The Mechanisms Of Lily Bulblet Initiation And Development Based On The In Vitro Model System

Flower bulbs,also called ornamental geophytes,represent a significant segment of the global floriculture industry,and are utilized for forced fresh-cut flowers,potted plants,and for landscaping,including commercial and private gardening.Unfortunately,due to low bulb multiplication rates or long juvenile phases,introduction of newly bred cultivars requires a very long period from seed to obtain sufficient materials for commercial purposes,i.e.lily for 12?15 yr,tulip for 20?25 yr.Additionally,only those bulbs above a critical size come to bloom and larger bulbs usually produce more vigorous plants with higher flower quality.As such,how to speed up the propagation process obtaining larger storage organ is crucial for breeding and mass production.Bulb initiation and development is of great importance to solve this problem,which can also make progress with the domestication of bulbs production in China.Lilium were chosen to study the bulb formation event,we firstly established the fast propagation protocols for some wild lilies native to Zhejiang Province and an in vitro model system for bulbing process study using L.Oriental Hybrids 'Sorbonne'.Several exogenous plant growth substances were involved to gain insight into the physiological and biochemical mechanisms of bulblet growth under in vitro condition based on the model system.Transcriptome for 'Sorbonne' in vitro bulblet and differentially expressed gene libraries in different treatments were constructed as well.Accordingly,the key gene LohAGPS1 in starch metabolism was cloned and the preliminary study for a special germplasm L.brownii F.E.Brown ex Miellez var.giganteum G.Y.Li&Z.H.Chen characterized by huge underground bulb was carried out.The main results were showed as follows:1.Tissue culture for Lilium and the establishment of in vitro model system using L.Oriental Hybrids 'Sorbonne'(1)A three-step disinfection method including 0.3?1 h in 200?300 mg/L thiophanate methyl solution,70?75%Alc.for 20?30 s,and then 15?25 min in 0.1%?0.2%(w/v)HgCl2 containing 1?2 drops Tween-20 was confirmed the best for L.lancifolium Thunb(4? pre-cold treatment for 2 weeks before use),the contamination rate and death rate were 24.23%and 19.14%,respectively.The optimal induction medium was MS + 6-BA 1.0 mg/L+ NAA 0.5 mg/L with the average induction rate 54.55%and 4.8 shoots.The best multiplication medium was 6-BA 1.5 mg/L + NAA 0.1 mg/L with the proliferation ratio 265%.(2)L.speciosum Thunb.var.gloriosoides Baker grew best in MS + 6-BA 1.0 mg/L+NAA 0.1 mg/L.The germination percentage,inducing rate and average shoots number were 83.3%,88.9%and 2.5,separately.Over 92%seeds belonged to the epigeal germination type.Seeds without peeling treatment did not germinate in any medium at all.The contamination rate was controlled lower than 5%for seeds without peeling treatment while it was 0 for seeds with peeling treatment.The seeds induction plantlets were diploid(2n=2x=24)with no genetic variation.(3)L.Oriental Hybrids 'Sorbonne' was chose for the whole in vitro bulbing study.The scale was sterilized with 2%NaClO for 6 min and the contamination rate and death rate were 0 and 15.2%,respectively.The optimum medium was MS + 6-BA 1.0 mg/L + NAA 0.1 mg/L.We obtained an induction rate of 57.6%and 6.9 shoots.Thereafter,the shoots were transferred to the MS basal medium only containing 70 g/L and 8 g/L for multiplication and bulblet formation until enough bulblets were gained from the same mother material with similar physiological status.Those sterile bulblets(5 mm to 8 mm in diameter)were used to inoculate on above-mentioned optimal shoot induction medium.Thus,after 35 days,shoots(shoot length 1?2 cm,diameter 3?5 mm)were chosen and with the leaf-like part excised to guarantee material uniformity for later bulblet development survey.The shoot induction process was divided into four stages:cell dedifferentiation stage,apical meristem formation stage,leaflet primordium formation stage and shoot growth stage according to our histological observations.The shoot formation belongs to exogenous origin.Three vascular bundles(closed vascular bundle type)parallel distributed in the mature parenchymal cells throughout the whole scale with intersection at the acropetal direction.We proposed the starch grains in