Mol Biol Rep (2014) 41:6899–6908 DOI 10.1007/s11033-014-3576-9
Lily breeding by using molecular tools and transformation systems Xiaohua Liu • Jiahui Gu • Jingmao Wang Yingmin Lu
•
Received: 7 March 2014 / Accepted: 1 July 2014 / Published online: 19 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract In our review, we highlighted the progresses made in molecular breeding of lily’s flowering, including the ABCDE models, the gene cloning, the establishment of regeneration system, the gene transformation methods, the transgene technology application in lily. Meanwhile, questions that were met at present in molecular breeding in flowering of lily were underlined, and we provide viable solutions. Although many researches on lily literature had been published in the world, in our review, we provided a valuable and unique resource and spring-board from which to understand or further study the molecular breeding in flowering of lily. Keywords Lily Molecular breeding Floral development gene Gene cloning
Introduction Lilies, (Lilium spp.), one of the most important flowering crops due to its ornamental value as cut flowers, garden
X. Liu J. Gu J. Wang Y. Lu (&) College of Landscape Architecture, Beijing Forestry University, Beijing, People’s Republic of China e-mail: [email protected] X. Liu J. Gu J. Wang Y. Lu Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing, People’s Republic of China e-mail: [email protected] X. Liu J. Gu J. Wang Y. Lu China National Engineering Research Center for Floriculture, Beijing, People’s Republic of China
plants as well as pot plants [1]. Recently, researchers have made great progresses in the genomic analysis of plants with the sequencing of rice and Arabidopsis genomes and great progress has been made in molecular breeding of lily in recent years. Several genes have been isolated and cloned, in which some have been analyzed functionally. What’s more, great progress has been gained in the establishment of high efficient regeneration system and methods to insert foreign genes, which would lay a good foundation for the high quality and molecular breeding of lily. MADS box genes included in model plant species have been classified into five major groups based on sequences and functional similarities. An ABC model was established on the base of the interaction of MADS box genes [2]. In this model, model species Antirrhinum and Arabidopsis classifies three major groups including A, B, and C functional genes for floral development genes [3]. Firstly, the functions of A genes were to specify sepals identities in first whorl; the functions of A and B genes were to specify petals identity together in second whorl. While, the functions of B and C genes were to specify stamens identity in third whorl; the functions of C genes were to specify carpels identity in fourth whorl. Additionally, the functions of A and C genes were antagonists and they repressed the expression of each other [4]. Recently, it was proposed that the functions of D genes were to determine the ovule development and the E genes played important roles in the development of all floral organs. As a consequence, the ABC model was an extension of ABCDE model [5]. The formation of lily transformation system has been met with great favor for their genetic development, such as plant form, novel flower color, environmental stress tolerance, and herbicide resistance. Major efforts In the respect of lily transformation, many genes have been found by
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direct gene delivery methods such as electroporation [6], particle bombardment [7], and transgenic plants has recently been presented for Lilium longiflorum by particle bombardment [8]. It has been made possible to acquire plant materials free by the application of genes transfer techniques in lily flowers. According to the Lilium regeneration, the growth regulator type and concentration, the condition of donor plants, the physiological stage of donor plants, the nature of the inoculums and environmental conditions have affected the formation of plantlet [9]. In addition, it is possible to obtain from bulbs for flowering bulbs through somatic embryogenesis [10].
Gene cloning in lily Gene cloning related to lily floral development In recent years, the progress of molecular biology in lilies develops rapidly with the constant improvement of the flower development model hypothesis. Development of the molecular biology techniques have derived various molecular tools for isolating genes, including homologous clone strategy (RT-PCR), library screening, transposon tagging and map-based cloning. The fore two techniques are effective methods for plant homologue genes with known functions cloning and isolation, the latter two are powerful molecular tools for isolating genes with unknown functions. The lily genes cloned up to date are summarized and classified roughly into three classes listed in the Table 1. Tzeng and Yang [11] cloned B functional gene LMADS1 (Lily MADS Box Gene 1) from L. longiflorum, which is 1021 bp in full-length, encoding 228 amino acid, sharing high sequence homology with APETALA3 (AP3) of
Arabidopsis thaliana, but not completely equivalent in expression patterns. LMADS1 expresses in flower bud and four whorls of flower, and highest in petals and stamens but not expresses in leaves in spatial expression; whereas basically the same in the petal and stamen in every stage of flowering development in temporal expression. AP3 is a typical B functional gene, playing a crucial role in petals and stamens developments. LMADS1-MADS with C terminal playing a decisive role in dimerization has a strong ability to form homodimer, LMADS1 expresses in Arabidopsis thaliana abnormally, with the transgenic plant dwarf, low yield, premature flowering, and so on. LMADS2 (Lily MADS Box Gene 2), another gene cloned by Tzeng [12], sharing high sequence homology with floral binding protein of Petunia hybrid. LMADS2 transcribed mRNA and its expression was limited to the carpels, but was not found in other floral organs. It was detected in ovules, weak in stigmas. By genetic transformation, the transgenic Arabidopsis showed low yield, premature flowering, and the heterologous expression also exhibited AP2-like flowers. Beyond that, the second whorl petal was transformed into stamens. So, LMADS2 belonged to D functional gene of lily MADS-box. Furthermore, Tzeng et al. [13] isolated and analyzed two supposed SEPlike E genes functionally from the lily (L. longiflorum). Besides, the cDNA of LMADS3 gene was 1,012 bp in length and it encoded a 242-amino acid protein. The high sequence consistency between the SEP3 orthologs and LMADS3 indicated that LMADS3 was a lily SEP3 ortholog. The cDNA of LMADS4 gene was 1,088 bp in length and it encoded a 246-amino acid protein. The comparability between SEP and LMADS4 suggested that LMADS4 is a lily SEP-like gene. It was detected in different developmental flower buds of LMADS3 mRNA (from 2 to 30 mm in length) yet it was
Table 1 Floral development genes of lily Gene
Source
Clone method
Function
Register number
Reference
LMADS1
L.longiflorum
Real time PCR
B function gene
AF503913
[11]
AY522052
LMADS2
L.longiflorum
Real time PCR
D function gene, related to ovule
LMADS3
L.longiflorum
Real time PCR
SEPALLATA-Like gene
[12] [13]
LMADS4
L.longiflorum
Real time PCR
SEPALLATA-Like gene
LRDEF
L.regale
Real time PCR
B function gene
AB071378
[15] [13]
LRGLOA
L.regale
Real time PCR
B function gene
AB071379
[16]
LRGLOB
L.regale
Real time PCR
B function gene
AB071380
[16]
LGC1
L.longiflorum
Library screening
Related to fertilization
AF110779
[18]
LLSEP3
L.longiflorum
Real time PCR
E function gene
[16]
LelAG1
Elodie
Real time PCR
C function gene
[17]
LaphAG1
Aphrodite
Real time PCR
C function gene
[17]
LFAG1
L. formolongi
Real time PCR, RACE
B function gene
[17]
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lack in vegetative leaves and it was expressed highly in all four floral organs, which was different from SEP3 or its orthologs slightly, which expressed in stamen, petal and carpel, but not in sepal. However, LMADS4 mRNA was also expressed in other organs such as the inflorescence stem and vegetative leaf, which pattern was different from DOMADS3, which was only detected in the pedicel of the flower [14], or, SEP1/SEP2, which expressed only in four flower organs [15]. LRDEF, LRGLOA and LRGLOB were B functional genes cloned from Lilium regale [16]. DEF-like protein binded to DNA as a heterodimeric complex with GLO-like protein, while GLO-like protein was able to homodimerize. It was indicated that two steps after a gene duplication evolved heterodimerization to give rise to GLO- and DEFlike genes. It was found in Lilium regale, a monocotyledonous flowering plant, the capacity of DEF-like protein, both to heterodimerize and homodimerize with GLO-like proteins appeared as transitory states resulting in the obligate heterodimerization in higher eudicots. Winter et al. [17] from Netherlands Wageningen University cloned LLSEP3 with Lilium longiflorum flower bud, which was similar to SEP3 transcription factor of Arabidopsis thaliana. LLSEP3 expressed in the whole floral development, mature perianth, stamens and carpels, but not in leaves. Theoretically, LLSEP3 was E functional gene as the Arabidopsis of over-expression bloomed earlier and did not transform the homologous genes in floral organs abnormally. It was reported that a class C AG-like gene clomed from ‘Elodie’ (LelAG1) was expressed in whorls 3 and 4. In strong petaloidy flowers h, LelAG1 expressed in whorl 3 was significantly decreased, whereas, in weak petaloidy flowers, it was expressed in both whorls 3 and 4 strongly. It was concluded that AG-like genes in ‘Elodie’ correlated with the petaloidy degree of the stamens [18]. In addition, it was detected only in whorl 4, but not in whorl 3 as in L.9 formolongi (LFAG1) from ‘Aphrodite’ (LaphAG1), while other classes genes (B and D) expressed similarly in both genotypes.
including flower colour modification through genetic engineering [19], the focus of this section recently was not cite all original researches. Flavonoids and their colored derivatives played an important role in flower pigmentation and made major contribution to different kinds of colors, from blue to red to yellow. Large numbers of related genes have been identified and among of them, the best studied is chalcone synthase (CHS), catalyzing the first reaction for flavonoid biosynthesisis. Lily has different kinds of colors but lack blue because of the shortage of F30 50 H (flavonoid 30 ,50 -hydoxylase), an important enzyme to compose delphinidin [20]. Understanding the mechanisms of flower coloration could have a help for the new cultivars breeding. The enzymes of anthocyanin biosynthesis were primarily interactions between basic-helixloop helix (bHLH) and R2R3-MYB transcription factors and manipulated by the transcriptional level [22]. In Asiatic hybrid lily, LhMYB6 and LhMYB12, R2R3-MYB transcription factors, had been cloned, which regulated anthocyanin biosynthesis [23]. LhMYB6 and LhMYB12 were homologues to petunia AN2, which were identified from lily firstly. LhMYB6 participated in pigment accumulation in petal spots and sprouting shoots, LhMYB12 controlled petal pigmentation [23]. Masumi [24] isolated LhMYB12 from the Oriental hybrid lily ‘‘Sorbonne’’, which was indicated to participate in the anthocyanin biosynthesis in petals and petals spots. And above all, it was indicated that that the MYB transcription factor LhSorMYB12 newly identified participated in anthocyanin biosynthesis definitely in the Oriental hybrid lily petals. Although in the Asiatic hybrid lily, LhMYB6, took part in spot pigmentation, LhMYB12 regulated another R2R3-MYB and petal pigmentation, but in the Oriental hybrid lily, LhSorMYB12 took part in anthocyanin biosynthesis both in petal spots and petals; namely, in the Asiatic hybrid lily and the Oriental hybrid lily, LhMYB12 genes had a little different in functions. Lai [25] found that during flower bud development, the gene transcripts and accumulation of pigments were evaluated to determine genes regulated by LhMYB12.
