A hydroponic-based efficient hardening protocol for in vitro raised commercial kiwifruit (Actinidia deliciosa)

In Vitro Cellular & Developmental Biology - Plant https://doi.org/10.1007/s11627-020-10127-3 PROTOCOLS/METHODS A hydroponic-based efficient hardening protocol for in vitro raised commercial kiwifruit (Actinidia deliciosa) Sumit Purohit 1 & Janhvi Mihra Rawat 2 & Vivek Kumar Pathak 3 & Dinesh Kumar Singh 1 & Balwant Rawat 4 Received: 17 June 2020 / Accepted: 15 September 2020 / Editor: Prakash Lakshmanan # The Society for In Vitro Biology 2020 Abstract Actinidia deliciosa is a commercially important plant receiving recognition because of its high nutritive value. This study presents an efficient protocol for the hardening of in vitro raised Actinidia deliciosa plants using a hydroponic method. Leaf explants inoculated on Murashige and Skoog (MS) medium supplemented with 5.00 μM 6-benzylaminopurine (BAP) and 1.00 μM αnaphthaleneacetic acid (NAA) resulted in 5.28 ± 0.14 shoots per explants and 8.33 ± 1.80 cm average shoot length. Rooting was achieved through half-strength MS medium supplemented with 1.5 μM indolebutyric acid (IBA) and 0.6 μM BAP. Maximum root number (12.76 ± 1.08) and 6.78 ± 0.25 cm average root length were recorded from plantlets using half-strength MS medium supplemented with 1.5 μM indolebutyric acid (IBA) and 0.6 μM BAP. In hydroponic system, an average root length of 22.40 ± 0.59 cm, average root number of 21.50 ± 1.24, average leaf number of 4.50 ± 0.40, and average shoot length of 9.71 ± 0.29 cm were observed in Hoagland & Arnon solution. The early development of shoots, roots, and leaves through hydroponics was advantageous in establishment of micropropagated plants in a greenhouse. Complete 100% plant survival was found by following proper acclimatization using hydroponic method. The study underlines the efficient hardening of micropropagated plants of A. deliciosa through hydroponic technique in Himalayan region. Keywords Kiwifruit . Himalayan region . Hydroponic technique . Hardening protocol . Shoot regeneration Kiwifruit (Actinidia deliciosa), native to eastern Asia, is a deciduous fruit plant with several health benefits. This species made its way to become a commercial crop in the twentieth century because of its high nutritive and economic values (Ward and Courtney 2013). Kiwifruit is native to southwest China and is also known as goose berry. However, the name kiwifruit came into existence and became more familiar after its introduction in New Zealand and, to date, there are 52 known species having different ploidy levels. The fruit is an excellent source of vitamin C (720 mg 100 g−1 fresh weight) * Balwant Rawat balwantkam@gmail.com 1 Uttarakhand Council for Biotechnology, Biotech Bhavan, P.O. Haldi-263146, Pantnagar, District-U. S. Nagar, Uttarakhand, India 2 Botany Division, Forest Research Institute, Dehradun, Uttarakhand 248006, India 3 G. B. Pant University of Agriculture and Technology, Pantnagar, District- U. S. Nagar, Uttarakhand 263145, India 4 School of Agriculture Sciences, Graphic Era Hill University, Dehradun, Uttarakhand 248002, India and minerals and also enriched with high antioxidant capacity (Selman 1983; Wang et al. 1996; Ferguson and Ferguson 2003; Richardson et al. 2018). Due to the higher nutraceutical properties, kiwifruit is used for the preparation of many food products (Rugini and Gutierrez-Pesce 2003). The kiwifruit industry has become important in the last decade with female plant cultivar ‘Hayward’ being the most cultivated variety worldwide (80 to 95%) because of its fruit quality and good taste (Ward and Courtney 2013). A. deliciosa, considering crop value in terms of marketable gross production, is the sixth most valuable crop after citrus, apples, table grapes, peaches/nectarines, and pears. The production of A. deliciosa is approximately 1.8 million tons per year. However, the total fresh fruit production of kiwifruit is only 0.2 to 0.3% compared with the total fresh fruit production internationally (Guroo et al. 2017). Production of kiwifruit is restricted to a few countries, including China, Italy, and New Zealand which are cultivating it on large scale. In India, a few states including Himachal Pradesh, Sikkim, Tamil Nadu, Jammu and Kashmir, and Uttarakhand are the minor producer of kiwifruit. Although the fruit is highly nutritious, its cultivation has not spread throughout the world because of weather PUROHIT ET AL. conditions and lower awareness of importance of the fruit among farmers. In Uttarakhand, it is cultivated in the temperate region and surprisingly grows well with good productivity. Kiwifruits can be propagated by seeds as well as by vegetative propagation. The vegetative propagation methods (cuttings and