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Fulltext RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes expression prolongs the shelf life of Eksotika (Carica papaya L.) papaya fruit

Sekeli R, Abdullah JO, Namasivayam P, Muda P, Abu Bakar UK, Yeong WC, et al.

Molecules, 2014 Jun 19;19(6):8350-62.
PMID: 24950439 DOI: 10.3390/molecules19068350

The purpose of this study was to evaluate the effectiveness of using RNA interference in down regulating the expression of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Eksotika papaya. One-month old embryogenic calli were separately transformed with Agrobacterium strain LBA 4404 harbouring the three different RNAi pOpOff2 constructs bearing the 1-aminocyclopropane-1-carboxylic acid oxidase gene. A total of 176 putative transformed lines were produced from 15,000 calli transformed, selected, then regenerated on medium supplemented with kanamycin. Integration and expression of the targeted gene in putatively transformed lines were verified by PCR and real-time RT-PCR. Confined field evaluation of a total of 31 putative transgenic lines planted showed a knockdown expression of the targeted ACO1 and ACO2 genes in 13 lines, which required more than 8 days to achieve the full yellow colour (Index 6). Fruits harvested from lines pRNAiACO2 L2-9 and pRNAiACO1 L2 exhibited about 20 and 14 days extended post-harvest shelf life to reach Index 6, respectively. The total soluble solids contents of the fruits ranged from 11 to 14° Brix, a range similar to fruits from non-transformed, wild type seed-derived plants.
Fulltext Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene

Sekeli R, Abdullah JO, Namasivayam P, Muda P, Abu Bakar UK

ISRN Biotechnol, 2013;2013:958945.
PMID: 25969786 DOI: 10.5402/2013/958945

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.
Fulltext Malaysian Carica papaya L. var. Eksotika: Current Research Strategies Fronting Challenges

Sekeli R, Hamid MH, Razak RA, Wee CY, Ong-Abdullah J

Front Plant Sci, 2018;9:1380.
PMID: 30279695 DOI: 10.3389/fpls.2018.01380

Carica papaya L. or commonly known as papaya, is a major tropical crop consumed worldwide either as a vegetable or fresh fruit or processed products. In Malaysia, papaya was initially planted as a smallholder crop throughout the country. Eventually after 15 years of breeding and selection, a new variety, named C. papaya L. var. Eksotika, was released by the Malaysian Agricultural Research and Development Institute (MARDI) in 1987. This event changed the outlook of papaya planting from a smallholder crop to a plantation crop. Despite the blooming papaya business, the industry faced various disease issues that jeopardize its future. The most devastating was the papaya dieback disease, which affected approximately 800 hectares of plantation, destroyed approximately 1 million trees nationwide with total losses estimated at US$ 58 million. Even though Eksotika is a favored commercial variety with good eating and aesthetic quality fruit, its potential for more lucrative distant markets is tarnished with its short-shelf life fruits. Several strategies had been reported to address the challenges faced by Eksotika specifically against the dieback disease and the fruit's short shelf-life. This review focuses on C. papaya L. var. Eksotika particularly on the strategies to address the challenges faced in order to sustain the economic value of this crop plant, which had contributed significantly to the Malaysian economy.
Fulltext The first dataset of de novo transcriptome assembly of Heterotrigona itama (Apidae, Meliponinae) queen larva

Tamizi AA, Nazaruddin NH, Yeong WC, Mohd Radzi MF, Jaafar MA, Sekeli R

Data Brief, 2020 Apr;29:105235.
PMID: 32071998 DOI: 10.1016/j.dib.2020.105235

Heterotrigona itama is a species of stingless bee recently domesticated (or reared) for honey production in a few Southeast Asian countries namely Malaysia and Indonesia. Being categorized in the clade Corbiculata together with the honeybees (Apis spp.) and bumble bees (Bombus spp.), the stingless bees are highly social in which the colony members are subjected to labor division where a queen functions as the reproductive caste. In this data article, we provide a resource encompassing a transcriptome profile (de novo assembled) from H. itama queen larva - the first report of transcriptome assembly for this species. The generated data is pivotal for the characterization of important genes and biological pathways in order to further improve our understanding on the developmental biology, behavior, social structure and ecological needs of this eusocial hymenopteran insect from the molecular aspect. The raw RNA sequencing data is available at NCBI Sequence Read Archive (SAR) under the accession number SRP230250 and the assembled reads are deposited at DDBJ/EMBL/Genbank as Transcriptome Shotgun Assembly (TSA) under the accession GIIH00000000.
Agrobacterium-mediated in planta transformation of cut coleoptile: a new, simplified, and tissue culture-independent method to deliver the CRISPR/Cas9 system in rice

Tamizi AA, Md-Yusof AA, Mohd-Zim NA, Nazaruddin NH, Sekeli R, Zainuddin Z, et al.

