Shoot-tip Grafting Research Articles

Successful shoot tip grafting of cacao (Theobroma cacao L.) due to the application of plant growth regulators on various concentrations

Cacao plays an important role in the Indonesia economy. Therefore, its production and area expansion have to increase to achieve maximum cacao productivity by the quality and superior cacao clones. Shoot tip grafting is one of the propagation methods to obtain superior cacao plants by applying Plant Growth Regulators (PGRs). Research on the effect of several PGRs with various concentrations on cacao (Theobroma cacao L.) shoot tip grafting was conducted at the Integrated Innovation Farmer Group, Belubus, Sungai Talang Village, Guguak District, Lima Puluh Kota Regency, West Sumatra from February until June 2022. This study aimed to determine the interaction between PGR type and concentration on the grafting success and growth of cacao shoot tip grafted seedling. The research was designed by a Factorial Completely Randomized Design with three replications. The first factor was the type of PGR, i.e., synthetic PGR (BAP), young corn seed extract, shallot extract, and young coconut water. The second factor was the concentration of PGR, i.e., 0%, 25%, 50%, 75% and 100%. The results showed an interaction between PGRs with some concentrations on the number of shoots, shoot length, and leaf length on the grafted cacao seedling. The natural PGR from young coconut water at a concentration of 75% gave the best effect on the number of shoots (3.33 shoots) and shoot length (24.50 cm) of cacao seedling.

Inhibition of Citrus Huanglongbing Disease by Paenibacillus polymyx KN-03 and Analysis with Transcriptome and Microflora

Soil drench treatment using Paenibacillus polymyxa strain KN-03 was applied to citrus plants infected with Candidatus Liberibacter asiaticus (CLas). The infection status was assessed using PCR and a real-time quantitative PCR detection system (qPCR). The application of KN-03 resulted in a notable reduction in CLas levels in citrus plants. Specifically, by the 257th day post treatment commencement, following 24 KN-03 applications, the negative rates of CLas in the vein, root tip, and shoot tip were 50%, 0%, and 50%, respectively. After 24 cycles, KN-03 application significantly enhanced plant growth and stimulated reactive oxygen production in citrus leaves compared to control plants. Transcriptome analysis identified specific upregulated pathways. Furthermore, flora analysis revealed an increased abundance of microorganisms possessing potential utilization value, including Burkholderia-Caballeronia-Paraburkholderia, uncultured_bacterium_o_Acidobacteriales, uncultured_bacterium_f_Gemmatimonadaceae, and Rhodanobacter, in the root zone. Moreover, the BugBase analysis indicated that KN-03 treatment increased the abundance of beneficial rhizosphere bacteria associated with biofilm formation, element mobilization, and stress tolerance. These findings support the utility of Paenibacillus polymyxa KN-03 as an effective plant-growth-promoting bacterium for CLas management, with additional benefits for plant growth and soil health, specifically offering detoxification resources for shoot tip grafting.

Elimination of Citrus psorosis virus using shoot tip grafting and electrotherapy techniques

Elimination of Citrus psorosis virus using shoot tip grafting and electrotherapy techniques

Combining ex vitro thermotherapy with shoot-tip grafting for elimination of virus from potted apple plants

Combining <i>ex vitro</i> thermotherapy with shoot-tip grafting for elimination of virus from potted apple plants

Distribution and Genetic Diversity of Coguvirus eburi in South African Citrus and the Development of a Real-Time RT-PCR Assay for Citrus-Infecting Coguviruses

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is, however, incomplete. CiVA was detected in grapefruit (C. paradisi Macf.), sweet orange [C. sinensis (L.) Osb.], and clementine (C. reticulata Blanco) in South Africa, and a survey to determine the distribution, symptom association, and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in 'Delta' Valencia trees in six citrus production regions, and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral-encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the Delta Valencia, grapefruit, and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared with the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus-infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

Clean Citrus Planting Materials Production through Shoot tip Grafting and Biological Indexing

Huanglongbing (Citrus Greening) is the most devastating citrus disease caused by phloem-inhabiting bacteria carried by the insect vector, Asian citrus psyllid (Diaphorina citri). In Bhutan, the production and movement of citrus seedlings and new orchard establishment have been restricted after the Citrus Greening disease was detected in most citrus orchards. To address this problem, Agriculture Research and Development Centre-Wengkhar has initiated research on clean citrus propagation through shoot tip grafting. This study assessed the efficiency of shoot tip grafting for the production of clean planting materials. Sweet orange was used as an indicator plant to study disease transmission and symptom expression in some of the potential and released citrus varieties. A total of 15 plants, 3 plants of each cultivar (AREP-1, AREP-2, Aoshima, Wengkhar Tshelu-2 and Yoshida Ponkan) produced through shoot tip grafting were used for bio-indexing. The cultivars were grafted on indicator plants using different grafting techniques (T-budding, side grafting, wedge, and split grafting). A total of twenty-six samples with two samples per cultivar was sent to the National Plant Protection Centre for PCR analysis. The result of PCR showed negative result for Huanglongbing. Thus, shoot tip grafting could be one potential method for clean citrus planting material production.

