Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis

doi:10.21203/rs.3.rs-1710759/v1

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Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis

https://doi.org/10.21203/rs.3.rs-1710759/v1

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Citrus is one of the major horticultural crops having high economical and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, the Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency and poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration, modifying in-vitro micrografting protocol to combat loss of transgenic lines. As the transgenic citrus shoots are difficult to root, we also developed ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months with transformation efficiency of 30% and 22% for respective species.

Agrobacterium

Citrus aurantifolia

Citrus sinensis

callus

in-vitro micrografting

rooting

somatic embryogenesis

genetic transformation

The world population is projected at 7.9 million as of 2022 and is estimated to exceed 10 million by the end of 2050¹. In between this increasing population, fruits production has consistently been inadequate to meet the required demand. Unbalanced consumption of fruits in the human diet leads to inadequate intake of micro- and macronutrients, which causes different forms of malnutrition². Citrus is one of the major fruit crops grown in more than 140 countries across the globe and has high nutritional value with an exotic taste. Despite its global availability, citrus is sensitive to various environmental stresses³. For the past many years, traditional breeding methods have been used to improve fruit yield and quality; however, in the case of citrus, it is an uphill task due to the long juvenile period, pollen/ovule sterility, apomixis, and absence of desired traits in related species. Through recent scientific developments in genetic engineering, adding desired traits from any source to target plants is now possible and thus, makes crop improvement easier in a limited period^4–6.

The initial studies to genetically modify citrus were done by using protoplast as an explant. The first attempt was made by Kobayashi and Uchimiya, where protoplast-derived cultured cells showed the presence of foreign gene; however, Vardi et al. were the first to successfully develop transgenic citrus plants^7,8. Later various other protocols, including Agrobacterium-mediated, particle bombardment, electroporation, and RNA interference were reported from different research labs^9–10. As genus citrus is not a natural host for Agrobacterium tumefaciens, various citrus species are recalcitrant for Agrobacterium-mediated transformation, though many improvements have been made to increase the transformation efficiency^11,12. Despite the refinement in transformation protocols, Citrus aurantifolia (Key lime) and Citrus sinensis (Sweet orange) are, recalcitrant and difficult to transform¹³. To overcome this, we critically reviewed all the stages of Agrobacterium-mediated citrus transformation and introduced several needful and innovative improvements. For our study, we selected commercial cultivars, namely, ‘Pramalini’ (C. aurnatifolia) and ‘Mosambi’ (C. sinensis). We believe that the adoption of our protocol would increase the explant transformation and regeneration efficiency, reduce the loss of in-vitro micrografted shoots and enhance the rooting frequency in transgenic citrus shoots.

Explant and Agrobacterium preparation.

Appropriate epicotyl age is critical for highly efficient transformation. Epicotyls after 4–5 weeks of germination become suitable for transformation (Fig. 1a). Additionally, the type of cut at the epicotyl ends (oblique) is also vital for transformation. Oblique cuts are made to expose the cambial ring (Fig. 1b).

For the transformation, we used binary vector pCAMBIA2301, which contains the gusA reporter gene with intron in the coding region. The intron-GUS reporter system did not generate perceptible GUS activity in Agrobacterium tumefaciens cells but can be efficiently utilized by plant cells through intron splicing. The existence of the intron also increases the level of matured mRNA and provides obvious proof of a successful transformation^14,15. This vector also contains the nptII gene for plant selection and was introduced into Agrobacterium EHA105.

Epicotyl pre-treatment and cocultivation.

Epicotyls were incubated with callus inducing supplements till the preparation of Agrobacterium secondary culture. Precultured epicotyls were then infected with Agrobacterium culture, co-cultivated on hormone enriched medium for 2/3 days and transferred on selection medium containing cefotaxime to control Agrobacterium and kanamycin for selection of transformed shoots. GUS signals could be observed immediately after co-cultivation, which subsequently increases with callus development. In this experiment, we stimulated the transformed cambial region to form callus and showed strong GUS signals during different phases of cambial callus development and shoot regeneration. We were successful in developing pronounced cambial callus covering the entire cut end of epicotyl which regenerates to give transgenic shoots (Fig. 2a-2d). A larger transformed area and well-developed callus would regenerate to give a greater number of transformed shoots thus, this step increased our transformation efficiency.

