In Vitro regeneration of bio-immunized banana cv. Grand Naine using a novel double-decker temporary immersion bioreactor

Abstract

Considering the havoc caused by Fusarium oxysporumf. sp. cubense (TR4) at the global level on banana cultivation, especially in India, there is a high demand for an economical bio-immunized tissue culture technique for the sustainable production of disease-resistant plantlets, wherein bio-immunization is achieved through treatment of tissue culture–derived plants with antifungal secondary metabolites to mitigate the impact of Fusarium wilt. In the current investigation, a novel, user-friendly, and cost-effective Double Decker Temporary Immersion Bioreactor (DDTIB) was designed and optimized for the mass multiplication of bio-immunized Grand Naine plantlets to cater to the huge demand of planting material of banana. An immersion frequency of six hours with three minutes duration of media immersion in DDTIB significantly improved the regeneration frequency (22.32 shoots/clump) as compared to the Semi Solid Tissue Culture System (SSTCS), which yielded only 7.86 shoots/clump. Additionally, after three consecutive multiplication cycles, DDTIB produced significantly higher biomass (1112.24 mg) as compared to SSTCS (996.36 mg). A novel primary hardening technique was also introduced in DDTIB-regenerated bio-immunized plantlets, resulting in a 91.52% survival rate. The genetic fidelity of DDTIB-regenerated plants was confirmed using ISSR markers and further confirmed by metabolomics analysis. The DDTIB developed in the current study would be the most suitable bioreactor for the rapid and large-scale commercial multiplication of bio-immunized Grand Naine plantlets.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42254-z.

Introduction

Banana (Musa sp.) is one of the major commercial fruit crops grown in tropical as well as subtropical parts of the world. It serves as a staple food for more than 400 million individuals¹ worldwide. It is important to remember that banana is major energy food items, most exported fruits, and one of the main fruit products which is transported across the globe frequently. Commercially, tissue culture is being utilized for the mass production of banana plantlets at an industrial scale worldwide to cater to the huge demand for planting material². In the recent past, intensive cultivation of Cavendish banana under subtropical zones of South Africa and Australia led to mutation of Foc Race 1 to Race 4, which caused severe wilting symptoms associated with Panama wilt³. The global banana industry is under serious threat of Fusarium Wilt (FW), a soil-borne fungal disease; caused by Fusarium oxysporum f. sp. cubense (Foc) Tropical Race 4 (TR4) pathogenic to Cavendish clones⁴. Its rapid spread generates huge international concerns about food security in the tropics and subtropics. Additionally, the international banana market, dominated by Cavendish clones affected severely by Panama wilt⁵, the Cavendish (AAA) group of cultivars, viz., Grand Naine and Robusta are the most widely traded bananas in India, accounting for 52% of the total banana-growing area and contributing up to 64% of the total bananas produced ⁶. Grand Naine cultivation in the subtropical region has also faced a serious threat because of the sudden outbreak of Fusarium wilt (TR4) in India, especiallyin Uttar Pradesh and Bihar^7,8. Farmers involved in banana cultivation in the hotspot region have been severely affected by this disease, significantly impacting thelivelihoods of numerous farmers⁹. To address the Panama wilt concern and to ensure sustainable production in the hot spot region as well as other banana cultivating pockets, a novel tissue culture technology, i.e., in vitro bio-immunization, was developed by Damodaran et al.¹⁰. Bio-immunized plants developed through engineering biomolecules in banana tissue culture plantlets during the organogenesis phase paved the way for the successful cultivation of banana cv. Grand Naine in disease-affected hotspot regions of Uttar Pradesh and Bihar. However, the application of bio-immunization technology in the existing tissue culture system (semi solid) for banana propagation is limited to the marginal growers due to its additional cost involvement¹⁰.Temporary immersion bioreactor systems (TIS) have emerged as the preferred option for fruit crop micropropagation¹¹, offering lower production costs, faster biomass production, higher multiplication rates, and reduced hyperhydricity issues due to improved gas exchange and automation¹². TIS systems such as RITA, SETIS, Plantform, and Twin glass airlift have been used to propagate a range of crops, including banana, date palm, strawberry, papaya, Citrus, grape, pineapple, apple, pear, plum, chestnut, pistachio nut, apricot, sweet cherry, and almond¹³. However, all TIS systems have been developed without considering the provision of light in the bioreactor. Light is mandatory for the morphogenesis or growth of the plants. All the developed TIS are kept on a rack with provision of illumination (4000 lx Light fitted on the rack. It results in low penetration of light within the tank. Secondly, one container cannot be kept above another container, leading to uneconomical utilization of lab space. Another constraint faced in available TIS systems (RITA, SETIS, Plantform is that they do not have detachable medium reservoirs, as a result, contaminated medium cannot be changed without impacting plants. Considering the facts above, a novel double-decker temporary bioreactor was developed and utilized for the production of bioimmunized plantlets of banana cv. Grand Naine. The present study focuses on the design of the cost-effective novel double-decker temporary bioreactor, evaluating the performance of regenerated plantlets in different stages, starting from in vitro to primary hardening, secondary hardening, and final field establishment stage, and comparative assessment of Double Decker Temporary Immersion Bioreactor (DDTIB) with Semi-Solid Tissue Culture System (SSTCS).

Materials and methods

Plant material

Sword suckers of banana cv. Grand Naine were collected from healthy mother plants maintained at the field gene bank of ICAR–Central Institute for Sub Tropical Horticulture, Lucknow. The explants were washed under running tap water, and corm segments (12 × 12 × 15 mm) were prepared. The segments were treated with Tween-20 and rinsed for 30 min, followed by prewashing in 0.1% carbendazim, 100 mg/L cefotaxime, and 100 mg/L ascorbic acid with shaking at 100 rpm for 60 min. After rinsing with distilled water, the explants were surface sterilized with 0.1% mercuric chloride for 8 min and washed six times with sterile distilled water. The explants were trimmed to 8 × 8 × 10 mm and inoculated on MS medium supplemented with 4.5 mg/L BAP.