grafts) have been used over the ages as an effective traditional method (Gjeloshi et al. 2014). Over the past five y, there is an increasing demand of the fruit and to meet the requirement in vitro micropropagation is required for the large-scale multiplication. In vitro micropropagation of A. deliciosa was established by Harada (1975), and since then, a range of in vitro propagation studies have been carried out (Velayandom et al. 1985; Rey et al. 1992; González et al. 1995; Rugini and Gutierrez-Pesce 2003; Akbaş et al. 2008). In addition, there are many other varieties of fruit which are being raised using the same method (Wang et al. 1982; Standardi 1983; Wessels et al. 1984; Monette 1986; Victoria et al. 1995). Hardening plays a key role in commercialization of any tissue culture–derived plantlets. Plants from long-term tissue culture do not survive during acclimatization due to weak root system, weak stomata, and poorly developed cuticle resulting in substantial numbers of in vitro raised plants that are unable to survive at the time of acclimatization (Mishra et al. 2006). To overcome this problem, a new technique, hydroponic cultivation system, has been introduced in a few plant species (Conn et al. 2013; Nguyen et al. 2016). Hydroponics is a very unique and advance technique for the growth of plants in water without any requirement of soil. Plants growing in hydroponic system require oxygen to be delivered to the roots, in addition to the water and nutrients supplied in growth solutions (Resh 2012; Nguyen et al. 2016). Without constant aeration, a hydroponics system will become anaerobic and inhibit the growth of most plants. Low availability of oxygen negatively affects the transport of metabolites to the plant body requiring a continuous and essential supply of oxygen (Gibbs et al. 1998; Nguyen et al. 2016). Hydroponics can be divided into two basic types depending on the method of aeration employed: (1) flood-drain systems and (2) continuous aeration. Both systems are routinely used in our research, and in the following sections, we will describe each system in more detail. The various types of hydroponic systems, such as drip and drain system, static system, nutrient flow system, and many other systems, are available subjected to the commercial supply (Zobel et al. 1976) . The quality of plantlets in hydroponic system is better in terms of shoot and root growth (Conn et al. 2013) as compared with the traditional way of hardening because the nutrient requirement of the plant is easily met. Hydroponic experiments have been proven unique to test plant phenotypes and responses to different nutrient availability (Pii et al. 2015), improved growth activities (Conn et al. 2013) and mass production of plant materials (Windsor and Schwarz 1990; Berezin et al. 2012; Conn et al. 2013; Alatorre-Cobos et al. 2014). Table 1. Effect of different concentrations of 2,4 D and TDZ on callus culture developed from the leaf explants of Actinidia deliciosa var. deliciosa ‘Hayward’ Serial number Plant growth regulator concentrations (μM) Callus formation 2,4-D TDZ 1 2 3 4 5 6 7 8 9 10 11 0.1 0.1 0.3 0.3 0.5 0.5 0.7 0.7 0.9 0.9 1.0 0.5 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5 Dry Dry Dry Compact yellow Compact greenish yellow Compact greenish Fragile yellow Yellow Dry Dry Dry 12 13 1.0 0.0 0.1 0.0 Dry Dry 2,4-D, 2,4-dichlorophenoxyacetic acid); TDZ, Thidiazuron The aeration and the nutrient system help in root respiration which in turn develops the overall quality of plantlets. This water-based technique makes it easy for the roots of plants in absorbing all the essential nutrients from water. Other than nutrients, roots also need continuous supply of oxygen. It is also recommended that to get optimal results in hydroponic technique, nutrient media should be saturated with air prior to its use. Apart from a few studies on hydroponically hardened Table 2. Effect of different concentrations and combinations of NAA and BAP on shoot regeneration from the callus culture of Actinidia deliciosa var. deliciosa ‘Hayward’ Serial number Plant growth Number of shoots Shoot length (cm) regulators (μM) NAA BAP 1 2 3 4 5 6 7 8 0.0 0.5 1 1.5 2 0.5 1 1.5 0.0 3.0 3.0 3.0 3.0 5.0 5.0 5.0 1.96 2.15 2.05 2.52 2.54 2.23 5.28 3.43 9 2 5.0 2.25 ± 0.19c ± ± ± ± ± ± ± ± 0.12c 0.7c 0.20c 0.30c 0.19c 0.21c 0.14a 0.20b 3.03 3.91 6.51 4.68 2.89 3.91 8.33 3.09 ± ± ± ± ± ± ± ± 0.19c 0.11c 0.64b 0.09bc 0.06c 0.11c 1.80a 0.05c 3.31 ± 0.10c Values are mean ± standard error. Mean values followed by the same letter(s) in a column are not significantly different (P < 0.05). NAA, αnaphthaleneacetic acid; BAP, 6-benzylaminopurine A HYDROPONIC BASED EFFICIENT HARDENING PROTOCOL plant species, use of hydroponics in in vitro raised plants to improve