Mol Biol Rep, 2023 Nov;50(11):9353-9366.
PMID: 37819494 DOI: 10.1007/s11033-023-08842-2

BACKGROUND: Agrobacterium-mediated transformation and particle bombardment are the two common approaches for genome editing in plant species using CRISPR/Cas9 system. Both methods require careful manipulations of undifferentiated cells and tissue culture to regenerate the potentially edited plants. However, tissue culture techniques are laborious and time-consuming.
METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.
CONCLUSION: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.
Fulltext Pluronic F-68 Improves Callus Proliferation of Recalcitrant Rice Cultivar via Enhanced Carbon and Nitrogen Metabolism and Nutrients Uptake

Kok AD, Mohd Yusoff NF, Sekeli R, Wee CY, Lamasudin DU, Ong-Abdullah J, et al.

Front Plant Sci, 2021;12:667434.
PMID: 34149763 DOI: 10.3389/fpls.2021.667434

Pluronic F-68 (PF-68) is a non-ionic surfactant used in plant tissue culture as a growth additive. Despite its usage as a plant growth enhancer, the mechanism underlying the growth-promoting effects of PF-68 remains largely unknown. Hence, this study was undertaken to elucidate the growth-promoting mechanism of PF-68 using recalcitrant MR 219 callus as a model. Supplementation of 0.04% PF-68 (optimum concentration) was shown to enhance callus proliferation. The treated callus recorded enhanced sugar content, protein content, and glutamate synthase activity as exemplified in the comparative proteome analysis, showing protein abundance involved in carbohydrate metabolism (alpha amylase), protein biosynthesis (ribosomal proteins), and nitrogen metabolism (glutamate synthase), which are crucial to plant growth and development. Moreover, an increase in nutrients uptake was also noted with potassium topping the list, suggesting a vital role of K in governing plant growth. In contrast, 0.10% PF-68 (high concentration) induced stress response in the callus, revealing an increment in phenylalanine ammonia lyase activity, malondialdehyde content, and peroxidase activity, which were consistent with high abundance of phenylalanine ammonia lyase, peroxidase, and peroxiredoxin proteins detected and concomitant with a reduced level of esterase activity. The data highlighted that incorporation of PF-68 at optimum concentration improved callus proliferation of recalcitrant MR 219 through enhanced carbohydrate metabolism, nitrogen metabolism, and nutrient uptake. However, growth-promoting effects of PF-68 are concentration dependent.
Fulltext Growth promoting effects of Pluronic F-68 on callus proliferation of recalcitrant rice cultivar

Kok AD, Wan Abdullah WMAN, Tan NP, Ong-Abdullah J, Sekeli R, Wee CY, et al.

3 Biotech, 2020 Mar;10(3):116.
PMID: 32117677 DOI: 10.1007/s13205-020-2118-5

This study was undertaken to evaluate growth-promoting effects of Pluronic F-68 (PF-68) on recalcitrant MR 219 rice callus. Our study shows that calli grown on Murashige and Skoog medium supplemented with 0.04% PF-68 significantly increased callus proliferation by 58.80% (fresh weight) and 23.98% (dry weight) while root formation from callus was enhanced by 28.57%. Enhanced callus proliferation was supported by biochemical analysis, whereby highest amount of soluble sugar (1.77 mg/mL) and protein (0.17 mg/mL) contents were recorded in calli grown on 0.04% PF-68. Furthermore, enhanced expression of sucrose synthase (2.65-folds) and NADH-dependent glutamate synthase (1.86-folds) genes in calli grown on 0.04% PF-68 also correlates with enhanced callus proliferation. In contrast, high concentration of PF-68 (0.10%) recorded highest amount of phenolic (0.74 mg/mL), flavonoid (0.08 mg/mL), and hydrogen peroxide content (0.06 mg/mL) as compared to other treatment groups indicates activation of plant defence mechanism towards stress. Similarly, high expression of 4-coumarate:CoA ligase 3 (1.28-folds), chalcone-flavonone isomerase (1.65-folds) and ascorbate peroxidase (1.61-folds) genes were observed in calli grown on 0.10% PF-68 further supports increasing stress caused by the high concentration of PF-68. Taken together, our study revealed that optimum concentration of PF-68 could improve recalcitrant rice callus proliferation via enhanced sugar metabolism and amino acid biosynthesis which are crucial towards plant growth and development. However, at high concentration, PF-68 induces stress in plant which enhance the production of secondary metabolite to maintain cellular homeostasis.