Evaluation and Agronomic Potential of Androgenic Pepper Genotypes Derived from Piran (Capsicum annuum L. cv. Piran)

Aims: This study was to evaluate and estimate the agronomic potential of two androgenic genotypes via important plant, fruit and production traits compared to the mother genotype Piran. Place and Duration of Study: Strumica, Faculty of Agriculture, Goce Delcev University-Stip, Republic of Macedonia, four-years experiment. Methodology: The experiment was set up in random block design in four replications. Evaluation of the androgenic genotypes was performed according to morphological traits given in Descriptor for Capsicum spp. The analysis of plant traits was conducted on 20 randomly chosen plants (5 plants per replication) in maximum growth stage from each androgenic genotype and the control Piran. The analysis of fruit traits was conducted on 40 randomly chosen fruits (10 fruits per replication) in ripe stage from each androgenic genotype and the control. Results: Androgenic genotype P3 was characterized as tall pepper type with medium-thick stem and large leaves. According to the fruit weight (41.45 g) and the fruit index higher than 4, the Original Research Article Trajkova and Gudeva; JEAI, 16(4): 1-12, 2017; Article no.JEAI.33807 2 genotype is classified as long, horn-shaped, middle-size fruit pepper. The fruit flesh of 77.05% and the pericarp thickness 0.26 cm characterized this fruit as medium-fleshy. Androgenic genotype P4 was characterized as very tall pepper type with medium-thick stem and medium-large leaves. The fruit weight was 42.84 g, contained averagely 7.37% dry matter, 76.71% fruit flesh and 0.25 cm pericarp thickness. P4 fruit was long, horn-shaped, medium-size and medium-fleshy. Fruits of both genotypes were suitable for fresh and preserved consumption. Conclusion: Analysis of morphological and productivity traits of androgenic genotypes provides valuable information for future breeding. Androgenic genotype P3 differ from the initial genotype for four traits, while the P4 genotype for six traits. The results of this research prove the advantage of pepper androgenesis as a tool for enrichment of biodiversity and fast development of valuable pepper genotypes for future breeding.


INTRODUCTION
Pepper (Capsicum annuum L.) is an important crop that can be improved by utilization of the methods of classical genetics, but also by using plant tissue culture and plant biotechnology tools which can shorten breeding process of many crops [1][2].
Androgenesis is one of the methods utilized for creation of haploid and spontaneous dihaploid plants in in vitro culture [3][4][5]. Androgenesis can be defined as a set of biological processes leading to an individual that genetically originated exclusively from a male nucleus. The main objective of haploid research is to obtain dihaploid homozygous lines with high practical value for breeders, but experiments in this area also have theoretical importance for fundamental biological sciences (genetics, embryology, cytology, and molecular biology). After spontaneous or induced genome doubling, dihaploids that are completely homozygous can be utilized differently in fundamental and paratactical research programs. From the standpoint of plant breeding, androgenesis has power to reduce the typical 7-9 inbreeding generations necessary to stabilize a hybrid genotype to only one. It is the key advantage of dihaploid technology in the context of plant breeding. Within this same context of plant breeding, dihaploids, which are homozygous for all of their loci, create a powerful tool for the study of the genetic basis of quantitative characteristics, including the genetic mapping of complex traits such as production or quality, the most agronomically interesting, and difficult to be addressed by other approaches [3].
There are reports about a high frequency of anther-derived embryos of different Capsicum species, but few of them developed into normal plants. Despite the considerable number of studies conducted with pepper anther culture, the reported effectiveness of the process is still low. Additionally, pepper is characterized as third culture of fam. Solanaceae which is "reluctant" on androgenic methods, after tomato and eggplant [3]. Even there are androgenic embryoids, they either cannot be regenerated into fully developed plant or are infertile. Critical factors for production of haploid/dihaploid embryoid and their regeneration into fertile plants are growth conditions of donor-plants, selection of flower buds in appropriate microspore stadium, stress, medium composition, genetic background of mother genotypes and environmental culture conditions [4]. On the other hand, breeding programs require availability of a considerable number of genetically stable plants. In this regard, the most important achievement is development and optimization of protocols for haploid and dihaploid production and regeneration of pepper under laboratory conditions and utilization of the novel di-haploid lines in practice [5].
Gained pepper haploids and dihaploids have certain genetic potentials which are quickly phenotypically expressed because they origin from a haploid cell [6][7]. These androgenic plants, before being included in breeding process, must be studied for their growth and development stages, their genetic and morphological characteristics compared to the mother genotype which give information about similarities and differences between androgenic and mother genotypes [8][9][10].
This study was conducted to evaluate and estimate the agronomic potential of two androgenic genotypes P3 and P4 derived from initial genotype Piran via important morphological and production traits.

