Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 9-19, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1248 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Federico Martinelli, Anna Perrone, Abhaya M. Dandekar (2021) Development of a protocol for genetic transformation of Malus spp. Caryolo- gia 74(3): 9-19. doi: 10.36253/caryolo- gia-1248 Received: March 11, 2021 Accepted: August 10, 2021 Published: December 21, 2021 Copyright: © 2021 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Development of a protocol for genetic transformation of Malus spp Federico Martinelli1,*, Anna Perrone2, Abhaya M. Dandekar3 1Department of Biology, University of Florence, Sesto Fiorentino, Florence, 50019, Italy 2Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze, Palermo, 90128, Italy 3Department of Plant Sciences, University of California, One Shields Avenue, Mail Stop 4, Davis, CA 5616, USA *Corresponding author. E-mail: federico.martinelli@unifi.it Abstract. A protocol to produce transgenic shoots of Malus X domestica cv Greens- leaves was optimized using two gene constructs previously used to create parthe- nocarpic tomato, Ino-IaaM and DefH9-IaaM. The aim was to obtain sufficient nº of transgenic shoots for in vitro multiplication, transfer to soil, grafting and testing for parthenocarpy in the next years. We investigated the effects of two modifications of a previous published protocol: 1) co-transformation with an Agrobacterium containing “VIP” genes in the gene construct and 2) two different hormones or hormone combi- nations. More shoot regeneration was obtained with a combination of three hormones (BA:NAA:TDZ) during co-cultivation instead of IBA and no co-transformation was performed using the VIP gene. For the DefH9-IaaM transgene, 21.04% regeneration was achieved for this treatment instead of 8.95% achieved with “IBA treatment” and 4.42% with the Agrobacterium co-transformation treatment. More shoot regeneration occurred with the combination of three hormones (BA:NAA:TDZ) instead of with only IBA and no co-transformation was performed using VIP gene. Experiments using Ino-IaaM confirmed the results shown for the DefH9-IaaM transgene. The regener- ated shoots were multiplied in selective media containing kanamycin and roots were obtained. Keywords: apple, Greensleaves, genetic transformation, Malus, organogenesis, TDZ INTRODUCTION Traditional genetic improvement in woody fruit species used selection and breeding, resulting in relatively few genotypes and a restricted germ- plasm base. This genetic uniformity has increased vulnerability of woody crops to insect pests and pathogens and caused excessive use of chemicals (Norelli et al. 1994) Genetic transformation provides an alternate approach through introduction of genes encoding desirable traits (Jia et al. 2019), bypassing the long periods required for genetic crosses and selection. Once a useful transgenic plant is isolated (assuming the transgene expression is stable), vegetative propagation allows rapid production of the desired trans- 10 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar genic line. Genetic improvement of an elite cultivar can occur because there is no sexual reproduction. Since production of most fruit tree species is based on a few cultivars, the impact of genetically transforming them is important.The characterization of induced overll metab- olism changes using omic tools has been previously done (Tosetti et al. 2010; Rizzini et al. 2010). The most widely produced commercial transgenic tree crop is papaya (Carica papaya L.) resistant to PRSV (Papaya Ringspot Virus), while transgenic apple is not yet on the market. This is partially due to the absence of efficient regeneration protocols for important commercial cultivars of Malus X domestica. Protocols developed for one cultivar are often not suitable for other cultivars of the same species. In some cases, genetic transformation has been obtained only from seedling material (Mante et al. 1991). The time required for transformation and evaluation of phenotype is generally much longer for tree crops (three to 20 years) than for herbaceous spe- cies. Space requirements can be large and evaluation of transgenic tree crops, expensive and time-consuming. However, conventional breeding for new cultivars has the same requirements. Among molecular genetic approach- es, genetic transformation is probably the most important tool to increase the speed of cultivar creation, because it avoids some disadvantages of conventional breeding, like loss of desirable characteristic in the offspring. In addi- tion, the small number of cultivars produced for each woody species increases the impact of genetic improve- ment of one of them. For example, over 50% of world and United States apple production is based on Red Delicious, Golden Delicious, Granny Smith, Gala and Fuji. An improvement of one of these cultivars can have a signifi- cant impact on total production. Methods for plant transformation fall into three main groups:1) biological vectors (virus- or Agrobacte- rium-mediated transformation; 2) direct DNA transfer (chemical-, electrical- or microlaser-induced permeabil- ity of protoplasts or cells; and 3) non-biological vector systems (microprojectiles, microinjection or liposome fusion). The availability of an efficient protocol for regen- eration is an important step for recovery of transgenic plants. There are efficient regeneration systems for many herbaceous species (tomato, Arabidopsis, tobacco). How- ever, systems