Inducing mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation

Journal Pre-proof Inducing mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation Margarita Pérez-Jiménez, Carlos Ignacio Tallón, Olaya Pérez-Tornero PII:

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DOI:

https://doi.org/10.1016/j.apradiso.2019.109030

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ARI 109030

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Applied Radiation and Isotopes

Received Date: 29 April 2019 Revised Date:

26 August 2019

Accepted Date: 25 December 2019

Please cite this article as: Pérez-Jiménez, M., Tallón, C.I., Pérez-Tornero, O., Inducing mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation, Applied Radiation and Isotopes (2020), doi: https://doi.org/10.1016/j.apradiso.2019.109030. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Inducing Mutations in Citrus spp.: Sensitivity of different sources of plant material to gamma radiation Margarita Pérez-Jiménez, Carlos Ignacio Tallón, Olaya Pérez-Tornero* Equipo de Mejora Genética de Cítricos, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), 30150, Murcia, Spain *

Corresponding author: [email protected]; telephone: (+34) 968 36 67 57

Abstract Gamma radiation represents an alternative for improving genotypes for which breeding through hybridization involves many difficulties because of their complex reproductive biology, as in the case of citrus. In this study in vitro and ex vitro mutagenesis was induced by gamma radiation in several citrus species (‘Alemow’ and sour orange as citrus rootstocks, lemon cv. ‘Fino 49’ and ‘Verna 51’, mandarin cv. ‘Nova’ and lime cv. ‘Bearss’). Three different sources of materials - seeds, budwoods and nodal segments from in vitro explants, were tested. Seeds and budwoods were germinated or induced to sprout, and de novo regeneration was obtained from in vitro nodal segments from which preexisting buds were previously removed. Budwoods were tested in summer and winter in order to assess their capacity for mutation and further sprouted in different seasons. Seeds were seen to be more resistant to gamma radiation (LD50 of 127 Gy in ‘Alemow and 156 Gy in sour orange) than budwoods (LD50 about 50 Gy for all cultivars) and nodal segments (LD50 about 25 Gy for both lemon cultivars), the last being the most sensitive material tested. Similar LD50 were found for budwoods collected in winter and summer for all the genotypes. All the tested plant material can be considered suitable for gamma

1

irradiation, although budwood is the most widely available and tissue culture material needs the least degree of expertise. Keywords: breeding, lemon, lime, mandarin, physical mutagenesis, rootstocks.

2

1

1. Introduction

2

The genus Citrus comprises some of the most commercially important fruits in the world

3

such as mandarins, sweet oranges, lemons, grapefruits and limes (Hazarika et al., 2014). In

4

recent decades, economic competition in the citrus market has increased interest in growing

5

better and more competitive cultivars that are adapted to different crops areas and resistant

6

to biotic and abiotic stresses (Navarro et al., 2006).

7

However, citrus breeding programs using conventional methods involve several difficulties

8

associated with long juvenile periods, high heterozygosity, polygenic traits and complicated

9

genetic systems (Kayim and Koe, 2006), which have led breeders to try mutation and

10

genetic transformation techniques. Although transformation appears to be the solution to

11

the problems of citrus breeding and transgenic approaches have resulted in some potentially

12

interesting cultivars, none of them can be released onto the global market in the near future

13

(Ge et al., 2015) due to restrictions placed on GMO in the major markets. Thus, mutation

14

breeding is increasingly considered a powerful alternative for the generation of genetic

15

variations for plant breeding in citrus.

16

Mutation induction techniques, such as radiation or chemical mutagens are valuable tools

17

for increasing variability in crop species because spontaneous mutations occur with an

18

extremely low frequency (Sutarto et al., 2009), and it is possible to increase variability of

19

an economically important cultivar by altering only few genes while conserving overall

20

genetic background (Gulsen et al., 2007). Among different mutation methods, gamma

21

radiation is probably the most used in fruit trees because of their easy availability and

22

power of penetration (Moussa, 2006). The success of any mutation induction exercise is

23

closely dependent on the strategies adopted. The rate of induced mutations may be

1

determined by critical factors such as the type of mutagen, the dose administered and the

2

method of treatment including genotype, choice of materials, plant physical and biological

3

factors or pre- and post-treatment handling. These issues need to be carefully assessed and

4

consequently executed according to the project objectives and available resources.

