Open access peer-reviewed chapter

Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods

By Mousa Mousavi and Mohsen Brajeh Fard

Submitted: May 6th 2018Reviewed: September 7th 2018Published: October 23rd 2019

DOI: 10.5772/intechopen.81373

Downloaded: 384


Biolistic is a special high-performance method for direct delivery of foreign DNA, RNA, or protein into plant cells. This method has less physiological risk on plant cell since there is no need for microbial intermediaries (Agrobacterium strains) and requires less additional DNA. Moreover, it can adapt for both monocotyledon and dicotyledonous plants. Recently, this method has also been successfully used to plant genome editing. Therefore, in this chapter, we discuss the application of this method for genetic improvement of some commercially important of tropical and subtropical fruit trees including banana, date palm, citrus, mango, olive, and pineapple. Also, we explain the details of biolistic protocols used for transient and stable gene expression in these fruit trees.


  • gene gun
  • gene delivery
  • microprojectile bombardment
  • tropical fruits

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, 2, 3, 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 Agrobacteriummediate, but the plasmids that are harboring desired DNA materials will deliver to the plant cells via physical or chemical means [5]. However, indirect gene transformation to plant tissues is usually achieved by mediated Agrobacteriumstrains. At the present time, in the most laboratories, gene delivery to plant tissues is achieved by mainly two means including biolistic and Agrobacteriummethods [2, 4]. However, most of the gene transformation studies on fruit trees have been mediated by Agrobacteriumstrains, and biolistic has been less frequently used [6]. Infection with Agrobacteriummay 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 with the biolistic method, the following are needed:

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

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

  3. Optimized bombardment condition (biological parameters, as well as physical parameters, should been optimized)

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

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.


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 Agrobacteriumto 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 uidA gene in banana cells using a special gene gun is illustrated by [14]. The tungsten particles coated with various plasmids harboring uidA 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 uidA gene along with hphas a selectable marker gene (conferring resistance to hygromycin) and cultured on the medium contain 50 mg/L hygromycin.

Stable transformation of the Cavendish banana (Musaspp. AAA group) cv. Grand Nain was also reported by [16], using uidA and potential virus-resistance (BBTV) genes along with nptII gene as selectable marker gene using various plasmids (Table 1).

Plant nameExplant typePlasmid (s)Reporter gene(s)/promoter (s)Selectable gene (s)/promoter (s)Helium pressureParticle size (μm)/typeTarget distance (cm)Osmoti-cumTransfor-mation efficiencyReference
Musaspp. (ABB group) BluggoeEmbryogenic suspension cellspBI364, pBI426, pBI505, pEmuGN, pAHC27, pWRG1515uidA/35S, Emu, UbiHygromycin (hph)4.5 barTungsten430%[14]
Musaspp. (AAB group) MaçãImmature male flowerspBI426, pFF19, pCAMBIA1303uid-A/neo/70S; uid-A/70S; uid-A/35SHygromycin1100 psiTungsten9Best result was obtained with uid-A/neo/70S[18]
Musaspp. (AAB group) Grand NainImmature male flowerpBT6.3-Ubi-NPT, pUbi-BTintORF1, pUbi-BTutORF5, pUGR73, pDHkanBBTV intO1/Ubi pro, BBTV utO5/Ubi pro, uidA/UbipronptII/BT6.3 pro, nptII/CaMV 35S pro550 KPa1.0/gold7.511%[16]
Musa acuminatacv. Mas (AA)Immature male flowerpCAMBIA-1301gus/CaMV 35S1100–1350 psi1.0/gold6[17]
Musa acuminataL. (AA group, cv. Mas) GongjiaoFloral apicespCAS04uidA, nptII/Ubi pro, actin pro1300 psi0.6/gold49.8%[15]
Musa sapientumcv. Rastali (AAA)BudpBI333-EN4-RCC2, pMRC1301, pROKLa-Eg, pGEM.Ubi1-sgfps65T (GFP)nptII/nopaline synthase gene, gusA and chitinase/rice actin 1, nptII/nopaline synthase gene (nos) promoter, soybean β-1,3-endoglucanse/CaMV 35S, gfp/maize polyubiquitin 1 (Ubi1)1100 psi94–7.5%[20]
Citrus reticulataBlanco × Citrus paradisiMacf. cultivar PageEmbryogenic cells from suspension culturesgusnptIITungsten0.3 M sorbitol + 0.3 M mannitol[26]
Carrizo citrange (Citrus sinensis(L.) Osbeck × Poncirus Trifoliate (L.) Raf.), sweet orange (Citrus sinensis(L.) Osbeck)cv. PeraThin epicotyl sectionspE2113-GUSuidA/CaMV 35SnptII/NOS promoter1550 psiTungsten M-25 (1.7)60.2 M sorbitol + 0.2 M mannitol[24]
Citrus macrophylla(C-mac)Second and third newest leavesCTV CP-CP interacting BiFC plasmidsgfp/35S260–280 psi0.6/gold[8]
Olea europaeaL. cv CaninoSomatic embryogenesispZ085 and pCGUδ0gus/Ubi (sunflower)580 kPaTungsten or gold[44]
Olea europaeacv. “Picual”Embryogenic calluspCGU∆1gus/Ubi (sunflower)nptII900 psi1.0/gold60.2 mannitol72.7%[41]
Ananas comosus“Phuket” and “Pattavia”Leaves of micropropagated shootsAHC25gus/maize Ubibar/maize ubiquitin promoter1350 psiGold70.2 M mannitol66.7–86.4%[29]
Ananas comosusL. cv. “Smooth Cayenne”CalluspDH-kanR, pBS420, pART7.35S.GUS, pBS247.SCSV4.GUS, pGEM-Ubi-GFPgus/35S or SCSV4, gfp/maize Ubi-1 and ppogene (isolated from pineapple, for control of blackheart) under the control of 35S or maize Ubi-1nptII/35S or SCSV41000 kPa1.0/gold180.21–1.5%[32]
Mangifera indica “Carabaoand “Kensington Pride”Nucellar proembryonic massespBI426, pBINgfp-Sergus, gfp /CaMV 35SnptII/CaMV35S125 psi7/tungsten150.2 M mannitol1101 foci per microgram of DNA[46]
Phoenix dactyliferaL.
Emberyogenic calluspAct1-Dgus/5′ region of the rice actin 11100 psi1.6/gold90.4 M mannitol1383 GUS blue spots/bombard-ment[39]
Phoenix dactyliferaL.
Somatic embryospAct1-Dgus/5′ region of the rice actin 11350 psi0.6/gold60.4 M mannitol6–12 blue spots/bombard-ment[39]
Phoenix dactyliferaL.
Emberyogenic calluspBC4Cholesterol oxidase gene/35S, gus/35SKan resistance/35S1300 psiTungsten90.2 M mannitol[40]