MATERIALS AND METHODS
Detailed description of the methodology and the process of creation of androgenic genotypes is presented in different publications [11][12][13][14].
In this research plants and fruits of two androgenic genotypes P3 and P4 created from the variety Piran were tested in greenhouse conditions at the Faculty of Agriculture, Goce Delcev University -Strumica, Republic of Macedonia, during four-years experiment. The variety Piran, used as mother genotype in anther culture, was used as control (standard) during evaluation of the androgenic genotypes P3 and P4. It was obtained from the gene bank of Faculty of Agriculture, Goce Delve University -Stip [15].
Piran is Macedonian variety which can be cultivated in open field and under protected conditions. The average plant height is 80 cm, fruits are large with average weight of 100-120 g, yellow in unripe stage and red when ripe. Piran average yield is 60-80 t/ha [14,16].

Research Design and Dynamics
During the first experimental year seeds obtained from fertile androgenic plants were utilized as starting material in the experiment. Seeds from selected typical fruit from each androgenic genotype of the previous experimental year were used in the subsequent experimental years.
In each experimental year seeds of studied genotypes were saw in the beginning of April in non-heated greenhouse. At the end of May, the seedlings were transplanted into pots (d=22 cm) filled with mixture of soil, sand and peat. The experiment was design in four replications, in random block design. From each androgenic genotype and control genotype Piran, 40 plants (10 per replication) were transplanted in plastic pots, which were placed in the greenhouse with 30cm/100cm distance scheme. Plants were cultivated according to standard cultivation practices under protected conditions. Irrigation and nutrition were performed as fertigation with drip system. Selection of parameters and evaluation of genotypes was performed according to recommendations given in Descriptor for Capsicum spp. [17]. The analysis of plant traits was conducted on 20 randomly chosen plants A classification of plant, leaf, fruits and productivity of androgenic genotypes was made based on the results of studied morphological and production traits according to Jankuloski (1983Jankuloski ( , 1997. It is the most suitable and acceptable classification of peppers by morphological traits which are bred and cultivated in the Western Balkans [18][19].

Biometric Analysis of Characteristics
Statistical analysis of variance is applied for the evaluation of the experiment in general and each of the characteristics among all tested genotypes (One-Way ANOVA test).
For evaluation of the differences among examined genotypes Duncan's multiple range test is utilized for each trait in each experimental year on level 0.05% and for the four-year average on level 0.05% and 0.01%.
The statistical analyzation of the results was performed by IBM SPSP Statistics Software 19.0.

Morphological Traits of Androgenic Plants
Morphological traits of pepper plants as total plant height and plant height to the first branching, leaf shape, stem thickness, fruit size and shape are traits which give basic marks of a pepper variety. However, they are dependent on many external factors as temperature, air humidity and cultivation practices [1,14]. The mean values of morphological traits of plants in maximum growth stage during the four-years research of androgenic lines P3, P4 and the mother genotype Piran are presented in Table 1. Other researchers also reported differences in plant height between androgenic lines and initial variety [20][21][22]. It was found that all antherderived lines were with higher values of plant height compared to the initial variety Hebar [20]. Diploid plants derived from minipaprika red showed more variation in plant height than the plants of minipaprika yellow and orange.
Similarly, researchers observed variation in leaf size, petiole and internode length, flower bud size, nodal color, leaf color, and leaf within the diploid lines of each minipaprika [21]. Differences by plant traits between anther-derived genotypes and the initial variety were observed in the field conditions [22].
Stem thickness of tested genotypes varied from 0.65 cm (P3, I year) to 1.14 cm (P4, I year), where the lowest average value was measured in IV experimental year and the highest in III experimental year. Averagely for the whole experimental period, the highest average value for stem thickness was registered for P4 genotype (1.01 cm) and it was significantly different from the control Piran (0.89 cm).
The mean internode length of studied genotypes has not shown statistically significant difference. Longer internodes were measured during the first two experimental years as compared to internode length in the third and the forth experimental year. Many plants, including pepper, have internodes length which is determined by day-night temperature variations, which is mainly due to increased cell elongation than due to increasing of cell number. Peppers have shorter internodes when difference between day and night temperatures is smaller [23]. Morphological characterization of anther derived plants in minipaprika showed variation in leaf size, petiole and internode length, flower bud size, nodal color, leaf color, and leaf shape of diploid lines of each minipaprika. As expected, the researchers recorded reduced plant height, leaf size and internodes for haploid regenerants [21,24]. The results of total plant height and plant height to the first branching show that plant growth between the flowering phase and maximum growth phase is happening above the first branching, consequently the plant grows in height with development and growth of new branches. The growth in stem base is very small. This characteristic is very important for cultivation of different pepper varieties under protected conditions because a lush growth will need stem and branches staking in order they to sustain fruit load [14]. This is the reason for development of different systems for pepper plant support with temporary or permanent staking construction [25].