for many woody fruit crops are either not available or suitable only for juvenile material of zygot- ic origin, which makes them useless for transforming elite cultivars. Dandekar (1992) considered two impor- tant conditions for regenerating transgenic plants:1) the regenerating cells must be accessible to Agrobacterium and 2) the regenerated plants must originate from single cells. Direct adventitious regeneration is preferred to intermediate proliferation of callus because callus can be a source of somaclonal variation, requiring extensive field tests to ensure that regenerated plants are true to type. Also, a pluricellular origin for regenerated plants can produce chimeric plants with variable expression. Genetic transformation of single cells or protoplasts can overcome this situation (Oliveira et al. 1994; Hidaka and Omura, 1993). Previous work on genetic transformation of apple has focused on genes to improve two kinds of traits: 1) disease resistance against viruses, bacteria, insects and fungi and 2) modification of agronomic phenotypic fea- tures, such as columnar growth, rooting ability, freez- ing tolerance or toxin resistance. Plant resistance to a pathogen is often caused by a hypersensitive response, involving elicitor recognition that activates a cascade of host genes and eventually leads to a generalized response known as systemic acquired resistance (SAR). Previous studies attempted to confer disease resistance by intro- ducing specific resistance genes rather than by activating plural defence mechanisms (Schuerman and Dandekar, 1993). Most research has focused on virus-induced disease. Some used genes encoding viral coat proteins to increase tolerance to specific viruses such as PRSV (Papaya Ringspot Virus) in papaya (Fitch et al. 1993) and CTV (Citrus Tristeza Virus) in Citrus (Ghorbel et al. 2001). In apricot, the regenerated plants were of zygotic origin and resistance has not yet be recovered from transformed commercial cultivars. Resistance to insects, bacteria and fungi has been developed in Actinidia deliciosa against Botrytis cinerea (Nakamura et al. 1999) and in wal- nut against Cydia pomonella (Dandekar et al. 1998). A Japanese persimmon cultivar was transformed with the CryIA (c) from Bacillus thuringiensis and biossays with two different lepidopteran pests showed significative resistance to these pathogens. Pear, like apple, is sever- ally affected by fire blight (Erwinia amylovora) and pear cultivars with increased resistance were recovered that expressed D5C1 (Puterka et al. 2002). The Rol A, B or C genes were used to improve root- ing in kiwifruit (Rugini et al. 1991) and in apple root- stocks such as M26 (Welander et al. 1998). In Citrus, the juvenile phase was shortened and precocious flowering was promoted using floral genes such as LEAFY (LFY) and APETALA1 (AP1) from Arabidopsis (Pena et al. 2001). Progeny of the transgenic LFY and AP1 trees had a generation time of one year from seed to seed, but only the AP1 trees had fully normal development. In peach, greater branching and shorter internodes were obtained using strains of Agrobacterium with a silenced auxin 11Improved apple transformation protocol synthesis gene and intact ipt gene for cytokinin synthe- sis (Smigocki and Hammerschlag, 1991). Among tree fruits, apple is used frequently for transgenic research because optimized transformation protocols exist for the elite cultivars Greesleaves (James et al. 1993) and Delicious (Sriskandarjah et al. 1994). Recently, transgenic apple trees with reduced scab sus- ceptibility were obtained by introducing a gene for puroindoline-b from wheat, effective against new rac- es of scab that are resistant to the Vf gene (Faize et al. 2004). Other researchers transformed apple using genes from the biocontrol fungus Trichoderma atroviride encoding the antifungal proteins endochitinase or exochitinase (N-acetyl-beta-D-hexosaminidase) driven by a modified CaMV35S promoter (Bolar et al. 2001). Exochitinase was less effective than endochitinase and the enzymes acted synergistically to reduce disease. The level of expression of endochitinase correlated negatively with apple tree growth, while exochitinase had no con- sistent effect on growth. Transgenic lines, especially one expressing low levels of endochitinase activity and mod- erate levels of exochitinase activity, were selected for high resistance in growth chamber trials and negligible reduction in vigor (Bolar et al. 2000, 2001). Other researchers used T4 lysozyme, attacin or cecropin MB39 genes to enhance resistance of trans- genic “Royal Gala” apple trees against Erwinia amylo- vora (Liu et al. 2001). Transgenic trees were evaluated for fire blight resistance, delayed fruit softening and scab resistance (Bolar et al. 2000). Apple fruit shelf life was improved by altering ethylene biosynthesis using sense or antisense cDNA encoding ACC-synthase and ACC-oxidase (Dandekar et al. 2004). Ethylene biosyn- thesis was also down-regulated in Gala apple using a SAM-k gene encoding a S-adenosylmethionine hydro- lase (SAMase). Resistance to codling moth was obtained using a chemical version of the Bacillus thuringiensis cryAC gene (Dandekar et al. date). Another important objective of genetic improvement in apple is regulation of tree growth. Apple growth has been modified using RolA genes isolated from Agrobac- terium rhizogenes. Apple rootstock M26 transformed with RolA had reduced internode length, dry matter and leaf area. When the scion Gravestein was grafted onto