Short Communication: Variations of morphology, anatomy, and metabolite profiles of Citrus reticulata Blanco cv. Tawangmangu grafts produced by shoot tip grafting using several rootstocks

Abstract. Nuryandani E, Susandarini R, Indrianto A, Nuringtyas RT, Fathima AM, Subandiyah S. 2020. Short Communication: Variations of morphology, anatomy, and metabolite profiles of Citrus reticulata Blanco cv. Tawangmangu grafts produced by shoot tip grafting using several rootstocks. Biodiversitas 21: 4671-4676. Cultivation of Citrus reticulata Blanco cv. Tawangmangu, one of leading mandarin cultivars in Indonesia, remains challenging due to the limitations of suitable rootstocks. The use of suitable rootstocks will greatly determine the success of grafting and affect the performance of the scion. This study aimed to evaluate the morphological, anatomical, and metabolic profiles of grafts between Tawangmangu and rootstocks from various species using shoot tip grafting. Observations of morphological, anatomical, and metabolite variation were carried out eight weeks after the beginning of the growth of the scions. The morphological characters were observed on leaves and stems of the scions. Anatomical data were obtained from the examination of longitudinal sections of grafts prepared using paraffin-embedded method. Analysis of metabolite profiles was performed using LC-MS. The results showed that for two of the three rootstocks used, C. ichangensis × C. reticulata cv. Austera (Japansche Citroen (JC)) and Citrus (Fortunella) japonica (Fortunella) scion growth was vigorous and resulted in a large number of leaves and long stems. Morphological variation was found in the leaf shape, as indicated by wider and wavy leaves of grafts with Fortunella, while narrower and straighter leaves were found in grafts with JC. The grafting onto the third rootstock, C. hystrix, induced dwarfing of the scions. Variation in the anatomical profiles was found in vascular vessels, and in exodermic and endodermic cell sizes. Differences in metabolic profiles of the grafts were found compared to scion and rootstocks. Results of this study provide an important contribution to the successful improvement of Tawangmangu.

Acquisition of Virus Eliminated Apple Plants by Thermotherapy and the Factors Influenced the Eliminating Efficiency

The aim of this study was to establish reliable and efficient detection and elimination methods for five apple viruses and viroids, including Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Prunus necrotic ringspot virus (PNRSV) and Apple scar skin viroid (ASSVd) from infected apple plants using thermotherapy. Based on multiple alignments of available virus or viroid characteristic genes, compatible degenerate primer pairs were designed and an efficient and sensitive detection method was developed using quantitative real-time PCR (qPCR) and was further verified by short interfering RNA (siRNA) deep sequencing. Coupled with root protecting and shoot growth promoting measures, six cultivars of potted apple plants were subjected to thermotherapy for 30 days, followed by virus detection of new emerged shoots and shoot tip grafting. The results showed that the virus elimination efficiency was over 30% for the five viruses tested for ‘Kudowu’, ‘Fuji 2001’, and ‘Devil Gala’, but only 10% for ‘Red Jonathan’ and ‘Shizuka’, while no virus-free plants were detected for ‘Shinano Gold’. Correlation analysis of the factors influencing the virus elimination showed that the elimination efficiency and the virus free plant number were significantly related to the shoot growth rate, implying a vital role of the shoot growth rate in virus elimination using thermotherapy. This study provides a new qPCR assay for five apple viruses and stresses the importance of cultivar characteristic in virus thermotherapy elimination.

