1 Actinidiaceae 1.1 Actinidia Spp. Kiwifruit

Home , Actinidia, Actinidiaceae, Actinidia arguta, Actinidia deliciosa, Actinidia kolomikta, Actinidia polygama

Manuel Rey,1,2 Yolanda Ferradás,3,4 Óscar Martínez1 and María Victoria González3 1Departamento Biología Vegetal y Ciencia del Suelo, Facultad de Biología, Campus Universitario, Universidad de Vigo, Vigo, Spain; 2CITACA, Agri-Food Research and Transfer Cluster, Campus da Auga, Universidad de Vigo, Ourense, Spain; 3Departamento Biología Funcional, Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Sur, Santiago de Compostela, Spain; 4Present address: Instituto de las Ciencias de la Vid y del Vino (Gobierno de La Rioja-Consejo Superior de Investigaciones Cientificas-Universidad de La Rioja), Logroño, Spain

1. Introduction stamens release sterile pollen because viability is lost by programmed cell death (PCD) during microspore development 1.1. Botany and history (Coimbra et al., 2004; Falasca et al., 2013). Floral evocation occurs during one growth season while The genus Actinidia, commonly known as kiwifruit, is included in floral initiation occurs in the following season (Walton et al., the Actinidiaceae family together with Clematoclethra (Franch.) 1997). During the reproductive process, pollen dispersion Maxim. and Saurauia [Saurauja] Willdenow. This genus com- takes place by wind and, mainly, bees (Costa et al., 1993). prises c.54 species and 75 taxa (Wang and Gleave, 2012; Huang Some Actinidia species are characterized by a high repro- et al., 2013). Paleobiology studies have shown that Actinidia is at ductive success, being able to produce one fruit per flower least 20–26 million years old (Qian and Yu, 1991). Actinidia plants (Biasi and Costa, 1984). Abundant secretions released into the are perennial, deciduous, climbing and/or scrambling vines. intermembrane space throughout the pistil by transmitting They are often reticulate polyploids with a basic chromosome tissue cells are involved in pollen nutrition and guidance, dis- number of x = 29 (McNeilage and Considine, 1989). The genus is appearing after pollen tube passage (González et al., 1996). unusual for the extensive variation in ploidy, meaning that dip- Despite its reproductive success, kiwifruit has a short ef- loid, tetraploid, hexaploid and even octoploid individuals can be fective pollination period which is limited by the stigmatic re- found (Ferguson and Huang, 2007). All Actinidia species appear ceptivity relying on stigmatic papillar integrity (González to be dioecious, which means that a single genetic determinant et al., 1995a,b). This short effective pollination period seems controls sex with a unique set of X/Y chromosomes together to be due to PCD processes occurring in the secretory tissue, with a sex-neutral (XX)n chromosome set, where n depends on which are eventually accelerated by pollination (Ferradás the ploidy levels (McNeilage, 1997; Testolin et al., 1999; Fraser et al., 2014). et al., 2009). Sometimes gender-inconstant variants can occur as Actinidia fruits are defined as berries, with great diversity in well; in this case, staminate flowers usually are smaller than pistil- size, shape, hairiness, skin toughness and palatability, external and late flowers (McNeilage, 1991a,b). Flowers are usually arranged internal colour, flavour, flesh chemical composition and storage in cymes, although they sometimes can appear solitary. Identity of capacity (Ferguson and Huang, 2007). Kiwifruits have a high nu- floral organs is determined by the (A)BCE-like floral model, tritional value and are an excellent source of vitamins C and E, where the A function is unclear (Varkonyi-Gasic et al., 2011). The folate and potassium, with high antioxidant capacity (Wang et al., staminate flowers in male vines develop viable pollen and a rudi- 1996; Ferguson and Ferguson, 2003; Drummond, 2013). mentary ovary without ovules. On the other hand, the pistillate Actinidia chinensis is the most important crop species of the flowers in female vines bear a functional ovary, which is formed by genus, although Actinidia arguta, Actinidia kolomikta or the fusion of many carpels, leaving the radiating styles free. The Actinidia eriantha also have some importance (Ferguson and

Huang, 2007). Planchon (1847) described kiwifruit as a unique for the future of this crop. Controlling excessive vegetative species. Further studies revealed differences in the level of vigour and improving the percentage of fruit set are im- ploidy and in morphological characteristics among individuals portant. Italy has been utilizing the clonal rootstock ‘D1’ for (Liang, 1975; Liang and Ferguson, 1984), and two separate calcareous soils (Vizzotto et al., 1999), and ‘Bruno’ and species were recognized, A. chinensis and Actinidia deliciosa, ‘Kaimai’ are important in New Zealand (Oliveira and Fraser, with diploid and hexaploid individuals, respectively (Liang and 2005). ‘Bruno’ seedlings have been used to support vines Ferguson, 1984). Currently, they are recognized as two varieties that grow rapidly and with greater productivity (Lawes, of the same species, A. chinensis var. chinensis and A. chinensis 1990). ‘Kaimai’, a clonal rootstock of Actinidia hemsleyana var. deliciosa, due to the presence of clines between both var- Dunn, stimulates doubling the number of flowers on each ieties, as well as extensive introgressive hybridization where shoot of the scion (Wang et al., 1994b). they cohabit (reviewed in Ferguson, 2016). The Actinidia genus is distributed throughout much of breeding accomplishments Analyses of A. arguta and eastern Asia, with most species and intraspecific taxa occurring . different rootstock–scion combinations of A. arguta have dem- in south central China (Liang, 1983). In 1904, some seeds of onstrated that both rootstock and scion affect actinidin activity were introduced into New Zealand (Ferguson, A. chinensis and free sugar and organic acid content in fruit (Boyes et al., 2004), with the establishment of the first commercial plantation 1996, 1997). Interspecific rootstocks of Actinidia can have a in the 1930s (Ferguson and Stanley, 2003; Ferguson and major effect on the amount of vegetative growth on A. chinensis Huang, 2007). New Zealand was responsible for the domesti- var. chinensis ‘Hort16A’ vines, dramatically altering plant size, cation and commercialization of kiwifruit. The inherent or- leaf area and scion vigour (Clearwater et al., 2006, 2007). Thorp ganoleptic, nutritional and storage qualities make kiwifruit a et al. (2007) reported that interspecific clonal rootstocks had a widely accepted and popular fruit crop for producers and con- substantial effect on the accumulation and concentration of sumers. For these reasons, commercial kiwifruit growing areas inorganic nutrients in the fruit, leaves and stem sap of ‘Hort16A’ have expanded rapidly in recent decades. In 2017, the global vines. The use of rootstocks of other species of Actinidia, e.g. kiwifruit growing areas had reached over 247,794 ha, with A. hemsleyana, A. eriantha and Actinidia rufa, reduced the level China (165,728 ha), Italy (26,403 ha), New Zealand (11,705 of floral abortion and increased bud burst in ‘Hayward’ (Wang ha) and Iran (10,771 ha) as the main producers accounting for et al., 1994a). The flower-promoting rootstocks have more starch about 87% of world kiwifruit plantings; global kiwifruit pro- grains, more and larger xylem vessels and idioblasts with raphides duction represents 0.62% of total production for major fruit and mucilage (Wang et al., 1994b). Powell and Santhanakrishnan crops (FAOSTAT, 2017). A. chinensis var. deliciosa is the most (1986) showed that ‘Hayward’ on mycorrhiza-infected rootstocks commercialized kiwifruit, and female ‘Hayward’ is the main had increased shoot length and fruit production. cultivar (Ferguson, 1999). However, different species and varieties have been increasing in importance in the last few years. 1.2.2. Scions

