In vitro shoot organogenesis in sweet orange (Citrus sinensis L.) cv. Mosambi and the effect of ethylene adsorbents on micro-shoot quality

An improved organogenesis protocol for multiplication of sweet orange cv. Mosambi has been standardized by the use of different PGRs, basal media and ethylene adsorbents. Incorporation of cytokinins, BAP (8.8 µM) and kinetin (6.97 µM) resulted in best shoot organogenesis with the highest response (81.40%), no. of micro-shoots/explant (2.06), mean micro-shoot length (1.30 cm) and no. of leaves/micro-shoot (3.55) owing to complementary effect of these factors. However, the regenerated micro-shoots failed to establish due to 100% leaf abscission of micro-shoot. To retard the effect of ethylene accumulation on the regenerated micro-shoots, two types of ethylene adsorbents carrying silver ions, namely, AgNO3 and Ag2S2O3 and two gelling agents (agar–agar and Phytagel™) were tested at different concentrations. Addition of AgNO3 (5.88 µM) to the medium containing Phytagel™ along with cytokinin (BAP 8.8 µM and kinetin 6.97 µM) led to significant reduction in shoot abscission rate (4.20), while 17.66 µM AgNO3 supplementation improved no. of micro-shoots/explants (2.19) and micro-shoot length (3.36 cm) whereas Ag2S2O3 at 20 µM enhanced the total chlorophyll content (3.47 mg g−1 FW) three times as compared to control. Similarly, among the tested basal media, MS basal medium induced best response on shoot organogenesis. Rooting of micro-shoots was highest (81.12%) with the supplementation of NAA (5.37 µM), which also affected no. of roots/explant (4.52) and mean root length (5.26 cm). The supplementation of ethylene adsorbents during in vitro micro-shoot multiplication significantly improved their quality, which provides ideal rooting for development of complete plantlets in sweet orange cv. Mosambi. Ethylene accumulation in glass containers inhibited micro-shoot establishment of Citrus sinensis L. Use of silver ions enhanced culture establishment and quality of the micro-shoot.


Introduction
Citrus is one of the important fruit crops of the world and rightly, renowned as the "world fruit" for its high socio-economic and nutritional attributes. Among different citrus groups, sweet orange (Citrus sinensis L.) tops the world production with 48.8 million metric tones (FAS 2021). In India, amongst the cultivated sweet orange cultivars, 'Mosambi' has gained momentum in terms of area and production due to its desirable attributes like high sweetness, TSS, vitamin C, minerals, total polyphenols and antioxidant properties.
During the recent years, the demand of planting material having desirable traits such as tolerance to various biotic (virus free planting material) and abiotic stresses (drought, salinity, thermal stress etc.) and particularly the low seeded varieties have touched the new heights. To achieve the said objective within a shorter span of time, plant tissue culture emerged as a boon in the field of plant biotechnology (George et al. 2008). Production of propagules under controlled environmental condition offers an additional advantage for round the year production and supply of planting material as compared to the conventional propagation (Marutani-Hert et al. 2011). Standardization of in vitro regeneration protocol is a basic requirement for an experiment to proceed. The successful standardization of an efficient plant regeneration protocol is genotype-dependent and explants specific. Again, it dependent on the media constituents, such as growth regulators and their concentration, explants preparation methods, different surface sterilization treatments, their duration and environmental condition of the culture room.
Among the different components that contribute for the success of culture establishment, ethylene, a gaseous hormone has a regressive effect. During the various steps of micro-propagation, ethylene produced by different stress treatments (high, low light intensity, wounding) (Fehér et al. 2003) affects the physiological activities of the explant (Lemos and Blake 1996). The effect of ethylene on response to cultured plant cell are diverse depending on plant species, cultivar, explants and the different culture conditions followed (Hu et al. 2006). Ethylene has been shown to have a positive effect on callus induction, organogenesis, culture growth in suspension, root formation, embryogenesis, and secondary metabolite production in vitro. In contrast, ethylene accumulation has been found to inhibit regeneration in several plant species, including inhibition of shoot formation, leaf elongation, hypertrophy, leaf epinasty, senescence, and foliar area reduction (Kumar et al. 1998). The leaf abscission in micro-shoot is the major limiting factor for the successful establishment of plantlets through nodal segments. Our initial observation while working on sweet orange cv. Mosambi showed similar throwback effect which might be a consequence of ethylene accumulation. To overcome ethylene effect, various researchers have attempted its regulation with the use of different adsorbents and inhibitors. Ethylene production in the plants is inhibited by weak antagonists like CO 2 and powerful antagonists like silver compounds. General properties of silver ion carrying compound silver nitrate (AgNO 3 ) with good water solubility, specificity, and stability make it ideal for a variety of applications in plant growth regulation and morphogenesis under in vivo and in vitro. For its faster mobility and less phytotoxicity, silver thiosulfate (Ag 2 S 2 O 3 ) is more effective in tissues than AgNO 3 . The present investigation was therefore aimed to study the counteract effect of ethylene accumulation by using ethylene adsorbent carrying Ag + like AgNO 3 and Ag 2 S 2 O 3 at different concentrations. Interaction effect of the silver compounds was also investigated to establish a reliable protocol for in vitro control of micro-shoot necrosis/leaf fall in sweet orange cv. Mosambi.

