An Improved Protocol for Rapid in Vitro Multiplication of Clonal Apple Rootstock MM106

Investigation of responses of some apple (Mallus x domestica Borkh.) cultivars grafted on MM106 and M9 rootstocks to lime-induced chlorosis and oxidative stress

In vitro culture at the rooting stage the aft ereff ect of the use of earlier (during proliferation) growth regulators with cytokinin activity (6-benzylaminopurine and thidiazuron) and the eff ect of lamps of diff erent spectral composition on the percentage of rooting and biometric parameters of the development of the roots of clonal apple rootstocks were studied. The following forms of apple clonal rootstocks were taken as objects of research: 54-118, 57-490, 57-545, ММ 106 and М 26. The research methods used at the stages of in vitro culture studies corresponded to those generally accepted for this section of research. Studies were performed on two variants of explants, one part was cultured at the proliferation stage on media with 6-benzylaminopurine and the other on media with tidiazurone. It was found that explants that were cultivated at the stage of proliferation on media with thidiazuron had a tendency to a subsequent increase in the number of roots (at the stage of rooting) by one microscopic cut, in comparison with cultivation on media with 6-benzylaminopurine, by 1.1-1.3 times, and this regularity was observed in all studied clonal rootstocks of the apple tree. It was noted that when explants were illuminated, explants had a greater percentage of rooting when illuminated with phytolamps in comparison with lighting with lamps with a color temperature of 6500K by 1.1-1.4 times in all studied apple rootstocks, and an increase in the number of roots per one microstalk was observed (for excluding rootstock 57-490), by 1.1-1.9 times. During the research, the specificity of rooting of clonal apple rootstocks was observed under the infl uence of the composition of light and the use of 6-benzylaminopurine and thidiazuron at the stage of proliferation, which was expressed in a higher ability to rhizogenesis of some rootstocks compared to others. The rootstocks MM 106 (83.3-93.9 %) and M26 (87.8-92.7 %) had a greater percentage of rooting, according to the average number of roots per one microcut, rootstocks 57-545 (15.4 pcs.), MM 106 (11.2 pcs.), and M 26 along the average root length (27.0 mm).

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

ABSTRACTThe specific in vitro conditions cause a modified structure and physiological functions of the photosynthetic apparatus which result in poor survival rate of plantlets during acclimatization. The type of vessel closure affects the gaseous composition and hence affects the growth of plantlets in culture. In tightly closed vessels concentrations of metabolically generated gases increase because their rate of diffusive escape is depressed. Different approaches have been proposed to enhance the photosynthetic ability of plants in vitro in striving to favour the success of further acclimatization to conditions ex vitro. The aims of the present research are to find the closure material which is gas permeable and to study the effect of the improved gas exchange on the growth and the photosynthetic ability of the micropropagated apple rootstock MM 106. The polyester wool—one of the tested types of closure, provides good gas exchange rate and preserves the culture from contamination. The data of this investigation show that there is a positive correlation between the improved gas exchange and accumulation of dry matter, light harvesting pigments as well as the ability of plantlet to assimilate 14CO2. In plantlets, cultured with polyester wool, the relative part of the labelled low -molecular photosynthetic products (sugars, amino acids, organic acids) decreases, while the part of labelled high-molecular fractions (lipids, pigments and starch) increases. The improvement of gas exchange leads to rise of the content of sorbitol and the ratio sorbitol: saccharose. This might be a sign of a better carbon metabolism of in vitro cultivated apple root-stock MM 106.

Apple replant disease (ARD) is a complex soilborne disease syndrome that often causes problems when renovating old orchard sites. Soil fumigants sometimes control ARD, but biological and cultural alternatives are needed. In this study the growth of two widely used clonal apple (Malus domestica) rootstocks (M7 and M26) were compared to three new rootstocks from the Cornell-Geneva series (CG16, CG30 and CG210 (a.k.a. CG6210)) in an orchard site with a history of ARD, in Ithaca, NY. Trees were planted in two distinguishable positions – the previous tree rows versus the old inter-row grass lanes. Additionally, we compared the effects of compost amendment and fumigation with dichloropropene plus chloropicrin on tree growth on each replant rootstock. Rhizosphere bacteria and actinobacteria were assessed using PCR-DGGE for the rootstocks M7, M26, CG30 and CG210. Tree growth on the rootstocks M7, M26 and CG16 was suppressed in the old tree rows relative to grass lanes, while trees on CG30 and CG210 rootstocks grew equally well in both positions. The species composition of rhizosphere bacteria and actinobacteria differed significantly between the planting positions and between the rootstocks M7 and M26 compared to CG30 and CG210. In contrast, the preplant compost or fumigation soil treatments had no effect on tree growth and little impact on rhizosphere bacterial community composition.