acclimatization and survival is still limited. Therefore, the present study was undertaken to develop a reliable and efficient hardening protocol to improve the survival percentage and better production of in vitro propagated plants of A. deliciosa using hydroponic technique. Mature (10-yr-old), field-grown, female kiwifruit (‘Hayward’) plant material was collected from ICARNBPGR Regional station Bhowali, Niglat District Nainital in Uttarakhand, India (1600 m asl; 29° 20′ 16.0″ N, 79° 30′ 57″ E). For callus culture, fresh leaf explants were taken from a mother plant of A. deliciosa (Fig. 1a) and sterilized for five min using Bavistin solution (0.1%, w/v; a systemic fungicide; BASF, Mumbai, India) with 1% Tween-20 (HiMedia, Mumbai, India, v/v) and mercuric chloride (0.1%, w/v; HiMedia; Mumbai, India). Finally, all the treated explants were washed five times with sterile distilled water under sterile conditions. The leaves were excised into 1 × 1 cm pieces (length × width) using a surgical blade, holes punched in explants using a sterilized stainless steel needle of size 0.55 × 25 mm (DISPO VAN, Hindustan Syringes & Medical Devices LTD, Ballabgarh, Faridabad, India) before being placed on full-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962; Rawat et al. 2013) supplemented with 3% sucrose, 0.2% (v/v) antifungal supplement, 0 to 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0 to 0.5 μM thidiazuron (TDZ) and solidified with 0.8% agar for callus induction (Table 1 and Table 2). The pH of the medium was adjusted to 5.8, and approximately 20 mL medium was dispensed in individual jam bottles and autoclaved (1.05 kg cm−2 at 121°C for 20 min). These explants were cultured on these media combinations maintained in a culture room at 25 ± 1°C, with a 16-h light and 8-h dark cycle, with low irradiance (42 mol μm−2 s−1; inside jam bottle) by cool white LED tube (Philips; 20 W). After four to six wk, explants were subcultured on fresh MS media with same PGR combinations (Rawat et al. 2013). After 60 d culture, there was notable appearance of callus of different shapes and colors arising from the explants (Table 1). Further, green color calluses (Fig. 1b-c) were transferred to MS medium supplemented with NAA (0.5 to 2.0 μM) and BAP (3.0 to 5.0 μM) and, after two mo incubation, multiple shoot buds were observed (Fig. 1d) which eventually formed shoots (Table 2). These shoots, when four to five cm long, were subcultured (Fig. 1e) and placed on rooting media consisting of ½ MS medium containing 0.0 to 1.5 μM IBA and 0.0 to 0.9 μM BAP (Table 3; Fig. 1f). Further, well rooted healthy regenerated plants (Fig. 1g) were taken out from ½ strength MS medium (Fig. 1h) for hardening and acclimatization. In the control treatment, the in vitro rooted plantlets were transplanted in a potting media of sand: soil: FYM (Farm Yard Manure) in ratio 2:1:1 filled to seedling tray, covered with polythene sheet a b c d e f g h i Figure 1. In vitro propagation of Actinidia deliciosa var. deliciosa ‘Hayward’ (kiwi) using leaf explants. (a) Mature kiwi trees in natural habitat, (b) in vitro callus culture, (c) in vitro sprouting in leaf explants, (d) leaf regeneration on Murashige and Skoog (MS) medium supplemented with BAP (6-benzylaminopurine) 5.00 μM and NAA (αnaphthaleneacetic acid) 1.00 μM, (e, f) multiple shoots development on MS media, (g, h) root induction on in vitro raised shoot, (i) in vitro raised plant growing in plastic root trainer cell. PUROHIT ET AL. a b c d e f Figure 2. Hydroponic system. (a) Representative scheme of the hydroponic system used for the acclimatization in tower-based hydroponic system, (b) a readymade 20 planter hydroponic system consisting of five floors, (c) storage tank at the bottom fitted with water pump, (d, e) movement of water through central pipe to top floor facilitating downward water movement, ( f ) hydroponic setup with plants. (Fig. 1i), and kept in greenhouse conditions with humidity of 30 to 40% for 14 d prior to transfer to a nursery. After 14 d, plants were transferred to polythene bags containing holes and were irrigated every four d. Polythene bags were removed after a period of 42 d to transfer the hardened plants into earthen pots containing garden soil. Prior to shifting in open sunlight, the earthen pots were kept under shade in a mist chamber for 35 d. Considering the increasing demand of hydroponic technique in commercial as well as research field, a variety of different hydroponic systems are available. Readymade twelve hydroponic vertical grow towers (Poorna Agri System, Noida, India) were used to grow plants in the present study which were kept in a hi-tech polyhouse (Fig. 2a). Each tower is a five-floor setup consisting of four residential units or holes on the sides