Promoting Effect And Mechanism Of Polyamines During The Conversion Of Protocorm-like Bodies Of Dendrobium Huoshanense To Shoots

Dendrobium huoshanense is an endangered orchid species with a very restricted distribution in Huoshan County,Anhui Province.It has long been valued for its medicinal value and has not only been used in the traditional Chinese medicines as a Yin tonic to nourish the stomach,promote the secretion of body fluid,prevent the development of cataract and relieve throat inflammation and fatigue,but also in the health care.However,it has fallen into serious danger of extinction because of poorly natural propagation,excessive collection for growing medicinal demands and deterioration of its natural habitats.Micropropagation has been an effective approach to produce clonal plantlets from wild plant,but only low rate of plant regeneration was achieved.Moreover almost all the micropropagation has been carried out with the plant growth regulators,and the remained plant growth regulators in the clonal plantlets will further harm to health.So it is difficult to provide safe raw materials for the development of medicine and functional food.In this paper,the high efficient system of conversion of protocorm-like bodies(PLBs) of D.huoshanense to shoots was established on the plant growth regulator-free MS medium and the mechanisms of enhanced shoot conversion from PLBs by exogenous PAs was investigated with the methods of morphology,physiology and biochemistry.The main results and conclusions obtained are as follows.1.Time-course changes in free PAs during shoot morphogenesis from PLBs of D. huoshanense showed that the endogenous levels of free Putrescine(Put) and Spermidine(Spd) exhibited high levels during the culture period,while Spermine(Spm) remained a relative stable and low level.The level of total endogenous PAs was high.2.A high efficient shoot morphogenesis system from PLBs in D.huoshanense was established by selecting the different kind of PAs.Both the exogenous addition of PAs,mainly Spd and Put at 2.0 mmol/L significantly promoted the frequency of conversion of PLBs to shoots. Addition of PA biosynthetic inhibitors,involvingα-difluoromethylornithine(DFMO) for Put and methylglyoxal(bis)-guanylhydrazone(MGBG) for Spd and Spm,decreased the shoot conversion from PLBs ofD.huoshanense.3.Endogenous levels of indole acetic acid(IAA) and total cytokinins(CTKs),including isopentenyladenine(iP),isopentenyladenine 9-riboside(iPR),zeatin(Z) and ZR(zeatin riboside) were quantified by means of enzyme-linked immunosorbent assay(ELISA).At the 21^st day,a marked increase in the level of total CTKs was detected,which were 1.7 times(Spd) and 2.2 times (Put) of the control,separately.Furthermore,IAA level was decreased by both Spd and Put.Hence, total CTKs/IAA ratios in Spd or Put treatments were 2.4 times and 4.6 times of the control on the 21^st day.During shoot morphogenesis,the cytokinins oxidase(CKX) activities showed similar changes in the control and in the PAs treatments.CKX activities increased rapidly,and peak on the 21^st day and the CKX activities in the control was 1.2 times and 1.8 times of the Spd and Put addition,separately.Addition of DFMO and MGBG decreased the ratio of total CTKs to IAA.This inhibition could be partly reversed by the application of exogenous Put or Spd.4.During the process of conversion of PLBs to shoots,the exogenous PAs significantly enhanced hydrogen peroxide(H₂O₂) content,especially on the 10^th day after inoculation the H₂₂ content in PAs treatments were 175.6%(Spd) and 180.6%(Put) than that in the control.Exogenous PAs enhanced the polyamine oxidase(PAO) at the prophase while catalase(CAT) activities at the anaphase,but had no obvious effect on superoxide dismutase(SOD) and peroxidase(POD) activities. Dimethylthiourea(DMTU),the scavenger of H₂O₂,inhibited conversion of PLBs to shoots and the inhibition could be counteracted partly by exogenous PA.Hence,the linker between the redox status and shoot morphogenesis is H₂O₂ which could affect the morphogenesis as the second messenger.5.Molecular mechanism of PAs enhancing conversion of PLBs to shoots was further investigated.A putative cytokinin oxidase gene,Dendrobium huoshanense cytokinin oxidase (DhCKX),was cloned by reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE) of total RNA isolated from shoot apices of D.huoshanense plantlets in the presence of 20μnol/L 6-Benzyladenine(6-BA).The full-length cDNA contains an open reading frame(ORF) encoding a putative protein of 537 amino acids with high homology to known CKX genes and with a predicted molecular mass of 60.383 kDa.Compared with the CKX sequences from other species,the DhCKX shows high identity with Dendrobium hybrid "Sonia", 92.2%in nucleotide sequence and 89.9%in amino acid sequence,respectively.RT-PCR analysis indicated the changes of the DhCKX gene expression were consistent to CKX activities during the enhanced conversion of PLBs to shoots by exogenous PAs.