Introduction
The Citrus genus, along with related genera such as Fortunella, Poncirus, Eremocitrus, and Microcitrus, is part of the Aurantioideae subfamily within the Rutaceae family These plants are cultivated extensively across tropical and temperate regions, with Southeastern Asia—particularly southern China, northeastern India, and Malaysia—recognized as the primary center of origin for citrus species Secondary centers include the Mediterranean and Caribbean regions Citrus fruits are economically significant, grown in over 146 countries, and are highly sought after for their vitamins and nutritional value Key commercially important fruits in this genus include mandarin, sweet orange, grapefruit, lemon, and lime.
In 2016, global citrus fruit production was estimated at 124.2 million tons (FAOSTAT, 2016), but this output has been significantly impacted by both biotic and abiotic diseases Notably, citrus tristeza and citrus greening disease, also known as Huanglongbing (HLB), are two of the most destructive biotic threats facing citrus growers (Warghane et al., 2017; Ayazpour et al., 2011; Lin et al.).
In 2018, it was noted that pathogens can easily spread to healthy trees through methods such as grafting and pruning, as well as by specific insect vectors One significant threat is Tristeza disease, caused by the Citrus tristeza virus (CTV), which is a phloem-limited, single-stranded RNA virus that adversely impacts citrus production.
Citrus production faces significant challenges due to various diseases, notably Citrus Tristeza Virus (CTV) and Huanglongbing (HLB) CTV has affected over 100 million citrus trees across North and South America and the Mediterranean, leading to severe quality issues even in resistant rootstocks (Moreno et al., 2008; Donkersley et al., 2018) HLB, caused by the phloem-limited bacterium Candidatus liberibacter, has devastated around 60 million trees in tropical and subtropical regions, severely impacting the Asian citrus industry (Ghosh et al., 2015) Additionally, the Satsuma dwarf virus (SDV) poses further threats to citrus health and productivity.
Satsuma dwarf disease gives negative effect on the cultivation of satsuma mandarins (C unshiu Marc.) and sweet oranges in many areas of China, Japan and Turkey (Iwanami et al., 2001)
In Vietnam, citrus fruits, including oranges, mandarins, and pummelos, are a vital agricultural crop, with an estimated cultivation area of 112.6 thousand hectares and a total production of 948.1 thousand tons (Statistical summary book of Vietnam, 2017) However, citrus orchards face significant threats from diseases like CTV, HLB, and root rot For instance, in Chau Thanh district of Hau Giang province, over 4,929 hectares of 'King' mandarin are affected, with approximately 2,715 hectares showing symptoms of "yellow shoot" and tree mortality (Hoahoi, 2015) Similarly, in Dong Thap province, more than 2,000 hectares (25% of the citrus cultivation area) are impacted by varying levels of citrus diseases (Vietnamese agriculture, 2019) The widespread use of low-quality or uncertified seedlings has exacerbated these issues, as they often carry CTV and HLB While fungicides can help manage diseases like citrus canker and root rot, they cannot eliminate viruses, viroids, or HLB from infected trees.
To ensure healthy citrus production, it is essential to introduce virus- and viroid-free plants at specific intervals However, the lack of expertise, facilities, and equipment has made it challenging to implement effective protocols for producing pathogen-free seedlings in the region Consequently, identifying a suitable technique for generating pathogen-free citrus seedlings is crucial for enhancing citrus cultivation.
The production of disease-free plants from polyembryonic or apomictic Citrus cultivars can be achieved using nucellar seedlings derived from virus-infected trees; however, this method is time-consuming, resulting in thorny seedlings that exhibit morphological fruit variations (Singh et al., 2018) Grafting mature shoots of woody perennials onto juvenile rootstocks has been shown to induce juvenile traits in the grafted shoots (George et al., 2008) In the case of Citrus, micrografting does not promote juvenility in adult scions, leading to the development of thornless plants that quickly reach the flowering stage when grafted onto juvenile rootstocks (Navarro, 1988).