transformed M26, the scion showed reduced stem and internode length without altered leaf area and relative growth rate (Zhu and Welander, 1999). RolB promotes rooting through increased auxin sensitivity (Delbarre et al. 1994). This gene has been successfully inserted into the apple rootstocks M26 (Welander et al. 1998) and Jork9 (Sedira et al. 2001). Self-incompatibilty restricts fertilization and fruit set in apple and makes pollinator plants necessary for orchard productivity. Transgenic plants created with deleted pisitil S-RNase proteins, which are responsible for self-incompatibility, produced normal fruit and seeds after selfing (Broothaerts et al. 2004). This work tested different hormone combinations and co-cultivation with different Agrobacterium har- boring VIP genes to improve regeneration of transgenic apple shoots. We used two plasmid constructs contain- ing ovule-specific promoters to induce expression of the IaaM gene, which is involved in auxin biosynthesis. The resulting trees will be evaluated for the presence of seeds, since these gene constructs were used successfully to cause parthenocarpy in tomato cv Micro-Tom. MATERIALS AND METHODS Binary vectors, plant materials and treatments - Two binary vectors were used to transform apple cv Greens- leaves. The first, pDU04100, contained the IaaM gene (involved in auxin biosynthesis) in a sense orientation, under the control of ovule-specific promoter “Ino,” iso- lated from Arabidopsis ovary integument (Meister et al. 2004). The second, pDU04160, contained IaaM in a sense orientation under the control of another ovule- specific promoter, Def H9, isolated from Anthirrium majus ovary (Martinelli et al. 2019). Apple cv Greensleaves was cultured in vitro in shoot multiplication medium (A17) under controlled tempera- ture (18 to 25°C) and 16-hour photoperiod (fluorescent light) with no bacterial or fungi contamination. The plants were subcultured and separated every ? months. The effects of several treatments on transformation efficiency were studied: - BA:NAA:TDZ (5:1:1, (mg/L)) - IBA (3 mg/L) - cotransformation with Agrobacterium containing the VIP1 gene construct as described (Escobar and Dan- dekar 2003, Raman et al. 2019). The A17 shoot multiplication medium consisted of 30 g/L sorbitol, 431 g/L MS salts (macro- and micronu- trients), 100 mg/L myo-inositol, 1 mL/L 1000x MS vita- min stock, 1 mL/L of 1mg/mL IBA, 1 mL/L of 1 mg/mL BA and 8 g Bactoagar, pH 5.8. Rooting of shoots The apple cv Greensleaves shoots used for genetic transformation were rooted using a two-phase method: root induction and root emergence. Shoots were trans- 12 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar ferred from A17 medium to RI medium and placed under a 16-hour photoperiod for two to five days (fluorescent light). Next the shoots were transferred to RE medium without cutting off the base and placed under a 16-hour photoperiod (fluorescent light) for four to five weeks until roots emerged and leaves were fully expanded. Root induction media (RI) was identical to A17 medium, except the BA was omitted. RE medium omit- ted both BA and IBA. Agrobacterium preparation - Agrobacterium from frozen stock was inoculated into YEP medium contain- ing 50 mg/mL Rifampicin, 50 mg/mL kanamycin sul- fate and 20 mg/mL gentamicin sulfate and incubated overnight at 28ºC. The next day, five mL YEP medium was inoculated with bacteria from the plate and incu- bated with shaking at room temperature for two to three hours. Afterward, 10 μL Tetracycline were added to five mL YEP medium, swirled, combined with agro-YEP sus- pension and incubated overnight at room temperature with shaking. The OD at A420 was determined using 100 μL bacterial suspension from the overnight growth and 900 μL YEP. The bacterial cells were centrifuged at 5000 g for 15 min at room temperature, resuspended in IM medium to OD420 = 0.5 and incubated at room tempera- ture with shaking for five hrs. Agrobacterium growth medium (YEP) consisted of 5 g/L Bacto yeast extract, 10 g/L Bacto peptone and 10 g/L NaCl, pH 7.2. Virulence induction medium (IM) consisted of 431 g/L MS salts, 1 ml/L 1000 x MS vitamins, 2% sucrose, 100 mg/L myo-ino- sitol, 1 mM proline and 100 μM acetosyringone, pH 5.2. Genetic transformation protocol Leaf discs were cut from leaves of shoots grown in RE media for four to five weeks and placed immediately in Petri dishes containing co-cultivation medium solu- tion with no hormone. The leaf discs were incubated with Agrobacterium suspension for 10 to 20 minutes, blotted onto sterile Whatman filter paper to remove excess bacteria, then transferred to co-cultivation medi- um supplemented with 200 μM acetosyringone and 1 mM proline (24 discs per plate). Plates were incubated in the dark at 21ºC for three days and transferred to regen- eration medium. Plates were checked weekly for regener- ants and the explants were transferred to fresh medium monthly. As soon as they appeared, regenerated shoots were transferred to A17 medium supplemented with 200 μg/mL cefotaxime and 100 μg/mL kanamycin and incu- bated under 16 hours photoperiod at room temperature. The regenerated shoots were divided grown separately in single tubes (20 mL) in fresh selective A17 until suffi- cient material was produced for biochemical and molec- ular analyses. The first co-cultivation medium (CC) was composed of 30 g/L sorbitol, 431 g/L MS salts (macro- and micro-elements), 100 mg/L myo-inositol, 1 mL 1000 x MS vitamin, 3 mL/L 1 mg/mL IBA, and 3 g/L Gelrite, pH 5.8. The