Designing a novel double decker temporary immersion bioreactor (DDTI bioreactor)

The novel Double Decker Temporary Immersion bioreactor (DDTIB) consisted of two transparent polycarbonate chambers capable of withstanding steam sterilization at 121 °C and 20 psi for 15–20 min cycle. The upper chamber measured 186 × 166 × 134 mm, and the lower chamber measured 186 × 166 × 50 mm. Air was passed through 0.2 µm sterile filters connected by silicone tubes to the inlets and outlets. A middle filter was connected via a silicone tube to the lower chamber.

The bottom of the upper chamber had five nipples. One nipple extended through both chambers, with 10 mm length on each side, while the remaining four opened only into the lower chamber. These four nipples facilitated the transfer of nutrient medium from the lower to the upper chamber. Twin side nipples delivered sterile air into the lower chamber, enabling the liquid nutrient medium to rise efficiently into the upper chamber when pressure was applied. The upper chamber had an in-built sealing system at its base that inserted securely into the lower chamber (Fig. 1). It was closed with an air-tight lid fitted with a silicone seal inside the groove. An LED box was placed on top of the lid to provide cool white fluorescent light (4000 lx) to the plantlets in the upper chamber. The 0.2 µm filters were connected to two timers and two pneumatic pumps via silicone tubes to regulate gas exchange. When air pressure was applied through the middle filter, the nutrient solution was forced upward to immerse the plant material in the upper chamber; upon pressure release, the solution drained back through the holes at the bottom into the lower chamber. A solenoid valve was fitted in the air outlet line connected to the middle filter to facilitate air release. The immersion interval and frequency were controlled by the timers. Since the DDTIB bioreactor had its own lighting system, multiple units could be conveniently stacked vertically (Figs. 1 and 3).

Fig. 1.

Design of DDTIB utilized for the multiplication of bio-Immunized Grand Naine banana plantlets: A.Complete bioreactor body comprising an upper and lower chamber, where upper chamber was utilized for plantletmultiplication and lower chamber was utilized for media storage. B. Side view of DDTIB. C. Upper and lowerchambers where three inlets are placed at upper chamber, air inlet connected with 0.22 micron fi lter for O2 supply(X), an inlet for media immersion (Y), inlet for gaseous exchange (Z).

Fig. 3.

Demonstrating the DDTIB utilized for the rapid multiplication of bio-immunized TC plantlets at its thirdstage of growth. A. Complete view of the self-illuminated DDTIB. B. Side view of non-self-illuminated DDTIB; C.Regeneration of plantlets using DDTIB. D. Showcasing regeneration from SSTCS.

Nutrient media

MS medium fortified with 4.5 mg/L BAP, 1.0 mg/L IAA , 60 mg/L Mg SO4, 100 mg/L M-Inositol, 100 mg/L Glutamin and30 gm/L Sucrose (Hi Media Pvt. Ltd., Mumbai, India) was sterilized in an autoclave at 121 °C temperature and 15 psi pressure. The medium was allowed to cool and kept for 72 h prior to utilization¹⁴. Approximately, 500 ml liquid nutrient medium was poured in a lower chamber of each DDTIB under a laminar air flow unit for shoot multiplication, whereas MS medium supplemented with 0.5% bioimmune s(ICAR-CISH, Lucknow) + 2 mg/L IBA (Hi Media Pvt. Ltd., Mumbai, India) + 20 gm/L sucrose (Hi Media Pvt. Ltd., Mumbai, India) was utilized for root induction¹⁰

Bio-immunization of banana cv. Grand Naineusingalipo-polypeptide molecule

A lipopeptide-based biomolecule derived from Trichodermareesei (“BIO-IMMUNE”) was developed and patented (Patent File No. 202111003761). The in vitro bio-immunization protocol for banana tissue culture plantlets during organogenesis and rhizogenesis was developed at ICAR–CISH, Lucknow. The BIO-IMMUNE formulation (0.5%) was incorporated into the MS shoot multiplication and rooting media, and all explants were cultured exclusively on the bio-immune–fortified medium¹⁰.

Growing media

Cocopeat (coconut coir dust) was used as the growing medium for primary and secondary hardening. The dried cocopeat cubes were submerged in water. The cocopeat was autoclaved at 121 °C, 15 PSI, for 20 min to prevent contamination. Once brought down to room temperature, the cocopeat matrix was enriched with a 50% strength MS salt solution containing the right nutrients necessary for plant growth¹⁵.

Primary and secondary hardening

Rooted plants derived from DDTIB were shifted to polypropylene bags filled with approximately 500 g of autoclaved cocopeat mixture fortified with 1 ml/L MS (1964) salt solution. A total of 30 tissue-cultured banana plantlets were carefully transferred from the DDTIB and SSTC containers to each PP bag, ensuring minimal disturbance to the root system. These bags were subsequently stored for an initial 15 days at 25 ± 5 °C in a growth room equipped with fluorescent tubes to achieve a light intensity of 4000 lx. Relative humidity in the chamber was maintained at 80–85% using a humidifier. At this stage, the PP bags were moved to a shade net house with 75% shade, 75% RH, and 30–35° C temperature for 30 days, at the end of which, small holes were made at the top of the bag to allow gaseous exchange and acclimatization to the ambient humidity. The bio-immunized plants in PP bags were further shifted to poly bags containing potting mixture as soil + cocopeat + sand (2:1:1) under 75% shade with 75% RH and 30–35 °C temperature for 30 days for full development of healthy plants.

