A protocol for Agrobacterium-mediated transformation of Japanese cedar, Sugi (Cryptomeria japonica D. Don) using embryogenic tissue explants.

Sugi (Cryptomeria japonica D. Don) is the most important afforestation coniferous tree in Japan. Coniferous trees normally have a long juvenile period and require a long cultivation time for breeding. Through a traditional breeding project that began in the 1950s, first generation plus trees with excellent traits were selected primarily from artificial forests and used as seedlings. Recently, the second generation plus trees obtained by crossing between plus trees have been selected. In light of this situation, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period compared with conventional crossing-dependent approaches. There are three key points to an efficient Agrobacterium-mediated transformation system: (1) establishment of explants with high regeneration ability, (2) optimal co-cultivation conditions for explants and Agrobacterium, and (3) efficient elimination of Agrobacterium. Here we describe a protocol for Agrobacterium-mediated transformation of Sugi that meets the above criteria using embryogenic tissues as explants isolated from immature seeds obtained by crossing.


Introduction
Widely distributed in Japan, Cryptomeria japonica D. Don (Sugi, Japanese cedar) is one of the major afforestation coniferous species there. Sugi had been previously classified in the family Taxodiaceae, but as a result of recent molecular phylogenetic studies it has been integrated into the family Cupressaceae sensu lato (Gadek et al. 2000;Kusumi et al. 2000). C. japonica var. sinensis, previously recognized as C. fortunei, is distributed in eastern China (Fu et al. 1999), but there are no differences in chloroplast DNA sequences between C. japonica and C. japonica var. sinensis (Kusumi et al. 2000;Tsumura et al. 1995). In Japan, there are two main lines locally known as Omote-sugi (C. japonica distributed on the Pacific Ocean side) and Ura-sugi (C. japonica var. radicans distributed on the Sea of Japan side), which are distinguished by differences in morphological characteristics (Murai 1947), diterpene components (Yasue et al. 1987), reproductive system (Kimura et al. 2013), and DNA variation (Tsumura 2011;Tsumura et al. 2014), which may have contributed to their local adaptation. Since Sugi trees may exceed 50 m in height, and grow with a straight bole of soft wood making processing easy, it is popular for house construction, paneling, flooring, carpentry, joinery, and furniture making (Farjón 2010;Ohba 1993). Around 1950, due to a decline in demand for fuelwood and an increase in demand for timber associated with residential construction during the period of high economic growth, reforestation to convert natural forests into artificial forests was actively promoted and afforestation areas were enlarged. As a result, Sugi has been planted widely throughout Japan and now covers an area of 4.44 million ha, accounting for approximately 44% of all Japanese artificial forests (Forestry Agency 2017). However, allergic rhinitis caused by Sugi pollen (Sugi pollinosis) began to be reported in 1963 (Horiguchi and Saito 1964), since when the prevalence has continued to rise year by year, and now Sugi pollinosis has become a severe public health concern in Japan ( Yamada et al. 2014). Therefore, the production of Sugi with a no-pollen trait has become an important breeding objective.
Genetic transformation, and genome editing technology based on it, can be a powerful tool for shortening the breeding period of trees whose generations are many years apart. A large number of expressed sequence tags obtained from vegetative buds, inner bark, female strobili, male strobili, pollen, sapwood, xylem, cambium, and roots, have been identified (Fukuda et al. 2018;Futamura et al. 2006Futamura et al. , 2008Kurita et al. 2011;Mishima et al. 2014Mishima et al. , 2018Nose and Watanabe 2014;Tsumura et al. 1997;Ueno et al. 2019;Ujino-Ihara et al. 2000Yoshida et al. 2007Yoshida et al. , 2012, and various genetic linkage maps and DNA markers have been developed (summarized by Tsumura 2011). Genetic transformation is also an essential tool for taking advantage of these abundant resources for molecular breeding and identification of gene function by reverse genetic analysis. Several stable transformation methods of coniferous trees have been reported in the family Pinaceae, including the genera Picea, Larix, and Pinus (summarized by Malabadi and Nataraja 2007). However, stable transformation in Cupressaceae has only been reported for Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc. ;Ishii 2002;Taniguchi et al. 2005) and Sugi Maruyama et al. 2000;Taniguchi et al. 2008).
