Optimization of Hormonal Compositions of Media in In Vitro Propagation of Orange Cultivars from Shoot Tip Nodal Segments

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Introduction
Te Citrus can be propagated by conventional methods like budding and grafting. Terefore, there is a possibility of virus transmission from the mother plant to the propagated plant. Despite substantial genetic diversity and interspecifc fertility, the genus Citrus includes some of the most difcult species to breed [1]. Tis is often due to several obstacles to conventional breeding including large plant size, self-and cross-incompatibility and pollen and/or ovule sterility, extended juvenility, and especially to nucellar embryony, and high polyembryony since sweet oranges generally contain one to several adventive nucellar embryos, and most species are highly heterozygous and produce progeny that segregates widely for several characters when crosses are made. Genetic transformation is an alternative to overcome these difculties. For a successful transformation, regeneration of whole plants from the transformed cells may be a prerequisite [2].
Sweet orange (Citrus sinensis (L.) Osbeck) is the most grown Citrus species [3]. Zygotic sweet orange hybrids are difcult to obtain, are often weak, and do not produce fruit that resembles sweet orange. It is generally accepted that commonly grown sweet orange cultivars probably originated from the selection of a chance seedling well-adapted to a particular area or a mutation in a particular cultivar or seedling [2,4]. Mutations visible as bud or limb sport or sectors on chimeric fruits occur frequently in citrus [5].
A great number of viruses and other graft transmissible infectious agents were known to afect and endanger the citrus industry worldwide [6]. Obtaining pathogen-free citrus plants is one of the most important steps in the citrus breeding program. Numerous methods are developed to recover virus-free plants. Shoot-tip grafting is the most efective technique for the elimination of all major viruses and virus-like pathogens, including those not eliminated by thermotherapy. Plants obtained by shoot tip grafting are true-to-type and do not have juvenile characters. Tus, these plants might be used for budwood production after they are indexed [7].
Sustainable development of the citrus industry is especially dependent on the continuous supply of new and improved cultivars. Te genetic improvements of perennial woody fruit crop plants often take a few years using traditional plant-breeding methods [8]. Plant tissue culture made it easy to enhance citrus against diferent abiotic stresses, low yield, and conserve important citrus genotypes through exploiting somaclonal variations, vegetative cell hybridization [9,10], and the transformation of highyielding cultivars and disease-free plants [11].
Micropropagation of citrus ofers rapid propagation of fruit crops in limited space and time under controlled conditions around the year [12]. Multiple shoot induction and regeneration are potentially useful for the genetic improvement of fruit crops [7]. It is known that the tissues obtained from young plant parts have relatively more regenerative capacity than old tissues. Te basis for bud formation in tissue culture is dependent on a specifc equilibrium between the auxins and the cytokinins, gibberellins, and cytokinins ratio, which controls the shoot and leaf development [8,13]. Sweet orange and mandarin are polyembryonic, whereas lemon is monoembryonic. Tese species are generally cross-pollinated. Tey are highly heterozygous, and zygotic embryos, whether or not produced by selfng, would difer from the maternal parents. Te plant regenerated from seeds usually exhibits prolonged juvenility in citrus fruits. Although sweet orange widely grows, some introduced cultivars are being found in limited farm areas in Ethiopia. Tere are no well-established citrus breeding programs and seed production found in the country despite huge consumption and market demand. Terefore, this study was undertaken to investigate the optimization of growth regulators' composition of media in in vitro propagation of orange cultivar from nodal segment explants.

Sample Collection.
Te nodal segment explants samples were collected from three orange cultivars including Washington Naval, Valencia, and Tangelo from Tony Farm in Dire Dewa City, Ethiopia. Te nodal segments close to the shoot tip were cut with scissors at about 30 cm. Ten, the explant samples were brought to Plant Biotechnology Lab, in the School of Plant Sciences, Haramaya University.

Media Preparation.
Te MS Murashiege and Skoog [14] medium was manually prepared as per the standard procedure. Te pH of the media was adjusted to 5.5-5.7 using 0.1 N HCl and 0.1 N NaOH, and fnally, 0.8% (w/v) agar-agar was added as the gelling agent. Ten, the agar was allowed to dissolve with the medium in microwave. Forty (40) ml of molten MS media was dispensed into each fask plugged with aluminum foil and autoclaved at 121°C and 15 lb/in 2 pressure for 15-20 min in 150 ml Erlenmeyer fasks. Te sterilized medium was kept for 1-2 days before inoculations to screen for inherent contamination. Te vitamins and hormone supplements were flter sterilized and poured onto the prepared MS media.