1.2. Breeding and genetics Major breeding objectives. Fruit size has been an important criterion for selecting wild species from China with commercial potential (Ferguson and Huang, 2007). Kiwifruit could Actinidia is a genus of recent domestication. Initially, the kiwifruit make an important contribution to human health (Singletary, industry was based on cultivars of A. chinensis var. deliciosa, and since 1975, ‘Hayward’ has been the dominant cultivar (Ferguson 2012; Skinner et al., 2013); the fruits have outstanding nutritional qualities and are a good source of vitamin C, minerals, folic acid and Huang, 2007). Another important cultivar is A. chinensis var. and fibre (Ferguson and Stanley, 2003). However, it can trigger chinensis ‘Hort16A’, known commercially as Zespri® Gold kiwifruit (Wang and Gleave, 2012). The rapid expansion of kiwifruit allergic responses in some consumers (Lucas et al., 2003). Other cultivation has revealed the need to obtain new cultivars or do- important breeding objectives include time of maturity and mestication of new species that are better adapted to different ripening, handling and storage (Ferguson, 1990). growing conditions. A distinctive feature of the genus is the struc- Diseases can affect the production of kiwifruit orchards. tured reticulate pattern of diploids, tetraploids, hexaploids and Reported kiwifruit diseases are mostly of fungal origin, i.e. octoploids in diminishing frequency, associated with geographic Armillaria novae-zelandiae in New Zealand (Horner, 1992), separation of ploidy races (Ferguson and Huang, 2007). This Phomopsis sp. in Greece (Elena, 2009), Cadophora melinii in ploidy variation between taxa and the existence of ploidy races Italy (Prodi et al., 2008) and Verticillium albo-atrum in Chile (Auger ., 2009). Blouin . (2012) identified two viti within taxa can make crossing in Actinidia difficult. However, Li et al et al viruses from in New Zealand. Bacterial canker et al. (2010) observed that the type of fruit hairs, fruit flesh colour A. chinensis and fruit weight are related to ploidy. Therefore, breeders can caused by Pseudomonas syringae pv. actinidiae (Psa) is by far obtain a large range of kiwifruits. the most serious disease worldwide (McCann et al., 2013). Introduction of genotypes of Actinidia resistant to Psa is of critical importance. 1.2.1. Rootstocks Major breeding objectives. Kiwifruit was initially not breeding accomplishments. Breeding programmes have grafted in commercial orchards, but expansion of the industry to been initiated wherever the industry has been dependent on regions with poor soil or environmental features is essential ‘Hayward’, e.g. New Zealand and Italy, to develop new cultivars Actinidia spp. Kiwifruit 3

that are better adapted. Selection was initially focused on Fraser et al., 2004; Liu et al., 2016). Single nucleotide poly- openly pollinated seedlings in the wild or from named cultivars. morphisms (SNPs) are valuable for characterizing genetic The red-fleshed cultivar ‘Hongyang’ was selected from 3213 variation in evolutionary and ecological studies of natural seedlings of A. chinensis var. rufopulpa (Wu and Li, 1993; Wang kiwifruit populations (Zhou et al., 2011). Recent studies pro- et al., 2003). ‘California Male’ and ‘Chico Male’ were selected vide new insights into the use of cpDNA markers in studies of from seedlings of A. chinensis var. deliciosa (Ferguson and phylogenetic, biogeography and introgression in Actinidia (Li Huang, 2007). Several A. chinensis var. deliciosa-derived culti- et al., 2013b). The North American collection of cold-hardy vars have been obtained in New Zealand, i.e. ‘Abbott’, ‘Allison’, kiwifruit has been deconvoluted through genotyping by sequen- ‘Bruno’, ‘Gracie’, ‘Hayward’ and ‘Monty’ (Ferguson, 1997). cing (GBS) (Melo et al., 2017). Most cultivars have been derived from the original introduc- Some of these molecular markers can be used for marker tion of seeds into New Zealand (Ferguson and Huang, 2007). assisted selection (MAS), especially for early sex identification. Gill Only a few selections have been derived from controlled et al. (1998) transformed two sex-linked RAPD markers into crosses: ‘Tomua’ (New Zealand) is a cross of ‘Hayward’ with an sequence-characterized amplified regions (SCAR) markers that early flowering male (Muggleston et al., 1998). ‘Summerkiwi™’ are now routinely used to identify and discard male plants at the (Italy) was obtained from the cross of ‘Hayward’ with a fruiting seedling stage in breeding programmes in New Zealand (Testolin male. Other A. chinensis var. deliciosa cultivars originating from and Cipriani, 2016). SSR markers have also been identified for dis- controlled crosses include ‘Katiuscia’, ‘Silvia’ and ‘Stefania’ criminating between males and females in progenies (Scaglione (Italy) and ‘Skelton’ (New Zealand), but none of these has been et al., 2015; Zhang et al., 2015b) and for molecular studies of widely grown (Ferguson and Huang, 2007). With respect to hermaphroditism and self-compatibility (Sakellariou et al., 2016). A. chinensis var. chinensis, ‘Hort16A’ (New Zealand) is the most successful cultivar obtained from controlled crosses. It was selected from a cross between a male and a female derived from 2.2. Gene cloning two seedling accessions from different parts of China (Muggleston et al., 1998; Ferguson et al., 1999). Molecular studies of Actinidia species initially focused on cloning Fairchild (1927) performed the first interspecific cross in several genes of interest, especially those expressed in fruit tis- Actinidia, crossing a female A. arguta with pollen of A. chinensis sues. Most of the early information was reviewed in the first edi- var. deliciosa. Since then, several other interspecific crosses tion of this chapter (Oliveira and Fraser, 2005). More recently, the have been made with reasonable success (Ferguson and Huang, characterization of new genes, in many cases cloned from a kiwi- 2007). Some interesting selections include ‘Kiri’, from a cross fruit expressed sequence tag (EST) database and involved in dif- of A. arguta × A. deliciosa backcrossed to A. deliciosa (White ferent aspects of fruit development, have been reported. These and Beatson, 1993), ‘Kosui’, a hybrid of A. chinensis and A. rufa, include six lipoxygenase enzyme (LOX) genes related to aroma ‘Shinzan’, from A. arguta × A. deliciosa, and ‘Sanryoku’, a hybrid production (Zhang et al., 2006), two lycopene beta-cyclase (LCY-β) of A. deliciosa and A. chinensis (Kokudo et al., 2003). genes associated with carotenoid accumulation (Ampomah- Although most of the cultivars obtained by breeding pro- Dwamena et al., 2009), 14 xyloglucan endotransglucosylase/ grammes have involved A. chinensis var. chinensis and A. chinensis hydrolase (XTH) genes involved in fruit ripening (Atkinson et al., var. deliciosa, other kiwi species have also been used, i.e. 2009) and an l-galactose guanyltransferase gene encoding an A. arguta, A. kolomikta and A. rufa (Ferguson and Huang, 2007). enzyme involved in ascorbate biosynthesis (Laing et al., 2007). Two glycosyltransferase genes, F3GT1 and F3GGT1, involved in the anthocyanin and flavonoid biosynthetic pathway were identified from red-fleshed kiwifruit (Montefiori et al., 2011), 2. Molecular Genetics and an l-galactose dehydrogenase (AlGalDH) gene was cloned from Actinidia latifolia (Shang et al., 2009). The recent publica- 2.1. Marker-assisted selection tion of a transcriptome profiling during fruit development and ripening (Tang et al., 2016) will encourage further studies. Molecular markers, i.e. restriction fragment length polymor- Several aspects of flowering, a critical developmental process phisms (RFLPs), random amplified polymorphic DNAs for kiwifruit, have gained interest. Walton et al. (2001) identified (RAPDs) and amplified fragment length polymorphisms fragments with homology to the floral meristem identity genes (AFLPs), are being used for genetic characterization (Palombi LFY and AP1, and nine MADS-box genes (FUL-like, FUL, and Damiano, 2002; Oliveira and Fraser, 2005; Prado et al., AP3-1, AP3-2, PI, AG, SEP1, SEP3 and SEP4) required for 2006), somaclonal variation evaluation of in vitro regenerated floral meristem and floral organ specification have been identi- plants (Palombi and Damiano, 2002; Prado et al., 2007), pre- fied and functionally characterized (Varkonyi-Gasic et al., diction of male plant flowering times (Novo et al., 2010) and 2011). Also, SVP-like (short vegetative phase) genes have been the characterization of genetic and epigenetic diversity with identified, with potentially distinct roles in bud dormancy and respect to ploidy levels in A. chinensis (Liu et al., 2015). Genetic flowering (Wu et al., 2012b, 2017). The genes encoding SOC1 diversity was also analysed using short sequence repeats (micro- and FT, integrators of endogenous and environmental signals to satellites or SSRs) (Huang et al., 1998; Zhen et al., 2004; promote flowering, have also been identified and characterized Fraser et al., 2005; Korkovelos et al., 2008; Man et al., 2011; (Varkonyi-Gasic et al., 2013; Voogd et al., 2015, 2017). Other Wang and Zhang, 2012; Kwon et al., 2013), and SSRs have aspects of flower development that have been addressed include been used for creating genetic maps (Testolin et al., 2001; the characterization of two terpene synthases that could 4 M. Rey et al.

