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Amenability of an Agrobacterium tumefaciens-mediated shoot apical meristem-targeted in planta transformation strategy in Mango (Mangifera indica L.)

ABSTRACT Mango (Mangifera indica L.) is one of the most popular tropical fruits in the world owing to its rich taste, flavor, color, production volume and diverse end usage. Conventional mango breeding practices are unable to withstand the demand for improved varieties as it is time consuming and requires heavy investment. However, problems associated with traditional plant breeding can be curtailed through genetic transformation. Nevertheless, major limitation of transgenic development has been its recalcitrant nature toward tissue culture practices involving latent microbial infection, phenol exudation, etc. This opens wide scope for tissue culture-independent in planta transformation approaches These strategies have proved to be easy to execute and cost effective in producing large number of transformants. One such apical meristem targeted in planta approach was successfully exploited to demonstrate its utility in transforming a tree species. Mango variety Amrapali was transformed with two visual marker gene vectors GFP::hptII in pCAMBIA1302 and GUS::nptII in pCAMBIA2301 individually, to demonstrate its amenability. Preliminary confirmations identified 65.0% of GFP and 57.14% of GUS plants to be transformed. Further, molecular characterization of these primary transformants demonstrated transgene integration at genomic and transcript level in some of the plants. This established protocol holds good for functional gene validation and knock in/out studies and aid in mango improvement programs.


Introduction
Mango (Mangifera indica L.) belonging to family Anacardiaceae is an allopolyploid (2 n = 40) with medium genome size (~439 Mbp). It is the most widely grown fruit crop in India and acclaimed as "King of fruits." India is one of the largest grower and exporter of mango, yielding foreign earnings of 39.6 million US dollars (https://www.statista.com/). 1 In India, mango is being cultivated in an area of 2315 thousand hectares with annual production of 20899 thousand metric tonnes (https://nhb.gov.in/). 2 Mango cultivation deals with biennial bearing habit, large tree size, susceptibility to major diseases (mango malformation, anthracnose, powdery mildew, bacterial black spot); pests (mango hopper, mealy bug, fruit fly, stone weevil); short-post-harvest life and physiological disorders (spongy tissue, jelly stone) being the major constraints. 3,4 Conventional breeding of woody perennial fruit crops such as mango is difficult due to their long juvenile phase, existence of self-incompatibility, high degree of cross-pollination, low fruit set, high fruit drop, development of single seed per fruit, polyembryony, allopolyploid nature, highly heterozygous genetic background and lack of information about inheritance pattern of important quantitative traits. 4,5 Moreover, improving popular mango cultivars by introducing genes from other wild species through interspecific hybridization has also been inadequate due to cross incompatibility barriers. 6 Genetic transformation facilitates the introduction of a desired gene into the plant genome to overcome problems associated with traditional plant breeding. 7 Mango micropropagation has not achieved much economic success than compared to other horticultural crops. This is due to several challenges that are associated with mango in vitro culture, including latent microbial infection, phenol exudation, culture medium discoloration, explant browning, in vitro recalcitrance of tissues either singly or in combination imperil the entire tissue culture attempts. 4 Non-availability of in vitro regeneration protocols is mainly due to the basic barriers which involve excessive phenolic exudation post excision of explants (activation of oxidative enzyme system), explant browning (necrosis), culture media discolorations, deep-seated microbial contamination; slow and sporadic in vitro response of mango to tissue culture. 8 Few studies have demonstrated genetic transformation of mango using Agrobacterium tumefaciens 6,[9][10][11] and gene gun 12 with varying levels of success. Further, in vitro regeneration is genotype-dependent, time resilant and prone to somaclonal variations. 13,14 Thus, to overcome the concerns associated with difficult-toregenerate crops, the need of in planta approaches have begun to gain importance. 15,16 Tissue culture-independent in planta transformation has been demonstrated in many crops such as Brassica rapa, 17 B. napus, 18 B. campestris, 19 Arabidopsis thaliana, 20 Medicago truncatula, 21 Raphanus sativus, 22 Solanum lycopersicum, 23 Glycine max, 24 Melilotus alba, 25 Zea mays, 26 Oryza sativa, 27 Citrus maxima 28 and Passiflora edulis. 29 Several in planta transformation strategies have been developed using different tissues, i.e., seed, epicotyl, shoot apical meristem, flower, fruit etc. 30 The advantages of in planta approaches are that they are cost effective, easy to execute and can produce a large number of transformants in a short period of time. Several reports have confirmed high transformation efficiencies in different crops. 31 Among several in planta transformation techniques, apical meristem mediated transformation targets T-DNA to the growing shoot apical meristematic regions in vitro and allows the development of plants ex vitro. The methodology has been unequivocally proved in different crops like field bean, 32 groundnut, 33 capsicum, 34 chili, 35 pigeon pea, 36,37 flax 38 and cotton. 39,40 Genetic transformation of mango holds significant potential, which can give leads in solving the problem of flowering, alternate bearing habit, development of parthenocarpy varieties and tolerance to different biotic and abiotic stresses. Furthermore, there is no information available on in planta transformation of mango. In the present study, our team has developed a successful strategy for transforming mango with the apical meristemtargeted in planta genetic transformation protocol. This strategy is expected to provide an alternate approach over tissue culture mediated transformation to develop genetically modified mango genotypes, which can hasten and shorten the varietal improvement programs.

