Stanislava Grozeva*,Velichka Todorova
Maritsa Vegetable Crops Research Institute,32 Brezovsko Shosse Str.,4003 Plovdiv,Bulgaria.
The regeneration answer of hypocotyl and cotyledon explants of two pepper genotypes on MS0 medium content of macro- and micro- nutrients – ½ MS0, 1 ¼ MS0, 1 ½ MS0 1 ¾ MS0 and MS0 (control) with 3.0 mgL-1 BA, 0.3 mgL-1 IAA, 0.3 mgL-1 GA3 was studied. It has been found higher frequency of indirect regeneration in control culture medium while differentiation and elongation of obtained shoots has been established in culture medium variant with 1 ½ MS0 + 0.2 mgL-1 AgNO3 and 0.3 mgL-1 GA3. In R0 generation the regenerants were characterized with reduced plant height, leaf size, fruit weight and seeds per fruit. Statistical analysis of fruits morphology and productivity per plant in the next R1 generation indicated variation between lines and compared to parents. Most lines were with shorter, but wider fruits with thicker pericarp and lower productivity per plant. For breeding purposes the complex of traits is of interest for following investigation deserve lines 3/13 and 2-1/13 from initial variety Yasen F1. Observed differences confirm the possibility for using of somaclonal variation as a method for improving and enriching the diversity in pepper
Cotyledons; Hypocotyls; Macronutrients; Micronutrients; Somaclonal variation.
BA - N6-benzyladenine; IAA - Indolil-3-acetic acid; GA3 - Gibberellic acid; TDZ - Thidiazuron; AgNO3 - Silver nitrate.
Capsicum spp. belongs in recalcitrant plant species to in vitro manipulation. Several main factors affect the regeneration rate of pepper as the genotype,explant type and culture medium [1-3]. Many investigations have been conducted to optimize the regeneration process in pepper,but the major difficulties remain induction of rosettes of distorted leaves which was not developed to normal shoots . Verma et al.  regenerated shoots from profuse rosettes after transfer for elongation to MS medium supplemented with 2.25 mgL-1 BA and 2.0 mgL-1 GA3. Joshi and Kothari  also used GA3 for elongation of obtained shoot buts,but combine with 30 time’s increased CuSO4 levels.
For plant breeding especially using somaclonal variation or genetic transformation for creation of useful genetic diversity developing of efficient plant regeneration protocol are need. The studies about gene control on in vitro regeneration in pepper is limited. According to Mezghani et al.  CDKA gene expression may be linked to dedifferentiation during adventitious organogenesis in pepper tissues cultivated in vitro and it can be used as a molecular marker for in vitro regeneration in this recalcitrant species.
Regardless of the existing difficulties and lack of effective genotype-independent protocol pepper lines different by agronomic performance and mineral content in plants and fruits were obtained [8,9]. The aim of this research work was to study the effect of basal medium concentration of shoot induction in two pepper varieties and morphological evaluation of plant-regenerants.
Seeds of two pepper varieties Yasen F1 and Kurtovska kapia 1619 were sterilized in 5% calcium hypochlorite solution for one hour and rinsed three-times in sterile dH2O. For germination the seeds were grown on basal medium containing macro- and micronutrients ,vitamins ,3% Sucrose and 0.7% Agar (MS0). The pH of all culture medium was adjusted to 5.8 before autoclaving (121º C,20 min).
