Introduction

Eggplant is a popular household vegetable crop in India and Bangladesh, cultivated mainly for cash and as a source of
nutrition by resource-poor small and marginal farmers. Even though this crop occupies about 25% of Bangladesh’s total
vegetable cultivation area, its productivity (9.2 t/ha) is found to lag much behind the global average. Incidences of viral,
bacterial, and fungal diseases coupled with abiotic stresses are major reasons behind the reduction of eggplant
production in Bangladesh. However, it is well known that the success rate of genetic transformation is affected by a wide
range of factors such as explants sources, genotype, or genetic background, pH of media, temperature of growth
condition, Agrobacterium cell density on liquid suspension culture and co-cultivation medium, time duration of co-
cultivation, antibiotic selection pressure, hormonal composition of shoot elongation or regeneration medium etc.
Therefore, a comprehensive study of eggplant transformation and regeneration is important for understanding these
crucial factors affecting genetic transformation efficiency. Genetic background of different cultivated varieties and source
of explant is an important factor that affects the different response to eggplant regeneration and transformation
efficiency. Also, problems like tissue hyperhydricity and low rooting frequency were found to severely affect the
efficiency of eggplant regeneration. however, no study has reported the solutions to these critical issues. In the present
study, we have first attempted to develop an easy, quick, reproducible and highly efficient in vitro regeneration protocol
addressing some of the important parameters such as genotype, explants sources, media and hormone composition and
pH, and also focused on tissue hyperhydricity and rooting frequency.

Eggplant, a popular household vegetable crop in India and Bangladesh, is lagging behind the global average in
productivity due to various factors such as viral, bacterial, and fungal diseases and abiotic stresses. The success rate
of genetic transformation is affected by factors such as explant sources, genotype, media pH, temperature,
Agrobacterium cell density, time duration, antibiotic selection pressure, and hormonal composition of shoot
elongation or regeneration medium. A comprehensive study of eggplant transformation and regeneration is crucial
to understand these factors affecting genetic transformation efficiency. Genetic background and source of explant
also affect the response to regeneration and transformation efficiency. Problems like tissue hyperhydricity and low
rooting frequency severely affect eggplant regeneration efficiency. No study has reported solutions to these critical
issues. This study aims to develop an easy, quick, reproducible, and highly efficient in vitro regeneration protocol,
focusing on tissue hyperhydricity and rooting frequency.
Agrobacterium-mediated transformation is a widely used method for genetically engineering crops, including
eggplant (Solanum melongena L.), to introduce beneficial traits and improve productivity. This approach allows for
the delivery of foreign genes, knockdown of negative regulatory genes, and precise genetic modifications. Eggplant
is a significant vegetable crop in India and Bangladesh, but it faces challenges like pests, diseases, and low
productivity. To address these issues, researchers are exploring genetic engineering techniques such as
transgenesis and CRISPR-based gene editing. However, the success of these methods relies on an efficient
Agrobacterium-mediated transformation protocol, which has been lacking in eggplant. This study aims to establish
a comprehensive transformation and regeneration protocol for popular eggplant varieties in Bangladesh, enabling
advanced genetic studies and the development of stress-resistant eggplant varieties through molecular breeding.
Materials and methods

2.1. Plant material, surface sterilization and growth condition

Three high-yielding eggplant varieties, BARI begun 2, 4, and 6, were collected from the Bangladesh Agricultural
Research Institute. Seeds were washed twice with sterile distilled water, sterilized with 1% bavistin and 0.1%
HgCl2 solution, air-dried, and inoculated on agar-solidified MS medium with 3% sucrose. In vitro cultures were
maintained under fluorescent illumination for 16 hours at 25 ± 2C. Cotyledon, hypocotyl, and root from 12-14 day-
old seedlings were used as explants for regeneration study and Agrobacterium transfection. Each segment was cut
into two or three segments, optimized for in vitro regeneration and subsequent Agrobacterium mediated
transformation.

2.2. Standardization of media composition for in vitro regeneration

The study involved two to three-slices of cotyledon, hypocotyl, or root explants cultured on MS medium
supplemented with different concentrations of cytokinin and auxin for shoot regeneration. The concentrations
ranged from 1.0 to 3 mg/l, with 0.2 and 0.5 mg/l concentrations applied with BAP and zeatin. Casein hydrolysate
and sorbitol were added to the shoot regeneration medium to test their effects on reducing hyperhydricity. Root
induction media involved excising 3,4 cm long shoots and cultured on MS medium supplemented with different
concentrations of IBA, IAA, and NAA. The cultures were incubated at a controlled temperature with a 16-hour
photoperiod, and sub-culturing was done at intervals of 12-14 days. The well-rooted plantlets were then
transplanted to earthen pots containing a mixture of soil and compost.