Comparisons were made of the fresh weights of trees on apple rootstocks MM 111, MM 106 and M.9 and of the weights of trees with interstocks of ‘Red Delicious’ (RD), MM 106 and M.9 of 2.5, 7.5, 12.5 or 18 cm lengths on MM 111 rootstocks. All scions were RD and the experiment covered the 1969-1972 growing seasons. Weight of all plant parts increased over the 4 years but the percentage of leaves and new growth decreased from 1970 to 1972, whereas the increase in scion and interstock weight tended to be a greater percentage of total plant weight increase (TPI) each year. Treatments MM 106 and M.9 rootstocks and M.9 interstock dwarfted the trees 37, 40, and 40 percent, respectively. M.9 rootstock and interstock and MM 106 interstock increased in weight proportionately more than comparable parts on the least dwarfing graft combinations. M.9 interstock also induced a proportionately larger root system than the other 2 interstocks. The faster growth rate of plants on MM 111 rootstock resulted from more rapid growth of woody tissues; leaf weights were similar. Plants with an RD or MM 106 interstock showed numerous variations in growth over the 4 years, but those with an M.9 interstock were uniform in growth habit. Plants with a 12.5 cm interstock weighed the least of all 3 interstock types. A comparison of weight of plants with a 2.5 or 7.5 cm interstock with those with a 12.5 or 18 cm interstock showed a reduction in growth due to the longer interstems only on plants with an M.9 interstock.

Background. Clonal apple rootstocks are one of the main components of intensive gardening. The degree of rootstock damage by fire blight affects the resistance of the variety–rootstock combination. The paper presents a study on marking quantitative trait loci (QTL) of resistance to fire blight Erwinia amylovora in clonal apple rootstock.Materials and methods. A collection of 20 rootstock forms was analyzed. For the study, SCAR markers GE-8019, AE10-375 and microsatellite marker CH-F7-FB1 were used.Results. Polymorphism was observed for all three markers, and their various combinations in one genotype were revealed. It was previously noted that genotypes that carry all three markers were more resistant than those that lack them. The presence of all three markers was observed only in forms 62-396 (В10), 16-1 and 2-9-102. The other genotypes did not have the GE8019 marker. The AE10-375 marker was identified in eight clonal rootstocks. Microsatellite marker CH-F7-FB1 was present in all tested rootstocks. However, polymorphism was detected there. Most genotypes had a 174 bp fragment, but a 210 bp fragment was identified in two of the 20 forms. Clonal rootstock 70-20-21 proved heterozygous for this marker. The analyzed collection also included samples that had only the microsatellite marker: G16, Malysh Budagovskogo, Paradizka Budagovskogo (B9), 54-118 (В118), 57-491, 70-20-20 (В119), 70-20-21, 71-7-22, 76-3-6, 83-1-15, 87-7-12, and 2-12-10. The study of rootstock forms on the basis of resistance to metabolites of the fire blight pathogen was carried out under laboratory conditions using the E. amylovora culture filtrate in vitro on leaf explants. Most of the studied genotypes had different combinations of markers. However, the experiments showed that forms 62-396 and 14-1 with two out of three markers (AE10-375 and CH-F7-FB1) phenotypically manifested the trait of resistance to metabolites of E. amylovora.

Surface sterilization is crucial in preparing viable and uncontaminated explants for tissue culture. Utilizing the right sterilizing agent in this procedure can efficiently eliminate the majority of surface contaminants. The present study aimed to establish the best sterilization procedure for clonal apple rootstock MM106 using shoot tips and nodal segments. Two important sterilizing agents, mercuric chloride (ranging from 0.05% to 0.3%) for various durations (2 to 6 minutes) and sodium hypochlorite (ranging from 3.0% to 12%) for different durations (10 to 30 minutes), were employed. The results indicated that the maximum aseptic conditions (80.00% and 80.67%) and survival rates of the cultures (60.66% and 67.33%) for shoot tips and nodal segments, respectively, were achieved through surface sterilization with 0.1% mercuric chloride for 4 minutes, specifically during April, in comparison to March and May of 2021 due to low phenol content and high meristematic activity. The results yielded valuable insights for efficiently propagating clonal apple rootstocks on a local scale, and they have the potential to offer guidance for establishing commercial facilities dedicated to producing these specific clonal apple rootstocks and varieties.

