Research advances on watermelon tissue culture and genetic transformation

Abstract:Watermelon is one of the economically significant cucurbits in China for its production yieldand areas rank the first in the world. There are many agricultural problems in the cultivation of watermel⁃on for which traditional breeding method often does not have already explained, such as lacking of resis⁃tance cultivar against viral diseases. However, there are inadequate germplasm resources to meet theneeds of watermelon breeders. Other important issues requiring improvement are fruit quality, nutrition,flavour, tolerance of storage of fruits and resistance to abiotic stresses. The above problems are difficult tosolve through time-consuming conventional breeding methods, but biotechnology shed more light on solv⁃ing this issue. Transgenic technology is a powerful tool for gene functional validation and genetic improve⁃ment of plants. Tissue culture and genetic transformation are the two basic components for that. Tissue cul⁃ture is a prerequisite to successful genetic transformation which introduce significantly interest genes intothe plant genome while preserving genetic identity of plants. Recent advances in this technology have re⁃sulted in successful development of commercially disease and herbicide resistant plants which enhancedtolerance to environment stresses, increased crop productivity and reduced the usage of harmful pesti⁃cides. The plants have been engineered for safe and inexpensive production in large quantities producedin transgenic plants, as well as plants which possess enhanced nutritional traits to date. In this paper, weanalyzed the major factors affecting watermelon tissue culture which is aimed to regenerate explants of cot⁃yledons, hypocotyls, apical buds, anthers, ovaries, protoplasts and leaves. But most watermelon tissue culture usedcotyledonsastheexplants,inductionofadventitiousshootswasobtainedfromproximalpartsofthecoty⁃ledonincubated on MS medium containing different concentrations of benzylaminpurine 1-7 mg·L^-1 and low⁃er concentrations of auxin 0-3 mg·L^-1 or 2 mg·L^-1 of 2,4-D. Regeneration of watermelon in tissue culturehas been achieved for various watermelon cultivars. The various factors that influenced tissue culture ofwatermelon including the seeds storage time, genotypes, age of seedlings, type of explants, composition ofculture medium and environmental conditions. However, an efficient system for plant regeneration is essen⁃tial for in vitro techniques which is useful in procedure of transgenic genes. Recently most transgenic wa⁃termelon researches focused on improving regeneration of plantlets from callus and adventitious shoots di⁃rectly, in addition to pursuing efficient transformation methods of foreign genes into watermelon. Agrobacte⁃rium-mediated genetic transformation currently was the common method in the watermelon transgenictechnology. In 1994, Choi in Korea reported the first transgenic watermelon plants regenerated from cotyle⁃dons explants by Agrobacterium inoculation. Since then, by using modified Agrobacterium-mediated genet⁃ic transformation, resistance to various plant diseases has been achieved in transgenic watermelon throughthe incorporation of genes coding for the coat protein gene of various plant viruses, including Zucchini yel⁃low mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV) and Cucumbergreen mottle mosaic virus (CGMMV). Other researchers have reported the development of genetically engi⁃neered watermelon plants through transformation with ACC oxidase antisense gene, antifungal proteinsand chitinases etc. Agrobacterium-mediated genetic transformation technology is a highly complex andevolved process involving genetic determinants of both the bacterium and the host plant cell. The watermel⁃on is still acknowledged recalcitrant crop for transformation. Its transformation efficiency is still very lowso far. Here we summarized key factors influencing Agrobacterium-mediated watermelon genetic transfor⁃mation, including genotypes of the plant, types of explants, plasmid vectors, bacterial strains, the selectionmarkers genes, the Agrobacterium strains, the pre-culture time, the infection concentrations and immers⁃ing time of Agrobacterium, the co-culture time. A number of researches indicated that three to ten days cot⁃yledons are used as suitable explants for watermelon transformation, kanamycin is the usual selectionmarker, and herbicide resistance Bar gene and hygromycin B resistance gene are also used. Some Agrobac⁃terium strains are applied to produce genetic transformation watermelon, such as EHA105, LBA4404,EHA101 and GV3101. There is no evidence to prove which strain is more effective. One to five days precultureor without can produce adventitious shoots, the co-cultivation time of watermelon explants withAgrobacterium from one to five days also produce adventitious shoots. The efficiency of plant transforma⁃tion can depend also on the Agrobacterium density, ranging from 1×106 to 1×1010 cfu·mL-1 can increasetranscient transformation, however, it is not always correlated with higher stable transformation. It also dis⁃cussed that some questions about tissue culture and genetic transformation of watermelon. It has not yetbeen solved that vitrification, yellowing, the top necrosis phenomenon in the process of subculture. Cotyle⁃dons is the mainly used explants affects the ploidy level of watermelon regenerated from tissue culture thathigh frequency of tetraploid regenerants is a common phenomenon. Some fruit characters are significantlydifferent in morphology, and there is gene escape in transgenic watermelon plants system. The disorganiza⁃tion of meristematic structures following the exposure of explants to Agrobacterium remains the major ob⁃stacle to develop efficient transformation technology. These problems seriously restrict the usage of watermelon transgenic technology. In 2013, a powerful tool CRISPR (clustered regulatory interspersed short pal⁃indromic repeat)/Cas9 (CRISPR associated proteins) gene editing technology in plants was developed. Since then, the CRISPR/Cas9 system has been used in various plant species for targeted genome editing,such as arabidopsis, tobacco, wheat, rice, maize, cucumber and watermelon. It is able to achieve highlyflexible and specific targeting. This system can edit multiple target genes simultaneously. We also expectnew watermelon germplasm with novel desired traits will be created by this tool in coming years.