2.3. Construction of expression vector(s) used for eggplant transformation

This study focuses on the genetic transformation of eggplant using Agrobacterium tumefaciens strain EHA105. Two
expression vectors, pMDC100 and pCAMBIA1302, were used to study the plant selection markers for eggplant
resistance. The pMDC100 vector contains a neomycin phosphotransferase II (nptII) gene for kanamycin resistance,
while pCAMBIA1302 contains a hygromycin-B-phosphotransferase II gene (hptII) for hygromycin B resistance.
Both plant selection markers were controlled by a cauliflower mosaic virus (CaMV35S) promoter and CaMV35S
poly-A tail terminator. Four different expression constructs were used to study the parameters of Agrobacterium
mediated transformation into eggplants.
The first construct was amplified from eggplant total cDNA pool using nested PCR primers and cloned into pCR-4-
TOPO vector. The sHSP gene was sub-cloned into a modified Gateway compatible entry vector 1 and transferred
into the binary expression vector pMDC100. The final construct was transformed into Agrobacterium tumefaciens
strain EHA105 by electroporation. Two expression constructs were generated with different reporter genes: green
fluorescent protein (mGFP) and β-glucuronidase (GUS) genes. The GUS expression vector under kanamycin
selection was amplified from pCAMBIA1301 plasmid and transferred into the pMDC100 binary vector. A separate
plant expression cassette was prepared by cloning the spCas9 gene under the regulation of the CaMV35S promoter
and Nos terminator.
This study evaluated factors related to Agrobacterium transformation in eggplant to determine the maximum
frequency for Agrobacterium mediated transformation. Factors included the density of Agrobacterium cell
suspension used for infection, eggplant compatible antibiotic selection pressure, infection time, and co-cultivation
duration. Agrobacterium was inoculated in yeast extract-mannitol broth with appropriate antibiotics, and then
inoculated into a secondary culture. Acetosyringone was added to the suspension and co-cultivation medium to
induce Agrobacterium vir genes, facilitating T-DNA transfer into plant cells. Newly germinated eggplant seedlings
were collected and dipped in the acetosyringone-treated Agrobacterium suspension. Explants were then
transferred to the co-cultivation medium and incubated for 48 hours. After three rounds of selection, transformed
cells developed green shoots. The shootlets were then transferred into different rooting mediums and planted in
soil:compost mixtures. The plants were allowed to acclimatize to the greenhouse condition for the first 12 days and
then transplanted in earthen pots for further growth and development observation.2.4. Preparation of
Agrobacterium culture for transformation into eggplant

2.5. GUS staining and GFP fluorescence assay

The transformation efficiency of eggplant cell suspension culture was determined by comparing the number of GUS
staining and expression of the green fluorescent fusion gene (mGFP) in control and transformed cotyledons,
hypocotyls, and root explants. The GUS assay was conducted using pMDC100:GUS construct and eggplant cell
suspension culture was prepared by inoculating 2-3 pieces of friable calli derived from both transfected and control
explants with pCAMBIA1302:sHSP::mGFP fusion construct selected with 10 mg/l hygromycin. The calli were
incubated with autoclaved liquid MS media for one week, then centrifuged and washed three times with fresh
sterile water. The pellet was resuspended in water and prepared for slide preparation.

2.6. Molecular confirmation of putative transgenic plants

Genomic DNA was extracted from putative T0 transgenic lines developed with three independent expression
vectors, pMDC100: sHSP, pMDC100:spCas9, and pCAMBIA1302:sHSP::mGFP, along with control eggplant leaves.
PCR screening was conducted to confirm transgene insertion for both expression cassettes. The first generation of
stable transgenic plants (T1 generation) were selected by germination of T0 seeds in 150 mg/l kanamycin for
pMDC100:sHSP and pMDC100:spCas9 constructs and 10 mg/l hygromycin for pCAMBIA1302:sHSP::mGFP. These
T1 transgenic plants were used for transgene integration and copy number determination by Southern blot
analysis. Restriction enzymes, NdeI and EcoRV were used for overnight digestion of genomic DNA. Fragments of the
digested DNA were separated by electrophoresis in 0.8% agarose gel, and Hybond™ nylon positive membrane was
used for capillary transfer of DNA fragments. Construct specific digoxigenin-labelled probes (DIG probe) were used
for hybridization.
Result and Discussion

3.1. In vitro regeneration protocol of eggplant

The study found that most explants initiation of multiple shoots within four to five weeks of culture duration. The
highest number of regenerated plants was obtained for the media composition of MS+2.5 mg/l BAP. Hyperhydricity
was a major issue for most shoots in all three varieties. To minimize hyperhydrated shoot formation, casein
hydrolysate and sorbitol were used in the same media composition. The best elongation was obtained using MS +
BAP + casein hydrolysate + sorbitol. After root induction, 80% of shoots rooted well within 9 days of culture.