Fig. 1. Plants and fruits of variety Piran used as control genotype in the experiment
The average leaf length of androgenic genotypes was 11.15 cm (P3) and 10.73 (P4). The only statistically proven difference in leaf length was in II experimental year, when the leaf length of P3 genotypes (11.09 cm) was longer as compared to P4 genotype (11.09 cm) and the control Piran (10.21 cm). The leaves of P3 (5.41 cm) and P4 (5.22 cm) were significantly narrower than the control leaves (5.76 cm) which is proven during the whole experimental period. The average leaf index for androgenic genotypes P3 and P4 is 2.07, which was significantly higher than the control Piran (1.88). Leaves of P3 genotype were classified as large-size leaves, while P4 as medium-size leaves. Although they have the same leaf index, the first one were longer.
Other researchers presented results of leaves dimensions of diploid androgenic plants smaller than the mother genotype which is opposite to our results [24,26].

Morphological Traits of Fruits from Androgenic Plants
Morphological traits of pepper fruit vary in wide ranges. The most authors agree that variation degree of morphological trait is genetically dependent and inherited, but they are always influenced by the complex of external factor, cultivation technology and practices, under protected conditions or open field conditions [18,25]. When making pepper characterization it is obvious that the most variations are observed in fruits, an organ which determines taxonomy and variety of the species [1,6,14,20,27,28]. F size and shape, pericarp thickness and yield fruit are quantitative traits which are governed by polygenes, as well influenced by environment. Yet, these are important characters that should not be omitted   [18,25]. When making pepper characterization it is obvious that the most variations are observed in fruits, an organ which determines taxonomy and variety of the species [1,6,14,20,27,28]. Fruit size and shape, pericarp thickness and yield fruit are quantitative traits which are governed by polygenes, as well influenced by the environment. Yet, these are important plant characters that should not be omitted in the process of evaluation of pepper dihaploids [21].
The average values for fruit length of androgenic genotypes and the control in the experimental period did not show significant difference for P=0.05 and P<0.01, which indicat control and androgenic genotypes are characterized by stability and good equability of fruit length (Table 3). Fruit length have not shown significant difference during I and II experimental year. In III experimental year the fruit length of the control genotype is significantly different compared to P4 androgenic genotype, while in  in the process of evaluation of pepper dihaploids The average values for fruit length of androgenic genotypes and the control in the experimental period did not show significant difference for <0.01, which indicates that the control and androgenic genotypes are characterized by stability and good equability of fruit length (Table 3). Fruit length have not shown significant difference during I and II experimental year. In III experimental year the fruit length of e control genotype is significantly different compared to P4 androgenic genotype, while in   Other authors reported a genetic analysis of anther culture-derived diploids of Capsicum spp. which showed that seventy-four percent of studied androgenic plants differ from the donor plant phenotype in ripe fruit color, pericarp structure and taste. Other nine diploid androgenic plants showed differences from the mother genotypes in fruit weight, wall thickness and dry matter content. In the study, statistical analysis of seven valuable traits (fruit length, width, weight, pericarp weight, pericarp portion of fruit weight, wall thickness, dry matter content) confirmed the significant differences between all the plants studied and their donor plants. The authors concluded that recombined microspores and not somatic cells of anthers were the origin of these diploids, thus they proved the androgenic origin of diploid plants [6]. Higher visual and taste quality, fruit firmness, dry matter content, total soluble content, titratable acidity, phenolic content, and antioxidant activity were exhibited in pepper dihaploid fruits [24]. In regards to fruit characteristics of androgenic lines, there is a report for selection of 5 superior dihaploid lines and observation of considerable variation in plant and fruit traits among other lines [26]. Other research group reported androgenic pepper lines of R2 progeny with improved yield characteristics and dry matter content in fruits [27]. Androgenic lines with positive traits were found after detailed characterization of morphological and production parameters of androgenic pepper fruits [28].
The results of androgenic lines study showed that they were characterized with uniformity and higher values of fruit length, fruit weight and wall thickness compared to the initial mother variety Hebar [20]. Other researchers found that diploid androgenic plants were characterized by big size fruit with high individual fruit weight and volume. Based on analysis of diploid populations in comparison to control variety Boogie, they selected some elite diploid lines for development of sweet pepper varieties. Similarly, it was suggested that existing variation within diploid population might be due to the anther culture and it is a naturally produced variation [26]. Variation in plant and fruit characters was observed in anther derived spontaneous diploid population of minipaprika. The authors concluded that such variation in quantitative traits had an additive character. Nevertheless, they recorded lower mean values of fruit characters in androgenic diploid plants of each group than the standard varieties [21].

