**2. Banana**

Several limitations had been reported for the breeding of banana cultivars through traditional methods mainly including long regenerating time, polyploidy, and male sterility [14, 15]. The biolistic method was successfully used for banana transformation so that several genes were transferred to different banana tissues for different purposes. However, this method may be integrated with the *Agrobacterium* to increase the efficiency of the gene transformation.

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 cultured on the medium contain 50 mg/L hygromycin.

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

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 the tungsten particles using 20 μL of spermidine and 50 μL of CaCl2 and then accelerated 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 pressure and 6–9 cm target distance (**Table 1**).

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

### **2.1. Plant-based vaccine**

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].

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

**2.** Proper tissue (eases to regeneration as well as pretreatment prior to bombardment)

**3.** Optimized bombardment condition (biological parameters, as well as physical parameters,

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 encod-

One of the more permissible applications of the biolistic method is using it for genome editing

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

Several limitations had been reported for the breeding of banana cultivars through traditional methods mainly including long regenerating time, polyploidy, and male sterility [14, 15]. The biolistic method was successfully used for banana transformation so that several genes were transferred to different banana tissues for different purposes. However, this method may be integrated with the *Agrobacterium* to increase the efficiency of the gene transformation.

with the biolistic method, the following are needed:

100 Transgenic Crops - Emerging Trends and Future Perspectives

should been optimized)

ing the economical traits).

or CRISPR in plants [13].

biolistic method.

**2. Banana**

**1.** Appropriate construct (the type of genes and promoter)

**4.** Detecting of the insertion (integrated to the genome)

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


**Plant name** *Citrus reticulata*

Embryogenic

*gus*

*npt*II

cells from

suspension

cultures

Blanco × *Citrus* 

*paradisi* Macf.

cultivar Page

Carrizo citrange

Thin epicotyl

pE2113-GUS

*uid*A/CaMV 35S

*npt*II*/*NOS

1550 psi

Tungsten

6

0.2 M

[24]

sorbitol +

0.2 M mannitol

M-25 (1.7)

promoter

(*Citrus sinensis* (L.)

sections

Osbeck ×

*Poncirus* 

*Trifoliate (L.) Raf.)*,

sweet orange

*(Citrus sinensis* (L.)

Osbeck*)* cv. Pera

*Citrus macrophylla*

Second and third

CTV CP-CP

*gfp/*35S

*—*

260–

0.6/gold

—

—

[8]

280 psi

interacting

BiFC plasmids

newest leaves

(C-mac)

*Olea europaea* L. cv

Somatic

pZ085 and

*gus*/Ubi

580 kPa

Tungsten

[44]

or gold

(sunflower)

pCGUδ0

embryogenesis

Canino

*Olea europaea* cv.

Embryogenic

pCGU

∆1

*gus*/Ubi

*npt*II

900 psi

1.0/gold

6

0.2

72.7%

[41]

mannitol

(sunflower)

callus

"Picual"

*Ananas comosus*

Leaves of

AHC25

*gus/*maize Ubi

*bar/*maize

1350 psi

Gold

7

0.2 M

66.7–86.4%

[29]

Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods

mannitol

ubiquitin

promoter

micropropagated

shoots

"Phuket" and

"Pattavia"

*Ananas comosus*

Callus

pDH-kanR,

*gus/*35S or SCSV4,

*npt*II*/*35S or

1000 kPa

1.0/gold

18

—

0.21–1.5%

[32]

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

103

SCSV4

*gfp/*maize Ubi-1

and *ppo* gene

*(*isolated from

pineapple,

for control of

blackheart) under

the control of 35S

or maize Ubi-1

pBS420,

pART7.35S.

GUS, pBS247.

SCSV4.GUS,

pGEM-Ubi-GFP

L. cv. "Smooth

Cayenne"

**Explant type**

**Plasmid (s)**

**Reporter gene(s)/**

**Selectable** 

**Helium** 

**Particle** 

**Target** 

**Osmoticum**

**Transfor- mation** 

**Reference**

**pressure**

**size (μm)/**

**distance** 

**type**

Tungsten

0.3 M

[26]

sorbitol +

0.3 M mannitol

**(cm)**

**efficiency**

**gene (s)/**

**promoter (s)**

**promoter (s)**

**Plant name** *Musa* spp. (ABB

Embryogenic

pBI364, pBI426,

*uid*A/35S, Emu,

Hygromycin

4.5 bar

Tungsten 4

(*hph*)

suspension cells

pBI505,

Ubi

pEmuGN,

pAHC27,

pWRG1515

*Musa* spp. (AAB

Immature male

pBI426, pFF19,

*uid-*A/neo/70S;

Hygromycin

1100 psi

Tungsten 9

Best

[18]

102 Transgenic Crops - Emerging Trends and Future Perspectives

result was

obtained

with

*uid*-A/*neo/70S*

*uid-*A/70S;

*uid-*A/35S

pCAMBIA1303

flowers

group) Maçã

*Musa* spp. (AAB

Immature male

pBT6.3-Ubi-NPT, pUbi-BTintORF1,

BBTV intO1/Ubi

*npt*II/BT6.3

550 KPa

1.0/gold

7.5

11%

[16]

pro, *npt* II/

CaMV 35S

pro, BBTV utO5/

Ubi pro, *uid*A/*Ubi*

pUbi-BTutORF5,

pro

pro

pUGR73,

pDHkan

*Musa acuminata*

Immature male

pCAMBIA-1301

*gus*/CaMV 35S

—

1100–

1.0/gold

6

—

[17]