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 2020-2022. 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. Before collection of explants, the mother-plants grown in the field were sprayed with fungicides 0.1% Carbendazim (Bavistin BASF, 50% WP, Mumbai, India) mixed with 0.1% monocrotophos 36% SL (Agrolinz, Inc. Bharat Pulverizing Mills Ltd. India) (to control disease/pest infestation) 15 days prior to the collection of explants. 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™ (Rolex-Laboratory-Reagent, Mumbai, India, 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 h. 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 (Changshu Yangyuan chemical analytical reagent, China) (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 (150 × 25 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 (Molychem, Mumabi, India) (4.44, 6.66, 8.88 and 11.1 µM) and kinetin (Sigma Aldrich chemicals, St. Louis, USA) (4.65, 6.97, 9.3 and 11.62 µM) 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 × 25 mm 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:
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-shootquality
This experiment aimed to improve the micro-shoot quality and prevention of shoot fall with the use of two factors, (1) ethylene adsorbents (2)  . 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 weeks) for multiplication. The data regarding the experiment were recorded on the elicited micro-shoot 1 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.

Rooting of micro-shoots and acclimatization of plantlets
In this experiment, rooting of micro-shoots was initiated using two different auxins; NAA (Sigma-Aldrich, St. Louis, USA-2.68, 5.37, 10.74 µM) and IBA (Sigma-Aldrich, St. Louis, 4.92,9.84 µM) in MS medium with and without auxin and activated charcoal (control). Rhizogenesis data was recorded 2 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 halfstrength liquid MS medium basal salts solution and covered with polythene bags (1 week) to maintain the relative humidity. Initially, spraying of water was carried out using 0.1% carbendazim 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 (54 µmol/m 2 /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.

Results
The salient finding of the experiments for mass multiplication of micro-shoot and improvement of micro-shoot quality are represented as below.

Effect of PGRs on culture establishment/shoot organogenesis
Nodal explants inoculated on solid MS medium supplied with different PGRs at different concentrations and their combinations were symbolized as T 1 -T 24 . The optimal regeneration in-terms of direct shoot organogenesis (%), days taken for organogenesis, mean no. of micro-shoot/explant, mean micro-shoot length, mean no. of leaves/explant were documented (Table 1). PGR treatments significantly influenced the morphological characters. The highest shoot organogenesis (81.40%) was noted in treatment combination T 18 (8.8 µM BAP + 6.97 µM kinetin) followed by T 2 (6.6 µM BAP) (66.68%). The lowest shoot organogenesis (26.45%) was recorded in treatment T 5 (11.62 µM kin), which did not differ statistically with treatment T 8 . Among different treatments followed, kinetin at all concentrations showed the poor response with regard to shoot organogenesis.
Shoot productivity and shoot forming index response among different treatments have been depicted (Fig. 1). This figure showed significant response among different treatments on shoot productivity and shoot forming index. The highest shoot productivity (2.70) and shoot forming index (1.68) were registered in T 18 (8.8 µM BAP + 6.97 µM kinetin), while the lowest productivity (0.66) was registered in treatment T 15 (6.6 µM BAP + 9.3 µM kinetin) 1 3 and minimum shoot forming index (0.32) was noted in T 5 (4.65 µM kinetin). However, these micro-shoots failed to maintain their quality due to the 100% leaf abscission thereby affecting shoot proliferation and rooting. As evident, the combinations of BAP and kinetin treatments were found to be superior in comparison with their individual levels (Fig. 2).