Cloning of genes related to flower color
Gene cloning related to lily pollen development
Flower color was a crucial concerned factor in consumer choice for ornamental plants with flower. Flower color is markedly because of three pigment types: flavonoid, carotenoid and betalain, among which, flavonoid is the most common and contributes to a series of colours from blue to red to yellow. In recent researches, the flower colour modification via genetic engineering generally has focused on the flavonoid pathway which was conserved among plant species generally (Fig. 1). Although it has been reviewed repeatedly in different perspectives
Pollen, used as a natural transformation vector to introduce foreign traits simply by pollination, could be transformed by methods of common plant transformation genetically. In the Antirrhinum majus pollens, particle bombardment method was been first used [26]. Available from cultivated plants abundantly, lily pollen was a convenient tool to research pollen gene expression, pollen tube growth, and pollen germination [27]. Due to its large anther, rich and large pollen, easy collection, lily is the model plant for the biological study of anther [28].
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Fig. 1 Flower colour generated from flavonoid biosynthesis pathway [21]. (Color figure online)
Wang et al. [29] got LLA23 from the cDNA library of L. longiflorum mature pollen. Through reverse nothern blotting, LLA23 expressed pollen specificity, and only expressed in the later mature pollen. The collection of LLA23 protein is also associated with dryness, and expressed in pollen cytoplast from subcellular localization. Presumably, LLA23 protein has a protective function to the pollen. Zhang [30] got the full cDNA of casein kinase CK2afrom lily pollen, which is similar to CK2a gene, contributing to the research of its physiological and biochemical functions in pollen. Furthermore, CK2a sequence is determined by two different PCR primers. Okada et al. [31] isolated the promoter of histones gch3 gene from lily andro gamete and analyzed it by gene gun, finding that animals and plants have about the same specific mechanisms in the expression of andro gamete. Xiang [32] reported a full length cDNA encoding plant gelsolin/fragmin and demonstrated that L1ABP29 is an alternative mRNA product of lily villin. Meanwhile, the
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homologous protein was found in the total pollen protein. Furthermore, as a new member of villin/gelsolin/fragmin supergene family, L1ABP29 is maybe the downstream of some signal pathway and has played important roles in regulating pollen tube growth and pollen germination by a response to regulate the dynamic change of microfilament cytoskeleton.
Gene regulation pathway in flowering Genes controlling flowering are distributed into organ identity genes and floral meristem identity genes at different stages. Control of the timing of flowering is divided into four pathways: floral repression pathway, autonomous promotion pathway, photoperiodic promotion pathway and vernalization promotion pathway. The first two pathways regulated the internal state, the other two pathways transmitted signals from external environment.
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Floral repression pathway
Table 2 Summary of the autonomous promotion of flower induction pathway genes in model species
In this pathway, some genes act to repress flowering while others promote flowering through response to the photoperiod, such as elf1, elf2, clf, elg, pef1, pef2, esd4, phyB. It was considered that EMF genes played a major role in repressing flowering as emf1 and emf2 mutants flower with no preceding vegetative phase essentially [33]. However, emf mutations resulted in late flowering combined with fca and other mutations [34]. TFL1, cloned recently based on its similarity to its Antirrhinum ortholog CENTRORADIALIS (CEN) by T-DNA tagging, is another floral repressor [35, 36] which mutant flowers early. Ordinarily, TFL1 must function to delay the transition from vegetative to reproductive development at the apex and suppress flower formation. Overexpression of TFL1 extends the inflorescence growth and vegetative and phases greatly [37]. The reason for it is likely that TFL1 gene exerts this flowering delay via repressing FPA, FVE, and FCA genes, operating in the autonomous promotion pathway. The reason for this is that the late-flowering phenotype awarded by mutations is epistatic to tfl1 [38].
Gene Locus
Gene Name
Function of proteins
AT4G02560
LUMINIDEPENDENS (LD)
Homeodomain proteins
AT4G16280
FCA
RNA-binding proteins
AT5G13480 AT2G43410
FY FPA
polyadenylation factor RNA-binding proteins
AT3G10390
FLOWERING LOCUS D (FLD)
Proteins related to HDAC
AT2G19520
FVE
MSI homologous protein
AT3G04610
FLOWERING LATE KH MOTIF (FLK)
RNA-binding proteins
Autonomous promotion pathway Under suitable environmental conditions, external environmental factors can induce flowering, but if the conditions are not suitable, some plants will bloom when the vegetative growth reaches a certain stage, this approach is called autonomous pathway [39]. Autonomous pathway is regulated by plant endogenous signals independently, do not rely photoperiod pathway and gibberellin pathways. Facultative long-day plant Arabidopsis under long-day will soon bloom, and will eventually blossom under short-day in non-inducing conditions, but late compared to long day photographic [38]. At present, the known autonomous pathway genes are FCA. LUMINIDEPENDENS (LD), FPA, FY, FVE, REF6, FLK, and FLOWERING LOCUS D (FLD) [39–41]. These genes have some remarkable characteristics, as is shown in Table 2. Proteins encoded by FPA, FLK and FCA, genes belong to RNA-binding proteins, FLK contains 3 KH domains, FPA and FCA genes contain RRM domain [40, 41]; FY is a gene homologous with yeast polyadenylation factor Pfs2p gene; LD is a transcription factor for homeodomain with unknown function; FVE and FLD are the chromatin remodeling proteins [40, 41], that FVE protein belongs to the family of MSI1 homolog, FLD may be a lysine-specific histone demethylase LSD1 homologous gene [40]. All gene expressions through autonomous pathway accelerate flowering via indirectly suppressing the FLC expression [42]. The autonomous pathway genes play a vital role in
the pathway mainly through the regulation of chromatin and RNA, so the mutant showed a delay in blossom, and some unique phenotypes, such as female gametophyte of FCA and FPA double mutants and developmental defects in early embryo. Photoperiodic promotion pathway Photoperiod is the main effect factors that affect the flowering time [43]. Photoperiod pathway in Arabidopsis thaliana begins with phototraduction signals in the photoreceptors. Now there are three types of known plant photoreceptors: phytochrome, cryptochrome and unknown photoreceptors. Cryptochrome feels blue and ultraviolet light; phytochrome feels the red and far-infrared light [42] and unknown photoreceptors only feel UV [44]. So far, at least 3 phytochrome genes have been found in rice, Arabidopsis, namely PHYTOCHROME A (PhyA), PhyB, PhyC, PhyD and PhyE [35], 3 cryptochrome genes, namely CRYPTOCHROME1 (CRY1), CRY2 and CRY3 [35, 45], they produce circadian rhythm through feeling the length of day and night and the intensity of light [35, 45]. Sufficient conditions for plants blossom induced by photoperiod are appropriate circadian rhythm, genes influencing circadian rhythm are CCA1, LHY, ELF3, LKP2, FKF1, TOC1, ZTL, LUX, GI and CDF1 genes, which are the upstream genes in photoperiod pathway, circadian change will make the change of the expressions, and will transfer the signals to leaves, activating the expression of CO gene [35, 45]. The Arabidopsis flowering key gene CO induced by the photoperiod pathway belongs to the plant-specific CCT transcription factor [41], The transcript level of CO is regulated controlled by the circadian gene GI and, CDF1, the protein level is regulated by phytochrome and cryptochrome [41]. The study found, the phenomenon of late flowering phenomenon of Arabidopsis mutant (CO) in the long-day conditions is most obvious in long-day conditions, CO overexpression shows early flowering. Therefore,
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Fig. 2 Integration of genes involved in flowering time pathways [48]
CO is the bridge between the outside light cycle and blossom, taking important part in the pathway of photoperiod [46], which can directly activate downstream gene of FT to promote blossom [47]. MiR172 gene belongs to the transcription factors of AP1 family, can inhibit the activity of flower gene FT (FLOWERING LOCUS T integration), playing a very important role in floral transformation. MiR172 is regulated by SPLs, SPLs is regulated by miR156 (Fig. 2). Vernalization promotion pathway In addition to light, temperature is another major environmental factors affecting flowering time. Some plants flower or early flower through a period of cold treatment, this phenomenon is known as vernalization [48]. Vernalization
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has two characteristics, one is that plant is required to continuously exposed to a period of time at low temperatures in order to bloom in the spring or summer; second is that the vernalization effect can not be passed on to offspring, progeny flowering still need vernalization [49]. Vernalization process is regulated by a series of genes. Through the study of different Arabidopsis ecotypes, FLC is found one of the key genes in vernalization process, which is a transcription factor with MADS-box structure [48], FLC interacts with CArG boxes of FT, SOC1 and LFY to inhibit their activity, and inhibit flowering induction. FLC is mainly expressed in root and stem tip, after vernalization of Arabidopsis mutant, the mRNA and protein of FLC increase, resulting in the down-regulation of FT, LFY and SOC1 genes in flower pathways and inhibit the blossom
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The induction frequency of lily embryo is low, and the related reports are less. Haensch [52] got four embryoid of body cell after vaccinating lily on MS medium of 2,4-D and Picloram.