5

Multiple types of material have been used in mutagenesis, from budwood or seeds collected

6

in the field, to protoplasts, calli or other plant/derived material obtained from tissue culture

7

(Suprasanna et al., 2011). The importance of the plant material lies in the sensitivity of its

8

cells to gamma irradiation (Surakshitha et al., 2017) and also the tendency of the plant

9

material to produce chimeras instead of complete mutated organisms. Thus, the more

10

developed the structure to be irradiated, the greater the possibility of obtaining chimeras

11

from our irradiation experiments. This is a key issue in perennial cultivars since long

12

juvenile periods may mean that years may pass before a plant is discarded, and chimeras

13

are an important loss of resources, time and money.

14

The first step in a breeding program through physical mutagenesis by radiation is to

15

evaluate the sensitivity of the plant material in order to determine the optimal dose

16

(Brunner, 1995) that will achieve optimal mutation frequency with minimal collateral

17

damage, as estimated from radio-sensitivity tests (van Harten, 1998). Mutagenic treatments

18

with high doses may destroy the promoters of growth being lethal retaining few plants for

19

selection, which, in turn, limits the success of artificial selection in the subsequent mutation

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generations to identify useful mutants (Surakshitha et al., 2017). Likewise, low doses of

21

radiation could produce chimeric plants preventing from solid mutant obtaining. Thus, to

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perform a preliminary study on the specific sensitivity of each type of material, it is

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essential to select the correct dose that will induce a higher mutation rate in a target trait 4

1

with minimal effects on the remaining genetic background. In this respect, LD50 (LD50 =

2

lethal dose 50%) is described as the dose that results in a 50% reduction in germinating

3

seeds, buds sprout, growth or viable plants. This dose is obtained from a sensitivity curve

4

that varies with the species and the genotype, with the physiological condition of plants and

5

organs, and with the manipulation of the irradiated material before and after mutagenic

6

treatment (Suprasanna et al., 2011). LD50 guarantees an increased mutation rate with

7

adequate survival in the mutation events (Saamin and Thompson, 1998).

8

Gamma radiation has been used in citrus to produce seedless cultivars or modify fruit

9

maturation dates (Gulsen et al., 2007, Spiegel-Roy et al., 2007, Uzun et al., 2008).

10

However, despite the success achieved in different species with gamma irradiation after

11

calculating the LD50 (Surakshitha et al., 2017, Nikam et al., 2014, Hossain et al., 2005),

12

only a few studies in citrus have reported on sensitivity curves and LD50 calculations

13

(Gulsen et al., 2007, Gonzaga et al., 2011).

14

With the above in mind, the authors propose a study in which plant material of different

15

citrus species are submitted to mutagenesis, studying three different types of material -

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budwoods, seeds and nodal segments - from in vitro explants. Seeds and budwoods were

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germinated or induced to sprout, while de novo regeneration was induced in nodal

18

segments, from which pre-existing buds were completely removed. The objective of this

19

study is the optimization of the dose for mutation induction by gamma irradiation through

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the study of the effect of irradiation (radio-sensitivity) on different types of material from

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several citrus species, which will be useful for further developments on mutagenesis

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experiments.

5

1

2. Material and Methods

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2.1. Plant Material

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2.1.1. Ex vitro Experiments

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Hard wood cuttings, of uniform size and with 8-10 buds per cutting (Fig. 1a), from the

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lemon cultivars ‘Fino 49’ (F49) and ‘Verna 51’ (V51), the mandarin cultivar ‘Nova’ (NO)

6

(C. clementina Hort x (C. paradisi Macf. x C. tangerina Hort)) and the lime cultivar

7

‘Bearss’ (BE) (C. aurantifolia), were obtained from adult trees growing on different citrus

8

farms in Murcia, Spain, in February (winter; W) and June (summer; S). Plant material was

9

collected at the first sign to sprouting, when budwoods were starting to swell, to avoid the

10

formation of chimaera.

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2.1.2. In vitro Experiments

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Mature fruits from ‘Alemow’ (AL) (C. macrophylla) and sour orange (SO) (C. aurantium)

13

rootstocks were collected. Seeds were removed from the endocarp, surface sterilized in a

14

solution of 20% (v/v) sodium hypochlorite and 0.1% (v/v) Tween20 for 10 minutes, and

15

then rinsed three times with sterile distilled water under a laminar flow hood. After

16

sterilization, the seed coat was removed and seeds were maintained in sterile conditions

17

until radiation treatment. The level of moisture of the seeds was 50%.