Optimization of shoot regeneration and GUS histochemical assay.

To get the best regeneration condition, we compared the regeneration efficiency of C. aurantifolia and C. sinensis on different nutrient media. Regeneration efficiency was observed after 45 days of epicotyl culture. For C. aurantifolia, the combination of ‘higher cytokinin with lower auxin levels’ gave the best regeneration, whereas the medium having only cytokinin, gave poor results (Fig. 3a-3d). We tested four medium MRNB1, MRNB2, MRNB3, and MRNB4 (for composition see supplementary information). However, in the case of C. sinensis, the combination of higher cytokinin along with lower auxin levels did not work, but the ‘lower cytokinin alone’ gave the maximum number of shoots per epicotyl (Fig. 4a-4d). Here, the four tested mediums were, ERB1, ERB2, ERB3, and ERB4 (for composition see supplementary information).

To provide auxin and cytokinin during epicotyl pre-treatment and co-cultivation, we used same hormone concentration as of the screened medium along with 2,4-D, for both of the citrus species. We got maximum number of regenerated shoots on MRNB1 medium (C. aurantifolia; Fig. 5a)/ ERB1 medium (C. sinensis; Fig. 5b) after Agrobacterium- mediated transformation. So, we recommend to use the same hormone concentration as of MRNB1 medium/ERB1 medium with additional 2,4-D for epicotyl pre-treatment and co-cultivation of C. aurantifolia/C.sinensis. We also observed stages of somatic embryogenesis in the regenerating epicotyls after transformation (Fig. 6a-6d).

After two biweekly subcultures, epicotyls with proliferating callus were shifted to 16-h photoperiod for 7–10 days (Fig. 1c-1d). After the emergence of a sufficient number of shoots from the cut ends, GUS screening should be started soon after (Fig. 1e-1f). Every regenerated shoot was separated from the mother explant and longer (≥ 5–6 mm) and smaller shoots (< 5 mm) were processed as described in experimental procedures (see screening of transgenic shoots). Both longer and smaller shoots (after elongation) showed similar transformation efficiency. No GUS activity was observed in the basal portion of the non-transgenic shoot; however, a strong GUS activity was detected in transgenic shoot (Fig. 1g-1h). Expression of gusA gene was used to calculate the transformation efficiency (%). Calculations were done using the formula number of GUS positive shoots/total number of evaluated shoots × 100.

In vitro micrografting and rooting of transgenic shoots.

To combat the loss in whole transgenic plant recovery, we did 2–3 separate in-vitro micrografting of single elongated transgenic shoot (see materials and methods: In-vitro micrografting and rooting of transgenic shoots). We were successful to recover almost every transgenic line, making negligible loss at this step. Additionally, we observed similar graft acceptance rate in shoot tip and nodes. Callus like structure between scion and rootstock could be observed third day onwards in the in-vitro micrografting system. New leaves emerge from the scion after ten days of culture. We recommend to change ML medium biweekly as it provides fresh nutrients to the micrograft system and facilitate graft union (Fig. 1i-1m).

We observed 45% and 28% rooting in C. aurantifolia and C. sinensis respectively (Fig. 1r-1u). Rooting efficiency was calculated (%) as total number of successfully rooted plantlet/total number of shoots cultured on rooting medium × 100.

Hardening of transgenic plantlets.

We slowly acclimatized in-vitro developed plantlets by covering the pot with cling film using elastic bands around its rim to maintain high humidity. Few holes were done in the cling film to allow gaseous exchange. After 10–12 days, we removed the elastic band and placed the cling film to covers the plantlet from the top but lifted it from one side. This was done to avoid sudden humidity change for plantlets. After 2–3 days, we completely removed the cling film. We observed almost 100% recovery rate of whole transgenic plantlets during greenhouse acclimatization (Fig. 1n-1q; Fig. 1v).