Estimation of the efficiency of DDTIM through the surface response model

Experiments were conducted to optimize immersion interval and duration for enhanced shoot proliferation in a liquid culture system. Nine treatment combinations were evaluated using immersion interval (3, 6, and 12 h) and immersion time (2, 3, and 4 min) as independent variables. The number of shoots per clump and shoot length (cm) were recorded as response variables under controlled conditions. The effects of the treatments were analysed using response surface methodology (RSM) in Statistica 12¹⁶.

Estimation of photosynthetic pigmentation

Photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids) were quantified in plantlets regenerated through the DDTIB and SSTCS systems. Leaf samples were collected at different developmental stages, including in vitro (after the third subculture cycle), primary hardening, and secondary hardening. Chlorophyll content was estimated using the method of Burnison (1962) with minor modifications. Absorbance was recorded at 663 nm for chlorophyll a, 645 nm for chlorophyll b, and 480 nm for carotenoids using a VS 2100 UV–Vis spectrophotometer (Chemito Instruments Pvt. Ltd.)¹⁷.

Genetic fidelity assessment by using omics tools

Molecular fingerprinting of Grand Naine banana regenerated through DDTIB and SSTCS using ISSR primer

Mother plants, DDTIB-derived plantlets, and semi-solid tissue culture (SSTC)-derived plantlets were evaluated for genetic fidelity using ISSR marker UCB 840 (primer sequence: 5′-GAGAGAGAGAGAGAGAYT-3′). DNA was extracted from individual plant samples using a Plant DNA Purification Kit (Qiagen) according to the manufacturer’s protocol. The quality and quantity of genomic DNA were assessed using a microvolume spectrophotometer (NanoDrop, Thermo Fisher Scientific), and samples were standardized to 40 ng/µl. PCR amplification was performed in a 25 µl reaction mixture containing 40 ng template DNA, PCR master mix (Takara), and 10 pmol ISSR primer (Sigma). The amplification protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 2 min, annealing at the primer-specific temperature (Table 2) for 30 s, and extension at 72 °C for 2 min, with a final extension at 72 °C for 12 min, using a thermocycler (ProFlex, ABI). PCR products were separated on 2% agarose gel in 1 × TAE buffer and stained with ethidium bromide. Bands were visualized and documented using a gel documentation system (Syngene), and amplicon sizes were estimated by comparison with a 1 kb DNA ladder (Fermentas)¹⁸.

Table 2.

Comparative assessment of two TC systems (DDTIB vs SSTC system) on primary hardening.

Systems	FW (mg)	DW (mg)	Pl Height (cm)	No. of leaves	Plant width	No. of roots	Length of roots	Fw of roots	DW of roots	Survivability %
DDTIB	3691.89 ± 17.37	950.59 ± 3.87	20.16 ± 0.12	5.31 ± 0.07	4.64 ± 0.06	18.13 ± 0.08	10.95 ± 0.03	361.13 ± 2.4	183.65 ± 0.82	91.51 ± 0.03
SSTCS	2047.38 ± 8.1	658.88 ± 4.55	14.36 ± 0.10	5.91 ± 0.08	3.88 ± 0.05	13.91 ± 0.06	9.67 ± 0.06	244.57 ± 1.87	153.05 ± 0.55	90.93 ± 0.15
t value	85.74***	48.73***	35.29***	6.79***	20.45***	40.45***	17.39***	38.13***	− 30.60***	3.79*

Values are mean ± SE from five independent replications per system; traits were compared between DDTIB (double decker temporary immersion bioreactor) and SSTCS (semi-solid tissue culture system) using two-tailed t-tests per trait. *** P < 0.001.

Metabolic fingerprinting of bio-immunized Grand Naine plants regenerated from DDTIB vs. SSTCS using LC–MS

Liquid chromatography coupled with mass spectrophotometry (LC–MS) analysis was performed in the bio-immunized Grand Naine banana regenerated from DDTIB and the plants regenerated from SS-TC systems. Leaf samples were collected from the third leaf of the banana plants and washed with sterile distilled water, and then shade-dried for 30 days. The standard protocol for sample preparation was followed as prescribed by CSIR-Central Drug Research Institute, Sophisticated Analytical Instrumentation Facility (SAIF), Lucknow, Uttar Pradesh, India.

A total of 250 g of completely dried leaf was crushed into a fine powder and extracted using 70% ethanol and further purified with Ethyl acetate. A small fraction of the solvent-free plant extract was analyzed using ESI-LC–MS (Micromass Quattro II triple quadrupole mass spectrometer with a JASCO PU-980 HPLC Pump) using ESI/MS mode. The water absorption ODS (250_4.6mm_5I) column was used with acetonitrile: water + 0.1% formic acid solvent system, gradient elution was performed at 1.0 ml/min. The photodiode array was monitored at 200–650 nm and recorded at 220 nm. The mass spectra were scanned in the range 80–1000 DA in 2.5 S. The ESO capillary was set at 3.5 kV and the cone voltage at 40 V. The m/z spectral chromatographs were analyzed, key metabolites were predicted based on already published reports, the m/z database, and presented for comparative analysis of the status of secondary metabolites in the banana leaf samples¹⁰.

Statistical analysis

All experiments were conducted following a completely randomized design (CRD) to evaluate the performance of in vitro and ex vitro plantlets of the bio-immunized Grand Naine banana regenerated through the Double Decker Temporary Immersion Bioreactor (DDTIB) and the Semi-Solid Tissue Culture System (SSTCS). Data from the in vitro experiments were expressed as mean ± standard error (SE) and subjected to analysis of variance (ANOVA) using the CRD framework. Treatment means were compared using Duncan’s Multiple Range Test (DMRT) at the 5% level of significance (p < 0.05) to determine statistical differences among subculture cycles and between the two culture systems (Table 1).

Table 1.

Comparative assessment of different morphological characters of bio-immunized Grand Naine banana plantlets regenerated through DDTIB and SSTC system.