second co-cultivation medium (CC) was the same, except that the hormones were 5 mL/L 1mg/ mL BA, 1 mL/L 1 mg/mL NAA and 1 mg/mL TDZ. In regeneration medium (RG) the hormone were 5 mL/L 1 mg/mL BA, 1 mL/L 1 mg/mL NAA, 1 mL/L 1 mg/mL TDZ, 200 μg/ml cefotaxime and 100 μg/mL kanamycin. Histochemical MUG assay Fifty to 100 mg tissue was ground in 100 μL extrac- tion buffer in a microcentrifuge tube using a plastic pel- let pestle and centrifuged five to 10 min at 14000 rpm at 4 ºC at room temperature. Fifty μL supernatant was transferred to microcentrifuge tubes containing 450 μL of extraction buffer. Two hundred μL 4 mM MUG were added, mixed and immediately added to 800 μL .02 M Na2CO3 (Time 0). Time 0 and remaining sam- Figure 1. Objective and scheme of the two ovary-specific gene constructs used for genetic transformation of ‘Micro-Tom’ tomato. Role of gene IaaM in auxin biosynthesis. Also the mechanism to induce parthenocarpy is described briefly. 13Improved apple transformation protocol ples were incubated at 37ºC for 30 min. Afterward, 200 μL of the remaining supernatant was added to 800 μL .02 M Na2CO3 and mixed (Time 30). Samples were analyzed under ultraviolet light and the fluorescence of Times 30 and 0 were compared to a control with a fluorometer. Dilutions were made to read fluorescence using .02 M Na2CO3. The extraction buffer consist- ed of 50 mM NaPO4, pH 7; 10 mM EDTA, pH 8; 01% Triton X-100; .01% sodium luryl sarcosine; 7μL/10 mL 2-β-mercaptoethanol; .02 M Na2CO3 and 4 mM MUG (4-methylumbellifery glucoronide). Rooting and soil transfer of transgenic shoots The same procedure described previously to generate shoots used for genetic transformation was also used to root transgenic shoots, although RI and RE media were supplemented with 200 μg/mL cefotaxime and 100 μg/ mL kanamycin. Transgenic shoots produced expanded roots and were acclimated. Statistical analysis For each treatment, 20 petri dishes containing 12 explants were used. Three parameters were calculated for each petri dish: 1) the percentage of regeneration (explants forming on at least one shoot/total explants used), 2) the nº of regenerated shoots/total explants used, 3) the nº of groups of shoots/ total explants used. Means were calculated for each treatment and SPSS statistical software was used to analyse the data with ANOVA uni- variate and Duncan t-test (P=005). RESULTS Different hormone combinations were used to improve the genetic transformation protocol, using two constructs containing the IaaM gene driven by Def H9 or Ino, two ovule-specific promoters previously used to transform tomato. Different in vitro plant culture factors were studied for each construct. Two different hormone combinations were used during co-cultivation with the Def H9-IaaM construct. The first was the same combina- tion of hormones used for regeneration (BA:NAA:TDZ at 5, 1 and 1 mg/L, respectively). The second was 3 mg/L IBA to induce callus formation before regeneration. The effect of co-transformation with two Agrobacte- rium strains was tested: 1) with a construct containing a “VIP1” gene, and 2) with Agrobacterium containing the Ino-IaaM construct. The VIP1 gene increases the number of transformed cells and also their regeneration capacity. Co-cultivation was also studied in two experi- ments using the construct Ino-IaaM. For all transfor- mation experiments, the regeneration percentage and nº single shoots regenerated were measured to determine transformation efficiency. The number of shoot groups were also counted, although it was unclear whether such groups derived from one or several transformation events. Generally, each group formed two to six shoots, of which only one was maintained in culture for confir- mation of transformation. IBA in co-cultivation or co-transformation pro- duced fewer regenerants, a lower percentage of regenera- tion, and fewer shoots (single or groups) than BA-NAA- TDZ treatment (Table 1, Figures 2 and 3). Leaf discs transformed with Ino-IaaM showed similar results: more Table 1. Transformation of “Greensleaves” apple using construct DefH9-IaaM. The construct, date and number assigned and a description of the experiments are included. Percentage of regen- eration and number of single or grouped shoots regenerated were determined. The letters on the side of the numbers in the same col- umn indicate significative differences calculated using the Duncan test (P=005). Treatment % regeneration Nº of shoots Nº of group of shoots Control 21.04 b 1.52 b 0.72 b IBA 8.95 a 0.78 a 0.04 a VIP 4.42 a 0.47 a 0 a Figure 2. A) Genetic transformation of ‘Greensleaves’ apple with the DefH9-IaaM gene construct. Percentage of regeneration (explants forming at least one shoot/total explants) for each treatment. B). Genetic transformation of ‘Greensleaves’ apple with the DefH9-IaaM gene construct. Number of shoots/total explants and number of group of shoots/total explants are indicated for each treatment. 