Confirmation of transgenic lines.

To confirm transgenic lines, we isolated genomic DNA and total RNA from leaves of wild type and putative transgenic plantlet. We performed conventional PCR from genomic DNA to check the presence of marker genes (Fig. 7a-7b). For detecting the expression of nptII and gusA gene, we carried out semiquantitative RT-PCR from cDNA, prepared from total RNA taking cytochrome oxidase (cox) as housekeeping gene (Fig. 8a-c). The complete workflow of Agrobacterium- mediated transformation of C. aurantifolia and C. sinensis is depicted below (Fig. 9).

The efficiency of the Agrobacterium-mediated transformation in citrus is limited by various factors. In this report, we implemented several improvements in the protocol that contributed to our success in efficient development of transgenic citrus. Seed age and quality are some of the silent but significant aspects of the efficacy of this protocol. The overall health status of donor trees and fruits at the time of seed harvesting is also critical for the quality of raised explants and their regeneration¹⁶. We also recommend the use of mature and freshly harvested seeds as we observed poor/delayed germination in older seeds, so longer storage must be avoided. We also recommend the use of 4–5 weeks older epicotyls for transformation experiments. Older epicotyl responds poorly to Agrobacterium-mediated transformation⁶.

Many citrus cultivars have poor transformation and regeneration competency. To develop an efficient transformation protocol, competency limitations must be addressed. In case of citrus, the cells competent for transformation and regeneration are localized in the cambial region. Additionally, shoots which regenerated after prominent cambial callus development are more likely to be transgenic. So, callus inducing treatment increases transformation frequency¹⁷. We focused on giving treatments favouring the callus development during pre-culture and co-cultivation of epicotyls. We pre-cultured and co-cultivated epicotyl in the medium having the combination of auxin and cytokinin along with acetosyringone. Together with auxin, cytokinin increases the competence of cells for transformation and contributes positively in callus development ^18,19. The exogenous cytokinin also increases the Agrobacterium viable cell number during the infection period²⁰. We also added malt extract in culture medium which is known to induce somatic embryogenesis in citrus²¹.

Overnight culture of Agrobacterium usually reaches to late exponential phase and has lost virulence. To achieve high virulence, Agrobacterium should be in the early log phase. So, we subcultured Agrobacterium in the presence of acetosyringone on the day of transformation^4,13. Gentle shaking while infecting the epicotyl with Agrobacterium culture provides proper exposure of every explant to the culture, thus increasing the transformation efficiency.

To establish an efficient transgenic development protocol, optimized shoot regeneration conditions are needed. Hence, we tested four different mediums for both C. aurnatifolia and C. sinensis to get the best regeneration condition. For C. aurnatifolia, BAP along with NAA gives the maximum number of shoots per explant. In many cultures, a high level of BAP and a low level of NAA have shown the positive impact on cell division and regeneration. For C. sinensis, BAP alone was sufficient to induce regeneration.

After the GUS screening of longer shoots (≥ 5 mm), we elongated smaller regenerated shoots (< 5 mm), before testing for for GUS activity. The GUS positive shoots were further cultured on kanamycin free medium to minimize the possible negative effect of antibiotic on shoot elongation and recovery, however, we do not recommend the elimination of cefotaxime at this stage.

We standardized both methods of recovering whole transgenic plants in citrus, in-vitro micrografting and rooting. For in-vitro micrografting, we used three-week-old seedling of C. jambhiri as rootstock. Older rootstock shows poor graft expectance. Generally, the success of shoot tip grafting in citrus was reported in between 30–50%²². Every in-vitro micrografted shoot would not survive, leading to the loss of many transgenic shoots. To tackle this loss, we separately in-vitro micrografted the apical portion and nodes of elongated transgenic shoot. Using our modified in-vitro grafting practice, almost every transgenic line survived, hence making this loss almost negligible. We also added a substantial amount of sucrose while culturing the scion-rootstock system., as it has been reported that increasing sucrose concentration increases the number and size of new leaves and lateral roots ²³. The use of liquid medium for culturing micrograft allowed easy culture handling and media replenishing, efficient nutrient absorption, and lesser oxidative stress on explant²⁴. For rooting, we performed three biweekly subcultures on the rooting medium.

