Study On Phalaenopsis Ssp.and Oncidium Ssp.in Tissue Culture And Chemical Induction In Vitro | | Posted on:2010-07-28 | Degree:Doctor | Type:Dissertation | | Country:China | Candidate:G R Cui | Full Text:PDF | | GTID:1223330374995123 | Subject:Ornamental horticulture | | Abstract/Summary: |  PDF Full Text Request | | The aims of this article were to establish the technique system of chemical induction in vitro for Phalaenopsis and Oncidium. The techniques about protocorm-like bodies(PLBs) propagation in liquid culture and embryo regeneration from thin cell layers(TCLs) of Phalaenopsis and Oncidium PLBs, and efficient somatic embryogenesis from leaf explants of Phalaenopsis in vitro culture were established and optimized. The technique systems of chemical induction with colchicines, NaN3and EMS for2kinds of orchids were established on the bases of these in vitro culture systems. A lot of polyploids of Phalaenopsis and Oncidium were got and the polyploids were identified in the morphological and cell. The regenerated plants treated with NaN3and EMS were tested by RAPD.1. Phalaenopsis and Oncidium tissue culture in vitroThe effects of cytokinins N6-benzyl adenine (6-BA) was better than adenine sulphate (Ad) in embryoid induction. The auxin a-naphthaleneacetic acid (NAA) combined with given concentrations of6-BA and Ad could effectively raise the frequency of somatic embryogenesis. The optimum plant growth regulator combination for somatic embryogenesis was6-BA4.0mg/L+Ad2.0～4.0mg/L+NAA1.0～2.0mg/L.20～40g/L sucrose (w/v) and100～200ml/L coconut water (v/v) added in the medium was benefit for somatic embryogenesis. The results of histological observations indicated that somatic embryos originated from upper epidermis and mesophyll single cell nearby stoma. The course of somatic embryos development was similar to other plants. Finally, the somatic embryos became protocorm-like body.The concentrations and combinations of hormones have a great effect on phalaenpsis PLBs propagation and growth in the liquid culture. The best combination of hormones was 2.0mg/l6-BA+1.0mg/l Ad+0.5mg/l NAA. The concentrations of sucrose have also great effect on PLBs propagation and growth. The medium added10-40%(v/v) coconut palm juice can not only elevate PLBs propagation coefficient but also improve the quality of PLBs. The propagation time of PLBs in the liquid culture was best within4weeks. The suitable medium for Phalaenpsis PLBs propagation was1/2MS+2.0mg/l6-BA+1.0mg/l Ad+20g/l sucrose+10%(v/v) coconut palm juice. The PLBs growth curve showed collapsed "V" within5weeks. The time of shoots regenerated from PLBs cultured in the liquid medium was longer than PLBs cultured in the solid medium.The combinations of the given concentrations of three kinds of plant growth regulators were key factors in the PLBs regeneration from the TCLs in vitro. Additional coconut water in the given medium could raise the rate of PLBs induced from the TCLs effectively. Higher concentrations of sucrose in the mediums reduced the efficiencies of PLBs formation of oncidium but no effects on phalaenopsis. The growth states of PLBs inducted from TCLs had no obvious differences among all treatments. The optimal medium for two kinds orchids of PLBs regeneration from TCLs was1/2MS+4.0mg/L6-BA+2.0mg/L Ad+1.0mg/L NAA+100-200ml/L coconut water+10-20g/L sucrose+4.0g/L agar powder.The concentrations and combinations of hormones have great effect on Oncidium PLBs propagation in the liquid culture. The best combination of hormones was6-BA0.5mg/L+Ad0.05mg/L. The concentrations of sucrose have also great effect on PLBs propagation. The suitable medium for oncidium PLBs propagation in the liquid culture was7.5g/L. The medium added5ml/l coconut palm juice can not only elevate PLBs propagation coefficient but also improve the quality of Oncidium PLBs. The best medium for Oncidium PLBs propagation was MS+6-BA0.5mg/L+Ad0.05mg/L+sucrose7.5g/L+coconut palm juice5ml/L. The PLBs cultured in this medium grew fast, less browning. The PLBs growth curve showed "V" in this liquid medium, while seemed beeline in the solid medium. However, the growth state of shoots regenerated from PLBs cultured in the liquid medium