This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
A Protocol for Agrobacterium-mediated Transformation of Cucumber (Cucumis sativus L.) from cotyledon explants
zhenxian zhang
Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, College of Horticulture, China Agricultural University, Beijing, P. R. China.
Corresponding Author
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Genetic transformation is central to the discovery, delivery, and functional analysis of genes, and for associated crop improvement, and yet is technically challenging in many plant species. As an example, cucumber (Cucumis sativus L.) is a model species for the study of phloem characteristics, raffinose family oligosaccharide (RFO) metabolism and sex determination, but methods for its genetic transformation have generally had low efficiency, thereby limiting basic and applied studies. Here, we describe a rapid and efficient protocol for Agrobacterium-mediated transformation of cucumber cotyledon explants, using vacuum infiltration. The transformation efficiency of the regenerated plants was as high as 54%, and the final positive plantlet transformation efficiency was approximately 26% of the total number of infected explants and this is obviously higher than previously published protocols. Transgenic plantlets can be obtained in 3 to 4 months and the transgenic T1 seeds generated in the subsequent 3 to 4 months after self-pollination. Using this protocol, we have obtained more than 600 transgenic cucumber lines. This protocol is of great importance for studies of cucumber and of other Cucurbitaceae species, since it enables gene functional analysis, and so opens a pipeline for rapid cucumber variety improvement.
Keywords
cucumber, Agrobacterium-mediated, transformation
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Cucumber (Cucumis sativus L.), a crop with economic and nutritional importance that is cultivated worldwide, is a typical raffinose family oligosaccharides (RFOs)-transporting plant1-4, and a model species for studying phloem functional characteristics and sex determination. Cucumber phloem is very complicated, there is one set of vascular bundle (VB, a bicollateral bundle including the internal and external phloem) in the petiole, stem and peduncle, but four sets of VBs in the fruit, including the peripheral, main, carpel and placental VB (two bicollateral, two collateral). Cucumber is a monoecious species with unisexual flowers, and is a model species for the study of sex determination. It has seven types of sex phenotypes, including andromonoecious, monoecious, androecious, heterozygous-gynoecious, hermaphrodite, gynoecious and gynoecious-monoecious. In addition, it can be parthenocarpic. However, a lack of effective genetic transformation methods, especially those based on Agrobacterium, has severely limited cucumber gene functional analyses and remains a bottleneck for both developing cucumber as a model, and for its improvement as a crop.
The transfer and stable integration of genetic material into the cucumber genome will enhance the breeding of more disease resistant cucumber (Cucurbitaceae plants or cucurbits) varieties, as well as the characterization of cucumber genes. It offers the possibility to introduce new characters into a cucumber cultivar, which is difficult to achieve via conventional breeding techniques5. Recently, the genome sequences of both cucumber and watermelon, another cucurbit, were released (http://www.icugi.org/cgi-bin/ICuGI/index.cgi). Moreover, a range of studies have investigated diverse aspects of cucumber biology, such as the rapid fruit expansion that occurs after flowering, and some candidate genes that influence this important process have been identified6,7. The development of an efficient cucumber transformation system is therefore timely and critical for gene discovery and validation.
Many studies have reported successful cucumber transformation, but only a few have obtained a high transformation rate and T1 generation, and there are very few reports on functional analysis using transformed plants (Table 1). The first transformation of cucumber plants, which expressed the NPT II gene, was reported by Trulson et al. (1986)8. This transformation was achieved by infecting cucumber hypocotyl segments with Agrobacterium rhizogenes and regenerating the transformed issue into whole plants. This approach had a low plant regeneration frequency, which limited its utility (Table 1). Other reports have described the successful regeneration of cucumber plants from cotyledon explants after transformation using A. tumefaciens, but transformation efficiency was still < 12%, and only a few of these studies resulted in transgenic T1 plants (Table 1). Higher transformation rates (16-23%) have been reported using cotyledon explants9-11, but in these studies, the transformation rate was calculated as the identified positive shoots and not plantlets, and few of these lines resulted in a stably transformed T1 generation. The selection of cucumber transformants using antibiotic selection with kanamycin9,12, phosphinothricin11,13 and hygromycin14, have been reported; however the method with the high transformation efficiency (23%) reported by He et al. (2006) was based on mannose and sucrose resistance10 (Table 1).
Other regeneration protocols with various culture types have also been investigated, such as leaf explants15, petioles15,16, hypocotyls14,17, internodes17, protoplasts18 and suspension cultures19,20, but these all showed a lower transformation efficiency than using cotyledons as explants. Until now, Agrobacterium-mediated transformation methods using cotyledons as explants has been the most successful approach for cucumber transformation21. With this approach, the transformation frequency is dependent on several factors, including plant genotype, explant source and size, Agrobacterium strains and concentration, length of exposure to Agrobacterium, and selection system22,23, among others. It was reported that dissecting half of an adaxial cotyledon using a V-shaped cut resulted in a higher transformation rate24, while another report showed that using cotyledon nodes as explants resulted in regeneration at the junction of cotyledon and hypocotyl in the proximal regions of the cotyledons12. In this current protocol, we use similar explants, half adaxial cotyledons, but we have modified the protocol in that we break off the hypocotyl instead of using the V-shaped cut to detach the growing point. We also highlight the key factors in the procedure that can cause technical problems for the inexperienced researcher.
Our laboratory has established a rapid and efficient Agrobacterium-mediated transformation system for cucumber that differs from previously reported protocols, and we have identified critical factors that influence transformation efficiency. To date, we have produced more than 600 transgenic lines and functionally analyzed a range of cucumber genes using this protocol25-33. The method can be widely used in cucumber breeding, variety improvement, and the characterization of gene function, and can be a reference for the transformation of other cucurbits, such as Cucumis melo and C.metuliferus21,23.
Overview of the technique (Figure 1)
This protocol consists of seven major steps, which can be briefly summarized as follows: Stage I: preparation of sterilized seeds. Stage II: explant preparation, infection and co-cultivation with A. tumefaciens. Stage III: shoot regeneration. Stage IV: root regeneration. Stage V: acclimation and molecular identification of T0 plants. Stage VI: cultivation and self-crossing of T0 plants. Stage VII: seed harvest from T0 plants.
Key factors for successful transformation of cucumber
**Varieties amenable to _Agrobacterium-mediated transformation**_
It has been reported that the genotype of transformed cucumber influences its shoot regeneration frequency9,21, and relatively high regeneration rates have been reported for some cucumber genotypes, such as Poinsett 76, Jinyan No.7, Cengel Köy, Shinhokusei 1, cv. pickling genotype 3676, 3672, and Xintaimici (Table 1). However, in our analyses we observed no differences in the regeneration frequency between different cucumber genotypes. We obtained a high regeneration rate (80-97 %) for the Deltasta, Guonong25, cucumber line 3413, 3407, 3461, Chinese long: 461, 9930, R1407, R470, short 32x, and Xintaimici genotypes, and obtained transgenic plants from all these varieties. Even so, it is advisable to determine the regeneration rate before using a new cucumber genotype for Agrobacterium-mediated transformation, in order to optimize the chance of successful transformation. We note that homozygous varieties should be considered, since they can provide a homozygous transgenic T1 generation for subsequent gene functional studies.
Preventing contamination with endophytic bacteria
Different endophytes may be associated with different parts of the cucumber seed. Before soaking seeds in tap water for 2 hours, they should be sterilized with HCl and NaClO in a sealed container for 1-2 hours, to kill bacteria on the seed surface or inside the seed coat. Some endophytic bacterial species have been isolated from surface sterilized cucumber seeds, and most of these were located in the seedling radicles34,35. It is therefore important to cut off the radicles before detaching the cotyledons to prevent the contamination with endophytic bacteria, and then knocking or breaking off the hypocotyl on new sterilized filter paper.