14 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar regeneration was obtained when co-transformation was not used (Figures 3, 4). All shoots transformed with one of the two ovule-specific constructs were transferred into a selective propagation medium containing 100 mg/L kanamicin and 200 mg/L cefotaxime to select for transgenic shoots and avoid “escapes”. Each single shoot was separated and grown separately, except in groups of indistinct shoots, where only one was chosen and propa- gated. The shoots with healthy growth were analyzed with a MUG assay to confirm the presence of the marker gene “GUS” in the constructs. Transgenic shoots were more fluorescent than control shoots (difference between Time 30 and Time 0; Table 3; Fig. 5). DISCUSSION Transformation of woody fruit species express- ing marker genes has occurred in apple (James et al. 1993), Citrus (Vardi et al. 1990) and Vitis (Scorza et al. 1995). Perennial transgenic plants that express genes of agronomic interest have been obtained in Actinidia (Rugini et al.. 1991) and apple (Norelli et al. 1994). Usu- ally, Agrobacterium-based methods were used because Figure 3. Regeneration of shoots after genetic transformation of leaf discs. On the right (a) treatment with the combination BA:NAA:TDZ (5:1:1) during co-cultivation; on the left (b) treat- ment with Agrobacterium “VIP” in co-transformation. Table 2. Transformations of “Greensleaves” apple using construct Ino-IaaM. The construct, date, assigned number and description of the experiments are indicated. Percentage of regeneration and num- ber of single or grouped shoots regenerated were measured. The let- ters on the side of the numbers in the same column for the same date experiment indicate significative differences calculated using the Duncan test (P=005). Experiment n. Treatment % regeneration Nº of shoots Nº of groups of shoots 1 Control 32.73 b 1.44 b 1.33 b 2 VIP 17.67 a 0.84 a 0.33 a 3 Control 26.13 b 0.90 b 0.93 b 4 VIP 7.73 a 0.40 a 0.25 a Figure 4. Genetic transformation of ‘Greensleaves’ apple leaf discs using the construct Ino-IaaM. The explants were cultivated in MS medium containing the combination BA:NAA:TDZ (ratio 5:1:1) either during co-cultivation or regeneration. Figure 5. A) Transformation of ‘Greensleaves’ apple using a leaf disc infected by two Agrobacterium strains simultaneously: one con- taining the construct Ino-IaaM and the second with a “gene VIP” B) Transformation of ‘Greensleaves’ apple using a leaf disc infected by two Agrobacterium strains simultaneously: one containing the construct Ino-IaaM and the second with a “gene VIP”. 15Improved apple transformation protocol Table 3. Measurements of fluorescence of five single shoots regenerated from each transformation treatment and ten control Greensleaves cultured in vitro. The construct, treatment, nº assigned to the shoot, presence of fluorescence at UV (“+” means fluorescence, “-”not fluores- cence) and concentration at the beginning (Time 0) and end of the MUG assay (Time 30) were indicated. Construct Treatment nºpetri (nºplant) UV fluorescence Total Concentration Total concentration DefH9-IaaM 1 2 (4) + 23700 159000 1 1 (3) + 28200 463000 1 6 (2) + 31300 260000 1 5 (4) + 253000 241000 1 7 (3) + 276000 245000 2 2 (3) + 75500 102000 2 4(10) + 68600 542000 2 5(4) + 27100 451000 2 8(2) + 46700 532000 2 3 (1) + 77600 746000 3 4 (8) + 67200 442000 3 3(6) + 67500 578000 3 2(5) + 43500 876000 3 3(5) + 87100 783000 3 8(4) + 85000 903000 Ino-IaaM 1 3(5) + 11000 613000 1 2(3) + 41700 403000 1 5(10) + 76500 338000 1 7(3) + 76500 338000 1 13(4) + 13300 141000 2 2(5) + 13300 141000 2 2(7) + 31300 1650000 2 11(5) + 12800 418000 2 2(4) + 58800 157000 2 4(6) + 55300 223000 3 9(4) + 12300 183000 3 11(7) + 82800 197000 3 3(4) + 30300 841000 3 15(6) + 31400 229000 3 2 (3) + 56300 437000 4 7 (11) + 68300 649000 4 12 (2) + 63900 726000 4 13 (4) + 92600 968000 4 4 (5) + 74300 319000 4 15 (3) + 28500 274000 Control 1 - 312 347 2 - 367 386 3 - 396 455 4 - 474 606 5 - 452 537 6 - 573 612 7 - 627 429 8 - 391 621 9 - 482 430 10 - 619 329 16 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar of their greater transformation efficiency and more stable integration of the transgene into the host plant genome. Agrobacterium strain LBA4404 has been used widely and the kanamycin-sensitive strain EHA105 was used to transform walnut (Mcgranahan et al. 1990) and apple (Dandekar et al. 2004). The virulence of Agrobac- terium strains against different crops can vary. Different alleles of vir G genes can increase virulence (Ghorbel et al. 2001). The expression of vir genes is also stimulated by different environmental factors, like pH, tempera- ture and osmotic conditions. The length of in vitro co- cultivation of explants with bacteria influences transfor- mation efficiency, which generally increases with time. However, co-cultivation of more than three to four days can make it difficult to control Agrobacterium growth (Petri et al. 2004). The efficiency of transformation can be increased if the medium contains phenolic com- pounds like acetosyringone or osmoprotectants such as betaine phosphate and proline. These metabolites stimu- late induction of the virulence genes (James et al. 1993). Two gene constructs, Ino-IaaM and Def H9-IaaM, previously used to transform Micro-Tom tomato, were used to test different hormone combinations to improve a genetic transformation protocol for ‘Greensleaves’ apple. A secondary objective was to create transgenic plants that might be tested in the future for partheno- carpy, since this feature might counter the auto-incom- patibility of many apple cultivars. In addition, Malus spp. are sensitive to adverse environmental conditions for pollination and/or fertilization. A parthenocarpic apple orchard would have several benefits. No pollina- tion or fertilization would be needed for fruit set, mak- ing fruit set resistant to inclement weather, which would allow consistent production of high-quality fruit. There are currently transformation protocols for many apple cultivars, such as Greensleaves (James et al. 1993), Delicious (Sriskanadarjah et al. 1994), Royal Gala (Yao et al. 1995) and Marshal McIntosh (Bolar et al. 1999). However, these protocols would benefit from more efficient regeneration of transgenic shoots. While a protocol for transformation of apple cv Greensleaves has been developed (James et al. 1993), the transforma- tion rate is only one to three % of the total explants. A recent and reliable procedure for grape transformation has been developed using meristematic bulk (MB tis- sue) made using mechanical and chemical treatments. MB tissue has a high regenerative competence and can be transformed efficiently by Agrobacterium (Xie et al.. 2016). This protocol should be tried in apple. Our protocol used mature leaf discs. The develop- mental stage of the explant is an important factor influ- encing genetic transformation. Juvenile material regen- erated better than old material in Citrus (12 to 80% vs 6%; Cervera et al. 1998). In apple, genetic transformation rates are < 3% (Dandekar et al. 2004); in pear cultivars, < 1 to 43% depending on genotype (Zhu and Welander, 2000); while in Prunus, protocols that regenerate trans- formed buds from 30% of explants were obtained almost thirty years ago (Mante et al. 1991). Our protocol tested two hormones, BA (benzyl ade- nine) and NAA (naphthalene acetic acid) for their abil- ity to stimulate regeneration of transgenic shoots. Our work was based on preliminary evidence that ‘Green- leaves’ leaf explants regenerated three to four times more shoots per explant with diphenyl urea thidiazu- ron (TDZ) combined with other medium changes, such as concentration of silver nitrate. The concentration of TDZ used is critical because high concentrations may cause “condensed” axillary shoots that do not elongate or proliferate in culture. In these experiments, a com- bination of 1 mg/mL TDZ, 5 mg/mL BA and 1 mg/mL NAA were used to regenerate transgenic shoots. Co- transformation with an Agrobacterium strain containing “VIP” genes did not increased the percentage of trans- genic shoots regenerated. Using IBA instead of the com- bination BA:TDZ:NAA during co-cultivation increased the amount of callus without increasing regeneration of transgenic shoots. Other factors that can affect regeneration were evaluated. These included the biological source of the explants (leaf age, maturity and position on the stem, explant orientation) or environmental conditions (nitro- gen concentration, growth regulators, incubation time and temperature; Oliveira et al. 1996). Here, we used mature leaf discs. Young leaves are very useful as an explant source and morphogenesis occurred mainly at the cut edges of midribs, or in association with vascu- lar tissues. Regeneration ability may be affected by stress induced by genetic transformation itself (Oliveira et al. 1996). A factor that greatly affects the regeneration capa- bility is the amount, type and timing of the antibiotics used to kill Agrobacterium (Sain et al. 1994). Together with the gene of interest, other genes are transferred to allow selection of transformed cells. Among these, anti- biotic resistance genes are common, such as the neomy- cin phosphotransferase gene (nptII) that confers resist- ance to aminoglycoside antibiotics (Miki and McHugh, 2004). Carbenicillin and kanamycin are used widely as selection antibiotics and can yield quite different results in different species. For example, in Citrus, pear, wal- nut or olive, 100 mg/L kanamycin is used for selec- tion, but in Prunus, the concentrations are usually five to 10 mg/L. In apple, alternate periods of selection and non-selection, or selection applied only on the regener- 17Improved apple transformation protocol ated shoots, were used (James et al. 1993). Selection of transformed shoots is also complicated by the presence of escapes (non-transformed shoots) due to inactivation of antibiotics by transformed cells or by the persistance of Agrobacterium in the explants. Because of public concern with introducing antibiotic resistance genes into food, methods have been developed to eliminate them from the selection process (Zuo et al. 2002). For instance, a reporter gene such as Gus (β-glucoronidase gene) can be used to evaluate transformation efficiency by visual selection. To avoid bacterial contamination, Gus genes that cannot be spliced out by the host cells were used. In Prunus, this method is still complicated by intrinsic GUS-like activity of the plants. The number of transformants obtained is usually underestimated by at least 25% when based on the expression of screen- able marker genes (Oliveira et al. 1996). Kanamycin resistance is still a common strategy for selecting trans- genic shoots, but the strong selection required to avoid escapes or chimeras reduces the number of cells that both received the DNA and regenerated buds. An inno- vative approach has improved transformation efficiencies tenfold over kanamycin selection in recalcitrant species. This method is based on giving transformants a meta- bolic advantage, rather than on killing non-transformed cells (Joersbo, 2001). It is hypothesized that necrosis pro- duced by antibiotics in non-transformed tissues could inhibit regeneration from transformed adjacent tissues (Joersbo, 2001). Using regeneration-promoting genes, combined with hormone-free regeneration medium, could also substitute for traditional antibiotic marker genes. With no growth regulators, only transformed cells can regenerate, allowing simple screening for puta- tive transformants without using