73 PHYTOLOGIA BALCANICA 23 (1): 73 – 83, Sofia, 2017 Somaclonal variation in the in vitro regenerated pineapple (Ananas comosus): investigation of the cellular characteristics, biochemical specificities and ISSR markers Fatemeh Nouri Kohpaii 1 , Farah Farahani 2 & Zahra Noormohammadi 1 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 2 Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran; e-mail: farahfarahani2000@yahoo.com (corresponding author) Received: November 12, 2016 ▷ Accepted: December 27, 2016 Abstract. Several parameters of the in vitro regenerated, grown and adapted to the greenhouse plantlets were studied and compared (P ≤ 0.05). The mean length of stem and leaves of the in vitro regenerated pineapple significantly decreased under greenhouse conditions. The chlorophyll pigments content in the regenerated plantlets was increased and carotenoid, anthocyanins, carbohydrate, total phenols, and bromelain showed a relatively lower amount in the in vitro plantlets. Cluster analysis was performed on the basis of Dice genetic distance matrix for the thirty regenerated plants, as well as the mother plant. On the basis of the Neighbor Joining Cluster the plantlets were grouped into three main clusters. AMOVA test showed that 46 % of the genetic variance is within the groups, while 54 % of the rest belongs in-between the three groups. On the basis of this analysis a significant genetic difference was observed within the regenerated plantlets (P=0.01). Key word: ISSR, micropropagation, regenerated plantlet, somaclonal variation Introduction Pineapple belongs to the Bromeliaceae family. As a tropical fruit, the plant plays an important com- mercial role in the economy of a number of tropi- cal countries. The most generally applied method of pineapple propagation so far has been through vege- tative regeneration. Traditional propagation uses var- ious vegetative parts, organs, or tissues, such as suck- ers, hapas, leafy branches, slips, crowns, and butts or stumps from the mature plant (Rangan 1984). Aghion & Beauchesne (1960) were the first to report the use of in vitro micropropagation method. Numerous pa- pers have been published on this subject ever since. Plants were regenerated in vitro from the apex or ax- illary buds of the crown (Fitchet 1985), slips (Sita & al. 1974), lateral buds (Zepeda & Sagawa 1981), syn- carp (Wakasa 1979), leaf bud (Seow & Wee 1970), as well as the callus (Rao & al. 1981). Some of these methods have also induced variations (DeWald 1988; Wakasa 1979). The appearance of off-types was main- ly due to somaclonal variation (Larkin & Scowcroft 1981). These variants may provide useful character- istics, such as new leaf and fruit shape, leaf color, and spininess (Kiss & al. 1992). Maintenance of the geno- typic and phenotypic identity is an indispensable re- quirement for mass propagation. In order to obtain high-quality transplants at an increased rate by micropropagation, optimization of the culture parameters is of prime importance. Cul- ture condition, especially sucrose as a growth regula- tor, influences photosynthesis in the in vitro growing plants. Evaluation of the photosynthetic properties is essential for optimization of the culture condition, in order to achieve an efficient micropropagation. The chlorophyll content of the plant has been used as a