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Lemon nodal segments were derived from in vitro adult explant cultures of Citrus limon,

19

cultivars F49 and V51, developed previously in our laboratory (Pérez-Tornero et al., 2010).

20

Explant preparation was carried out from elongated, healthy green shoots, except basal

21

nodal segments, using standard procedures described formerly Navarro-García et al. 6

1

(2016). In a simplified way, leaves and buds of the explants were completely eliminated by

2

running a sharp scalpel parallel to the stem. Nodal explants were cut transversally into thin

3

3–5 mm segments and maintained in sterile conditions until receiving the radiation

4

treatment.

5

2.2. Physical Mutagenesis – Gamma Rays

6

Plant material was subjected to gamma irradiation using an IBL 437C cell irradiator at the

7

Centro de Hemodonación (Blood Donation Centre) in Murcia (Spain). Gamma irradiation

8

was derived from a 137Cs source. The dose rate of 0.125 kGy h−1 was determined with a

9

Fricke solution dosimeter. Seeds were irradiated on a cylinder rotating 360° in a cylindrical

10

radiation field.

11

2.2.1. Ex vitro

12

Plant material from F49, V51, NO and LB cultivars was irradiated with 0, 25, 50, 75 and

13

100 Gy. After exposure, treated buds were grafted onto one-year-old citrus rootstocks, one

14

bud per rootstock, and were kept in a semi-controlled greenhouse located at IMIDA, La

15

Alberca, Murcia, Spain.

16

2.2.2. In vitro

17

Seeds of AL were irradiated with 0, 50, 100 and 150 Gy and those of SO with 0, 50, 100,

18

150, 200 and 250 Gy. Irradiated and control seeds were cultured individually in

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150×20 mm test tubes containing 15 ml germination medium composed of salts and

20

vitamins of Murashige and Skoog medium (Murashige and Skoog, 1962), supplemented

7

1

with 30 g/l sucrose, and 6 g/l agar, pH 5.7. Seeds from both rootstocks were incubated at

2

25±1 °C in darkness for 21 days before exposure to light with a 16 h photoperiod.

3

In the case of the nodal segments, from which leaves and pre-existing buds of the explants

4

were completely eliminated, the irradiation doses applied were 0, 10, 20, 30, 40 and 50 Gy.

5

The irradiated and control nodal segments were cultured in plastic Petri dishes (9×1.5 cm)

6

and placed horizontally in contact with the media, with nodal section upwards, using the

7

regeneration medium described by Navarro-García et al. (2016). Nodal explants were

8

transferred to fresh medium every 4 weeks.

9

2.3. Experimental design and statistical analysis

10

The ex vitro experiment was developed with 100 buds per treatment for each of the

11

cultivars used in the study (F49, V51, NO and LB) in 2 seasons (W and S) of two different

12

years (2015 and 2016). Data were collected in autumn (October - November) in the case of

13

the buds irradiated in February (W experiment) and in spring (March - May) of the

14

following year for those irradiated in June (S experiment).

15

In the experiments using AL and SO seeds, each treatment comprised 40 seeds, and the

16

germination percentages were recorded 28 days after radiation (Fig. 1b).

17

In the in vitro experiments using F49 and V51 nodal segments, 5 replicates (Petri dishes)

18

per treatment were prepared, each containing at least 10-12 nodal segments. After 8 weeks

19

of incubation, the number of nodal explants forming adventitious buds and the regeneration

20

rate (number of buds formed/total number of explants) were recorded. The assessment of

21

nodal explants was carried out with the aid of a stereomicroscope. To prevent chimera 8

1

formation, nodal segments with adventitious buds regenerating within the first two weeks

2

were eliminated, and were considered as pre-existing meristems.

