Chaohong Feng

Cryopreservation of sweetpotato (Ipomoea batatas) and its pathogen eradication by cryotherapy

Sweetpotato (Ipomoea batatas) ranks as the seventh most important staple crop in the world and the fifth in developing countries after rice, wheat, maize and cassava. Sweetpotato is mainly grown in developing countries, which account for more than 95% of total production of the whole world. Genetic resources, including cultivated varieties and wild species, are a prerequisite for novel sweetpotato breeding in both conventional and genetic engineering programs. Various cryopreservation protocols have been developed for shoot tips and embryogenic tissues. The former explants are preferred for long-term conservation of sweetpotato genetic resources, while the latter are valuable for sweetpotato genetic improvement. This review provides update comprehensive information on cryopreservation of sweetpotato shoot tips and embryogenic tissues. Plant pathogens such as viruses and phytoplasma severely hamper high yield and high quality production of sweetpotato. Thus, usage of pathogen-free planting materials is pivotal for sustainable sweetpotato production. Cryotherapy of shoot tips can efficiently eradicate sweetpotato pathogens such as viruses and phytoplasma. The mechanism behind pathogen eradication by cryotherapy of shoot tips has been elucidated. Pathogen eradication by cryotherapy provides an alternative, efficient strategy for production of pathogen-free plants. In addition, cryopreserved tissues may also be considered to be safer for exchange of germplasm between countries and regions.

Novel and potential application of cryopreservation to plant genetic transformation.

The world population now is 6.7 billion and is predicted to reach 9 billion by 2050. Such a rapid growing population has tremendously increased the challenge for food security. Obviously, it is impossible for traditional agriculture to ensure the food security, while plant biotechnology offers considerable potential to realize this goal. Over the last 15 years, great benefits have been brought to sustainable agriculture by commercial cultivation of genetically modified (GM) crops. Further development of new GM crops will with no doubt contribute to meeting the requirements for food by the increasing population. The present article provides updated comprehensive information on novel and potential application of cryopreservation to genetic transformation. The major progresses that have been achieved in this subject include (1), long-term storage of a large number of valuable plant genes, which offers a good potential for further development of novel cultivars by genetic transformation; (2), retention of regenerative capacity of embryogenic tissues and protoplasts, which ensures efficient plant regeneration system for genetic transformation; (3), improvement of transformation efficiency and plant regeneration of transformed cells; (4), long-term preservation of transgenic materials with stable expression of transgenes and productive ability of recombinant proteins, which allows transgenic materials to be stored in a safe manner before being analyzed and evaluated, and allows establishment of stable seed stocks for commercial production of homologous proteins. Data provided in this article clearly demonstrate that cryo-technique has an important role to play in the whole chain of genetic transformation. Further studies coupling cryotechnique and genetic transformation are expected to significantly improve development of new GM crops.

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips.

Production of Pathogen-Free Horticultural Crops by Cryotherapy of In Vitro-Grown Shoot Tips

Horticultural crops are economically valuable for sustainable agricultural production. Plant diseases caused by Pathogens including virus, phytoplasma and bacterium have been a great threat to production of horticultural crops. The efficient use of pathogen-free plant materials has overcome the menace of plant diseases and has sustained crop production. Cryotherapy of shoot tips, a novel application of cryopreservation technique, has become a new plant biotechnology tool for plant pathogen eradication. When compared with the traditional methods, cryotherapy of shoot tips produces high frequency of pathogen-free plants, which is independent of shoot tip size and cryogenic methods. Cryotherapy of shoot tips has six major steps to produce pathogen-free plants: (1) introduction of infected plant materials into in vitro cultures; (2) excision of shoot tips; (3) cryotherapy; (4) post-culture for plant regeneration; (5) indexing of pathogens in regenerated plants after cryotherapy; and (6) establishment of pathogen-free nuclear stock plants. The key steps 2, 3, and 4 are similar to cryopreservation, and play a major role in obtaining high pathogen eradication frequency.

First Discovery of Acetone Extract from Cottonseed Oil Sludge as a Novel Antiviral Agent against Plant Viruses

A novel acetone extract from cottonseed oil sludge was firstly discovered against plant viruses including Tobacco mosaic virus (TMV), Rice stripe virus (RSV) and Southern rice black streaked dwarf virus (SRBSDV). Gossypol and β-sitosterol separated from the acetone extract were tested for their effects on anti-TMV and analysed by nuclear magnetic resonance (NMR) assay. In vivo and field trials in different geographic distributions and different host varieties declared that this extract mixture was more efficient than the commercial agent Ningnanmycin with a broad spectrum of anti-plant-viruses activity. No phytotoxic activity was observed in the treated plants and environmental toxicology showed that this new acetone extract was environmentally friendly, indicating that this acetone extract has potential application in the control of plant virus in the future.

COMPLETE GENOMIC SEQUENCE ANALYSES OF WATERMELON MOSAIC VIRUS ISOLATES FROM CHINA

The complete genomic sequences of Watermelon mosaic virus (WMV) of a Chinese isolate (WMV-CHN) and a Shaanxi isolate (WMV-Shaanxi) were each determined from 10 overlapping fragments. Excluding the poly(A) tail, the genome of the two isolates were 10,037 and 10,046 nucleotides in length and contained one single open reading frame of 9,651 and 9,657 nucleotides, respectively. The identity frequencies between the two complete genomes were 96.5% at the nt level and 98.5% at the amino acid level. The two complete genomic sequences shared 89.7-94.8% identity with other characterized WMV isolates at the nucleotide level and 94.6 to 97.1% at the amino acid level.

Rapid detection of apple stem grooving virus by reverse transcription loop-mediated isothermal amplification.

Apple stem grooving virus (ASGV) is one of the relevant apple viruses worldwide. In this study, a simple, sensitive one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of ASGV, using primers designed in the coat protein gene. The RT-LAMP assay identified 53 infected apple leaf samples while RT-PCR identified only 49 infected samples. To the best of our knowledge, this is the first report on detection of ASGV by RT-LAMP.