Elimination of apple necrosis mosaic virus from potted apple plants by thermotherapy combined with shoot-tip grafting

Apple necrosis mosaic virus (ApNMV), a newly found virus in apple, is a pathogenic factor associated with apple mosaic disease. In this study, we evaluated the elimination efficiencies of ApNMV by thermotherapy combined with shoot-tip grafting. Four potted apple varieties, Xiangfu (XF), Xiangji (XJ), Longkou no. 1 (LK), and Longwei (LW) were used as the original materials. ApNMV was detected in XF, XJ, and LK by reverse transcription-polymerase chain reaction (RT-PCR) prior to treatment. These three apple varieties also harbored apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple stem pitting virus (ASPV). The four apple varieties were subjected to thermotherapy at 38 ± 1 °C. The ApNMV titer in LK during thermotherapy was quantified by quantitative real-time RT-PCR. The results showed that the titer of ApNMV markedly decreased after only 1 day of 38 °C treatment and could hardly be detected after the end of thermotherapy, but the virus was able to recover after the plants began to grow normally. The shoot tips were grafted onto rootstock after thermotherapy. The survival rate across all grafted shoot tips was 66.7% (52/78). The average elimination rate of ApNMV was 89.5% (34/38). The four viruses were ranked, from highest elimination efficiencies to lowest, ACLSV > ApNMV > ASPV > ASGV. This combination of thermotherapy and shoot-tip grafting will be helpful for the elimination of ApNMV.

In vitro micrografting using three diverse indigenous rootstocks for the production of Citrus tristeza virus-free plants of Khasi mandarin

The present work reports the prospective applicability of three indigenous rootstocks belonging to different species viz. Nemutenga, Tayum and Tasi cultivars of north eastern Himalayan region for producing Citrus tristeza virus (CTV; a viral species of the Closterovirus genus) free quality planting materials of Khasi mandarin (Citrus reticulata Blanco) through micrografting or shoot-tip grafting (STG). The development of disease-free plants through STG is essential as CTV is in vivo transmissible through infected bud sticks. The technique of STG along with the diverse culture parameters like effect of sucrose, plant growth regulators, pre-treatment of scion and stock, effect of scion size after thermotherapy and grafting method was analysed. Among the three indigenous tested rootstocks, Nemutenga was found superior showing maximum STG success of 58.5%. STGs using scion size ranging from 0.3 to 0.5 mm, survival was up to 42.0% and completely virus-free. Pre-treatment of scion and stock with kinetin (1.0 mg L−1) was found more suitable than N6-benzylaminopurine (BAP) and further increased the rate of micrografts. For further growth of micrografts, Murashige and Skoog medium fortified with 0.5 mg L−1 BAP and 0.1 mg L−1 indole-3-acetic acid (IAA) along with 5% sucrose resulted in maximum (56.8%) response. Considering the two techniques applied for STG, cleft grafting was found more suitable than inverted T grafting. Viral assessment with enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR) revealed negative results for all plants from 0.3 to 0.5 mm scion while 0.7-mm scion-raised micrografts showed 60% negative with RT-PCR for CTV.

ウンシュウミカンにおける茎頂接ぎ木による3種ウイロイドの効率的無毒化法

ウンシュウミカンにおける茎頂接ぎ木による3種ウイロイドの効率的無毒化法

Genetic Transformation of Commercially Important Mature Citrus Scions

ABSTRACTA Mature Citrus Facility (MCF) was established at the University of Florida's Citrus Research and Education Center (CREC) to produce transgenic citrus (Citrus spp.) tolerant to canker and greening bacterial diseases. Plants produced from mature tissue should flower and fruit early. Budsticks of ‘Hamlin’, ‘Valencia’, and ‘Pineapple’ sweet orange [C. sinensis (L.) Osbeck] and ‘Ray Ruby’ grapefruit (C. paradisi Macfad.) were obtained from the Florida Division of Plant Industry (DPI) and introduced into the facility through shoot‐tip grafting (STG). These plants were tested for bacteria, phloem‐limited Huanglongbing (HLB), and indexed for viruses to ensure freedom from disease. Mature buds were grafted onto immature rootstock and the first two flushes of mature scion used in transformation feasibility studies with Agrobacterium strain EHA105 harboring pCAMBIA2301 and strain EHA101 harboring pTLAB21, which carry the β‐glucuronidase (GUS) and green fluorescent protein (GFP) reporters, respectively. β‐glucuronidase assays or GFP fluorescence was used to identify transgenic shoots, which developed through organogenesis from stem explants. Transgenic shoots were micrografted onto immature rootstock, and secondarily micrografted onto 4‐ to 5‐mo‐old rootstock. NPTII enzyme expression in older trees was confirmed using the immunostrip and enzyme‐linked immunosorbent assays (ELISA). Southern blots of a representative group of transgenics showed that most were low copy number with 1 to 3 copies of the T‐DNA integrated into the genome. There were a large number of escapes on kanamycin, and many transgenic shoots were lost in micrografting. Several transgenic trees flowered 18 to 19 mo after cocultivation with Agrobacterium. Transformation of mature citrus with disease resistance genes would be a feasible way to produce citrus tolerant to canker and greening.

