**3. Citrus**

**Plant name** *Mangifera indica* 

Nucellar

pBI426,

*gus, gfp /*CaMV

*npt*II*/CaMV*

125 psi

7/tungsten 15

0.2 M

1101 foci per

[46]

microgram

of DNA

mannitol

35S

pBINgfp-Ser

35S

proembryonic

*"*Carabao*"* and

"Kensington

masses

Pride"

*Phoenix dactylifera*

Emberyogenic

pAct1-D

*gus*/5′ region of

1100 psi

1.6/gold

9

0.4 M

1383 GUS

[39]

104 Transgenic Crops - Emerging Trends and Future Perspectives

mannitol

blue spots/

bombardment

the rice actin 1

callus

L.

"Estamaran"

*Phoenix dactylifera*

Somatic embryos

pAct1-D

*gus*/5′ region of

1350 psi

0.6/gold

6

0.4 M

6–12 blue

[39]

mannitol

spots/

bombardment

the rice actin 1

L.

"Estamaran"

*Phoenix dactylifera*

Emberyogenic

pBC4

Cholesterol

Kan

1300 psi

Tungsten 9

0.2 M

[40]

mannitol

oxidase gene/35S,

resistance/35S

*gus*/35S

Description of gene transformation to some economically important tropical and subtropical fruit trees through biolistic method.

callus

L.

"Siwy"

**Table 1.**

**Explant type**

**Plasmid (s)**

**Reporter gene(s)/**

**Selectable** 

**Helium** 

**Particle** 

**Target** 

**Osmoticum**

**Transfor- mation** 

**Reference**

**pressure**

**size (μm)/**

**distance** 

**type**

**(cm)**

**efficiency**

**gene (s)/**

**promoter (s)**

**promoter (s)**

The citrus breeding by traditional methods has some limitations including lengthy period of juvenility (8–10 years), polyembryony, incompatibility, parthenocarpy [22, 23], and high heterozygosity [24]. Molecular methods and gene transformation could be an alternative for breeding of the citruses and rapid regeneration with less time consumption. Currently, gene delivery into the epicotyl segments by *Agrobacterium*-mediated transformation is the most widely used method for gene transformation of the citruses. However, this approach has several drawbacks including the high number of chimeric or non-transformed plants due to the requirement for larger explant and gradient concentrations of the selective agent to the explant [24] and low regeneration frequency of stably transformed cells and recalcitrant of some citrus genotypes to *Agrobacterium* infection [23]. On the other hand, the biolistic method provides several advantages over *Agrobacterium*-mediated transformation such as high transformation efficiency, simplicity of the plasmid constructs which allows for the integration of larger inserts, the co-transformation of more than one construct, and less biological damage to the explant [23–25].

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 method (**Table 1**).

compared to the higher temperature (20°C) [33]. Also, the Del Monte Foods company introduces a red-fleshed pineapple "Rosé" by overexpression/suppression of some genes related

Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods

http://dx.doi.org/10.5772/intechopen.81373

107

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.

Gene transformation to olive cultivars is considered as a difficult task due to recalcitrant nature of their tissues to regeneration process in vitro condition; however, it stays the most promising technique in respect to conventional and unconventional and even some biotechnological methods such as protoplast and somaclonal variation techniques. Classical methods of the olive breeding are more time-consuming, with very low efficient, due to lengthy seedling juvenile phase, alternation bearing, low fruitfulness, and low seed germinability [41–43]. The same as the other tropical fruit trees, the most of olive gene transformation studies were conducted using *Agrobacterium*-mediated transformation. However, there are few reports on

to lycopene accumulation [34].

**5. Date palm**

**6. Olive**

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 transformation are transferred [27].

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.
