Abstract: This study describes the investigation on the technology of synthetic seed production using somatic embryos in two varieties (Loda and China) of eggplant. To standardize a media composition of artificial endosperm of synthetic seed, different concentrations and combinations of phytohormones with MS media were used in seed bead to achieve optimum germination rate on MS0 media. Among the different concentrations and combinations, MS+1.0 mg L-1 BAP+0.1 mg L-1 GA3 gave the highest germination rate and it was 81% for Loda and 70% for China variety. The influence of storage at 4°C and 0°C temperature on germination rate was also examined. This investigation indicates that synthetic seed could be stored at 4°C temperature for 45 days.
INTRODUCTION
Eggplant (Solanum melongena L.) is a vegetable crop of the family Solanaceae grown in the subtropics and tropics. It is one of the most popular vegetable in many parts of the world including Bangladesh. The crop is cultivated on small family firms and considerd to be important source of nutrition (Bose et al., 1993) and cash income for many resource-poor farmers. But in Bangladesh fruit fly (Leucinodes orbonalis), an insect which damaged upto 30% of total yield (Shukla and Upadhyay, 2000). For controlling the devastating insect pest farmers deliberately use the chenical insecticides in their crop fields resulting another disaster for health and environment. Educated people recently are in position to hesitate to buy eggplant from market due to liberal use of insecticides in eggplant cultivation. Development of insect resistant crop plant by genetic engineering can be an alternative measure to combat the problem. But in this case maintenance of resultant genotype (e.g, hybrid, meiotically unstable genotypes and genetically engineered genotypes) may be a great problem or inhibitor. Artificial seed in eggplant can offer a new avenue for supporting the program of genetic engineering providing an exciting asexual propagation bridge for readily multiplication of transformed plants. The present investigation was undertaken towards establishing an efficient protocol for production of synthetic seeds in eggplant in order to maintain proper genotype of resultant plant and its preservation.
MATERIALS AND METHODS
This research was conducted at the Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh in 2005.
Induction of Somatic Embryo
In this experiment, two varieties of Solanum melongena viz., Loda
and China were used. The seeds of two varieties were cultured on MS0 medium
for seedlings to have easy supply of cotyledon pair. Cotyledons were used as
explants and cultured on callus inducing medium (2.0 mg L-1 NAA+0.05
mg L-1 BA) for callus induction. Best calli derived from cotyledonary
explants of the two varieties were sub cultured separately on MS media having
different concentrations and combinations of phytohormone for somatic embryogenesis.
Somatic embryos formed within 7-28 days were used as explant/materials for preparing
synthetic seed in the subsequent experiments.
Preparation of Synthetic Seed
MS medium was used as the basal medium supplemented by phytohormone and
carbon sources. Sodium alginate was added to the medium and mixed well to make
a homogenous solution. The explants were taken in a beaker containing alginate
solution and the explants were dipped into the alginate solution. Explants dipped
in alginate solution were taken up by a forcep and placed to the beaker of CaCl2
and each explant then became a hard ball encoded by alginate.
Culture and Storage of Synthetic Seed
To examine the germination rate, the synthetic seeds were cultured on MS0
media and data were recorded. On the other hand, to examine their viability
range, the synthetic seeds were stored at 4°C and 0°C temperature in
refrigerator for 15, 30, 45, 60, 90 and 120 days. Following storage in refrigerator
under respective days regime seeds were cultured on MS0 medium.
RESULTS AND DISCUSSION
To establish a suitable media composition of artificial seed bead, different concentrations and combinations of phytohormones were added to synthetic seed bead media. For the this purpose, cytokinin (BAP and KIN) alone and in combination with auxin (NAA and IBA) and GA3 were used in artificial seed bead medium during the process of encapsulation (Fig. 1A). Data were recorded on germination rate, days to germination and shoot length and the results are represented in Table 1. Among the different concentrations and combinations, best result was observed when 1.0 mg L-1 BAP with 0.1 mg L-1 GA3 used in seed bead (Fig. 2). In this combination 81% synthetic seed of Loda variety was germinated within 4-5 days and 70% seed of China variety was germinated within 15-16 days of culture. Second highest germination rate was observed in seed bead supplemented with 1.0 mg L-1 BAP + 0.05 mg L-1 IBA. In this combination 73% seed of loda variety and 65% seed of China variety were germinated. But when high concentration of phytohormone was used in seed bead, callus was observed from somatic embryos in seed bead (Fig. 1F) instead of multiple shoot formation. Lowest germination rate was achieved, when synthetic seed was prepared without any growth regulator.