Gene transformation methods in lily Agrobacterium mediated genetic transformation
Fig. 3 Proposed pathways to flowering in Arabidopsis
[49]. Another vernalization related gene is FRI, which plays a significant role in Arabidopsis thaliana vernalization through the up-regulation of the expression of FLC gene. The activation of FRI and FRI-like gene on FLC is more significant, which can overcome the plant itself inhibitory effect on FLC. Besides, two genes VRN1 and VRN2, related to vernalization could keep the inhibitory action of FLC. Methylation in general was closely related to vernalization, which might influence the time of flowering by regulating FWA. Sheldon illustrated relevant relationships of vernalization genes by the following image in Fig. 3.
Agrobacterium-mediated transformation provides several benefits, such as preferential integration into transcriptionally active regions of the chromosome, potentially lowcopy number, and the defined integration of transgenes, compared with the method of direct gene delivery methods [54]. In the past several years, it has been reported that Agrobacterium-mediated transformation had been applied for some Liliaceous species, including Asparagus officinalis [53], Allium sativum [54], Allium cepa [55], Agapanthus praecox [56], and Muscari armeniacum [57] successfully, indicating that this transformation method could be applied for other species. Biological carriers of gene transformation are Agrobacterium tumefaciens and Agrobacferium rhizogene. The transformation of Agrobacterium as carrier us a broadest method for foreign genes transforming into plant cells with low price and high efficiency. In recent years, the domestic studies about the use of Agrobacterium tumefaciens transformation of lily are shown in Table 5. The genetic transformation of DNA directly into system
Establishment of regeneration system in lily In lilies, it has been reported effective methods for separating large quantities of protoplasts from calli initiated from bulb scale tissues, pollen, and generative cells. Regeneration for plant from protoplasts has been obtained [50], and a modified method for plants regeneration has been established subsequently [51]. Bud culture in Lilium spp. are used as acceptor materials for gene transformation because of its short time to regeneration, good fertility of regenerated plants, wide sources of explants, but chimera and great difference among regeneration, frequency of different genotype also exist. Scales and small leaves are easy to differentiate as explants for lily genetic transformation. The researches related to adventitious shoot regeneration are shown in Table 3, which provided theoretical basis for further research of lily genetic transformation. The technology for lily explants from dedifferentiation of tissue culture to redifferentiation of organ is mature, which is an important way of regeneration. Callus regeneration system and genetic transformation of lily are shown in Table 4.
Nishihara et al. [58] transferred GUS gene into lily pollen by gene gun, and found the transient expression of GUS by histochemistry. Watad et al. [7] got three positive plants after southern blot detection by transferring PAT gene into callus by gene gun. Miyoshi [59] imported GUS gene into lily protoplast by electroporation transformation and got transient expression but failed to get transformed plant from the protoplast culture.
Problems, challenges and future perspective Few genes have been cloned in lily, the functional genes such as color and type genes have not been cloned. In China, researches of regulating mechanism of anti-hot stress genes are of great significance for the majority of areas are plains with hot summers where lily grows slower because of the poor quality, serious diseases, small flower and soft stem. In addition, few reports are about floral genes with Asiatic lily fragrance-free but Oriental lily intense spicy fragrance, so the flower breeding aim is to
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Table 3 Adventitious bud regeneration system and genetic transformation of lily Material
Explant
Culture medium (mg L-1)
Lilium oriental
Leaf
Leave differentiation: MS ? BA 0.5 ? ZT 0.1 ? NAA 1.0;Root differentiation: 1/2MS ? NAA 0.2–1.0; Seedball growth: MS ? IAA 1.0
Lilium oriental
Scale and leaf
Scale differentiation: MS ? NAA 0.5 ? BA 1.0 ?2,4-D 0.1–1.0; Leave differentiation: MS ? IAA 1.0 ? BA 0.5
Kanamycin: scale 75, scale leaf 120, petiolule 75, mall leaf 50; Cephalosporin or carbenicillin: 250
Lilium oriental ‘Siberia’
Scale and regenerative bulb
Scale differentiation:MS ? 2,4-D 0.5 ? BA 0.5; Leave and bulb differentiation: MS ? BA 0.1 ? IAA 0.05
Kanamycin: leaf 100, small scale 125; Cephalosporin or carbenicillin: 300–400
Lilium oriental ‘Siberia’ and ‘Sorbonne’
Sterile leaf
Leave differentiation: MS ? BA 2.0 ? ZT 1.0; Rooting culture: MS ? NAA 0.75
G418:80; Glyphosate (PPT):1.75; Carbenicillin: 300–400
Lilium oriental ‘Sorbonne’ Lilium longiflorum
Scales and leaf
Scale differentiation: AS ? NAA 0.1 ? 6-BA 1.0; Scaly leaf differentiation: MS ? 2,4-D 0.3 ? 6-BA 1.0 Leaf differentiation: MS ? NAA 0.2 ? 6-BA 2.0; Subculture: MS ? NAA0.2 ? 6-BA 1.0; Root culture:1/ 2MS ? IBA 0.1
Kanamycin: petiole 80, scaly leaf 120; Carbenicillin:300 Kanamycin: leaf 50
Hygromycin: tissue culture 40
Leaf
Antibiotic concentration (mg L-1)
‘Star Gazer’ and ‘Siberia’ Lilium oriental ‘Siberia’
Tissue plantlet leaf scale
MS ? Picloram 3.0 mg L-1 ? sugar 20 g L-1 ? maltose 10 mg L-1 ? phytate 0.1 g L-1 Inducement: MS ? 2,4-D 2.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? 6-BA 0.5 mgL-1
Lilium longiflorum
pseudo-bulb
MS ? NAA 5.4 lmol L-1?TDZ 0.4 lmol L-1?CoCl2 0.05 lmol L-1?sugar 60gl L-1
Hygromycin: tissue culture 7.5
Table 4 Callus regeneration system and genetic transformation of lily Material
Explant
Culture medium
Antibiotic concentration (mg L-1)
Lilium brownie var.viridulum
scale
Inducement: MS ? 2,4-D 2.0 mg L-1?6-BA 0.2 mg L-1; Regeneration: MS ? NAA 0.05 mg L-1 ? 6-BA 1.0 mg L-1
Kanamycin: scale 150
Oriental hybrid lily, Lilium cv.Acapulco
Filament
Inducement: MS ? PIC 2.0 mg L-1?30 g L-1 sucrose ?3.0 g L-1 gellan gum; Regeneration: MS ? PIC 0.1 mg L-1 ? BA 0.01 mg L-1?30 g L-1 sucrose ?3 g L-1 gellan gum
Hygromycin: 75
Lilium longiflorum
Filament and pedicel
Inducement: MS ? NAA 1.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? KT 1.0 mg L-1
Kanamycin: scale 75. Hygromycin: 20;Cephalosporin:250
Lilium oriental ‘Sorbonne’
Scale,regenerated scale and regenerate petiole
Inducement: MS ? NAA 0.5 mg L-1 ? 6-BA 0.4 mg L-1?sugar 90 g L-1 ? VB14.0 mg L-1; Regeneration: MS ? 6-BA 0.5 mg L-1
Hygromycin: tissue culture 20
‘Star Gazer’ and ‘Siberia’
Tissue plantlet leaf
MS ? Picloram 3.0 mg L-1 ? sugar 20 g L-1 ? maltose10 mg L-1 ? phytate 0.1 g L-1
Hygromycin: tissue culture 40
Lilium oriental ‘Siberia’
scale
Inducement: MS ? 2,4-D 2.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? 6-BA 0.5 mg L-1
Hygromycin: tissue culture 7.5
Lilium longiflorum
pseudo-bulb
MS ? NAA 5.4 lmol L-1 ? TDZ 0.4 lmol L-1 ? CoCl2 0.05 lmol L-1 ? sugar 60 gl L-1
reduce the fragrance of Oriental lily and to increase the fragrance of Asiatic lily. Our country have multiple lily strains of fragrance, which could provide materials for flower breeding, such as Lilium longiflorum, Lilium regale,
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Lilium lancifolium, Lilium davidii, Lilium candidum, Lilium tsingtauense. In our review, we provided a valuable, focused, and indepth insight into molecular breeding of lily’ flowering,
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Table 5 Agrobacterium-mediated transformation of lily Lily
Materials
Strain
Transforming gene
Detection method
Lilium longiflorum Thunb
embryoid callus
EHA105
Pb1XPTAGus
PCR
Lilium formolongi
Scales of aseptic seedling
LBA4404
Zm401
PCR and PCR-Southern
Lilium longiflorum
leaves
EHA105
Mn2SOD
PCR
‘ButterPixie’
scales
LBA440p43300
rd29a-CBF1
PCR
‘Star Gazer’ and ‘Siberia’
Callus and leaves
LBA4404
pCAMBIA1301
PCR
Lilium oriental ‘Siberia’
Scales and callus
LBA4404
GV310p3CAMBIA130Gus
PCR
Lilium longiflorum ‘White-elegance’
leaves
EHA105
DHAR
PCR
Lilium longiflorum Thunb
scales
GV3103
pBHF2
PCR, Southern
Lilium oriental ‘Siberia’
Bulbs, scales and callus
EHA105GV3103
antisense recombinant plasmid of ACC oxidase
GUS, PCR, PCR-Southern
Lilium longiflorum
leaves
LBA4404
PBI121-1
PCR
which will permit a higher level for exploratory research to come up, either in using genes as a tool for other technologies including genetic transformation, molecular breeding, and bioreactors, or in gene cloning. At present, molecular breeding of lily is mostly focused on the gene transformation. Along with further development of lily genetic engineering techniques, further researches on exogenous gene integration and expression regulation, the rapid development of SNP markers based on sequence and the development of bioinformatics, improving lily traits by genetic engineering will become a reality. Acknowledgments Our research was sponsored by the China National Natural Science Foundation (Grant No. 31071815 and No. 31272204), ‘‘863’’ research program (Grant No. 2011AA10020804).
