Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene.

Rogayah Sekeli,Parameswari Namasivayam,Umi Kalsom Abu Bakar,Pauziah Muda,Janna Ong Abdullah

doi:10.5402/2013/958945

Rogayah Sekeli, Parameswari Namasivayam + Show 3 more

Open Access

https://doi.org/10.5402/2013/958945

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Abstract

A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets.

Highlights

E world production of papaya in 2008 was valued at 9.1 million tonnes [1], and Malaysia is listed as one of the top six major exporters. e most popularly grown cultivar and the main variety grown for export is Eksotika, which is a high yielding variety producing 50–70 tonnes per hectare over a two-year crop cycle [2]. e release of Eksotika papaya variety in 1987 had since resulted in an increase in its production areas in Malaysia
We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the eld
E pOpOff 2 vector [13] was used for all the three RNAi constructs, designated as pRNAiCACO, pRNAiACO1, and pRNAiACO2. is pOpOff 2 vector contains an nptII selectable marker gene, a gusA reporter gene, an inducible pOp6 promoter, a CaMV35S promoter, and a nos terminator. is inducible RNAi vector was obtained through an MTA agreement between the Malaysian Agricultural Research and Development Institute (MARDI) and the Commonwealth Scienti c and Industrial Research Organization (CSIRO) for use in this research. e pRNAiACO1 and pRNAiACO2 constructs contain the 3 UTR region of the respective acid oxidase (ACO), and the pRNAiCACO contains the conserved regions of both genes. e primers used for each gene constructs were designed using the Pearl Primer so ware (Sourceforge.net)

Summary

Introduction

E world production of papaya in 2008 was valued at 9.1 million tonnes [1], and Malaysia is listed as one of the top six major exporters. e most popularly grown cultivar and the main variety grown for export is Eksotika, which is a high yielding variety producing 50–70 tonnes per hectare over a two-year crop cycle [2]. e release of Eksotika papaya variety in 1987 had since resulted in an increase in its production areas in Malaysia. E two major problems faced in developing transgenic Malaysian Eksotika papaya plants are low rooting efficiency of regenerated shoots and low acclimatization rate of rooted transgenic papaya plants in the eld. Erefore, rooting efficiency and quality roots formation are critical in ensuring successful and continuous production of transgenic Eksotika papaya. E aim of the study reported here was to improve the rooting efficiency and roots quality of tissue-cultured Malaysian Eksotika papaya shoots regenerated from embryogenic calli transformed separately with RNAi and antisense constructs of the ACO genes (ACO1 and ACO2). E improved rooting method presented here will be useful for the development of transgenic papaya plants facing rooting problem (poor root quality) a er a genetic manipulation event Morphological and histological analyses were carried out to assess the quality of the roots produced. e improved rooting method presented here will be useful for the development of transgenic papaya plants facing rooting problem (poor root quality) a er a genetic manipulation event

Materials and Methods

Results and Discussion

Conclusions