Agrobacterium-Mediated Genetic Transformation of Larix kaempferi (Lamb.) Carr. Embryogenic Cell Suspension Cultures and Expression Analysis of Exogenous Genes
Round 1
Reviewer 1 Report
Reviewer comments
The manuscript brings essential information and techniques for propagating high-value wood through Somatic embryogenesis. The improvement and optimization for SE transformation are paramount for conservation strategies and the selection of elite genotypes. In my opinion, the manuscript has important implications but still needs some improvement. Also, the amplification test for virG was a clever solution for checking Agrobacterium contamination.
Specific points to be improved
Line 157: Is the Hygromycin resistance gene mutant? Why is it in lowercase letters?
What is HYG? I didn't see any mention of this gene, only the acronym. Please describe what HYG is. The GUS and HYG are the exogenous genes....explain Gus and HYG...
Line 168: Was the RNA extracted from different tissues? Which ones? I got confused because calli can bring a chimeric structure. Please, explain what tissues were used for this analysis.
Line 173-185: the cloning step in the middle of qPCR procedures for standardization needs to be clarified. Why are you doing this procedure? Please describe it better.
Line 186: Why the relative quantifications were normalized against the expression of EF. Please, clarify.
Line 192-194: One of the manuscript's objectives is reproducibility. Please rewrite the sentences - The putatively transformed cell lines were stained by incubation overnight at 37 °C. Chlorophyll was removed by soaking in 70% ethanol.
Line 212-215: Are you using water in the Agrobacterium cultivation? I don't think so. Please avoid the word water stain; use: when the turbidity of the culture becomes....
206-207: Instead of: In most experiments performed, a 46–120 h co-cultivation period was conducive to cell recovery and to the acquisition of hygromycin-resistant cell lines. Use: In most experiments, the co-cultivation period of 46–120 h was conducive to cell recovery and acquiring hygromycin-resistant cell lines.
Fig 1 I couldn't see any difference among the samples. This is because the resolution was shallow.
232: I suggested removing the sentence: Different larch embryogenic cell lines differ in sensitivity to hygromycin. In the following sentence, the authors declare that the sensitivity of the S287 line was evaluated.
Fig 4b. Relative expression? Log fold change? Please, include the units on the axis.
Figure 6 is of low quality and is difficult to understand.
329-332 Are you testing reference genes? Are these reference genes suitable for those samples?
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear Authors,
The manuscript presents relevant results to support research in the forestry genetic breeding sector and shows advances with the species Larix kaempferi that contribute to the study with gymnosperms in general and can help further works with genetic transformation based on somatic embryogenesis.
The manuscript is well written and the results are well supported by the data obtained. If possible, it would be interesting to determine the number of copies in each event, to support one of the hypotheses raised by the authors in the discussion.
From my point of view, the work should, however, be improved in one point. Both in the results and in the discussion, the authors compare the transformation protocol presented with those currently used for conifers, without, however, having used these as a control. In the work, the protocols were not directly compared. For such comparison, the protocols should have been tested in the same experiment and not only for the same species, but also for the evaluated masses, and only than it could be affirmed that the proposed protocol is more efficient.
In addition, tests comparing the duration of co-cultivation and the method of removing agrobacteria were not shown. The way the transformation part is presented, it is described as an already established protocol.
Which means that tests were not presented to support what is stated in lines 372-373, nor what is stated in line 377. Nor what is stated in lines 447-449.
Despite this, I believe that the work should be accepted and published, regarding its relevance, once only a few reports for genetic transformation of Gymnosperms are found in the literature . However, I suggest a reformulation of the results an discussion part, so as not to make a direct comparison between protocols, but rather to cite the proposed one as another alternative transformation of Larix kaempferi that could even be tested for other conifers.
Furthermore, I suggest that in the figure 8, a, d, e and f - the structures should be referred as " embryogenic masses”; in b and c as "suspension cells" and in g as "somatic embryo maturation”.
Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript submitted for review is focused on the achievement of rapid breeding of superior larch cultivars by application of modern breeding technologies because of the long life history and complex genetic background of individual trees, the use of conventional breeding methods is difficult and propagation by seeds inevitably leads to segregation of desirable characteristics. The somatic embryogenesis is such method that may provides a novel procedure for rapid multiplication of commercial hybrid larch genotypes. However, somatic embryogenesis in conifers presents a number of challenges, such as difficulties with embryonal-suspensor mass induction, a high frequency of abnormal embryo development during the maturation stage, and difficulties with in vitro synchronization. Overcoming these problems requires knowledge of the molecular mechanisms of crucial genes that regulate the different developmental stages of somatic embryogenesis. Thus, an efficient transformation procedure is a prerequisite for functional studies of the aforementioned genes. Somatic embryogenesis is the basis of the genetic transformation system used for the majority of gymnosperms. In the present study, a simple and efficient genetic transformation system for Larix kaempferi was established using A. tumefaciens strain GV3101 in conjunction with the binary vector pCAMBIA1301. In result, a simple and efficient protocol for genetic transformation of Japanese larch (Larix kaempferi) was developed.
The manuscript is well written and presented. The research methods applied are appropriate, comprehensive and sufficient. The illustrative material (tables and figures) are representative and of good quality.
However, some comments need to be made:
Abstract:
The abstract exceeds the defined in the Instruction for authors 200 words as maximum and must to be shortened and revised.
Introduction:
The last paragraph (lines 86-96) is more appropriate for conclusion.
There is no defined the aim of the study. According to the Instruction for authors, the Introduction must finish with briefly mention the main aim of the work and highlight the main conclusions. Therefore, my suggestion for the cited paragraph is as follow: The present study was aimed to establish a simple and efficient genetic transformation system for Larix kaempferi using A. tumefaciens strain GV3101 in conjunction with the binary vector pCAMBIA1301. This novel transformation system may accelerate the development of genetically modified L. kaempferi and promote further molecular functional studies. Тhe rest of the paragraph to put in the conclusions, as follows:
In the present study, a simple and efficient protocol for the laboratory-scale genetic transformation of Larix kaempferi was established using A. tumefaciens strain GV3101 in conjunction with the binary vector pCAMBIA1301.
Several factors affecting Agrobacterium-mediated genetic transformation of Japanese larch were adjusted, including the infection method and duration, and the bacterial elimination process. The liquid medium-based suspension subculture of larch embryogenic cells can provide large numbers of high-quality materials with vigorous growth for genetic transformation within a short period. Altering the infection method and duration, as well as eliminating the pre-culture of the embryogenic suspension and bacterial mixture, resulted in a reasonably improved efficiency of Japanese larch transformation.
The latter vector harbors the hygromycin phosphotransferase gene as a selection marker and the GUS reporter gene driven by the CaMV 35S promoter. In addition, the endogenous pLaTCTP drove the constitutive expression of GUS in the ESM and somatic embryos at different developmental stages. A relatively stable transformation efficiency was achieved by simplifying the infection method and improving the bacterial removal procedure. The bacterial removal process was simplified by modifying the vacuum filter. The transcript abundances of the exogenous genes HYG and GUS driven by the CaMV 35S promoter ranged from 101to 104, which was equivalent to genes with moderate and low abundances, respectively, in Japanese larch. In addition, the GUS reporter gene, with and without introns, was normally expressed in larch. The transcript abundance of GUS driven by the pLaTCTP promoter in Japanese larch verified the aforementioned results, which might be related to the insertion site and GUS copy number in the genome, or to large genome of Japanese larch. A higher level of GUS expression was not detected, which may be due to the rela- tively small number of transgenic samples. Therefore, it is necessary to develop a new vector construction strategy or efficiently expressed promoter that can drive the ectopic expression of crucial genes to facilitate studies of gene function and quality modifications in Japanese larch.
Materials and Methods:
In the line 111 ‘embryonic’ to be corrected as ‘embryogenic’
Results
To specify the description of Figure 7 to make it more informative, namely: “Histochemical detection of the GUS staining in somatic embryos from transgenic cell lines pLaTCTP:GUS at different developmental stages detected in 7 days (7 days, 14 days, 21 days and 42 days).
In the captions of the figures, the letters with which the individual subfigures are designated (a, b, c, …) should be placed in brackets: (а), (b), (c), ….
The caption of Figure 5 must be modified as follows: Histochemical detection of the GUS staining in somatic embryos at different developmental stages from transgenic cell lines P2 and P5. (a) Embryogenic tissues, (b), (c) Early embryos cultured for 15 days: (b) mature somatic embryos; (c) embryos cultured for 42 days on differentiation medium supplemented with abscisic acid.
Author Response
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Author Response File: Author Response.docx