References 1. Theissen G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Bio. 4:75–85 2. Krizek BA, Fletcher JC (2005) Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet 6:688–698 3. Bowman JL, Symth DR, Meyerowitz EM (1991) Genetic interactions among floral homeotic genes of Arabidopsis. Development 112:1–20 4. Ditta G, Pinyopich A, Roble P, Pelaz S, Yanofsky MF (2004) The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Curr Biol 14:1935–1940 5. Miyoshi H, Usami T, Tanaka I (1995) High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts. Sex Plant Reprod 8:205–209 6. Tsuchiya T, Takumi S, Shimada T (1996) Transient expression of a reporter gene in bulbscales and immature embryos of three Liliumspecies is affected by upstream sequences and culture conditions. Physiol Plant 98:699–704 7. Watad AA, Yun DJ, Matsumoto T, Niu X, Wu Y, Kononowicz AK, Bressan RA, Hasegawa PM (1998) Microprojectile bombardment-mediated transformation of Lilium longiflorum. Plant Cell Rep 17:262–267 8. Yamagishi M (1995) Effect of cold treatment, BA and GA3 on enlargement and leaf emergence of in vitro cultured bulblets of Lilium japonicum Thumb. J Jap Soc Hort Sci 64:367–373
9. Tribulato A, Remotti PC, Loffler HJM, Van Tuyl JM (1997) Somatic embryogenesis and plant regeneration in Lilium longiflorum Thumb. Plant Cell Rep 17:113–118 10. Tzeng TY, Yang CH (2001) A MADS-box gene from lily(Lilium longiflorum)is sufficient to generate dominant negative mutation by interacting with PISTILLATA(PI) in Arabidopsis thliana. Plant Cell Physiol 42:1156–1168 11. Tzeng TY, Chen HY, Yang CH (2002) Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis. Plant Physiol 130:1827–1836 12. Tzeng TY, Hsiao CC, Chi PJ (2003) Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis. Plant Physiol 133(3):1091–1101 13. Yu H, Goh CJ (2000) Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiol 123:1325–1336 14. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405:200–203 15. Winter KU, Weister C, Kerstin K, Arend B, Charlotte K, Akira K, Heinz S, Gu¨nter T (2002) Evolution of class B floral homeotia proteins: obligate heterodimerization originated from homodimerization. Mol Biol Evol 19(5):587–596 16. Akita Y, Nakada M, Kanno A (2011) Effect of the expression level of an AGAMOUS-like gene on the petaloidy of stamens in the double-flowered lily, ‘Elodie’. Sci Hortic 128(1):48–53 17. Winter KU, Bhalla PL (2003) Isolation and characterization of a flowering plant male gametic cell-specific promoter. FEBS Lett 542:47–52 18. Martens S, Forkmann G, Britsch L, Wellmann F, Matern U, Lukacin R (2003) Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley. FEBS Lett 544:93–98 19. Martens S, Knott J, Seitz C, Janvari L, Yu SN, Forkmann G (2003) Impact of biochemical pre-studies on specific metabolic engineering strategies of flavonoid bisynthesis in plant tissues. Biochem Eng J 14:227–235 20. Shimada Y, Ohbayashi M, Nakano-Shimada R, Okinaka Y, Kiyokawa S, Kikuchi Y (2001) Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-30 50 -hydroxylase: specific switching of the pathway in petunia. Plant Cell Rep 20:456–462 21. Yoshikaza T, Yukihisa K, Filippa B, John M (2005) Genetic engineering in floriculture. Plant Cell, Tissue Organ Cult 80:1–24 22. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242
123
6908 23. Yamagishi M, Shimoyamada Y, Nakatsuka T, Masuda K (2010) Two R2R3-MYB genes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flower sepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol 51(3):463–474 24. Lai YS, Shimoyamada Y, Nakayama M, Yamagishi M (2012) Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily. Plant Sci 193:136–147 25. Lord E (2000) Adhesion and cell movement during pollination: cherchez la femme. Tren Plant Sci 5:368–373 26. Bariniva I, Zhexembekova M, Barsova E, Lukyanov S, HeberleBors E, Touraev A (2002) Antirrhinum majus microspore maturation and transient transformation in vitro. J Exp Bot 53:1119–1129 27. Mogami N, Shiota H, Tanaka I (2002) The identification of a pollen-specific LEA-like protein in Lilium longiflorum. Plant Cell Env 25:653–663 28. Wang ML (2004) Gene expression profiles of cold-stored and fresh pollen to investigate pollen germination and growth. Plant Cell Physiology. 45(10):1519–1528 29. Wang CS, Huang JC, Lin SM (1998) Characterization of a desiccation-related protein in lily pollen during development and stress. Plant Cell Physiol 39(12):1307–1314 30. Okada T, Bhalla PL, Singh MB (2005) Transcriptional activity of male gamete-specific histone gcH3 promoter in sperm cell of Lilium longiflorum. Plant Cell Physiol 46(5):797–802 31. Yang CH, Chen LJ, Sung ZR (1995) Genetic regulation of shoot development in Arabidopsis: role of the EMF genes. Dev Biol 169:421–435 32. Haung MD, Yang CH (1998) EMF genes interact with lateflowering genes to regulate Arabidopsis shoot development. Plant Cell Physiol 39:382–393 33. Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflorescence commitment and architecture in Arabidopsis. Science 275:80–83 34. Ohshima S, Murata M, Sakamoto W, Ogura Y, Motoyoshi F (1997) Cloning and molecular analysis of the Arabidopsis gene terminal flower 1. Mol Gen Genet 254:186–194 35. Ratcliffe OJ, Amaya I, Vincent CA, Rothstein S, Carpenter R, Coen ES, Bradley DJ (1998) A common mechanism controls the life cycle and architecture of plants. Development 125:1609–1615 36. Ruiz-Garcı´a L, Maduen˜o F, Wilkinson M, Haughn G, Salinas J, Martı´nez-Zapater JM (1997) Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis. Plant Cell 9:1921–1934 37. Yaron YL, Caroline D (1998) The transition to flowering. Plant Cell 10:1973–1990 38. Srikanth A, Schmid M (2011) Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci 68:2013–2037 39. Jack T (2004) Molecular and genetic mechanisms of floral control. Plant Cell 16(Suppl 1):S1–S17 40. Marquardt S, Boss PK, Hadfield J et al (2006) Additional targets of the Arabidopsis autonomous pathway members, FCA and FY [J]. J Exp Bot 57(13):3379–3386 41. Feng W, Jacob Y, Veley KM et al (2011) Hypomorphic alleles reveal FCA-independent roles for FY in the regulation of FLOWERING LOCUS C [J]. Plant Physiol 155(3):1425–1434 42. Michaels SD, Amasino RM (2001) Loss of FLOWERING LOCUS C activity eliminates the late-flowering phenotype of
123
Mol Biol Rep (2014) 41:6899–6908
43. 44.
45.
46.
47.
48. 49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
FRIGIDA and autonomous pathway mutations but not responsiveness to vernalization [J]. Plant Cell 13:935–941 Jackson SD (2009) Plant responses to photoperiod. New Phytol 181:517–531 Guo HW, Yang HY, Mocker TC et al (1998) Regulation of flowering time by Arabidopsis photoreceptor. Science 279(5355):1360–1363 Onouchi H, Igen˜o MI, Pe´rilleux C, Graves K, Coupland G (2000) Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes. Plant Cell 12:885–900 Valverde F, Mouradov A, Soppe W et al (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303:1003–1006 Samach A, Onouchi H, Gold SE et al (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288:1613–1616 Tanaka I (1988) Isolation of generative cells and their protoplasts from pollen of Lilium longiflorum. Protoplasma 142:68–73 Mii M, Yuzawa Y, Suetomi H, Motegi T, Godo T (1994) Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Liliumformolongi by utilizing meristematic nodular cell clumps. Plant Sci 100:221–226 Lee H, Suh SS, Park E, Cho E, Ahn JH, Kim SG, Lee JS, Kwon YM, Lee I (2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14:2366–2376 Godo T, Kobayashi K, Tagami T, Matsui K, Kida T (1998) In vitro propagation utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of Lilium 9 formolongi. Hort Sci Hortic 72(3–4):193–202 Hiei Y, Komari T, Ishida Y, Saito H (2000) Development of Agrobacterium-mediated transformation method for monocotyledonous plants. Breed Res 2:205–213 Kisaka H, Kameya T (1998) Fertile transgenic asparagus plants produced by Agrobacterium-mediated transformation. Plant Biotechnol 15:177–181 Kondo T, Hasegawa H, Suzuki M (2000) Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium mediated gene transfer. Plant Cell Rep 19:989–993 Eady CC, Weld RJ, Lister CE (2000) Agrobacterium tumefaciens mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.). Plant Cell Rep 19:376–381 Suzuki S, Supaibulwatana K, Mii M, Nakano M (2001) Production of transgenic plants of Liliaceous ornamental plant Agapanthus praecoxssp. Orientalis (Leighton) Leighton via Agrobacterium mediated transformation of embryogenic calli. Plant Sci 161:89–97 Suzuki S, Nakano M (2002) Agrobacterium-mediated production of transgenic plants Muscari armeniacum Leichtl. ex Bak. Plant Cell Rep 20:835–841 Nishihara M, Ito M, Tanaka I, Kyo M, Ono K, Irifune K, Morikawa H (1993) Expression of the [beta]-Glucuronidase gene in pollen of lily (Lilium longiflorum), tobacco (Nicotiana tabacum), Nicotiana rustica, and peony (Paeonia lactiflora) by particle bombardment. Plant Physiol 102(2):357–361 Miyoshi H (1995) High level of GUS gene expression driver by pollen specific promoters in electroporated lily pollen protoplasts. Sex Plant Rep 8(4):205–209
Lily breeding by using molecular tools and transformation systems Xiaohua Liu • Jiahui Gu • Jingmao Wang Yingmin Lu
•
Received: 7 March 2014 / Accepted: 1 July 2014 / Published online: 19 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract In our review, we highlighted the progresses made in molecular breeding of lily’s flowering, including the ABCDE models, the gene cloning, the establishment of regeneration system, the gene transformation methods, the transgene technology application in lily. Meanwhile, questions that were met at present in molecular breeding in flowering of lily were underlined, and we provide viable solutions. Although many researches on lily literature had been published in the world, in our review, we provided a valuable and unique resource and spring-board from which to understand or further study the molecular breeding in flowering of lily. Keywords Lily Molecular breeding Floral development gene Gene cloning
Introduction Lilies, (Lilium spp.), one of the most important flowering crops due to its ornamental value as cut flowers, garden
X. Liu J. Gu J. Wang Y. Lu (&) College of Landscape Architecture, Beijing Forestry University, Beijing, People’s Republic of China e-mail: [email protected] X. Liu J. Gu J. Wang Y. Lu Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing, People’s Republic of China e-mail: [email protected] X. Liu J. Gu J. Wang Y. Lu China National Engineering Research Center for Floriculture, Beijing, People’s Republic of China
plants as well as pot plants [1]. Recently, researchers have made great progresses in the genomic analysis of plants with the sequencing of rice and Arabidopsis genomes and great progress has been made in molecular breeding of lily in recent years. Several genes have been isolated and cloned, in which some have been analyzed functionally. What’s more, great progress has been gained in the establishment of high efficient regeneration system and methods to insert foreign genes, which would lay a good foundation for the high quality and molecular breeding of lily. MADS box genes included in model plant species have been classified into five major groups based on sequences and functional similarities. An ABC model was established on the base of the interaction of MADS box genes [2]. In this model, model species Antirrhinum and Arabidopsis classifies three major groups including A, B, and C functional genes for floral development genes [3]. Firstly, the functions of A genes were to specify sepals identities in first whorl; the functions of A and B genes were to specify petals identity together in second whorl. While, the functions of B and C genes were to specify stamens identity in third whorl; the functions of C genes were to specify carpels identity in fourth whorl. Additionally, the functions of A and C genes were antagonists and they repressed the expression of each other [4]. Recently, it was proposed that the functions of D genes were to determine the ovule development and the E genes played important roles in the development of all floral organs. As a consequence, the ABC model was an extension of ABCDE model [5]. The formation of lily transformation system has been met with great favor for their genetic development, such as plant form, novel flower color, environmental stress tolerance, and herbicide resistance. Major efforts In the respect of lily transformation, many genes have been found by
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direct gene delivery methods such as electroporation [6], particle bombardment [7], and transgenic plants has recently been presented for Lilium longiflorum by particle bombardment [8]. It has been made possible to acquire plant materials free by the application of genes transfer techniques in lily flowers. According to the Lilium regeneration, the growth regulator type and concentration, the condition of donor plants, the physiological stage of donor plants, the nature of the inoculums and environmental conditions have affected the formation of plantlet [9]. In addition, it is possible to obtain from bulbs for flowering bulbs through somatic embryogenesis [10].
