**1. Introduction**

In the recent years, the scientists believe that the molecular methods have high potential for making gene delivery or genome editing possible in different plant species without altering their phenotypes. This capability is particularly valuable for fruit trees that have lengthy generation time and high levels of heterozygosity. In fact, the biotechnology methods now became as routine tools in biology research and plant transformation. So various methods had been introduced by the scientist for gene delivery to the plant cells (which may not be achievable by the traditional breeding methods), and subsequently they successfully regenerated without serious limitations [1–4]. Gene transferring to plant tissues can be achieved by two means: direct or indirect methods. In the direct methods, there is no need to *Agrobacterium* mediate, but the plasmids that are harboring desired DNA materials will deliver to the plant cells via physical or

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

chemical means [5]. However, indirect gene transformation to plant tissues is usually achieved by mediated *Agrobacterium* strains. At the present time, in the most laboratories, gene delivery to plant tissues is achieved by mainly two means including biolistic and *Agrobacterium* methods [2, 4]. However, most of the gene transformation studies on fruit trees have been mediated by *Agrobacterium* strains, and biolistic has been less frequently used [6]. Infection with *Agrobacterium* may potentially produce unpredictable effects on the plant cells when transformed with T-DNA [7, 8]. The biolistic is a more applicable method for gene transformation in a wide range of plant cells and tissues [9], even those that could not transform by other transformation methods. In this method the precipitated DNA on gold or tungsten particles is transferred directly into the plant cells and tissues. Therefore, it is possible to introduce new traits with lower risk of the GMO effect with high reproducibility and no significant damage or artifacts [8]. Further, this method can be more adapted for breeding of plant species with a high degree of heterozygosity [10].

Embryogenic cells initiated from different tissues including immature male flowers [16], immature embryos [17], male inflorescence, and buds [18] were reported with high potential to gene transformation in banana and plantain. A protocol optimized for transient and stable transformation of the *uid*A gene in banana cells using a special gene gun is illustrated by [14]. The tungsten particles coated with various plasmids harboring *uid*A gene including pEmuGN (with Emu promoter), pBI-364, pBI-426, pBI-505 (with 35S promoter), and pAHC27 (with Ubi promoter) were delivered into banana cells and then comprised among them based on the level of transient expression after assayed with X-Gluc and MUG. The highest transient transformation was obtained with the pAHC27 plasmid. Also, the stable transformation was achieved after the bombardment of banana cells with pWRG1515 plasmid harboring *uid*A gene along with *hph* as a selectable marker gene (conferring resistance to hygromycin) and

Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods

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Stable transformation of the Cavendish banana (*Musa* spp. AAA group) cv. Grand Nain was also reported by [16], using *uid*A and potential virus-resistance (BBTV) genes along with *npt*II

In other experiment [18], researchers transferred to the *Musa* spp. (AAB group) cv. Maçã the three plasmid constructions harboring *uid*A gene including pBI426 (70S promoter), pFF19 (70S promoter), and pCAMBIA1303 (35S promoter). The plasmids had been precipitated on

to penetrate callus tissue located at 9 cm from stopping screen using 1100 psi helium pressure force. The transient expression was observed for all constructs, but the best result was obtained for pBI426 due to achievement of the highest regenerated plant after 3 months.

For obtaining a successful transformation through the biolistic method, it is important to reduce physical stress entered on target tissues promoted by bombardment shock waves. Bombarded tissues may reduce their regeneration potential especially in the case of embryogenic callus and immature tissues. Therefore, such sensitive tissues should bombard with lower helium pressures and target distance. In most studies on gene transformation of banana by biolistic methods, it had been found that best results were obtained in 1100–1350 psi helium

Another strategy for increasing the transformation frequency in biolistic method is the integrating biolistic with *Agrobacterium*-mediated transformation especially with monocotyledons plants. It has been found that the infection of the Gongjiao (*Musa acuminata* L. AA group, cv. Mas) floral apices with *Agrobacterium tumefaciens* (AGL1 contains pCAS04) suspension for 30 min after bombardment thrice with pCAS04 plasmid coated on the 0.6 μm gold particles under 1300 psi helium pressure force was increased transformation frequency 1.6- and 3.3-

Hepatitis B virus (*HBV*) is a worldwide disease causing chronic and acute infections in the human liver. Therefore, needful to produce a vaccine for this disease is very important. On the other hand, production of vaccines required a high cost whereas may not be possible to secure the large segment of the population in the world. An attempt was made by [19], to transfer the *HBsAg* gene, coding hepatitis B surface antigen to banana cv. Williams to make

fold higher than that of gene gun and *Agrobacterium* methods, respectively [15].

and then accelerated

cultured on the medium contain 50 mg/L hygromycin.

pressure and 6–9 cm target distance (**Table 1**).

**2.1. Plant-based vaccine**

gene as selectable marker gene using various plasmids (**Table 1**).

the tungsten particles using 20 μL of spermidine and 50 μL of CaCl2

The biolistic method was introduced for the first time by Sanford [11]. Optimization of the transformation condition is very critical for achievement of an efficient protocol with high transformation frequency [12]. This strongly depends on the construct and promoter type and optimization of the physical and biological parameters. In order to achieve the best results with the biolistic method, the following are needed:


The biolistic method has a potential use for breeding of several tropical and subtropical fruit trees so that different genes were transferred to these trees for different purposes. Most of these genes are selectable and scorable marker genes which were used for the establishment of the optimized transformation protocols and some other genes of interest (which are encoding the economical traits).

One of the more permissible applications of the biolistic method is using it for genome editing or CRISPR in plants [13].

In this chapter, we explain the different gene transformation procedures introduced by scientists for various economically important tropical and subtropical fruit trees by the use of the biolistic method.