of each floor that house each plant (Fig. 2b). In total, one grow tower houses 20 plants at a time. The height of the grow tower may vary according to the requirement of the study. The system illustrated is constructed with a lower tank for storage and pumping 10 L of growth solution and upper grow tower (Fig. 2c). The storage tank is fitted with a pump (18 W) at the bottom for regular mixing of hydroponic nutrients by repeatedly pumping the solution into the growing vessel and allowing it to drain and return into the storage tank (Fig. 2a). A central pipe passing through all the floors takes the nutrients to the uppermost floor consisting of holes which facilitate the supply of nutrients downwards (Fig. 2d–f). Continuous movement of solution during the pumping/drainage cycles facilitates delivery of oxygen to the roots. The volume of growth solution required and the length of the drainage/pumping cycle will depend upon the size and design of the setup. Each 3 ft. 15 cm vertical grow tower used in this study allows the movement of 3-L solution through all the floors and storage of 7-L solution in the storage tank at a time. A HYDROPONIC BASED EFFICIENT HARDENING PROTOCOL a b c d e f Figure 3. In vitro raised Actinidia deliciosa var. deliciosa ‘Hayward’ (kiwi) plant transfer to hydroponic system. (a) Taking plant out of in vitro medium with sterile forceps, (b) cleaning the plants to remove agar and Table 3. Effect of different concentrations of IBA and BAP on Actinidia deliciosa var. deliciosa ‘Hayward’ root development Serial number 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. treat with bavistin solution; (c) place the plants into foam incision, (d) insert the foam plug into the net pot, (e) place the net pot in hydroponic system, ( f ) a completed hydroponic set up with plants. Plant growth regulator concentrations (μM) IBA BAP 0.5 1 1.5 0.5 1 1.5 0.5 1 1.5 0.0 0.3 0.3 0.3 0.6 0.6 0.6 0.9 0.9 0.9 0.0 Number of Root Root length (cm) 0.73 ± 0.06c 3.97 ± 0.09b 3.37 ± 0.08b 4.03 ± 0.15b 4.14 ± 0.09b 12.76 ± 1.08a 3.11 ± .011b 3.09 ± 0.13b 2.41 ± 0.6b 0.00 ± 0.00c 0.86 ± 0.07e 4.03 ± 0.13bc 4.33 ± 0.21b 4.17 ± 0.14bc 3.19 ± 0.02cd 6.78 ± 0.25a 3.22 ± 0.23cd 2.69 ± 0.10d 3.20 ± 0.09cd 0.00 ± .0.00 e Values are mean ± standard error. Mean values followed by the same letter(s) in a column are not significantly different (P < 0.05). IBA, indolebutyric acid; BAP, 6-benzylaminopurine PUROHIT ET AL. Table 4. Different nutrient solutions and their concentration in hydroponic system Nutrient Hewitt (1966) mg L−1 Hoagland & Arnon (1950) mg L−1 Cooper (1975) mg L−1 Steiner (1984) mg L−1 NH4NO3 KH2PO4 KNO3 CaCl2.2H2O MgSO4 Iron EDTA CuSO4 ZnSO4 MnCl2 H3Po3 168.00 41.00 156.00 160.00 36.00 2.80 0.064 0.065 0.54 0.54 210.00 31.00 234.00 160.00 34.00 2.50 0.02 0.05 0.50 0.50 200.00 60.00 300.00 170.00 50.00 12.00 0.10 0.10 2.00 0.30 168.00 31.00 273.00 180.00 48.00 24.00 0.02 0.11 0.62 0.44 Na2MoO3 0.04 0.01 0.20 0.01 Plants have a specific nutritional requirement which varies according to its biochemical processes. Successful growth of kiwifruit has been observed using the nutrient solutions shown in Table 4. Water content of salts used for preparation of nutrients may vary with the supplier that does not affect the properties of the nutrient solution (Nguyen et al. 2016). Different types of self-prepared hydroponic solutions as described by Hoagland and Arnon 1950, Hewitt 1966, Cooper, 1975, and Steiner 1984 were used for acclimatization (Table 4). The macroelements used for preparation of solution include nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S), while the microelements (trace elements) include manganese (Mn), zinc (Zn), boron (B), copper (Cu), molybdenum (Mo), and iron (Fe). Stock solution A including macronutrients and stock solution B including micronutrients were prepared in sterile bottles. In the case of stock B, IronEDTA was added last while mixing the solution. 20× stock solution for macronutrients (A) and 200× stock solution for micronutrients (B) were prepared prior to experiment initiation and autoclaved and store at 4°C. Stock solutions A and B were brought to room temperature before use. Both A and B of stock solutions were prepared in double distilled water. The micro-propagated plants were gently removed from the culture medium inside the flask using sterile forceps Table 5. (Fig. 3a). Prior to hardening in hydroponics system, agar and other dead tissues were removed from the well rooted plants (Fig. 3b). All the plants were dipped into Bavistin solution (0.1 mg 100 mL−1) for 1 h and washed with tap water. Each plant along with roots was laid along the incision of each foam plug (Fig. 3c) and individually inserted into each net pot and placed into hydroponic system so that