mother scale,from apical to basal,from abaxial to final adaxial new shoot producing position,in a programmed response way,was an essential carbon source and energy guarantee of successive shoot growth.2.The effects of exogenous plant growth substances application to 'Sorbonne' bulblet development and the physiological and biochemical mechanisms(1)PBZ application suppressed the growth of leaves and roots simultaneously with the increasing PBZ doses.The leaf number was 0 and root number was 1.3 at HPBZ(5×10-2mM).Accordingly,the relative bulblet weight enhanced with the doses,the highest reached 100%.On a weight basis,the most promotive effect of bulblet production was obtained with 396 mg for weight and 10.7 nm for diameter at low PBZ(5×10-4mM,LPBZ).The relative bulblet weight for LPBZ ranged between 69.86?85.38%which guaranteed the balance within aboveground and bulblet.PBZ usage accelerated the accumulation of soluble carbohydrates and starch content with increasing PBZ doses.The major reason for the starch increase was ascribed to the enhancement of GBSS enzyme which is responsible for the amylose synthesis.Herein,the activity of AGPase for LPBZ kept at a high level indicating adequate ADPG substrate supply.PBZ stimulated IAA level during the early growth stage,demonstrating PBZ might be favorable to the cell division and expansion.High APX,CAT and GR activities over the control at the early development stage(DAT15),implicating a possible role to eliminate ROS to a proper physiological status.(2)HA promoted bulblet growth from 0.2 mg/L to 20 mg/L by 2.9-,1.8-,1.8-,respectively,over the control at the final stage.The most promotive effect of bulblet production was obtained with 468 mg for weight and 11.68 mm for diameter at low HA(0.2 mg/L,LHA).Higher doses HA(?0.2 mg/L)caused excessively aboveground biomass accumulation while the inhibitory effect in root length was observed as well.LHA was beneficial to the root growth with root number 14.5 and root length 5.75 cm at DAT 75.The relative bulblet decreased with HA doses increase which affected source-sink balance.A pronounced increase in bulblet sucrose,total soluble sugar and starch content were detected with increasing HA doses.Accordingly,three major starch enzyme activities enhanced dramatically after HA application especially LHA.AGPase and GBSS were greater,indicating amylopectin mainly accounted for starch increase.LHA significantly decreased GA content and a pronounced balance within phytohormones(promotive/inhibitive)during the whole bulbing process which might be beneficial to transportation of assimilates from the shoot to sink organ(bulblets),followed by starch accumulation.Oppositely,GA content was greater for higher HA treatments which corresponded well with the vigorous aboveground biomass growth.Intriguingly,LHA increased SOD,POD,APX,CAT,GR activities over the control at the early development stage(DAT 15),implicating a possible role to eliminate ROS which is beneficial to cell expansion and development.(3)Different CPPU concentrations varied significantly in bulbing capacity.When treated with high CPPU(5×10-2mM),the bulbing ratio was 0%.The shoot was swollen at the base without any scale which we called it shoot-like pseudo-bulblet.Lower CPPU(?5×10-3mM)enhanced the aboveground growth while it was repressed at high concentration.All CPPU treatments inhibited the root growth.CPPU strengthened sink activity and accelerated the translocation of assimilate to the bulblet.As a result,the soluble carbohydrates accumulated more with the doses increase.Nevertheless,photosynthesis for HCPUU was less by the fact of abnormal leaves,resulting in low accumulation.Overall,CPPU promoted AGPase,GBSS and SSS to different extent while we observed low starch content surprisingly.The average starch content was 52.37 mg/g FW during the whole development for HCPPU,and we speculated that starch synthesis was probably accompanied by starch degradation.CPPU decreased endogenous CTKs content while increased ABA and IAA.ABA/GA climbed while IAA/GA declined for HCPPU during the swelling process.Those peaks were highest in all treatments which gave rise to its special physiological state.CPPU increased the antioxidant enzymes.At 45 DAT,almost all the endogenous hormones and antioxidant enzymes emerged peak values,which might be a turning point for former 30%bulblet ratio and final 0%.