3

All the data obtained were analyzed with the statistical software SPSS (v.21, IBM®). Data

4

were tested first for homogeneity of variance and normality of distribution. The effect of

5

the irradiation treatments on the regeneration, germination and bud sprout percentage was

6

analyzed by means of a maximum likelihood ANOVA. When a significant χ2 was obtained,

7

specific maximum likelihood contrasts were designed to examine differences between

8

treatments. ANOVA was used to analyze the effect of the treatments on the regeneration

9

rate (number of buds formed/total number of explants) and to identify differences between

10

various treatments the Least Significant Difference test (LSD) was used. In all cases (nodal

11

segments, seeds or buds), LD50 was calculated as the dose of γ radiation that reduced the

12

percentage of regeneration (for nodal segments), germination (for seeds) or sprouted buds

13

(for budwood) of irradiated explants to 50% of unirradiated control explant levels, based on

14

linear regression as described by Wu et al. (1978).

15

3. Results and Discussion

16

Mutation techniques have significantly contributed to plant improvements worldwide, and

17

have had an outstanding impact on the productivity and economic value of some crops

18

(Ahloowalia et al., 2004). Nonetheless, sensitivity to these methods varies with genetic

19

origin, with the physiological state of the plant and organs and with manipulation of the

20

irradiated material before and after the mutagenic treatment (D’Amato, 1992, Tullmann-

21

Neto et al., 1994, Gonzaga et al., 2011). Thus, the estimation of the LD50, as described by

9

1

Meyer (1996), is necessary as a way to reach a balance between mutation induction and

2

tissue survival.

3

3.1. Determination of lethal dose of gamma radiation in ex vitro experiments

4

Budwood is the most reported plant material in terms of breeding mutation in citrus

5

(Spiegel-Roy et al., 2007, Gulsen et al., 2007, Uzun et al., 2008) and in other woody plant

6

species (Elhiti et al., 2016, Surakshitha et al., 2017). However, differences between the

7

seasons they were collected have never been studied.

8

In this study, increases in the dose of gamma radiation caused a very significant downward

9

trend (P<0.001, for all cultivars) in the sprouting percentage of hardwood cuttings in all

10

cultivars (Fig. 2). This reduction in sprouting was particularly evident following the

11

application of 50 Gy in all the genotypes: 67% decrease in the budbreaking percentage in

12

W and 63% in S in NO budwoods, the most affected cultivar, and 22% in W in F49, the

13

least affected (compared with the non-radiated control) (Fig. 2). The application of 75 Gy

14

caused the biggest drop in the bud sprout, which was strongly reduced in BE and

15

completely inhibited in all other cultivars (Fig. 2). No bud sprouting was observed when

16

100 Gy were applied.

17

The LD50 was very similar among all cultivars and between the two seasons (Fig. 2), which

18

wides the number of mutagenesis attempts per year in these species in those climates where

19

more than one sprouting occur per year. This indicates that season, and therefore weather,

20

do not change the sensibility of the buds to gamma radiation. In previous reports, Gulsen et

21

al. (2007) and Uzun et al. (2008) established an LD50 of above 50 Gy for budwood of citrus

22

using 60Co as a source. Nevertheless, in the present study, similar results were only 10

1

observed in W budwoods of F49, while the LD50 obtained for the other cultivars, for both

2

W and S budwoods, was equal to below 50 Gy. This agrees with the observations of

3

Tulmann-Neto et al. (1994) in budwoods of sweet orange, and also similar to the results

4

reported for other woody plant species (Sanada and Amano, 1998, Surakshitha et al., 2017)

5

all of whom used 60Co as source as well. Although radio-sensitivity depends on the source

6

of irradiation, the dose rate and the plant material (FAO/I IAEA, 2018), in this case the

7

explant seems to have a high weight on sensibility, since different woody plant species,

8

sources and doses showed results that were similar between them and to those obtained in

9

this study. This could be related to a similar composition in their structural tissue, which

10

could confer a comparable degree of sensitivity to gamma rays due to wood formation.

11

3.2. Determination of lethal dose of gamma radiation in in vitro experiments

12

Mutagenesis in seeds induce variations in descendants through the changes that occur in

13

after exposing plant seeds to radiation (Beyaz et al., 2016). Due their ease of manageability

14

and high storage capability, seeds are considered a good alternative to budwood for

15

mutagenesis experiments both in vitro and ex vitro. In our experiment, in vitro procedures

16

were chosen to ensure a high germination rate.

