In the case of citrus, for instance, a globally important fruit crop that is often heterozygous for important trait genes, protoplast regeneration can be achieved only if protoplasts are derived from juvenile tissues. However, plants generated from juvenile citrus tissues require years to reach a mature fruit production stage, and this delay is an impediment to the efficient evaluation of gene functions in fruit and for other genes pertinent to citrus breeding programs. On the other hand, tissues from mature citrus trees, such as shoot segments, can be transformed with Agrobacterium. Jacobs et al.discussed the possibility of using Agrobacterium-mediated transient expression of Cas9 and sgRNA genes to produce non-transgenic mutant plants. Iaffaldano et al.reported the production of CRISPR/ Cas9-mediated mutant plants using Agrobacterium without any selection of stable transgenic plants. However, Iaffaldano et al.did not characterize whether their mutant plants were transgenic or non-transgenic, nor did they calculate the percentage of each type. The nontransgenic mutation rate reported in this study is lower than mutation rates for the previously reported protoplast micro-injection methods but higher than for particle bombardment methods. However, Agrobacterium can be a more versatile tool because of its ability to efficiently infect many different tissues types across most plant species, and it is the most widely used method for plant transformation. Thus, protocols for Agrobacterium infection and subsequent callus and shoot regeneration are readily available for many crop plant species. Furthermore, even when other methods are applicable, Agrobacterium infection procedures are relatively simple and easy to perform for many plant species. Thus,draining pot the method presented here provides an alternative method for the production of non-transgenic mutant plants.
Our method of using Agrobacterium to transiently express CRISPR/Cas9 genes, without the use of chemical selection such as kanamycin, also offers some other advantages. The lack of a chemical selection agent following Agrobacterium infection allows for even greater rates of plant regeneration compared to when chemical selection is used and therefore likely enhances mutant callus/shoot production. This is because selection agents, such as kanamycin, hygromycin, and various herbicides, can suppress shoot regeneration for many plant species, even when those shoots express relevant resistance genes. Furthermore, our high-throughput screening method, based on a novel DNA sequencing strategy in combination with HRM analysis, makes mutant identification fast and easy to perform. The drawback of our method is that there is a risk of creating transgenic mutant plants; however, identifying and discarding these plants does not add any significant time to the screening process. We have observed that the threshold of mutant plant detection using the Illumina sequencing method can be much more sensitive than the detection of one mutant out of 42 plants described in this study. The use of the 42-plant sample pools, therefore, assures that the presence of a mutant plant can be readily detected in pooled samples. Additionally, the threshold of HRM analysis to detect mutant plants can be as low as 1 of 19 plants, but we chose one out seven . Again, this pooling choice provides a safe margin for error by reducing the chance of missing mutant plants due to false negatives. Furthermore, a small number of false positives from the sequencing analysis of 42-plant pools are not a concern because they can be easily identified in the subsequent step of screening. Thus, our method, which combines a unique DNA sequencing strategy with HRM analysis, provides a reliable protocol to screen for CRISPR/Cas9-mediated mutant shoots from a population of shoots regenerated in the absence of selection pressure. The overall protocol for mutant identification is schematically represented in Supplementary Figure 4.
Using this protocol, it would take ~14 days to identify all mutants from 1000 independent shoots that were subjected to Agrobacterium-mediated transient expression of CRISPR/Cas9 genes. In conclusion, we have developed a highly useful method utilizing Agrobacterium-mediated transient expression of Cas9 and sgRNA genes combined with multi-step pooled screening, enabling the reliable production and efficient identification of non-transgenic mutants regenerated in the absence of selection pressure. Due to the versatility of CRISPR/Cas9 and Agrobacterium-mediated infection, as well as the ease of plant regeneration from leaf, shoot, or root explants, this method is applicable to many economically important plant species, particularly heterozygous perennial plant species that are recalcitrant to regeneration from protoplasts or following biolistic bombardment.Urban trees are garnering increasing attention in this era of interest in biodiversity and urban sustainability. There is growing recognition that urban areas are largely the result of human decisions and actions – they are constructed spaces. When cities are designed and built, for example, local vegetation is usually removed, and the earth recontoured and/or excavated to facilitate construction.The species chosen can be culturally, historically, or functionally significant, but it is logical to think that species planted are generally representative of species available from the local nursery industry at the time and may or may not draw from the native flora of a regional ecosystem . Regional nursery catalogs as a data source for urban biodiversity have not been evaluated prior to this research. Yet, the horticultural industry has been shown to be an important contributor to regional biodiversity with regard to invasive species distributions . This paper contributes to the literature on urban biodiversity by suggesting that plants offered by nurseries are a source of yetto-be-explored diversity in cities. We hope that this research will encourage the further consideration of the role of nurseries in urban biodiversity. The significant historical legacy of tree introductions by European and Eastern U.S. settlers in California likely accounts for the initial urban tree diversity of Los Angeles and Southern California. The early 17th-century missionaries came with a culture of planting both crop-yielding and ornamental trees and this tree palate was substantially supplemented with the advent of the intercontinental railroad and increased oceanic traffic connecting Southern California with the world in the 19th Century.