Gene cloning in lily Gene cloning related to lily floral development In recent years, the progress of molecular biology in lilies develops rapidly with the constant improvement of the flower development model hypothesis. Development of the molecular biology techniques have derived various molecular tools for isolating genes, including homologous clone strategy (RT-PCR), library screening, transposon tagging and map-based cloning. The fore two techniques are effective methods for plant homologue genes with known functions cloning and isolation, the latter two are powerful molecular tools for isolating genes with unknown functions. The lily genes cloned up to date are summarized and classified roughly into three classes listed in the Table 1. Tzeng and Yang [11] cloned B functional gene LMADS1 (Lily MADS Box Gene 1) from L. longiflorum, which is 1021 bp in full-length, encoding 228 amino acid, sharing high sequence homology with APETALA3 (AP3) of
Arabidopsis thaliana, but not completely equivalent in expression patterns. LMADS1 expresses in flower bud and four whorls of flower, and highest in petals and stamens but not expresses in leaves in spatial expression; whereas basically the same in the petal and stamen in every stage of flowering development in temporal expression. AP3 is a typical B functional gene, playing a crucial role in petals and stamens developments. LMADS1-MADS with C terminal playing a decisive role in dimerization has a strong ability to form homodimer, LMADS1 expresses in Arabidopsis thaliana abnormally, with the transgenic plant dwarf, low yield, premature flowering, and so on. LMADS2 (Lily MADS Box Gene 2), another gene cloned by Tzeng [12], sharing high sequence homology with floral binding protein of Petunia hybrid. LMADS2 transcribed mRNA and its expression was limited to the carpels, but was not found in other floral organs. It was detected in ovules, weak in stigmas. By genetic transformation, the transgenic Arabidopsis showed low yield, premature flowering, and the heterologous expression also exhibited AP2-like flowers. Beyond that, the second whorl petal was transformed into stamens. So, LMADS2 belonged to D functional gene of lily MADS-box. Furthermore, Tzeng et al. [13] isolated and analyzed two supposed SEPlike E genes functionally from the lily (L. longiflorum). Besides, the cDNA of LMADS3 gene was 1,012 bp in length and it encoded a 242-amino acid protein. The high sequence consistency between the SEP3 orthologs and LMADS3 indicated that LMADS3 was a lily SEP3 ortholog. The cDNA of LMADS4 gene was 1,088 bp in length and it encoded a 246-amino acid protein. The comparability between SEP and LMADS4 suggested that LMADS4 is a lily SEP-like gene. It was detected in different developmental flower buds of LMADS3 mRNA (from 2 to 30 mm in length) yet it was
Table 1 Floral development genes of lily Gene
Source
Clone method
Function
Register number
Reference
LMADS1
L.longiflorum
Real time PCR
B function gene
AF503913
[11]
AY522052
LMADS2
L.longiflorum
Real time PCR
D function gene, related to ovule
LMADS3
L.longiflorum
Real time PCR
SEPALLATA-Like gene
[12] [13]
LMADS4
L.longiflorum
Real time PCR
SEPALLATA-Like gene
LRDEF
L.regale
Real time PCR
B function gene
AB071378
[15] [13]
LRGLOA
L.regale
Real time PCR
B function gene
AB071379
[16]
LRGLOB
L.regale
Real time PCR
B function gene
AB071380
[16]
LGC1
L.longiflorum
Library screening
Related to fertilization
AF110779
[18]
LLSEP3
L.longiflorum
Real time PCR
E function gene
[16]
LelAG1
Elodie
Real time PCR
C function gene
[17]
LaphAG1
Aphrodite
Real time PCR
C function gene
[17]
LFAG1
L. formolongi
Real time PCR, RACE
B function gene
[17]
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lack in vegetative leaves and it was expressed highly in all four floral organs, which was different from SEP3 or its orthologs slightly, which expressed in stamen, petal and carpel, but not in sepal. However, LMADS4 mRNA was also expressed in other organs such as the inflorescence stem and vegetative leaf, which pattern was different from DOMADS3, which was only detected in the pedicel of the flower [14], or, SEP1/SEP2, which expressed only in four flower organs [15]. LRDEF, LRGLOA and LRGLOB were B functional genes cloned from Lilium regale [16]. DEF-like protein binded to DNA as a heterodimeric complex with GLO-like protein, while GLO-like protein was able to homodimerize. It was indicated that two steps after a gene duplication evolved heterodimerization to give rise to GLO- and DEFlike genes. It was found in Lilium regale, a monocotyledonous flowering plant, the capacity of DEF-like protein, both to heterodimerize and homodimerize with GLO-like proteins appeared as transitory states resulting in the obligate heterodimerization in higher eudicots. Winter et al. [17] from Netherlands Wageningen University cloned LLSEP3 with Lilium longiflorum flower bud, which was similar to SEP3 transcription factor of Arabidopsis thaliana. LLSEP3 expressed in the whole floral development, mature perianth, stamens and carpels, but not in leaves. Theoretically, LLSEP3 was E functional gene as the Arabidopsis of over-expression bloomed earlier and did not transform the homologous genes in floral organs abnormally. It was reported that a class C AG-like gene clomed from ‘Elodie’ (LelAG1) was expressed in whorls 3 and 4. In strong petaloidy flowers h, LelAG1 expressed in whorl 3 was significantly decreased, whereas, in weak petaloidy flowers, it was expressed in both whorls 3 and 4 strongly. It was concluded that AG-like genes in ‘Elodie’ correlated with the petaloidy degree of the stamens [18]. In addition, it was detected only in whorl 4, but not in whorl 3 as in L.9 formolongi (LFAG1) from ‘Aphrodite’ (LaphAG1), while other classes genes (B and D) expressed similarly in both genotypes.
including flower colour modification through genetic engineering [19], the focus of this section recently was not cite all original researches. Flavonoids and their colored derivatives played an important role in flower pigmentation and made major contribution to different kinds of colors, from blue to red to yellow. Large numbers of related genes have been identified and among of them, the best studied is chalcone synthase (CHS), catalyzing the first reaction for flavonoid biosynthesisis. Lily has different kinds of colors but lack blue because of the shortage of F30 50 H (flavonoid 30 ,50 -hydoxylase), an important enzyme to compose delphinidin [20]. Understanding the mechanisms of flower coloration could have a help for the new cultivars breeding. The enzymes of anthocyanin biosynthesis were primarily interactions between basic-helixloop helix (bHLH) and R2R3-MYB transcription factors and manipulated by the transcriptional level [22]. In Asiatic hybrid lily, LhMYB6 and LhMYB12, R2R3-MYB transcription factors, had been cloned, which regulated anthocyanin biosynthesis [23]. LhMYB6 and LhMYB12 were homologues to petunia AN2, which were identified from lily firstly. LhMYB6 participated in pigment accumulation in petal spots and sprouting shoots, LhMYB12 controlled petal pigmentation [23]. Masumi [24] isolated LhMYB12 from the Oriental hybrid lily ‘‘Sorbonne’’, which was indicated to participate in the anthocyanin biosynthesis in petals and petals spots. And above all, it was indicated that that the MYB transcription factor LhSorMYB12 newly identified participated in anthocyanin biosynthesis definitely in the Oriental hybrid lily petals. Although in the Asiatic hybrid lily, LhMYB6, took part in spot pigmentation, LhMYB12 regulated another R2R3-MYB and petal pigmentation, but in the Oriental hybrid lily, LhSorMYB12 took part in anthocyanin biosynthesis both in petal spots and petals; namely, in the Asiatic hybrid lily and the Oriental hybrid lily, LhMYB12 genes had a little different in functions. Lai [25] found that during flower bud development, the gene transcripts and accumulation of pigments were evaluated to determine genes regulated by LhMYB12.
