Overexpression ofβ-1,4-Glucanase Gene EuEG1 Improves Micrografting of Eucommia ulmoides

Adventitious root formation poses a major constraint on the tissue culture and genetic transformation of Eucommia ulmoides.Micrografting offers a new method for the transplantation of genetic transformation,and its success depends on the formation of graft unions.This study used transgenic rootless test-tube seedlings as scions and seedlings from seed as rootstocks during micrografting to avoid the rooting issues that occur during tissue culture and to investigate the role of the EuEG1 gene in the graft healing process.We found that the EuEG1 gene is a vital regulator of graft,and its overexpression contributes to the survival of Eucommia ulmoides micrografting.The EuEG1 gene transgenic plants(TP)used as scions for micrografting presented a significantly higher survival rate than the wild type(WT)and empty vector(EV)regenerated scions.During the grafting healing process,the expression of the EuEG1 gene was higher during the period of callus proliferation,suggesting that the EuEG1 gene was involved in the graft healing process.Histological observation revealed that more calluses tissue appeared at the junction of transgenic scions,and the connection with the rootstock was stronger,which benefits wound healing.These results provide new insights into Eucommia ulmoides micrografting and indicate that the EuEG1 gene can promote wound healing and improve the micrografting survival rate.

Treatment of Gray Hair in Vitiligo Patients by Direct Melanocytes Transplant Using Needling Micrografting and Dermabrasion Techniques

Background: Melanocytes transplant for treatment of vitiligo is a common therapy using different surgical procedures. But there was no interest in repigmentation of grayness of hair in the treated vitiliginous area. Objective: To do melanocytes transplant from donor area into the recipient vitiliginous area with associated gray hair. Patient and Methods: This is a case interventional study was done in Department of Dermatology/Baghdad Teaching Hospital from February 2011-March 2012. Eleven patients were enrolled in this study, six males and five females with vitiligo in association of gray hair. Their ages ranged from 8 - 35 years with a mean ±SD of 20.90 ± 7.006. Melanocytes transplant in patients with vitiligo using needling micrografting technique for twelve patches and direct melanocytes transplant from normal donor area into vitiliginous recipient area by dermabrasion technique for eleven patches. Dressing was applied and patients were seen every two weeks for the first month and monthly for one year. Results: Repigmentation of the vitiliginous area was started after two weeks and was obvious at one month that progressed over time. The repigmentation of hair appeared usually after few months and was obvious after four months and the repigmentation of gray hair was quicker in patients with micrografting technique than those with dermabrasion technique. The mean rate of repigmentation was 18.3% at six months and 37.5% at twelve months in micrografting technique while the mean rate of repigmentation was 9.15% at six months and 18.55 at twelve months in dermabrasion technique. Conclusions: Direct transplant of melanocytes from normal donor area into recipient vitiliginous area with associated white hair is an effective procedure to induce repigmentation of gray hair.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained.Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

Photochemical Efficiency during the Establishment and Consolidation Phases of in Vitro Pinus radiata Micrograft Made from Scions of Different Ontogenetic Age

The aim of the present study was to evaluate the applicability of maximal photochemical efficiency of photosystem II (Fv/Fm) as an early estimate of P. radiata micrografts viability coming from different position (basal vs. apical) in the ortets. We hypothesize that Fv/Fm variation is a good indicator of micrograft’s viability and phenological stage during micrograft development. The micrografts were established in QL medium supplemented whit 0.1 mg·L-1 IBA and 1 mg·L-1 BAP and cultured at 25°C ± 2°C and 80 μmol photons m-2s-1 of photosynthetic active radiation by 16 h per day. During the establishment and consolidation phase, we found significant differences in Fv/Fm with respect to time and buds positions provenience. During establishment, basal shoot tips have lower Fv/Fm than apical shoot tips, which agrees with the lowest viability (35%). However, during the consolidation phase, the trend changed and basal shoot tips presented higher Fv/Fm than apical shoot tips and showed an increase in ETR and NPQ, with respect to apical shoots and ortet. Although the measurement of fluorescence parameters implies the insertion of the fluorometer sonde in vitro, this implies aseptic considerations, but always conveies a contamination risk. We conclude that fluorescence (Fv/Fm, ETR, NPQ) can be indicators of the micrograft’s development according to the shoot tips position in the ortet and can be useful early-indicators of the scions’ physiological condition during micrograft transition from establishment to consolidation.