Hydrogel polymer in emergency and early growth of citrus rootstocks

The development of techniques for rapid and uniform emergence of seeds is the alternative to the diversification of citrus rootstocks that were little used due to low vigor compared to ‘Rangpur’ lime, the main rootstock used in Brazil, but it has shown susceptible to serious disease problems, such as the citrus sudden death (CSD). The addition of the hydrogel polymer to the substrate can increase the uniformity of seed emergence and reduce the time for the production of rootstocks due to the increase in water availability. Thus, the study was done to evaluate the formulation of the substrate with the hydrogel polymer on the uniformity of emergence and early growth of citrus rootstocks. The factorial scheme 3 × 2 × 2 was used, with the factors: Three rootstocks [‘Rangpur’ lime (Citrus limonia Osbeck), Poncirus trifoliata (L.) and ‘Sunki’ mandarin (Citrus sunki Hort. ex Tanaka); two formulations of substrate (with and without 0.4 g per container of Hidroplan-EB/HyB-M polymer and two environments (greenhouse with an average maximum temperature of 36.6°C and minimum of 17.2°C and growth chamber programmed with temperature to 25°C and a photoperiod of 12 h, distributed in a completely randomized design with four replications and 35 seeds per plot. The emergency velocity index (EVI), the percentage of emergence and early growth of rootstocks were evaluated. Data were subjected to analysis of variance and the means were compared by Tukey test at 5% of probability. The addition of the polymer to the substrate favored the percentage of emergence and EVI of ‘Rangpur’ lime and of mandarin ‘Sunki’ seeds in the growth chamber. The ‘Rangpur’ lime was the rootstock which presented the highest percentage of emergence and uniformity in both environments. The addition of the hydrogel polymer to substrate favored initial growth of citrus rootstocks.


INTRODUCTION
In the formation of citrus nursery trees, the seeds are used for the production of rootstocks (Siqueira et al., 2002).The main rootstock used in Brazil in recent years, 'Rangpur' lime (Citrus limonia Osbeck), has proved susceptible to serious disease problems (Moreira et al., 2010), such as the citrus sudden death (CSD), disease associated with a virus (Maccheroni et al., 2005).Therefore, the need to diversify the use of rootstocks has *Corresponding author.E-mail: amatomoreira@yahoo.com.br.Tel: +553835321200.Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License been identified.
One difficulty for the increased use of these mandarins, P. trifoliata (L.) and 'Swingle' citrumelo is the low drought tolerance.Another is at the nursery stage; the seed germination can be a limiting factor because the number of days to germination occurs, resulting in irregularity between seedlings and slowing the formation phase of the rootstocks.Among the stages of citrus nursery tree production, the production of rootstock is responsible for about 60% of the requested time.This delay is due to the period of germination, which can reach 60 days or more (Sousa et al., 2002).
The time of citrus nursery tree production can be from 10 to 15 months after sowing, with the appropriateness of the growth environment in several countries, including Brazil (Carvalho et al., 2005).For this it is necessary for the germination to be uniform and occurs as soon as possible.To promote germination, water availability is a major factor to guarantee ideal conditions for seed imbibition, and initial rapid growth of rootstocks are characteristics that contribute to advance the appropriate time of grafting point.
In order to improve the availability of water in the substrate, the addition of hydrogel polymer to the soil favors seed germination, root development, plant growth, and contribute to improving aeration and soil drainage, minimize nutrient losses by leaching (Azevedo et al., 2002).The polymers are arrangements of organic molecules (Fonteno and Bilderback, 1993) that exhibit granular when in dry form.When hydrated, these are transformed into a soft elastic gel (Prevedello and Loyola, 2007), capable of absorbing about a hundred or more times its weight in water.
Furthermore, the containers used for the production of rootstocks comprise small volume of substrate, which limits the availability of water to plants making necessary frequent irrigation, resulting in significant loss of some nutrients (Cruz et al., 2008).Thus, the use of the hydrogel polymer on substrate is an alternative to improve the process of production of citrus rootstocks, because it keeps the substrate humid, minimizes nutrient leaching and increases growth of plants.
The addition of hydrogel polymer to substrate showed satisfactory results for the rootstock 'Cleopatra' mandarin (Cruz et al., 2008) and propagation by cuttings of sweet passion fruit (Hafle et al., 2008).Also, it favored the highest percentage of survival, length of roots and shoots of blackberry cv.'Brazos' (Rubus sp.) (Moreira et al., 2012) and in reducing the frequency of irrigation for the production of passion fruit seedlings (Carvalho et al., 2013).
Based on the above, the study was done to evaluate the formulation of the substrate with the hydrogel polymer on the uniformity of emergence and early growth of citrus rootstocks.