Most transformation methods in coniferous trees use embryogenic tissues (e.g., embryogenic cells, embryogenic callus, embryogenic mass, embryonalsuspensor mass, etc.) derived from immature seeds as explants. Somatic embryogenesis has long been studied in coniferous trees because it is an effective procedure not only for providing target tissue for genetic transformation but also for mass propagation of breeding strains showing preferred traits (summarized by Konagaya and Taniguchi 2016). One of the key factors for successful coniferous transformation is the use of embryogenic tissues of cell lines with high regeneration efficiency for somatic embryos, necessitating selection of many cell lines. It has been shown that the induction rate of embryogenic tissues from immature seeds in Sugi is affected by genotype and embryonic developmental stage (Igasaki et al 2003;Maruyama et al. 2000;Taniguchi and Kondo 2000). Furthermore, it has been revealed that the frequency of somatic embryogenesis from embryogenic tissues is also affected by genotype (Taniguchi 2007; see the chapter by Taniguchi et al.). Somatic embryo maturation is generally promoted by abscisic acid, osmotic penetrants (sugars, sugar alcohols, etc.) or nonpenetrating osmoticants (e.g., polyethylene glycol), which induce water stress (Lipavská and Konrádová 2004). Mature somatic embryos in Cupressaceae trees have also been efficiently formed on a medium containing a combination of 100 µM abscisic acid, 30 g/l maltose as the sugar, and 150 g/l polyethylene glycol (Maruyama et al. 2002;Taniguchi 2007;Taniguchi et al. 2004a).
Genetic transformation of Sugi was first attempted by particle bombardment with, for example, gene transfer of the luciferase (Mohri et al. 2000) and green fluorescent protein (GFP; Taniguchi et al. 2004b) genes in zygotic embryos, but only transient expression was observed. Maruyama et al. (2000) reported bud formation from embryos after introducing the β-glucuronidase gene.
The Agrobacterium tumefaciens (recently reclassified as Rhizobium radiobacter; Young et al. 2001; formerly, A. tumefaciens. We have used A. tumefaciens for convenience in this review because the majority of transgenic research has used this name)-mediated transformation method in Sugi was first successfully achieved by Taniguchi et al. (2008) and further improved by Konagaya et al. (2013). The improvements are described below. Bacterial overgrowth during co-cultivation in Agrobacterium-mediated transformation may lead to undesirable effects, such as tissue necrosis, which can reduce transformation efficiency. Ozawa (2009) showed that liquid medium-moistened filter paper wicks regulated the growth rate of Agrobacterium in an effective manner, improving cell viability in the transformed callus obtained from rice. Thus, as a result of determining the optimal co-cultivation conditions with filter paper wicks in Sugi, transformation efficiency was greatly improved (>17-fold; Konagaya et al. 2013) despite the mean number of Agrobacterium cells obtained in liquid medium conditions being significantly less than that obtained on solid media using conventional methods (Taniguchi et al. 2008). Similar improvements have been reported in rice, cucumber, kabocha squash, Jatropha, and safflower transformation (Nanasato et al. 2011(Nanasato et al. , 2013(Nanasato et al. , 2015Ozawa 2009;Rani et al. 2018). These results support the utility of filter paper wicks in co-cultivation procedures.
Recently, it has been reported that meropenem, a novel β-lactam antibiotic, eliminates Agrobacterium very effectively at low concentrations. Also, meropenem is more efficient for transformation in some crops than other β-lactam antibiotics, such as cefotaxime, carbenicillin, and cefbuperazone (Li et al. 2011;Mii 2004, 2007). Meropenem treatment during Sugi transformation increased transformation efficiency approximately 2-fold compared with carbenicillin using the conventional method and did not affect growth. In transformation experiments using our improved method (co-cultivation on filter paper wicks and Agrobacterium elimination with meropenem), the mean transformation efficiency was approximately 105 GFP-positive colonies per gram of co-cultivated embryogenic tissues, approximately 30 times more than the conventional method . Currently, an independent transgenic Sugi tree of 10-50 lines has been obtained from 1 g of embryogenic tissues established from cross progeny between plus trees Nanasato et al. unpublished data). In this review, we introduce our modified Sugi transformation protocol.     Table 3.