(4)The promotive treatments LHA(0.2 mg/L)and LPBZ(5×10-4mM)were involved by removing the sucrose to determine the importance of sucrose in the medium.We found that LPBZ and LHA partially exerted its beneficial effects,e.g.,the height control for PBZ and leaf/root promotion for HA.The number of leaf was 15.5 for LHA-SF,4.2 for LPBZ-SF and 9.7 for CON,which were 4.85-fold,1.75-fold and 4.43-fold,separately,compared with sucrose addition treatments.The relative bulblet weight was only 50.32%for those sucrose-free treatments while it was 75.97%for sucrose ones.The fresh weight was 88 mg and 82 mg for LHA-SF and LPBZ-SF,respectively at DAT60.The results demonstrated that sucrose was a necessity for in vitro bulbelt development and played as not only the carbon source but also the signal to regulate the relative metabolism pathways and assimilate partitioning.3.The molecular regulation pathways and key genes mining for in vitro bulblet formation(1)A total of 64,794 unigenes with an average length of 776 bp were assembled from 4.10 Gb clean reads based on the Illumina HiSeq2000 platform using RNA-Seq.At least 33,255 unigenes(51.32%)were functionally annotated and assigned with gene descriptions,conserved protein domains,or GO terms according to public databases.A set of 2,598 unigenes with 2,732 potential SSR motifs were also identified by MISA and the most abundant was tri-motif.Functional classification and KEGG pathways(10.37%)revealed great importance of starch and sucrose metabolism.Also,phytohormones like zeatin,auxin and abscisic acid merged significantly indicating the function of endogenous hormone signaling.Transcription factor family analysis showed bHLH and ERFs TF family were possibly attributable to bulblet induction and/or swelling.Moreover,bulblet might also participate in photosynthesis under in vitro 12/12 photoperiod cycle due to most highly expressed LHCb transcripts,implying the putative factor in bulblet growth regulation.(2)HCPPU(Failure in bulbification type),LPBZ(Enlargement in bulbification type)and CON(Normal in bulbification)were used to construct three separate transcriptom libraries.3,663 DEGs were acquired including 6 different expression patterns and clusters.Light-harvesting chlorophyll a/b protein complex LHCB6 and LHCA4 were highly expressed in LPBZ and CON while early light-induced protein(ELIPs)showed high expression in HCPPU.The expression of AVP1(Inorganic H pyrophosphatase)which is responsible for the auxin transportation and GST(Gutathione S-transferase)were high in bulblet forming types.By contrast,auxin-responsive CH3(Gretchen Hagen 3)and JAZ(Jasmonate-zim-domain protein)which is the transcription inhibitor in JA metabolism pathway showed high expression in HCPPU.CKX(Cytokinin oxidase)specially expressed in HCPPU,implying CTKs might be very important for bulblet formation,and ZT(Zeatin)was the critical one among others.The highly enriched GO pathway oxidation-reduction process and response to oxidative stress might be involved in the elimination of ROS.SUS3(Sucrose synthase)expressed higher in HCPPU than in LPBZ,which indicated the lacking of carbohydrates(especially starch)was the direct evidence of failure in bulbification for HCPPU.(3)Modified CTAB method was optimal for lily bulblet RNA extraction,and the quality was suitable for further molecular experiments such as gene cloning.We cloned a small subunit of AGPase using the RACE technique,designated LohAGPSl(GenBank accession number:KX398951).The full-length cDNA of LohAGPS1 was 1,929 bp encoding a protein of 522 amino acids.The protein sequence covered those putative conserved regions:ATP-binding site,catalytic site,Glc-1-phosphate binding and activator site.4.L.brownii var.giganteum-a natural mutant for bulblet development study0.1%HgCl2 for 8 min and the contamination rate were 40.00%for L.brownii var.giganteum.The optimum induction medium was MS + 6-BA 1.5 mg/L+ NAA 0.5 mg/L.We obtained an induction rate of 100.00%and 5.0 shoots.The iron content was 5.84 mg/100g.The selenium content was 0.014 ?g/kg which was 2.92 fold compared with the famous edible lily L.lancifolium.Therefore,L.brownii var.giganteum probably could be used as the raw material for designing functional foods.All in all,L.brownii var.giganteum can be used as a natural mutant for bulblet swelling event research with the giant bulb characteristic.