Subculture cycles (Nos.)	Fresh weight of plantlets (mg)	Dry weight of plantlets (mg)	Shoot height of plantlets (cm)	Shoot Diameter (mm)	Length of roots (cm)	Fresh weight of roots (mg)	Dry weight of roots (mg)
Double Decker Temporary Immersion Bioreactor (DDTIB)
I	1011.64 ± 18.26c	69.05 ± 1.25f.	6.54 ± 0.12d	3.19 ± 0.06 d	3.01 ± 0.05e	346.62 ± 1.86d	160.03 ± 2.00c
II	1088.66 ± 19.65b	85.33 ± 1.54c	7.37 ± 0.13b	3.78 ± 0.07b	3.85 ± 0.07c	443.58 ± 1.87a	168.33 ± 3.09b
III	1112.24 ± 20.08a	90.63 ± 1.64a	7.89 ± 0.14a	3.94 ± 0.07 a	5.77 ± 0.10a	439.94 ± 3.14a	171.32 ± 1.63a
Semi solid tissue culture system (SSTCS)
I	946.23 ± 17.08d	79.35 ± 1.43e	6.20 ± 0.11f.	2.85 ± 0.05e	3.31 ± 0.06d	303.79 ± 3.17e	152.06 ± 2.74e
II	928.63 ± 16.76e	80.35 ± 1.45d	6.47 ± 0.12e	2.86 ± 0.05e	3.28 ± 0.06d	407.80 ± 4.07b	154.05 ± 2.52d
III	996.36 ± 17.98f.	86.32 ± 1.56b	6.74 ± 0.12c	3.49 ± 0.06c	4.78 ± 0.09b	381.18 ± 1.38c	153.71 ± 2.77de

Values are presented as mean ± SE (n = 5), derived from three replications with three biological units per replication. Means followed by the same letter are not significantly different according to Duncan’s multiple range test (DMRT) at p < 0.05, based on acompletely randomizeddesign (CRD).

For comparisons involving only two systems (DDTIB vs. SSTCS) across physiological stages—namely primary hardening, secondary hardening, and field performance, the data were analyzed using two-tailed Student’s t-tests for independent samples (Tables 2, 3, 4, 5). Each treatment consisted of five independent replications per system unless otherwise specified. Field data (Table 5) were evaluated in the same way with the identical replication number (10 plants), and equivalent management practices for both systems to allow comparison.

Table 3.

Comparative assessment of two TC systems (DDTIB and SSTCS) on secondary hardening.

TC systems	Plant height (cm)	Root height (cm)	Pl. girth (cm)	No. of leaves	Survivability (%)
DDTIB	22.57 ± 0.75	23.94 ± 0.33	5.11 ± 0.11	6.00 ± 0.58	96.50 ± 0.17
SSTCS	21.23 ± 0.51	23.07 ± 0.43	4.78 ± 0.15	5.33 ± 0.33	96.37 ± 0.09
t value	1.47NS	1.60NS	1.71NS	1.0NS	0.68NS

Values are mean ± SE from five independent replications per system; traits were compared between DDTIB (double decker temporary immersion bioreactor) and SSTCS (semi-solid tissue culture system) using two-tailed t-tests per trait. NS, non significant.

Table 4.

Physiological assessment of plantlets derived through DDTIBbioreactor and SSTCS system during in-vitro and primary hardening.

	Chla	Chlb	Total chlorophyll		Chla/ Chlb	Carotenoid
In-vitro
DDTIB	4.50 ± 0.03	0.18 ± 0.01	4.68 ± 0.01		24.36 ± 0.10	1.20 ± 0.01
SSTCS	4.10 ± 0.02	0.13 ± 0.00	4.28 ± 0.01		31.11 ± 0.01	1.00 ± 0.01
t value	10.42***	22.04***	20.63***		− 63.57***	27.71***
Primary hardening
DDTIB	5.31 ± 0.03	0.20 ± 0.00		5.53 ± 0.04	30.83 ± 0.09	1.39 ± 0.01
SSTCS	4.81 ± 0.02	0.15 ± 0.00		4.90 ± 0.02	26.33 ± 0.25	1.08 ± 0.01
t value	13.18***	15.17***		11.63***	− 16.46***	27.34***

Values are mean ± SE from five independent replications per system; traits were compared between DDTIB (double decker temporary immersion bioreactor) and SSTCS (semi-solid tissue culture system) using two-tailed t-tests per trait. *** P < 0.001.

Table 5.

Field performance of plants raised through DDTIB and SSTCS.

	Plantheight (cm)	Pl. Girth (cm)	No. of leaves	Leaf length (cm)	Leaf breadth (cm)	Bunch weight (kg)	Yield/ha (tonnes)
DDTIB	224.6 ± 3.95	63 ± 1.25	10.8 ± 0.29	204.0 ± 6.54	81.9 ± 1.31	28.81 ± 0.59	46.18 ± 0.68
SSTCS	215.6 ± 2.33	62.6 ± 0.84	10.1 ± 0.23	197.0 ± 8.35	79.1 ± 0.99	28.21 ± 0.26	46.78 ± 0.55
t value	1.95 NS	0.39 NS	1.87 NS	0.65 NS	1.70NS	0.916 NS	0.67 NS

Values are means ± standard error of the mean for field-grown plants produced via DDTIB and SSTCS at harvest maturity; each treatment comprised identical replication size (10 plants) and plot management under the same orchard conditions. NS indicates the Student’s t test was not significant at for the corresponding traits. For clarity, t values are shown row-wise for each trait comparing DDTIB vs SSTCS.

Metabolomics (Table 6) were descriptively analyzed and identification of compounds relied on the distinctive combination of retention time (RT), mass/charge ratio (m/z) and intensity (%), supported by established references in literature and databases. All statistical analyses including 3D plot were performed using Statistica 12, StatSoft, Inc.¹⁶.

Table 6.

Metabolic profiling of bio-immunized plantlets developed through two TC systems.

