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Exploring the Development of Gametophyte –mediated Genetic Transformation Systems in Crop Plants (2019--2020)

M.A.Y. Akhond
Biotechnology Division, BARI, Joydebpur, Gazipu

K. Nahar
Biotechnology Division, BARI, Joydebpur, Gazipu

N. Bilkish
Biotechnology Division, BARI, Joydebpur, Gazipu

Roland Schafleitner
Biotechnology Division, BARI, Joydebpur, Gazipu

Abstract/ Executive Summary:

A genetic transformation is a powerful tool for plant improvement programmes. Most of the transformation methods developed are based on Agrobacterium-mediated transformation and biolistic bombardment which are time- and labour-intensive. Therefore, it is desirable to develop alternate simple and rapid transformation protocols for the development of transgenic plants without a prolonged tissue culture and regeneration process. Although it has been shown that plant gametophytes could be a potential target for transformation as an alternative to producing transgenic plants, a reproducible method is still lacking in most of crop plants. To fulfill the objectives of the experiment, tobacco microspores as a model system were used for Biolistic transformation where GUS expression was observed in bombarded microspores. Results obtained in this experiment could lead the way for an optimized system for gametophyte-mediated transformation for crop plants in the future.

Keywords: Genetic transformation, Agrobacterium, Plant improvement, Transgenic plants

Location:

Start Date: 00-00-2019

End Date: 00-00-2020

Program Area: Variety and Species

Commodity: Tobacco

Objectives:

This experiment was conducted to find out the possibility of developing gametophyte–mediated genetic transformation systems in crop plants.

Methodology:

To fulfill the objectives of the experiment, tobacco microspores as a model system were isolated from various sizes of flower buds to correlate their developmental stage with bud size. Isolated microspores were stained with DAPI solution (Vergne et. al. 1987) and observed under an epifluorescence microscope to determine the developmental stages. Anthers from five selected flower buds were extracted and cultured according to Touraev et. al., 1997. Observations were taken from sealed Petri dishes with an inverted microscope to check the progression of maturity over the culture period. Microspores were bombarded immediately after isolation with pCAMBIA1301 plasmid DNA containing LAT52-GUS, LAT56-GUS and LAT59-GUS gene using a PDS-1000/He Biolistic^TMParticle Delivery System (BIO-RAD) as described by Akhond and Machery (2009) for transient expression analysis. After bombardment in vitro matured pollens along with controls were cultured in a germination medium containing 0.3 mM X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid; Sigma). The plates were cultured for 24-48 hours at 25ºC in dark and then observed under an inverted microscope for pollen grains expressing β-glucuronidase (GUS) showing the characteristic blue colour.

Source of Information: Annual Research Report 2019-2020, Biotechnology Division, BARI, Joydebpur, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Tobacco microspores were isolated from various sizes of flower buds to correlate their developmental stage with bud size and flower buds of about 12-13 mm in size were found to contain late uninucleate microspores. The high percentage of maturity of tobacco microspores into pollen grains was observed with satisfactory pollen germination in-vitro for the advancement of the experiment. GUS expression was observed in microspores transformed with LAT52 and LAT59 construct. No expression was observed in LAT56-GUS bomberded microspores. However, sporadic expression was observed in microspore bombarded with only gold and pCAMBIA

Knowledge Management: Report/Proceedings