account for the major floral sesquiterpene volatiles observed in eight of these AdERF genes as highly associated with kiwifruit both male and female A. deliciosa flowers (Nieuwenhuizen et al., ripening and softening, including four putative activators and 2009) and an (S)-linalool synthase gene encoding a terpene four putative repressors (Zhang et al., 2016). synthase related to floral aroma in A. arguta (Chen et al., 2010). Yang et al. (2007) reported that α-expansin genes are The identification of genes involved in sex determination upregulated by exposure to ethylene or expressed during fruit has been an early research goal. The accumulating genetic evi- softening (Richardson et al., 2011). On the other hand, the dence for sex determination suggests that an active Y system study of the expression profiles of several lignin-related genes functions in Actinidia with maleness determined by two linked indicated that lignin accumulates during kiwifruit cold storage and dominant loci mapped in a 1-Mb subtelomeric region on (Li et al., 2017), thus reducing fruit edibility and quality. Hu chromosome 25 (Fraser et al., 2009; Zhang et al., 2015b). The et al. (2016) identified and characterized five genes as being genes contained in this region are unknown, although Kim et al. potentially involved in starch degradation during kiwifruit (2010a) identified, among 15 differentially expressed genes in postharvest ripening. male and female flowers, two with known functions. These Flavour, colour and vitamin content are important traits encode a pectin methyl esterase, with functions in pollen devel- related to kiwifruit ripening. Günther et al. (2011) investigated opment, and an adenosine diphosphate (ADP)-ribosylation changes in steady-state transcript levels of Actinidia acyl- factor, with essential roles in vesicle trafficking. transferases (ATs) to select sequences, potentially encoding for alcohol ATs that could be involved in ester biosynthesis. They evaluated their involvement in flavour-related fruit ester formation, identifying increased transcript levels specific in ripe 2.3. Genomics fruit and regulated by ethylene. These authors also found that the production of aroma-related esters decreased in response to Early molecular studies reported the expression of a limited cold storage with time, but they were restored by ethylene treat- number of genes that were differentially expressed during a ment (Günther et al., 2015). Terpenes and their derivatives are particular developmental process or phase. Available genomic related to kiwifruit aroma. Nieuwenhuizen et al. (2015) cor- tools, i.e. an extensive kiwifruit EST database (Crowhurst et al., related terpinolene production in ripe A. arguta fruit with 2008) and the first draft of the kiwifruit genome sequence (Huang expression of terpene synthase1 (AaTPS1). Comparative pro- et al., 2013), have begun to generate more data. An international moter analysis allowed them to identify transcription factors as consortium was formed in 2015 to generate new information key for TPS expression. C6-aldehydes have been also reported for this draft (Crowhurst et al., 2016). Other recent genomic as active flavour notes in kiwifruit; their production is mediated resources include a restriction site-associated DNA (RAD)-based by LOX. Zhang et al. (2009) described how several LOX linkage map (Scaglione et al., 2015), a comparative database for encoding genes that are related to ripening and sensitive to kiwifruit genomics (Yue et al., 2015) and the first complete ethylene are related to the release of fruit ripening-associated chloroplast genome sequence in this family (Yao et al., 2015), aromatic compounds. whose evolution was recently analysed (Wang et al., 2016). With High content of vitamin C is an important feature of some of this information, Avsar and Aliabadi (2015) identified kiwifruit. Bulley et al. (2009) determined that the high concen- putative microRNAs and Li et al. (2015) established the expres- tration of ascorbate found in the fruit of A. eriantha is probably sion profiles of anthocyanin biosynthesis and accumulation genes. due to the expression of GDP-mannose-3′,5′-epimerase An important part of functional genomic studies is the use (GME) and GDP-l-galactose guanyltransferase (GGT) genes. of quantitative polymerase chain reaction (qPCR) analysis to de- Overexpression of GGT in Arabidopsis or transient expression termine the expression at the transcriptional level of those genes in tobacco leaves of GME and GGT together confirm that identified using genomic tools. For obtaining accurate relative GGT catalyses a major control point of ascorbate biosynthesis. expression data in qPCR, appropriate reference genes for nor- Another enzyme involved in ascorbate synthesis is GDP-galactose malization must be used. Petriccione et al. (2015) assessed nine phosphorylase (GGP). Li et al. (2014) found some correlation candidate reference genes in A. deliciosa leaves during the first between the expression of GGP and ascorbate concentration period of interaction (13 days post inoculation) with Psa. Ferradás during early fruit development. et al. (2016) conducted a systematic study of eight primer pairs New kiwifruit cultivars with different flesh colour have been corresponding to four candidate reference genes into five plant introduced, and red-fleshed cultivars are probably the most sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh interesting. Anthocyanins, carotenoids and chlorophylls are re- and seeds) commonly used in kiwifruit gene expression analyses. sponsible for different flesh colours. Montefiori et al. (2011) The most extensive studies of functional genomics were identified two glycosyltransferases (F3GT1 and F3GGT1) as related to fruit development and ripening, e.g. the role of ethylene responsible for much of the anthocyanin diversity. While during ripening. Synthesis and signalling of ethylene in relation F3GGT1 is responsible for the end product of the pathway, to these processes (in particular kiwifruit ripening) have been F3GT1 is the key enzyme regulating the accumulation of addressed (Yin et al., 2008, 2009, 2010; Atkinson et al., 2011; anthocyanin in red-fleshed cultivars. Li et al. (2015) conducted Murakami et al., 2014; McAtee et al., 2015; Nieuwenhuizen a phylogenetic analysis of the kiwifruit genome followed by et al., 2015). From the draft sequence of the kiwifruit genome examination of the transcript levels of putative anthocyanin (Huang et al., 2013), 105 novel ERF genes were identified, genes, finding differential expression levels during kiwifruit belonging to different subfamilies, but all of which displayed a development. They also used phylogenetic analysis to identify single AP2/ERF domain. Gene expression analysis identified nine transcription factors potentially involved in anthocyanin Actinidia spp. Kiwifruit 5

