Ex Vitro Rooting and Simultaneous Micrografting of the Walnut Hybrid Rootstock ‘Paradox’ (Juglans hindsi × Juglans regia) cl. ‘Vlach’
, João Cruz 1, Hélia Cardoso 2, João Mota Barroso 3 and Augusto Peixe 3,*Round 1
Reviewer 1 Report
The research was generally carried out in accordance with the material method.
Could Protko be explained in a little more detail?
The practical use of the research results was seen as weak.
another rootstock should have been used as a witness. In this aspect, the research was found to be incomplete. What is the explanation of the authors about this?
What is the difference from the research of vahdati et al. (2004,20079)? What are the advantages/differences? not understood.
Author Response
We would like to thank the reviewer for the detailed appreciation of the manuscript and the comment made, which we will try to answer, and will for sure help to improve it.
Comment: Could Protko be explained in a little more detail?
Answer: We believe that by PROTKO the reviewer means PROTOCOL. We have done our best to describe all the steps of the process, including the type of explant used, the containers, the culture media, and substrates for each step, as well as the environmental conditions. A schema of all the process steps with the duration of each one was presented. So, it is difficult for us to understand how it can be described in more detail. Nevertheless, after a careful read of the manuscript materials and methods, we decided to improve it by adding a few more images that can help to better understand the text.
Comment: The practical use of the research results was seen as weak, another rootstock should have been used as a witness. In this aspect, the research was found to be incomplete. What is the explanation of the authors about this?
Answer: Walnut grafting is being done in nurseries, mainly over seedling rootstocks, with variable success rates every year, due to its dependency on the climate conditions, but usually with an average success rate of about 60 to 65%. Here, we present a technique for walnut grafting using clonal materials for rootstocks and scions, with success rates for grafting and acclimatization higher than 80% on average, using a procedure that allows plant production all around the year. So, we believe that this is important for walnut propagation from a practical point of view.
Concerning the use of another rootstock as a witness. Is not possible to have a witness in a protocol that is being tested for the first time. The only way to prove that a new protocol works in a given condition is by repeating it and we have made this 5 times throughout the year, achieving similar results in all the replicates. To have another rootstock, not as a witness, but to prove that the protocol can be used with different rootstock, would have been good, but, unfortunately, from the Paradox rootstocks available, ‘Vlach’ is the only one not patented. Others, like RX1 or VX211, are subjected to UCDavis authorization to be multiplied, and we don’t have this authorization.
Comment: What is the difference from the research of Vahdati et al. (2004,20079)? What are the advantages/differences? not understood.
Answer: By Vahdati et al. (2004,20079) we believe that the reviewer means Vahdati et al. (2004,2009). The main purpose of our manuscript is to present a micrografting procedure for walnut, using in vitro growing explants both, for rootstock and scion. On the contrary, the work of Vahdati (2004), dials with the micropropagation (rooting and acclimatization) of 3 Juglans regia cultivars, and the work of Vahdati (2009), presents an improved culture media formulation based on evaluation of the mineral content of walnut seeds. By saying that, we believe that is not possible to compare this work and the Vahdati (2004, 2009) publications, because their main goals are totally different.
Nevertheless, apart from presenting a micrografting protocol, this manuscript also proved that the rooting and acclimatization protocol proposed by the corresponding author in 2015 for the ‘Vlach’ rootstock (Peixe et al, 2015), is efficient. On this subject, our trials for rooting and acclimatization can be compared with those proposed by Vahdati (2004, 2009), even if one deals with rooting of a rootstock and the others deal with the rooting of walnut cultivars. The differences are evident. Following a procedure proposed by Jay Alemand , Vahdati (2004, 2009) rooted the in vitro explants in aseptic conditions in DKW culture medium added with vermiculite and transferred the rooted plants with bare roots for acclimatization. Here, rooting is made in non-aseptic conditions using an organic substrate and transferred with protected roots into a new organic substrate for acclimatization. The main advantages are the work on non-aseptic conditions immediately after root induction, and the easiest plant acclimatization process due to the manipulation of the explants with protected roots.