Plant Material and Binary Vectors Used for Transformation
In the present study, mango variety Amrapali, was used for the development of transformants. Seeds were surface sterilized with Ridomil Gold® (Syngenta Basal, Switzerland), seed coat was removed to facilitate germination and sown in plastic bags containing sterile potting media (cocopeat, vermiculite and perlite, 3:1:1). These bags were maintained under controlled conditions (26 ± 1° C; RH 65-75%; 16/8 h photoperiod of 57 µmole m −2 s −1 ) till transformation. Two-week-old seedlings were used as explants for Agrobacterium infection.
Agrobacterium tumefaciens strain EHA105 harboring binary vectors pCAMBIA1302 carrying GFP (Green Fluorescent Protein) gene and hptII as antibiotic gene; pCAMBIA2301 carrying GUS (β-glucuronidase) marker gene which contains a 5' extension of modified castor bean catalase intron (190 bp) to facilitate expression in plants but not in bacteria and nptII selectable marker gene were used for transformation of mango seedlings individually (Fig. 1a, b).

Development of Transgenics through an Apical meristem-targeted in planta Transformation Strategy
Axenic culture of Agrobacterium harboring 35S:: GFP and 35S::hptII in pCAMBIA1302 and 35S:: GUS and 35S:: nptII in pCAMBIA2301 from freshly streaked culture plate was inoculated into 5 ml LB medium (pH 7.0) containing 50 mg/L kanamycin, 10 mg/L rifampicin and incubated overnight at 28° C. The 5 ml starter culture on the next day was transferred to 200 ml of LB broth suplemeneted with antibiotics, which was later inoculated into 1 L of Winans' AB minimal medium (pH 5.2) 41 and incubated for 18 h at 28°C; 220 rpm. Two-week-old mango seedlings with emerging plumules were punctured 15-20 times with an insulin syringe at the apical meristem and incubated in AB minimal medium previously supplemented with crushed mature tobacco leaf extract 42 maintained at 28°C; 50 rpm for 5 h. The plants were later allowed to grow under controlled conditions until they recovered from injury and resumed their growth (Figs. 2 and 3).

GFP Expression
Seedlings transformed with pCAMBIA1302 were preliminarily confirmed using a fluorescence microscope with a 488 nm excitation wavelength. GFP expression in primary transformants, 36 h post infection was observed under a fluorescence microscope (ZEISS SteREO Discovery V20 microscope, Oberkochen, Germany). An excitation wavelength of 488 nm and 505-530 bandpath filter (which permits visualization of GFP by blue light) to separate GFP and a 560 long-pass filter to determine chlorophyll fluorescence were used. 16

GUS Histochemical Analysis
Seedlings transformed with pCAMBIA2301 were initially confirmed by GUS histochemical analyses. Excised tissues (leaf and stem) after 76 h of Agrobacterium infection were incubated overnight in GUS assay buffer (0.1 M phosphate buffer, pH 7.0, 2 mM X-Gluc, 5 mM each of potassium ferricyanide and potassium ferrocyanide and 0.1% Triton X100) at 37°C in water bath. Chlorophyll present in the tissues were later destained using 75% ethanol (v/v). 43 GUS expression at cellular level was observed using binocular microscope (Olympus, CX33, Shinjuku, Tokyo, Japan). 44