1350 psi

cv. Mas (AA)

*Musa acuminata*

Floral apices

pCAS04

*uid*A, *npt*II/Ubi

—

1300 psi

0.6/gold

4

9.8%

[15]

pro, actin pro

L. (AA group, cv.

Mas) Gongjiao

*Musa sapientum*

Bud

pBI333-

*npt*II/nopaline

1100 psi

9

4–7.5%

[20]

synthase gene,

*gus*A and

chitinase/rice actin

1, *npt*II/nopaline

synthase gene

(*nos*) promoter,

soybean β-1,3-

endoglucanse/

CaMV 35S,

*gfp*/maize

polyubiquitin 1

(Ubi1)

EN4-RCC2,

pMRC1301,

pROKLa-Eg,

pGEM.Ubi1-

sgfps65T (GFP)

cv. Rastali (AAA)

flower

group) Grand

flower

Nain

group) Bluggoe

**Explant type**

**Plasmid (s)**

**Reporter gene(s)/**

**Selectable** 

**Helium** 

**Particle** 

**Target** 

**Osmoticum**

**Transfor- mation** 

**Reference**

**pressure**

**size (μm)/**

**distance** 

**type**

**(cm)**

**efficiency**

30%

[14]

**gene (s)/**

**promoter (s)**

**promoter (s)**


**Table 1.** Description of gene transformation to some economically important tropical and subtropical fruit trees through biolistic method. an alternative plant-based oral vaccine. After the bombardment of the banana meristems with pBHsAg plasmid vector harboring *bar* gene (inactivates phosphinothricin) as a selectable marker and *HBsAg* gene, they had detected the expression of antigen in banana which may

Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods

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

105

*Fusarium* wilt race 1 is one of the limitation factors in banana production, caused by *Fusarium oxysporum* cubense f. sp. Due to cell wall of these fungi mainly made from chitin and β-1, 3-glucan, therefore, presence of chitinase and β-1,3-glucanase in banana tissues can increase the level of resistance to this disease. This was achieved by banana gene transformation with chitinase and β-1,3-glucanase genes using biolistic method [20]. They also transferred reporter genes *gfp* and *uid*A along with the chitinase and β-1,3-glucanase genes to detection of the transformation occurrence and subsequent expression in buds of Rastali cultivar (*Musa* spp.

Black Leaf Streak Disease (BLSD) is another worldwide banana disease caused by *Mycosphaerella fijiensis*. The fungi induce streaks on the banana leaves which may lead to reduced fertility and may destroy the whole trees. On the other hand, infested plants usually produce a high level of free radicals, causing more challenges. In a research with goal to increase the level of banana tolerance to the BLSD reported by [21], they transferred two genes, including endochitinase (*ThEn-42*) and grape stilbene synthase (*StSy*) antifungal genes (with synergistic effect) together with chloroplastic (*chl*) Cu, Zn superoxide dismutase gene (*Cu*, *Zu-SOD*) (scavenging of free radical) to embryogenic callus of Cavendish banana (*Musa* spp. AAA group) cv. Grand Nain. After 4 years, the infection of the transgenic banana with

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

these three genes was significantly reduced without any decrease in yield.

have a potential to use it for security against this disease.

**2.2. Disease resistance**

AAB group).

**3. Citrus**

to the explant [23–25].

an alternative plant-based oral vaccine. After the bombardment of the banana meristems with pBHsAg plasmid vector harboring *bar* gene (inactivates phosphinothricin) as a selectable marker and *HBsAg* gene, they had detected the expression of antigen in banana which may have a potential to use it for security against this disease.

#### **2.2. Disease resistance**

*Fusarium* wilt race 1 is one of the limitation factors in banana production, caused by *Fusarium oxysporum* cubense f. sp. Due to cell wall of these fungi mainly made from chitin and β-1, 3-glucan, therefore, presence of chitinase and β-1,3-glucanase in banana tissues can increase the level of resistance to this disease. This was achieved by banana gene transformation with chitinase and β-1,3-glucanase genes using biolistic method [20]. They also transferred reporter genes *gfp* and *uid*A along with the chitinase and β-1,3-glucanase genes to detection of the transformation occurrence and subsequent expression in buds of Rastali cultivar (*Musa* spp. AAB group).

Black Leaf Streak Disease (BLSD) is another worldwide banana disease caused by *Mycosphaerella fijiensis*. The fungi induce streaks on the banana leaves which may lead to reduced fertility and may destroy the whole trees. On the other hand, infested plants usually produce a high level of free radicals, causing more challenges. In a research with goal to increase the level of banana tolerance to the BLSD reported by [21], they transferred two genes, including endochitinase (*ThEn-42*) and grape stilbene synthase (*StSy*) antifungal genes (with synergistic effect) together with chloroplastic (*chl*) Cu, Zn superoxide dismutase gene (*Cu*, *Zu-SOD*) (scavenging of free radical) to embryogenic callus of Cavendish banana (*Musa* spp. AAA group) cv. Grand Nain. After 4 years, the infection of the transgenic banana with these three genes was significantly reduced without any decrease in yield.