Explant Preparation and Surface Sterilization.
Te shoot tip nodal segments of adult orange cultivars were used as explants sources. Following the removal of leaves with sterile scissors, the nodal segments (15−20 cm in length) were washed with distilled water in 1000 ml beakers for 5 min. Ten, the explants were successively surface sterilized in 75% (v/v) alcohol (30 sec), 0.1% (w/v) mercuric chloride plus 2 drops of Tween-20 per 100 ml disinfectant solution (10 min) in a laminar hood. To remove all traces of detergents and Tween-20 from the surface, explants were rinsed in sterile double distilled water 3-4 times. Te exposed cut ends of explants were trimmed of with sharp secateurs to eliminate all toxic efects of mercuric chloride. All experiments were conducted in two replications.

Shoot Initiation and Proliferation.
Te culture media for in vitro shoot initiation consisted of the basic salts and vitamins of Murashige and Skoog (MS) culture medium at full strength (basal medium) supplemented with kinetin at 1.50, 2.00, and 2.50 mg/L, IAA at 1.00 and 2.00 mg/L. Te media was also supplemented with 3.5 percent (w/v) sucrose (as carbon and energy source) and bactoagar at 0.8 percent (w/ v) as a solidifcation medium. Two g/L ascorbic acids were used to minimize explant browning by adsorbing phenolics, inactivating polyphenol oxidases, and peroxidases. About 15 cm nodal segment explants were inoculated into a culture jar containing 100 ml MS growth medium with three explants per jar. Te cultured jars were plugged with nonabsorbent cotton and placed in a growth chamber room at 26 ± 2°C and under fuorescent light receiving 16 hrs illumination followed by 8 hrs dark period. A similar procedure was used for shoot proliferation with half-strength MS medium. Data were recorded for shoot response, the average number of shoots/explants, the average length of proliferated shoots/explants, average and number of leaves/ explants after about three weeks.

Rooting of Regenerated Shoots.
Te basic salts and vitamins of MS at half strength were used for rooting media. During the growing period, healthy regenerated shoots were excised and transferred individually under aseptic conditions and cultured vertically in glass jars (9 × 4.5 cm) each containing 30 ml basal medium supplemented with 0.30, 0.50, 1.00, and 1.50 mg/L 1-naphthalene acetic acid (NAA), and 0.30, 0.50, 1.00, and 1.50 mg/L indole-3-butyric acid (IBA) were added solely or in combinations, amended with sucrose at 30 g/L and 7 g/L bactoagar. Te rooting media pH was adjusted to 5.8 before the addition of agar. Te cultured jars were capped with aluminum foil and autoclaved at 121°C for 20 min, then left to cool, and harden for fve days before being used. Te cultured jars were incubated at 25 ± 1°C and exposed to photoperiod high light intensity for 16 hrs (1500 lux) and 8 hrs darkness.
Young shoots, when subjected to root initiating treatment, invariably callused at the base, and over some time callus mass increased and root initials were barely visible. A two-step strategy for in vitro rooting was adopted. In the frst step, microshoots were implanted in basal MS medium supplemented with various IBA concentrations. Maximum root initiation and least callusing were observed on fullstrength MS medium supplemented with 14.70 μM IBA. Exposure to root initiation medium was not allowed for more than 11 d since microshoots were overwhelmed by callus mass, and root development was arrested. Indeed, root development was apparent in course of as brief as 5 days of exposure of microshoots to the rooting medium. In the second step, immediately after root initiation, the microshoots were reimplanted on a PGR-free half-strength MS medium for root development. Te root formation was maximal when organics were supplied at full strength. Data were recorded for rooting rate, root number per plantlet, and root length per plantlet after 5 weeks of culturing.

Data Analysis.
Te data in Tables 1-4 (supplementary materials fle) about shoot regeneration and rooting were subjected to a one-way analysis of variance (ANOVA), and the diferences among means were compared based on the least signifcant diference (LSD) t-test using SAS software version 9.2.

Optimization of MS Medium for Shoot Proliferation of
Orange Cultivars. Te efects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin on shoot proliferation rate, shoot number per explants, shoot length, and leaf and leaf number are indicated in Table 1 and Figure 1. Signifcantly, the highest shoot response was recorded for Washington naval orange with maximum shoot proliferation rate (99.75%), shoot number per explant (3.10), shoot length (10.70 cm), and leaf number per explants (12.50) after three weeks of culture. Te last shoot responses were recorded for the Valencia cultivar with shoot proliferation rate (68.75%), shoot number per explant (1.85), shoot length (6.85 cm), and leaf number per explant (7.90).
Te basal MS mediums without any supplements were used as a control. In all experiments, no growth was observed for the basal MS medium. Phytohormones combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Te shoot responses were found to be increasing with an increase in kinetin concentration with IAA concentration at 1.20 mg/L. Al-Teha et al. [15] suggested that somatic embryogenesis and plantlet regeneration were achieved in callus cultures of nucellus tissues derived from undeveloped ovules of immature fruits of local orange C. sinensis using half-strength MS medium supplemented with BAP and 2,4-D. Te highest (70%) shoot formation was obtained from BARI Malta-1 (Citrus sinensis) seeds without seed coat treated with MS basal media + BAP 1.0 mg/L while kinetin showed no response for shoot formation in any supplemented concentration.