Cotyledon (0.5 cm) and hypocotyl (1.0 cm) explants were excised from 5-7 days old in vitro grown seedlings and cultivated in Petri dishes on basal medium MS0 containing 3.0 mgL-1 BA,0.3 mgL-1 IAA,0.3 mgL-1 GA3,(as a control) and four variant of MS0 different in concentration of basal medium:
1. ½ MS0
2. 1 ¼ MS0
3. 1 ½ MS0
4. 1 ¾ MS0
After 40 days the explants were transferred to the same culture medium without BA and IAA,but supplemented with 0.2 mgL-1 AgNO3 and 0.3 mgL-1 GA3. Cultures were incubated in a growth chamber: temperature 26ºC±1ºC,a photosynthetic proton flux density (PPFD) of 200 μmol m-2 s-1 and 14/10 h photoperiod and sub-cultured at intervals of 20 days to the same medium variants. Well-developed shoots (2-3 cm) were isolated from explants and transferred on rooting medium consisting of ½ MS0 and 0.1 mgL-1 IAA
The experimental design for regeneration was: three replication with 20 explants of each,twice,120 explants per treatment – culture medium variant,explant type and genotype. The callusogenesis,organogenesis and regeneration frequency (% explants with response) and number of regenerants per explant (compared to reacted with organogenesis explants) were examined for a period of 90 days.
Regenerants 4-5 cm in height were extracted from the culture medium,the roots were rinsed with running water and planted in 0.5 L pots in mixture peat moss and perlite in the ratio 1:3 (v/v) for 10-12 days. Successfully adapted plants were transferred in 5 L containers mixture peat moss and perlite in the ratio 1:1 (v/v) and grown in glasshouse. As a control was used in vitro germinated and adapted plants from initial genotypes.
The following traits between plants derived both from in vitro culture and from seeds from R0 progeny were recorded: plant height (cm); leaf blade – length and width (cm),leaf colour,pollen fertility (squashing in 4% acetocarmine and glycerin (1:1). After self-pollinated 9 R1 lines were compared by fruit weight (g),length (cm) and width (cm),usable part of the fruit (g) and pericarp thickness (mm),productivity per plant (g),number of fruits per plant and seeds per fruit (number). These traits were evaluated on five plants per replication and three fruits per plants.
The plants for R1 were grown under field conditions in two replications with 15 plants of each line and initial variety on furrow surface by 70/15 cm scheme. The plants were grown according to the adopted technology for mid-early field production.
The results of regeneration frequency and morphology traits were are given as means ± standard deviation (SD). Data were subjected to Duncan’s Multiple Range Test to evaluate the statistical significance among the means. Three-way analysis of variance for influence of variation factors on the regeneration was studied.
Hypocotyl and cotyledon segments showed induction of callus of the cut edges after 10 day of cultivation in all culture medium. Callus morphology in cotyledons was friable and whitish with intensive growth. In hypocotyls callus was compact,white and hard with slower growth (Figure 1a,b). Initial formation of organogenic structures was about 15-20 days of cultured. Organogenic frequency in cotyledons varied from 3.3% (medium variant 4) to 100% (control medium) in variety Yasen F1 and from 20% (medium variant 4) to 100% (control medium) in variety Kurtovska kapia 1619 (Figure 2 ). On the other hand organogenic reaction in hypocotyls was registered only in Yasen F1 in control culture medium and variant 3. However,after 2 or 3 subcultures of the fresh medium the explants were covered by more callus tissue and subsequent differentiation and elongation of shoot regeneration decreased (Figure 1c,d). Verma et al.  also observed more shoots buds during initial stages of callus proliferation and decreased shoot regeneration during later stages of callus grown.
Differences in the regeneration potential were observed depending on the Genotype (Factor A),Explant type (Factor B) and Culture medium (Factor C) (Figure 3 ). Statistical data showed the strongest influence of Culture medium (38.13%) followed by interaction Genotype x Culture medium (21.57%),Explant type x Culture medium (14.95%) and Culture medium (14.00%). Significantly lower were effects of Genotype (3.16%) and interaction Genotype x Explant type x Culture medium (2.61%).