In vitro shoot tip grafting or micrografting is a technique firstly carried out by Murashige et al (1972) and developed by Navarro et al (1975) According to Navarro
Since 1988, micrografting has been utilized for several key purposes: obtaining virus-free plants, isolating virus and virus-like organisms in mixed infections, examining graft compatibility between scions and rootstocks, and facilitating the importation of plant material under quarantine Additionally, in vitro micrografting proves to be an effective method for generating plants from irradiated and transgenic shoots, as well as for producing stable tetraploid plants from non-apomictic genotypes Notably, it has been reported that shoot tip grafting can successfully eliminate virus and virus-like pathogens.
Micrografting has been effectively utilized in citrus and avocado to eliminate viruses and virus-like pathogens, often in combination with thermotherapy or chemotherapy The size of the shoot apex is crucial for successful grafting; smaller apices are preferred for virus elimination However, this technique can yield unreliable results due to poor contact between the rootstock and scion, necessitating proper training for effective application While larger shoot tips may increase graft success rates, they also enhance the potential for pathogen spread Therefore, determining the optimal shoot apex size for successful micrografting and virus eradication is essential Virus elimination is influenced by the concentration of viruses in plant tissue and the viability of shoot tips, with specific virus-host combinations affecting virus distribution Research indicates that an optimal scion size of 0.2-0.3 mm, corresponding to a meristem with three leaf primordia, can effectively eliminate CTV through in vitro micrografting without the need for thermotherapy In trials, micrografting small shoot tips (0.3 mm) onto Troyer citrange rootstock seedlings achieved a CTV elimination rate of 42%.
Rootstock is an important factor of production systems for many tree fruits The usage of superior rootstock can determine the success of citrus fruit production In
Rootstocks play a crucial role in citrus cultivation by preventing biotic diseases such as Phytophthora root rot and citrus tristeza virus, while also providing tolerance to abiotic stresses like freezing, drought, flooding, and high salinity (Albrecht et al., 2019) The success of micrografting, whether in vitro or in vivo, is significantly influenced by the choice of rootstock genotypes, as different rootstocks exhibit varying responses with scions (Sanabam et al., 2015; Chand et al., 2016) Additionally, the age of rootstocks affects micrografting success; older rootstocks tend to have reduced grafting success due to increased cytodifferentiation and lignification, along with diminished wound-healing capabilities (Sanjaya et al., 2006) Research by Takahara et al (1986) demonstrated that using 12- to 14-day-old trifoliate orange seedlings as rootstocks for micrografting various Citrus cultivars resulted in virus-free plants, especially when employing thermotherapy on the scions.
Special techniques like the application of plant hormones, etiolation treatments, antioxidants, and high sucrose concentrations have been utilized to enhance the success rate of micrografting (Hussain et al., 2014) Plant growth regulators play a crucial role in all aspects of plant growth and development, influencing the timing and formation of vascular bridges between the stock and scion (Lu et al., 1996) Numerous studies have explored the effects of various plant growth regulators to improve micrografting success, highlighting the significant relationship and interactions between plant hormones (Farsi et al., 2018).
Soaking of the cut ends of rootstocks into the low concentration of IAA or IBA (10 -
Research indicates that treating grafted cucumber seedlings with a 20 ppm solution for a few seconds can enhance growth parameters (Balliu and Sallaku, 2017) In the initial grafting stages, a rise in endogenous indole-3-acetic acid levels in both the scion and rootstock is crucial for establishing a vascular connection (Zheng et al., 2010) Conversely, Kose and Guleryuz (2006) found that kinetin and benzyladenine (BA) promote rapid callus proliferation between the scion and rootstock, while NAA and IBA enhance root development when the cut surfaces of four different grapevine varieties are treated with these solutions.
Micrografting, traditionally performed in vitro, requires acclimatization of grafted plants to their natural environment Recent findings indicate that aseptic conditions may not be essential, allowing shoot apices to be grafted onto rootstocks grown in vivo Although the success rates of these grafts appear lower than those achieved in vitro, the challenges of acclimatization are significantly reduced.
2008) Ohta et al (2011) thought that it is too difficult to get high success rates using micrografting technique for untrained people, because it required expert skill and aseptic manipulation
Citrus seedlings often develop adventitious shoots on the cut ends of epicotyls, making it challenging to differentiate between scion shoots and rootstock adventitious shoots To address this issue, trifoliate orange and its hybrids have been utilized for micrografting, enabling the identification of adventitious shoots based on leaf shapes However, the use of trifoliate orange for micrografting is complicated during the summer months and in tropical and subtropical regions.