In the conditions of the Southern Urals, sowing of seeds of clonal apple rootstocks obtained from open pollination of cultivated varieties growing in the garden of the variety-rootstock trial was carried out. Over the years of selection work on the creation of clonal apple rootstocks for the Volga-Ural region with its sharp continental climatic conditions, it was possible to obtain forms with increased drought resistance and frost resistance of the root system, unpretentious to soil differences, already at the first stage. Compared with the control, the selected forms stand out by increased productivity (by 1.5–2.0 times) in the garden and a high coefficient of rootstock reproduction (Ural 1, Ural 5, Volga 3, Volga 12). Among all the available forms, second-generation rootstocks have been obtained at the present time (OB series and a number of other forms not included in the Register of Breeding Achievements, requiring testing in the garden).

The taxonomic structure of tissue endophytic bacterial microbiome was comparatively studied in microplants (undifferentiated explant callus tissues, passage 25) and 5-year clonal apple rootstocks 57-490 and 54-118 cultured from corresponding tissues (passage 1) on sod-podzolic soils with variant granulometry, chemical, physical and physicochemical properties. Proteobacteria (91.6 %) predominated in vitro tissues among other endophytic bacterial phyla in rootstock 57-490, while Proteobacteria (52.5 %) and Firmicutes (47.4 %) — in rootstock 54-118. The endophytic Firmicutes ratio vs. in vitro tissues decreases (0.7-2.0 %) in roots and more severely (0-0.2 %) in leaves. Endophytic Actinobacteriota are revealed in 11.7 % in roots of the study rootstock in heavy loam soil, whilst in medium loam their ratio drops to 2.74.1 % in roots and 0.1-0.2 % in leaves. The phylogenetic diversity indices estimation for main endophytic bacterial phyla in apple rootstock tissue recovers their essentially lower diversity and evenness in culture endosphere (Shannon index 0.42-1.00) vs. open soil roots (1.34-2.08). The leaves Shannon index is typically low (0.06-0.13) indicating poor diversity and evenness of the main endophytic bacterial phyla.

Within the framework of the joint program of the FSBSI NCFSCHVW and the Stavropol experimental station of horticulture – Branch of Federal State Scientific Institution «North-Caucasian FNAC», for the breeding of apple tree rootstocks, hybrids of apple-tree rootstocks of the ST series were assessed in terms of their adaptability to the conditions of the summer vegetation period in the central subzone of the Prikuban horticultural zone (Krasnodar city). The resistance of apple rootstocks to the stresses was determined by the water content and water-holding capacity of the leaves, which characterize the ability of plants to maintain water homeostasis during stress. In the nursery of offspring rootstocks, the drought resistance of apple tree rootstocks was determined by the laboratory-field method during the period of the greatest intensity of the water regime of plants. The zoned rootstock MM-106 was used as a control. The leaves of rootstocks CT 20-2 and CT 18-2 retained water better, in which the water loss for 2 hours of exposure was 28.3 and 30.3 % compared to 34.7 % for the control rootstock MM-106. The high water-holding capacity of the ST 20-2 rootstock was previously noted in the dry August 2020 – water loss in 4 hours was 31.5 % compared to 53.0 % for rootstock MM-106. The most productive were trees of the Prikubanskoye variety on rootstocks SK 2U, ST 20-4, 106-3, ST 13-3. The best specific productivity for five years of fruiting was in trees on rootstocks SK 2U, SK 5 and ST 13-3 hybrid (5.28; 4.74 and 4.22 kg/m2 ). Selected and recommended f or participation in breeding programs as a source of drought resistance rootstock ST 20-2 (MM-106 x M 9) and as a source of tree productivity in the garden, rootstock ST 13-3 (rootstock 58-250 x free pollination).