metabolism from transcriptome data. It has been shown that Several traits related to biotic and abiotic stresses have storage at low temperature stimulates anthocyanin accumula- been recently addressed, e.g. bacterial canker of kiwifruit caused tion in kiwifruit by regulating structural and regulatory genes by a virulent strain of Psa. Psa affects mainly kiwifruit cultivars involved in anthocyanin biosynthesis (Li et al., 2017). with gold flesh, although it can also affect green-fleshed culti- Studies of flowering regulation are extremely important vars (‘Hayward’). A sample of genes representing both the basal due to the dependence of this species on pollination. Walton et al. defence and the resistance gene-mediated defence pathways (2001) identified homologues of the Arabidopsis floral meristem were characterized by Fraser et al. (2015). Li et al. (2016) identity genes LEAFY and APETALA1, and in situ hybridiza- identified nucleotide-binding sites (NBS)-encoding resistance tion analysis revealed that flowering occurs through two growing genes in the kiwifruit genome. Corsi et al. (2017) detected seasons. Later, Varkonyi-Gasic et al. (2011) described floral changes in the transcription profiles of two genes involved development in kiwifruit at the molecular level, identifying in the metabolic pathway of systemic acquired resistance nine Actinidia MADS-box genes as genetic markers for inflor- (SAR) (pathogenesis-related proteins 1 and 5) in response to escence and flower development, potentially having impact on chitosan treatment of kiwifruit plants inoculated with Psa. kiwifruit growth, phase change and time of flowering. The role Wang et al. (2017) identified circular RNAs as a mechanism of of kiwifruit FT, described in other species as a floral integrator, the species-specific response to Psa; Yin et al. (2012) showed but also CEN, BFT or FD genes were explored by Varkonyi- that AdERF genes differentially respond to abiotic stresses Gasic et al. (2013) and Voogd et al. (2017), suggesting their roles during fruit postharvest storage, and some of them have been in the integration of developmental and environmental cues identified as acetylsalicylic acid-responsive genes (Yin et al., that affect dormancy, bud break and flowering. However, func- 2013). Li et al. (2013a) established that GGP expression in two tional and expression analyses of kiwifruit SOC1-like genes, kiwifruit species is regulated by light or abiotic stress via the also described as a floral integrator, suggested that they may relative cis-elements in their promoters. Waterlogging tolerance not have a role in the transition to flowering but may affect the can be important in fruit production; Zhang et al. (2015a) have duration of dormancy (Voogd et al., 2015). Other kiwifruit identified up to 14,843 unigenes differentially expressed during MADS-box genes with homology to Arabidopsis SVP have waterlogging, representing an important resource for under- been identified by Wu et al. (2012b), suggesting that they may standing the molecular mechanisms of the waterlogging re- have distinct roles during bud dormancy and flowering. A kiwi- sponse. Zhang et al. (2016) also cloned and established the fruit protein inversely correlated with spring bud break has implication of pyruvate decarboxylase 1 gene (AdPDC1) in been identified (Wood et al., 2013). waterlogging stress. Liu et al. (2016) cloned two dehydroascor- Wu et al. (2014) demonstrated that SVP3 reduced antho- bate reductases, correlating them with increased l-ascorbic cyanin accumulation in petals of transgenic A. eriantha in a acid concentration and tolerance of salinity. quantitative manner. The underlying mechanism was the reduced expression of the key structural gene F3GT1 (Montefiori et al., 2011) and the transcriptional repression of regulatory 3. Micropropagation MYB transcription factors, i.e. MYB110a, a key gene required for kiwifruit petal pigmentation (Fraser et al., 2013). Kiwifruit micropropagation was first described by Harada The regulation of the timing of flowering is necessary to (1975) and has been reviewed by several authors (Revilla et al., ensure that sexual reproduction occurs at an appropriate time 1992; Ferguson et al., 1996; Kumar and Sharma, 2002; Rugini to enable cross-pollination. In many plant species, the timing of and Gutiérrez-Pesce, 2003; Oliveira and Fraser, 2005; Wang flowering depends on seasonal cues, i.e. photoperiod and tem- and Gleave, 2012). Nodal segments or shoot apices are disin- perature. With kiwifruit, exposure to winter temperatures is fested with sodium hypochlorite (up to 1.5%, w/v) with a few essential for synchronized bud break and efficient flowering drops of detergent and then cultured on MS-based medium (Walton et al., 2009), but it is less clear if photoperiod plays any (Murashige and Skoog, 1962) with several phytohormone com- role in flowering. Ferradás et al. (2017) identified genes en- binations (for details see Revilla et al., 1992). Other culture coding kiwifruit phytochromes and cryptochromes and deter- media used are B5 medium (Gamborg et al., 1968; Barbieri and mined their daily and annual expression patterns, showing that Morini, 1987) or Quoirin medium (Quoirin and Lepoivre, they were significantly highly expressed from late floral devel- 1977) as modified by Standardi (1981). Zeatin is widely used opment until full bloom and fitting with floral evocation, for shoot proliferation because benzyladenine (BA) causes closely matching the peaks of expression of AcFT- and AcCO- hyperhydricity of leaves (Marino and Bertazza, 1990); however, like genes. another study of the effect of BA on kiwifruit apical shoot cul- Kiwifruit usually has >1000 seeds in each fruit. Studies on tures did not show hyperhydricity (Moncaleán et al., 2001). pollen–pistil interaction have determined that PCD occurs in Thidiazuron and m-topolin have also successfully been used female tissues after anthesis and is accelerated by pollination for micropropagating two male cultivars (Prado et al., 2005). The (Ferradás et al., 2014). Several genes related to ethylene biosyn- best proliferation rates are five shoots per explant in several re- thesis and signalling show a correlation with PCD signs in un- ports (Standardi, 1983; Standardi and Catalano, 1985). pollinated and pollinated flowers. The only exception is ACO5 Inhibition of ethylene in apical segments using aminoethoxy that is only expressed at the end of effective pollination period vinylglycine (AVG), 1-methylcyclopropene (1-MCP) or its or after pollen tube passage, indicating its possible implication reduction in the culture atmosphere with ventilated flasks in regulating senescence processes in other organs, i.e. ovaries increases the number and length of shoots produced per ex- or petals (Ferradás et al., in press). plant (Arigita et al., 2003, 2004). 6 M. Rey et al.

