On 2015 Feb 20, Jaime A. Teixeira da Silva commented:

The authors developed some alternative methods for cryopreserving a single chrysanthemum cultivar ‘Escort’ while developing an ultra-rapid freezing protocol for nine chrysanthemum cultivars. In the controlled rate freezing protocol, the authors do not indicate how many shoot tips are placed into each cryovial nor for how long explants are maintained in LN. The same lack of detailed information exists for the encapsulation-dehydration (ED) protocol. In the vitrification protocol, the authors claim that shoot tips were added to a small drop of PVS2 on a sheet of aluminium that was then placed in cryovials and then plunged into LN. However, what was the function of adding the aluminium foil to cryovials? Only for the vitrification protocol do the authors state that “The vials were cooled from underneath to liquid nitrogen temperature to prevent boiling of the liquid nitrogen in the vials.” But it is not clear if such caution was exercised in the other cryopreservation protocols. The density or ratio of explants to medium is never indicated for any protocol. The commercial source of the cryovials and the reagents and equipment is also not described thus the experiment cannot be reliably reproduced. Throughout the manuscript, the authors use two terms, shoot tips and apical shoots to describe the exact same experimental explant, which could be confusing to amateur readers. Much of the description of the protocols is very repetitive and poorly written, and most of the “noise” in the protocol caused by this overlapping information could have been considerably reduced had the authors used suitable acronyms, e.g., SIM for shoot induction medium rather than defining all constituents every time. Fig. 1 legend contains a mistake: it should indicate –1°C but instead it is written as –0.1°C. In Fig. 2 legend, the claim that “There were no statistically significant differences between the different preculture procedures” might be incorrect, considering the wide span between 31% and ~45%. No statistical analyses were conducted on data in Table 1 and Fig. 3, so there is an inconsistent use of statistics and thus interpretation of the data set. All data is represented as only shoot % regeneration, which is not very informative. Shoot numbers would have also been useful. Even though the authors claimed to assess shoot % regeneration from shoot tips after 4 weeks or from callus after 8 weeks, none of the figures or tables indicate the source of the shoots (i.e., from callus or from shoot tips?). Each figure represents different sample sizes, with no apparent explanation, and there is absolutely no discussion about shoot regeneration from callus, which is an undesirable form of clonal propagation for chrysanthemum and could result in somaclonal variation.

On 2015 Feb 20, Jaime A. Teixeira da Silva commented:

The authors developed some alternative methods for cryopreserving a single chrysanthemum cultivar ‘Escort’ while developing an ultra-rapid freezing protocol for nine chrysanthemum cultivars. In the controlled rate freezing protocol, the authors do not indicate how many shoot tips are placed into each cryovial nor for how long explants are maintained in LN. The same lack of detailed information exists for the encapsulation-dehydration (ED) protocol. In the vitrification protocol, the authors claim that shoot tips were added to a small drop of PVS2 on a sheet of aluminium that was then placed in cryovials and then plunged into LN. However, what was the function of adding the aluminium foil to cryovials? Only for the vitrification protocol do the authors state that “The vials were cooled from underneath to liquid nitrogen temperature to prevent boiling of the liquid nitrogen in the vials.” But it is not clear if such caution was exercised in the other cryopreservation protocols. The density or ratio of explants to medium is never indicated for any protocol. The commercial source of the cryovials and the reagents and equipment is also not described thus the experiment cannot be reliably reproduced. Throughout the manuscript, the authors use two terms, shoot tips and apical shoots to describe the exact same experimental explant, which could be confusing to amateur readers. Much of the description of the protocols is very repetitive and poorly written, and most of the “noise” in the protocol caused by this overlapping information could have been considerably reduced had the authors used suitable acronyms, e.g., SIM for shoot induction medium rather than defining all constituents every time. Fig. 1 legend contains a mistake: it should indicate –1°C but instead it is written as –0.1°C. In Fig. 2 legend, the claim that “There were no statistically significant differences between the different preculture procedures” might be incorrect, considering the wide span between 31% and ~45%. No statistical analyses were conducted on data in Table 1 and Fig. 3, so there is an inconsistent use of statistics and thus interpretation of the data set. All data is represented as only shoot % regeneration, which is not very informative. Shoot numbers would have also been useful. Even though the authors claimed to assess shoot % regeneration from shoot tips after 4 weeks or from callus after 8 weeks, none of the figures or tables indicate the source of the shoots (i.e., from callus or from shoot tips?). Each figure represents different sample sizes, with no apparent explanation, and there is absolutely no discussion about shoot regeneration from callus, which is an undesirable form of clonal propagation for chrysanthemum and could result in somaclonal variation.