a marker gene. Much work is devoted to identifying regenerating- promoting genes, presumably related to cytokinin syn- thesis, that enable the embryogenic or organogenic transition (Zuo et al. 2002). The Ipt gene, from Agrobac- terium, must be used under the control of a inducible promoter, because constitutive over-expression of this gene can cause phenotypic growth disorder (Kunkel et al. 1999). CONCLUSIONS Explants transformed with either Ino-IaaM or Def H9-IaaM transgenes regenerated more shoots on combination of three hormones (BA:NAA:TDZ) than on IBA and co-transformation had no effect. In experi- ments using Def H9-IaaM, the percentage of regenera- tion for the hormone combination was significatively greater than for the other two treatments (21.04% vs 8.95 and 4.42%, respectively). The number of transgenic shoots was also greater with the hormone combination (1.52% vs 0.78 and 0.47%, respectively). Experiments using Ino-IaaM confirmed these results. Co-transfor- mation with Agrobacterium containing VIP genes was deleterious to production of regenerants, possibly due to a lower concentration of Agrobacterium containing the Ino-IaaM or Def H9-IaaM transgene during infection. Most shoots regenerated in selection medium con- taining 100 mg/L kanamycin at were transgenics with significantly greater fluorescence in the MUG assay than untransformed, regenerated Greensleaves. This sug- gests that this concentration of kanamycin provided a good balance between selection of transgenic shoots and allowing reasonable regeneration efficiency. AUTHOR CONTRIBUTIONS MF and AMD designed and conceived the research work. MF performed the experimental work and statisti- cal analysis. MF mainly wrote the article. AP and AMD reviewed and discussed results. All authors contributed significantly on the writing of the manuscript. REFERENCES Bolar J.P., Norelli J.L., Harman G.E., Brown S.K., Ald- winckle, H.S. 2001. Synergistic activity of endochi- tinase and exochitinase from Trichoderma atroviride (T-harzianum) against the pathogenic fungus (Ventu- ria inaequalis) in transgenic apple plants. Transgenic Research, 10 (6): 533-543. Bolar J.P., Norelli J.L., Wong K.W., Hayes C.K., Harman G.E., Aldwinckle H.S. 2000. Expression of endochi- tinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology, 90 (1): 72-77. Broothaerts W., Keulemans J., Van Nerum I. 2004. Self- fertile apple resulting from S-RNase gene silencing. Plant Cell Reports, 22 (7): 497-501. Cervera M., Pina J.A., Juraez J., Navarro L., Pena L. 1998. Agrobacterium-mediated transformation of citrus:factors affecting transformation and regenera- tion. Plant Cell Reports, 18 (3-4): 271-278. Dandekar A.M., 1992. Transformation. In:Biotechnology of Perennal Fruit Crops. Hammerschlag FA Litz RE eds CAB International, 141-168. Dandekar A.M., Teo G., Defilippi, B.G., Uratsu, S.L., Pas- sey, A.J., Kader, A.A., Stow, J.R., Colgan R.J.,, James 18 Federico Martinelli, Anna Perrone, Abhaya M. Dandekar D.J. 2004. Effect of down-regulation of ethylene biosyn- thesis on fruit flavor complex in apple fruit. Transgen- ic Research, 13 (4): 373-384. Dandekar A.M., McGranahan G.H., Vail P.V., Uratsu S.L., Leslie C.A., Tebbets J.S. 1998. High levels of expression of full-length cryIA(c) gene from Bacillus thuringiensis in transgenic somatic walnut embryos. Plant Science, 131 (2): 181-193. Delbarre A., Muller P., Imhoff V., Barbierbrygoo H., Maurel C., Leblanc N., Perrotrechemann C., Guerin J. 1994. The RolB gene of Agrobacterium-rhyzogenes does not increase the axin sensitivity of tobacco proto- plasts by modifying the intracellular auxin concentra- tion. Plant Physiology, 105 (2): 563-569. Donzella G., Spena A., Rotino G.L.. 2000. Transgenic parthenocarpic eggplants:superior germplasm for increased winter production. Molecular breeding, 6 (1): 79-86. Faize M., Sourice S., Dupuis F., Parisi L., Gautier M.F., Chevreau E. 2004. Expression of wheat puroindoline-b reduces scab susceptibility in transgenic apple (Malus x domestica Borkh). Plant Science, 167 (2): 347-354. Fitch M.M.M., Manshardt R.M., Gonsalves D., Slightom J.L. 1993. Transgenic papaya plants from agrobacte- rium-mediated transformation of somatic embryos. Plant Cell Reports, 12: 245-249. Ghorbel R., La-Malfa S., Lopez M.M., Petit A., Navarro L., Pena L. 2001. Additional copies of virG from pTi- Bo542 provide a super-transformation ability to Agro- bacterium tumefaciens in citrus. Physiological and Molecular Plant Pathology, 58 (3): 103-110. Hidaka T., Omura M. 1993. Transformation of citrus pro- toplasts by electroporation. Journal of the Japanese Society for Horticultural Science, 62 (2): 371-376. James D.J., Uratsu S., Cheng J.S., Negri P., Viss P., Dan- dekar A.M. 1993. Acetosyringone and osmoprotectants like betaine or praline synergistically enhance agrobac- terium-mediated transformation of apple. Plant Cell Reports, 12 (10): 559-563. Jia D., Fan L., Shen J., Qin S., Li F., Yuan Y. 2019. Genet- ic transformation of the astaxanthin biosynthetic genes bkt and crtR-B into apple tree to increase photoox- idation resistance. Scientia Horticulturae, 3: 428-433. Joersbo M. 2001. Advances in the selection of transgenic plants using non-antibiotic marker genes. Physiologia Plantarum, 11 (3): 269-272. Kunkel T., Niu Q.W., Chan Y.S., Chua N.H. 1999. Induc- ible isopentenyl transferase as a high-efficiency marker for plant transformation. Nature Biotechnology 17 (9): 916-919. Liu Q., Ingersoll J., Owens L., Salih S., Meng R., Ham- merschalg F., 2001- Response of