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An Improved Protocol for Multiple Shoot Regeneration from Seedlings and Mature Explants of Citrus Macroptera Mont.

Md. Nesawar Miah
Institute of Life Sciences, National University, Gazipur

Shahina Islam
Tissue Culture Section, BCSIR Laboratories, Dhanmondi, Dhaka

Syed Hadiuzzaman
Department of Botany, University of Dhaka

Abstract/ Executive Summary:

Efforts have been made to establish a protocol for direct multiple shoot regeneration from both in vitro grown seedlings and mature plants of Citrus macroptera. Both nodal and shoot tip explants taken from in vitro grown seedlings were cultured in MS supplemented with different concentrations of BAP and Kn either singly or in combinations. Both these explants are capable to regenerate and produce in vitro multiple shoots. Maximum number of shoots were obtained from nodal explants in MS supplemented with 1.0 mg/l BAP. BAP alone was found superior to Kn. On the other hand, only nodal explants from mature plants were used and 1.0 mg/1 BAP was also found best suitable for shoot induction and multiplication. Ex vitro rooting in pot soil (mixed with biogas slurry derived from cow-dung) was most successful compared to in vitro rooting in half strength of MS supplemented with different concentrations of NAA and IBA.

Keywords: In vitro, Shoot regeneration, Citrus macroptera

Location:

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Satkara

Objectives:

To develop protocol for direct regeneration and multiplication from the explants of seedlings and mature plant.

Methodology:

Nodal segments and shoot tips were collected from six - eight weeks old in vitro grown seedlings of Citrus macroptera Mont. var. annamensis and cut into 10-12 mm long tips. These segments were cultured on MS supplemented with different concentrations of of BAP and Kn. BAP and Kn alone at 0.5, 0.1 and 1.5 mg/1 and combinations BAP and Kn at 0.5 + 0.5, 1.0 + 0.5 and 0.5 + 1.0 were used for direct shoot regeneration. The regenerated shoots were cultured on the same medium for further multiplication. The data from both shoot induction and multiplication were recorded after four - five weeks interval. Stem twigs of mature plants were collected from the field and washed thoroughly under running tap water for 30 min to remove dust and surface contaminants. Then they were treated in 70% alcohol for five min and were surface sterilized in 4% Na(OCL) solution with two - three drops of Tween 20 for 15 min. During this process the liquid was continuously stirred on a magnetic stirrer under a vacuum pump to facilitate expulsion of all air bubbles followed by surface sterilization of all exposed surfaces. Finally they were washed four times in sterile distilled water under laminar airflow cabinet. Then the stem was cut into 10-15 mm segments each containing a single node. The prepared explants were cultured on MS containing BAP and Kn for auxillary bud initiation at 0.5, 1.0, 1.5 and 2.0 mg/1. The regenerated shoots were transferred into the same media for further multiplication. Each regenerated shoot was cultured on a freshly prepared medium containing half strength of MS with NAA and IBA alone at 0.1, 0.5 and 1.0 mg/1 for adventitious root initiation. At the same time, a number of shoots were cultured on MS without growth regulator for further elongation. Thereafter, elongated shoots were directly transferred to sterilized plastic posts filled with sterile soil and cow-dung derived from biogas slurry (1 : 1) for adventitious root formation.

Source of Information: Plant Tissue Cult. & Biotech. 18(1): 17-24, 2008 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

However, in this investigation direct rooting into pot soil was found best. After four weeks a single root developed that was 2.5 - 3.5 cm long; and after eight weeks two - three adventitious roots (5 - 7 cm) developed with several branching. So far there is no report on direct rooting of citrus in in vitro grown microshoots into soil. The induction of ex vitro rooting from cultured shoots may be more economical besides producing better and strong roots. Since the in vitro rooting of the plantlets was very poor they were not suitable for transfer to soil for establishment. However, successful achievement of direct ex vitro rooting encouraged transfer of the shoots directly into soil for their establishment. The survival rate of the transferred shoots was also high (85 - 90%). Better survival rate was observed in case of more aged shoots. The rooted plantlets also showed healthy and steady growth

Knowledge Management: Journal