Effect of different basal media on shoot organogenesis
A comparative analysis among MS, MT, DKW, WPM and B5 with supplementation of T 18 -8.8 µM BAP + 6.97 µM kinetin (proved best for shoot organogenesis from the first experiment) was conducted to identify the most effective basal medium for shoot organogenesis, micro-shoot growth and proliferation ( Table 2). The highest shoot organogenesis (87.05%), and number of micro-shoot/explant (2.04) were documented for MS in the shortest time (7.6 days). The minimum shoot organogenesis (49.62%) and number of micro-shoots (1.49) were recorded on WPM medium compared to other basal media. The longest (1.60 cm) and the shortest (1.41 cm) micro-shoots were recorded on DKW and B5 medium respectively. The results clearly revealed the superiority of MS basal medium in shoot organogenesis in Mosambi.

Effect of ethylene adsorbents
The results on the effect of ethylene adsorbent and gelling agents on shoot organogenesis, foliar abscission and micro-shoot quality are shown in Table 3. Supplementation of ethylene adsorbents in the form of AgNO 3 and Ag 2 S 2 O 3 exhibited significant effect on micro-shoot development,  control of micro-shoot/leaf abscission and quality of the micro-shoots. Ethylene absorbents and their interaction with gelling agent significantly affected the different parameters, though gelling agent individually had non-significant effect on different parameters except micro-shoot/leaf abscission rate. Among gelling agents, Phytagel™ was most effective for minimizing micro-shoot/leaf abscission (3.51), number of micro-shoot (1.82), and total leaf chlorophyll content (2.61 mg g −1 FW), except micro-shoot length (2.84 cm) which was documented highest on agar-agar. Among the tested ethylene adsorbents, AgNO 3 was noted superior to Ag 2 S 2 O 3 in inducing increased number and length of the regenerated micro-shoots. In contrast, Ag 2 S 2 O 3 controlled leaf abscission and improved the quality of micro-shoots with higher total leaf chlorophyll content. The highest number of micro-shoots (2.14) was noted for treatment 17.66 µM AgNO 3 followed by 5.88 µM AgNO 3 (1.85), which were significantly different compared to other treatments. The mean micro-shoot length was recorded maximum (3.20 cm) for the treatment 17.66 µM AgNO 3 followed by 29.43 µM AgNO 3 (3.04 cm). The control rate of micro-shoot/leaf abscission (3.96) and leaf chlorophyll content (3.40 mg g −1 FW) were recorded highest with 20 µM Ag 2 S 2 O 3 followed by 40 µM (3.93, 3.20 mg g −1 FW, respectively) and was significantly superior over other treatments. Contrary to this, the lowest micro-shoot number (1.40), micro-shoot length (1.43 cm), leaf chlorophyll content (1.17 mg g −1 FW) and highest micro-shoot/leaf abscission rate (1.09) were recorded in control.
Interaction among ethylene adsorbents and gelling agent levels were statistically similar except the highest and the lowest values which differed than others (Table 4). The highest no. of micro-shoot/explant (2.19) was recorded in the treatment combination 17.66 µM AgNO 3 + Phytagel™, whereas micro-shoot length (3.36 cm) was recorded highest in 17.66 µM AgNO 3 + agar-agar. The lowest micro-shoot/  (Fig. 3). The increase in frequency of sub-culture enhanced the regeneration of higher number of micro-shoots from the base of the primary micro-shoot. The result on multiplication in number of micro-shoot varied significantly among each-other. The number of micro-shoots increased to 3.06 in first subculture which was further goes on increasing (7.67) with the increase in number of subcultures i.e., fifth subcultures (Fig. 4).