17

In the in vitro experiment, AL and SO seeds were treated with different dose of gamma

18

radiation to determine the LD50 for each genotype. As a consequence of the radiation, a

19

very significant reduction in germination (P<0.0001 for both genotypes) was evident (Fig.

20

3), with a negative relationship between this parameter and increasing doses of gamma

21

rays. Indeed, seed germination fell by 50% at 129 Gy in AL seeds and at 156 Gy in SO

22

seeds. The germination percentage was higher than 80% for both rootstocks in the control 11

1

treatment (0 Gy) and decreased to 35% in AL or 15% in SO, when doses of 150 Gy or

2

250 Gy, respectively, were applied (Fig. 3).

3

According to the results, SO seeds presented less sensitivity to gamma radiation than AL.

4

Nevertheless, both results are coincident with previous reports in citrus. Hearn (1984),

5

using germination to calculate LD50, obtained a LD50 of between 100 and 150 Gy in sweet

6

orange and grapefruit seeds, results that closely reflect ours. Similar results have also been

7

reported in seeds of other citrus species (Spiegel-Roy 1990). Notwithstanding, Ling et al.

8

(2008) gamma-irradiated in vitro sweet orange seeds using 137Cs as a source, and reported

9

that, based on the height increment, the LD50 of the plantlets was achieved at 27 Gy, which

10

is 5-6 times lower than that obtained in AL and SO seeds in this study. On the other hand,

11

results also varied in studies of seeds from other genera, the LD50 ranges widely - between

12

25 Gy in ornamental foliage plants (Huang et al., 2017) and 1500 Gy in seeds of African

13

grass (Álvarez-Holguín et al., 2018). As can be observed, seeds of related species have

14

similar sensitivities to gamma radiation provided the chosen method to measure LD50 is the

15

same. This is similar to what had been seen in budwoods above.

16

The use of tissue cultured material as a target tissue in mutagenesis is infrequent, even

17

though tissue culture allows complete availability of explants, the capacity to handle large

18

populations and, therefore, increased mutation induction efficiency, the possibility of

19

mutant recovery and the speedy cloning of selected variants (Predieri and Gatti, 2000).

20

While the number of mutagenesis studies in woody plants developed in seeds is low, the

21

reports about mutagenesis using in vitro culture explants are practically non-existent.

22

However, in vitro cultured explants have been described as the optimal plant material for

23

successful gamma ray irradiation mutagenesis (Li et al., 2010). 12

1

Our results using in vitro nodal segments showed that radiation produced a very significant

2

decrease in the regeneration percentage (P < 0.001 in both cultivars). The LD50 for the

3

nodal segments of lemon cultivars was 26 Gy for F49 and 25 Gy for V51 (Fig. 3). The

4

highest response was obtained in non-irradiated control explants (58% and 71%

5

regenerating explants for F49 and V51, respectively), falling gradually as the dose

6

increased; the lowest regeneration percentage (<10%) was obtained in the explants

7

irradiated with a dose of 50 Gy (Fig. 3). The in vitro regeneration rate (buds/explant) was

8

also significantly affected (P<0.0001 for both cultivars) by the radiation dose in a dose-

9

responsive way (Fig. 4). The regeneration rate obtained in F49 nodal explants after

10

eliminating the adventitious buds that arose within the first two weeks was 1.29 for control

11

explants (Fig. 1c) and 0.95 for those nodal segments irradiated with 10 Gy, which meant a

12

reduction of 26%. A huge decrease in the regeneration rate was observed as explants were

13

irradiated from 20 to 50 Gy, ranging from 0.38 to 0.036 adventitious buds per nodal

14

segment, a reduction of 71 to 97%. In V51 no significant differences for the regeneration

15

rate were observed between non-irradiated explants (1.91) and those irradiated with 10 or

16

20 Gy (0.94 and 1.05) (Fig. 4). However, significant differences were found between this

17

group and segments irradiated with doses of 30-50 Gy (0.37-0.15), with a sudden reduction

18

in the regeneration rate of nearly 70%. These results point to the lower sensitivity to gamma

19

radiation of V51 than F49 nodal segments. Many explants died when 50 Gy were applied to

20

nodal explants of both cultivars (Fig. 1d). Tallón et al. (2015) studied the sensitivity of AL

21

nodal segments to gamma radiation using the same methodology, obtaining 29.2 Gy as

22

LD50 and these results agree with ours in the present study for both lemon cv. F49 and V51.

