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Cells Structure and Morphogenesis of Embryogenic Aggregates in Suspension Culture of Bitter Melon (momordica Charantia L.)

Rubaiyat Sharmin Sultana
Department of Botany, University of Rajshahi, Rajshahi 6205, Bangladesh

Md. Mahabubur Rahman
Research Institute of Sustainable Humanosphere, Kyoto University, Uji, Kyoto 611-0011, Japan

Abstract/ Executive Summary:

Cell suspension culture from leaf-derived callus of Bitter melon (Momordica charantia L.) was established. The callus could be induced from leaf segments on agarified Murashige and Skoog (MS) medium containing 1.0 mgl^-1 2,4- dichlorophenoxyacetic acid (2,4-D), which consequently used for suspension culture. In the establishment of cell suspension culture, the highest performance in the growth of cells was observed in liquid MMS (addition of 0.5 mgl^-1 folic acid and 0.05 mgl^-1 biotin to MS medium) medium containing 2,4-D (1.5 mgl^-1) when callus was subcultured. The S-shaped growth curve with three typical phages (lag, exponential and stationary phases) was found in the batch culture. Cells in suspension culture underwent division as result both free cells and cell aggregates were formed. The number of cell aggregation affected by the initial amount of cells. When the initial amounts of cell were increased from 1 to 5 ml sedimented cell volume (SCV), the number of cell aggregates was increased gradually. The number of aggregates decreased with further increasing initial amount of cells from 5 ml SCV. Aggregate formation at the highest level when 5 ml (SCV) initial cells subcultured. The duration of subculture was found to be effective for cell proliferation in suspension and proliferated cell structure. The cell growth in culture was lower when suspension maintained up to 8 weeks without subculturing. The elliptical cell structure was mostly found in suspension maintained up to 8 weeks without subculturing. The most of cells were spherical-shaped and cell growth was high in suspension when suspension culture changed to fresh medium at 7-week-interval. The established system on the cell suspension culture might be used for establishing an efficient somatic embryogenesis method of bitter melon.

Keywords: Bitter melon, Callus, Cell aggregate, Cell suspension, Morphogenesis, Proembryogenic structures.

Location: Department of Botany, University of Rajshahi, Rajshahi 6205, Bangladesh

Start Date: 00-00-2012

End Date: 00-00-2012

Program Area: Variety and Species

Commodity: Bitter Melon

Objectives:

The objective of the present study was to establish efficient cell suspension culture for bitter melon from the leaf-derived callus. In the present study, the growth of cells, cells structure and development of cell aggregates in suspension culture were studied under the various media and culture conditions.

Methodology:

Plant materials- Seeds of bitter melon were collected from local market and used for further investigation. Preparation of basal medium and conditions of in vitro culture- MS (Murashige and Skoog, 1962) basal medium were used for callus induction and MMS (additionally used 0.5 mgl^-1 folic acid and 0.05 mgl^-1 biotin to MS medium) for the establishment of cell suspension culture. The callus induction was carried out on agarified medium. The medium was solidified with 0.8% (w/v) agar (Type M, Sigma). The liquid medium was applied for cell suspension culture. All media were adjusted to pH 5.7 ± 0.1 and autoclaved at 121°C for 20 min. The medium without plant growth regulators (PGRs) received as control for all experiments. The cultures were maintained at 27 ± 1°C under a 16-h photoperiod (35 µmol m^-2 s^-1), 16-hour light and 8-hour dark. Callus induction- Leaves of ten-day-old seedling were used as plant materials in the present study. The seeds collection, disinfection and in vitro germination procedures were described in our previous report (Sultana and Miah, 2003). The leaves of aseptically growing seedling were harvested and sliced into approximately 1 cm x 1 cm pieces and placed on MS medium containing different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4- D) at 0.5, 1.0, and 2.0 mgl^-1 alone or combinations of 6- Benzylaminopurine (BAP) (1.0, 5.0 and 10.0 mgl^-1) and 1-Naphthaleneacetic acid (NAA) (0.1 and 0.5 mgl^-1) for callus induction. Data on percentage of explants that induced callus were recorded after 4 weeks of culture initiation. Initiation of cell suspension culture- The induced callus (5 g fresh weight) on MS medium fortified with 1.0 mgl-1 2,4-dichlorophenoxyacetic acid (2,4-D) was transferred to each 300-ml Erlenmeyer flask containing 50 ml of liquid MMS medium fortified with 1.0, 1.5, 2.0, and 3.0 mgl^-1 2,4-D alone. The flasks were sealed with Aluminum foil, wrapped with parafilm, and then they were placed on a rotary shaker (95 rpm). Callus cells were proliferated in suspension cultures for 2 weeks. The cell growth in each PGR treatment was measured. For measurement of cell growth, the proliferated cells in an Erlenmeyer flask were dispensed in sterile 100 ml measuring cylinders and allowed to sediment for 30 min after that measured total cell amount by sedimented cell volume (SCV) as milliliter (ml). Cell growth by batch culture- Cell proliferation in suspension culture was examined by batch culture. The total amount of proliferated cells at the end of each subculture was determined by SCV (ml). For batch culture, 5 ml SCV of cells collected from 2-week-old suspension culture using callus, and they were subcultured in 300 ml Erlenmeyer flasks containing 50 ml MMS liquid medium fortified with 1.5 mgl^-1 2,4-D. The cultures were routinely transferred at 1-week-interval to a fresh medium and the total amount (ml) SCV of cell per flask measured prior to transfer in each subculture. The suspension cultures were maintained up to 8 weeks. Effect of initial cell amounts on cell proliferation and cell aggregates development- Cells from 2-week-old suspension were subcultured initially at different volumes, 1, 3, 5, 10, 15, and 20 ml SCV in 300 ml Erlenmeyer flasks containing 50 ml of MMS liquid medium supplemented with 1.5 mgl^-1 2,4- D. The cultures were transferred into fresh medium routinely in a week. After 4 weeks of culture, the proliferated cells in an Erlenmeyer flask were dispensed in sterile 100 ml measuring cylinders and additional cell amount measured with SCV (ml). In above all suspension cultures, the number of cell aggregates was measured using hemacytometer. For counting the number of cell aggregates, 10 µl of liquid cultures were used. The number of cell aggregates was counted using the manual counter in each grid of hemacytometer and the total number was calculated per 10 µl suspension and then total number of cell aggregates was counted from 1 ml suspension. Effect of subculture duration on cell size- Two-week-old 5 ml SCV cells (on MMS containing 1.5 mgl^-1 2,4-D) were subcultured in MMS containing 1.5 mgl^-1 2,4-D. Cell structure in suspension culture were observed after 8-week-old culture without transferring to the fresh medium and in 8-week-old culture with regularly transferring at 7-day-interval under the light microscope. The structures of cell were compared between these two conditions of cell suspension culture. Statistical analysis- All experiments were repeated at least three times. The experiments were conducted with a completely randomized design and the data were analyzed by analysis of variance with Tukey’s multiple comparison test using JMP Statistical Discovery Software (SAS Institute, Cary, NC, USA). The least significance difference (LSD) test was used to distinguish differences among the mean value in treatments at 5% (p ≤ 0.05) level.

Source of Information: International Journal of Biosciences; Vol. 2, No. 3, p. 97-105, 2012

Article Link (Online Journal): http://www.innspub.net

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, cell suspension culture of bitter melon was established in the present study. The medium and culture conditions for the free cells and different stages of proembryogenic structures in the suspension were optimized. The present study will accelerate the establishment of somatic embryogenesis through cell suspension culture and also will be helpful to establish single-cell origin of bitter melon plant.

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