Protocols
Step 1: Establishment of embryogenic tissues 1. From late June to mid-July of the previous year, soak or spray branches in 100 ppm gibberellic acid (GA 3 ; Kyowa-Hakko, Japan) aqueous solution to promote setting of male strobili. From late July to early August of the same year, treat with GA 3 in the same way to promote setting of female strobili. In March of the following year, perform artificial crossing or open pollination and collect immature female cones from late June to early July. Tip: Although the above-mentioned period for GA 3 treatment is effective for the main Sugi planting areas in Japan (Honshu, Sikoku, and Kyushu islands), optimization of the treatment date in advance depending on other areas may be required because it is affected by environmental conditions (Nagao et al. 1989). In order to prevent insect damage, wrap the branches with a knitted fabric bag (bag size 30×45 cm, width×length; AS ONE,) as an insect screen in April when pollination is complete. Immature zygotic embryos before differentiation of the cotyledonary primordium are suitable for induction of embryogenic tissues (Igasaki et al. 2003 Figure 1A). Then soak the cones in 70% ethanol for 10 min and rinse three times with distilled water. 3. Make a cut in the proximal region of each female cone with a scalpel and split it in half lengthwise ( Figure 1B, C), release the immature seeds from separated individual ovuliferous scales, and drop them in a small beaker.
4. Rinse the immature seeds with 70% ethanol for 1 min, sterilize with 6% hydrogen peroxide for 5 min, and rinse 3 times with sterile water. 5. Under a stereomicroscope, aseptically remove the megagametophytes containing immature precotyledonary zygotic embryos from the sterilized immature seeds (Figure 1D, E) and place them on initiation medium (1/2MD′ medium; Figure 1F). Tip: Cut the seed coat vertically with a scalpel, and lightly press it from above with tweezers to expose the megagametophyte ( Figure 1E). Peel off the nucellus coating the megagametophyte. 6. After incubation in the dark at 25°C for 1 month, transfer onto maintenance medium (solid 1/2MD medium), and then subculture every 2 weeks in the same medium ( Figure 1G). 7. Transfer embryogenic tissues from megagametophytes onto solid 1/2MD medium. After proliferation, divide into pieces ∼5 mm in diameter and repeat the subculture to obtain embryogenic tissues for transformation ( Figure 1H, I).

Tip: Since the efficiency of somatic embryo induction from embryogenic tissues is expected to affect the genetic background of the parental family, it is necessary to test with multiple crossing combinations and select the cell line with high somatic embryo induction efficiency. Since the ability to differentiate from embryogenic tissues to somatic embryos is often lost within one year by repeated subculture, it is desirable to cryopreserve the embryonic tissue (see the chapter of Taniguchi et al.).
Step 2 resuspend in co-cultivation medium to obtain a final OD 600 =0.15.
Step 3: Co-cultivation of embryogenic tissues 1. Grow the embryogenic tissue on solid 1/2MD medium for 1 week and transfer 1 g of fresh weight (approximately 10 masses of 8 mm in diameter; Figure 1H and I) to 20 ml of the bacterial suspension in a 50 ml tube (Figure 2A).

Resuspend the embryogenic tissues by vigorously
shaking 10-20 times. 3. Incubate the tube horizontally on an orbital shaker at 100 rpm for 20 min at 25°C. 4. Place a sterile filter paper (7-cm diameter, No. 7, Advantech, Japan, cat. no. 01701070) in a Büchner funnel on a suction bottle, pour in a little cocultivation medium to moisten, and aspirate briefly. 5. Pour approximately 10 ml (containing 0.5 g of embryogenic tissues) of the culture in a Büchner funnel ( Figure 2B), and filter the liquid completely using a vacuum pump (3-5 s, 0.03 MPa). 6. For co-cultivation, lay the filter paper with the embryogenic tissue on a stack of three pieces of sterile filter paper (8.5-cm diameter, No. 2, Advantech, cat. no. 00021085) containing 5.5 ml co-cultivation medium in a 90×20 mm petri dish ( Figure 2C). Repeat step 5 and 6 for the remaining culture. 7. Co-culture for 2 days at 25°C in the dark.
Step 4: Eliminating Agrobacterium contamination 1. Immerse the upper filter paper (from the stack of filter papers) with co-cultured tissue (0.5 g fresh weight) in 40 ml liquid decontamination medium in a 50 ml tube ( Figure 2D). 2. Resuspend the tissue by vigorously shaking 10-15 times and then discard the filter paper. Tip: If embryogenic tissues remain on the filter paper, dislodge them with a spatula ( Figure 2E). 3. Combine the embryogenic tissues from two pieces of Copyright © 2020 The Japanese Society for Plant Cell and Molecular Biology filter paper (total 1g fresh weight) into 40 ml liquid decontamination medium. 4. Wash the tissues using one of the following methods: (i) Vacuum method i-1. Pour 40 ml of the culture containing 1 g of embryogenic tissues onto a sterile filter paper (7-cm diameter) in a Büchner funnel, and filter the liquid completely using a vacuum pump. i-2. Pour 50 ml liquid decontamination medium and gently swirl until the tissues are resuspended.
i-3. Filter the liquid completely using a vacuum pump. Repeat step i-2 three times. Finally resuspend in 40 ml of liquid decontamination medium. i-4. Collect the resuspended tissue into a 50 ml tube and fill up to 40 ml with liquid decontamination medium. i-5. Resuspend tissues by vigorously shaking 5-10 times.

