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Regeneration of asparagus officinalis l. through Embryogenic callus

Mustafa Abul Kalam Azad
Department of Botany, University of Rajshahi, Rajshahi--6205, Bangladesh

Muhammad Nurul Amin
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

A plant regeneration system was established from hypocotyl explants of in vitro grown seedlings of A. officinalis and in vitro proliferated shoots, respectively through somatic embryogenesis and embryogenic calli. Somatic embryogenesis was significantly influenced by the types of plant growth regulators. Embryogenic calli with somatic embryos developed well in MS supplemented with 2.0 - 4.0 μM BAP and 1.0 - 4.0 μM 22,4-D, NAA or IBA. The highest frequency (95.3%) of embryogenic calli and 55.2 somatic embryos formation were obtained when the MS was amended with 4.0 μM BAP and 2.0 μM 2,4-D. The best embryo germination occurred in 1.0 μM BAP supplemented MMS. The highest 97.2% of shoot proliferation was observed in embryogenic calli in MS medium containing 2.0 μM BAP and 1.0 μM IBA. In vitro grown shoots were rooted in MMS with 1.0 - 2.0 μM IBA. Regenerants were transferred to vermicompost and successfully established under an ex vitro environment in garden soil with 80% survival rate.

Keywords: Somatic embryo, Hypocotyl explants, Asparagus officinalis

Location: Rajshahi, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Plant

Objectives:

To developing an efficient propagation protocol for the production of clonally uniform plants through somatic embryogenesis and organogenesis method (Makris and Rossiter 2001).

Methodology:

Seeds of Asparagus officinalis were collected from Japan. These seeds were surface sterilized with 70.0% EtOH for 3 min. Sterilized seeds were germinated on hormone' free MS. The seeds germinated within 3 weeks and gave rise to shoots that developed two to three nodes five to six weeks later. Shoots were then propagated by subculturing single-node cuttings at 4 week intervals in MS with BAP (2.0 - 4.0 μM). Hypocotyl explants were prepared from 6-week old aseptically grown seedlings. These explants were cultured in MS containing different concentrations of BAP (2.0 - 4.0 μM) in combination with either 2,4-D (1.0 - 4.0 μM), NAA or IBA for the induction of embryogenic calli and somatic embryos. Hypocotyl explants were also cultured in B5 (Gamborg et al. 1968), MMS (half strength of MS basal salts), and WPM (Lloyd and McCown 1981) media, with the addition of 4.0 μM BAP plus 2.0 μM 2,4-D, NAA or IBA to determine the effects of media on somatic embryogenesis. For the germination of somatic embryos (SEs), the individual embryos were cultured in an MMS basal medium with 2.0% sucrose and a low concentration (0.5 - 2.0 μM) of BAP and Kn, 10 - 30% CW (Coconut water) or without the addition of any plant growth regulators. The embryogenic calli (approx. 0.5 - 1.0 g) were transferred to MS medium supplemented with various concentrations and combinations of BAP (2.0 - 4.0 μM) and NAA or IBA (0.5 - 2.0 μM) for adventitious shoot regeneration. The data were recorded after eight weeks of culture initiation: percentage of shoot formation, number of total shoots per culture, and average length of shoots per culture. Embryogenic calli derived shoots were rooted in MMS supplemented with different concentrations (0.5 - 4.0 μM) of IBA, NAA, or IAA. All culture media were adjusted to pH 5.7 ± 0.1, fortified with 3.0% sucrose (w/v) (except embryo germination medium), and gelled with 8.0% agar. The cultures were grown at 25 ± 1°C under the illumination of cool' white fluorescence tubular lamp with a light intensity of 50 µmol⋅m-2⋅s-1 for a 16 hrs photoperiod. Twenty replicates of all experiments were conducted. Experiments were repeated four times. The effects of different treatments were quantified, and the data were analyzed using ANOVA. Tukey’s multiple comparison was used to distinguish differences between treatments.

Source of Information: Plant Tissue Cult. & Biotech. 27(1): 21?31, 2017 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The highest frequency (95.3%) of embryogenic calli and 55.2 somatic embryos formation were obtained when the MS was amended with 4.0 μM BAP and 2.0 μM 2,4-D. The best embryo germination occurred in 1.0 μM BAP supplemented MMS. The highest 97.2% of shoot proliferation was observed in embryogenic calli in MS medium containing 2.0 μM BAP and 1.0 μM IBA. In vitro grown shoots were rooted in MMS with 1.0 - 2.0 μM IBA. Regenerants were transferred to vermicompost and successfully established under an ex vitro environment in garden soil with 80% survival rate.

Knowledge Management: Journal