23

Nevertheless, Gonzaga et al. (2011) obtained a slightly higher LD50 of between 22 and

24

35 Gy when in vitro segments of epicotyl of different citrus species were irradiated with 13

1

gamma rays. By contrast, much higher doses were observed in experiments involving

2

single node cuttings of St. Augustine grass, where the LD50 was fixed at 48.5 Gy (Li et al.,

3

2010). Although the authors used a different radiation source and dose, the differences

4

between these three LD50 might be mainly attributable to the different calculation of this

5

indicator in all the experiments. While Tallón et al. (2015) used the regeneration percentage

6

to calculate LD50, the same method as in this paper, Gonzaga et al. (2011) based their

7

results on bud sprouting and Li et al. (2010) used the percentage of surviving explants. As

8

can be seen, there is a lack of uniformity in the calculation of LD50, which together with the

9

scarce number of studies makes difficult to compare results in gamma radiation

10

experiments in vitro.

11

As observed above, seeds can endure higher doses of gamma rays than other tissues (Noor

12

et al., 2009). The LD50 in seeds were three times higher than in budwoods and 4-5 times

13

higher than in nodal explants cultured in vitro. This is probably due to the low moisture

14

content of seeds. One of the most important targets of gamma rays is the water molecule,

15

and ionized water molecule and OH and H radicals result from the primary reactions of

16

excitation and ionization (Marcu et al., 2013). This leads to secondary free radicals that can

17

damage and/or modify certain components of the plant cells (Esnault et al., 2010) and the

18

morphology, anatomy, biochemistry and physiology of plants, the extent of which depends

19

on the radiation dose (Ashraf et al., 2003). This may have been the case in our work, where

20

no damaged seeds were observed after irradiation, while some nodal segments were

21

damaged (Fig. 1d).

22

4. Conclusions

14

1

According to our observations, all the explants studied in these experiments can be

2

considered a valid option for gamma irradiation experiments, although tissue culture

3

explants have the advantage of being more convenient in terms of manageability and space.

4

Despite this, the greater degree of expertise needed means that ex vitro explants have

5

historically been preferred.

6

Although different genotypes have been used in these experiments, the biggest differences

7

that have been found among tissues are probably due to their water content. Thus,

8

budwoods collected in W and in S did not show big differences neither among species nor

9

between seasons. The biggest differences were found in F49, and they were minimal

10

(57 Gy in W, 49 Gy in S). No great changes were evident in V51 (43 Gy in W and 48 Gy in

11

S), Nova (44 Gy in W, 49 Gy in S) and Bearss (44 Gy in W and 50 Gy in S). On the other

12

hand, the biggest differences between tissues were found in the in vitro experiments. The

13

LD50 obtained in seeds was of 156 Gy for SO, while the one obtained for AL was of

14

129 Gy. In the nodal segments, the values were much lower: 26 Gy in F49 and 25 Gy in

15

V51.

16

Bearing all the above in mind, it is clear that the different degrees of sensitivity of explants

17

to gamma radiation must be assessed by accurate experiments in order to determine the

18

most suitable dose to obtain the best result from each irradiation event. Irradiation variables

19

must be adjusted to the chosen method, explant and genotype, since small variations can

20

make all the difference between success and failure.

21

Acknowledgements

22

The authors thank Mr. Fernando Córdoba and Mr. Antonio J. López-Pérez for technical

23

assistance in the laboratory. This work was supported by the Ministerio de Ciencia, 15

1

Innovación y Universidades through the project RTC-2016-5758-2 and the European

2

Regional Development Fund.

3

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A

B

C

D

Fig. 1.