Explant preparation
The selection and preparation of explants is critical factor in the success of the transformation pipeline. In our protocol, the embryonic cotyledons are used as the explants. The cucumber seeds should be soaked in water for 2 hours to facilitate the removal of the seed coats and to obtain a high germination rate. The de-coated seeds should then be surface sterilized with 75% (vol/vol) alcohol for no more than 1 minute.
After germination for approximately 2 days on MS/B5 medium, a radicle of 1-1.5 cm and root hairs are visible, and the cotyledons can be detached from the embryos. Half of the adaxial cotyledon is then used as the explant. Seeds have a better germination rate on B5 medium than on MS medium; however, different varieties may have a difference in germination time and media requirements, as well as in how readily the cotyledons can be detached. Forceps, rather than a knife, should be used to detach the cotyledons from the radicle growing point (Figure 1, Explant preparation) in order to obtain a higher regeneration rate.
Previous studies have shown a high regeneration frequency with cotyledon nodes as explants36, and we also found that breaking off the hypocotyl to detach the cotyledon, rather than cutting it, is important to obtain high regeneration frequency. However, care needs to be taken to cut off the growing point of the seedlings to prevent negative plants growing.
Influence of abscisic acid (ABA) and 6-benzylaminopurine (BAP) levels on shoot organogenesis during shoot initiation and outgrowth
It has been reported that the hormones ABA and BAP can induce the regeneration of cucumber cotyledon explants36, 38, and most of these studies focused on the influence of BAP levels on cucumber shoot regeneration. In this study we have investigated the combined effects of BAP and ABA levels on shoot regeneration from cotyledon explants (Table 2, Supplementary Figure S1). We observed that the highest shoot regeneration rate were 76% and 86%, using a ABA/BAP concentration of 1.0/0.8-1.5 mg/l (Table 2), and noted that the regenerated shoots grew more slowly with higher ABA and lower BAP concentrations (Supplementary Figure S1 A). We therefore chose an ABA/BAP concentration of 1.0/0.8-1.5 mg/l, as this resulted in healthy and fast growing plants, as well as a high regeneration rates with the Agrobacterium-mediated transformation system. We compared efficacy of the MS medium and B5 medium for shoot regeneration with a 1.0 mg/l ABA concentration and different BAP concentration, and found that the average number of regenerated shoots per explant in MS and B5 media was 2.5 and 1.0, respectively (Table 3, Supplementary Figure S1 B). We concluded that use of the MS medium resulted in the production of more shoots than the B5 medium (Table 3), while the B5 medium was more effective for seed generation than the MS medium.
Agrobacterium concentration and infection time
The Agrobacterium concentration and the infection time were found to be important factors in preventing explant turning necrosis and improving transformation efficiency. We determined that the optical density (OD, at 600 nm) of the Agrobacterium culture should be adjusted to approximately 0.25 (Table 4), and that the infection time should be about 15 minutes to avoid explant necrosis, and to obtain the highest regeneration rate of 86% (Table 4).
Agrobacterium vacuum infiltration
It has been reported that vacuum infiltration can improve the Agrobacterium-mediated transformation of cucumber12, and we also found this to be the case. Since we had found that the infection time and Agrobacterium concentration were important in obtaining a high transformation rate, we vacuum infiltrated with Agrobacterium solution and explants in beakerflask (OD600=0.25) twice for 5 min, and then move them into shaker for an additional 5 min.
Orientation of the explants on the co-cultivation medium
After Agrobacterium infection, placed the cotyledon explants on co-cultivation medium with the adaxial surface facing upward, so that the abaxial surface was in contact with the culture medium. We found this to be essential for high transformation efficiency. After 2 days of co-cultivation, we transferred the cotyledons to the shooting medium, and again made sure that the abaxial surface of the cotyledon was touching the medium.
Shoot regeneration and rooting
We determined that when regenerated shoots have grown to 1 cm, they should be excised from the cotyledon explants and transferred to rooting medium. Embryoids that appear on the cut end surface should be detached. Regenerated shoots will then grow rapidly and the leaves will expand immediately after transfer to rooting medium. The un-rooted regenerated shoot end shoot then be re-cut and the unfolded leaves and the shoots transferred into new rooting medium once per week for the next 2-3 weeks, ensuring that the lower part of the regenerated shoots are inserted in the rooting medium. We found this to be an effective way to remove residual ABA/BAP from the shoots and to be important for efficient rooting.
Antibiotic Selection
The use of kanamycin selection has previously been reported for cucumber transformation37,38, and we found it to be more effective than hygromycin since the latter affects plant development. Kanamycin concentrations of 50-150 mg/l are commonly used14,22,39, and it has been shown that a higher concentration will slow growth at the rooting stage and suppress regeneration24. Here, 50 mg/l was used during shoot initiation, shoot outgrowth and the rooting stage to encourage the regeneration of transgenic plants. In addition to antibiotic selection, PCR should be used to confirm the presence of the targeted transgene in each transformant at each generation.
Plant growth conditions
Since cucumber is a warm-season crop, the tissue cultures should be kept at 25°C, and after plant acclimation in the soil, the temperature should be maintained at 28°C/18°C with a 16 h/8 h light/dark cycle, which are typical cucumber cultivation conditions.
REAGENTS
Cucumber (Cucumis sativus L.) seeds
Agrobacterium strain
Sodium hypochlorite, 13% (wt/vol) (NaClO; Chem-Supply, CAS 7681-52-9)
Hydrochloric acid, HCl, 36.0-38.0% (Sinopharm Chemical Reagent Co., Ltd, 80070591)
B5 Medium (Waryong，GT213250B)
Waryong Phytagel plantcell Ⅱ, (Waryong，GT21026M)
Agar (Biowest, 9012-36-6) for LB plates
Tryptone (Oxoid, cat. no. 91079-40-2)
Yeast extract (Oxoid, cat. no. 8013-1-2)
Sodium chloride (NaCl; Chem-Supply, GB/T 1266-19867520, cat. no. 10019792)
Sodium hydroxide (NaOH; Chem-Supply, cat. no.1310-73-2)
Sterile distilled water
Ethanol (Chem-Supply, cat. no. 10009292)
Ethanol 75% (Vol/Vol) in distilled water
Sucrose (C12H22O11; Chem-Supply, cat. no. 10021492)
6-Benzylaminopurine (BAP, C12H11N5l; Sigma, cat. No. B3408)
Abscisic acid (ABA, C15H20O4; Bjxjksw, cat. no. 21293-29-8)
Rifampicin (C43H58N4O12; Bjxjksw, cat. no. 13292-46-1)
Kanamycin monosulfate (C18H36N4O11﹒H2SO4)
Carbenicillin disodium salt (C17H16N2O6SNa2; MP Biomedicals, cat. No. 195092)
Murashige and Skoog basal medium with vitamins (PhytoTechnology Laboratories, cat. No. M519)
Gellan gum powder (PhytoTechnology Laboratories, cat. No. G434)
PCR Primerstar mix (Takara Biotechnology, Cat. no. R045A)
REAGENT SETUP
LB liquid medium for Agrobacterium preparation
Dissolve 5 g of yeast extract, 10 g tryptone and 5 g sodium chloride in 1 liter of distilled water; pH 7.0 autoclave. Add 50 μl of 100 mg/ml rifampicin stock and 50 μl of 50 mg/ml kanamycin stock to 50 ml LB solid medium.
Murashige and Skoog (MS) liquid medium for Agrobacterium suspension Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8 autoclave
Murashige and Skoog (MS) plates for seed germination (germination medium)
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
B5 plates for seed germination (germination medium)
Dissolve 30.21 g B5 medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Cocultivation medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water, add 750 μl 2 mg/ml BAP; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 500 μl 2 mg/ml ABA, and pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Shooting medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water, add 750 μl 2 mg/ml BAP; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 500 μl 2 mg/ml ABA, 50 μl of 50 mg/ml kanamycin and 2.5 ml of 200 mg/ml carbenicillin. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Rooting medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 50 μl of 50 mg/ml kanamycin and 1 ml of 200 mg/ml carbenicillin. Pour about 40 ml of medium into 100 ml glass beaker under laminar flow hood.