Optimization of MS Medium for Root Initiation of Orange
Cultivars. Te root inductions of microshoots generated for the three sweet orange cultivars were conducted by supplementing basal MS medium with phytohormones including IAA and NAA as in Tables 2 and 3. Root induction was observed after 15 days of culturing. Signifcant rooting rate, root number, and root length were observed for all treatments. Among the cultivars, signifcantly, the highest rooting rate (81.255), root number (4.95), and root length (2.95 cm) were recorded for Washington naval orange cultivar while the least rooting rate (48.45%), root number (3.55), and root length (2.26 cm) were observed for Valencia cultivar. As the concentration of IAA within the medium increases, the root responses including root initiation rate, root number, and root length also increase with maximum rooting observed at 1.5 mg/L. No root growth was observed in the control medium (MS basal medium).
Several factors have been observed to be associated with the rooting of the microshoot that includes the nature of cuttings, rooting cofactor, the synergistic role of exogenously applied growth hormone and endogenously present cofactors in the rooting, the relative efciency of diferent auxins, their combination, and methods of application [16][17][18][19]. High light intensity also induces better rooting and causes hardening of the plant which renders them more tolerant to moisture stress and diseases. A low salt medium is found satisfactory for rooting of shoots in a large number of plant species [20]. Te addition of activated charcoal in the root expression medium improved the overall rooting capacity in Pinus panaster [21].
Te efect of MS medium supplemented with NAA on root response rate, root number, and root length of microshoot generated from nodal segment explants of three sweet orange cultivars is presented in Table 3. Te microshoots from Washington naval orange cultivar have demonstrated signifcantly the highest rooting rate (84.90%), root number per microshoot (5.20), and root length (3.05 cm) for MS medium supplemented with 1.5 mg/L NAA. On the other hand, the least root response in terms of rooting rate (58.95%), root number per microshoot (4.86), and root length (2.68 cm) was recorded for the Valencia cultivar.
It was observed from the result of the present study that root response was increased as the concentration of the growth regulator increased from 0.3 to 1.5 mg/L. Te comparison of diferent concentrations of IAA and NAA on root induction of microshoots from nodal segments of sweet orange cultivars as in Figure 2 and Table 5 (in Supplementary material) demonstrated signifcantly higher efects of NAA than IAA. Signifcant diferences were observed between rooting rates due to NAA and IAA for all three

15.55b
Means followed by the same letter within a column were not signifcantly diferent at a 0.05 probability level based on the least signifcance diference (LSD) test. Small letters: signifcance within a column. 4 International Journal of Agronomy tested cultivars. NAA has presented a signifcantly higher rooting rate (84.90%) than the rooting rate due to IAA (81.25%) at the maximum concentration of the growth regulators. However, for other parameters including root number and root length, no signifcant diferences were observed between both phytohormones.  Means followed by the same letter within a column were not signifcantly diferent at a 0.05 probability level based on the least signifcance diference (LSD) test. Small letters: signifcance within a column.

Conclusion
In all experiments, no growth was observed for the basal MS medium. Phytohormones combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Te shoot responses were found to be increasing with an increase in kinetin concentration with IAA concentration at 1.20 mg/L. As the concentration of IAA within the medium increases, the root responses including root initiation rate, root number, and root length also increase with maximum rooting observed at 1.5 mg/L. No root growth was observed in the control medium (MS basal medium). It was observed from the result of the present study that root response was increased as the concentration Root response due to IAA or NAA Figure 2: Comparison of the efects of MS medium supplemented with IAA and NAA on rooting of microshoots from nodal segment explants of three orange cultivars. Where RRI: rooting rate due to IAA supplement; RRN: rooting rate due to NAA supplement; RNA: root number due to NAA supplement; RNA: rooting number due to NAA supplement; RLI: rooting length due to IAA supplement; RLA: root length due to NAA supplement.
of the growth regulator increased from 0.3 to 1.5 mg/L. Te comparison of diferent concentrations of IAA and NAA on root induction of microshoots from nodal segments of sweet orange cultivars demonstrated NAA as a more efective hormone than IAA.

Data Availability
Te data used to support the fndings of this study are included within the supplementary material fle.

Conflicts of Interest
Te authors declare that they have no conficts of interest.