FINO 49-W

FINO 49-S 100 BUDBREAKING (%)

BUDBREAKING (%)

100 75 50 25 0 -25

LD50= 57 Gy

0

25

50 25 LD50= 49 Gy

0

y = 109.04 - 1.055x R² adj. = 0.880

-25

75

-25 50 DOSE (Gy)

75

100

y = 106.6 - 1.168x R² adj. = 0.870

-25

0

25

50 DOSE (Gy)

75

VERNA 51-W

VERNA 51-S 100 BUDBREAKING (%)

BUDBREAKING (%)

100 75 50 25 LD50= 43 Gy

0 -25

0

75 50 25 LD50= 48 Gy

0

y = 91.262 - 0.96x R² adj. = 0.930

-25

25

-25 50 DOSE (Gy)

75

100

y = 101.4 - 1.12x R² adj. = 0.900

-25

0

25

50 DOSE (Gy)

75

NOVA-S 100 BUDBREAKING (%)

BUDBREAKING (%)

75 50 25 LD50= 49 Gy

0

0

75 50 25 LD50= 44 Gy

0

y = 104 - 1.124x R² adj. = 0.870

-25

-25 25

50 DOSE (Gy)

75

100

y = 91.302 - 1.0353x R² adj. = 0.900

-25

0

25

50 DOSE (Gy)

75

BEARSS-W BUDBREAKING (%)

BUDBREAKING (%)

100

75 50 25

-25

100

BEARSS-S

100

0

100

NOVA-W

100

-25

100

LD50= 44 Gy

0

50 25 0

y = 82 - 0.736x R² adj. = 0.997

-25

75

-25 25

50 DOSE (Gy)

75

100

LD50= 50 Gy y = 73.366 - 0.7462x R² adj. = 0.986

-25

0

25

50 DOSE (Gy)

75

100

Fig. 2.

GERMINATION (%)

GERMINATION (%)

80 60 40 20 0

0

50 DOSE (Gy)

100

FINO 49

100

60 40

0

10

LD50= 156 Gy y = 87.47 - 0.289x R² adj.= 0.985

0

40

50

100 150 DOSE (Gy)

200

250

80 60 40

0 20 30 DOSE (Gy)

50

VERNA 51

20

LD50=26 Gy y = 43.958 - 0.798x R² adj. = 0.908

-10

40

0 -50

80

0

60

150

REGENERATION (%)

REGENERATION (%)

100

20

80

20

LD50= 129 Gy y = 84.86 - 0.336x R² adj. = 0.952

-50

SOUR ORANGE

100

ALEMOW

100

60

LD50= 25 Gy y = 66.435 - 1.239x R² adj. = 0.874

-10

0

10

20 30 DOSE (Gy)

40

50

60

Fig. 3. FINO 49

1.8

1.6

a

1.4 1.2

VERNA 51

1.8

REGENERATION RATE

REGENERATION RATE

1.6

b

1.0 0.8 0.6

c

c

0.4

cd

0.2

d

0.0

1.4

a

1.2

a

a

1.0 0.8 0.6

b

0.4

b

b

40

50

0.2 0.0

0

10

20 30 DOSE (Gy)

40

50

0

10

20 30 DOSE (Gy)

Fig. 4.

Fig. 1. Hard wood cuttings from mandarin cultivar ‘Nova’ (A); germinated seed of ‘Alemow’ (B); nodal segment of ‘Fino 49’ with adventitious regeneration (C); nodal segment of ‘Verna 51’ necrotised by the radiation effect (D).

Fig. 2. Average budbreaking percentages of budwoods of ‘Fino 49’, ‘Verna 51’ lemon cultivars, mandarin cultivar ‘Nova’ and lime cultivar ‘Bearss’, subjected to different gamma radiation doses both in Winter (W) buds and Summer (S) buds. Linear trend from the regression analysis, equation for the linear regression, adjusted R2 and LD50 obtained are shown. Fig. 3. Average germination percentages of seeds of ‘Alemow’ and ‘Sour Orange’ and regeneration percentage of nodal segments of ‘Fino 49’ and ‘Verna 51’, subjected to different gamma radiation doses. Linear trend for the regression analysis, equation for the linear regression, adjusted R2 and LD50 obtained. Fig. 4. Regeneration rates (number of buds formed/total number of explants) of nodal segments of ‘Fino 49’ and ‘Verna 51’ subjected to different gamma radiation doses. Data are means ± standard error. Different letters within each cultivar indicate significant differences (P<0.001).

Highlights •

The different degrees of sensitivity of explants to gamma radiation must be assessed by accurate experiments to set LD50.

•

Irradiation variables must be adjusted to the chosen method, explant and genotype.

•

Tissue culture explants are more convenient in terms of manageability and space for gamma irradiation experiments.

•

The explant that needs less degree of expertise is budwoods and its sensitivity to gamma radiation is not dependent of the season.