the roots come in contact with the nutrient solution (Fig. 3d–f). Apart from the internal factors such as genetics, external factors including environment and nutrients determine the basic quality of plants. The excess use of nutrients in a hydroponic system lowers the quality of plant material; therefore, the optimum use of the nutrients is suggested for the better growth of plants (Abou-Hadid et al. 1996). The pH of the hydroponic solution was adjusted to 5.8 ± 1 using 1 N H2SO4. Electrical conductivity (EC) was set on 2 as EC level between 1.5 and 2.5 dS m−1 has been found optimal for growth of plant under hydroponic system (Abou-Hadid et al. 1996; Ding et al. 2018). Due to evaporation of water as well as the absorption of nutrients by plants, fluctuation/reduction in pH and EC levels are well understood. Every 15 d, nutrient solution in each tower was replaced by fresh solution to avoid any salt imbalance. To maintain optimum pH (5.8 ± 1), drops of 1 N H2SO4 were added to the solution while pH and EC were Comparison of growth performances of plants in hydroponic system with different nutrient solutions and control Serial number Nutrient solution Average root length (cm) Average root number Average leaf number 1 2 3 4 5 Hewitt Hoagland & Arnon Cooper Steiner Control 12.70 ± 0.93b 22.40 ± 0.59a 10.33 ± 0.44d 13.18 ± 0.27c 7.34 ± 0.30e 8.30 ± 0.95c 21.50 ± 1.24a 15.60 ± 0.99b 7.30 ± 0.94c 6.90 ± 0.77c 3.10 4.50 3.20 3.50 2.80 ± ± ± ± ± 0.38b 0.40a 0.33b 0.17b 0.25b Average shoot length (cm) 3.97 9.71 7.18 3.99 2.84 ± ± ± ± ± 0.17c 0.29a 0.26b 0.26c 0.22d Values are mean ± standard error. Mean values followed by the same letter(s) in a column are not significantly different (P < 0.05); all growth parameters were recorded after 30 d of transfer from in vitro system A HYDROPONIC BASED EFFICIENT HARDENING PROTOCOL a b c d e f Figure 4. In vitro raised Actinidia deliciosa var. deliciosa ‘Hayward’ (kiwi) plant acclimatized in hydroponic system. (a) Acclimatization of micropropagated A. deliciosa plant under aerated hydroponic system, (b) hydroponically raised plant in net pot, (c) well grown kiwi plants with developed root and shoot system, (d) transfer of hydroponically grown plant in sand:soil:FYM mixture, (e, f) acclimatization of hydroponic raised plants in greenhouse system for further growth. regularly checked with a HANNA Groline pH/EC/TDS meter (Hanna Euipments Pvt. Ltd. Kharghar, Navi Mumbai, Maharashtra, India). After hardening in hydroponic system for 30 d, the plantlets were transferred to earthen pot containing potting mixture of sand:soil:FYM (2:1:2) and kept in mist chamber for 15 d prior to shifting in open sunlight. For the present hardening investigation using hydroponic technique, each treatment was repeated three times with 60 explants (n = 20 in each replicate). Analysis of variance using SPSS version 7.5 was used to analyze the data obtained from various treatments. The level of significance was determined (P ˂ 0.05), and Duncan’s multiple range test (DMRT) was used to separate mean when values were significantly different. The data is presented as mean value ± standard error (SE). Leaf explants of A. deliciosa when placed on MS medium supplemented with 2,4-D gave differing responses in terms of callus culture (Fig. 1b). Optimal green compact calluses developed from leaf segments cultured two mo on MS medium supplemented with 0.5 μM 2,4 D and 0.1 μM TDZ (Table 1). Greenish compact calluses produced multiple shoots two mo after transfer (Fig. 1c) to MS media containing 1.0 μM NAA and 5.0 μM BAP (Fig. 1d). Results showed that the concentration of PGRs in MS medium also affected the shoot proliferation. The average length and number of the shoots produced were recorded after 30 d of culture (Fig. 1e-f Table 2). The shoot segments under aseptic conditions responded well by initially multiplying within the first week of culture, followed by multiple shoot induction within 3 wk. MS media supplemented with 5.0 μM BAP and 1.0 μM NAA (Table 2) was found to be the best combination in terms of shoot length (8.33 cm) and number of shoots (5.28). BAP at 5.0 μM in combination with 2.0 μM NAA (Table 2) in the media negatively affected the growth and multiplication of the plants. Root initiation was observed after 25 d on IBA and BAP medium (Fig. 1g). On average, 12.76 roots and 6.78-cm root length were observed from regenerated shoots after 6 wk on PUROHIT ET AL. Figure 5. Summary of suggested protocol showing various steps of regeneration and acclimatization of Actinidia deliciosa var. deliciosa ‘Hayward’ (kiwi) fruits plants; 2,4-D, (2,4dichlorophenoxyacetic acid); MS (Murashige and Skoog1962); BAP (6-benzylaminopurine); NAA (α-naphthaleneacetic acid); IBA (Indolebutyric acid); TDZ (thidiazuron). Leaf