Rapid water loss could lead to the death of transgenic plantlets while shifting to in vivo conditions. We recommend the slow acclimatization with high humidity maintenance while shifting the in vitro established plantlets to greenhouse conditions. Confirmation of transgenic plantlets were done by conventional PCR and semiquantitative RT-PCR of nptII and gusA reporter genes.

Various reports showing the transgene integration inability and recalcitrant behaviour of C. aurantifolia and C. sinensis by Agrobacterium- mediated transformation are available²⁵. In a study, C. aurantifolia was found to have maximum transformation efficiency of 8%¹¹. By using this protocol, we found transformation efficiency of C. aurantifolia Cv. ‘Pramalini’ and C. sinensis Cv. ‘Mosambi’ to be 30% and 22% respectively. We found these citrus species compatible for transformation and in-vitro micrografting.

We minimized the loss of transgenic shoot/plantlet at different stages and developed several hundred transgenic plants by using this protocol. Our protocol is not only suitable for the development of disease/stress resistant citrus but also for functional genomics studies on citrus.

Plant material.

Trees of C. aurantifolia Cv. ‘Pramalini’ and C. sinensis Cv. ‘Mosambi’ used in this study are maintained in ICAR- Central Citrus Research Institute, Nagpur, India. Mature and freshly harvested seeds were dipped in 70% v/v ethanol for one minute before washing in sterile distilled water (SDW). After air-drying and removing the outer coat, the seeds were sterilized with 2% v/v sodium hypochlorite for 15 minutes. Disinfected seeds were rinsed three to four times in SDW and blotted dry on autoclaved filter paper. The sterilized seeds were cultured on SG medium for germination and incubated for 3–4 weeks at 25 ± 2°C in dark, followed by a 16-h photoperiod for another one week. The light green epicotyls with 12–15 cm height were ideal for transformation.

Epicotyl pre-culture.

After removing the root, hypocotyl, cotyledon, and apical hook from the etiolated seedlings, the light green epicotyls were obliquely cut into ~ 2 cm² long segments. Epicotyls with the exposed cambial ring were pre-cultured into 60 ml of LM2NB medium (C. aurantifolia)/LE2B medium (C. sinensis) till the preparation of Agrobacterium suspension with gentle shaking (100 r.p.m.) at room temperature.

Agrobacterium infection, co-cultivation and selection of transgenic shoots.

Agrobacterium strain EHA105 harboring binary vector pCAMBIA2301 was cultured on YEM agar plates containing 50 mg l^− 1 kanamycin and 25 mg l^− 1 rifampicin for 2 days at 28°C in dark. On the day of transformation, one loop of Agrobacterium culture was inoculated into 60 ml of liquid YEM and cultured to OD₆₀₀ of approximately 0.3 at 28°C in dark with shaking at 200 r.p.m. At the time of inoculation, 100 µM of acetosyringone was also added along with appropriate antibiotics. Agrobacterium cells were harvested by centrifugation at 5000xg for 8 min at 25°C and resuspended in MSA medium to a final OD₆₀₀ of 0.3 before use. Approximately 80–100 pre-cultured epicotyls were incubated with resuspended Agrobacterium culture for 15 minutes with gentle shaking at room temperature. Infected epicotyls were blotted dry on sterilized filter paper to remove the excess bacterial suspension. The dried epicotyls were co-cultivated on SMA2NB medium for 2 days (C. aurantifolia)/SEA2B medium for 3 days (C. sinensis) at 25 ± 2°C in dark with the oblique cut surface facing upward. Co-cultivated epicotyls were transferred to MRNB1 (C. aurantifolia) medium/ERB1 (C. sinensis) medium for 4 weeks at 25 ± 2°C in dark for callus development and shoot regeneration. After the emergence of shoots from proliferating cambial callus at the cut end, epicotyls were shifted to a 16-h photoperiod for 7–10 days.

