ABSTRACT
Various steps of micropropagation include selection of suitable explant, establishment of adventitious shoot induction cultures, proliferation, rooting, and acclimatization of the resulting plantlets. A systematic protocol is provided for the micropropagation and Agrobacterium tumefaciens-mediated genetic transformation of a fast growing, multipurpose tree, Paulownia elongata. Our studies show that optimum shoot induction is on half leaf with petiole explant on MS medium supplemented with 25 µM thidiazuron and 10 µM indole-3 acetic acid. Micropropagation protocols provided here are applicable to explants collected from the primed in vitro raised seedlings on MS medium containing 2.5 µM 6-benzylaminopurine (BAP) or actively growing shoots collected from greenhouse or field growing plants. We also discuss a possible role of "Python" script guided protocol optimization for higher and consistent multiplication of shoots that can be very helpful for scaled up production in commercial settings. To facilitate future plant improvement and gene editing possibilities, an A. tumefaciens based genetic transformation protocol and molecular identification of transgenic plants using Polymerase Chain Reaction (PCR) and Reverse Transcriptase-PCR (RT-PCR) techniques have also been optimized.
Subject(s)
Lamiales/genetics , Plant Breeding/methods , Agrobacterium tumefaciens/drug effects , Culture Media , Indoleacetic Acids/pharmacology , Lamiales/growth & development , Phenylurea Compounds/pharmacology , Plant Growth Regulators/pharmacology , Plant Leaves/drug effects , Plant Roots/drug effects , Plant Shoots/drug effects , Seedlings/drug effects , Thiadiazoles/pharmacology , Tissue Culture Techniques/methods , Transformation, Genetic/genetics , Transformation, Genetic/physiology , Trees/geneticsABSTRACT
OBJECTIVE: To define concomitant risk factors, treatment, and outcomes for patients with nonnephrogenic calciphylaxis (NNC). PATIENTS AND METHODS: A retrospective review of Massachusetts General Hospital (MGH) medical records (January 1, 2014, through February 29, 2016) and a systematic literature review of PubMed, Google Scholar, EMBASE, MEDLINE, and CENTRAL (August 1, 1970, through July 31, 2016) were performed. Demographic characteristics and concomitant features were summarized and compared between patients with different lesion characteristics. Outcomes (lesion improvement and mortality) and their predictors were analyzed. RESULTS: Nine patients (median age, 72 years [interquartile range (IQR), 44-82 years]; 78% women; 89% white race) were identified through MGH records. The literature review identified 107 patients (median age, 60 years [IQR, 49-72 years]; 77% women; 86% white race). Vitamin K antagonism and obesity were the most common concomitant factors. In the literature review, lower age (P<.001) and higher body mass index (P=.03) were associated with the central location of lesions, whereas vitamin K antagonism was associated with the peripheral location (P=.009). In the MGH series, median survival was 24.0 months (95% CI, 7.8-36.0 months), and 33% (95% CI, 14%-60%) had lesion improvement by 6 months. In the literature review, median survival was 4.2 months (95% CI, 1.9-5.9 months), median time to lesion improvement was 5.9 months (95% CI, 3.9-8.9 months), and none of the treatments were associated with lesion improvement or survival. CONCLUSION: This description of concomitant traits may augment an earlier recognition of NNC. Future research is needed to investigate NNC pathogenesis and treatments.