Treatments	Tentative compound indentified	Peak value (mz)	Intensity (%)				References
			SSTCS		DDTIB
			RT	Intensity	RT	Intensity
Metabolites expressed in the Grand Naine banana	Trans zeatin	219.2	15.99	50%	15.99	50%	Wyrepkowski et al. (2014)
	Trihydroxy methylene-di-oxyflavone	611.3	15.0	58%	14.11	09%	Scigelova et al. (2016), Singh et al. (2021)
	Cryptochlorogenic acid	353.3	26.84	65%	15.58	18%	Mz cloud database
	Quercetin	301.3	21.45	09%	21.62	15%	Drapal et al. (2019)
	Flavonoid quercertin	303.3	23.42	30%	15.58	100%	Drapal et al. (2019)
	Caffeoyl-malate	295.4	16.00	23%	16.00	21%	Kozukue et al. (2004)
	Cryptochlorogenic acid	353.4	21.05	8%	26.56	7%	Drapal et al. (2019)
	Arachidonic Acid	441.4	22.1	4%	27.98	10%	Podolskaya et al. (2018)

Results

Mass multiplication of bio-immunized plantlets through DDTIB

Standardization of immersion interval and immersion frequency in the DDTIB bioreactor

DDTIB was utilized for the rapid multiplication of the bio-immunized banana cv. Grand Naine. The results clearly indicated that both immersion interval and immersion time significantly influenced the regeneration frequency, as measured by the number of shoots/clump and the length of the shoots. When shoots were immersed for 3 min in shoot multiplication medium after every 6-h interval, the highest number of shoots per clump (22.31 cm) with maximum shoot length (7.86 cm) was obtained. An immersion frequency of 3 min with reduced interval time (3 h) gave 16.33 shoots/clumps having 6.67 cm shoot length. Frequent immersion of explants led to the development of hyperhydricity, which hampered regeneration frequency. Conversely, the 12-h immersion interval produced the lowest shoot number (11.25 cm) owing to less nutrient availability to growing explants underthe bioreactor system. These results suggest that moderate immersion intervals, particularly 6 h, with an immersion time of 3 min, promoted optimal shoot proliferation and growth, while prolonged intervals (12 h) negatively affect both parameters (Fig. 2). The growth performance of plantlets varied significantly in two different tissue culture systems, viz., DDTIB and SSTCS, across different subculture cycles. Incremental morphogenesis was recorded in DDTIB as compared to the SSTC system. Notably, at third subculture cycle, the highest fresh and dry weights (1112.24 mg and 90.63 mg, respectively), shoot height (7.89 cm), shoot diameter (3.94 mm), root length (5.77 cm), and root biomass (439.94 mg) fresh weight and (171.32 mg) dry weight were recorded in DDTIB. In contrast, the SSTC system consistently recorded lower values for all parameters across the cycles. While slight improvements were observed from cycle I to III, the overall performance was superior in the DDTIB system. For instance, in SSTC at cycle III, the fresh and dry weights of plantlets were 996.36 mg and 86.32 mg, respectively, with a shoot height of 6.74 cm and root length of 4.78 cm. These results indicated that the DDTIB not only enhances regeneration frequency but also facilitates higher biomass accumulation (Table 1, Figs. 2, 3).

Fig. 2.

Comparative assessment of DDTIB vs SSTC system on morphogenetic potential of bio-immunized bananacv. Grand Naine through 3D Surface Plot A. length of shoot against immersion interval (hours) and immersion time(minutes); B. no. of shoots/ clump against immersion interval (hours) and immersion time (minutes).

Impact of illuminated top on plant biomass

The DDTIB was fitted with an LED top consisting of 4000 lx of light, along with a non-illuminated top, and placed on normal culture racks equipped with cool, white fluorescent light to assess its effectiveness. Data revealed that self-illuminated DDTIB produced a higher plant biomass (1133.33 mg) as compared to the non-illuminated DDTIB (1025.33 mg). Self-illuminated TIB systems can be stored in dark rooms or on culture shelves with no illumination in the growth room. This will reduce the cost of electricity, as light is provided only in the required area instead of illuminating the entire rack. Closure fitting of LED strips provides therequisite illumination, which helps in the accumulation of higher plant biomass (Fig. 4).

Fig. 4.

Total biomass accumulation under self-illuminated vs non-illuminated DDTIB bioreactor tops, shown asmean ± SE; groups differ significantly (t = 3.68, P = 0.021).

Acclimatization of bio-immunized plantlets

Comparative assessment of two TC systems on primary hardening

The results revealed significant differences between the DDTIB and the SSTCS across most growth and physiological parameters. The DDTIB produced significantly higher fresh weight (3691.89 mg) and dry weight (950.59 mg) compared to the SSTCS (2047.38 mg and 658.88 mg, respectively). Plant height, number of leaves, plant width, number and length of roots, as well as fresh and dry weight of the root, were all significantly greater in the DDTIB, which could be attributed to the fact that more surface area of the explant is available for nutrient medium absorption in the bioreactor system. Liquid media absorbs quickly by a large surface area as compared to SSTCS, where only a part of the explant is exposed to the nutrient medium. However, no significant difference was observed in survivability percentage between the two systems, with both treatments maintaining high survival rates (91.51% and 90.93% for DDTIB and SSTC systems, respectively). These findings suggest that the DDTIB supports superior plant growth performance compared to the SSTC system, without compromising plant survivability (Table 2; Fig. 5).

Fig. 5.

Demonstrating the innovative hardening technique associated with the DDTIB raised bio-immunized GrandNaine plantlets: A. DDTIB regenerated plantlets are under novel hardening technique in lab conditions. B. Primaryhardening of DDTIB raised TC banana plantlets in polypropylene bags; C. Plantlets transferred to polybag having soilmixture; D. Demonstration of bio-immunized DDTIB regenerated plantlets at the secondary hardening stage.