Reviewer 2 Report
The submitted manuscript titled „Ex vitro rooting and simultaneous micrografting of the walnut hybrid rootstock ‘Paradox’ (cl. ‘Vlach’)” is an interesting paper about a new approach of the Persian walnut propagation. There is a keen interest in grafted walnut trees worldwide, the growers are always searching for them. The successful propagation rate is approx. 50 to 60 % in the best nurseries, so it is important to increase this number. The grafting among sterile conditions (avoiding usage of wet saw dust) can ensure the best successful rate.
There are detailed information about the steps and the conditions how to make the whole process. I have just some questions to improve the quality of this excellent paper. Were the stem cuttings collected from virus-free trees? When did those cuttings collected?
The grafted walnut trees can be sold, when they reach at least 1.5 m based on the standards. I took another year after hand grafting to have this tree size. Was there any difference in growing up period between micrografting and reaching sold able tree size? Did the micrografted walnut trees grow faster than the normal hand grafted trees?
Author Response
Please see the answers in blue text
The submitted manuscript titled „Ex vitro rooting and simultaneous micrografting of the walnut hybrid rootstock ‘Paradox’ (cl. ‘Vlach’)” is an interesting paper about a new approach of the Persian walnut propagation. There is a keen interest in grafted walnut trees worldwide, the growers are always searching for them. The successful propagation rate is approx. 50 to 60 % in the best nurseries, so it is important to increase this number. The grafting among sterile conditions (avoiding usage of wet saw dust) can ensure the best successful rate.
Answer: The authors thanks to the reviewer for these comments. To increase walnut successful grafting rates and the use of clonal scions and rootstocks was the main purpose of this work.
There are detailed information about the steps and the conditions how to make the whole process. I have just some questions to improve the quality of this excellent paper. Were the stem cuttings collected from virus-free trees? When did those cuttings collected?
For this work, the initial explants were already cultured in vitro.
Nevertheless, the original explants were collected for orchard plants with 5 years old, in winter, and forded to sprout in plant growth chambers, being the new epicormic shoots used to start the in vitro culture process. Concerning virus, both, PCR and ELISA texts for CLRV are being performed in routine to the in vitro plant material and all the results were negative.
The grafted walnut trees can be sold, when they reach at least 1.5 m based on the standards. I took another year after hand grafting to have this tree size. Was there any difference in growing up period between micrografting and reaching sold able tree size? Did the micrografted walnut trees grow faster than the normal hand grafted trees?
To achieve 1,5 m grafted trees is not the goal. The new mechanization orchard planting techniques are changing this paradigm. Actually in the USA the growers prefer small plants, with 30-50 cm, supplied in plant trays with protected roots to start a new orchard. This is the type of plant that the presented protocol aims to produce. We did not grow the plants until 1,5 m long, but we don't believe that in vitro grafted plants will grow bigger and faster than the nursery plants, because they are growing in a limited space. Nevertheless, as previously stated this is not the point.
Reviewer 3 Report
The study describes a new approach to obtain high quality clonal walnut grafted plants independently of the external weather conditions in a significantly shorter time. The design is appropriate and the method was adequately described. Moreover, the results of the manuscript are adequately supported by the results
Author Response
Comment: The study describes a new approach to obtain high quality clonal walnut grafted plants independently of the external weather conditions in a significantly shorter time. The design is appropriate and the method was adequately described. Moreover, the results of the manuscript are adequately supported by the results.
Answer: The authors are extremely pleased with this comment of the reviewer and will continue to work in order to make walnut clonal propagation materials the standard for new walnut orchards. Grafting over seedlings is a non-sense in a culture where production is becoming more and more based on mechanization.
Reviewer 4 Report
Comments
The paper is generally well written and structured and the subject is very interesting for walnut industry. However, in my opinion the paper has some shortcomings in some parts. The introduction and discussion and be enriched using more relevant literature.
The methodology and associated data analyses seemed appropriate.
My main concern is the originality and novelty of this work. In line71 you mentioned “two references could be found in the literature describing micrografting applied to Juglans” What is the advantage of your work over them?
In line 104, you cited,” scions and rootstocks were obtained from explants growing under similar in vitro conditions”, was the physical condition the same? do you use the same medium culture? why? please explain this.
Another question is about the medium culture, it was prepared with 1.5x micronutrients. Why you use extra micronutrients?
Photos: The authors should add some photos of plants during multiplication, induction, root expression, acclimatization and hardening stages. Also a photo showing different plugs and containers is needed.