Sterile 50 ml plastic tubes (Corning, cat. no. 430828)
Autoclave (Cheng YI, LS-B35 Type; cat. no. 09J-3298)
Controlled tissue culture rooms 25 °C/25 °C, 16/8 h light/dark regime
Controlled environmental rooms (16 h photoperiod) or glasshouse facilities for whole plant culture
Orbital incubator shaker (Pei Ting, THZ-C-1 Type)
Tabletop orbital shaker
Vacuum pump (DA-60D; ULVAC, Kanagawa, Japan) was connected to a desiccator (VM-C; AS ONE, Osaka, Japan)
PH meter (PHSJ-4A Type)
Laminar flow hood for plant culture work
Glassware (100 ml glass beaker, graduated cylinders, glass Petri dish, Duran bottles and Erlenmeyer flasks)
Filter paper 9 cm diameter (15-20 μm)
Parafilm (Bemis Flexible Packaging)
230 mm pot (230 mm in diameter nd 190 mm in height) for cucumber cultivation
Forceps and scalpel
Surgical blades
Plant growth culture vessels
Pipettes: P1000, P200, P20, P10
Sterile pipettes tips (Axygen Scientific: 1–200 μl, cat. no. T-200Y; 1–1,000 μl, cat. no. T-1000B; 1–20 μl, cat. no. T-20Y; 1–10 μl, cat. no. T-10W)
Centrifuge
Spectrophotometer (SFZ0806011544 Type)
Mini Vortexer (QL-901 Type)
Remove the seed coats, surface sterilization and germination of cucumber seeds •TIMING 3 hours for handling, 2 days for cultivation (seeds germination)
- Use Cl2 (add 2 ml HCl to 15 ml NaClO in a seal container, please be careful) sterile the cucumber seeds for 1-2 hours, not for long. Soak cucumber mature seeds in tap water for 2 h at room temperature in Petri dish.
- Remove the seed coats from seeds with forceps by pinch the pointed end of seeds first and then push aside the seed coats.
- Sterilize 20-50 seeds for 30 s with 20 ml of 75% ethanol in a sterile Petri dish with a lid. Drain ethanol and rinse with 20 ml of 3% sodium hypochlorite solution. Gently shake the seeds on a shaker for 15 min. Rinse three times with sterile deionized water. From this step to step 18 should be performed in a laminar flow hood.
- Place surface-sterilized seeds with forceps onto Petri plates containing seed germination medium, allowing 15-25 seeds per plate. Germinate the seeds in the dark at 28°C for 2 d. Inoculum preparation •TIMING 30 min for handling, 2 days for cultivation
- On the day after Step 4, inoculating 2 ml of LB liquid medium containing rifampicin (100 mg/l) and Kanamycin (50 mg/l) with a single Agrobacterium colony carrying the appropriate construct. Culture at 28°C in a shaker at 200 rpm for 24 h.
- Subculture 200 μl of 2 ml culture from Step 6 into 50 ml of LB liquid medium containing rifampicin (100 mg/l) and Kanamycin (50 mg/l). Culture at 28°C in a shaker at 200 rpm until the OD at 600 nm to 0.6-0.8.
- Spin the Agrobacterium culture down (4000g or 5000g) for 10 min at RT (room temperature). Remove the supernatant and rinse the pellet in 1 ml liquid MS liquid medium. Dilute the MS medium containing Agrobacterium to OD600=0.25 using MS liquid medium. Explant preparation, Agrobacterium infection and co-cultivation •TIMING 4 hours for handling, 2 days for cultivation (co-cultivation)
- Pull the seedlings (from Step 5) out of the germination medium and place in an empty sterile Petri plate dishes with a stack of sterile filter paper.
- Cut the radicle first, and then cut off 1/2-1/3 the end of cotyledon off and remove the endopleura off at the same time. Detach the first cotyledon by using the sterile scalpel to wipe the upside cotyledon off. Knocking the hypocotyl over to detach the downside cotyledon and growing point using sterile forceps and scalpel (Fig. 1).
- Collect the detached cotyledons into MS liquid medium (without Agrobacterium) in a 50 ml sterile glass beaker.
- Pour out the MS liquid medium (without Agrobacterium), and pour into MS liquid medium (with Agrobacterium OD600=0.25 from Step 8) and shake gently. Cover the sterile glass beaker with sterile culture container sealing film and put into the desiccator which connected with the vacuum pump. After 2 sessions of vacuum infiltration were applied for 5 min, explants were leave for another 5 min in Agrobacterium.
- Pour out the MS liquid medium (with Agrobacterium), put a sterile filter paper onto cocultivation medium and touch the medium exactly. Place the infected explants on the filter paper and keep the adaxial surface of the cotyledons upward using forceps (Fig. 1), allowing about 20-35 explants per Petri dish plate. Seal the plate and incubate in dark at 28°C for 2d. Shoot initiation •TIMING 0.5 hours for handling, 2-3 weeks for cultivation (shoot regeneration)
- Transfer explants onto shooting medium containing 50 mg/ml kanamycin and 500 mg/ml carbenicillin to select the transgenic shoot and inhibit the growth of Agrobacterium, allowing 5-6 explants per Petri dish plate, as the explants will grow up. Incubate under light (150 μmol m-2 s-1, 16 h) at 25°C for 2-3 weeks. Regeneration of transgenic plants •TIMING 3 hours for handling, 4-6 weeks for cultivation (root regeneration)
- After the shoots growth out from the explants (Fig. 1), pull the explants out and place in an empty sterile Petri plate dishes with a stack of sterile filter paper.
- Cut the shoots off using sterile forceps and scalpel. Remove the embryoids on the end part of the shoots. Transfer shoots to 100 ml glass Erlenmeyer flasks or glass tissue culture vessels containing rooting medium with kanamycin (50 mg/l) and carbenicillin (500 mg/l). Place three to four shoots per Erlenmeyer flasks. Incubate under light (150 μmol m-2 s-1, 16 h) at 25°C for 1-2 weeks.
- After 1-2 weeks, take out the no rooting plants and recut the unfolded leaves and the end part of the shoots using sterile forceps and tweezers and transfer shoots to new rooting medium. Repeat this procedure one times per week in the next 2-3 weeks. Place one to two shoots per glass Erlenmeyer flasks. Incubate in under light (150 μmol m-2 s-1, 16 h) at 25°C for 2-4 weeks. Plant acclimation •TIMING 3 hours for handling, 3-5 weeks for cultivation
- After the roots grow strongly, open the cover of the glass Erlenmeyer flasks. Put the cover on the top of the Erlenmeyer flasks loosely. Incubate in dim light (10-50 μmol m-2 s-1) at 25°C for 3 days.
- Knock the bottom of the Erlenmeyer flasks gently and make the medium not firmed. Pull out the plants from the Erlenmeyer flasks gently using forceps. Wash off the medium with running tap water gently.
- Fill the pots with wet compost mixture, place the regenerated plants into the compost by making a hole. Compact the soil and water slightly.
- Cover the plants with zip bags and tight to the pot using rubber band to keep moisture. Incubate in good condition chamber for cucumber growth under light (gradually from 300 μmol m-2 s-1 to 500 μmol m-2 s-1, 16 h) at 25°C/18°C for 1-2 weeks. Add water into the tray under pots when the soil is getting dry.