7 because the mature seeds have dormancy and germinate after encountering chilling temperature in autumn and winter
The gradient for adventitious shoot formation seen in the cut edge of epicotyls in
Citrus and Poncirus accessions offer an effective strategy for promoting rapid seedling recovery after the loss of shoots above the epicotyls Research by Costa et al (2004) indicates that in vitro culture of epicotyl sections from Rangpur lime and grapefruit seedlings shows an increased potential for adventitious shoot formation as the distance from the cotyledonary node increases This potential is influenced by factors such as seedling age, plant material, and incubation conditions However, the extent of adventitious shoot formation in decapitated seedlings of Citrus and Poncirus species at various growth stages remains poorly understood.
Potential for adventitious shoot and cotyledon axillary shoot
Elimination of viruses in satsuma mandarin ( C unshiu Marc.) by in vivo micrografting with effective Citrus rootstock seedlings in summer
Citrus trees, essential for global nutrition and cultivated in diverse climates, face significant threats from various viruses, viroids, and bacteria that lead to substantial economic losses One of the most damaging pathogens is the Citrus tristeza virus (CTV), which infects the phloem tissues of the trees and is primarily spread by aphid vectors and contaminated propagating materials Effective management of these diseases is crucial to protect citrus production worldwide.
Satsuma dwarf virus (SDV) causing satsuma dwarf disease has tremendous negative impact on Citrus cultivars grown in Japan and East Asia (Karasev et al., 2001;
Soil transmission is the primary method for the spread of satsuma dwarf disease (Iwanami et al., 2001; Kusano et al., 2007) While fungicides and pesticides can effectively manage harmful pathogens, controlling virus and virus-like diseases remains a significant challenge with these chemicals.
Research indicates that virus and virus-like pathogens in Citrus can be effectively eliminated through techniques such as shoot tip grafting and micrografting (Abbas et al., 2008; Chand et al., 2013; Singh et al., 2008; Juarez et al., 2015) Additionally, the combination of micrografting and chemotherapy has proven successful in eradicating SDV in various Citrus cultivars (Ohta et al., 2011) and Grapevine.
The successful eradication of viruses in grapevine plants, such as the 45 rupestris stem pitting-associated virus, can be achieved through thermotherapy and shoot-tip grafting (Hu et al., 2018; Hu et al., 2015) Factors influencing the success of in vitro culture of shoot apices include the choice of rootstocks, the size of shoot apices, the growth conditions of the mother plant, and the composition of the culture media (Sanjaya et al., 2006; Chand et al., 2016; Thimmappaiah et al., 2002; Onay et al., 2003; Singh et al., 2008; Sanabam et al., 2015) In many micrografting studies, approximately two-week-old etiolated seedlings of rootstock cultivars, such as trifoliate orange, citranges, and rough lemon, were used for initial micrografting, with successful scions being regrafted onto older, vigorous seedlings 4-6 weeks later to promote growth (Chand et al., 2013; Juarez et al., 2015).
In vivo micrografting in Citrus was first demonstrated by Takahara et al (1986), who utilized 12- to 14-day-old trifoliate orange seedlings as rootstocks, successfully producing virus-free plants from shoot apices measuring 0.2-0.4 mm and 0.8-1.0 mm in length, combined with thermotherapy treatment for the scions However, Sanjaya et al (2006) found that the success rate of in vivo micrografting diminished as the age of rootstock seedlings increased, while Chand et al (2016) reported a lower success rate in younger rootstock seedlings.
Trifoliate orange is recognized as a primary rootstock for satsuma mandarin in Japan, characterized by a dormancy period where buds sprout after 1,000 hours of exposure to chilling temperatures below 7°C (Takishita, 2016) Additionally, mature seeds typically germinate only after experiencing chilling temperatures during autumn and winter Consequently, utilizing trifoliate orange seedlings for micrografting in summer or tropical regions poses challenges Therefore, alternative Citrus rootstocks are recommended for such conditions.