In-vitro germination of rootstock.

After transferring the co-cultivated epicotyls to the regeneration medium, Citrus jambhiri (Rough lemon) seeds were in-vitro germinated (see part; Experimental procedures- Plant material). Three-week-old C. jambhiri seedlings were used as rootstock for in-vitro micrografting of transgenic shoots.

Screening of transgenic shoots.

A small Stem basal portion of individually separated shoot (≥ 5–6 mm) was assayed histochemically for GUS activity^26,27. GUS positive shoots were further cultured for the recovery of whole transgenic plantlet and GUS negative shoots or escapes were discarded straight away. Smaller shoots (< 5 mm) were transferred to MEGK medium (C. aurantifolia)/EEGK medium (C. sinensis) for 3–4 days at 25 ± 2°C with a 16-h photoperiod, for elongation before being tested for the GUS activity.

In-vitro micrografting and rooting of transgenic shoots.

For in-vitro micrografting, GUS positive shoots were transferred for elongation (without kanamycin), MEG medium (C. aurantifolia)/EEG medium (C. sinensis) at 25 ± 2°C with a 16-h photoperiod for 12–15 days. A Transgenic shoot with 2–3 well-developed nodes was placed on a sterilized 90 mm Petri dish, and a cut was made after the shoot tip and every node. Separated shoot tip and each node were treated as an individual scion and grafted individually on rootstocks. The cotyledon, apical hook, lateral secondary, and tertiary roots were removed from three weeks old rootstock seedlings (Citrus jambhiri). Also, the main taproot was shortened to a final length of 4–5 cm. A longitudinal slit of 0.5 cm was made in the decapitated rootstock. After the preparation of rootstock, a sharp V-shaped cut was made in the scion, exposing its cambial ring. The scion was fitted in the slit of prepared rootstock and cultured in ML medium for 3–4 weeks at 25 ± 2°C with a 16-h photoperiod. The whole micrograft system was supported by M- shaped filter paper bridge having a hole at the center.

For rooting, GUS positive shoots were transferred to MR medium (C. aurantifolia)/EIR medium (c. sinensis) for 3–4 weeks at 25 ± 2 °c with a 16-h photoperiod. The transgenic shoots failed to develop roots even after three biweekly subcultures were in-vitro micrografted.

Hardening and analysis of transgenic plantlets.

Successfully in-vitro micrografted or rooted plantlets were gently pulled out from the culture medium. Plantlets were transferred to 2” pots filled with a 1:1 mixture of steam-sterilized garden soil and soilrite and acclimated under greenhouse conditions.

After 2 months of hardening, genomic DNA and RNA were extracted from leaves of putative transgenic plantlet using DNeasy® plant mini kit, Qiagen and RNeasy® plant mini kit, Qiagen (follow the manufacturer's protocol), respectively. cDNA was prepared using the iScript™ cDNA synthesis kit (Bio-Rad), according to the procedure recommended by the manufacturer. For the confirmation of transgenic plantlets, we detected the presence and expression of marker genes (nptII and gusA) by conventional PCR and semiquantitative RT-PCR, respectively.

Statistical analysis.

Statistical comparison among different regeneration media was compared by one-way analysis of variance (ANOVA; P < 0.05). Statistical analysis of the data was performed in Microsoft Office Excel 2019. Error bars represent in the graphs are standard deviation (SD). Experiments were repeated 3 times.

We declare that the research in this publication followed institutional biosafety guidelines and procedures.

Acknowledgements and Funding

SS and ZT thanks MHRD for Junior and Senior research fellowships. This work was partly supported by a faculty initiation grant (FIG-100677) by IITR to HC.

Author contribution statement

SS and HC contributed to experimental design and conceived the study. SS and ZT performed the experiment. SS and HC wrote the manuscript. HC and AKS supervised the experiment and reviewed the manuscript. SK and DKS gave input during experiments. All authors read and approved the manuscript.

Competing interests

The authors have not disclosed any competing interests.

Data Availability Statements

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis

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