DNA Isolation
The leaves of transgenic and wild-type mango plants were crushed in liquid nitrogen to isolate genomic DNA using a CTAB (Cetyl Trimethyl Ammonium Bromide) method 44 with minor modifications (added 1% PVP w/v for removal of phenols). For purification of DNA, 2 µl RNase A (10 mg/ml) was added per 200 µl of crude DNA solution and incubated for 1 h at 37°C, then treated with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and then precipitated with ethanol. The concentration and quality of DNA was estimated using NanoDrop™ (Thermo Scientific, Waltham, Massachusetts, USA) at 260 nm and by electrophoresis on 0.8% agarose gel.

Polymerase Chain Reaction (PCR) Analyses
The presence of transgenes and Agrobacterium specific VirD1 gene in the genome of putative mango transformants was assessed through PCR. The PCR reaction mixture (25 µl) containing 1 U Taq DNA polymerase (GeNie, Bengaluru, Karnataka, India), 1X assay buffer (10 mM pH 9.0 Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin), 2.5 µM of each dNTP, 0.5 µl of each forward and reverse primer (Table 1)   DNA was used to amplify the transgenes. PCR amplification was carried out in a thermal cycler (Applied Biosystems® Veriti® 96-Well Fast Thermal Cycler, Waltham, Massachusetts, USA) programmed with a hot start of initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 65°C for GFP, 55°C for hptII & GUS, 61°C for VirD1 and 58°C for nptII for 1 min, extension at 72°C for 1 min, final extension at 72°C for 10 min. Amplified gene products of size 571 bp (GFP), 700 bp (hptII), 438 bp (VirD1) 750 bp (nptII) and 1 kb (GUS) were visualized by gel electrophoresis.

Genomic Southern Analysis
For identification of T-DNA copy number in transgenic plants developed using both the binary vectors, 10 µg of genomic DNA from transgenic and wild-type plants was digested with HindIII (NEB high fidelity, New England Biolabs, Ipswich, Massachusetts, USA) overnight and separated on 0.8% agarose gel in 1X TAE buffer at constant voltage of 40 V. Restricted fragments were transferred onto a positively charged nylon membrane (Amersham™ Hybond™-N + ) by capillary movement using 20× SSC and the membrane was later UV cross-linked. The membrane was hybridized with a DIG labeled 571 bp GFP and 750 bp nptII gene fragment for their corresponding transgenic plants.
The blot was further processed with washing, blocking, and development as per manufacturer's instructions (Roche Holding AG, Basel, Switzerland). The membranes were exposed to Xray film for 1 h in dark and later observed for hybridization signal.

Analysis of Transgenic Plants for Transcript Accumulation by sqRT-PCR
Total RNA was isolated from transgenic and wildtype mango plants using a total RNA isolation kit (Spectrum TM , Sigma Aldrich, St. Louis, MO, United States). Further, the isolated RNA was quantified using Nanodrop™ 3300 (ThermoFisher Scientifc, Carlsbad Waltham, Massachusetts, USA) and transcribed to cDNA using SuperScript® (VILO TM , Invitrogen, Carlsbad, CA, USA). sqRT-PCR was performed with 100 ng of diluted cDNA as a template.
M. indica actin 1 (MiACT1) was used as an internal control gene. 45 (Table 1) and extension at 72°C for 30s. The final extension was carried out at 72°C for 7 min to amplify specific gene products. "Blank" was devoid of cDNA, wild type contained 1 µL of cDNA of wild type, and positive control contained 25 ng of pCAMBIA2301/ pCAMBIA1302. The amplified gene products were analyzed on a 2.0% agarose gel.

Results and Discussion
Genetic transformation involves introduction of foreign genes to modify horticultural traits in perennial plants without changing their phenotype. Though mango genetic transformation has great potential, 46,47,48,11 it is negatively affected by the non-availability of regeneration protocols and recalcitrance to tissue culture. Despite several attempts made by researchers to regenerate mango using leaf 49,50 and shoots explants, 51,52 it was proved inefficient and established mango in the category of hard to deal with tissue culturebased approaches. Unavailability of an efficient regeneration protocol makes transformation more difficult in mango. However, in planta transformation strategies provide an alternative to evade all the steps involved in tissue culture. 15,16 The current study provides evidences for the development of transgenics in mango by an apical meristem targeted in planta transformation protocol.

