Identification of somaclonal variation using inter simple sequence repeats (ISSR) markers and optimization of types of fertilizers in acclimatization of in vitro regenerated pineapple (Ananas Comosus (L.) MERR. VAR. MD2) / Nur Aishah Ab Rahman

Ananas comosus (L.) Merr. is propagated asexually from its crowns, slips, hapas, and suckers using the traditional method. In comparison to traditional propagation, the plant tissue culture approach is used to mass-produce clonal pineapple in a shorter time. However, the existence of variation in clonal seedlings var. MD2 prevented it to be planted in the field. Somaclonal variation was referred to the changes in the electrophoretic profile of accessions belonging to the same botanical variety. Hence, this research aims to identify the effect of different types of plant growth regulators on the formation of somaclonal variation and to identify the existence of somaclonal variation in traditional propagated suckers and in vitro plantlets. This research also aims at optimizing the types of fertilizers for the acclimatization of clonal pineapple ex vitro. The in vitro regeneration was carried out by adding different concentration of 6- Benzylaminopurine (BAP) ranging from 5.0 – 10.0 mg/l in the first stage treatments. Later, the plantlets produced was sub-cultured into a various concentration of Indole 3-Acetic Acid (IAA) ranging from 1.0 – 5.0 mg/l as the second stage of treatments. Inter simple sequence repeats (ISSR) markers were used to assess genetic variation. Genomic DNA samples was extracted from young leaves' traditional propagated suckers (one sample), six (6) samples from the young leaves from the plantlets produced in the first stage, and 30 samples were extracted from the young leaves of the plantlets that had been sub-cultured into second stage. A dendrogram showing genetic distance among pineapple was constructed based on polymorphic bands using the Darwin Software (version 6). For the acclimatization, the treatments were applied on secondary acclimatization with two different types of fertilizers served as treatments. The fertilizers used were compound fertilizers, NPK green with (15:15:15) ratio and controlled-release fertilizer AJIB CRF with NPKMg ratio (10:5:18:3). The planting media for clonal seedlings in secondary acclimatization consisted of topsoil, sand, and compost composition in a ratio of 2:1:2. As a result, 5.0 mg/l BAP produced the best result with the second highest for the mean of leaves number (6.07±0.23), the highest mean for leaf length (cm) and plantlet height (cm) with 3.29±0.10 and 5.57±0.07, respectively. In second stage treatments, 4.0 mg/l IAA produced the highest mean for roots length (cm) with 2.16±0.04 while 5.0 mg/l IAA produced the highest mean for root number with 7.67±0.33. Out of 10 primers screened only six (6) primers (Primer 3, Primer 4, Primer 5, Primer 6, Primer 7, and Primer 9) were amplified and showed 45.85% of polymorphism with 188 bands. A total of 410 DNA fragments amplified, varying in size from less than 3.0kb to 6.0kb. Based on the results from the dendrogram, two major clusters were formed for both stages of treatments when compared to the control treatments. The genetic distance ranging from less than 0.04 to 0.42. Meanwhile, the acclimatization method found that controlled-release fertilizer (AJIB CRF), NPKMg (10:5:18:3) showed about 90% of survival rate. The mean value for the number of leaves and leaves length (cm) in acclimatized clonal pineapple using Controlled-release fertilizer (AJIB CRF) were 5.48±0.96 and 6.00±1.04, respectively. Hence, the use of inter simple sequence repeats (ISSR) markers are suitable to detect somaclonal variation in pineapple. For acclimatization, the used of controlled-released fertilizer (AJIB CRF) optimized the growth performance of clonal pineapple in field.