS medium, 1% (10 mg L-1) sucrose showed the best result for germination of synthetic seed made of nodal segment (Fig. 1F). In this sucrose level, 95% seeds of Loda variety and 90% of China variety were germinated on artificial medium. Second highest result was observed, when 2% (20 g L-1) sucrose was used but germination rate was gradually decreased when the amount of sucrose was increased. It was observed under the increased dose of sucrose, germination period was delayed and the growth of shoot was decreased.
On the other hand, when somatic embryos were used as explant for synthetic seed production, 3% (30 g L-1) sucrose level showed the better performance for seed germination (Fig. 1C). In this concentration, 67% seeds of Loda variety were germinated within 5-7 days of culture and 50% seeds of China variety were germinated within 16-21 days of culture. The optimal concentration of sucrose was 1% for synthetic seed containing nodal segment as explant and in case of somatic embryo it was 3%. In this experiment it was also found that in absence of carbon source, no germination was initiated. Lakshmana and Singh (1991) conducted similar types of experiments with eggplant using different levels of alginate concentrations in encapsulating somatic embryo and 3% sodium alginate and 75 mM calcium chloride were found to be optimum in plantlet regeneration. In their case the frequency of plantlet regeneration varied from 27.0-49.7% in vitro condition. But in our investigation, in case of somatic embryo, 70% germination was obtained using 4% sodium alginate with 1.4% calcium chloride. They used phytohormones in culture medium but we used phytohormone in seed bead medium and cultured on hormone free MS; here is the basic difference between these two investigations.
Artificial seeds consisting of somatic embryos enclosed in protective coating have been proposed as a low-cost, high volume propagation system (Radenbaugh et al., 1986) and somatic embryo possess shoot and root primordia and are usually able to develop directly into complete plant without any pretreatment (Ara et al., 2000). In addition to using somatic embryos, axillary buds, adventitious buds and shoot tips have also been used in preparation of synthetic seeds (Redenbaugh, 1993; Bapat and Rao, 1988, 1990; Ganapathia et al., 1992, 1994). Among the different encapsulating agents, sodium alginate was used for encapsulation of somatic embryo due to it’s solubility at room temperature and ability to form completely permeable gell with calcium chloride (Bapat et al., 1987). It was found in carrot or celery that a suitable synthetic endosperm was required to provide nutrients including carbon sources necessary for optimum germination and conversion (Redenbaugh et al., 1988). Kinoshita and Satto (1990) also suggest that sucrose is essential for the germination of encapsulated buds.
In the present investigation, three types of carbon source viz., sucrose; sorbitol, mannitol and their combination in the ratio 1:1 were used in alginate bead (Table 1).
| Fig. 1: | (A) Encapsulated nodal segments, (B) Encapsulated somatic embryos, (C) Germination from seed bead containing low concentration of sucrose. (Somatic embryo) (D) Germination from seed bead containing high concentration of sucrose. (Somatic embryo), (E) Germination of seed from seed bead containing high concentration of sucrose. (Nodal segment) and (F) Germination from seed bead containing low concentration of sucrose. (Nodal segment) |
Nieves et al. (1998) also used two types of carbon source viz., corn starch and sucrose in alginate seed bead of Cleopatra tangerine. Artificial endosperm of Cleopatra tangerine assays showed that mannitol with abscisic acid delayed germination and conversion of artificial seed where zygotic embryos were used as explant for encapsulation. In contrast of Cleopatra tangerine, the present investigation also suggest that mannitol is less desirable among the three carbon sources because it gave lowest germination rate of synthetic seed. In vitro propagation of mulberry by encapsulating auxillary buds also reported by Machii (1992) obtained 10% seed germination using 20 mg L-1 (2%) sucrose in both seed bead and culture medium. But when he used only distilled water in seed bead germination rate was zero supporting the result of this investigation. On the other hand Kinoshita and Satto (1990) used abundant amount of sucrose in gell matrices of encapsulated axillary buds of Japanese White Birch to examine whether or not the beads germinate and concluded that the sucrose concentration in alginate gell should be as much as 20%. But the germination rate slows down when gell matrices contain 5% sucrose. The present investigation indicated that similar to White Birch, eggplant also required low concentration of sucrose in seed bead medium for better germination. This result suggests that if one wants to produce artificial seed using nodal segment with axillary buds as explant in eggplant, should use sucrose as carbon source and in case of somatic embryo combination of sucrose and sorbitol would be recommended.