Rooting of microshoots has been achieved by a short (15–20 s) A. chinensis var. deliciosa (green kiwifruit, hexaploid) (Ferguson, pulse with a high indole-3-butyric acid (IBA) concentration 2016). Oliveira and Pais (1992) reported the induction of som- (up to 5 mM), followed by transfer to half-strength Cheng atic embryos from in vitro ‘Hayward’ leaves, although germin- (1975) culture medium (González et al., 1995c) or to a sterile ation was not achieved. In both reports, a pretreatment of cold substrate mixture for ex vitro rooting (Monette, 1986, 1987). and starvation was required for embryogenic culture induction. Kiwifruit microshoots have also been rooted with lower concen- Induction from in vitro leaves occurred in explants submerged trations of IBA for a longer period and transfer to medium with in the culture medium, indicating that the anaerobic conditions activated charcoal (Harada, 1975; Revilla and Power, 1988). could be beneficial (Oliveira and Pais, 1992). Acclimatization can be achieved by gradually reducing relative The induction of embryogenic cultures from stigmatic humidity (Marino and Bertazza, 1990; González et al., 1995c). arm explants can occur (Ferradás et al., unpublished data). Reduced light during acclimatization can improve survival Induction medium (SEIM4) consisted of Nitsch and Nitsch (Shen et al., 1990; Pedroso et al., 1992). Carbon dioxide enrich- (1969) salts, Murashige and Skoog (1962) vitamins, 0.1% casein ment (up to 600 μmol/mol) of the culture atmosphere of kiwi- hydrolysate and 6% sucrose, 1 μM 2,4-dichlorophenoxyacetic fruit explants also improves acclimatization (Arigita et al., 2002). acid (2,4-D) and 4.5 μM thidiazuron (TDZ) (Acanda et al., Conventional propagation of kiwifruit is being supple- 2013) and supplemented with 0.5 mM sodium butyrate (NaB). mented by micropropagation due to increasing demand for Somatic embryos germinated in vitro (Fig. 1.1.1). plant material (Oliveira and Fraser, 2005). Micropropagated plants show a 1-year delay in flowering and lower efficiency in energy uptake and partitioning of photoassimilates (Oliveira 5.1.2. Organogenesis and Fraser, 2005) that has been ascribed to tissue culture-induced The organogenic potential of different explants is characteristic juvenility (Díaz Hernandez et al., 1997). of kiwifruit and related species (Revilla et al., 1992; Ferguson et al., 1996; Oliveira and Fraser, 2005; Wang and Gleave, 2012). The response is dependent on the explant type and the level and 4. Micrografting combination of growth regulators used. Cytokinins are critical for the induction of the organogenic cultures. Shoot induction Apple stem grooving virus (ASGV) was first identified in a New was obtained using zeatin or BA, although other cytokinins are Zealand quarantine of a kiwifruit import from China (Clover effective, e.g. TDZ (Nayak and Beyl, 1987; Suezawa et al., 1988). et al., 2003). Since then, up to 13 viruses have been identified Interactions between salt dilutions in the culture medium and the and classified into three groups depending on the specificity cytokinin concentrations were observed with nodal segments to kiwifruit, host range and the ability to damage the crop of ‘Hayward’ (Rey et al., 1992); reducing both factors increased (Blouin et al., 2013). However, protocols for virus eradication shoot bud induction. Auxins, e.g. indoleacetic acid (IAA), at have not been reported. Ke et al. (1993) used ex vitro micro- low levels also had a positive role (Rey et al., 1992). When au- grafting for accelerating growth and maturity of kiwifruit xins, i.e. naphthaleneacetic acid (NAA), are used at higher con- clones derived from protoplasts. centrations, the organogenic response is improved (Suezawa et al., 1988), whereas, 2,4-D has a negative effect (Oliveira, 1992, cited in Oliveira and Fraser, 2005). 5. Somatic Cell Genetics Kim et al. (2007) described an efficient regeneration protocol using cross sections of shoots (800 μm thick) as explants; sev- 5.1. Regeneration eral explants are thereby obtained from a single stem with high shoot proliferation rates. The type of callus formed is a critical 5.1.1. Somatic embryogenesis factor for indirect organogenesis induction (Suezawa et al., 1988; Rey et al., 1992; González et al., 1995c). Fraser and Harvey (1986) described somatic embryogenesis The organogenic potential of explants from male and female from cultures that originated from anther walls in three tissues differs and is related to the different endogenous cyto- A. chinensis genotypes: A. chinensis var. chinensis (yellow kiwi- kinin and auxin levels (Oliveira and Fraser, 2005). Female stem fruit, diploid), male (‘Matua’) and female (‘Hayward’) and segments of A. deliciosa showed higher organogenic potential

(a) (b)

Fig. 1.1.1. Somatic embryogenesis from stigmatic arms of kiwifruit ‘Hayward’. (a) Cluster of somatic embryos from embryogenic culture that originated in stigmatic arms on SEIM4 (Acanda et al., 2013) medium supplemented with 0.5 mM NaB. Bar = 1 mm. (b) Plantlet formation from somatic embryos cultured on germination medium (Acanda et al., 2013). Bar = 1 cm. Actinidia spp. Kiwifruit 7

than male explants (Gui, 1979); whereas, male stamens produced induced from mature endosperm was reported by Goralski et al. callus and shoots but female ones produced roots (Brossard- (2005). Callus was induced on an MS medium containing 23.3 Chriqui and Tripathi, 1984, cited in Ferguson et al., 1996). μM kinetin; shoots developed from callus obtained on a medium Explanted roots of the male ‘Tomuri’ regenerated shoots on containing 2.27 μM TDZ. Flow cytometry analysis of leaves in- medium with N6-2-isopentenyladenine, but not of the female dicated the presence of triploid plants. ‘Hayward’ (Chiariotti et al., 1991). Hirsch and Bligny-Fortune (1979) reported the loss of chlorophyll in cell suspensions in 5.1.5. Protoplast isolation and culture the presence of 2,4-D derived from stem segments of female genotypes, whereas this did not occur with male genotypes. In Breeding of Actinidia spp. is restricted by dioecism and polyploidy, ‘Hayward’ petioles, organogenic callus formation was restricted and somatic hybridization could be useful to produce material to the cut ends of the explant in contact with the induction with desirable traits from two species in this genus (Wang and medium (González et al., 1995c), but petioles from male culti- Gleave, 2012). Methods for protoplast isolation have relied on vars responded by forming callus over the entire surface of the use of cell wall-degrading enzymes, i.e. Cellulase R-10 and the explants (Prado et al., 2007). Other factors, e.g. the level of Macerozyme R-10, etc. (Table 1.1.1). Success in plant regener- sugar in the culture medium and the light quality, can affect or- ation from protoplasts has been very limited (Table 1.1.1). ganogenesis (Muleo and Morini, 1990; Leva and Muleo, 1993). Tsai (1988) isolated protoplasts from callus derived from leaves on MS medium with 4.5 μM zeatin. Protoplasts were isolated following incubation for 4–5 h at 28°C in a solution con- 5.1.3. Haploid recovery taining 2% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.5 M mannitol and 3 mM MES. Isolated protoplasts were cul- Germanà (2006), Fraser and Harvey (1986) and Oliveira and tured in a series of differentiation media (Tsai, 1988; Revilla Fraser (2005) reported that haploid recovery has been unsuc- et al., 1992) for cell wall regeneration, callus formation and shoot cessful in Actinidia. Trihaploid plants have been recovered in bud induction. Protoplasts from A. eriantha leaves were also ‘Hayward’ by parthenogenic induction though pollination with obtained (Zhang et al., 1998) using 1% Cellulase R-10, 0.5% gamma-irradiated pollen (Pandey et al., 1990; Chalak and Macerozyme R-10 and 0.05% Pectolyase Y-23. Subsequently, Legave, 1996, 1997). After pollination of ‘Hayward’ with ir- callus was recovered and shoots were regenerated on MS radiated pollen from a male plant (‘D uno’), four trihaploid medium with 2.5 μM zeatin and 0.5 μM IAA. plants and a double trihaploid plant were recovered (Chat et al., Oliveira and Pais (1991) used thin slices of callus derived 2003). Chromosome doubling was also obtained following from petioles as a source of protoplasts after incubation with cel- treatment with oryzalin and adventitious regeneration (Chalak lulase and macerozyme and purification by filtration and centri- and Legave, 1996). As A. chinensis var. deliciosa may be an allo- fugation in a 0.6 M sucrose solution. Protoplasts were cultured polyploid, the double trihaploids obtained by chromosome using a two-step procedure with a SH-based (Schenk and doubling (spontaneous or induced) are not necessarily homozy- Hildebrandt, 1972) medium in the presence of a complex mix- gous for a number of genetic loci (Oliveira and Fraser, 2005). ture of phytohormones (NAA, 2,4-D, kinetin and BA). Culture Chat et al. (2003) analysed the trihaploid and the double trihap- on an MS-based medium with IAA and zeatin stimulated the loid plants and determined that the chloroplast genome of the recovery of shoot buds (Oliveira and Pais, 1991). Raquel and male pollen donor plant was transmitted to the trihaploid pro- Oliveira (1996) improved the yield of protoplasts by preculturing geny and that all the nuclear SSR loci tested were of maternal the leaves for 5–6 days on a zeatin-containing medium. origin in the progeny. Fraser and Harvey (1988) isolated protoplasts from microspores obtained at the tetrad stage of pollen development. Protoplasts from male plants remained viable for several weeks 5.1.4. Triploid recovery in culture, but those isolated from female plants did not survive Kiwifruit plants were first regenerated by somatic embryogenesis more than 2 days, suggesting that those pollen grains were from endosperm (Gui et al., 1982 and Huang et al., 1983, both already not viable. cited in Revilla et al. 1992). High doses of zeatin (13.7 μM) in MS It is likely that interest in protoplast technology could be medium were used for induction. The production of triploid revived with the advent of new genomic technologies such as plants (87 chromosomes) was reported by Huang et al. (1983), CRISPR-Cas9. with regenerants having characteristics intermediate between the diploid var. chinensis and the hexaploid A. deliciosa (Oliveira and Fraser, 2005). Gui et al. (1993) also regenerated triploid plantlets 5.2. Genetic manipulation from organogenic callus induced from A. chinensis endosperm. Triploid plants in the field showed variation in fruit size and 5.2.1. Mutation induction and somaclonal variation shape, seed number, quality components, and chromosome number. However, correlation between the number of seeds or ‘Tomuri’ and ‘Clone A’ showed increased genetic variation based chromosomes and fruit size was not observed (Gui et al., 1993). on AFLP analysis following an in vitro cycle in comparison with With the same zeatin concentration with LS (Linsmaier field-grown plants (Prado et al., 2005, 2007). Chiariotti et al. (1991) and Skoog, 1965) medium, regeneration of plants from organo- detected isoenzyme variation among plants regenerated from genic callus induced from immature endosperm of interspecific in vitro roots of ‘Tomuri’. Boase and Hopping (1995) obtained kiwifruit hybrids was reported by Kin et al. (1990). Chromosome dodecaploid plants after axillary bud culture of hexaploid counts of these plants revealed the presence of extensive mixo- plants. Marino and Bertazza (1998) regenerated leaf-derived ploidy. The regeneration of plants from organogenic callus somaclones of A. deliciosa under different pH variations. They 8 M. Rey et al.