APPLICATIONS OF CITRUS SHOOT-TIP GRAFTING IN VITRO

The main application of shoot-tip grafting in vitro (STG) is to control graft-transmissible pathogens that requires the use of healthy trees in the new plantings. The routine application of STG using 0.1-0.2 mm shoot tips is very efficient for elimination of all citrus graft-transmissible pathogens from local or imported varieties. It has allowed the recovery of hundreds of healthy cultivars and the planting of hundreds of millions of healthy certified trees worldwide. Only in Spain about 140 million certified nursery plants propagated from micrografted plants have been planted. STG is also a very useful technique for regeneration of elite genotypes in several research areas. In vitro grafting for these purposes may be done using larger shoots (up to 1 cm). STG is being routinely used for the following purposes: (i) Regeneration of somatic hybrids from embryos difficult to germinate; (ii) Regeneration of plants from irradiated shoots to produce seedless varieties; (iii) Regeneration of plants from haploid embryos that are very difficult to germinate. STG was used to regenerate the ‘Clemenules’ haploid plant that has been used by the International Citrus Genome Consortium to sequence the whole citrus genome; (iv) Production of stable tetraploid plants of non-apomictic genotypes, which are very useful for triploid breeding; (v) Regeneration of transgenic plants from shoots that are very difficult to root in vitro. STG has become a routine application in citrus genetic transformation.

Efficiency of virus elimination from potted apple plants by thermotherapy coupled with shoot-tip grafting

Four varieties of potted apple were subjected to thermotherapy (37 ± 1 °C) coupled with shoot tip grafting. Using reverse transcription-polymerase chain reaction assays prior to treatment, mixed infections with Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV) and Apple scar skin viroid (ASSVd) were detected in 95.8 % of treated plants. It was found that the sprouts of Dahongrong (DHR) and Qiyuetianxian (QYT) variety buds were inhibited by high temperature, although some buds of these two varieties were able to sprout, the growth was slow. The average survival rate of shoot tips cut after thermotherapy was 41.2 % (61/148). The survival rate of DHR (63.2 %) was highest among the four varieties. Sixty-one surviving apple plants were detected over two periods, indicative of an average elimination rate of 65.6 % (40/61) for new leaves (June) and 37.7 % (23/61) for dormant branches (November). The difference of elimination rate for DHR between the two periods was the most obvious. Detections rates of viruses were also different in the two periods; ASGV in dormant branches were 27.8 % higher than those in new leaves, and ASPV (90.6 %) remained the same over the two periods.

The Citrus Sanitation Center of the Estación Experimental Agroindustrial “Obispo Colombres”, Tucumán, Argentina

Argentina is the largest lemon producing country in the world and Tucumán province leads in lemon production. Knowing that disease free citrus propagating materials prevent the spread of diseases and are the basis of a profitable industry, the Estación Experimental Agroindustrial Obispo Colombres of Tucumán (EEAOC) established the Citrus Sanitation Center (CSC) in 2004. The goal of the CSC is to provide the important citrus varieties and rootstocks true to type and free of graft-transmissible pathogens as primary sources of propagating material for citrus growers and researchers in northwest Argentina. A national citrus certification program became mandatory in 2010 and enclosure of all commercial nurseries in January 2011.&nbsp; At present, most of the main citrus varieties and rootstocks of commercial interest have been recovered through the standard procedure of shoot-tip grafting in vitro. Mother trees and increase blocks are maintained in insect proof greenhouses to supply budwood for rapid nursery multiplication. Regular testing of mother trees by biological, serological and molecular methods is performed for different virus, viroids, CVC, citrus canker and now HLB. Current surveys report that Tucuman is free of the vector, Diaphorina citri, and the HLB pathogen. The CSC will also perform HLB testing during quarantine entry of imported varieties to provide a program for safe accession of citrus germplasm and provision of clean budwood.