explants from in vitro callus culture In vitro callus culture formation (2,4 -D 0.5 µM and TDZ 0.1 µM) after two mo Shoot regeneration on MS media BAP (5.00 µM) and NAA (1.00 µM) after two mo In vitro rooting (IBA 1.5 µM and 0.6 µM BAP) six wk Acclimatization Traditional method Rooted plantlets washed in running tap water sterile coco peat in net pot covered with transparent plastic sheet greenhouse 14 d plant transfer in polythene bags Sand:Soil:FYM (2:1:2) green mist house 25° ± 3°C; 50% shade 42 d plant transfer in earthen pots Sand:Soil:FYM (2:1:2) green mist house 35 d medium containing 1.5 μM IBA and 0.6 μM BAP (Table 3; Fig. 1h). All well rooted in vitro raised plants, after taken out of the medium and washed with water, were individually inserted into foam pieces and plugged into the net pot and eventually transferred to hydroponic system as described earlier (Fig. 3). Growth performance of hydroponically raised plants is shown in (Fig. 4a-f), and hardening of plants from control and hydroponic system treatments was compared after 30 d (Table 5). The quality of shoots, leaves, and roots from A. deliciosa plantlets from the hydroponic system treatments were better as compared with traditional (control) method (Fig. 4b). The best results in terms of average root length (22.40 cm), average root number (21.50), average leaf number (4.50), and average shoot length (9.71 cm) were observed in Hoagland solution (Hoagland and Arnon 1950) (Table 5, Fig. 4c). Since the shoots and roots were well developed in hydroponics system, subsequent growth of A. deliciosa plants grown in sand:soil:FYM mixture was faster as compared with plantlets from the established control (Fig. 4e-f). Minimum growth on the basis of abovementioned parameters, including the average root length (7.34), average root number (6.90 cm), average leaf number (2.80), and average shoot length (2.84 cm), was recorded in control plants with 60% survival after 30 d, which showed slow growth during the first 20 d, but improved after the next 10 d. After a complete hardening period of 90 days, the survival of control plantlets was 50% while plants from the hydroponic system reached 100% plant survival after 30 d. Plant survival remained at 100% after a complete hardening period of 45 d as well as after 90 d when plants were grown in open sunlight (Fig. 4f). The present study Hydroponic method Rooted plantlets washed in running tap water dipped in bavistin (0.1 mL in 100 mL) for 8-12 h hydroponic system contain nutrient adjusted pH 5.8 ± 1 green mist house 25°C ± 3°C; 50% shade plant acclimatization 30 d plant transfer in earthen pots Sand:Soil:FYM (2:1:2) green mist house 15 d demonstrated the efficient hardening protocol for in vitro raised plants using a hydroponic technique (Fig. 5). Leaf explants of A. deliciosa were used to initiate callus by culture on MS medium containing 0.5 μM 2,4-D and 0.1 μM TDZ which produced green compact calluses that developed multiple shoots. MS media supplemented with BAP (5.0 μM) and NAA (1.0 μM; Table 2) were found to be the best combination in terms of shoot length (8.33 cm) and number of shoots (5.28). Similar results were recorded on cv. ‘Hayward’ kiwifruit, where BAP showed optimal results in regeneration of shoots from callus (Revilla et al. 1992; Moncaleon et al. 1999, Purohit et al. 2016, 2017). In another study, González et al. (1995) reported the combination of IAA (0.1 μM) and zeatin (4.5 μM) with 2% sucrose was suitable for the callus induction. In addition, Marino and Bertazza (1990) reported that the presence of BAP in medium gave superior results in terms of shoot proliferation and node production in A. deliciosa cultivars ‘Hayward’ and ‘Tomuri’ followed by the enhanced callus growth on zeatin-enriched media. The present study advocates the use of inexpensive and reliable readymade hydroponic setups which are effective in the mass production of plant materials raised through in vitro tissue culture propagation. The overall shoot and root development from hydroponics was superior as compared with traditionally used hardening methods due to the continuous supply of nutrients and aeration (Fig. 2e-f). Further, transplanting the hydroponically grown plants in potting mixture of sand:soil:FYM (2:1:2) showed better growth when compared with green mist chamber conditions over a period of 15 d and there was no mortality which was exceptionally better than traditional method (50% plant survival followed by overall A HYDROPONIC BASED EFFICIENT HARDENING PROTOCOL less growth). Improved growth and physiological activities of Arabidopsis thaliana have been recorded using hydroponic system (Conn et al. 2013). Sufficient plant materials in lettuce, tomato, turmeric and tobacco have been generated using