MATERIALS AND METHODS
The experiment was conducted at the division of fruit crops of Federal University of Jequitinhonha and Mucuri Valleys (UFVJM) in Diamantina, Minas Gerais, Brazil, located at 18° 14 '56 "S and 43° 36' 0" W, from December 2012 to March 2013.
For the evaluation of the seed emergency was used factorial scheme 3 × 2 × 2, with the factors: three rootstocks ['Rangpur' lime (C.limonia Osbeck), P. trifoliata (L.) and 'Sunki' mandarin (C.sunki Hort.ex Tanaka); two formulations of substrate (with and without 0.4 g per container of Hidroplan-EB/HyB-M ® polymer and two environments (greenhouse with an average maximum temperature of 36.6°C and minimum of 17.2°C and growth chamber, programmed with temperature to 25°C and a photoperiod of 12 h, distributed in a completely randomized design with four replications and 35 seeds per plot.To monitor the temperature variations during the experiment, a digital termohygrometer (Model MT 240 MINIPA) inside the greenhouse was installed (Figure 1).
The hydrogel polymer was incorporated to the substrate Bioplant ® , by the homogenization of the mixture before filling the containers.The Hidroplan-EB/HyB-M ® is a mixed product of a copolymer of acrylamide and potassium acrylate used to absorb and retain large quantities of water.
The seeds of rootstocks were sowed in containers of 50 ml (one seed per container).During the emergency the frequency of irrigation was used to keep the humid substrate and to ensure uniform seed emergence.For this, in the growth chamber the irrigation was performed every 2 days and in the greenhouse daily.
At 25 days after sowing the emergence started, using as criteria for emergence the radicle protrusion of 1 mm.To evaluate the emergency velocity index (EVI), it was counted the number of seedlings that emerged every 2 days until the 66th day, and it was calculated using the formula suggested by Maguire (1962): [EVI = (G1 / N1 + G2/N2 + ... + Gn / Nn)]; G = number of seeds emerged and N = number of days that have evaluated the emergence.
After the end, the emergence period was done in the thinning of rootstocks, because of polyembryony, leaving only one seedling per container.From this time the nitrogen fertilization of rootstocks was done, applying 10 ml of a solution (1% of N) per container weekly.At 60 days after emergence the germination percentage, the shoot length (cm), the stem diameter (mm), the number of leaves per plant, the leaf area (cm 2 ), the root length (cm), the number of lateral roots, the dry shoot mass (g) and the dry root mass (g) of each rootstock was evaluated.
The shoots and the roots were washed in water running and placed to dry in an oven with forced air circulation at 65°C, until constant mass to determine dry mass, on electronic scale accurate to 0.001 g.
Leaf area was determined using the methodology of Benincasa (2003), which the leaf discs were obtained with a puncher of known area.These disks and the leaves were placed to dry in an oven with forced air circulation at 65°C in paper bags to determine its mass.Leaf area was calculated according to the equation: [LA = (dry mass of leaf × disc area / dry mass of the disks)].
In order to compare, the growth was adopted the 3×2 factorial scheme, with three rootstocks: 'Rangpur' lime; P. trifoliata and 'Sunki' mandarin and two substrate formulations: with and without hydrogel polymer, this is because after emergence all plants in  growth chamber were taken to the greenhouse.Data were subjected to analysis of variance and the means were compared by Tukey test at 5% of probability.