Standardization of protocol for in-vitro shoot tip grafting in Kinnow mandarin (Citrus deliciosa)

An experiment to standardize the rootstocks and PGRs for success and survival of in-vitro shoot tip grafting in Kinnow mandarin (Citrus deliciosa) was conducted during the year of 2007-08 at Department of Horticulture, Agricultural Research Station, Sriganganagar, Rajasthan. For this, in-vitro generated and 2-3 weeks etiolated old seedlings of cleopatra, rough lemon and carrizo were used as rootstocks. The seedlings were grafted by shoot tips measuring = 1 mm in length containing apical meristem and one or two leaf primordial which were excised from in-vitro generated shoots and grafted on rootstocks. Among different rootstocks used, twelve days old seedlings of Carrizo recorded the maximum success (56.60 %) and length of shoot (1.43 cm). All the concentrations of the PGRS tried to accelerate the growth of new shoot and survival per cent of STG, BAP @ 1.0 mg/l was found to be best in minimizing the time taken to bud break (12.12 days) and recorded maximum shoot length (3.10 cm). The results of the study revealed that the maximum survival (66.90 %) was observed with 2, 4-D @ 3.0 mg/l in all the PGRs tried for growth promotion of scion shoots. It is also revealed from the study that the 90 % grafted plants survival in greenhouse at after 45 days after shoot tip grafting. Based on the observations, the Carrizo as rootstocks, BAP and 2, 4-D were found to be the best protocol for shoot tip grafting in Kinnow mandarin.

English

Micrografting is an in vitro grafting technique which involves the placement of a meristem or shoot tip explant onto a decapitated rootstock that has been grown aseptically from seed or micropropagated cultures. Following early experiments of micrografting in ivy and chrysanthemum, the technique has been used in woody species, especially fruit trees. Major work was carried out in different Citrus species for the elimination of various viral diseases. In vitro micrografting has been used for improvement and multiplication of fruit trees as the technique has potential to combine the advantages of rapid in vitro multiplication with the increased productivity that results from grafting superior rootstock and scion combinations. Successful micrografting protocols have been developed for various fruit crops including almond, apple, cherry, chestnut, Citrus, grapes, mulberry, olive, peach, pear, pistacio, walnut, etc. Special techniques have been used for increasing the percentage of successful micrografts with the use of growth regulators, etiolation treatments, antioxidants, higher sucrose levels, silicon tubes, etc. The technique has great potential for improvement and large scale multiplication of fruit plants. It has been used on commercial scale for production of virus-free plants in fruit crops and viroid free plants in Citrus. Micrografting has also been used in prediction of incompatibility between the grafting partners, histological studies, disease indexing, production of disease-free plants particularly resistant to soil borne pathogens and multiplication of difficult to root plants. &nbsp; Key words: Fruit crops, graft incompatibility, crop improvement, micrografting, propagation, shoot tip grafting.

Recent advances in Citrus psorosis virus.

Psorosis is a globally devastating disease of citrus caused by an infectious filamentous ophiovirus, Citrus psorosis virus (CPsV), which causes annual losses of about 5% and a progressive decline of trees by affecting the conductive tissues. The disease can be harboured asymptomatically in many citrus species. In the field, the most characteristic symptoms of the disease in adult trees are bark scaling in the trunk and main branches and also internal staining in the underlying wood. The virus has a tripartite single-stranded RNA genome, and has been inadvertently spread to most citrus growing areas through the movement of citrus propagative material. No natural vectors have been identified except in limited citrus areas in some cases. Management strategies for CPsV involving shoot-tip grafting and thermotherapy or somatic embryogenesis from stigma and style cultures have been successfully used to eliminate CPsV from plant propagating material. Molecular pathogen-mediated strategies have been used to produce citrus plants. Such a strategy protects against infections by the virus from which the resistance gene and promising resistance may emerge from trials. Certification programs are among the best established means of increasing phytosanitary health, and some of those for citrus are among the oldest in the world. In conjunction with quarantine and clean stock programs, they remain important weapons in the ongoing fight against citrus diseases. One of the elements essential for successful certification programs to produce such propagation material is the availability of sensitive and effective diagnostic methods. In this review, we discuss an updated status of CPsV disease.

In vitroShoot tip Grafting of Patharnakh [Pyrus pyrifolia(Burm F.) Nakai] Pear on Kainth Rootstock

In vitro propagated Patharnakh shoot-tips were grafted on in vitro propagated Kainth rootstock. Graft success, occurrence of necrosis and vigour were found to be influenced significantly by scion length, grafting technique and medium used for shoot tip grafting. Graft success was maximum (33.71%) by using L2 (5–10 mm scions), G1 (wedge grafting) and M2 (MS liquid media + 20 g/l sucrose). However, necrosis was least (0.0%) in many treatment combinations. Maximum vigour (2.68) was found by using L2, G1 and M2.

Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus

To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.