| Populus tomentosa is widely used for forest, landscape and pulp industry due to itsbeautiful shape, dense canopy cover, weel woody texture. But the pollen produced bymale plants and the hair on seeds produced by female plants scattered everywherewhich bring a lot of inconvenience to pedestrians, causing problems for security, andpolluting the environment seriously. In this study, we have estalished indirect in vitroregeneration system use Populus tomentosa petioles and aseptic leaves as explants,through the way we have studied the impact of various factors, including explantsspecies, plant growth regulators and other aspects in detail. And then we haveestablished the transformation system by optimizing the related factors. The Barnasegene conversion aims to achieve the pure vegetative growth plant by closing thereproductive growth through Agrobacterium-mediated way, so as to solve theproblem of seed hair flying of Populus tomentosa.1Establishment of regeneration systemIn the process of establishing regeneration system, analyzed the influence of threedifferent explants on regeneration process and screened to establish the optimumregeneration system explants eventually, by comparing the effects of differentdisinfection methods on the rate of brown and dirt, analyzing the influence of explanttypes on callus induction. The results found that when using aseptic leaves as explants,in addition to its induction frequency is better than the outdoor leaves, while there isno significant difference with the petiole, but the callus induction frequency andcallus division speed of it is higher than that of petiole explants.In callus inductionrate, found that when useing cytokinin and auxin, the effect is significantly higherthan them useing separately by comparing the different plant growth regulators oncallus induction. And the comparation of medium for petiole inducing callus is1/2MS+NAA0.5mg/L+BA0.5mg/L, while using aseptic leaves as explants, thecomparation of medium for callus induction is1/2MS+NAA0.25mg/L+BA0.25mg/L+ZT0.25mg/L,and the good condition callus induction frequency is100%. And all of it can meet the needs of genetic transformation in the next part. At the end, we have finalized the establishment of regeneration system of Populustomentosa in aseptic leaves as explants.When using the callus induced from aseptic leaves to formated shoots, we hadanalysed the influence of TDZ, and had recorded the process of the adventitious budformation steps. Results show that TDZ can improve the frequency adventitious budinduction: when adding0.0001mg/L TDZ, more vitrification adventitious budformation, while adding no TDZ into the medium, no vaccine callus formation ofadventitious bud induction has been achieved100%; When using the petiole ascontrast, we found that0.0015mg/L TDZ can improve adventitious bud inductionrate to92.0%, and adventitious bud to grow strong and healthy.We had analyzed the MS salt concentration and plant growth regulator (IBA andNAA) on the induction frequency and quality of rooting. Results show that low saltconcentration (1/2)of the MS culture medium and collocated with0.5mg/L NAA and1.0mg/L IBA, all of the plants can be induced roots, and the average root length of97.6mm, and this time there is no vaccine robust growth.2Establishment of the genetic transformation systemWith aseptic leaves as explant, we had not only analyzed the effects ofAgrobacterium bacterium concentration and the infection time on transformation, butalso determined the concentration of bacterial antibiotic Cef and plant screeningmaterial PPT from the degree of injury and infection respectively. Results showedthat1/2MS is best which compared with the other two different medium (YEB andZBN) from bacteria growth and the impact of foreign implant activity. Then based on1/2MS medium, the polishing liquid coated on YEB solid medium which selectedfrom infested explants, we found that engineering bacteria is with the largest cellviability and cell number is (OD600value was1.0abs) more enough for meettingAgrobacterium infection.When determined the concentration of bacteria antibiotic（Cef）and non-transformedplants screenes (PPT), we found that the Agrobacterium grwoth well whenconcentration of Cef is200mg/mL, and the untransformed plants are killed whenadding25mg/L PPT into the medium. And at last, we detected41plants whichsurvived, among them there are5resistant plants,13semi-positive plants,23false positives plants finalized. The resistant and semi-positive plants were validated byPCR, and we found that there are2plants with Bar gene, and this means these twoplants successfully transformaed. But both of them did not survive, thus proving thattransformation way with Barnase genes to Populus tomentosa is relatively difficult. |
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