- Take the zip bags off after the regeneration plants grown in a good condition. Watering the plants when the soil is getting dry. Incubate in under light (500 μmol m-2 s-1, 16 h) at 25°C/18°C for 1-2 weeks. Examination of transgenic plants •TIMING 1 day for handling
- Number the regeneration cucumber plants and cut a peace of the mature leaves, extract the genome DNA using the cetyltrimethy ammonium bromide (CTAB) method40.
- Confirm the transgenic plants has been inserted in the T-DNA using the specific primers designed on the T-DNA and do PCR amplification and gel electrophoresis assay. Seed production from transgenic plants •TIMING 1 hour for handling, 8-12 weeks for cultivation
- Transfer the molecular identified transgenic plants to greenhouse, to give seedlings normal environment condition with the temperature no higher than 33°C and no lower than 16°C, light intensity 500 μmol m-2 s-1, relative humidity 70-80%.
- The transgenic cucumber plants require normal cultivation management and artificial self-pollination when plants flowering. On the day before the female and male flower blooming, clip the both the female and male flowers on the same plants, and then do artificial pollination using the same plants male flowers to the female flowers on the second morning, and then clip the female flowers to keep away the pollen from the other plants.
- When the epidermis of autocarps are turned yellow and indicated that the seeds are fully mature, pick them up and place under the cool, dry place for 1-2 weeks for the after-ripening of seeds.
- Open the cucumber fruit using knife and collect the seeds. Wash the seeds clean with tap water and store in mesh bags under the cool, dry place to dry the seeds.
- The transgenic cucumber seeds of T0 can germinated directly in soil or in cultivation containers filled soil and used for further analysis of gene biology after molecular identified the exit of T-DNA insertion. T-DNA identification refer to Step 25 and Step 26.
Steps 1–4, Remove the seed coats, surface sterilization and germination of cucumber seeds: 3 hours for handling, 2 days for cultivation (seeds germination)
Step 5-7, Inoculum preparation: 30 min for handling, 2 days for cultivation
Step 8-12, Explant preparation, Agrobacterium infection and co-cultivation: 4 hours for handling, 2 days for cultivation (co-cultivation)
Steps 13, Shoot initiation: 0.5 hours for handling, 2-3 weeks for cultivation (shoot regeneration)
Steps 14–16, Regeneration of transgenic plants: 3 hours for handling, 4-6 weeks for cultivation (root regeneration)
Steps 17–21, Plant acclimation: 3 hours for handling, 3-5 weeks for cultivation.
Steps 22–23, Examination of transgenic plants: 1 day for handling
Steps 24–28, Seed production from transgenic plants: 1 hour for handling, 8-12 weeks for cultivation
Troubleshooting advice can be found in Table 5
The average plant regeneration rate and transformation frequencies are affected by cucumber genotypes, explants types, explants preparation methods, the Agrobacterium infection methods, the concentration and ratio of ABA and BAP and culture methods (Table 1, Table 2, Table 3, Table 4 and figure 1). Different Agrobacterium species (EHA105, C58, LBA4404 AGL1 and GV3101) contained a series of plant binary vectors (pBI121, pCAMBIA 2301, pCAMBIA1391, pCAMBIA1305.1 and pFGC1008) has been used to transform different cucumber varieties (Deltasta, Guonong25, cucumber line 3407, 3413, 3461, Chinses long: 461, 9930, R1407, R470, short 32x, and Xintaimici,) and obtained the transgenic plants successfully25-33. The pure line should be used when considering for obtain T1 generation and for gene function analysis.
This protocol has improved the frequency of transformation to 26%, and we have successfully obtained over six hundred cucumber transgenic lines and published some papers of gene function analysis using this protocol25-33. We believe that this protocol is and will play important role in gene discovery, gene delivery, gene functional analyses and variety improvement for cucumber and Cucurbitaceae plants (need to be slightly modified) in the future study.
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12 Nanasato, Y., Konagaya, K., Okuzaki, A., Tsuda, M. & Tabei, Y. Improvement of Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) by combination of vacuum infiltration and co-cultivation on filter paper wicks. Plant Biotechnol Rep 7, 267-276 (2013).
13 Vengadesan, G., Anand, R. P., Selvaraj, N., Perl-Treves, R. & Ganapathi, A. Transfer and expression of nptII and bar genes in cucumber (Cucumis sativus L.). In Vitro Cell Dev Biol 41, 17-21 (2005).
14 Nishibayashi, S., Kaneko, H. & Hayakawa, T. Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants. Plant Cell Rep 15, 809-814 (1996).
15 Punja, Z. K., Abbas, N., Sarmento, G. G. & Tang, F. A. Regeneration of Cucumis sativus var. hardwickii, C. melo, and C. metuliferus from explants through somatic embryogenesis and organogenesis. Plant Cell Tiss Org 21, 93-102 (1990).
16 Raharjo, S. H. T., Hernandez, M. O., Zhang, Y. Y. & Punja, Z. K. Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens. Plant Cell Rep 15, 591-596 (1996).
17 Lou, H. & Kako, S. Somatic embryogenesis and plant regeneration in cucumber. Hortscience 29, 906-909 (1994).
18 Colijnhooymans, C. M., Bouwer, R., Orczyk, W. & Dons, J. J. M. Plant regeneration from cucumber (Cucumis sativus) protoplasts. Plant Sci 57, 63-71 (1988).
19 Wehner, T. C. & Locy, R. D. In vitro adventitious shoot and root formation of cultivars and lines of cucumis sativus L. Hortscience 16, 759-760 (1981).
20 Schulze, J. et al. Biolistic transformation of cucumber using embryogenic suspension cultures: long-term expression of reporter genes. Plant Sci 112, 197-206 (1995).
21 Wang, S. L. et al. Current status of genetic transformation technology developed in cucumber (Cucumis sativus L.). J Integr Agr 14, 469-482 (2015).
22 Chee, P. P. Transformation of Cucumis sativus tissue by Agrobacterium tumefaciens and the regeneration of transformed plants. Plant Cell Rep 9, 245-248 (1990).
23 Lin, Y. T. et al. In Vitro regeneration and genetic tansformation of cucumis metuliferus through cotyledon organogenesis. Hortscience 46, 616-621 (2011).
24 Rajagopalan, P. A. & Perl-Treves, R. Improved cucumber transformation by a modified explant dissection and selection protocol. Hortscience 40, 431-435 (2005).
25 Cheng, J. T. et al. Down-regulating CsHT1, a cucumber pollen specific hexose transporter, inhibits pollen germination, tube growth, and seed development. Plant Physiol 168, 635-647 (2015).
26 Wang, H. Y. et al. Antisense suppression of cucumber (Cucumis sativus L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance. Plant Cell Environ 37, 795-810 (2014).
27 Ding, L. et al. HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber (Cucumis sativus L.). J Exp Bot 66, 7075-7087 (2015).
28 Chen, C. H. et al. The WD-repeat protein CsTTG1 regulates fruit wart formation through interaction with the homeodomain-leucine zipper I protein Mict. Plant Physiol 171, 1156-1168 (2016).
29 Liu, B. et al. Silencing of the gibberellin receptor homolog, CsGID1a, affects locule formation in cucumber (Cucumis sativus) fruit. New Phytol 210, 551-563 (2016).
30 Wang, W. J. et al. Cucumis sativus L. WAX2 plays a pivotal role in wax biosynthesis, influencing pollen fertility and plant biotic and abiotic stress responses. Plant Cell Physiol 56, 1339-1354 (2015).
31 Wang, W. J. et al. Cucumber ECERIFERUM1 (CsCER1), which influences the cuticle properties and drought tolerance of cucumber, plays a key role in VLC alkanes biosynthesis. Plant Mol Biol 87, 219-233 (2015).
32 Zhang, Y. et al. A GAMYB homologue CsGAMYB1 regulates sex expression of cucumber via an ethylene independent pathway. J Exp Bot 65, 3201-3213 (2014).