transgenic Royal Gala apple (Malus x domestica Borkh) shoots carrying a modified cecropin MB39 gene, to Erwinia amylovora. Plant Cell Reports, 20 (4): 306-312. Mante S., Morgens P.H., Scorza R., Cordts J.M., Callahan A.M. 1991. Agrobacterium-mediated transformation of plum (Prunus domestica l) hypocotil slices and regen- eration of transgenic plants. Bio/Technology, 9: 853- 857. Martinelli F., Uratsu S.L., Reagan R.L., Chen Y., Tricoli D., Fiehn O., Rocke D.M.., Gasser C.S., Dandekar A.M., 2009. Gene regulation in parthenocarpic toma- to fruit. Journal of Experimental Botany, 60 (13): 3873-3890. McGranahan G.H., Leslie C.A., Uratsu S.L., Dandekar A.M., 1990. Improved efficiency of the walnut somat- ic embryo gene-transfer system. Plant Cell Reports, 8 (9): 512-516. Meister R.J., Williams L.A., Mona M.M., GallAgher T.L., Kraft E.A., Nelson C.G., Gasser C.S. 2004. Definition and interactions of a positive regulatory element of the Arabidopsis INNER NO OUTER promoter. The Plant Journal, 37: 426-438. Miki B., McHugh S., 2004. Selectable marker genes in transgenic plants:applications, alternatives and biosafe- ty. Journal of biotechnology, 107 (3): 193-232. Nakamura Y., Sawada H., Kobayashi S., Nakajima I., Yoshikawa M. 1999. Expression of soybean beta- 1,3-endoglucanase cDNA and effect on disease toler- ance in kiwifruit plants. Plant Cell Reports, 18 (7-8): 527-532. Norelli J.L., Aldwinckle H.S., Destefanobeltran L., Jaynes J.M. 1994. Transgenic malling-26 apple expressing the attacin-E gene has increased resistance to Erwinia amylovora. Euphytica, 77 (1-2): 123-128. Petri C., Alburquerque N., Garcia-Castillo S., Egea J., Burgos L. 2004. Factors affecting gene transfer efficien- cy to apricot leaves during early Agrobacterium-medi- ated transformation steps. Journal of Horticultural Science and Biotechnology, 79 (5): 704-712. Puterka G.J., Bocchetti C., Dang P., Bell R.L., Sorza R. 2002. Pear transformed with a lytic peptide gene for disease control affects nontarget organism, pear psylla (Homoptera; Psyllidae). Journal of economic ento- mology, 95 (4): 797-802. Oliveira M.M., Miguel C.M., Raquel M.H. 1996. Trans- formation studies in woody fruit species. Plant Tissue Culture and Biotechnology, 2: 76-92. Rizzini F.M., Bonghi C., Chkaiban L., Martinelli F., Tonutti P. 2010. Effects of postaharvest partial dehy- dration and prolonged treatments with ethylene on transcript profiling in skins of wine grape berries. Acta horticulturae 877: 1099 -1104. 19Improved apple transformation protocol Sain S.L., Oduro K.K., Furtek D.B. 1994. Genetic-trans- formation of co-cua leaf-cells using agrobacterium- tumefaciens. Plant Cell and Organ Culture, 37 (3): 243-251. Sedira M., Holefors A., Welander M. 2001. Protocol for transformation of the apple rootstock Jork 9 with the rolB gene and its influence on rooting. Plant Cell Reports, 20 (6): 517-524. Smigocki A.C., Hammerschlag F.A. 1991. Regeneration of plants from peach embryo cells infected with a shooty mutant strain of agrobacterium. Journal of the Ameri- can society for Horticultural Science, 116 (6): 1092- 1097. Raman V., Anand A., Vasudevan B., Morsy R.M., Pant B.D., Lee H.-K, Tang Y., Mysore K.S. 2019. Over- expression of  VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium- mediated plant transformation and abiotic stresses. Scientific Reports, 9: 13503. Rugini E., Pellegrineschi A., Mencuccini M., Mariotti D., 1991. Increase of rooting ability in the woody species kiwi (Actinidia deliciosa  A Chev) by transformation with  Agrobacterium rhizogenes rol  genes. Plant Cell Reports, 10: 291-295. Schuerman P.L., Dandekar A.M. 1993. Transformation of temperature woody crops-progress and potentials. Sci- entia horticulturae, 55 (1-2): 101-124. Scorza R., Cordts J.M., Ramming D.W., Emershad R.L. 1995. Transformation of grape (Vitis vinifera L) zygot- ic-derived somatic embryos and regeneration of trans- genic plants. Plant Cell Reports, 14 (9): 589-592. Sriskandaraiah S., Mullins M.G., 1981. Micropropagation of Granny Smith apple-factors affecting root-formation in vitro. Journal of Horticultural Science, 56 (1): 71-76. Sriskandarajah S., Goodwin P.B., Speirs J., 1994. Genetic- transformation of the apple scion cultivar delicious via agrobacterium-tumefaciens. Plant Cell Tissue and Organ Culture 36 (3): 317-329. Tosetti R., Martinelli F., Tonutti P., Barupal D.K. 2010. Metabolomics approach to studying minimally pro- cessed peach (Prunus persica) fruit. Acta Horticultu- rae 1017-1021. Yao J.L., Cohen D., Atkinson R., Richardson K., Morris B. 1995. Regeneration of transgenic plants from the com- mercial apple cultivar Royal gala. Plant Cell Reports, 14 (7): 407-412. Vardi A., Bleichman S., Aviv D. 1990. Genetic transforma- tion of citrus protoplasts and regeneration of transgenic plants. Plant Science, 69 (2): 199-206. Xie X., Aguero C., Wang Y., Walker A. 2016. Genetic transformation of grape varieties and rootstocks via organogenesis. Plant Cell, Tissue and Organ Culture, 126: 541-552. Welander, M., Pawlick, N., Holefors, A., Wilson, F. 1998. Genetic transformation of the apple rootstock M26 with the RolB gene and its influence on rooting. Jour- nal of Plant Physiology, 153 (3-4): 371-380. Zhu L.H., Welander M. 1999. Growth characteristics of apple cultivar Gravenstein plants grafted onto the transformed rootstock M26 with rolA and rolB genes under non-limiting nutrient conditions. Plant Science, 147 (1): 75-80. Zuo J.R., Niu Q.W., Ikeda Y., Chua N.H. 2002. 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