Table 1.

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

In other experiment [18], researchers transferred to the Musaspp. (AAB group) cv. Maçã the three plasmid constructions harboring uidA 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 acuminataL. 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 Agrobacteriummethods, respectively [15].

2.1 Plant-based vaccine

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 HBsAggene, coding hepatitis B surface antigen to banana cv. Williams to make an alternative plant-based oral vaccine. After the bombardment of the banana meristems with pBHsAg plasmid vector harboring bargene (inactivates phosphinothricin) as a selectable marker and HBsAggene, 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

Fusariumwilt race 1 is one of the limitation factors in banana production, caused by Fusarium oxysporumcubense 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 gfpand uidA along with the chitinase and β-1,3-glucanase genes to detection of the transformation occurrence and subsequent expression in buds of Rastali cultivar (Musaspp. 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 (Musaspp. 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.

3. Citrus

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 Agrobacteriuminfection [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, 24, 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 uidA/nptII genes to embryogenic cell suspension of citrus Tangelo (Citrus reticulataBlanco × C. paradisiMacf.) cultivar “Page,” the researchers [26] used biolistic transformation method (Table 1).

Also, in other research [24], the uidA/nptII 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 gfpand also nptII 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.

4. Pineapple

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/nptII genes and then confirmed the gene insertions at one to three loci by Southern hybridization. After that, the published results [29, 30, 31] indicated that the pineapple cv. Phuket to herbicide Basta® X (with glufosinate ammonium as the active component) can be resisted by transforming with bargene 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 bargene in to the genome of the transformed plants was confirmed with PCR, RT-PCR, and Southern analyses after 380 days.

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 AcGA2oxgene and reduce GA4 levels 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 to lycopene accumulation [34].

5. Date palm

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 gusgene in different helium pressures (900, 1100, and 1350 psi) and target distances (6, 9, and 12 cm). The results indicated that highest gusexpression 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 gusblue spot per bombardment); however, somatic embryos present very lower potential of transient expression (9 ± 3 gusblue 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 gusmarker gene under control of 35S promoter and confirmed the insertions by gusassay, ELISA, and PCR.


6. Olive

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, 42, 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 the gene transformation by means of biolistic method in which most of them were down to optimization of scorable and selectable marker genes. Successfully transferred gusgene under the control of sunflower ubiquitin promoter in to small somatic embryos of Canino olive cultivar by biolistic method was reported by [44]. Afterward [45] bombarded the embryogenic tissues of Picual cultivar with three different plasmid constructs harboring gusgene under control of 35S, 35S with enhancer and sunflower ubiquitin promoters, and found that the ubiquitin promoter could significantly enhance the gusgene expression in olive.

More recently, [41] introduced an optimized protocol for transformation of olive cv. Picual embryogenic callus with gusgene under the control of sunflower ubiquitin promoter and nptII selective gene (Table 1) and achieved 72.7% transformation efficiency for embryogenic calli.

7. Mango

The result of [46] study reported an optimized protocol for transient and stable transformation of mango “Carabao” and “Kensington Pride” by biolistic method. They successfully optimized different bombardment parameters (Table 1), whereas more than thousand foci were observed per each nucellar proembryonic masses bombarded with a μg plasmid DNA. Afterwards [47], genetically transformed somatic embryos of the three mango varieties Haden, Madame Francis, and Kent with pCAMBIA 3201 construct harboring gusand bargenes by particle bombardment. After 3 months, only 4% embryos of Kent variety survived, while the other varieties did not survive. They confirmed integration of gusand bargenes by means of gusassay and PCR.

8. Conclusion

Gene transfer to tropical fruit trees via biolistic method can lower GMO risk. Therefore, it is recommended to use this method to gene transformation and particular genome editing via CRISPR technique. So plants can be genetically modified with low risk for humans and the environment.


The authors thank the IntechOpen Editorial Board for this publication and also would thank Mr. Muhammad Sarwar Khan for the invitation to write this chapter. There was no financial support for this study.


Conflict of interest

The authors declare that they have no conflict of interest.

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

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Mousa Mousavi and Mohsen Brajeh Fard (October 23rd 2019). Genetic Improvement of Tropical and Subtropical Fruit Trees via Biolistic Methods, Transgenic Crops - Emerging Trends and Future Perspectives, Muhammad Sarwar Khan and Kauser Abdulla Malik, IntechOpen, DOI: 10.5772/intechopen.81373. Available from:

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