33 Lu, J. et al. Suppression of cucumber stachyose synthase gene (CsSTS) inhibits phloem loading and reduces low temperature stress tolerance. Plant Mol Biol, doi:10.1007/s11103-017-0621-9 (2017).
34 Hallmann, J., Quadt-Hallmann, A., Rodrı́guez-Kábana, R. & Kloepper, J. W. Interactions between Meloidogyne incognita and endophytic bacteria in cotton and cucumber. Soil Biology and Biochemistry 30, 925-937 (1998).
35 Yan, X., Huang, L. L., Tu, X., Gao, X. N. & Kang, Z. S. Saccharothrix yanglingensis sp. nov., an antagonistic endophytic actinomycete isolated from cucumber plant. Antonie van Leeuwenhoek 101 (2012).
36 Wang, J. et al. Agrobacterium-mediated transformation of cucumber (Cucumis sativus L.) using a sense mitogen-activated protein kinase gene (CsNMAPK). Plant Cell Tiss Org 113, 269-277 (2013).
37 Tabei, Y. et al. Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea). Plant Cell Rep 17, 159-164 (1998).
38 Sarmento, G. G., Alpert, K., Tang, F. A. & Punja, Z. K. Factors influencing Agrobacterium-tumefaciens mediated transformation and expression of kanamycin resistance in pickling Cucumber. Plant Cell Tiss Org 31, 185-193 (1992).
39 Ganapathi, A. & Perl-Treves, R. Agrobacterium-mediated transformation in Cucumis sativus via direct organogenesis. Acta Hortic 510, 405-408 (2000).
40 Lodhi, M. A. , Ye, G. N., Weeden, N. F., Reisch, B. I. A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis. Plant Mol Biol Rep. 12, 6-13 (1994).
We thank Dr. Xingfang Gu (Chinese Academy of Agricultural Sciences) for the gift of the “Xintaimici” cucumber seeds. This work was supported by the National Natural Science Foundation of China (Grant No. 31471876 to Z.Z.) and the Ministry of Agriculture of China (Project No. 2013ZX08009)
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Supplementary Figure S1 Supplementary Figure S1 ∣ Combined effect of different hormones and base media on cucumber regeneration using the Xintaimici and Chinese long 461 genotypes. A, Combined effect of ABA and BAP on cucumber regeneration using Xintaimici after 15 days of shoot regeneration culturing. B, Combined effect of MS and B5 base media on cucumber regeneration using 1.0 mg/l ABA and different BAP concentrations and the Chinese long 461 genotype after 20 days of shoot regeneration culturing.
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Associated Publications
Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development
by Jintao Cheng, Zhenyu Wang, Fengzhen Yao, +4
PLANT PHYSIOLOGY (28 July, 2017)
Antisense suppression of cucumber ( Cucumis sativus L.) sucrose synthase 3 ( CsSUS3 ) reduces hypoxic stress tolerance
by HONGYUN WANG, XIAOLEI SUI, JINJU GUO, +5
A GAMYB homologue CsGAMYB1 regulates sex expression of cucumber via an ethylene-independent pathway
by Yan Zhang, Xiaolan Zhang, Bin Liu, +6
HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber ( Cucumis sativus L.)
by Lian Ding, Shuangshuang Yan, Li Jiang, +7
Suppression of cucumber stachyose synthase gene (CsSTS) inhibits phloem loading and reduces low temperature stress tolerance
by Jianguo Lü, Xiaolei Sui, Si Ma, +3
Plant Molecular Biology (29 July, 2017)
Method Article
A Protocol for Agrobacterium-mediated Transformation of Cucumber (Cucumis sativus L.) from cotyledon explants
zhenxian zhang
Beijing Key Laboratory of Growth and Developmental Regulation for Protected Vegetable Crops, College of Horticulture, China Agricultural University, Beijing, P. R. China.
Corresponding Author
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Genetic transformation is central to the discovery, delivery, and functional analysis of genes, and for associated crop improvement, and yet is technically challenging in many plant species. As an example, cucumber (Cucumis sativus L.) is a model species for the study of phloem characteristics, raffinose family oligosaccharide (RFO) metabolism and sex determination, but methods for its genetic transformation have generally had low efficiency, thereby limiting basic and applied studies. Here, we describe a rapid and efficient protocol for Agrobacterium-mediated transformation of cucumber cotyledon explants, using vacuum infiltration. The transformation efficiency of the regenerated plants was as high as 54%, and the final positive plantlet transformation efficiency was approximately 26% of the total number of infected explants and this is obviously higher than previously published protocols. Transgenic plantlets can be obtained in 3 to 4 months and the transgenic T1 seeds generated in the subsequent 3 to 4 months after self-pollination. Using this protocol, we have obtained more than 600 transgenic cucumber lines. This protocol is of great importance for studies of cucumber and of other Cucurbitaceae species, since it enables gene functional analysis, and so opens a pipeline for rapid cucumber variety improvement.
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Cucumber (Cucumis sativus L.), a crop with economic and nutritional importance that is cultivated worldwide, is a typical raffinose family oligosaccharides (RFOs)-transporting plant1-4, and a model species for studying phloem functional characteristics and sex determination. Cucumber phloem is very complicated, there is one set of vascular bundle (VB, a bicollateral bundle including the internal and external phloem) in the petiole, stem and peduncle, but four sets of VBs in the fruit, including the peripheral, main, carpel and placental VB (two bicollateral, two collateral). Cucumber is a monoecious species with unisexual flowers, and is a model species for the study of sex determination. It has seven types of sex phenotypes, including andromonoecious, monoecious, androecious, heterozygous-gynoecious, hermaphrodite, gynoecious and gynoecious-monoecious. In addition, it can be parthenocarpic. However, a lack of effective genetic transformation methods, especially those based on Agrobacterium, has severely limited cucumber gene functional analyses and remains a bottleneck for both developing cucumber as a model, and for its improvement as a crop.
The transfer and stable integration of genetic material into the cucumber genome will enhance the breeding of more disease resistant cucumber (Cucurbitaceae plants or cucurbits) varieties, as well as the characterization of cucumber genes. It offers the possibility to introduce new characters into a cucumber cultivar, which is difficult to achieve via conventional breeding techniques5. Recently, the genome sequences of both cucumber and watermelon, another cucurbit, were released (http://www.icugi.org/cgi-bin/ICuGI/index.cgi). Moreover, a range of studies have investigated diverse aspects of cucumber biology, such as the rapid fruit expansion that occurs after flowering, and some candidate genes that influence this important process have been identified6,7. The development of an efficient cucumber transformation system is therefore timely and critical for gene discovery and validation.
Many studies have reported successful cucumber transformation, but only a few have obtained a high transformation rate and T1 generation, and there are very few reports on functional analysis using transformed plants (Table 1). The first transformation of cucumber plants, which expressed the NPT II gene, was reported by Trulson et al. (1986)8. This transformation was achieved by infecting cucumber hypocotyl segments with Agrobacterium rhizogenes and regenerating the transformed issue into whole plants. This approach had a low plant regeneration frequency, which limited its utility (Table 1). Other reports have described the successful regeneration of cucumber plants from cotyledon explants after transformation using A. tumefaciens, but transformation efficiency was still < 12%, and only a few of these studies resulted in transgenic T1 plants (Table 1). Higher transformation rates (16-23%) have been reported using cotyledon explants9-11, but in these studies, the transformation rate was calculated as the identified positive shoots and not plantlets, and few of these lines resulted in a stably transformed T1 generation. The selection of cucumber transformants using antibiotic selection with kanamycin9,12, phosphinothricin11,13 and hygromycin14, have been reported; however the method with the high transformation efficiency (23%) reported by He et al. (2006) was based on mannose and sucrose resistance10 (Table 1).