Successful micrografting requires seedlings with high success and scion growth rates, as demonstrated by studies showing rates comparable to trifoliate orange in various Citrus rootstock genotypes (Sanabam et al., 2015; Singh et al., 2018) However, these studies did not explore the potential of seedlings from pummelo, sour orange, mandarin, and padeda accessions that could be found in tropical regions.
Auxin, a key plant hormone, plays a crucial role in wound healing and establishing vascular connections between the shoot tip and rootstock (Goldschmidt, 2014) Various studies have shown differing outcomes in in vitro micrografting techniques For instance, soaking the cut end of cucumber rootstock in an IBA solution for a few seconds enhances compatibility between the rootstock and scion (Balliu and Sallaku, 2017) Additionally, pretreatment with kinetin at a concentration of 1.0 mg/L has been found to improve the success rate of in vitro micrografting in Khasi mandarin (Singh et al.).
Most research has concentrated on in vitro micrografting, which demands specialized facilities and expertise Therefore, it is essential to identify appropriate rootstocks and micrografting techniques to produce virus-free plants in tropical regions.
This study explored the successful micrografting of satsuma mandarin using seedlings from seven Citrus accessions The success rate was enhanced by micrografting two shoot apices onto a single rootstock seedling and incorporating auxin (NAA) prior to the procedure Additionally, the research evaluated the effects of shoot tip size, the age of rootstock seedlings, and seasonal variations on the efficacy of in vivo micrografting and virus elimination The elimination of viruses in the micrografted plants was confirmed through immunochromatographic assays.
Seven Citrus accessions used to produce rootstock seedlings were monoembryonic
The study involved various citrus cultivars, including the Hirado-buntan pummelo, polyembryonic Bilolo, Natsudaidai tangelo, Zadaidai, Kabusu, Variegated Daidai sour oranges, and Shiikuwasha No 3 mandarin, along with a control polyembryonic trifoliate orange known as 'Flying Dragon.' The research was conducted using fifteen 50-year-old satsuma mandarin trees for virus detection, all cultivated at the Kyushu University Experimental Farm in Fukuoka, Japan.
Perfect seeds from seven Citrus cultivars were collected from open-pollinated mature fruits The embryos were placed on wet filter paper in a covered petri dish and incubated at 25 ± 2 ℃ for approximately two weeks Vigorously germinating embryos, with roots around 20 mm long, were then transplanted into pots containing a soil mixture of Kanuma pumice, pumice, Akadama soil, and leaf mold in equal volumes A total of six seedlings were grown per 600 mL pot in a greenhouse Seedlings at various stages—2 weeks, 4 weeks, 8 weeks, and 4 months post-germination—were utilized for the study Notably, seeds from the 'Variegated Daidai' cultivar produced a high number of albino seedlings alongside a few normal green-leaved zygotic seedlings.
48 week-old albino seedlings were used in this study, since their growth was getting worse from four weeks after germination and they died later
Seeds of trifoliate orange were collected from mature fruit in October and directly sown in wet sand, where they underwent natural winter stratification to break dormancy for spring germination The germinated seeds were then transplanted into pots In this study, 2-week-old albino seedlings were primarily used as controls, as older seedlings (4-week-old and beyond) exhibited declining growth and ultimately died Additionally, 8-month-old and 1-year-old seedlings of the variety "Hirado-buntan" were included to assess the impact of seedling age on the success of micrografting.
In the summer, micrografting was performed using three to four well-developed axillary buds, approximately 5 cm in length, from the upper half of the spring shoots of satsuma mandarin Shoots were sourced from trees No 1 and No 3 to identify effective rootstock cultivars with a high success rate in micrografting, while trees No 8 and No 13 were selected for their potential to eliminate Citrus tristeza virus (CTV).
The Satsuma dwarf virus (SDV) was studied through in vivo micrografting techniques Using a stereoscopic microscope, shoot apices containing 2, 4, and 6 leaf primordia were carefully excised from the axillary buds of spring shoots The shoot tips with 2 or 4 leaf primordia were accurately identified, while those with 6 leaf primordia were estimated based on a length measurement of 0.6 mm.