Effect of auxins on rooting
Micro-shoots obtained from the fifth sub-culture were used for in vitro rooting trials. The micro-shoots cultured on halfstrength MS medium supplemented with different auxins did not give root initiation even after 6 weeks of culture onto rooting media (Data not presented). The observations on rhizogenesis of micro-shoot in full-strength MS medium supplemented with different auxins, however, had significant effects. The highest rooting (81.12%) was observed on MS medium supplemented with 5.37 µM NAA followed by 10.74 µM NAA (72.26%), while in control (without auxin supplementation) the rooting were not observed. The highest no. of roots/micro-shoot (4.52) and mean root length (5.26 cm) were documented for 5.37 µM NAA followed by 9.84 µM IBA for number of micro-shoot (3.82) and 4.92 µM IBA for micro-shoot length (4.70 cm), respectively (Table 5).

Discussion
In vitro approach is the most pre-requisite sophisticated procedure for the generation of disease-free plantlets as culture contamination is still a major concern that frequently leads to the loss of the entire batch of cultures. Therefore, sterilization techniques are employed against potential pathogens by use of various disinfectants that resulted satisfactory performance and also increases the efficacy of culture survival. Among the disinfectant, mercuric chloride has a significant phytotoxic impact and possible antiseptic or disinfecting properties. In essence, it oxidizes the peptide bonds thereby denaturing the microbial protein. The use of mercuric chloride (0.1%) with best survibility was reported in banana (Titov et al. 2006), strawberry (Jan et al. 2013), and sweet orange (Sujata and Syamal 2010). Moreover, ethanol has an inherent antibacterial characteristic that functions by denaturing and coagulating proteins and rupturing their cell walls, therefore killing the microbes.
Plant growth regulators supplemented in the MS medium were successfully stimulate bud break and shoot organogenesis. Different BAP levels in the treatments produced good outcomes, although kinetin supplements in the therapies were shown to be less efficient than other treatments. The reaction on shoot organogenesis, shoot number, and shoot length were improved with the rise in concentration up to an optimal level (BAP 8.8 µM and kinetin 6.97 µM), but additional increase in the concentration resulted in a drop in shoot/leaf number and also shoot length. The effect of BAP in increment in shoot organogenesis may be attributed to the increase in nucleic acid and protein content, thereby leading to enhanced enzymatic activity within the cell and the ultimate increase in cell division, micro-shoot multiplication, micro-shoot length and number of leaves. These proteins and enzymes are capable of quantitative rheostat-like signal transduction, enabling long-lasting, continually changeable signals that are also reliant on hormone concentration (Heyl and Schmulling 2003). The findings of the study are in consonance with those reported by Rattanpal et al (2011) in C. jambhiri andAl-Bahrany (2002) in C. lemon in which they stated that the presence of kinetin and NAA without supplementation of BAP did not show any shoot elongation, thus clearly explaining the role of BAP in inducing and stimulating cell division. Similar variable response of BAP was also reported in different lemon genotypes at varying concentration by Navarro-García et al. (2016b). They also reported deterioration in quality, was the response of toxicity effect due to increase in concentration of BAP than the optimal range, which was also observed in the present study. Our results on number of micro-shoot/explants supports the earlier result of Tallon et al. (2013) who reported that maximum number of buds (2.5-3.0 buds/explant) with BAP concentration varying from 4.4 to 13.2 µM. Cervera et al. (2008) reported the same with 13.2 µM BAP and Almeida et al. (2003) with 4.4 µM for different C. sinensis genotypes. The supremacy of BAP over kinetin might be due to rapid transport and uptake by the explants thereby activating the quiescent cell or meristem to function more efficiently.