Other regeneration protocols with various culture types have also been investigated, such as leaf explants15, petioles15,16, hypocotyls14,17, internodes17, protoplasts18 and suspension cultures19,20, but these all showed a lower transformation efficiency than using cotyledons as explants. Until now, Agrobacterium-mediated transformation methods using cotyledons as explants has been the most successful approach for cucumber transformation21. With this approach, the transformation frequency is dependent on several factors, including plant genotype, explant source and size, Agrobacterium strains and concentration, length of exposure to Agrobacterium, and selection system22,23, among others. It was reported that dissecting half of an adaxial cotyledon using a V-shaped cut resulted in a higher transformation rate24, while another report showed that using cotyledon nodes as explants resulted in regeneration at the junction of cotyledon and hypocotyl in the proximal regions of the cotyledons12. In this current protocol, we use similar explants, half adaxial cotyledons, but we have modified the protocol in that we break off the hypocotyl instead of using the V-shaped cut to detach the growing point. We also highlight the key factors in the procedure that can cause technical problems for the inexperienced researcher.
Our laboratory has established a rapid and efficient Agrobacterium-mediated transformation system for cucumber that differs from previously reported protocols, and we have identified critical factors that influence transformation efficiency. To date, we have produced more than 600 transgenic lines and functionally analyzed a range of cucumber genes using this protocol25-33. The method can be widely used in cucumber breeding, variety improvement, and the characterization of gene function, and can be a reference for the transformation of other cucurbits, such as Cucumis melo and C.metuliferus21,23.
Overview of the technique (Figure 1)
This protocol consists of seven major steps, which can be briefly summarized as follows: Stage I: preparation of sterilized seeds. Stage II: explant preparation, infection and co-cultivation with A. tumefaciens. Stage III: shoot regeneration. Stage IV: root regeneration. Stage V: acclimation and molecular identification of T0 plants. Stage VI: cultivation and self-crossing of T0 plants. Stage VII: seed harvest from T0 plants.
Key factors for successful transformation of cucumber
**Varieties amenable to _Agrobacterium-mediated transformation**_
It has been reported that the genotype of transformed cucumber influences its shoot regeneration frequency9,21, and relatively high regeneration rates have been reported for some cucumber genotypes, such as Poinsett 76, Jinyan No.7, Cengel Köy, Shinhokusei 1, cv. pickling genotype 3676, 3672, and Xintaimici (Table 1). However, in our analyses we observed no differences in the regeneration frequency between different cucumber genotypes. We obtained a high regeneration rate (80-97 %) for the Deltasta, Guonong25, cucumber line 3413, 3407, 3461, Chinese long: 461, 9930, R1407, R470, short 32x, and Xintaimici genotypes, and obtained transgenic plants from all these varieties. Even so, it is advisable to determine the regeneration rate before using a new cucumber genotype for Agrobacterium-mediated transformation, in order to optimize the chance of successful transformation. We note that homozygous varieties should be considered, since they can provide a homozygous transgenic T1 generation for subsequent gene functional studies.
Preventing contamination with endophytic bacteria
Different endophytes may be associated with different parts of the cucumber seed. Before soaking seeds in tap water for 2 hours, they should be sterilized with HCl and NaClO in a sealed container for 1-2 hours, to kill bacteria on the seed surface or inside the seed coat. Some endophytic bacterial species have been isolated from surface sterilized cucumber seeds, and most of these were located in the seedling radicles34,35. It is therefore important to cut off the radicles before detaching the cotyledons to prevent the contamination with endophytic bacteria, and then knocking or breaking off the hypocotyl on new sterilized filter paper.
Explant preparation
The selection and preparation of explants is critical factor in the success of the transformation pipeline. In our protocol, the embryonic cotyledons are used as the explants. The cucumber seeds should be soaked in water for 2 hours to facilitate the removal of the seed coats and to obtain a high germination rate. The de-coated seeds should then be surface sterilized with 75% (vol/vol) alcohol for no more than 1 minute.
After germination for approximately 2 days on MS/B5 medium, a radicle of 1-1.5 cm and root hairs are visible, and the cotyledons can be detached from the embryos. Half of the adaxial cotyledon is then used as the explant. Seeds have a better germination rate on B5 medium than on MS medium; however, different varieties may have a difference in germination time and media requirements, as well as in how readily the cotyledons can be detached. Forceps, rather than a knife, should be used to detach the cotyledons from the radicle growing point (Figure 1, Explant preparation) in order to obtain a higher regeneration rate.
Previous studies have shown a high regeneration frequency with cotyledon nodes as explants36, and we also found that breaking off the hypocotyl to detach the cotyledon, rather than cutting it, is important to obtain high regeneration frequency. However, care needs to be taken to cut off the growing point of the seedlings to prevent negative plants growing.
Influence of abscisic acid (ABA) and 6-benzylaminopurine (BAP) levels on shoot organogenesis during shoot initiation and outgrowth
It has been reported that the hormones ABA and BAP can induce the regeneration of cucumber cotyledon explants36, 38, and most of these studies focused on the influence of BAP levels on cucumber shoot regeneration. In this study we have investigated the combined effects of BAP and ABA levels on shoot regeneration from cotyledon explants (Table 2, Supplementary Figure S1). We observed that the highest shoot regeneration rate were 76% and 86%, using a ABA/BAP concentration of 1.0/0.8-1.5 mg/l (Table 2), and noted that the regenerated shoots grew more slowly with higher ABA and lower BAP concentrations (Supplementary Figure S1 A). We therefore chose an ABA/BAP concentration of 1.0/0.8-1.5 mg/l, as this resulted in healthy and fast growing plants, as well as a high regeneration rates with the Agrobacterium-mediated transformation system. We compared efficacy of the MS medium and B5 medium for shoot regeneration with a 1.0 mg/l ABA concentration and different BAP concentration, and found that the average number of regenerated shoots per explant in MS and B5 media was 2.5 and 1.0, respectively (Table 3, Supplementary Figure S1 B). We concluded that use of the MS medium resulted in the production of more shoots than the B5 medium (Table 3), while the B5 medium was more effective for seed generation than the MS medium.
Agrobacterium concentration and infection time
The Agrobacterium concentration and the infection time were found to be important factors in preventing explant turning necrosis and improving transformation efficiency. We determined that the optical density (OD, at 600 nm) of the Agrobacterium culture should be adjusted to approximately 0.25 (Table 4), and that the infection time should be about 15 minutes to avoid explant necrosis, and to obtain the highest regeneration rate of 86% (Table 4).
Agrobacterium vacuum infiltration
It has been reported that vacuum infiltration can improve the Agrobacterium-mediated transformation of cucumber12, and we also found this to be the case. Since we had found that the infection time and Agrobacterium concentration were important in obtaining a high transformation rate, we vacuum infiltrated with Agrobacterium solution and explants in beakerflask (OD600=0.25) twice for 5 min, and then move them into shaker for an additional 5 min.
Orientation of the explants on the co-cultivation medium
After Agrobacterium infection, placed the cotyledon explants on co-cultivation medium with the adaxial surface facing upward, so that the abaxial surface was in contact with the culture medium. We found this to be essential for high transformation efficiency. After 2 days of co-cultivation, we transferred the cotyledons to the shooting medium, and again made sure that the abaxial surface of the cotyledon was touching the medium.
Shoot regeneration and rooting
We determined that when regenerated shoots have grown to 1 cm, they should be excised from the cotyledon explants and transferred to rooting medium. Embryoids that appear on the cut end surface should be detached. Regenerated shoots will then grow rapidly and the leaves will expand immediately after transfer to rooting medium. The un-rooted regenerated shoot end shoot then be re-cut and the unfolded leaves and the shoots transferred into new rooting medium once per week for the next 2-3 weeks, ensuring that the lower part of the regenerated shoots are inserted in the rooting medium. We found this to be an effective way to remove residual ABA/BAP from the shoots and to be important for efficient rooting.