Comparative assessment of two TC systems on secondary hardening

The performance of different tissue culture systems showed significant variations in most growth parameters measured. The DDTIB facilitated higher plant height (22.57 cm), root length (23.94 cm), and no. of leaves (6.00), compared to the SSTCS (21.23 cm, 23.07 cm, and 5.33 respectively). However, the number of leaves and the survivability percentage were found to be quite similar between the systems. DDTIB and SSTCS survivability rates of 96.50% and 96.37%, respectively (Table 3; Fig. 5).

Physiological assessment during in-vitro and primary hardening stage

The bio-immunized plantlets grown during the organogenesis phase and primary hardening were assessed for physiological parameters. The photosynthetic pigment chlorophyll (“a”, “b”, and total) and carotenoid content varied significantly between the DDTIB and SS-TC system in both in-vitro and primary hardening and secondary conditions. In the in-vitro stage, the DDTIB exhibited higher chlorophyll “a” (4.50), chlorophyll “b” (0.18), and carotenoid content (1.20) compared to the SSTCS (4.10, 0.13, and 1.0, respectively). The Chl “a”/Chl “b”ratio was significantly lower in the DDTIB (24.36) than in the SSTCS system (31.11). Similar trends were observed during primary hardening, where plants from the DDTIB bioreactor had higher chlorophyll “a” (5.31), chlorophyll “b” (0.20), and carotenoid content (1.39) compared to those from the SSTCS (4.81, 0.15, and 1.08, respectively). However, the Chl “a”/Chl “b” ratio was higher in the DDTIB (30.95) than in the SSTCS (26.18). The study revealed that photosynthetic pigmentations were higher under bioreactors as compared to the conventional tissue culture system, which may be due to higher absorption of liquid nutrient medium by the explants and better physiological function (Table 4; Figs. 3, 5).

Field evaluation of bioreactor regenerated plantlets

The plants regenerated through DDTIB and SSTC systems were planted in the experimental block at ICAR-CISH, R.B. Road campus, Lucknow, India for assessment of the growth and yield potential of both systems. The results indicated that there were no significant differences between plants propagated through both systems. Plant height was slightly higher in DDTIB (224.6 cm) as compared to SSTCS (215.6 cm). Similarly, plant girth, number of leaves, and leaf breadth showed comparable values between the two treatments, with no significant variation. Bunch weight was slightly higher in DDTIB (28.81 kg) than in SSTCS (28.21 kg), which resulted in a marginally higher yield per hectare for DDTIB (46.18tonnes) compared to SSTCS (46.78 tonnes), but these differences were also not statistically significant. Overall, the data indicate that plants produced through both the TC systems performed at par in terms of yield (Table 5; Fig. 6).

Fig. 6.

Field assessment of Grand Naine plants raised through DDTIB. The fi gure illustrates the fi eld evaluation ofbanana (Grand Naine) plantlets regenerated by DDTIB at R. B. Road campus, Telibagh, Lucknow, Uttar Pradesh, India.A. Overview of the banana plantation with well-spaced rows in between two banana plants (2.5 x 2.5 cm). B. Close-up view of the plantation showing plants at a slightly advanced growth stage. Some plants display early-stage fruitformation, demonstrating good adaptation to the fi eld environment.

Genetic fidelity assessment through multi-omics tools

Genetic fidelity through ISSR markers

Eight samples of mother plants of the banana cv. Grand Naine was compared with propagules regenerated through DDTIB and SSTCS using IISR marker UBC 840 on 1% agarose gel. All the samples were amplified using the ISSR primer sequence 5′-GAGAGAGAGAGAGAGAYT-3′. The result demonstrates that the plants generated from both the systems (DDTIB and SSTCS) are genetically similar or true to type, clones from the mother plant (Fig. 7).

Fig. 7.

Genetic fi delity analysis of mother plants, plantlets derived through DDTIB and SSTCS system. ISSR marker(UBC-840) was utilized to screen the populations. Lane denoted with L: 1000 kb Ladder; Lane M1-M8: mother plantsof grand Naine banana; Lane B1-B8: plantlets raised by using DDTIB bioreactor system; Lane S1-S9: plantletsderived through SSTCS. Leaf samples (randomly chosen from 5 diff erent plants pooled and considered as onesample) were collected and the experiment was performed with a total of 100 plants. A. B. C: Gel images showingthe reproducibility of the results with multiple repetitions.

Genetic fidelity through metabolic profiling

Metabolite profiling of plantlets derived through both tissue culture systems was done in order to ascertain the similarity of metabolites in the propagules. It is clearly indicated that all nine metabolites, viz., Transzeatin, Trihydroxy methylene-di-oxyflavone, Cryptochlorogenic acid, Quercetin, Flavonoid quercertin, Caffeoyl-malate, Cryptochlorogenic acid, Arachidonic Acid were expressed in plantlets regenerated from both the systems (DDTIB and SSTCS). Thisconfirms similar metabolic imprints in plantlets developed through two tissue culture systems (Table 6, Fig. S1).

