Plant material and explant preparation
The experiment was carried out at Central Tissue Culture Laboratory, ICAR-National Institute of Plant Biotechnology, ICAR-IARI, New Delhi during 2020 to 2022. The explants (nodal stem segments, 10-15 cm) were collected from the mother plant of sweet orange cv. Mosambi, maintained at the Experimental Fruit Orchard of Division of Fruits and Horticultural Technology, ICAR-IARI, New Delhi. The collected explants were transported to laboratory in an icebox and immediately washed under running tap water followed by washing in distilled water with the addition of 1-2 drops of Tween-20™ (surfactants) to remove the contaminants. The washed nodal segments were trimmed from both the ends and leaves were excised keeping a short petiole intact. The nodal stem segments were treated with 0.1% carbendazim (Bavsitin BASF, India) + 0.1% metalaxyl + mancozeb (Ridomil Gold®, Syngenta, India) suspension and kept on a horizontal shaker (60 rpm) for 1 hour. The nodal segments were then shifted to the laminar and subjected to surface sterilization with freshly prepared sterile 0.1% mercuric chloride solution for 10 min. Thereafter, a quick 70% ethanol dip (10 s) was followed by three rinsing with sterile double-distilled water for 5 min. The surface sterilized explants were trimmed with a sterile scalpel and cut to 3-5 cm segments having one or two nodes and then used for in vitro culture into the test-tubes (150x25 mm) containing different medium treatment combinations.
Effect of BAP and kinetin on culture establishment/shoot organogenesis
This set of first experiment comprised of two cytokinins, i.e., BAP (6-benzylaminopurine) (4.44, 6.66, 8.88 and 11.1µM) and kinetin (N6-furfuryladenine) (4.65, 6.97, 9.3 and 11.62µM) each at four different concentrations. The combinations were supplemented with basal MS medium (Murashige and Skoog 1962) (Hi-Media, Mumbai PT021).The nodal explants were then transferred onto the glass test tubes of 150×25mm containing 15 ml of MS medium supplemented with different levels of growth regulators. After 4 weeks of culture, data regarding the response of nodal segments to different growth regulators and their combinations (shoot organogenesis (%), days taken for organogenesis, No. of micro-shoots/explant, mean micro-shoot length (cm), mean No. of leaves per micro-shoot, productivity and shoot forming index) were recorded using the following formula:
Effect of basal medium on shoot organogenesis
In the second experiment, 5 different basal media, i.e., MS (Murashige and Skoog 1962), MT (Murashige and Tucker 1969), DKW (Driver and Kuniyuki 1984), WPM (Lloyd and Mc Cown 1980) and B5 (Gamborg et al. 1968) were fortified with BAP 11.1µM and kinetin 9.3µM, 5% sucrose, 200 mg L-1 activated charcoal (Qualigens, Mumbai) and 0.7% agar-agar (Hi media PCT0412) to study the comeback effect of different medium on nodal explant proliferation, shoot organogenesis and the data were recorded after 4 weeks of culture.
Effect of ethylene adsorbents and gelling agents on micro-shoot quality
This experiment aimed to improve the micro-shoot quality and prevention of shoot fall with the use of two factors, (i) ethylene adsorbents (ii) gelling agents. Different ethylene adsorbents (AgNO3- 5.88, 17.66 and 29.43µM) and (Ag2S2O3- 20, 40 and 60 µM) were supplemented at different concentrations to MS medium and without any ethylene adsorbent solidified with agar-agar and Phytagel™ (Sigma-Aldrich, St. Louis, USA) as control. These ethylene adsorbents were added in the MS medium supplemented (BAP 11.1µM and kinetin 9.3µM) individually with agar-agar (0.7%) and Phytagel™ (0.25%). For preparing 10mM STS (Ag2S2O3), 100mM stock solution of Ag2NO3 was added to 100mM Ag2S2O3 (1:4 M) (Navarro-García et al. 2016a). The stock solutions of ethylene adsorbents were added using sterile micro-filter (0.22 µm; Sartorius, USA). The gelling agents were used as a factor as they affect the physical properties (e.g., water potential) of the media. The micro-shoots obtained from the experiment were used for sub-cultures (4 week) for multiplication. The data regarding the experiment were recorded on the elicited micro-shoot one month after the initiation of culture. Total leaf chlorophyll contents were estimated using DMSO (Dimethyl sulfoxide, AR grade) as per the method suggested by Hiscox and Israelstom (1979). The absorbance was read at 645 and 663 nm wavelengths and total chlorophyll were calculated by using the following formula and the value was expressed as mg g-1 of fresh weight of leaves.
Micro-shoot/leaf abscission control rate was rated as 1 = all leaves dropped, 2 = ≥ 70% of leaves dropped, 3 = 30–70% of leaves dropped, 4 = ≤ 30% of leaves dropped, 5 = no leaves dropped (Marutani-Hert et al. 2011).
Rooting of micro-shoots and acclimatization of plantlets
In this experiment, rooting of micro-shoots was initiated using two different auxins; NAA (2.68, 5.37, 10.74 µM) and IBA (2.46, 4.92, 9.84 µM) in MS medium with and without auxin and activated charcoal (control). Rhizogenesis data was recorded two months after root initiation. After the initiation of roots, micro-shoots were transferred from the rooting media to auxin-free basal media for the elongation of roots.
After 5 weeks, the rooted plantlets were washed with sterile double-distilled water to remove the agar and transferred to the glass jars filled with autoclaved potting medium (coco peat: vermiculite and perlite in 1:1:1) moistened with half-strength liquid MS medium basal salts solution and covered with polythene bags (one week) to maintain the relative humidity. Initially, spraying of water was carried out using 0.1% carbendazim (Bavsitin BASF, India) to prevent fungal infection and thereafter with half-strength sterilized liquid MS medium to prevent mortality of the in vitro raised plantlets.
Media preparation and culture maintenance
All the media used in the experiments were supplied with 5% sucrose (w/v) except for rooting the sucrose was supplemented at 3% (w/v). The pH of the culture media in all the four experiments was adjusted to 5.7±0.5 with the addition of 1N NaOH before the addition of gelling media and after that, the medium was sterilized at 121°C for 20 min. Each treatment comprised of 20-25 test tubes. All the cultures were maintained in the controlled culture room (24±2°C). The culture maintenance room was programmed to maintain a 16/ 8 h light/ dark cycle using cool white fluorescent lights (54µmol/m2/s).
Experimental design and statistical analysis
The statistical analysis of the first two and fourth experiment comprising of different treatment combination and four replications were analyzed in a completely randomized design (CRD) using statistical analysis system software, SAS package (9.3 SAS Institute, Inc. USA), followed by a t-test (LSD). The third experiment with two factors, i.e., ethylene adsorbents and gelling agents was analyzed in two factors CRD. P-values ≤0.05 were considered significant. Regression analysis was done to analyze trends and relationships between leaf abscission rates with other growth parameters using ethylene adsorbents. The treatment means were computed by Pearson’s simple correlation.