Antibiotic Selection
The use of kanamycin selection has previously been reported for cucumber transformation37,38, and we found it to be more effective than hygromycin since the latter affects plant development. Kanamycin concentrations of 50-150 mg/l are commonly used14,22,39, and it has been shown that a higher concentration will slow growth at the rooting stage and suppress regeneration24. Here, 50 mg/l was used during shoot initiation, shoot outgrowth and the rooting stage to encourage the regeneration of transgenic plants. In addition to antibiotic selection, PCR should be used to confirm the presence of the targeted transgene in each transformant at each generation.
Plant growth conditions
Since cucumber is a warm-season crop, the tissue cultures should be kept at 25°C, and after plant acclimation in the soil, the temperature should be maintained at 28°C/18°C with a 16 h/8 h light/dark cycle, which are typical cucumber cultivation conditions.
REAGENTS
Cucumber (Cucumis sativus L.) seeds
Agrobacterium strain
Sodium hypochlorite, 13% (wt/vol) (NaClO; Chem-Supply, CAS 7681-52-9)
Hydrochloric acid, HCl, 36.0-38.0% (Sinopharm Chemical Reagent Co., Ltd, 80070591)
B5 Medium (Waryong，GT213250B)
Waryong Phytagel plantcell Ⅱ, (Waryong，GT21026M)
Agar (Biowest, 9012-36-6) for LB plates
Tryptone (Oxoid, cat. no. 91079-40-2)
Yeast extract (Oxoid, cat. no. 8013-1-2)
Sodium chloride (NaCl; Chem-Supply, GB/T 1266-19867520, cat. no. 10019792)
Sodium hydroxide (NaOH; Chem-Supply, cat. no.1310-73-2)
Sterile distilled water
Ethanol (Chem-Supply, cat. no. 10009292)
Ethanol 75% (Vol/Vol) in distilled water
Sucrose (C12H22O11; Chem-Supply, cat. no. 10021492)
6-Benzylaminopurine (BAP, C12H11N5l; Sigma, cat. No. B3408)
Abscisic acid (ABA, C15H20O4; Bjxjksw, cat. no. 21293-29-8)
Rifampicin (C43H58N4O12; Bjxjksw, cat. no. 13292-46-1)
Kanamycin monosulfate (C18H36N4O11﹒H2SO4)
Carbenicillin disodium salt (C17H16N2O6SNa2; MP Biomedicals, cat. No. 195092)
Murashige and Skoog basal medium with vitamins (PhytoTechnology Laboratories, cat. No. M519)
Gellan gum powder (PhytoTechnology Laboratories, cat. No. G434)
PCR Primerstar mix (Takara Biotechnology, Cat. no. R045A)
REAGENT SETUP
LB liquid medium for Agrobacterium preparation
Dissolve 5 g of yeast extract, 10 g tryptone and 5 g sodium chloride in 1 liter of distilled water; pH 7.0 autoclave. Add 50 μl of 100 mg/ml rifampicin stock and 50 μl of 50 mg/ml kanamycin stock to 50 ml LB solid medium.
Murashige and Skoog (MS) liquid medium for Agrobacterium suspension Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8 autoclave
Murashige and Skoog (MS) plates for seed germination (germination medium)
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
B5 plates for seed germination (germination medium)
Dissolve 30.21 g B5 medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Cocultivation medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water, add 750 μl 2 mg/ml BAP; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 500 μl 2 mg/ml ABA, and pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Shooting medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water, add 750 μl 2 mg/ml BAP; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 500 μl 2 mg/ml ABA, 50 μl of 50 mg/ml kanamycin and 2.5 ml of 200 mg/ml carbenicillin. Pour about 25 ml of medium into sterile Petri dishes under laminar flow hood.
Rooting medium
Dissolve 4.43 g murashige and skoog basal medium (with vitamins) powder and 30 g sucrose in 1 liter of distilled water; PH 5.8. Add 2.5 g gellan gum powder, autoclave. Add 50 μl of 50 mg/ml kanamycin and 1 ml of 200 mg/ml carbenicillin. Pour about 40 ml of medium into 100 ml glass beaker under laminar flow hood.
Sterile 50 ml plastic tubes (Corning, cat. no. 430828)
Autoclave (Cheng YI, LS-B35 Type; cat. no. 09J-3298)
Controlled tissue culture rooms 25 °C/25 °C, 16/8 h light/dark regime
Controlled environmental rooms (16 h photoperiod) or glasshouse facilities for whole plant culture
Orbital incubator shaker (Pei Ting, THZ-C-1 Type)
Tabletop orbital shaker
Vacuum pump (DA-60D; ULVAC, Kanagawa, Japan) was connected to a desiccator (VM-C; AS ONE, Osaka, Japan)
PH meter (PHSJ-4A Type)
Laminar flow hood for plant culture work
Glassware (100 ml glass beaker, graduated cylinders, glass Petri dish, Duran bottles and Erlenmeyer flasks)
Filter paper 9 cm diameter (15-20 μm)
Parafilm (Bemis Flexible Packaging)
230 mm pot (230 mm in diameter nd 190 mm in height) for cucumber cultivation
Forceps and scalpel
Surgical blades
Plant growth culture vessels
Pipettes: P1000, P200, P20, P10
Sterile pipettes tips (Axygen Scientific: 1–200 μl, cat. no. T-200Y; 1–1,000 μl, cat. no. T-1000B; 1–20 μl, cat. no. T-20Y; 1–10 μl, cat. no. T-10W)
Centrifuge
Spectrophotometer (SFZ0806011544 Type)
Mini Vortexer (QL-901 Type)
Remove the seed coats, surface sterilization and germination of cucumber seeds •TIMING 3 hours for handling, 2 days for cultivation (seeds germination)
- Use Cl2 (add 2 ml HCl to 15 ml NaClO in a seal container, please be careful) sterile the cucumber seeds for 1-2 hours, not for long. Soak cucumber mature seeds in tap water for 2 h at room temperature in Petri dish.
- Remove the seed coats from seeds with forceps by pinch the pointed end of seeds first and then push aside the seed coats.
- Sterilize 20-50 seeds for 30 s with 20 ml of 75% ethanol in a sterile Petri dish with a lid. Drain ethanol and rinse with 20 ml of 3% sodium hypochlorite solution. Gently shake the seeds on a shaker for 15 min. Rinse three times with sterile deionized water. From this step to step 18 should be performed in a laminar flow hood.
- Place surface-sterilized seeds with forceps onto Petri plates containing seed germination medium, allowing 15-25 seeds per plate. Germinate the seeds in the dark at 28°C for 2 d. Inoculum preparation •TIMING 30 min for handling, 2 days for cultivation
- On the day after Step 4, inoculating 2 ml of LB liquid medium containing rifampicin (100 mg/l) and Kanamycin (50 mg/l) with a single Agrobacterium colony carrying the appropriate construct. Culture at 28°C in a shaker at 200 rpm for 24 h.
- Subculture 200 μl of 2 ml culture from Step 6 into 50 ml of LB liquid medium containing rifampicin (100 mg/l) and Kanamycin (50 mg/l). Culture at 28°C in a shaker at 200 rpm until the OD at 600 nm to 0.6-0.8.
- Spin the Agrobacterium culture down (4000g or 5000g) for 10 min at RT (room temperature). Remove the supernatant and rinse the pellet in 1 ml liquid MS liquid medium. Dilute the MS medium containing Agrobacterium to OD600=0.25 using MS liquid medium. Explant preparation, Agrobacterium infection and co-cultivation •TIMING 4 hours for handling, 2 days for cultivation (co-cultivation)
- Pull the seedlings (from Step 5) out of the germination medium and place in an empty sterile Petri plate dishes with a stack of sterile filter paper.