Table 1.1.1. Summary of results of protoplast isolation of different Actinidia genotypes. Genotypes were corrected from the original papers and named according to the current taxonomy of the genus Actinidia (Ferguson, 2016).

Enzymes for protoplast Plant Genotype Explant source of protoplasts isolation regeneration References A. chinensis var. Leaves from in vitro grown 1.5% Cellulase R-10 No; formation of Cossio and Marino deliciosa, ‘Hayward’ plants; callus from stem 0.3% Macerozyme R-10 small colonies (1983) slices and cell suspensions

A. chinensis var. Callus from stems and petioles 1.5% Cellulase R-10 No; formation of Pedroso et al. (1988) deliciosa, ‘Hayward’ and cell suspensions 1% pectinase small colonies

A. chinensis var. Cell suspensions from petiole 0.5% Cellulase R-10 No; formation of Revilla and Power deliciosa, ‘Hayward’ callus 0.5% Driselase small colonies (1988) 0.5% hemicellulase 0.5% Macerozyme R-10

A. chinensis var. Callus from in vitro leaves 2% Cellulase R-10 Ye s Tsai (1988) deliciosa 0.5% Macerozyme R-10

A. chinensis var. Microspores 1% crude extract from No Fraser and Harvey deliciosa snail digestive gland (1988)

A. chinensis var. Cell suspensions from stem 3% Cellulase R-10 Ye s Mii and Ohashi (1988) deliciosa, ‘Abbott’ callus 1% Macerozyme R-10

A. chinensis var. Petiole callus 1.5% Cellulase R-10 Ye s Oliveira and Pais deliciosa, ‘Hayward’ 0.5% Macerozyme R-10 (1991)

A. chinensis var. Leaves from in vitro shoots 1.5% Cellulase R-10 Ye s Raquel and Oliveira deliciosa, ‘Hayward’ 0.5% Macerozyme R-10 (1996)

A. chinensis genotype Callus from cotyledons 2% Cellulase R-10 Ye s Xiao et al. (1992)

A14N7 0.5% Macerozyme R-10

A. deliciosa genotype Callus from cotyledons 2% Cellulase R-10 Ye s Xiao and Han (1997)

A16N1 0.5% Macerozyme R-10

A. eriantha Leaves from in vitro shoots 1% Cellulase R-10 Ye s Zhang et al. (1998) 0.5% Macerozyme R-10 0.05% Pectolyase Y-23

showed that high pH produced plants more tolerant of high genotypes were obtained (Shen et al., 1990b, cited in Ferguson pH. Protoplast culture can also produce genetic variation (pro- et al., 1996). Fraser et al. (1991) and Harvey et al. (1995) tried toclonal variation) in different traits of regenerated plants, e.g. to achieve chromosome doubling via colchicine treatment, cytology, morphology and resistance (reviewed by Liu et al., but very few doubled plantlets were identified. Wu et al. 2003). He et al. (1995, cited in Liu et al., 2003) reported that (2011) recovered tetraploid plants from five different chromosome numbers of plants regenerated from protoplasts genotypes of A. chinensis using colchicine treatment. In this varied between 59 and 310, with several different ploidy levels work, small shoot buds induced from petioles were as aneuploids, diploids, tetraploids or hexaploids. In the same immersed in a colchicine solution (0.05 or 0.1%) for 4 h, work, changes in sex were reported, since a third of the plants washed with sterile water and recultured for shoot induction. regenerated from female A. deliciosa were male. Differences in Treatment with 0.05% colchicine was the optimum treatment internode length, leaf length and width and petiole length for producing tetraploid shoots. Fruit of the autotetraploids among different plants regenerated from protoplasts of A. deliciosa induced from three female red-fleshed kiwifruit selections were also reported (Cai et al., 1992, cited in Liu et al., 2003). were 50–60% larger than fruit from their diploid progen- Gamma-irradiation of axillary and adventitious buds was itors, and this characteristic was stable over four consecutive attempted for mutation breeding, but no clearly improved years (Wu et al., 2012a). Actinidia spp. Kiwifruit 9

