Development of protoplast isolation and DNA free CRISPR-CAS9 genome editing protocols for banana (Musa acuminata cv Berangan) / Leh Lin San

Leh , Lin San (2022) Development of protoplast isolation and DNA free CRISPR-CAS9 genome editing protocols for banana (Musa acuminata cv Berangan) / Leh Lin San. Masters thesis, Universiti Malaya.

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Abstract

Banana is an important crop in the tropics and subtropics and is ranked the fourth most important crop after rice, wheat and maize. In Malaysia, Musa acuminata cultivar Berangan (with an AAA genome) is the most widely cultivated banana cultivar due to its sweet taste and high nutritional value. However, banana production is seriously affected by environmental stress. Soil salinity has become an emerging challenge for banana farmers as most banana plants are grown in marginal soil with high salinity. Hence, developing a cultivar with enhanced salinity tolerance in bananas is imperative. Sugar Transport Protein 13 (STP13) maybe a valuable target for banana improvement due to its essential stress response role in plants. However, improving bananas via modern breeding and conventional genetic engineering is often limited by the recalcitrance and complexity of the genetic system of bananas. Hence, this study aimed to establish efficient protoplast isolation and DNA-free CRISPR/Cas9 ribonucleoprotein-mediated transformation protocols for banana cultivar Berangan. In this study, immature male flowers and bracts of bananas were used for protoplast isolation. The results showed that immature male flower buds produced more protoplasts (1.54 × 107 protoplasts/g FW) than bracts in an enzymatic mixture of 1% cellulase RS, 1% macerozyme and 0.15% pectolyase. Therefore, immature male flower buds were used for the subsequent experiments. The efficiency of isolating protoplasts under different vacuum infiltration times and mannitol concentrations were then determined. The application of 10 minute-vacuum infiltration twice and 0.5 M mannitol significantly increased the number of protoplasts. To optimise the protoplast transfection procedures, the isolated protoplasts were transfected with pCAMBIA1304-GFP using polyethylene glycol solution for different incubation times. About 76.89% of the pCAMBIA1304-GFP-transformed protoplasts were GFP-positive after 15 minutes of transfection. Next, STP13 digested by different molar ratios of CRISPR/Cas9 ribonucleoprotein complex targeting the banana STP13 gene. A 3:1 ratio resulted in the highest percentage of cleavage at 12.5%. The protoplasts were transfected with 3:1 Cas9: STP13gRNA were then sampled for targeted deep sequencing. The results showed that mostly indels (-1 bp) occurred at the target sites of STP13 with a mutation rate of 4.40-4.90%, indicating that the established protocols can efficiently modify the genomes of recalcitrant plants like bananas.

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