Apical meristem-targeted in Planta Transformation
Two-week-old mango seedlings were subjected to transformation with pCAMBIA1302 and pCAMBIA2301 ( Fig. 1a-b). The emerging shoots with brown-green coleoptiles were pricked in the upper region of the epicotyl, near the apical meristem with an insulin syringe (Fig. 2a-d). The punctured seedlings were co-cultivated with Agrobacterium strains (pCAMBIA1302/ pCAMBIA2301) in AB minimal medium containing wounded tobacco leaf (mature, yellowishgreen) extract to increase the virulence of Agrobacterium (Fig. 2e). Pricked seeds were incubated on a rotatory shaker at 28 ο C with 50 rpm for 2 h. After infection, seeds were thoroughly washed with autoclaved double distilled water and transferred to polybags containing sterilized growing media (cocopeat, vermiculite and perlite, 3:1:1 ratio) and maintained under diffused light initially and later transferred to direct light in growth chambers maintained at 25°C with 16 h light and 8 h dark photoperiod ( fig. 2f). Diffused light has positive effect on the shoot regeneration and dark incubation was always found useful in regeneration and transformation experiments. 28,29,53,54 Seedlings took 4-5 weeks for recovery during which, they were regularly irrigated with sterile-double distilled water and Hoagland solution (Fig. 2g). Recovered plants were later transferred to transgenic glasshouse as per regulatory guidelines (Fig. 2h, i). The overview of apical meristem targeted in planta transformation has been provided in a flow chart (Fig. 3).
Tissue culture mediated regeneration and transformation have been proven to be disadvantageous in several plant species due to low transformation efficiencies, genotype dependence and are time consuming. [55][56][57] However, the in planta transformation approach does not demand any sterile growth conditions, phyto-hormones, is less timeconsuming, needs low-cost inputs and also genotype independent.

Preliminary Screening of Transformants
Detection of visible scorable markers like GUS and GFP provide an early signal of transformability and/ or successful transformation of infected tissues. 38 Therefore, in this study, GFP expression and GUS histochemical analysis were performed in their specific transformants to identify primary transformants.
As Agrobacterium infection is a random event, GFP expression analysis in the pricked apical meristematic regions (Fig. 4a) identified GFP expression in some transformed seedlings (Fig. 4b iii-v) indicating that few of the infected seedlings were found to be positive toward the transformation strategy. Some transgenic plants lacked expression in the pricked regions indicating the chances of transformation as random (Fig. 4b ii). Further, GFP expression was absent in wild type seedlings (Fig. 4b i). Microscopic observation under UV illumination revealed that out of 40 seedlings taken for visualization, 26 seedlings displayed the presence of green fluorescent protein (GFP) and were selected as primary transformants (T0 plants) of which, 24 plants were transferred in transgenic glasshouse. This precisely demonstrates a preliminary confirmation of 65.0% of the seedlings having GFP expression and putatively transgenic ( Table 2).
Another batch of plants where GUS gene was used as scorable marker identified primary transformants which demonstrated the presence of GUS expression in the pricked regions. Out of 30 primary transformants, GUS histochemical assay was performed using 7 plants and remaining plants were allowed to grow normally. It was found that out of 7 plants, 4 showed GUS expression at the pricked regions (Fig. 4b, viii). However, wild type plants did not show any color in stem and its dissections as they lacked the GUS transgene (Fig. 4b  vi, vii). Presence of GUS expression at cellular level was further confirmed by observing the GUS-stained tissue sections under microscope (Fig. 4b ix, x). Since the binary vector used for transformation had a GUS gene with a catalase intron, the histochemical analysis provided evidences for the integration of the T-DNA into the genome of mango plants. Nearly 57% of the plants showed GUS expression. Based on post pricking recovery and initial evidences of transformability in mango, the remaining 15 seedlings were transferred to the transgenic glasshouse and were allowed for growth.
A successful genetic transformation system seeks an appropriate visualization marker gene for the identification of transgenic plants. 58 In our experiment, GFP and GUS were used as screenable marker genes and similar studies have been reported in mango, 6 pummelo, 28 and passion fruit. 29