hydroponic systems (Zapata et al. 2003; Berezin et al. 2012; Conn et al. 2013; Alatorre-Cobos et al. 2014). Reduced hardening period by 45 d with 100% plant survival is a new observation in this study, especially when compared with the control treatment where only 50% plant survival was observed. This new application will boost commercialization of tissue culture plants, as the growth of the plantlet is enhanced and the hardening time period and cost can be reduced considerably. The results of present study indicate a striking relationship in the percentage of acclimatization and root system development suggesting the applicability of the hydroponic technique. This is the first successful report of ex vitro acclimatization of tissue culture–raised plantlets using hydroponic techniques. The established protocol can be used for in vitro propagation and acclimatization of A. deliciosa and potentially other species using this hydroponic method. Further, efficient production of elite planting material which in turn is required for promotion of cultivation in hilly areas of Uttarakhand can be achieved through this protocol. Acknowledgments The authors are thankful to the Director of Uttarakhand Council for Biotechnology, Haldi, and Vice Chancellor, Graphic Era Hill University, Dehradun, for the institutional facilities. Efforts of Ms. Babeeta Singh (JRF), Mr. Lalit Mishra, and other lab workers are highly acknowledged. Society for the conservation of Nature, Anantpur, MP, is thanked for help during study design and analysis. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. References Abou-Hadid AF, Abd-Elmoniem EM, El-Shinawy MZ, Abou-Elsoud M (1996) Electrical conductivity effect on growth and mineral composition of lettuce plants in hydroponic system. Acta Hortic 434, 59:– 66. https://doi.org/10.17660/actahortic.1996.434.6 Akbaş F, Işikalan C, Namli S (2008) Callus induction and plant regeneration from different explants of Actinidia deliciosa. Appl Biochem Biotechnol 158:470–475. https://doi.org/10.1007/s12010-0088401-2 Alatorre-Cobos F, Calderón-Vázquez C, Ibarra-Laclett E, YongVillalobos L, Pérez-Torres C-A, Oropeza-Aburto A, MéndezBravo A, González-Morales SI, Gutiérrez-Alanís D, ChacónLópez A, Peña-Ocaña B-A, Herrera-Estrella L (2014) An improved, low-cost, hydroponic system for growing Arabidopsis and other plant species under aseptic conditions. BMC Plant Biol 14:69. https://doi.org/10.1186/1471-2229-14-69 Berezin I, Elazar M, Gaash R, Avramov-Mor M, Shaul O (2012) The use of hydroponic growth systems to study the root and shoot ionome of Arabidopsis thaliana, hydroponics - a standard methodology for plant biological researches. In: Asao T (ed) Hydroponics - a standard methodology for plant biological researches. InTech publishers, Karnal Conn SJ, Hocking B, Dayod1 M, Xu B, Athman A, Henderson S, Aukett L, Conn V, Shearer MK, Fuentes S, Tyerman SD, Gilliham M (2013) Protocol: optimising hydroponic growth systems for nutritional and physiological analysis of Arabidopsis thaliana and other plants. Plant Method 9:1–11. https://doi.org/10.1186/1746-4811-94 Cooper AJ (1975) Crop production in recirculating nutrient solution. Sci Hortic 3:251–258. https://doi.org/10.1016/0304-4238(75)90008-4 Ding X, Jiang Y, Zhao H, Guo D, He L, Liu F, Zhou Q, Nandwani D, Hui D, Yu J (2018) Electrical conductivity of nutrient solution influenced photosynthesis, quality, and antioxidant enzyme activity of pakchoi (Brassica campestris L. ssp. Chinensis) in a hydroponic system. PLoS ONE 13(8):e0202090. https://doi.org/10.1371/ journal.pone.0202090 Ferguson AR, Ferguson LR (2003) Are kiwifruit really good for you? Acta Hort 610:131–138 Gibbs J, Turner DW, Armstrong W, Darwent MJ, Greenway H (1998) Response to oxygen deficiency in primary maize roots. I. Development of oxygen deficiency in the stele reduces radial solute transport to the xylem. Funct Plant Biol 25:745–758 Gjeloshi G, Thomai T, Vrapi H (2014) Influence of supporter substrate on the rooting percentage of kiwifruit cuttings (Actinide Delicious cv. Hayward). Int Ref J Eng Sci 12:10–14 González MV, Rey M, Rodríguez R (1995) Plant regeneration from petioles of kiwifruit microshoots. HortSci 30:1302–1303 Guroo I, Wani SA, Wani SM, Ahmad M, Mir SA, Masoodi FA (2017) A review of production and processing of kiwifruit. J Food Process Technol 8:1–6. https://doi.org/10.4172/2157-7110.1000699 Harada H (1975) In vitro organ culture of Actinidia chinensis as a technique for vegetative multiplication. J Hort Sci 50:81–83 ISSN 00221589 Hewitt EJ (1966) Sand and water culture methods used in study of plant nutrition, 2nd edn. England, England, Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, p 549 Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil, University of