Effect of basal media
The majority of micro-propagation papers emphasize the impact of PGR combinations but neglect the value of various basal medium compositions and macro/micro salt concentrations. Although all the reported regeneration protocol on citrus was based on the compositions of MS and MT media, Kotsias and Roussos (2001) proposed the superior response of DKW for micro-propagation of lemon. Full-strength MS medium and DKW were shown to be superior to the other media tested in our study. The varying quantities of minerals, such as micronutrients, vitamins, and amino acids, are primarily responsible for the differential response of various basal medium treatments.
The high ammonia level (20.6 mM), which must have enhanced protein and nucleic acid synthesis and resulted in higher expression of genes involved with ideal regeneration, could be the cause of the greatest shoot organogenesis on MS and the second-highest on B5. Along with the ammonia content, the vitamins thiamine, pyridoxine, and nicotinic acid in the different media MS (0.00037, 0.0029, 0.00406 mM), MT (0.037, 0.059, 004 mM), WPM (0.0037, 0.0029, 0.004 mM), and B5 (0.037, 0.0059, 0.00812 mM) must have made a significant contribution to the improvement of organogenesis. According to the current observation, WPM had the lowest shoot organogenesis, which is consistent with the findings of Navarro- García et al. (2016b), who found that MS was the most successful among the three treatments-MS, DKW, and WPM and that WPM was the least effective. Similarly, the superiority of MS medium compared to WPM for morphogenesis and growth stimulation was earlier reported by Oliveira et al. (2010). In contrast to the effectiveness of WPM over MS basal medium was reported for in vitro organogenesis of Alemow and sour orange (Tallon et al. 2013), which signifies that the different tissue requires distinct nutrients, vitamin and minerals for different responses which could not be supplied alone in MS medium.

Effect of ethylene adsorbents
Among the ethylene adsorbents tested, none of the combinations recorded complete inhibition of micro-shoot/leaf abscission, however Ag 2 S 2 O 3 (STS) was comparably more effective than AgNO 3 since it enhanced the quality of the micro-shoots and raised the chlorophyll content. Ag 2 S 2 O 3 must have neutralized the negative effects of ethylene by reactivating the chlorophyllase gene, which led to an increase in chlorophyll concentration and leaf hue (Veen and Van de Geijn 1978). Similar findings were made by Mahmoud et al. (2020), who also observed that Ag 2 S 2 O 3 was more effective than AgNO 3 in controlling leaf abscission in Australian finger lime. Moreover, the explants reaction is concentration-dependent. The best response of Ag 2 S 2 O 3 on regeneration percentage and no. of buds/explant were noted at 20 µM for lemon genotypes V51 and F49 (Navarro-García et al. 2016a;b). The usefulness of Ag 2 S 2 O 3 in boosting the regeneration percentage was also reported for plum (Petri and Scorza 2010) and gloxinia (Chae et al. 2012). In Annona squamosa L., Lemos and Blake (1996) observed 100% leaves abscission within 4 days of culture onset. Although there is a paucity of literature on the pattern of citrus leaf abscission, the current observation on the effect of silver compounds revealed delayed abscission that occurred 8 days from the start of the culture, and the plants perished within 10 days. AgNO 3 has a stronger inhibitory effect on ethylene action than Ag 2 S 2 O 3 , which might have improved shoot organogenesis, as evidenced by its superior ability to increase the number of micro-shoots. Numerous crop species, including Punica granatum (Naik and Chand 2003), Manihot esculenta (Zhang et al. 2001), Coffea canephora (Sridevi et al. 2010), and Hevea brasiliensis (Sirisom and Te-chato 2012), have shown that AgNO 3 has a positive impact on better plant regeneration. The declining reaction on the number of shoots and length with high concentrations of AgNO 3 may be caused by AgNO 3 toxicity, which increased the ethylene content and had a negative impact on shoot organogenesis. Comparable inhibitory effects of AgNO 3 have been seen in A. annua, and high concentrations (35.82 µM) of this compound cause plant toxicity (Lei et al. 2014). In the current investigation, Phytagel™ outperformed agar-agar in terms of controlling leaf abscission. Although the outcomes differed widely between adsorbents, they were statistically comparable among gelling agents. According to Mahmoud et al. (2020), the gelling agent had no appreciable impact on the regulation of leaf abscission.
The increment in subcultures had a major effect on the multiplication of micro-shoot and also varied significantly. The number of micro-shoots increased four-fold more than the primary subcultures, because the plantlets limit the growth after a certain period due to the depletion of media constituents thereby requiring further subculture. Again the proliferation and multiplication in number of micro-shoots was due to transfer of the plantlets to the fresh medium containing ample nutrients.