Discussion

Banana plants were first domesticated in Southeast Asia and have since evolved from being the first cultivated fruit to becoming the most consumed and exported fruit in the world (ITC^19,20). Bananas, along with plantains, are the fourth most important staple fruit crop worldwide and are essential to maintaining food and nutritional security among 400 million people in producing countries²¹.The banana sector is estimated to be a 25 billion USD industry, projected to expand at a compound annual growth rate (CAGR) of 4.5% between 2022 and 2027^22,23. Gross Michel was the most popular banana variety until the 1950s. It was devastated in Central and South America by the Fusarium wilt Tropical Race 4, a deadly fungus found in soils, and gradually replaced by the Cavendish group of banana²⁴. The Cavendish group of bananas is now facing similar threats from emerging pests and diseases, including Tropical Race 4 (TR-4), which could wipe it out in the coming years^20,24.Globally, India is renowned as the largest producer of bananas, cultivating 0.96 million ha with a total production of 35 million tons (GOI, 2022). Among the banana, Grand Naine and Dwarf Cavendish cultivars account for almost 47% of total Cavendish banana production worldwide²⁵. In the recent past, commercial cultivation of Grand Naine banana has been severely threatened by the outbreak of a virulent strain of Fusarium wilt tropical race 4^7,8. This sudden outbreak of Fusarium wilt in Grand Naine banana creates huge losses among the major cultivation areas of Uttar Pradesh and Bihar^7,26, and it prompted a group of scientists to urgently develop sustainable management strategies to combat Fusarium wilt and restore the banana industry to full functionality in subtropical regions. Till now, the most viable commercial approach of banana micro-propagation is through tissue culture techniques. Therefore, novel approaches could facilitate the incorporation of bio-molecules in the nutrient media itself for sustainable resistance or to produce healthy and immune plantlets for cultivation. Finally, a novel approach called bio-immunization through patent-protected lipo-polypeptide-based molecule “Bio-immune” was developed and implemented in the TC media during organogenesis and rhizogenesis^7,10,26. This novel method was developed and validated for infusing with a secondary metabolite-based bio-molecule during the in vitro stage in order to generate FOC TR4-tolerantplantlets. The technological incorporation in the tissue culture media fortified with 0.5% of ‘bio-immune’ showed a marked improvement in the shoot and root biomass, including other physiological parameters clearly described by¹⁰. The application of ‘bio-immune’ may direct the production of protective enzymes such as chitinase, β-1, 3-glucanase, peroxidase, and polyphenol oxidase, along with pathogenesis-related proteins that help resist pathogens. At the same time, key metabolic pathways are stimulated, leading to the synthesis of flavonoids, antioxidants, and antifungal compounds that strengthen the plant’s immunity. The combined effect enhances lignification of the cell wall, provides a hypersensitive response to stop pathogen spread, and induces systemic acquired resistance that offers long-term protection. This process equips the plant with a strong primary immune response, making it more resilient against destructive pathogens like Fusarium oxysporum f. sp. cubense TR4^10,26.

In order to develop a cost-effective tissue culture technology for bio-immunized banana plantlets, we designed and developed a novel prototype of the DDTIB system and parameterized bioreactor conditions for commercial production¹³. The complete process of mass multiplication of bio-immunized Grand Naine banana has been visualized in the Figs. 6 and S2, which comprises collection of healthy suckers followed by surface sterilization, bio-immunization, multiplication in bioreactor system, where they undergo rapid shoot multiplication using controlled conditions there after multiplied plants were shifted to innovative primary hardening facilities followed by secondary hardening facilities for acclimatization of the bio immunized DDTIB regenerated plantlets. Finally, secondary hardened DDTIB raised plants were field evaluated. The required timeline for the whole process of mass multiplication of bio-immunized Grand Naine banana has been described in Fig. S2 to cater to the routine operations involved, along with the timeline. There are few temporary immersion bioreactor systems currently available in the market with their own pros and cons for the mass multiplication of TC-generated plantlets. The different types of bioreactors available in the market include RITA, PlantForm, and SETIS¹³. Use of bioreactors in plant propagation was initially reported in 1981 for begonia culture by ^21,27. Further, Tisserat and Vendercook et al.²⁸ designed the first automated plant tissue culture system (APCS) for commercial use. Temporary immersion bioreactors are simple and inexpensive to run²⁹. TIS bioreactors are advantageous as they reduce hyperhydracity, consumption of chemicals, levels of ethylene and CO₂ in vessels, energy and labour requirements¹³, and augment shoot proliferation and plant growth due to higher exposure of explants surface to liquid media³⁰. The use of a temporary immersion system has been reported to improve the shoot multiplication in banana^31,32 and other monocot crops such as sugarcane³³. However, most bioreactors are not designed in such a way that they separate the media tank for refilling, take up less space, and are self-illuminated. RITA® (https://www.cirad.fr, CIRAD, France) bioreactor system, consisting of an autoclavable polypropylene container having two compartments separated by a column supported with a grid and a central plastic pipe, is being used commercially. However, inadequacies of nutrient exchange, limited ventilation, and inappropriate positioning of the central pipe create handling problems while loading culture in the chamber³¹. A major drawback of this system is the risk of media contamination at any stage of growth, which can result in the loss of the entire tissue culture batch. The problem has been properly addressed in the Plantform bioreactor, which is extensively utilized in the TC labs for mass multiplication³⁴. However, this bioreactor could not address the media contamination issue due to its single-unit system¹³. Later, Uma et al.³⁵ developed a TIB based on borosilicate glass, which is quite risky in terms of handling and transport. Moreover,the basement of the reactor is round in shape, which will create a space scarcity in culture rack. If the culture media becomes contaminated at any stage during culturing, there is no way to remove or replace it without disturbing the plantlets. These concerns and drawbacks in existing bioreactor designs must be addressed to ensure faster and healthier plantlet production¹³. Hence, in the current investigation, a prototype was designed and fabricated to address the existing problems of mass multiplication on bio-immunized plants of banana cv. Grand Naine (Fig. 1). The culture parameters, such as immersion time and immersion interval, play a significant role in shoot multiplication inside the bioreactor system, nutrient uptake, gas exchange, and hyperhydricity³². In the current DDTIB, 6 h of immersion frequency with 3 min of immersion time was found better in regeneration of shoots/ clump (22.31) and shoot length of 7.86 (Fig. 2). The experimentally found optimum range, roughly 7 h space and 3 min immersion, hence serves as a compromise between the physiological needs of proliferation and elongation. Overall, these results demonstrate that micropropagation efficiency can be improved by optimizing the liquid culture conditions using RSM (Response Surface Methodology). More experimental verification in the region of the predicted optimal conditions would substantiate these findings and then improve standard protocols. Our findings are in line with the findings of Uma et al.³⁵, who reported that 6 h of immersion frequency was found to be better (shoots /explants were 24.0) for the multiplication of Banana cv. Rasthali (AAB). Results are in accordance with earlier work conducted by Rico et al.³⁶ and Uma et al.³⁷. In the current investigation, itwas found that a six-hour interval with four immersion frequencies per day (12-min immersion per day) promotes 2.5 fold higher shoot multiplication of bio-immunized banana over a semi-solid tissue culture system. However, when the immersion frequency increases to every three hours, shoot multiplication frequency dwindles, leading to hyperhydricity, a condition caused by excessive nutrient absorption, leading to a glossy appearance in the plantlets. Longer immersion periods, even when reduced to twice a day, hamper the regeneration process due to the reduced nutrient supply, directly affecting the growth of the plantlets inside the bioreactor. In the initial three cycles conducted on a solid phase, BA (benzyladenine) concentrations were found to be higher in the Temporary Immersion System (TIS) compared to the Semi-Solid Temporary Culture (SSTC) system. This is attributed to the enhanced absorption capacity in TIS, facilitated by the controlled environment of the bioreactor where gaseous exchange is restricted, resulting in greater biomass accumulation. Morphologically, physically, and at the molecular level, the regenerated plants were true-to-type. Plantlets derived from both the DDTIB and SSTCS performed equally well in terms of vegetative growth and yield. Bioimmunized banana cv. Grand Naine regenerated from the DDTIB system yielded 46.18 tones/ha, while those from the SSTC system yielded 46.78 tones/ha, which shows that yields are at par with the conventional system under Lucknow, India conditions. Genetic fidelity assessments confirmed that both systems produced genetically identical plants.