California, College of Agriculture, Agricultural Experiment Station, Circular 347, Berkeley, USA Marino G, Bertazza G (1990) Micropropagation of Actinidia deliciosa cvs “Hayward” and “Tomuri.” Sci Hortic 45:65–74. https://doi.org/ 10.1016/0304-4238(90)90069-Q, Micropropagation of Actinidia deliciosa cvs. ‘Hayward’ and ‘Tomuri’ Mishra J, Nandi SK, Palni LMS (2006) Assessment of physiological, biochemical and genetic fidelity with a brief review on tissue culture of tea. Int J Tea Sci 5(1):61–80 Moncaleon P, Cañal MJ, Feito I, Rodriguez A, Fernandez B (1999) Cytokinins and mineral nutrition in Actinidia deliciosa (kiwi) shoots cultured In vitro. J Plant Physiol 155:606–612 Monette PL (1986) Micropropagation of kiwifruit using non-axenic shoot tips. Plant Cell Tiss Org Cult 6:73–82 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays of tobacco tissue cultures. Plant Physiol 15:473–497 Nguyen NT, McInturf SA, Mendoza-Cózatl DG (2016) Hydroponics: a versatile system to study nutrient allocation and plant responses to nutrient availability and exposure to toxic elements. J Vis Exp 113: e54317. https://doi.org/10.3791/54317 Pii Y, Cesco S, Mimmo T (2015) Shoot ionome to predict the synergism and antagonism between nutrients as affected by substrate and physiological status. Plant Physiol Biochem 94:48–56 Purohit S, Jugran AK, Bhatt ID, Palni LMS, Bhatt A, Nandi SK (2016) In vitro approaches for conservation and reducing juvenility of Zanthoxylum armatum DC: an endangered medicinal plant of PUROHIT ET AL. Himalayan region. Trees 31:1101–1108. https://doi.org/10.1007/ s00468-016-1494-2 Purohit S, Nandi SK, Palni PS, Mohd T, Palni LMS (2017) Micropropagation and genetic fidelity analysis in Amomum subulatum Roxb.: a commercially important Himalayan plant. J Appl Res Med Aromat Plants 4:21–26 Rawat JM, Rawat B, Agnihotri RK, Chandra A, Nautiyal S (2013) In vitro propagation, genetic and secondary metabolite analysis of Aconitum violaceum Jacq.: a threatened medicinal herb. Acta Physiol Plant 35:2589–2599. https://doi.org/10.1007/s11738-0131294-x) Resh HM (2012) Hydroponic food production: a definitive guidebook for the advanced home gardener and the commercial hydroponic grower, seventh edition (7th edition). CRC Press, p 560 Revilla MA, Rey MA, Gonzalez-Rio F, Gonzalez MV, Diaz-Sala C, Rodriguez R (1992) Micropropagation of kiwi (Actinidia spp). In: YPS B (ed) High-Tech and Micropropagation II. Biotechnology in Agriculture and Forestry, vol 18. Springer, Berlin, Heidelberg, pp 399–423 Rey M, Fernández T, González V, Rodriguez R (1992) Kiwifruit micropropagation through callus shoot-bud induction. In Vitro Cell Dev Biol-Plant 28:148–152 Richardson DP, Ansell J, Drummond LN (2018) The nutritional and health attributes of kiwifruit: a review. Eur J Nutr 57:2659–2676. https://doi.org/10.1007/s00394-018-1627-z Rugini E, Gutierrez-Pesce P (2003) Micropropagation of kiwifruit (Actinidia ssp). In: Jain SM, Ishii K (eds) Micropropagation of Woody trees and fruits. Kluwer Academic Publishers, Dordrecht, pp 647–669 Selman JD (1983) The vitamin C content of some kiwifruits (Actinidia chinensis planch., variety Hayward). Food Chem 11:63–75 Standardi A (1983) La “micropropagazione” nella moltiplicazione dell’actinidia. Frutticoltura 45:17–22 Steiner AA (1984) The universal nutrient solution, Proceedings of IWOSC 1984 6th International Congress on Soilless Culture, ISSN 9070976048. Wageningen, the Netherlands, pp 633-650 Velayandom L, Hirsch AM, Fortune D (1985) Tissue culture of nodal stem segments of Actinidia chinensis (L.) Planchon, as a method of micropropagation. C R Acad Sci III, Serie III. Sciences de la Vie (Life Sciences) 301:598–600 Victoria MG, Rey M, Rodríguez R (1995) Plant regeneration from petioles of kiwifruit microshoots. HortSci 30:1302–1303 Wang H, Cao GH, Prior RL (1996) Total antioxidant capacity of fruits. J Agr Food Chem 44:701–705 Wang JX, Li SZ, Li BW, Ren EY (1982) Propagation of Actinidia chinensis by tissue culture. Liaoning Agric Sci 1:32–34 Ward C, Courtney D (2013) Kiwifruit: taking its place in global food bowl. Adv Food Nutr Res 68:1–14. https://doi.org/10.1016/B9780-12-394294-4.00001-8 Wessels E, Nel DD, Von Staden DFA (1984) In vitro propagation of Actinidia chinensis cultivar Hayward. Deciduous Fruit Grower 34: 453–457 Windsor G, Schwarz M (1990) Soilless culture for horticultural crop production. FAO, plant production and protection. Paper 101, Roma, p 202 Zapata EV, Morales GS, Lauzardo ANH, Bonfil BM, Tapia GT, Sánchez ADJ, Valle MVD, Aparicio AJ (2003) In vitro regeneration and acclimatization of plants of turmeric (Curcuma longa L.) in a hydroponic system. Biotechnol Appl 20:25–31 Zobel RW, Del Tredici P, Torrey JG (1976) Method for growing plants aeroponically. Plant Physiol 57:344–346