Cloning of genes related to flower color
Gene cloning related to lily pollen development
Flower color was a crucial concerned factor in consumer choice for ornamental plants with flower. Flower color is markedly because of three pigment types: flavonoid, carotenoid and betalain, among which, flavonoid is the most common and contributes to a series of colours from blue to red to yellow. In recent researches, the flower colour modification via genetic engineering generally has focused on the flavonoid pathway which was conserved among plant species generally (Fig. 1). Although it has been reviewed repeatedly in different perspectives
Pollen, used as a natural transformation vector to introduce foreign traits simply by pollination, could be transformed by methods of common plant transformation genetically. In the Antirrhinum majus pollens, particle bombardment method was been first used [26]. Available from cultivated plants abundantly, lily pollen was a convenient tool to research pollen gene expression, pollen tube growth, and pollen germination [27]. Due to its large anther, rich and large pollen, easy collection, lily is the model plant for the biological study of anther [28].
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Fig. 1 Flower colour generated from flavonoid biosynthesis pathway [21]. (Color figure online)
Wang et al. [29] got LLA23 from the cDNA library of L. longiflorum mature pollen. Through reverse nothern blotting, LLA23 expressed pollen specificity, and only expressed in the later mature pollen. The collection of LLA23 protein is also associated with dryness, and expressed in pollen cytoplast from subcellular localization. Presumably, LLA23 protein has a protective function to the pollen. Zhang [30] got the full cDNA of casein kinase CK2afrom lily pollen, which is similar to CK2a gene, contributing to the research of its physiological and biochemical functions in pollen. Furthermore, CK2a sequence is determined by two different PCR primers. Okada et al. [31] isolated the promoter of histones gch3 gene from lily andro gamete and analyzed it by gene gun, finding that animals and plants have about the same specific mechanisms in the expression of andro gamete. Xiang [32] reported a full length cDNA encoding plant gelsolin/fragmin and demonstrated that L1ABP29 is an alternative mRNA product of lily villin. Meanwhile, the
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homologous protein was found in the total pollen protein. Furthermore, as a new member of villin/gelsolin/fragmin supergene family, L1ABP29 is maybe the downstream of some signal pathway and has played important roles in regulating pollen tube growth and pollen germination by a response to regulate the dynamic change of microfilament cytoskeleton.
Gene regulation pathway in flowering Genes controlling flowering are distributed into organ identity genes and floral meristem identity genes at different stages. Control of the timing of flowering is divided into four pathways: floral repression pathway, autonomous promotion pathway, photoperiodic promotion pathway and vernalization promotion pathway. The first two pathways regulated the internal state, the other two pathways transmitted signals from external environment.
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Floral repression pathway
Table 2 Summary of the autonomous promotion of flower induction pathway genes in model species
In this pathway, some genes act to repress flowering while others promote flowering through response to the photoperiod, such as elf1, elf2, clf, elg, pef1, pef2, esd4, phyB. It was considered that EMF genes played a major role in repressing flowering as emf1 and emf2 mutants flower with no preceding vegetative phase essentially [33]. However, emf mutations resulted in late flowering combined with fca and other mutations [34]. TFL1, cloned recently based on its similarity to its Antirrhinum ortholog CENTRORADIALIS (CEN) by T-DNA tagging, is another floral repressor [35, 36] which mutant flowers early. Ordinarily, TFL1 must function to delay the transition from vegetative to reproductive development at the apex and suppress flower formation. Overexpression of TFL1 extends the inflorescence growth and vegetative and phases greatly [37]. The reason for it is likely that TFL1 gene exerts this flowering delay via repressing FPA, FVE, and FCA genes, operating in the autonomous promotion pathway. The reason for this is that the late-flowering phenotype awarded by mutations is epistatic to tfl1 [38].
Gene Locus
Gene Name
Function of proteins
AT4G02560
LUMINIDEPENDENS (LD)
Homeodomain proteins
AT4G16280
FCA
RNA-binding proteins
AT5G13480 AT2G43410
FY FPA
polyadenylation factor RNA-binding proteins
AT3G10390
FLOWERING LOCUS D (FLD)
Proteins related to HDAC
AT2G19520
FVE
MSI homologous protein
AT3G04610
FLOWERING LATE KH MOTIF (FLK)
RNA-binding proteins
Autonomous promotion pathway Under suitable environmental conditions, external environmental factors can induce flowering, but if the conditions are not suitable, some plants will bloom when the vegetative growth reaches a certain stage, this approach is called autonomous pathway [39]. Autonomous pathway is regulated by plant endogenous signals independently, do not rely photoperiod pathway and gibberellin pathways. Facultative long-day plant Arabidopsis under long-day will soon bloom, and will eventually blossom under short-day in non-inducing conditions, but late compared to long day photographic [38]. At present, the known autonomous pathway genes are FCA. LUMINIDEPENDENS (LD), FPA, FY, FVE, REF6, FLK, and FLOWERING LOCUS D (FLD) [39–41]. These genes have some remarkable characteristics, as is shown in Table 2. Proteins encoded by FPA, FLK and FCA, genes belong to RNA-binding proteins, FLK contains 3 KH domains, FPA and FCA genes contain RRM domain [40, 41]; FY is a gene homologous with yeast polyadenylation factor Pfs2p gene; LD is a transcription factor for homeodomain with unknown function; FVE and FLD are the chromatin remodeling proteins [40, 41], that FVE protein belongs to the family of MSI1 homolog, FLD may be a lysine-specific histone demethylase LSD1 homologous gene [40]. All gene expressions through autonomous pathway accelerate flowering via indirectly suppressing the FLC expression [42]. The autonomous pathway genes play a vital role in
the pathway mainly through the regulation of chromatin and RNA, so the mutant showed a delay in blossom, and some unique phenotypes, such as female gametophyte of FCA and FPA double mutants and developmental defects in early embryo. Photoperiodic promotion pathway Photoperiod is the main effect factors that affect the flowering time [43]. Photoperiod pathway in Arabidopsis thaliana begins with phototraduction signals in the photoreceptors. Now there are three types of known plant photoreceptors: phytochrome, cryptochrome and unknown photoreceptors. Cryptochrome feels blue and ultraviolet light; phytochrome feels the red and far-infrared light [42] and unknown photoreceptors only feel UV [44]. So far, at least 3 phytochrome genes have been found in rice, Arabidopsis, namely PHYTOCHROME A (PhyA), PhyB, PhyC, PhyD and PhyE [35], 3 cryptochrome genes, namely CRYPTOCHROME1 (CRY1), CRY2 and CRY3 [35, 45], they produce circadian rhythm through feeling the length of day and night and the intensity of light [35, 45]. Sufficient conditions for plants blossom induced by photoperiod are appropriate circadian rhythm, genes influencing circadian rhythm are CCA1, LHY, ELF3, LKP2, FKF1, TOC1, ZTL, LUX, GI and CDF1 genes, which are the upstream genes in photoperiod pathway, circadian change will make the change of the expressions, and will transfer the signals to leaves, activating the expression of CO gene [35, 45]. The Arabidopsis flowering key gene CO induced by the photoperiod pathway belongs to the plant-specific CCT transcription factor [41], The transcript level of CO is regulated controlled by the circadian gene GI and, CDF1, the protein level is regulated by phytochrome and cryptochrome [41]. The study found, the phenomenon of late flowering phenomenon of Arabidopsis mutant (CO) in the long-day conditions is most obvious in long-day conditions, CO overexpression shows early flowering. Therefore,
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Fig. 2 Integration of genes involved in flowering time pathways [48]
CO is the bridge between the outside light cycle and blossom, taking important part in the pathway of photoperiod [46], which can directly activate downstream gene of FT to promote blossom [47]. MiR172 gene belongs to the transcription factors of AP1 family, can inhibit the activity of flower gene FT (FLOWERING LOCUS T integration), playing a very important role in floral transformation. MiR172 is regulated by SPLs, SPLs is regulated by miR156 (Fig. 2). Vernalization promotion pathway In addition to light, temperature is another major environmental factors affecting flowering time. Some plants flower or early flower through a period of cold treatment, this phenomenon is known as vernalization [48]. Vernalization
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has two characteristics, one is that plant is required to continuously exposed to a period of time at low temperatures in order to bloom in the spring or summer; second is that the vernalization effect can not be passed on to offspring, progeny flowering still need vernalization [49]. Vernalization process is regulated by a series of genes. Through the study of different Arabidopsis ecotypes, FLC is found one of the key genes in vernalization process, which is a transcription factor with MADS-box structure [48], FLC interacts with CArG boxes of FT, SOC1 and LFY to inhibit their activity, and inhibit flowering induction. FLC is mainly expressed in root and stem tip, after vernalization of Arabidopsis mutant, the mRNA and protein of FLC increase, resulting in the down-regulation of FT, LFY and SOC1 genes in flower pathways and inhibit the blossom
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The induction frequency of lily embryo is low, and the related reports are less. Haensch [52] got four embryoid of body cell after vaccinating lily on MS medium of 2,4-D and Picloram.
Gene transformation methods in lily Agrobacterium mediated genetic transformation
Fig. 3 Proposed pathways to flowering in Arabidopsis
[49]. Another vernalization related gene is FRI, which plays a significant role in Arabidopsis thaliana vernalization through the up-regulation of the expression of FLC gene. The activation of FRI and FRI-like gene on FLC is more significant, which can overcome the plant itself inhibitory effect on FLC. Besides, two genes VRN1 and VRN2, related to vernalization could keep the inhibitory action of FLC. Methylation in general was closely related to vernalization, which might influence the time of flowering by regulating FWA. Sheldon illustrated relevant relationships of vernalization genes by the following image in Fig. 3.