An innovative horticultural industry emerged in California which both catered to, and shaped the tastes of, settlers . Today Los Angeles has an exceptionally diverse tree assemblage where the imprint of this early period can still be detected. Referred to as the Garden of Eden, Paradise, and the Garden of the Hesperides throughout its relatively young history, images from this region spread worldwide . There are a number of historical and social factors that can shape and influence tree species composition and diversity in cities beyond biophysical ones such as climate, environment, and ecological factors . Cultural factors , and factors related to symbolic and representational associations of trees are also important. However, one possible critical factor in the planting and distribution of trees in the urban environment that has not been investigated previously, and is certainly related to the factors above, is the selection of tree species available from the nursery industry of the region. People obtain trees from purveyors,round plastic planters and thus this article examines the evolution of the selection of trees available from tree nurseries and seed availability. If there is no stock or seeds locally available, the burden of finding a desired tree is much higher. Thus we suggest that a window into which trees are found in the urban fabric may be by examining nursery catalogs over time. This paper describes results from a longitudinal study of nursery offerings of tree species from 1900 to 2010. Los Angeles County has around 6 million trees in a bio-region that naturally supported trees only along riparian corridors and along the foothills of the cityregion . As Schoenherr and Rundel and Gustafson have documented, in pre-colonial cismontane Southern California was dominated by chaparral and coastal sage scrub. Trees were mostly found in riparian corridors and along the foothills and included 14 native species. To understand the role of the landscape nursery industry in influencing diversity of the tree canopy cover, we developed three main areas of inquiry. First, we examined whether there has been a significant change in the number of tree genera and species offered by tree nurseries over time. Second, we asked whether there have been significant changes in the functional classifications of trees offered over time. Finally, due to the size of the data set we were curious to know whether some species had been offered consistently from the early 1900s to 2011.The limited historical documentation available from the California Department of Food and Agriculture was supplemented with archival catalogs. We located two nursery catalog collections in the region: Collection of Nursery Catalogs in the Department of Special Collections located in the Charles E. Young Research Library, University of California, Los Angeles, and the collection of catalogs available at the Los Angeles County Arboretum and Botanic Garden Library. The 2009 and 2011 catalogs of West Covina Nurseries and Monrovia Nurseries, both large and well known nurseries in the region, were accessed on the company’s websites respectively . We exclusively utilized catalogs of nurseries located in Los Angeles County. Data collection involved manually transcribing tree species information from each catalog into a database for further analysis. Species that were 12 feet and taller were classified as trees in this analysis. Images of the catalog pages were scanned and converted to text using optical character recognition software. The location of the nursery, the year of the catalog, scientific name, and names of varieties were recorded. Information was also collected for commercial fruit trees but is not included in this analysis. In order to classify the tree as deciduous or evergreen, native or non-native , angiosperm or gymnosperm, we used the Sunset Western Garden Book . Gymnosperms were classified as tree species belonging to Araucariaceae, Cupressaceae, Ginkgoaceae, Pinaceae, Podocarpaceae, Sciadopitayaceae, and Taxaceae families .
Tree fern species were classified as ferns. Catalogs were chosen to represent every decade from the 1900s to the 2010s. This 110-year period was split into four periods of 30 years each in order to conduct the statistical analysis, because the numbers of catalogs represented in each time period was different . A total of 18 unique nursery catalogs were sampled for this research. The number was restricted by availability in the archival collections. The number of catalogs sampled in each time period differed such that period one had three catalogs, period two had four catalogs, period three had six catalogs, and period four had five catalogs. We streamlined the study design by sampling at least one nursery catalog to represent each decade . While it is likely that having more catalogs, and in turn, having a larger sample size, especially in the earlier decades, would have yielded more robust trends and results for the earlier decades, it is unlikely this would have changed the overall results.The large data set was then analyzed using both Microsoft Excel 2011 and MATLAB 10. MATLAB was used to obtain specific numerical information . Using a statistical count model we were able to predict the number of species offered in each time period; we suspect that the difference in number of catalogs sampled would have little to no effect on the actual number of species offered, thus the overall result would still not change. The entries were optimized manually to account for spelling errors, alternative names, and repeats. In order to compare the time periods and the different numbers of catalogs sampled in each time period, we employed a negative binomial regression count model. This allowed for comparison between time periods by predicting the number of species offered in each time period. One goal of the analysis was to identify whether there was a statistically significant difference in the number of species throughout the time period, and we found there was with a 95% confidence interval. A polynomial contrast was used to detect linear and quadratic trends with time. An exponential model was also used to analyze the same data. However, unlike the count model, the exponential model did not take into consideration the difference in the number of catalogs used in each time period. While the results indicated that both models fit the data, we chose to use the negative binomial regression count model because it took into consideration the differences in the number of catalogs used in each time period.We also found that there were fifteen species offered consistently in every decade.We used the nursery catalogs to identify wholesale and retail nurseries, and city websites to identify cities that had municipal arborists.