**5. Date palm**

Evaluating the transient expression of a gene can provide valuable information in association with various properties of its produced protein, such as subcellular localization and intra−/ intercellular trafficking, stability and degradation, expression levels, and interactions with other proteins [8]. In order to initiate a procedure for transient and stable transformation of the *uid*A/*npt*II genes to embryogenic cell suspension of citrus Tangelo (*Citrus reticulata* Blanco × *C. paradisi* Macf.) cultivar "Page," the researchers [26] used biolistic transformation

Also, in other research [24], the *uid*A/*npt*II genes to thin epicotyl sections of the Carrizo citrange (*Citrus sinensis* (L.) Osbeck × *Poncirus trifoliata* (L.) Raf.) and sweet orange (*Citrus sinensis* (L.) Osbeck) cv. Pera were successfully delivered (**Table 1**). Recently the Carrizo immature epicotyl with another reporter gene *gfp* and also *npt*II gene as selectable marker through biolistic

Most reports on citrus gene transformation by biolistic were carried out on the transformation and expression detection of the selectable and scorable marker genes. However, result reported by [8] showed that the bombardment of the young leaves of the *Citrus macrophylla* (C-mac) with pSAT4-cEYFP-C1(B) harboring CPCTV-GFP using Bio-Rad Helios gene-gun could have been causing the express of CP in the cytoplasm and nuclei of the epidermal cells.

The first report on the using of the biolistic method for gene transformation of pineapple was published by [28]. They introduced an efficient system for transformation of protocorm-like bodies with *gus*/*npt*II genes and then confirmed the gene insertions at one to three loci by Southern hybridization. After that, the published results [29–31] indicated that the pineapple cv. Phuket to herbicide Basta® X (with glufosinate ammonium as the active component) can be resisted by transforming with *bar* gene using biolistic transformation method. The transgenic plants showed herbicide tolerance when they were sprayed with herbicide (with twice the routine dose which used in the field) and remained green and healthy after 7 months, whereas the non-transformed plants became necrotic and died after 21 days. The stable integration of the *bar* gene in to the genome of the transformed plants was confirmed with PCR, RT-PCR,

One of the physiological disorders which limited the industry of the pineapple in different area productions in the world such as Australia is the internal browning or blackheart. This disorder causes severe loss when appearing at conditions with day/night temperatures below 25/20°C with low light during fruit development and also during storage and shipment [32, 33]. To control the internal browning by the molecular breeding methods, an effort was made by [32] in order to obtain a transgene resistant to blackheart through biolistic method. The leaf callus of Smooth Cayenne cultivar was bombarded with gold particles coated with pART7 plasmid harboring PINPPO1 gene (pineapple polyphenol oxidase gene) which could successfully attain resistant plants to blackheart with an efficiency of 0.21–1.5% based on the PCR and Southern blot analysis (**Table 1**). Recent studies demonstrated that low temperature

(5°C) could reduce blackheart through upregulated *AcGA2ox* gene and reduce GA4

method (**Table 1**).

**4. Pineapple**

transformation are transferred [27].

106 Transgenic Crops - Emerging Trends and Future Perspectives

and Southern analyses after 380 days.

One most important challenge face to genetical improvement of date palm through gene transformation and genome editing methods is difficult to regenerate in vitro due to lack of an efficient procedure for raped embryogenic callus induction. However, numerous successful protocols have been developed for regeneration of palm dates in in vitro conditions [35]. At present, shoot tips and immature inflorescence are mostly used for callus induction; however, several months and high levels of auxins (such as 2,4-D with100 mg/L concentration) are necessary that may induce epigenetic variation. Among the different tissues of date palm, the embryogenic callus and somatic embryos had more competencies to gene transformation [36]. Fortunately, the first report on date palm gene transformation had been done with biolistic method [37]. In this study embryogenic callus and somatic embryos of Kabkab cultivar were bombarded with gold particle coated with plasmid DNA construct carrying *gus* gene in different helium pressures (900, 1100, and 1350 psi) and target distances (6, 9, and 12 cm). The results indicated that highest *gus* expression in embryogenic callus was achieved when bombarded with 1100 psi/6 cm (helium pressures/target distance), whereas in somatic embryos, it was obtained in 1350 psi/9 cm. Date palm embryogenic callus exhibits the highest potential of transient expression (1383 *gus* blue spot per bombardment); however, somatic embryos present very lower potential of transient expression (9 ± 3 *gus* blue spot per bombardment). But they were more competent for attainment stable transformation [36, 38]. Unfortunately, the regeneration potential of embryogenic callus was dramatically decreased after bombardment due to shock wave. Recently, we introduce an efficient and optimized protocol for stable transformation of date palm Estamaran (Sayer) cultivar through biolistic transformation method [39]. Also, [40] developed a procedure for delivering the insecticidal cholesterol oxidase (*ChoA*) gene to embryogenic callus of Siwy cultivar through particle bombardment (**Table 1**). They transferred ChoA gene along with *gus* marker gene under control of 35S promoter and confirmed the insertions by *gus* assay, ELISA, and PCR.
