Description of gene transformation to some economically important tropical and subtropical fruit trees through biolistic method.
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
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
The biolistic method was introduced for the first time by Sanford . Optimization of the transformation condition is very critical for achievement of an efficient protocol with high transformation frequency . 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:
Appropriate construct (the type of genes and promoter)
Proper tissue (eases to regeneration as well as pretreatment prior to bombardment)
Optimized bombardment condition (biological parameters, as well as physical parameters, should been optimized)
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 .
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.
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
Embryogenic cells initiated from different tissues including immature male flowers , immature embryos , male inflorescence, and buds  were reported with high potential to gene transformation in banana and plantain. A protocol optimized for transient and stable transformation of the
Stable transformation of the Cavendish banana (
|Plant name||Explant type||Plasmid (s)||Reporter gene(s)/promoter (s)||Selectable gene (s)/promoter (s)||Helium pressure||Particle size (μm)/type||Target distance (cm)||Osmoti-cum||Transfor-mation efficiency||Reference|
|Embryogenic suspension cells||pBI364, pBI426, pBI505, pEmuGN, pAHC27, pWRG1515||Hygromycin (||4.5 bar||Tungsten||4||30%|||
|Immature male flowers||pBI426, pFF19, pCAMBIA1303||Hygromycin||1100 psi||Tungsten||9||Best result was obtained with |||
|Immature male flower||pBT6.3-Ubi-NPT, pUbi-BTintORF1, pUbi-BTutORF5, pUGR73, pDHkan||BBTV intO1/Ubi pro, BBTV utO5/Ubi pro, ||550 KPa||1.0/gold||7.5||11%|||
|Immature male flower||pCAMBIA-1301||—||1100–1350 psi||1.0/gold||6||—|||
|Floral apices||pCAS04||—||1300 psi||0.6/gold||4||9.8%|||
|Bud||pBI333-EN4-RCC2, pMRC1301, pROKLa-Eg, pGEM.Ubi1-sgfps65T (GFP)||1100 psi||9||4–7.5%|||
|Embryogenic cells from suspension cultures||Tungsten||0.3 M sorbitol + 0.3 M mannitol|||
|Carrizo citrange (||Thin epicotyl sections||pE2113-GUS||1550 psi||Tungsten M-25 (1.7)||6||0.2 M sorbitol + 0.2 M mannitol|||
|Second and third newest leaves||CTV CP-CP interacting BiFC plasmids||260–280 psi||0.6/gold||—||—|||
|Somatic embryogenesis||pZ085 and pCGUδ0||580 kPa||Tungsten or gold|||
|Embryogenic callus||pCGU∆1||900 psi||1.0/gold||6||0.2 mannitol||72.7%|||
|Leaves of micropropagated shoots||AHC25||1350 psi||Gold||7||0.2 M mannitol||66.7–86.4%|||
|Callus||pDH-kanR, pBS420, pART7.35S.GUS, pBS247.SCSV4.GUS, pGEM-Ubi-GFP||1000 kPa||1.0/gold||18||—||0.21–1.5%|||
|Nucellar proembryonic masses||pBI426, pBINgfp-Ser||125 psi||7/tungsten||15||0.2 M mannitol||1101 foci per microgram of DNA|||
|Emberyogenic callus||pAct1-D||1100 psi||1.6/gold||9||0.4 M mannitol||1383 GUS blue spots/bombard-ment|||
|Somatic embryos||pAct1-D||1350 psi||0.6/gold||6||0.4 M mannitol||6–12 blue spots/bombard-ment|||
|Emberyogenic callus||pBC4||Cholesterol oxidase gene/35S, ||Kan resistance/35S||1300 psi||Tungsten||9||0.2 M mannitol|||
In other experiment , researchers transferred to the
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
2.1 Plant-based vaccine
Hepatitis B virus (
2.2 Disease resistance
Black Leaf Streak Disease (BLSD) is another worldwide banana disease caused by
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 . 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
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 . In order to initiate a procedure for transient and stable transformation of the
Also, in other research , the
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  showed that the bombardment of the young leaves of the
The first report on the using of the biolistic method for gene transformation of pineapple was published by . They introduced an efficient system for transformation of protocorm-like bodies with
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  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
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 . 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 . Fortunately, the first report on date palm gene transformation had been done with biolistic method . In this study embryogenic callus and somatic embryos of Kabkab cultivar were bombarded with gold particle coated with plasmid DNA construct carrying
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
More recently,  introduced an optimized protocol for transformation of olive cv. Picual embryogenic callus with
The result of  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 , genetically transformed somatic embryos of the three mango varieties Haden, Madame Francis, and Kent with pCAMBIA 3201 construct harboring
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.