Further, it was also observed that the fresh weight of DDTIB regenerated bio-immunized Grand Naine plantlets showed higher (1112.24 mg) than the SSTCS (996.36 mg) after the completion of III consecutive subculture cycles (Table 1; Fig. 3). This observation is a clear indication of higher biomass accumulation in DDTIB. Moreover, earlier studies also demonstrated a similar kind of results during the multiplication of banana plantlets through a bioreactor^35–37. Earlier reports are available on the significant effect of light-emitting diode (LED) on the biomass accumulation in SSTCS³⁸. Keeping in mind, the bioreactor top has been fabricated with LED light (4000 lx) for proliferated culture growth inside the bioreactor conditions. The DDTIB self-illuminated system was found to accumulate significantly higher biomass (1133.33 mg) than the non-self-illuminated DDTIB (1025.33 mg) (Fig. 4). Our results (Fig. 2) showed that moderate immersion periods and intermediate immersion intervals were most suitable for promoting shoot multiplication as well as elongation. Brief periods may be insufficient to absorb the necessary nutrients, whereas very long ones would induce stress or oxygen limitation. Similarly, short durations of immersion may fail to elicit sufficient response, while longer durations of immersion may result in tissue damage or oversaturation. The experimentally found optimum range, roughly 6 h space and 3 min immersion, hence serves as a compromise between the physiological needs of proliferation and elongation. Further, DDTIB-regenerated bio-immunized Grand Naine plantlets were shifted for environmental acclimatization. Acclimatization technology¹⁵ was utilized for bio-immunized Grand Naine banana plantlets regenerated through DDTIB to enhance the survival of the plantlets. It was estimated that 98% plants survived in PPE bags. Photosynthetic pigments were also found to be higher in the DDTIB system, which could be attributed to external airflow for the higher accumulation. Hassle-free gas exchange between the inside and outside environment could be the reason for greater photosynthetic activity. According to Aragon et al.³⁹, TIB allowed for increased ethylene, O₂, and CO₂ exchange. Temporary immersion systems also provided an excellent method to use a liquid medium and to control the gas environment at the same time⁴⁰. The gas exchange mechanism allowed DDTIB-regenerated plants to improve their mixo trophic capacity under ex vitro conditions⁴¹.

Conclusion

In conclusion, this newly developed Double Decker Temporary Immersion Bioreactor (DDTIB) was practicable and efficient for the mass production of bioimmunized Grand Naine banana plant lets. Optimum immersion interval for 6 h duration with 3 min of immersion time improved shoot multiplication, biomass accumulation and physiological performance over the conventional Semi-Solid Tissue Culture System (SSTCS). In addition, the self-illuminated DDTIB increased additional plant biomass and were energy efficient. Growth and yield performance of plantlets derived from two systems viz., DDTIB and SSTC were at par. Bio-immunization with antifungal secondary metabolites in tissue culture process can significantly enhance the plant resistance against Fusarium oxysporum f. sp. cubense TR4. Thus, the DDTIB system provides an accurate and affordable strategy for large-scale production of healthy and disease-free banana plantlets.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Maneesh Mishra (MM): Conceptualized the study, collected data, and wrote the initial draft of the manuscript, configure ideological figure and interpreted the data; PrasenjitDebnath (PD): Genetic fidelity study, field evaluation of plantlets,; Anil Kumar Verma (AKV): Designed DDTIB; Muthukumar M:writing and revising the manuscript, configure ideological figure PriyankaYadav (PY): Parameterizing immersion time and interval; Priti Sharma; S. K. Dwivedi (SKD): Physiological data observation; SwostiSubhdarshini Das (SSD): Evaluation of plants during primary and secondary hardening; ShailendraRajan (SR): Editing of manuscript and modeling of data; T. Damodaran (TD): Data analysis, editing of manuscript, ideological figure.

Funding

The funds for the research work was derived from In house project on bioreactor mediated micropropagation of horticultural crops.

Data availability

All the relevant data generated in the study has been reported in the manuscript and the raw data is available with the investigators (Dr. Maneesh Mishra, Principal Scientist, ICAR-CISH, Lucknow).

Declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval

For ethical permission was granted by Institutional Ethics Committee.