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In Vitro Callus Induction, Shoot Formation and Their Histological Study in Cotton (gossypium Sp.)

R. Sultana
Assistant teacher, Bogra Zilla School,Bogra

N. Hossain
Department of Botany,University of Rajshahi, Rajshahi

Abstract/ Executive Summary:

A study was conducted to standardize in vitro culture techniques for callus induction, direct shoot formation and to find out developmental pattern of induced calli and the successive stage of the direct organogenesis in different explants of Gossypium sp. Explants of seedling was established. Callus initiated from hypocotyl portion and nodal segment. Hypocotyl explant showed the highest degree of morphogenic potentiality than nodal explants. MS medium containing Auxin (2 4-D) alone or in combination with Auxin and Cytokinin induced callus and the induction varied with hormonal supplement. Low concentration of 2,4-D with high concentration of KIN showed the best induction of callus. Other than callus proliferation, these hormonal supplements did not induce any root shoot differentiation from primary callus. In vitro grown nodal explants produce a number of adventitious bud in MS medium containing 5mg/1 2IP. 2IP in combination with NAA also showed good production in this case. Shoot regeneration from shoot tip explant was highest in MS0 medium . Ms medium with 2,4-D+KIN concentration showed the clear observation of cell development stage, particularly near the wound zone than 2,4-D+BAP concentrated medium. Both hypocotyls and nodal portion derived callus showed same cell development pattern in long term callus. Disrupted vascular tissue was found in some section. In case of long time (75 days old) callus section showed large sized plastid. Callus initiate with the starting of meristematic activity. Further meristematic activity resulted the formulation of leaf trace which is connected with nodal tissue and also found shoot developing stage from node.

Keywords: In vitro, callus, organogenesis, hypocotyl, nodal and vascular tissue

Location: Rajshahi University

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cotton

Objectives:

1. The aim of this study was to establish in vitro culture techniques for callus induction and direct shoot formation in cotton.

Methodology:

The experiment was carried out at Rajshahi University. An indigenous cotton genotype’DL-90’ was used. Hypocotyl portion, nodal segment and shoot tip excised from in vitro grown seedlings. MS medium was used. Napthalene acetic acid (NAA), 2,4-dichlorophonoxy acetic acid(2,4-D), 2-isopentenyl adenine(2IP), Benzyl amino purine(BAP) and kinetin(KIN) was used as the plant growth regulators in different combinations and concentrations. 8gm of Carolina Biological supply co-agar was added for one litre of medium. Seedlings were collected from 15, 20, 25 and 30 days old plants used as explants for primary establishment of cultures. 3, 5 and 7 days old seedlings were used for hypocotyl explants. The hypocotyl portion was separated from seedlings. The portion was divided into small pieces. Each of hypocotyl portion contained 4-5 mm. Those segments were divided into two halves. 20, 25 and 30 days old sterile seedlings were used as nodal explants. Each nodal segment contained nodal portion 4-5mm above the bud and 2mm below the bud. Shoot apices were prepared from in vitro grown 5,7, and 9 days old seedlings. Shoot tip measuring 5-7 mm were excised from the top of the plant. Nodal portion (7-8mm) including auxiliary buds from in vitro grown plantlet were excised 4mm above the bud and 2mm below the bud. All the explants for callus induction were placed in petridishes and sealed with parafilm and they were incubated in dark cup board self in a temperature controlled room (25±2⁰c). When the callus attained at a size of 40-45mm diameter was cut into convenient size by the sterilized scalpel and again set on same or different hormone supplemented media. Data were recorded on % of explants induced callus, average number of shoots and average length of shoots (cm). Calli at different stages were fixed in a formalin aceto alcohol (FAA) solution contained 90cc ethyl alcohol (50%), 5cc Glacial acetic and 5cc formalin for microtome technique using paraffin method. The paraffin course involved fixing the plant material in fixative which is followed by washing, dehydrating, infiltrating, embedding and sectioning it much anticlinaly with the aid of microtome. The paraffin slides were then stained with safranin, orange G tanic acid, Iron-alum and zinc chloride. The slides were dehydrated through different grades of alcohol (50%, 60%, 70%, 80%, 90% and absolute alcohol. Then the tissue were passed through xylene and finally mounted in coverslip with the help of Canada balsum. The prepared slide was studied under a compound microscope.

Source of Information: BANGLADESH RESEARCH PUBLICATIONS JOURNAL; ISSN: 1998-2003, Volume: 5, Issue: 4, Page: 306-313, July -August, 2011

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Hypocotyl explant showed the highest degree of morphogenic potentiality than nodal explants. MS medium containing Auxin (2 4-D) alone or in combination with Auxin and Cytokinin induced callus and the induction varied with hormonal supplement. Low concentration of 2,4-D with high concentration of KIN showed the best induction of callus.

Knowledge Management: Journal