Agrobacterium-mediated transformation provides several benefits, such as preferential integration into transcriptionally active regions of the chromosome, potentially lowcopy number, and the defined integration of transgenes, compared with the method of direct gene delivery methods [54]. In the past several years, it has been reported that Agrobacterium-mediated transformation had been applied for some Liliaceous species, including Asparagus officinalis [53], Allium sativum [54], Allium cepa [55], Agapanthus praecox [56], and Muscari armeniacum [57] successfully, indicating that this transformation method could be applied for other species. Biological carriers of gene transformation are Agrobacterium tumefaciens and Agrobacferium rhizogene. The transformation of Agrobacterium as carrier us a broadest method for foreign genes transforming into plant cells with low price and high efficiency. In recent years, the domestic studies about the use of Agrobacterium tumefaciens transformation of lily are shown in Table 5. The genetic transformation of DNA directly into system
Establishment of regeneration system in lily In lilies, it has been reported effective methods for separating large quantities of protoplasts from calli initiated from bulb scale tissues, pollen, and generative cells. Regeneration for plant from protoplasts has been obtained [50], and a modified method for plants regeneration has been established subsequently [51]. Bud culture in Lilium spp. are used as acceptor materials for gene transformation because of its short time to regeneration, good fertility of regenerated plants, wide sources of explants, but chimera and great difference among regeneration, frequency of different genotype also exist. Scales and small leaves are easy to differentiate as explants for lily genetic transformation. The researches related to adventitious shoot regeneration are shown in Table 3, which provided theoretical basis for further research of lily genetic transformation. The technology for lily explants from dedifferentiation of tissue culture to redifferentiation of organ is mature, which is an important way of regeneration. Callus regeneration system and genetic transformation of lily are shown in Table 4.
Nishihara et al. [58] transferred GUS gene into lily pollen by gene gun, and found the transient expression of GUS by histochemistry. Watad et al. [7] got three positive plants after southern blot detection by transferring PAT gene into callus by gene gun. Miyoshi [59] imported GUS gene into lily protoplast by electroporation transformation and got transient expression but failed to get transformed plant from the protoplast culture.
Problems, challenges and future perspective Few genes have been cloned in lily, the functional genes such as color and type genes have not been cloned. In China, researches of regulating mechanism of anti-hot stress genes are of great significance for the majority of areas are plains with hot summers where lily grows slower because of the poor quality, serious diseases, small flower and soft stem. In addition, few reports are about floral genes with Asiatic lily fragrance-free but Oriental lily intense spicy fragrance, so the flower breeding aim is to
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Table 3 Adventitious bud regeneration system and genetic transformation of lily Material
Explant
Culture medium (mg L-1)
Lilium oriental
Leaf
Leave differentiation: MS ? BA 0.5 ? ZT 0.1 ? NAA 1.0;Root differentiation: 1/2MS ? NAA 0.2–1.0; Seedball growth: MS ? IAA 1.0
Lilium oriental
Scale and leaf
Scale differentiation: MS ? NAA 0.5 ? BA 1.0 ?2,4-D 0.1–1.0; Leave differentiation: MS ? IAA 1.0 ? BA 0.5
Kanamycin: scale 75, scale leaf 120, petiolule 75, mall leaf 50; Cephalosporin or carbenicillin: 250
Lilium oriental ‘Siberia’
Scale and regenerative bulb
Scale differentiation:MS ? 2,4-D 0.5 ? BA 0.5; Leave and bulb differentiation: MS ? BA 0.1 ? IAA 0.05
Kanamycin: leaf 100, small scale 125; Cephalosporin or carbenicillin: 300–400
Lilium oriental ‘Siberia’ and ‘Sorbonne’
Sterile leaf
Leave differentiation: MS ? BA 2.0 ? ZT 1.0; Rooting culture: MS ? NAA 0.75
G418:80; Glyphosate (PPT):1.75; Carbenicillin: 300–400
Lilium oriental ‘Sorbonne’ Lilium longiflorum
Scales and leaf
Scale differentiation: AS ? NAA 0.1 ? 6-BA 1.0; Scaly leaf differentiation: MS ? 2,4-D 0.3 ? 6-BA 1.0 Leaf differentiation: MS ? NAA 0.2 ? 6-BA 2.0; Subculture: MS ? NAA0.2 ? 6-BA 1.0; Root culture:1/ 2MS ? IBA 0.1
Kanamycin: petiole 80, scaly leaf 120; Carbenicillin:300 Kanamycin: leaf 50
Hygromycin: tissue culture 40
Leaf
Antibiotic concentration (mg L-1)
‘Star Gazer’ and ‘Siberia’ Lilium oriental ‘Siberia’
Tissue plantlet leaf scale
MS ? Picloram 3.0 mg L-1 ? sugar 20 g L-1 ? maltose 10 mg L-1 ? phytate 0.1 g L-1 Inducement: MS ? 2,4-D 2.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? 6-BA 0.5 mgL-1
Lilium longiflorum
pseudo-bulb
MS ? NAA 5.4 lmol L-1?TDZ 0.4 lmol L-1?CoCl2 0.05 lmol L-1?sugar 60gl L-1
Hygromycin: tissue culture 7.5
Table 4 Callus regeneration system and genetic transformation of lily Material
Explant
Culture medium
Antibiotic concentration (mg L-1)
Lilium brownie var.viridulum
scale
Inducement: MS ? 2,4-D 2.0 mg L-1?6-BA 0.2 mg L-1; Regeneration: MS ? NAA 0.05 mg L-1 ? 6-BA 1.0 mg L-1
Kanamycin: scale 150
Oriental hybrid lily, Lilium cv.Acapulco
Filament
Inducement: MS ? PIC 2.0 mg L-1?30 g L-1 sucrose ?3.0 g L-1 gellan gum; Regeneration: MS ? PIC 0.1 mg L-1 ? BA 0.01 mg L-1?30 g L-1 sucrose ?3 g L-1 gellan gum
Hygromycin: 75
Lilium longiflorum
Filament and pedicel
Inducement: MS ? NAA 1.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? KT 1.0 mg L-1
Kanamycin: scale 75. Hygromycin: 20;Cephalosporin:250
Lilium oriental ‘Sorbonne’
Scale,regenerated scale and regenerate petiole
Inducement: MS ? NAA 0.5 mg L-1 ? 6-BA 0.4 mg L-1?sugar 90 g L-1 ? VB14.0 mg L-1; Regeneration: MS ? 6-BA 0.5 mg L-1
Hygromycin: tissue culture 20
‘Star Gazer’ and ‘Siberia’
Tissue plantlet leaf
MS ? Picloram 3.0 mg L-1 ? sugar 20 g L-1 ? maltose10 mg L-1 ? phytate 0.1 g L-1
Hygromycin: tissue culture 40
Lilium oriental ‘Siberia’
scale
Inducement: MS ? 2,4-D 2.0 mg L-1 ? 6-BA 0.5 mg L-1; Regeneration: MS ? NAA 0.2 mg L-1 ? 6-BA 0.5 mg L-1
Hygromycin: tissue culture 7.5
Lilium longiflorum
pseudo-bulb
MS ? NAA 5.4 lmol L-1 ? TDZ 0.4 lmol L-1 ? CoCl2 0.05 lmol L-1 ? sugar 60 gl L-1
reduce the fragrance of Oriental lily and to increase the fragrance of Asiatic lily. Our country have multiple lily strains of fragrance, which could provide materials for flower breeding, such as Lilium longiflorum, Lilium regale,
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Lilium lancifolium, Lilium davidii, Lilium candidum, Lilium tsingtauense. In our review, we provided a valuable, focused, and indepth insight into molecular breeding of lily’ flowering,
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Table 5 Agrobacterium-mediated transformation of lily Lily
Materials
Strain
Transforming gene
Detection method
Lilium longiflorum Thunb
embryoid callus
EHA105
Pb1XPTAGus
PCR
Lilium formolongi
Scales of aseptic seedling
LBA4404
Zm401
PCR and PCR-Southern
Lilium longiflorum
leaves
EHA105
Mn2SOD
PCR
‘ButterPixie’
scales
LBA440p43300
rd29a-CBF1
PCR
‘Star Gazer’ and ‘Siberia’
Callus and leaves
LBA4404
pCAMBIA1301
PCR
Lilium oriental ‘Siberia’
Scales and callus
LBA4404
GV310p3CAMBIA130Gus
PCR
Lilium longiflorum ‘White-elegance’
leaves
EHA105
DHAR
PCR
Lilium longiflorum Thunb
scales
GV3103
pBHF2
PCR, Southern
Lilium oriental ‘Siberia’
Bulbs, scales and callus
EHA105GV3103
antisense recombinant plasmid of ACC oxidase
GUS, PCR, PCR-Southern
Lilium longiflorum
leaves
LBA4404
PBI121-1
PCR
which will permit a higher level for exploratory research to come up, either in using genes as a tool for other technologies including genetic transformation, molecular breeding, and bioreactors, or in gene cloning. At present, molecular breeding of lily is mostly focused on the gene transformation. Along with further development of lily genetic engineering techniques, further researches on exogenous gene integration and expression regulation, the rapid development of SNP markers based on sequence and the development of bioinformatics, improving lily traits by genetic engineering will become a reality. Acknowledgments Our research was sponsored by the China National Natural Science Foundation (Grant No. 31071815 and No. 31272204), ‘‘863’’ research program (Grant No. 2011AA10020804).