RESULTS AND DISCUSSION
Interaction among rootstocks, the addition of the polymer and the environment for the EVI and the percentage of emergence were observed (Tables 1 and 2).
It was observed that the 'Rangpur' lime was the rootstock with greater EVI in the two environments.The addition of the polymer favored emergence uniformity of 'Rangpur' lime and 'Sunki' mandarin seeds when they were sowed in the growth chamber.The behavior of 'Rangpur' lime was similar regarding the percentage of emergence, achieving the best results in both environments.89.01 and 82.75% of emergence with and without the addition of polymer in the growth chamber, respectively, and 87.00% without the use of hydrogel polymer in the greenhouse (Table 1).
The differences in the percentage of emergence and uniformity can be related to the seed coat, because the seed coat of P. trifoliata is more leathery than the other citrus rootstocks, favoring the rottenness of seeds during germination (Rouse, 1997).The integument is considered the main limiting factor for seed germination of these rootstocks, which possibly have low germination due to low water absorption, or loss of seeds caused by the long period that they remain in contact with the substrate.Significant increase in seed emergence of hybrid rootstock from P. trifoliata has been achieved with the removal of the seed coat by Moreira et al. (2010).
Comparing the environments, it was observed under the conditions of growth chamber that the addition of the hydrogel polymer favored the higher emergence uniformity for all rootstocks.However, without polymer, P. trifoliata showed greater emergence uniformity in greenhouse, possibly because in this environment the irrigation was performed daily (Table 2).
For the percentage of emergency, 'Rangpur' lime showed the highest values with 89% of seed germination with the use of the polymer on the substrate in growth chamber (Table 2), possibly because the greenhouse with the addition of the polymer provided excessive humidity in substrate, damaging its aeration and consequently the emergency, which did not occur in the growth chamber by irrigation have been held every 2 days.This behavior suggests that the irrigation should be performed less frequently with the use of polymer.Carvalho et al. (2013) observed that it is possible to irrigate the substrate in a greenhouse with an interval of 1 day with the use of hydrogel polymer on the substrate.
In general, it was observed that the addition of the hydrogel polymer promotes uniformity emergency when environmental conditions are not favorable.On the other hand, it is not enough to increase seed emergence, probably because even in the presence of humidity, low germination results from the presence of the seed coat.To increase germination the use of techniques for removal of the seed coat is necessary, because it makes possible to achieve more than 90% of emergence, while with seed coat the emergence does not reach 30%.This behavior was observed in 'Flying Dragon' (P.trifoliata var.monstrosa) and hybrids of P. trifoliata, both used as citrus rootstocks (Moreira et al., 2010).
The difficulty of obtaining high percentages of emergence in the nursery has been reported by many researchers, mainly because the number of days for seed emergence to occur, results in disuniformity among the seedlings, slowing the formation phase of rootstocks (Sousa et al., 2002).
In the initial growth of rootstocks, there was an interaction between application of the polymer substrate and rootstocks for all evaluated characteristics.
The incorporation of the polymer to the substrate favored the initial growth of rootstocks (Table 3).The plants of P. trifoliata showed greater growth in height, stem diameter, root number, leaf area, dry mass of shoot and root system with the addition of the polymer to the substrate.
For 'Rangpur' lime, the behavior was similar with better results in relation to the stem diameter, number of leaves, dry mass of shoot and root in the presence of the polymer (Table 3).
The development of stem diameter is important to anticipate the production of rootstocks because the Table 3. Height, stem diameter, number of leaves, number of roots, root length, leaf area, dry mass of shoot and dry mass of root of citrus rootstocks grown on substrate with and without the addition of polymer hydrogel in greenhouse at 60 days after emergence.greater development in diameter can anticipate the time of grafting (Schäfer et al., 2006).On the other hand, 'Sunki' mandarin did not differ from those grown without the incorporation of the polymer, except for the length of the root system that was longer in the absence of polymer, the same behavior was observed in other rootstocks (Table 3).This can be attributed to greater availability of water in the substrate, because the root system tends to become deeper when humidity conditions are scarce in the exploration zone of the root, leading the growth of the root system to increase efficiency in absorbing water.