5.2.2. Somatic hybridization of drought and salinity, accumulation of bioactive compounds important in human health, improving rootstocks and manipu- Breeding objectives. As polyploidy and dioecy in Actinidia lation of ethylene production in relation to fruit ripening restrict breeding, somatic hybridization could enable recovery (reviewed in Oliveira and Fraser, 2005; Table 1.1.2). of fertile hybrids, by combining genetic backgrounds within the same gender. After initial attempts (Lindsay et al., 1995), protocol Although most genetic transformations were somatic hybridization of A. deliciosa with A. chinensis (6x + 2x) . Agrobacterium mediated, other methods, i.e. particle bombard- and A. chinensis with A. kolomikta (2x + 2x) was achieved (Xiao ment and PEG mediated, have been reported. Qiu et al. (2002) and Han, 1997; Xiao et al., 2004). Protoplasts of A. deliciosa and transformed A. deliciosa cell suspensions using particle bom- A. chinensis were isolated from cotyledon callus, while those of bardment. They introduced the maize DHN1 gene (induced A. kolomikta were from the youngest fully expanded leaves of in response to abiotic stresses) fused to the green fluorescent micropropagated shoots. protein (GFP) reporter gene. GFP expression was localized in the nucleus after 10 h in response to increased osmotic stress. protocol. Purified protoplasts of A. deliciosa and A. chin- PEG-mediated permeabilization yielded high levels of ensis were mixed in equal volumes, with 0.2 ml of this mixture transient expression, especially in combination with heat shock being transferred into a glass tube with an equal volume of a (Oliveira et al., 1991). They used chloramphenicol acetyl trans- mixture (8:1:1) of a polyethylene glycol (PEG) solution (40% ferase (CAT) reporter gene to assess the conditions for PEG- PEG 6000, 60 mM CaCl2, 0.3 M sucrose), glycine buffer (pH mediated transfection of kiwifruit protoplasts. The best CAT 10.3) and dimethylsulfoxide (DMSO). After a short incubation, activity was obtained using 30% PEG with protoplasts that had 5 ml of a dilution solution (W5 with 50 mM 2-N-morpholine been exposed to heat shock (45°C, 5 min); however, no trans- ethane sulfonic acid, pH 5.6) was added. After 1 h, the proto- genic plants were recovered. To recover transformants, selec- plast mixture was centrifuged and briefly incubated in 2 ml of tion pressure was applied only after recovery of colonies, and the dilution solution, and the protoplasts were cultured in removed again to facilitate shoot multiplication and elongation fresh medium (Nitsch and Nitsch, 1969) supplemented with (Oliveira and Raquel, 2001). 4.5 mM 2,4-D, 1.1 mM zeatin, 200 mg/l casein hydrolysate, 1% There are several critical points in the Agrobacterium- sucrose, 0.2 M glucose and 0.4 M mannitol. mediated transformation system that determines efficiency: Some modifications were made for the hybridization of A. genotype, explant type, Agrobacterium strain, cocultivation condi- chinensis and A. kolomikta. The purified protoplasts were mixed tions and recovery/selection of the transformed cells. Although and diluted in W5 solution (154 mM NaCl, 5 mM glucose, 125 most of the transformation systems have been developed for mM CaCl ·2H O and 5 mM KCl, pH 5.6) in a ratio of 1:2 and two 2 2 A. deliciosa, A. chinensis and A. eriantha, there are also protocols drops of the protoplast mixture were plated with a spacing of 3–5 for A. arguta, A. kolomikta and A. latifolia (reviewed in Wang mm. After 5 min, two drops of PEG solution were added and in- and Gleave, 2012). Targeted explants are leaves (whole, discs or cubated for 5 min before two drops of W10 buffer (nine parts of strips), petioles and, less frequently, stems. stock A with one part of stock B, prepared freshly; stock A: 10% Agrobacterium strains LBA4404, A281, C58, EHA101 and DMSO, 0.06 M CaCl2 and 0.3 M mannitol; stock B: 1 M glycine– EHA105 have been used for transformation (Wang and Lin- NaOH buffer, pH 10.5) were added. The mixture was gradually Wang, 2007). Janssen and Gardner (1993) compared the effi- diluted (5 min) with 2 ml of dilution solution (W5 with 50 mM ciency of different Agrobacterium strains for transformation of MES). The solution was replaced 20 min later, and the proto- A. deliciosa. In general, strain A281 produced slightly higher plasts were cultured with 2 ml of fresh protoplast medium. gene transfer rates than C58 and EHA101. Strain A281 har- bours a tumour-inducing plasmid (pTiBo542) that contains an accomplishments. Somatic hybrids were obtained and extra copy of a transcription activator involved in the regulation confirmed by RAPD analysis, flow cytometry and intermediate of the vir genes (Wang and Lin-Wang, 2007). Fraser et al. morphology characters (Xiao and Han, 1997; Xiao et al., 2004). (1995) did not find marked differences between A281 and C58 Ploidy levels of A. chinensis + A. deliciosa hybrids were octoploid strains (both carrying the pKIWI105 plasmid) in transform- (8x), and one plant had RAPD banding patterns combining ations of A. chinensis. parental banding profiles. Four A. chinensis + A. kolomikta A comparison of four Agrobacterium strains (A281, GV3101, plants also had a combination of parental RAPD banding pro- EHA105 and LBA4404) for transformation efficiency of files, and the regenerated plants were triploid (3x), tetraploid A. chinensis was reported (Wang and Lin-Wang, 2007). The (4x) or pentaploid (5x). The somatic hybrids between A. chinen- highest callus initiation from inoculated leaf strips was sis and A. kolomikta were tetraploid (4x) and also showed a com- obtained with A281 strain (27%), followed by EHA105 (22.2%); bination of parental RAPD banding profiles. As A. kolomikta however, <20% of the transformants from A281 developed can grow in higher latitudes than A. chinensis, traits related to shoots and roots. In contrast, >70% of calluses derived from cold tolerance were screened. The somatic hybrids displayed strains EHA105, GV3101 and LBA4404 regenerated shoots and values close to A. kolomikta for these traits, suggesting acquisi- roots. With A. eriantha (Wang et al., 2006), similar results were tion of chilling tolerance. obtained except that no shoot regeneration was observed from calluses induced after the transformation with strain A281. Several reports (Wang et al., 2006; Han et al., 2010; Honda 5.2.3. Genetic transformation et al., 2011; Tian et al., 2011) demonstrated that time required Breeding objectives. Genetic transformation of Actinidia has for Agrobacterium infection and coculture were variable factors. focused on enhancing disease resistance, increased tolerance The infection time for leaves ranged from 2 min (Tian et al., 10 M. Rey et al.

Table 1.1.2. Summary of achievements of Actinidia genetic transformation. Genotypes were corrected from the original papers and named according to the current taxonomy of the genus Actinidia (Ferguson, 2016).

Genotype Gene of interest Achievements References

A. deliciosa Soybean β-1,3 endoglucanase Increased tolerance to a variety Nakamura et al. (1999) ‘Hayward’ of fungal diseases

A. deliciosa Na+/H+ antiporter gene AtNHX1 Improved salt tolerance Tian et al. (2011) ‘Qinmei’ from Arabidopsis

A. deliciosa Agrobacterium rhizogenes rol Increased tolerance to drought Rugini et al. (1991, 1999) ‘Hayward’ genes and osmotin gene Plants with dwarf phenotype Less susceptibility to Botrytis cinerea

A. deliciosa OSH1 rice homeobox gene Plants with dwarf phenotype Kusaba et al. (1999) ‘Hayward’

A. deliciosa Agrobacterium tumefaciens Plants with dwarf and branching Honda et al. (2011) ‘Hayward’ isopentenyl transferase (ipt) gene phenotype

A. deliciosa Citrus geranylgeranyl diphosphate Increased β-carotene and Kim et al. (2010b) ‘Hayward’ synthase, phytoene desaturase, lutein content ζ-carotene desaturase, β-carotene hydroxylase and phytoene synthase genes

A. deliciosa Human epidermal growth hEGF gene expression in leaves Kobayashi et al. (1996) ‘Hayward’ factor (hEGF)

A. deliciosa Vitis stilbene synthase genes Resveratrol genes expression Kobayashi et al. (2000) ‘Hayward’ in leaves

A. deliciosa Vitis vinifera VvmybA1 gene VvmybA1 gene expression Koshita et al. (2008) ‘Hayward’ Red pigmentation (potential use like reporter gene)

A. deliciosa Persimmon DkMyb4 gene Understanding the DkMyb4 role in Akagi et al. (2009) proanthocyanidins biosynthesis

A. chinensis ACC oxidase (ACO) gene Transgenic ACO knockdown lines Atkinson et al. (2011) ‘Hort16A’ knockdown Ripening behaviour altered

A. chinensis AcCCD8 gene knockdown Transgenic CCD8 knockdown lines Ledger et al. (2010) Plants with increased number of branches