was bad than shoots regenerated from solid medium.2. Phalaenopsis chemical induction in vitro and identificationThere were great effects of colchicines treatment on the PLBs growth, inner structure of tissue or cell and the trait of plantlets regenerated from these PLBs. The body of PLBs became large and the surface of PLBs was crude. The effects were stronger with higher concentration of colchicines and longer treatment time. The inner structural changes of tissue or cell of PLBs were related closely to the treatment time and concentration of colchicines. The time of shoot formation was longer and the rate of shoot formation was lower when the concentration of colchicines got higher or the time of treatment got longer. The rate of polyploid induction in each treatment was different. The highest rate of tetraploid induction could reach to40.0%. There were evident differences in morphological and histological characteristics between the diploid and polyploid. The nucleus got bigger and the number of chromosomes were doubled in the cell of polyploid.The mutagens NaN3and EMS had great effects on TCLs growth. Some TCLs got white or browning and died, the PLBs grew slowly and the number of regenerated plantlets were reduced.There was obvious injured effects on the explants, PLBs and plantlets regenerated from the TCLs. The injured effects got more and more serious with higher concentration of mutagens and longer time treatment. The mutagens both NaN3and EMS made the plantlets aberrance, leaves crimpled and got thicker. The results of10primers of RAPD reaction indicated that the molecule maps of RAPD had some polymorphisms which means the sequence of DNA had changed. The concentrations of3-6mmol/L NaN3treatment for2days and the concentration of0.4%EMS treatment for2-4days are suitable dosage for Phalaenopsis chemical induction in vitro.3. Oncidium chemical induction in vitro and identificationThe effects of different concentrations of colchicine treatments on PLBs formation and shoots regeneration from the TCLs in different time. The morphological and the histological characters of lower epidermal cell of the polyploidy plantlet leaves were compared with the diploid. Meanwhile, the chromosomes of root tip cell of polyploid plantlets were identified. The results showed that there were great effects of colchicines on PLBs formation and shoot regeneration from TCLs of Oncidium in vitro culture. The rate of PLBs formation from the TCLs and the number of shoots were obviously decreased in the longer time treatments with higher concentrations of colchicines. However, the rate of polyploidy plantlets was higher in the treatments with higher concentrations of colchicines in the longer time. The polyploidy plants were short and stout, and had thicker leaves. The structure of lower epidermal tissue, cell and stomata had some differences between the diploid and polyploidy. The cell nucleus of the polyploidy was bigger than the diploid and the number of chromosomes was doubled.Two kinds of mutagens NaN3and EMS had great effects on TCLs growth. Some TCLs got browning and died, the PLBs grew slowly and the number of regenerated plantlets were reduced.There was obvious injured effects on the explants, PLBs and plantlets regenerated from the TCLs. The injured effects got more and more serious with higher concentration of mutagens and longer time treatment. The mutagen NaN3inhibited plantlets growing but EMS had no evident effects. The results of10primers of RAPD reaction indicated that the molecule maps of RAPD had some polymorphisms which means the sequence of DNA had changed. The ratio of polymorphisms got higher with higher concentration of mutagens and longer time treatment. However, the ratio of RAPD polymorphisms had no obvious raised when the concentration of EMS reached to0.4%. The concentrations of6mmol/L NaN3treatment for2～4days and the concentration of0.4%EMS treatment for2～4days are suitable dosage for Oncdium chemical induction in vitro. | | Keywords/Search Tags: | Phalaenopsis, Oncidium, In vitro culture, Colchicines, NaN3, EMS, Chemical induction, RAPD | | PDF Full Text Request | Related items |
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