Effect of auxin on rooting
The MS medium without any supplementation of auxins recorded the lowest rooting percentage, number of roots and root length, which signifies the importance of auxin in root initiation. In the present study the observed effectiveness of NAA over IBA might be due to the greater stability and persistence of NAA than IBA. Our results contradict the general belief that IBA is a more potent auxin than NAA for rooting as it is transported faster than IBA in the cell (Epstein and Ludwig-Muller 1993). With increase in concentration of NAA the rooting percentage increased which may be due to the fact of increasing the endogenous auxin concentration at the rooting primordia thus inducing signal for root initiation. Similar result of NAA on rooting was also reported by Rashad et al (2005). Hence, it is convincing to say that 5.4 µM NAA is the optimum concentration for induction of roots for micro-shoot derived from nodal segments (Nwe et al. 2014). According to Duran Villa et al. (1989), the ideal concentration of NAA needed for root development in sweet oranges is 50.4 µM. Further, the depressing effect on rhizogenesis with increasing auxin concentration is in consonance with Al-Bahrany 2002 in Citrus aurantifolia (Christm.) Swing.

Correlation and regression analysis
The Fig. 5 depicts the correlation and regression analysis among the micro-shoot/leaf abscission control rate with no. of micro-shoot/explant, mean micro-shoot length and total chlorophyll content by use of ethylene adsorbents. The correlation study indicated a statistically strong significant correlation among the parameters undertaken. A significant positive correlation was observed between leaf abscission control rate with number of micro-shoot/explant (0.775), mean micro-shoot length (0.964) and total chlorophyll content (0.809) (Fig. 5A, B, C). The regression analysis between leaf abscission rate and other organogenesis parameters portrayed the highest R 2 value for the micro-shoot length (0.930) followed by chlorophyll content (0.654) and the lowest R 2 value was recorded for number of micro-shoot/explant (0.601). As evident in the regression equation, all were documented with positive equations. The value of the coefficient of x-variables for number of micro-shoot (0.162), micro-shoot length (0.560) and total chlorophyll content (0.183) were noted which signifies the value of y changes with an increase in 0.162, 0.560 and 0.183 of x-variable in number of micro-shoot, mean micro-shoot length and chlorophyll content, respectively.

Conclusion
The current study comes to the conclusion that the standardization of a protocol for the mass multiplication of sweet oranges while simultaneously enhancing the quality of the regenerated plants can be accomplished with the use of various PGRs, basal media, and ethylene absorbents (AgNO 3 and Ag 2 S 2 O 3 ). Inoculation of nodal segments in the medium containing the BAP and kinetin resulted in patronizing the effect on shoot organogenesis than the individual effects. Among the tested basal medium, MS medium comeback with effect of maximizing shoot organogenesis as compared to the other mediums. Both the ethylene absorbents contributed in preventing microshoot abscission at similar level but Ag 2 S 2 O 3 has a superior effect than AgNO 3 in controlling micro-shoot abscission and enhancing chlorophyll content of the leaves whereas AgNO 3 proved best for increasing number of micro-shoot/explants and micro-shoot length. In the context of rooting, NAA proved best in increasing the rooting efficiency of micro-shoots. The standardization of this protocol adds value to the existing protocol and to carry out the experiment in developing the plantlets controlling abscission. These results can further be tested for different genotypes to manage the leaf abscission and also establishment of a reliable protocol.