Recovery of Primary Transformants
Recovery of plants after genetic transformation and their establishment is essential for the success of the protocol. After transferring plants to glasshouse, 24 of 26 GFP and 12 of 15 GUS plants could survive. These plants were transferred to pots filled with soil, sand and farm yard manure 2:1:1 (FYM) and allowed to grow in glasshouse. Plants were continuously irrigated and supplemented with Hoagland's solution at regular intervals

Molecular Analyses for Transgene Integration in Mango
The purview of the study considering the perennial nature of mango (long juvenile phase of 6-8 years), deals with the demonstration of T-DNA integration in the T0 generation, despite the fact that the primary transformants produced through in planta transformation are chimeric. Similar kind of molecular characterization in the chimeric plants of other perennial tree species have been reported in pummelo, 28 and passion fruit. 29 In order to confirm the presence of transgenes in the transgenic mango plants, genomic DNA was isolated from primary transformants and wildtype mango plants. PCR analysis was carried out using GFP and hptII; GUS and nptII gene-specific primers in their respective transgenic plants.   Ai-ii, Bi-ii). Further, no amplification was found in wild type (WT) plants. The absence of Agrobacterium contamination in GFP transgenic plants were confirmed using PCR analysis of VirD1 gene (Fig. 5c). This unequivocally demonstrated that the GFP expression in the transgenic plants was due to the integration of the transgene and not due to the persisting bacteria. Transgenic plants that tested positive by PCR were further assessed at transcription level and expression of transgene was confirmed by semiquantitative RT-PCR analysis. Five transgenic plants and positive control showed the amplification of 124 and 130 bp GFP and hptII gene transcript fragments in the respective transgenic plants. However, amplification was absent in wild type (WT) plants (Fig. 5d i-ii). An internal control (MiACT1) authenticated amplification of 134 bp fragment in both transgenic and wild-type plants (Fig. 5d, iii). Finally, transgenic mango plants were assessed to determine the copy number of the transgene. Two transgenic mango plants (1 F and 15 F) were found to have a single copy of the transgene integrated in the genome and the absence of hybridization signal in the wild type providing proof for transgene integration (Fig. 5e).
Out of 12 plants tested for GUS and nptII genes, 8 plants showed PCR amplification of 1 kb GUS gene and 750 bp nptII gene fragments indicating the presence of transgenes in the primary transformants (Fig. 6a i-ii). Five GUS positive PCR plants were further tested for transgene accumulation that have shown amplification of 122 bp (GUS) and 67 bp (nptII) fragments verifying the presence of transcripts in transgenic plants and their absence in wild-type plants (Fig. 6b i-ii). Amplification of MiACT11 internal gene fragment in both transgenic and wild-type plants by sqRT-PCR further authenticated the results (Fig. 6b iii). Furthermore, transgene integration by Southern blotting identified single copy integration of the transgene in one transgenic plant (1 U) and the hybridization signal was absent in the wild-type plant DNA precisely confirming the transgenic nature of the plant (Fig. 6c).
Several researchers have tried to develop transformation protocols for mango using different methodologies. [9][10][11][12]46,59,60 However, in all the previous studies they were unable to unequivocally demonstrate transformability, more so the transgene integration by genomic Southern analysis. This study demonstrated gene introgression in mango genome using shoot apical meristem-targeted in planta transformation and the associated molecular analyses. Tree crops have always been found difficult to improve by conventional breeding tools, biotechnology complement the conventional breeding and amends the mango improvement programs 48 . Transgenic technology holds several promises and can open ways in tackling a multitude of problems in mango

Conclusion
This study demonstrates the amenability of mango to apical meristem targeted in planta transformation protocol in a genotype independent manner. This report is the first successful demonstration of transgenic mango development using in planta transformation, which can assure it to be a significant contribution toward advancement in the area of mango biotechnology. New Delhi for providing microscope facility. The authors also thank Mr. Rakesh Kumar Pandey (Late), PhD Research Scholar, Division of Fruits & Horticultural Technology, ICAR-IARI, New Delhi for the assistance during the experiment.

Disclosure Statement
The authors declare no conflict of interest. All the authors have read and approved the final draft of the manuscript.

Funding
The author(s) are thankful for funds recieved from ICAR-Indian Agricultural Research Institute, New Delhi, India.