References: There are some mistakes in writing and formatting the references. For example the following reference should be written as follow:
Vahdati, K., Aliniaeifard, S., 2017. Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants. Sci Hort. 224, 1-7. https://0-doi-org.brum.beds.ac.uk/10.1016/j.scienta.2017.04.023.
Correct form:
Asayesh ZM, Vahdati K, Aliniaeifard. Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants. Scientia Horticulturae. 224: 1-7. https://0-doi-org.brum.beds.ac.uk/10.1016/j.scienta.2017.04.023.
Author Response
The authors would like to thank the reviewer for the detailed analysis of the manuscript and the comments made, that will contribute to its improvement.
Comment: The paper is generally well written and structured and the subject is very interesting for the walnut industry. However, in my opinion, the paper has some shortcomings in some parts. The introduction and discussion and be enriched using more relevant literature.
Answer: We have made an exhaustive search for the latest published advances on the technique presented, not only on walnut, but also on other fruit trees, and we could not find other relevant literature. Concerning the introduction and discussion chapters, each researcher has his own way to introduce and discussing a subject. No other reviewer of the manuscript commented on this need. So, unless the reviewer wishes to specify how to enrich those chapters, we don’t understand the comment.
Comment: The methodology and associated data analyses seemed appropriate.
Answer: We appreciate this opinion, nevertheless, we tried to improve the methodology description by adding a few more images to the manuscripts as suggested not only by other reviewers but also by this reviewer in a comment presented later on the text.
Comment: My main concern is the originality and novelty of this work. In line71 you mentioned “two references could be found in the literature describing micrografting applied to Juglans” What is the advantage of your work over them?
Answer: The two references are Wang et al. (2010) and Liu et al. (2017). Wang et al. (2010), used seedling rootstocks and grafted them ex-vitro using in vitro produced explants as scions. In fact, this is more related to epicotyl grafting using in vitro microshoots as scions, than with a real micrografting technique. Besides, unlike our work, the rootstock arises from seeds with all the known problems of genetic variability associated. On the other reference, Liu et al. (2017), performed an autografting (‘Paradox’ over ‘Paradox’), using in vitro explants resulting from the evolution of somatic embryos and the only purpose was to evaluate the transference of m-RNA between scion and rootstock. Walnut propagation was never the goal. So, none of these works contributed to improving clonal propagation of walnut, unlike the work here presented. We used in vitro shoots resulting from initial explants taken from field-grown plants and a micrografting technique of Juglans regia over Paradox ‘Vlach’, the most important combination of scion and rootstock used worldwide by now, is presented. The results of graft consolidation and acclimatized plants are superior to the traditional nursery grafting techniques and the technique can be performed along all the year. So, we believe that this work is new, is different from other approaches tested until now for the species, and represents a valuable contribution for clonal propagation of the walnut, that clams for the use of other than seedling rootstocks.
In line 104, you cited,” scions and rootstocks were obtained from explants growing under similar in vitro conditions”, was the physical condition the same? do you use the same medium culture? why? please explain this.
Answer: The culture media were the same in fact, but the physical conditions during the multiplication phase for scions and rootstocks were a little different. The temperature on the plant growth chamber was higher for Chandler (28/24 ºC day/nigth) and lower for Vlach (24/22 ºC day/nigth). This has been corrected in the manuscript.
Another question is about the medium culture, it was prepared with 1.5x micronutrients. Why you use extra micronutrients?
Answer: Previous trials carried on in our laboratory, and based on results from leaf nutritional analysis, proved that this was the best macronutrient concentration. Also the research from Vahdati (2009) points in the same direction.
Comment: Photos: The authors should add some photos of plants during multiplication, induction, root expression, acclimatization and hardening stages. Also a photo showing different plugs and containers is needed.
Answer: Previously answered. New images were included in the manuscript.
Comment: References: There are some mistakes in writing and formatting the references. For example the following reference should be written as follow:
Answer: The indicated reference has been corrected:
Asayesh ZM, Vahdati K, Aliniaeifard. Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants. Scientia Horticulturae. 224: 1-7. https://0-doi-org.brum.beds.ac.uk/10.1016/j.scienta.2017.04.023.
Any other problems with bibliography uniformization, as indicated on the journal instructions for the authors, will be made after the manuscript acceptance.