(ii) Strainer method
ii-1. Pour 40 ml of the culture containing 1 g of embryogenic tissue into a 200-mesh tea strainer (Total Kitchen Goods, Japan, cat. no. 5-0706-0703) resting on a 200-300 ml beaker or a 450 ml culture bottle ( Figure  2F) and allow the liquid to strain through completely. ii-2. Gradually p our 50 ml liquid decontamination medium and allow the liquid to strain through completely. Repeat three times. ii-3. Pour approximately 10 ml liquid decontamination medium and quickly transfer the resuspended tissues to a new 200 ml beaker. Repeat four times and collect total 40 ml ( Figure 2G). Tip: Transformation efficiency between the vacuum method and the strainer method is equivalent. The strainer method is simpler than the vacuum method, but if it is difficult to get a high-density mesh strainer, use the vacuum method. 5. Pour 10 ml of the washed suspension containing 0.25 g of tissues onto a new filter paper (7-cm diameter) in a Büchner funnel and filter completely using a vacuum pump. 6. Place the filter paper with the tissues on solid decontamination medium, incubate in the dark at 25°C for 3 days, then transfer to new medium. Tip: When a binary vector containing a visualization marker (GFP, β-glucuronidase gene, etc.) is used, transient expression can be clearly observed by culturing for 3 days on a solid decontamination medium ( Figure 3A; Konagaya et al. 2013).
Step 5: Selection of transgenic embryogenic tissues 1. Transfer the filter paper with the embryogenic tissues to the selection medium approximately 2 weeks after infection (counted from the day when co-cultivation was started).
Tip: Slightly protruded cell masses (approximately 1-2 mm in diameter) observed by regrowth of the tissues is used as a criterion for transfer timing. 2. Incubate in the dark at 25°C and then subculture every 2 weeks in the same medium. 3. Transgenic colonies are formed about 1 to 3 months after infection ( Figure 3B, C, D). Pick up these colonies with tweezers and grow them on a new selection medium. Subculture every 2 weeks in the same medium. Step 6: Somatic embryogenesis and germination 1. Transfer ∼5 mm (diameter) of embryogenic tissues to somatic embryo maturation medium (COM medium; five masses per petri dish) and culture in the dark at 25°C. 2. Somatic embryos are formed after 6-8 weeks ( Figure  3E, F). Pick up somatic embryos with tweezers, transfer to germination medium (mGD medium), and culture at 25°C with a regime of 16 h light (70 µmol m −2 s −1 light intensity) and 8 h darkness ( Figure 3G). 3. Subculture every 1 month in the same medium.
Step 7: Shoot elongation and acclimatization 1. After 2-3 months, transfer plantlets (now ∼2-3 cm high) to 1/2WPM-f medium in a culture bottle ( Figure 3H). Tip: To promote growth, make a hole in the lid of the culture bottle and seal it with the Milliseal (Millipore, Germany, cat. no. FWMS01800) to improve ventilation. 2. Subculture every 1 month in the same medium.
After 3-4 months, carefully wash the roots of the elongated plantlets (now ∼10-15 cm high) and transfer to pots containing soil mixture consisting of peat moss-based soil (Jiffy Mix; Sakata Seed, Japan), akadama soil (granules of volcanic ash soil; Tachikawa Heiwa Nouen, Japan, cat. size middle, 7-14 mm diameter) and vermiculite in the ratio of 1 : 1 : 1 ( Figure 3I). 3. For acclimatization, cover the container containing the plantlets with a polythene sheet to maintain high humidity. Place in a closed glasshouse at 25°C and grow in the shade for the first few weeks. 4. After 5-7 days, gradually reduce the humidity by punching holes in the polythene sheet, increasing the number of holes daily. 5. Approximately 3 months later, transfer the Sugi plants to a semi-closed greenhouse and grow under natural conditions ( Figure 3J). Tip: It is possible to easily induce setting of male strobili by GA 3 treatment even for current year plants of about 15-20 cm height.

Concluding remarks
In order to enhance the efficiency of Agrobacteriummediated transformation of Sugi and simplify the procedure, we have improved the co-cultivation, antibiotics for eliminating Agrobacterium contamination, and washing method of embryogenic tissues. These effective methods could be applied to transformation of embryogenic tissues in other coniferous trees. Currently, we are developing no-pollen Sugi by genome editing via transformation. Using the present method, it is possible to acclimatize the seedlings within about 1 year of transformation (Nanasato et al., manuscript in preparation), and it is possible to determine pollenforming ability approximately 6 months after GA 3 treatment. Although it requires time to establish useful cell lines as explants and to establish a stable supply, cryopreservation technology ameliorates this problem. We hope that the advancement of molecular breeding and molecular genetics will be accelerated in many coniferous trees by the wide utilization of genetic transformation technology.