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In Vitro Callus Induction and Plantlet Regeneration Is Influenced by the Maturity Status of Embryos of Brassica Rapa Varieties

Sabrina Zisan
Dept. of Genetics & Plant Breeding, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

Arif Hassan Khan Robin
Dept. of Genetics & Plant Breeding, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

Ahasanul Hoque
Dept. of Genetics & Plant Breeding, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

Mohammad Rashed Hossain
Dept. of Genetics & Plant Breeding, Bangladesh Agricultural University (BAU), Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Brassica rapa (AA, 2n = 20) is a prime oilseed species in Bangladesh that can be improved via biotechnological approaches. Rescue of developing embryos after an interspecific hybridization, which otherwise usually tend to abort, is very important for improvement of this species, that require an efficient embryo culture protocol. The immature torpedo shaped embryos (451-700 µm) and mature walking-stick type (>700 µm) embryos of five B. rapa varieties namely, Safal, Agrani, BINA Sarisha-6, BARI Sarisha-6, BARI Sarisha-15 were cultured in basal MS media supplemented with 1 mgL^-1 BAP, 0.5 mgL^-1 NAA & 1 mgL^-12,4-D. The immature embryo culture followed indirect somatic embryogenesis process but the mature embryos followed direct organogenesis. The immature embryos induced callus within 11-15 days whereas the mature embryos regenerated plantlets through direct organogenesis within 8-11 days. The varieties Agrani, BARI Sarisha-6 and BINA Sarisha-6 induced the highest percentages of callus from immature embryos and the varieties Safal and BARI Sarisha-15 regenerated the highest percentage of plantlet from mature embryos. Safal and Agrani induced callus and regenerated plantlets comparatively earlier than other varieties. An increase in the concentration of BAP from 10 to 20 ppm in MS media + 5 ppm IBA decreased the size of the plantlets of all varieties except the Agrani. The data could be used in improving the existing embryo rescue protocols. The direct organogenesis process of mature embryos can potentially shorten the breeding cycles.

Keywords: Mustard, Embryo culture, Callus, Phytohormone, Plantlet, 2’4-D, MS media

Location: Mymensingh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Mustard

Objectives:

To optimize the in vitro culture system of immature and mature embryo of five selected B. rapa species.

Methodology:

Seeds of five B. rapa varieties viz. Safal, Agrani, BINA Sarisha-6, BARI Sarisha-6, BARI Sarisha-15 were collected from Bangladesh Agricultural Research Institute (BARI), Gazipur and Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Seedlings were raised in the field of BINA and siliquas of different ages were collected. Embryos of different shapes viz., globular, heart, torpedo and walking-stick type, as determined by the relative age, size and shape of embryos, were dissected using a stereoscopic dissecting microscope in a chamber under laminar flow. The torpedo shaped embryos were categorized as immature embryos (451-700 μm) and walking-stick type embryos were categorized as mature embryos (>700 μm). Excised mature and immature embryos of different ages from the five varieties, with five replicates for each, were cultured directly in MS (Murashige and Skoog, 1962) medium supplemented with sucrose and with different concentrations of hormones viz., 1 mgL^-1 6- Benzylaminopurine (BAP), 0.5 mgL^-1 1-naphthaleneacetic acid (NAA) & 1 mgL^-1 2,4-Dichlorophenoxyacetic acid (2,4-D) for callus induction following a Completely Randomized Design (CRD). The explants were incubated under fluorescent light with controlled temperature (22±2^oC), photoperiod (16 hrs) and relative humidity (75%). The cultures were observed regularly to record their callus formation and regeneration response. At every stage, the contaminated vials were immediately discarded from the stock. Almost after 14-15 days of inoculation, different morphological callus phenotypes were noticed. The immature embryos produced callus and the mature embryos produced plantlets directly. The calli, induced from immature embryos, attained convenient size, usually after 15-16 days after inoculation of explants, were sub-cultured aseptically on freshly prepared sterilized medium containing the same hormonal supplements. The vials showing signs of contamination were discarded on regular basis and data were collected from the growing calli. The plantlets, induced from mature embryos, of convenient size were transferred to shoot induction media (basal MS media supplemented with 4 ppm BAP to proliferate shoots. When these shoots grew about 3-4 cm in length, they were rescued aseptically from the cultured vials and were separated from each other and again sub-cultured individually on vials with freshly prepared shoot induction medium (MS). But this time MS medium was supplemented with two different hormonal combinations, one with 10 ppm BAP and 5 ppm Indole-3-butyric acid (IBA) and another with 20 ppm BAP and 5 ppm IBA to observe the effect of these hormonal combinations on the proliferation of plantlets. The vials containing shoots were incubated at 25±1°C under controlled environmental condition. The proliferated shoots were finally transferred to the rooting media (Basal MS media supplemented with 4 ppm IBA) to initiate root formation. Day to day observations was carried out to note the responses of shoot formation. The varietal performances in various callus parameters such as percentages of callus initiation, days to callus initiation, size, colour and texture of callus etc. were recorded from the immature embryo culture. While both the varietal performance and the effects of two different hormonal conditions were observed via various plant regeneration parameters such as percentage of plant regeneration, days required to plant regeneration and size of the plantlets etc. were studied from the regenerated plantlets from mature embryo culture. The analysis of variance for different characters was performed and means were compared by the Duncan's Multiple Range Test (DMRT). The data were analyzed using the MSTATc.

Source of Information: Journal of Bioscience and Agriculture Research, Vol. 06, Issue 01: 518-529 (2015), eISSN: 2312-7945

Article Link (Online Journal): http://www.journalbinet.com/jbar-060115-62.html

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Characterizing the embryo culture from mature and immature embryos of fives B. rapa varieties would be very helpful to improve embryo rescue protocols in future. As the torpedo shaped immature embryos induced callus and walking-stick type mature embryos directly regenerated into plantlets therefore the later protocol could be established as a ‘rapid regeneration protocol’. However, further research is needed to improve the embryo cultures techniques.

Knowledge Management: Journal