3.2. Optimization of antibiotic concentration for transgenic plant selection

The study aimed to determine the appropriate antibiotic concentration for screening putative transformed lines in
eggplant shoots and cotyledonary leaves. The shoot regeneration was inhibited by 100 mg/l of kanamycin and 10 mg/l of
hygromycin, and the right antibiotic concentration was determined for germination selection of T1 transgenic lines from
T0 seeds. Agrobacterium overgrowth was controlled in MS medium supplemented with different concentrations of
cefotaxime. The growth of Agrobacterium was effectively inhibited with 250 mg/l of cefotaxime, resulting in minimal cell
damage after co-cultivation.

3.3. Overview of eggplant genetic transformation for the generation of independent events

The study aimed to assess Agrobacterium-mediated transformation in eggplants using two vectors (pMDC100:GUS and
pCAMBIA1302:sHSP::mGFP) with kanamycin and hygromycin markers. Optimal conditions included an Agrobacterium OD
of 0.6 at 600 nm, 10-minute infection, and 2-day co-cultivation with lower acetosyringone concentration. Cotyledons and
hypocotyls from 10-day-old seedlings were used for transformation. After three selection rounds, 40% of transformed
shoots developed roots. Researchers worked on improving rooting strategies for higher success.

3.4. Molecular confirmation of the transgenic plants

Three stable transgenic lines with different expression vectors were developed for eggplant development. An average of
40 putative T0 transgenic eggplant lines were generated from these constructs using an optimized transformation
protocol. Primary PCR confirmation of transgene integration was performed using specific primer pairs for all three
constructs. On PCR screening for kanamycin and hygromycin genes, an average of 32 and 22% of transformation
efficiency was observed in T0 transgenic plants, respectively. Southern analysis confirmed the copy number of transfer
deoxyribonucleic acid insertions using sHSP and spCas9 cDNA specific probes.

4. Discussion

This study aims to address the low success rate of transformation and regeneration in eggplant cultivars from
Bangladesh by addressing various key factors. The study examines the shoot regeneration response of three high-
yielding eggplant varieties (BARI Begun 2, 4, and 6) under different auxin and cytokinin concentrations. The
researchers found that 2.5 mg/l BAP alone is the best supplement for maximum shoot regeneration of cotyledon,
hypocotyl, and root explants. The study also found that the application of antibiotic concentration on selecting
stably integrated transformed cells can inhibit the regenerative response of different explants. The researchers
found that 250 mg/l of cefatoxime is lethal for Agrobacterium but doesn't have significant adverse effect on callus
development and regeneration in BARI begun 2, 4, and 6. The study also found superior number of shoot induction
with optimized kanamycin concentration of 100 mg/l (4.16 shoots/explant) compared to hygromycin
concentration of 10 mg/l (2.16 shoots/explant). This is not unusual as similar observations have been reported for
different plant species and it is considered that non-transgenic cells can survive due to incomplete selection of
adjacent transformed cells. The study supports the finding that kanamycin is better for selecting eggplant
transformants than other selection agents.
The density of Agrobacterium suspensions used for eggplant infection is crucial for achieving high transformation
frequency. The highest transformation frequency was observed when the Agrobacterium suspension had an optical
density (OD) of about 0.6 at 600 nm. Infection and co-cultivation times with Agrobacterium are equally important
to achieve the highest transformation efficiency. Fresh-cut explants are more competent in inducing Agrobacterium
vir gene expression when only 50 µmol of acetosyringone is added to the re-suspension media.

Hyperhydricity and root induction frequency are serious problems in eggplant transformation. Hyperhydricity can
be overcome by better ventilation, manipulation of solidifying media, lower cytokinin and ammonium nitrogen
concentrations, frequent subculture, and adding anti-hyperhydricity agents. Casein hydrolysate and sorbitol can
significantly reduce hyperhydricity and accelerate the growth of in vitro tissues.

Successful rooting of transformed micro-shoots is another crucial step to facilitate their establishment in soil.
Different concentrations of IBA exhibit maximum root formation in eggplant. To optimize rooting efficiency, both in
vitro and ex vitro root induction strategies were applied. The best results were obtained when the basal portion of
a well-elongated micro-shoot is dipped into filter sterilized IBA solution before their transfer to the rooting
medium. This process can be considered a cost-effective strategy and can overcome problems encountered during
acclimatization.

Conclusion
This study highlights the importance of addressing hyperhydricity, rooting, and shootlet regeneration in
Agrobacterium-mediated eggplant transformation, especially for high-yielding varieties from Bangladesh. To
overcome these challenges, the study recommends enhancing the culture medium with casein hydrolysate and
sorbitol to reduce hyperhydricity. Additionally, using a 1.0 mg/l IBA shock significantly improves rooting in both in
vitro and ex vitro conditions. Optimal Agrobacterium culture density was found at OD600 = 0.6, with a 2-day
infection time in co-cultivation media. This protocol can be a valuable reference for enhancing transformation
efficiencies not only in eggplant varieties BARI begun 2, 4, and 6 but also in other plant species.