Production Traits of Androgenic Genotypes
The results which characterize production trait of pepper genotypes under this study are presented in   The total and standard yield of 6 androgenic lines was higher compared to initial variety Hebar and they surpassed the control by standard yield from 11.02% to 42.26% [20]. Eight double haploid lines of pepper were studied for agromorphological and molecular traits. Two of them derived a special attention regarding yielding because they were either at the level of the maternal form or surpassed both parental forms regarding yield level [29]. Eleven R2 generations under study differed considerably in yielding and fruit number. According to authors of this study, the high values of the coefficient of variation suggested that the yield and the number of fruit per plant depend significantly on the environmental conditions [30]. Finally, there was higher variation in fruit yield in spontaneous doubled haploids of minipaprika which is considered as androgenic variation under the nuclear control [21].

Androgenic genotype Р3
Androgenic genotype P3 was created from the Macedonian variety Piran via androgenesis in vitro conditions. In the phase of maximum growth, the plant reached height of 88.55 cm, while the mean stem height to the first branching was 44.29 cm with stem width of 0.94 cm. These traits classified P3 genotypes as tall pepper with medium-thick steam. The leaves were averagely 11.15 cm long and 5.41 cm width which characterized the genotype with large leaves. According to the fruit weight (41.45 g) and the fruit index higher than 4, the genotype is classified as long, horn-shaped, middle-size peppers. The fruit flesh of 77.05% and the pericarp thickness 0.26 cm characterize this fruit as medium-fleshy and suitable for consumption as fresh and preserved. Regarding productivity traits, this genotype was characterized by 5.65 fruits per plant, averagely weighting 222.73 g and total yield of 1.392 kg/m 2 . According to production traits, in the given agroecological and cultivation conditions, it is a low-yield pepper genotype.

Androgenic genotype P4
Androgenic genotype P4 was created from the Macedonian variety Piran via androgenesis in vitro conditions. In the phase of maximum growth, the plant height was 93.68 cm, while the mean stem height to the first branching was 38.62 cm which classified it as very tall pepper. The stem width was 1.01 cm. The height and steam thickness classified this genotypes as very tall pepper with medium-thick steam. The leaf index, a ratio of mean leaf length (10.73 cm) and mean leaf width (5.22 cm), was 2.07 which classified the leaves as medium-large.

. Plants and fruits of androgenic genotype P4
The fruit was characterized with weight of 42.84 g, contains averagely 7.37% dry matter, 76.71% fruit flesh and 0.25 cm pericarp thickness. P4 fruit is classified as long, horn-shaped, mediumsize and medium-fleshy pepper. The total yield gained in this research was 1.449 kg/m 2 . One plant was averagely loaded with 5.80 fruits weighting 231.90 g. According to production traits, in the given agroecological and cultivation conditions, it is a low-yield pepper genotype.
In regards to overall variability of traits of antherderived plants, it is sourced from a genetic diversity of microspores which is an effect of random gene segregation during meiosis and our results agree with this finding. The existing difference within the diploid lines might be due to the result of naturally produced variation. Besides, most of the agronomic variation could originate from the changes that occur in the DNA of the vegetative pollen grain cell [31]. Phenotypic homogeneity of an androgenic population can point to the high level of its homozygosity. Besides, due to a necessity of providing a biometrical description of the most important plant and fruit parameters, this method additionally allows for a simultaneous selection of the lines which present the characters of interest to breeders and for allocating them for further breeding work [27]. On the other hand, phenotypic variation of plant and fruit traits may be the result of environmental conditions, which may often modify plant morphology. It suggests that although the importance of biometrical characteristics of anther-derived lines is of great value, as it allows for an objective evaluation of the essential functional traits of plants, which is necessary for the selection of the most interesting materials for breeding programs, the biometrical analysis cannot be the main method for androgenic regenerant microspore origin evaluation. However, especially the total fruit yield, the weight and size of the fruit, as well as the pericarp thickness, appear to be the most important plant characters that should not be omitted while dihaploid lines are being evaluated [30].

CONCLUSION
Analysis of morphological and production traits of androgenic genotypes provides valuable information for future breeding. Androgenic genotype P3 differ from the control variety for the traits height to the first branching, leaf index, fruit length and fruit weight. Androgenic genotype P4 differ from the control variety for the traits height to the first branching, stem thickness, leaf index, fruit length, fruit weight and fruit dry matter content.
The most important application of anther-derived, androgenic cultures in pepper has been their use in breeding programs, finally aimed to produce commercial hybrids with maximum heterosis and new or improved traits. Our results prove the advantage of pepper androgenesis as a tool for enrichment of biodiversity and fast development of valuable pepper genotypes for future breeding. These androgenic genotypes are recommended for their inclusion in breeding programs for development of long sweet pepper varieties for fresh and processed consumption.