In this experiment, the influence of storage at 0 and 4°C on germination rate of synthetic seed was also examined. The seeds were stored at 0 and 4°C temperature for different time durations and cultured on MS0 media (Table 2). This investigation indicates that seeds can be stored at 4°C for 45 days. But synthetic seed could not be stored with viability at 0°C temperature, because at this temperature asexual embryos underwent crystal formation due to the lack of cryoprotectant.
In this experiment, somatic embryos were used as explant for artificial seed
production because artificial seeds consisting of somatic embryos enclosed in
protective coating have been proposed as a low-cost, high volume propagation
system (Redenbaugh et al., 1986). On the other hand production of synthetic
seed by encapsulating somatic embryo has been largely favoured since, somatic
embryo possess shoot and root primordia and are usually able to develop directly
into complete plant without any pretreatment (Ara et al., 2000). Among
the different encapsulating agent, 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).
| Fig. 1A: | Encapsulated somatic embryo (B) initial stage of seed germination (C) germinated seed of loda variety (D) germinated seed of china variety (E) plant obtained from synthetic seed and (F) callus was observed from somatic embryo in seed bead |
Rao and Singh (1991) also conducted similar type of experiment with eggplant. They used phytohormones in culture medium, but we used phytohormones in seed bead medium and cultured on free MS, here is the basic difference between these two investigations.
Here different concentrations and combinations of phytohormones were added to seed bead. Similar experiment was conducted for encapsulation of adventitious buds of Mulberry by Machii (1992) and he observed that synthetic seed grew vigorously when nutrient component and phytohormones were added during encapsulation.
| Table 1: | Effect of phytohormones in alginate bead on synthetic seed germination |
| Table 2: | Effect of preservation duration on the viability of synthetic seed |
Because germination of synthetic seed to plantlets appears to depend on the hormonal concentrations in bead medium (Soneji et al., 2002). In Morus alba it was found that BAP in seed bead gave desirable result in synthetic seed germination (Machii, 1992).
| Fig. 2: | Graph showing the effect of phytohormone on synthetic seed germination (Here phytohormone was used in mg L-1 unit) |
On the other hand best germination rate was observed using 1 μML-1 GA3 in seed bead, of Cleopatra tangerine, where's zygotic embryos were used as explant to standardize an artificial endosperm for synthetic seed production using somatic embryos (Nieves et al., 1998). But in the present investigation, BAP in combination with GA3 gave the highest germination rate than the BAP alone. This result further indicate that BAP in combination with IBA also gave desirable germination which supports the result of Soneji et al. (2002) because in his experiment, it was found that pretreatment of explant with IBA and cytokinine before encapsulation gave desirable germination rate. Bapat et al. (1987) also pretreated Mulberry axillary buds with liquid MS basal media supplemented with high concentration (2 mg L-1) of BAP for 48 h for the induction of multiple shoot prior to encapsulation and achieved multiple shoot. But in contrast to Mulberry, the present experiment indicates that, when high concentration was used, the encapsulated embryos under went callus formation instead of multiple shoot. Another significant observation in this study was that the encapsulated embryos did not lose the capacity for germination even after storing at 4°C for 45 days. Similar results also observed in Mulberry by Bapat et al. (1987) and in Japanese White Birch (Kinoshita and Saito, 1990). On the other hand, as it is belongs to the family solanaceae, roots were induced from germinated shoot within 30-35 days of culture.
The results of this study demonstrate that seeds can be germinated on MS0 media or even on agar beds containing distilled water which reduce the labour and cost. So it has opened a new area of advance research for developing the conservation strategies for eggplant genetic resources by the application of Biotechnological tools like chilling and Cryopreservation. This is why this article will play an important role in germplasm conservation with the development of appropriate storage technique. It includes the production of hybrid synthetic seed for those species where conventional technique are difficult or as a delivery system for genetically engineered genotypes produced by in vitro techniques. It will be also useful for exchange of sterile materials between laboratories thanks also to small capsule size and relative handle ease. Most important plant multiplication and nursery activities will take great advantage from this study.