- Cut the radicle first, and then cut off 1/2-1/3 the end of cotyledon off and remove the endopleura off at the same time. Detach the first cotyledon by using the sterile scalpel to wipe the upside cotyledon off. Knocking the hypocotyl over to detach the downside cotyledon and growing point using sterile forceps and scalpel (Fig. 1).
- Collect the detached cotyledons into MS liquid medium (without Agrobacterium) in a 50 ml sterile glass beaker.
- Pour out the MS liquid medium (without Agrobacterium), and pour into MS liquid medium (with Agrobacterium OD600=0.25 from Step 8) and shake gently. Cover the sterile glass beaker with sterile culture container sealing film and put into the desiccator which connected with the vacuum pump. After 2 sessions of vacuum infiltration were applied for 5 min, explants were leave for another 5 min in Agrobacterium.
- Pour out the MS liquid medium (with Agrobacterium), put a sterile filter paper onto cocultivation medium and touch the medium exactly. Place the infected explants on the filter paper and keep the adaxial surface of the cotyledons upward using forceps (Fig. 1), allowing about 20-35 explants per Petri dish plate. Seal the plate and incubate in dark at 28°C for 2d. Shoot initiation •TIMING 0.5 hours for handling, 2-3 weeks for cultivation (shoot regeneration)
- Transfer explants onto shooting medium containing 50 mg/ml kanamycin and 500 mg/ml carbenicillin to select the transgenic shoot and inhibit the growth of Agrobacterium, allowing 5-6 explants per Petri dish plate, as the explants will grow up. Incubate under light (150 μmol m-2 s-1, 16 h) at 25°C for 2-3 weeks. Regeneration of transgenic plants •TIMING 3 hours for handling, 4-6 weeks for cultivation (root regeneration)
- After the shoots growth out from the explants (Fig. 1), pull the explants out and place in an empty sterile Petri plate dishes with a stack of sterile filter paper.
- Cut the shoots off using sterile forceps and scalpel. Remove the embryoids on the end part of the shoots. Transfer shoots to 100 ml glass Erlenmeyer flasks or glass tissue culture vessels containing rooting medium with kanamycin (50 mg/l) and carbenicillin (500 mg/l). Place three to four shoots per Erlenmeyer flasks. Incubate under light (150 μmol m-2 s-1, 16 h) at 25°C for 1-2 weeks.
- After 1-2 weeks, take out the no rooting plants and recut the unfolded leaves and the end part of the shoots using sterile forceps and tweezers and transfer shoots to new rooting medium. Repeat this procedure one times per week in the next 2-3 weeks. Place one to two shoots per glass Erlenmeyer flasks. Incubate in under light (150 μmol m-2 s-1, 16 h) at 25°C for 2-4 weeks. Plant acclimation •TIMING 3 hours for handling, 3-5 weeks for cultivation
- After the roots grow strongly, open the cover of the glass Erlenmeyer flasks. Put the cover on the top of the Erlenmeyer flasks loosely. Incubate in dim light (10-50 μmol m-2 s-1) at 25°C for 3 days.
- Knock the bottom of the Erlenmeyer flasks gently and make the medium not firmed. Pull out the plants from the Erlenmeyer flasks gently using forceps. Wash off the medium with running tap water gently.
- Fill the pots with wet compost mixture, place the regenerated plants into the compost by making a hole. Compact the soil and water slightly.
- Cover the plants with zip bags and tight to the pot using rubber band to keep moisture. Incubate in good condition chamber for cucumber growth under light (gradually from 300 μmol m-2 s-1 to 500 μmol m-2 s-1, 16 h) at 25°C/18°C for 1-2 weeks. Add water into the tray under pots when the soil is getting dry.
- Take the zip bags off after the regeneration plants grown in a good condition. Watering the plants when the soil is getting dry. Incubate in under light (500 μmol m-2 s-1, 16 h) at 25°C/18°C for 1-2 weeks. Examination of transgenic plants •TIMING 1 day for handling
- Number the regeneration cucumber plants and cut a peace of the mature leaves, extract the genome DNA using the cetyltrimethy ammonium bromide (CTAB) method40.
- Confirm the transgenic plants has been inserted in the T-DNA using the specific primers designed on the T-DNA and do PCR amplification and gel electrophoresis assay. Seed production from transgenic plants •TIMING 1 hour for handling, 8-12 weeks for cultivation
- Transfer the molecular identified transgenic plants to greenhouse, to give seedlings normal environment condition with the temperature no higher than 33°C and no lower than 16°C, light intensity 500 μmol m-2 s-1, relative humidity 70-80%.
- The transgenic cucumber plants require normal cultivation management and artificial self-pollination when plants flowering. On the day before the female and male flower blooming, clip the both the female and male flowers on the same plants, and then do artificial pollination using the same plants male flowers to the female flowers on the second morning, and then clip the female flowers to keep away the pollen from the other plants.
- When the epidermis of autocarps are turned yellow and indicated that the seeds are fully mature, pick them up and place under the cool, dry place for 1-2 weeks for the after-ripening of seeds.
- Open the cucumber fruit using knife and collect the seeds. Wash the seeds clean with tap water and store in mesh bags under the cool, dry place to dry the seeds.
- The transgenic cucumber seeds of T0 can germinated directly in soil or in cultivation containers filled soil and used for further analysis of gene biology after molecular identified the exit of T-DNA insertion. T-DNA identification refer to Step 25 and Step 26.
Steps 1–4, Remove the seed coats, surface sterilization and germination of cucumber seeds: 3 hours for handling, 2 days for cultivation (seeds germination)
Step 5-7, Inoculum preparation: 30 min for handling, 2 days for cultivation
Step 8-12, Explant preparation, Agrobacterium infection and co-cultivation: 4 hours for handling, 2 days for cultivation (co-cultivation)
Steps 13, Shoot initiation: 0.5 hours for handling, 2-3 weeks for cultivation (shoot regeneration)
Steps 14–16, Regeneration of transgenic plants: 3 hours for handling, 4-6 weeks for cultivation (root regeneration)
Steps 17–21, Plant acclimation: 3 hours for handling, 3-5 weeks for cultivation.
Steps 22–23, Examination of transgenic plants: 1 day for handling
Steps 24–28, Seed production from transgenic plants: 1 hour for handling, 8-12 weeks for cultivation
Troubleshooting advice can be found in Table 5
The average plant regeneration rate and transformation frequencies are affected by cucumber genotypes, explants types, explants preparation methods, the Agrobacterium infection methods, the concentration and ratio of ABA and BAP and culture methods (Table 1, Table 2, Table 3, Table 4 and figure 1). Different Agrobacterium species (EHA105, C58, LBA4404 AGL1 and GV3101) contained a series of plant binary vectors (pBI121, pCAMBIA 2301, pCAMBIA1391, pCAMBIA1305.1 and pFGC1008) has been used to transform different cucumber varieties (Deltasta, Guonong25, cucumber line 3407, 3413, 3461, Chinses long: 461, 9930, R1407, R470, short 32x, and Xintaimici,) and obtained the transgenic plants successfully25-33. The pure line should be used when considering for obtain T1 generation and for gene function analysis.
This protocol has improved the frequency of transformation to 26%, and we have successfully obtained over six hundred cucumber transgenic lines and published some papers of gene function analysis using this protocol25-33. We believe that this protocol is and will play important role in gene discovery, gene delivery, gene functional analyses and variety improvement for cucumber and Cucurbitaceae plants (need to be slightly modified) in the future study.
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We thank Dr. Xingfang Gu (Chinese Academy of Agricultural Sciences) for the gift of the “Xintaimici” cucumber seeds. This work was supported by the National Natural Science Foundation of China (Grant No. 31471876 to Z.Z.) and the Ministry of Agriculture of China (Project No. 2013ZX08009)
arezoo
commented on 25 May, 2019
thanx
arezoo
commented on 07 January, 2020
thanx
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