True-to-type clonal fidelity is of paramount concern during micropropagation of crop species. Though, micropropagation has found an important role in propagation of apple rootstocks, commercial propagation may be constrained by the occurrence of genetic variations, especially when cultures are maintained in vitro for a long period. To determine genetic fidelity of micropropagated plants of apple rootstock MM 106 multiplied by enhanced axillary branching method, we screened 24 inter simple sequence repeat markers (ISSR). Of these, 11 primers reproducibly generated a total of 107 distinct bands. Apple rootstock Merton 793 and scion ‘Jonathon’ were analysed along with tissue culture raised progenies of MM 106 as outliers to rule out the possibility that the invariant banding pattern was on account of inefficiency of ISSR primers in detecting variations. The number of bands per primer varied from 5 to 9 with an average of 9.72 scorable bands per primer. Among 83 bands amplified in micropropagated plants of MM 106, 78 bands were monomorphic (93.9%) and 5 bands (6.02%) were polymorphic. The results showed that ISSR markers can be used to gain rapid and precise information about the genetic stability of micropropagated apple rootstock MM 106 and corroborated the fact that the cultures multiplied through pre-existing meristems (axillary branching) may also show genetic instability especially upon extended in vitro cultivation.

Surveys were conducted in the major apple growing regions of New York state to determine the incidence of borers infesting burrknots on clonal apple rootstocks. Dogwood borer, Synanthedon scitula (Harris), was generally prevalent throughout the state, but American plum borer, Euzophera semifuneralis (Walker), was limited to western New York apple orchards near infested stone fruit trees. Insecticides evaluated in the field for efficacy against both borers were chlorpyrifos, endosulfan, indoxacarb plus oil, methoxyfenozide, fenpropathrin, and kaolin clay. Also, white latex paint was tested alone and mixed with chlorpyrifos. One application of chlorpyrifos applied at the petal fall developmental stage was equivalent to chlorpyrifos applied at petal fall and again in mid-July, and it provided season-long control of dogwood borer and American plum borer. One application of chlorpyrifos applied any time between the half-inch green developmental stage and petal fall, or after harvest the previous season, controlled both overwintered and summer brood larvae of dogwood borer. Multiple applications of fenpropathrin, indoxacarb plus oil, and endosulfan applied during the dogwood borer flight period controlled the summer brood.

The paper presents the results of identification of quantitative trait loci (QTL) of Dw1 and Dw2 involved in the control of dwarf growth in clonal apple rootstocks using the molecular markers. In total, 14 forms of rootstocks were analyzed. The microsatellite markers Hi01c04, Hi04a08, CH03a09 (for the Dw1 locus), MDP0000365711, and MDP0000243703 (for the Dw2 locus) were used in the study. Analysis of the results did not reveal a relationship between the presence of a marker and the manifestation of a sign. Of the three markers of the Dw1 locus, only Hi01c04 amplifies a fragment of the expected size of 120 bp. It was found in 9 forms of rootstocks (PB-4, 57-491, 83-1-15, M9, G16, 2-12-10, 2-9-102, 4-6-5 and 70-20-20). The Hi01c04 marker was identified in both dwarf and medium-sized forms. Of the two markers of the Dw2 locus, the desired fragment is amplified in MDP0000365711. It is typical for almost all genotypes. The exception is 83-1-15, 2-12-10 and 70-20-20, which have a null allele. To assess the genetic diversity of the Dw1 and Dw2 loci, 6 microsatellite sequences Hi01c04, Hi04a08, CH03a09, CH02d08, MDP0000365711, and MDP0000243703 were used. In 14 studied samples, 29 allelic variants ranging in size from 102 bp. up to 170 bp were identified. The number of alleles per locus varied from 1 (for the MDP0000365711 locus) to 7 (for the Hi04a08 locus). No rare alleles were identified. All alleles were observed more than three times. Based on the analysis of SSR spectra, a dendrogram reflecting the similarity of the genotypes under study was built.

Surveys were conducted in the major apple growing regions of New York state to determine the incidence of borers infesting burrknots on clonal apple rootstocks. Dogwood borer, Synanthedon scitula (Harris), was generally prevalent throughout the state, but American plum borer, Euzophera semifuneralis (Walker), was limited to western New York apple orchards near infested stone fruit trees. Insecticides evaluated in the field for efficacy against both borers were chlorpyrifos, endosulfan, indoxacarb plus oil, methoxyfenozide, fenpropathrin, and kaolin clay. Also, white latex paint was tested alone and mixed with chlorpyrifos. One application of chlorpyrifos applied at the petal fall developmental stage was equivalent to chlorpyrifos applied at petal fall and again in mid-July, and it provided season-long control of dogwood borer and American plum borer. One application of chlorpyrifos applied any time between the half-inch green developmental stage and petal fall, or after harvest the previous season, controlled both overwintered and summer brood larvae of dogwood borer. Multiple applications of fenpropathrin, indoxacarb plus oil, and endosulfan applied during the dogwood borer flight period controlled the summer brood.