A. deliciosa A. chinensis SVP3 Overexpression of the gene Wu et al. (2014) and A. eriantha

2011) to 30 min (Wang et al., 2006; Han et al., 2010; Honda et al., bacterial culture medium before inoculation. However, Tian et al. 2011). The best coculture time was 2 days (Tian et al., 2011). (2011) showed a slight effect of AS on transformation efficiency. Janssen and Gardner (1993) and Matsuta et al. (1993) found Some species, e.g. A. arguta, show considerable browning that acetosyringone (AS) in the Agrobacterium growth and cocul- and necrosis during shoot regeneration (Wang and Lin-Wang, tivation medium increased β-glucuronidase expression in A. deli- 2007), and this may be caused by the additional stress during ciosa leaves. Wang et al. (2006) improved the transformation transformation and selection. Han et al. (2010) concluded that efficiency of A. chinensis and A. eriantha by using AS in the browning was significantly alleviated and plantlets could be Actinidia spp. Kiwifruit 11

regenerated using half-strength MS basal medium and low light and plunged them directly into liquid nitrogen. The range of intensity (3.4 μmol/m2/s). In general, the most common selection survival was between 85% and 95%. protocol employs 25–150 mg/l kanamycin, and Agrobacterium Wu et al. (2000) used encapsulated shoot tips of A. deliciosa removal is usually achieved with timentin (300 mg/l), carbeni- ‘Tomuri M’ and ‘Tomuri F’ and of A. chinensis precultured in cillin (200 mg/l) or cefotaxime (500 mg/l). daily increasing sucrose concentrations (0.5, 0.7 and 1.0 M) and dehydrated to 26% moisture content before plunging into liquid accomplishments. Koshita et al. (2008) transformed nitrogen. Desiccated shoot tips were cryopreserved either by A. deliciosa with an anthocyanin regulatory gene of grapevine two-step freezing (prefreezing at 0.2°C/min to between −20°C and (VlmybA1-2) that can be used as reporter gene without any −40°C followed by immersion in liquid nitrogen) or by direct staining procedure. Enhanced salt tolerance by the overex- immersion in liquid nitrogen. The two-step freezing procedure pression of an Arabidopsis Na+/H+ antiporter gene (Tian et al., provided better results, with regrowth ranging between 22% and 2011) and the modification of plant architecture by the intro- 56%. This protocol was also used by Zhai et al. (2003) who duction of the Agrobacterium tumefaciens isopentenyl trans- achieved c.45% regrowth using A. deliciosa ‘Tomuri F’ shoot tips. ferase (ipt) gene (Honda et al., 2011) have been reported. These authors also verified genetic stability using RAPD markers. The chemical composition of the fruit has been altered to Only two protocols related to the application of the vitrifica- increase quality and to produce bioactive compounds. Kim et al. tion method in Actinidia have been reported (Hakozaki et al., 1996; (2010b) increased the fruit content of β-carotene and lutein by Xu et al., 2006). Calluses from A. deliciosa ‘Hayward’ seedlings introducing several carotenoid biosynthetic Citrus genes with a were cultured on medium with 24% or 41% sucrose for 2 days, transformation protocol involving micro-cross sections of dehydrated twice (20 min each) in 60% and 100% PVS2 solution shoots (800 μm thick) as explants. (30% glycerol, 15% DMSO, 15% PEG and 13.7% sucrose) and Functional genomic studies employing genetic transform- immersed in liquid nitrogen. Frozen tissue was thawed at 37°C and ation include both the elaboration of knockdown lines for the washed in liquid medium with high sucrose content. Surviving genes encoding a carotenoid cleavage dioxygenase (AcCCD8, calluses were obtained after 15 days (Hakozaki et al., 1996). Ledger et al., 2010) and a 1-aminocyclopropane 1-carboxylate Xu et al. (2006) precultured shoot tips (2 mm long) of (ACC) oxidase (AcACO1, Atkinson et al., 2011), as well as for A. chinensis ‘Ganmi’ in MS basal medium supplemented with the overexpression of the genes (SVP3 and SVP2) encoding 5% DMSO and 5% sucrose for 4 days. The explants were then SVP protein genes (Wu et al., 2014, 2017). Actinidia was also dehydrated in a solution of PVS (30% glycerol, 15% ethylene transformed to be used as a heterologous system for functional glycol, 15% DMSO and 0.4 M sucrose) for 40 min at 0°C and analysis of the DkMyb4 persimmon gene to study its function frozen in liquid nitrogen. The regeneration rate achieved after in the biosynthesis of condensed tannins (Akagi et al., 2009). thawing the tissue at 40°C was 51.6%. The main goals and achievements of Actinidia transformation with genes of interest are summarized in Table 1.1.2. 6. Conclusions

Molecular maps, databases and the first draft of the kiwifruit 5.3. Cryopreservation genome (Huang et al., 2013) have created data amenable to be validated with other techniques, mainly genetic transformation Jian and Sun (1989) used the controlled rate cooling approach by protocols. This set of tools is allowing the detailed analysis of immersing surface-sterilized kiwifruit stem segments into a pre- many and very different aspects of kiwifruit biology. The ana- cooled (0°C) cryoprotectant solution and lowering the tempera- lyses range from taxonomy to several aspects related to floral ture to −10°C at a rate of 1°C/min followed by transfer to liquid biology, fruit quality, delivery of new cultivars and resistance to nitrogen. After 120 days of storage, the tissue was thawed to 40°C, new challenging pathogens. Other aspects of the biotechnology washed in MS salts and plants were regenerated. Encapsulation– of this crop remain to be improved. Critical technologies such as dehydration has been the most frequently employed vitrifica- somatic embryogenesis remain unexplored. New and promising tion-based technique for Actinidia (Susuki et al., 1997; Wu et al., genomic edition technologies such as TALEN’s or CRISPR- 2000; Bachiri et al., 2001; Zhai et al., 2003). Susuki et al. (1997) Cas will probably stimulate new research and discovery in the used 2-mm-long shoot tips pretreated with 37.8 μM abscisic acid field of plant regeneration from protoplasts. (ABA) for 10 days, or with 100 μM proline for 1 day, and then Oliveira and Fraser (2005) stated that the introduction into cultured the explants on media with increasing sucrose concen- the market of new cultivars of Actinidia, e.g. Zespri® Gold, trations prior to encapsulation in calcium–alginate beads with 0.5 would lead to the end of the ‘Hayward’ monoculture. Bacterial M sucrose. Shoot tips were then dehydrated on medium with 1 M canker caused by Psa can delay the development and release of sucrose for 16 h, desiccated over silica gel for 8 h, and immersed new cultivars, although biotechnology will be poised to address in liquid nitrogen. The survival rate was 30% with the ABA pre- this and other issues. treatment and 23% with the proline one. Bachiri et al. (2001) improved the procedure by using shoot tips of different Actinidia species and cultivars. They pre- Acknowledgements cultured the alginate beads containing the shoot tips in media with daily increasing sucrose concentrations (0.3, 0.5 and 0.75 Research on kiwifruit in the laboratory of the authors was sup- M), dehydrated them with silica gel to 20% moisture content ported by the regional Government of Galicia (Xunta de Galicia, 12 M. Rey et al.

north-western Spain) with grants PGIDT00-BIO29101PR, providing us with the plant material. This is a contribution of PGIDIT04RAG291002PR and 10PXIB203128PR, and by the the Interuniversity Research Group in Biotechnology and Spanish Government (CICYT-FEDER grant 1FD97-1208). Reproductive Biology of Woody Plants (BioVitAc Research We also thank Kiwi Atlántico S.A. for their collaboration in group, code 09IDI1705).

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