References 1. Theissen G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Bio. 4:75–85 2. Krizek BA, Fletcher JC (2005) Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet 6:688–698 3. Bowman JL, Symth DR, Meyerowitz EM (1991) Genetic interactions among floral homeotic genes of Arabidopsis. Development 112:1–20 4. Ditta G, Pinyopich A, Roble P, Pelaz S, Yanofsky MF (2004) The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Curr Biol 14:1935–1940 5. Miyoshi H, Usami T, Tanaka I (1995) High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts. Sex Plant Reprod 8:205–209 6. Tsuchiya T, Takumi S, Shimada T (1996) Transient expression of a reporter gene in bulbscales and immature embryos of three Liliumspecies is affected by upstream sequences and culture conditions. Physiol Plant 98:699–704 7. Watad AA, Yun DJ, Matsumoto T, Niu X, Wu Y, Kononowicz AK, Bressan RA, Hasegawa PM (1998) Microprojectile bombardment-mediated transformation of Lilium longiflorum. Plant Cell Rep 17:262–267 8. Yamagishi M (1995) Effect of cold treatment, BA and GA3 on enlargement and leaf emergence of in vitro cultured bulblets of Lilium japonicum Thumb. J Jap Soc Hort Sci 64:367–373
9. Tribulato A, Remotti PC, Loffler HJM, Van Tuyl JM (1997) Somatic embryogenesis and plant regeneration in Lilium longiflorum Thumb. Plant Cell Rep 17:113–118 10. Tzeng TY, Yang CH (2001) A MADS-box gene from lily(Lilium longiflorum)is sufficient to generate dominant negative mutation by interacting with PISTILLATA(PI) in Arabidopsis thliana. Plant Cell Physiol 42:1156–1168 11. Tzeng TY, Chen HY, Yang CH (2002) Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis. Plant Physiol 130:1827–1836 12. Tzeng TY, Hsiao CC, Chi PJ (2003) Two lily SEPALLATA-like genes cause different effects on floral formation and floral transition in Arabidopsis. Plant Physiol 133(3):1091–1101 13. Yu H, Goh CJ (2000) Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiol 123:1325–1336 14. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405:200–203 15. Winter KU, Weister C, Kerstin K, Arend B, Charlotte K, Akira K, Heinz S, Gu¨nter T (2002) Evolution of class B floral homeotia proteins: obligate heterodimerization originated from homodimerization. Mol Biol Evol 19(5):587–596 16. Akita Y, Nakada M, Kanno A (2011) Effect of the expression level of an AGAMOUS-like gene on the petaloidy of stamens in the double-flowered lily, ‘Elodie’. Sci Hortic 128(1):48–53 17. Winter KU, Bhalla PL (2003) Isolation and characterization of a flowering plant male gametic cell-specific promoter. FEBS Lett 542:47–52 18. Martens S, Forkmann G, Britsch L, Wellmann F, Matern U, Lukacin R (2003) Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley. FEBS Lett 544:93–98 19. Martens S, Knott J, Seitz C, Janvari L, Yu SN, Forkmann G (2003) Impact of biochemical pre-studies on specific metabolic engineering strategies of flavonoid bisynthesis in plant tissues. Biochem Eng J 14:227–235 20. Shimada Y, Ohbayashi M, Nakano-Shimada R, Okinaka Y, Kiyokawa S, Kikuchi Y (2001) Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-30 50 -hydroxylase: specific switching of the pathway in petunia. Plant Cell Rep 20:456–462 21. Yoshikaza T, Yukihisa K, Filippa B, John M (2005) Genetic engineering in floriculture. Plant Cell, Tissue Organ Cult 80:1–24 22. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242
123
6908 23. Yamagishi M, Shimoyamada Y, Nakatsuka T, Masuda K (2010) Two R2R3-MYB genes, homologs of petunia AN2, regulate anthocyanin biosyntheses in flower sepals, tepal spots and leaves of Asiatic hybrid lily. Plant Cell Physiol 51(3):463–474 24. Lai YS, Shimoyamada Y, Nakayama M, Yamagishi M (2012) Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily. Plant Sci 193:136–147 25. Lord E (2000) Adhesion and cell movement during pollination: cherchez la femme. Tren Plant Sci 5:368–373 26. Bariniva I, Zhexembekova M, Barsova E, Lukyanov S, HeberleBors E, Touraev A (2002) Antirrhinum majus microspore maturation and transient transformation in vitro. J Exp Bot 53:1119–1129 27. Mogami N, Shiota H, Tanaka I (2002) The identification of a pollen-specific LEA-like protein in Lilium longiflorum. Plant Cell Env 25:653–663 28. Wang ML (2004) Gene expression profiles of cold-stored and fresh pollen to investigate pollen germination and growth. Plant Cell Physiology. 45(10):1519–1528 29. Wang CS, Huang JC, Lin SM (1998) Characterization of a desiccation-related protein in lily pollen during development and stress. Plant Cell Physiol 39(12):1307–1314 30. Okada T, Bhalla PL, Singh MB (2005) Transcriptional activity of male gamete-specific histone gcH3 promoter in sperm cell of Lilium longiflorum. Plant Cell Physiol 46(5):797–802 31. Yang CH, Chen LJ, Sung ZR (1995) Genetic regulation of shoot development in Arabidopsis: role of the EMF genes. Dev Biol 169:421–435 32. Haung MD, Yang CH (1998) EMF genes interact with lateflowering genes to regulate Arabidopsis shoot development. Plant Cell Physiol 39:382–393 33. Bradley D, Ratcliffe O, Vincent C, Carpenter R, Coen E (1997) Inflorescence commitment and architecture in Arabidopsis. Science 275:80–83 34. Ohshima S, Murata M, Sakamoto W, Ogura Y, Motoyoshi F (1997) Cloning and molecular analysis of the Arabidopsis gene terminal flower 1. Mol Gen Genet 254:186–194 35. Ratcliffe OJ, Amaya I, Vincent CA, Rothstein S, Carpenter R, Coen ES, Bradley DJ (1998) A common mechanism controls the life cycle and architecture of plants. Development 125:1609–1615 36. Ruiz-Garcı´a L, Maduen˜o F, Wilkinson M, Haughn G, Salinas J, Martı´nez-Zapater JM (1997) Different roles of flowering-time genes in the activation of floral initiation genes in Arabidopsis. Plant Cell 9:1921–1934 37. Yaron YL, Caroline D (1998) The transition to flowering. Plant Cell 10:1973–1990 38. Srikanth A, Schmid M (2011) Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci 68:2013–2037 39. Jack T (2004) Molecular and genetic mechanisms of floral control. Plant Cell 16(Suppl 1):S1–S17 40. Marquardt S, Boss PK, Hadfield J et al (2006) Additional targets of the Arabidopsis autonomous pathway members, FCA and FY [J]. J Exp Bot 57(13):3379–3386 41. Feng W, Jacob Y, Veley KM et al (2011) Hypomorphic alleles reveal FCA-independent roles for FY in the regulation of FLOWERING LOCUS C [J]. Plant Physiol 155(3):1425–1434 42. Michaels SD, Amasino RM (2001) Loss of FLOWERING LOCUS C activity eliminates the late-flowering phenotype of
123
Mol Biol Rep (2014) 41:6899–6908
43. 44.
45.
46.
47.
48. 49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
FRIGIDA and autonomous pathway mutations but not responsiveness to vernalization [J]. Plant Cell 13:935–941 Jackson SD (2009) Plant responses to photoperiod. New Phytol 181:517–531 Guo HW, Yang HY, Mocker TC et al (1998) Regulation of flowering time by Arabidopsis photoreceptor. Science 279(5355):1360–1363 Onouchi H, Igen˜o MI, Pe´rilleux C, Graves K, Coupland G (2000) Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes. Plant Cell 12:885–900 Valverde F, Mouradov A, Soppe W et al (2004) Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science 303:1003–1006 Samach A, Onouchi H, Gold SE et al (2000) Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288:1613–1616 Tanaka I (1988) Isolation of generative cells and their protoplasts from pollen of Lilium longiflorum. Protoplasma 142:68–73 Mii M, Yuzawa Y, Suetomi H, Motegi T, Godo T (1994) Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Liliumformolongi by utilizing meristematic nodular cell clumps. Plant Sci 100:221–226 Lee H, Suh SS, Park E, Cho E, Ahn JH, Kim SG, Lee JS, Kwon YM, Lee I (2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev 14:2366–2376 Godo T, Kobayashi K, Tagami T, Matsui K, Kida T (1998) In vitro propagation utilizing suspension cultures of meristematic nodular cell clumps and chromosome stability of Lilium 9 formolongi. Hort Sci Hortic 72(3–4):193–202 Hiei Y, Komari T, Ishida Y, Saito H (2000) Development of Agrobacterium-mediated transformation method for monocotyledonous plants. Breed Res 2:205–213 Kisaka H, Kameya T (1998) Fertile transgenic asparagus plants produced by Agrobacterium-mediated transformation. Plant Biotechnol 15:177–181 Kondo T, Hasegawa H, Suzuki M (2000) Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium mediated gene transfer. Plant Cell Rep 19:989–993 Eady CC, Weld RJ, Lister CE (2000) Agrobacterium tumefaciens mediated transformation and transgenic-plant regeneration of onion (Allium cepa L.). Plant Cell Rep 19:376–381 Suzuki S, Supaibulwatana K, Mii M, Nakano M (2001) Production of transgenic plants of Liliaceous ornamental plant Agapanthus praecoxssp. Orientalis (Leighton) Leighton via Agrobacterium mediated transformation of embryogenic calli. Plant Sci 161:89–97 Suzuki S, Nakano M (2002) Agrobacterium-mediated production of transgenic plants Muscari armeniacum Leichtl. ex Bak. Plant Cell Rep 20:835–841 Nishihara M, Ito M, Tanaka I, Kyo M, Ono K, Irifune K, Morikawa H (1993) Expression of the [beta]-Glucuronidase gene in pollen of lily (Lilium longiflorum), tobacco (Nicotiana tabacum), Nicotiana rustica, and peony (Paeonia lactiflora) by particle bombardment. Plant Physiol 102(2):357–361 Miyoshi H (1995) High level of GUS gene expression driver by pollen specific promoters in electroporated lily pollen protoplasts. Sex Plant Rep 8(4):205–209