Rootstocks
The difference in the growth of rootstocks with the polymer can be attributed to the improved hydraulic properties of substrate, increasing the availability of water (Azevedo et al., 2002;Saad et al., 2009).
This improvement is important for the production of citrus nursery trees, because the conventional method of nursery trees production in controlled environments used small containers, which limits the availability of water (Cruz et al., 2008).For these reasons, the addition of hydrogel polymer to substrate is possible in order to reduce the frequency of irrigation, providing water and fertilizer saving.
Comparing growth among rootstocks, larger height and larger stem diameter for P. trifoliata was observed with and without the use of polymer.The stem diameter is directly related to plant height (Bernardi et al., 2000), and is the morphological characteristic of the rootstock which defines the possibility of grafting (Bernardi et al., 2008).This behavior may occur due to intrinsic differences of species, by the genetic characteristics to each rootstock.Another important aspect was noticed that the use of the polymer, the growth in relation to the number of leaves, number of roots, dry mass of shoot and root system of P. trifoliata was similar to 'Rangpur' lime, whereas in the absence of the polymer, 'Rangpur' lime showed higher growth (Table 3).This can be attributed to P. trifoliata to be a rootstock that has low drought tolerance, so with the greater availability of water it can be similar to 'Rangpur' lime which is a rootstock well accepted by nurserymen for its excellent vigor.
The difference of development is common to be found among the rootstocks, which may occur due to different genetic characteristics, which affect the light and CO 2 use, influencing the uptake, transport and interaction of nutrients in the plant (Fochesato et al., 2006).However, each species can develop mechanisms to grow under adverse conditions, such as the reduced availability of water, which is one advantage of 'Rangpur' lime compared to other rootstocks that without addition of the polymer, it showed a higher number of roots (Table 3).
The results of this study show that the addition of the hydrogel polymer to substrate for the production of rootstocks is presented as alternative to reduce the time in the nursery, because of better emergence uniformity and growth of plants.However, the use of appropriate techniques is necessary to optimize the percentage of emergence of species with potential for citrus rootstocks, which have the integument as a limiting factor.

Conclusions
The addition of the hydrogel polymer to the substrate favored the percentage of emergence and EVI of 'Rangpur' lime and of 'Sunki' mandarin seeds in the growth chamber.
The 'Rangpur' lime was the rootstock which presented the highest percentage of emergence and uniformity in both environments.
The addition of the hydrogel polymer to substrate favored early growth of citrus rootstocks.The hydrogel polymer can be added to substrate in order to reduce the irrigation frequency.

Figure 1 .
Figure 1.Average of minimum and maximum temperatures recorded in the greenhouse during the emergence and growth of rootstocks, Diamantina, Minas Gerais, Brazil.

Table 1 .
Rootstocks versus environment interaction of emergence velocity index (EVI) and percentage of emergence of citrus rootstocks grown on substrate with and without hydrogel polymer in greenhouse and growth chamber growth chamber.Means followed by different small letters in the line differ by F test and the capital letters in column differ by Tukey test at 5% probability of error.

Table 2 .
Environment versus polymer interaction of emergency velocity index (EVI) and percentage of emergence of citrus rootstocks grown on substrate with and without hydrogel polymer in greenhouse and growth chamber, inside each species.
Means followed by different capital letters in the column, different by F-test at 5% probability of error.