In both studied pepper genotypes the regeneration frequency varied from 0.0% to 48.3% (Table 1). The highest percentage of regenerated cotyledon explants in variety Yasen F1 was achieved in control medium variant (48.3%),while in variety Kurtovska kapia 1619 in variant 3 (33.3%) contained 1 ½ basal MS0 medium. Reaction with lower frequency was registered in culture medium variant 2 with 1 ¼ strength. When culture medium contained ½ and 1 ¾ strength (variants 1 and 4 respectively) regeneration process was absent. Regeneration in hypocotyls was obtained only in variety Yasen F1 under control medium.
After transfer of explants on elongation medium the highest number of regenerants per cotyledon explant in both genotypes was on 1 ½ (variant 3) followed by control medium (Figure 4 a,b). Hypocotyls reacted only on control medium in Yasen F1 (Table 2). To induce regeneration in pepper from different explants combinations of BA or TDZ with IAA are applied,but the main difficult is elongation of obtained shoot buds [1,12]. Cytokinins stimulate shoot proliferation and inhibit their elongation. To overcome the problem with shoot elongation culture medium without cytokinins was used [13,14]. Pishbin et al.  reported to better shoot elongation in culture medium contained low concentration of BA (0.1 mgL-1) and GA3 (0.5 mgL-1). In contrast fair elongation Verma et al.  obtained after shoots transfer on 2.25 mgL-1 BA and 2.0 mgL-1 GA3. The differences observed in this study may be due to the distinctions in concentration of macro- and microelements of basal medium. Increasing of concentration of basal elements stimulated shoot elongation and differentiation to plants but have no such effect on regeneration. In cherry Ruzic et al.  reported for the best multiplication on MS 2× and MS media which they have associated with the highest N and P content.
The morphological characterization of R0 in vitro regenerants and seed-derived plants from Yasen F1 and Kurtovska kapia 1619 is given in Table 3. Regenerants were with reduced plant height,leaf size,fruit weight and seeds per fruit compared to the seed-derived plants (Figure 4 c). Pollen fertility between regenerants and seed-derived plants were 70.1% and 91.7% in Yasen F1 and 68.9% and 87.3% in Kurtovska kapia 1619,respectively. The regenerants from variety Kurtovska kapia 1619 were distinguished with green leaves in contrast to dark green colour in control plants. After self-pollination of regenerants seeds from the next investigation were produced from 9 plants – 6 originated from variety Yasen F1 and 3 – Kurtovska kapia 1619.
The results from the characterization of the R1 lines of pepper showed that regenerants originating from variety Kurtovska kapia 1619 were distinguished with decreased values of the studied traits. Only the characters fruit width and pericarp thickness were with values close to the control plants (Table 4). However diversity among the lines obtained of variety Yasen F1 was higher. The average fruit weight in regenerants varied from 46.69 g to 95.68 g,while fruit length and width varied in narrow margins – 9.23/4.83 cm to 12.37/5.80 cm,respectively. The usable part of the fruit varied wider than the pericarp thickness. The variation interval was from 30.92 g to 68.04 g for usable part of the fruit and 3.67 mm to 4.67 mm – pericarp thickness. Only fruits from lines 2/13 and 5/13 were with the lowest seeds per fruits (30 and 57 number,respectively). Significant differences between lines were established by productivity per plant (from 390 g to 1130 g) and number of fruits per plant (from 7 to 17). It has been found that most regenerants from variety Yasen F1 were with shorter,but wider fruits with thicker pericarp and lower productivity per plant than parent. Only line 3/13 was with productivity per plant,close to the control. For breeding purposes by complex of traits interest for following investigation are lines 3/13 and 2-1/13 from variety Yasen F1 (Figure 4 d). On the other hand studies in other lines also need to continue because they may have other useful features,such as disease or pest resistance,fruit quality,etc. which contribute to enrich the population of pepper with new gene combination.
In Solanaceae family somaclonal variation as a method leading to creation of suitable genetic diversity and its involving in the breeding process is applied with success in tomato,potato and tobacco. Pepper belongs to recalcitrant species with respect to its in vitro regeneration ability. Little is known about the morphological and genetic variations in somaclones of pepper because of more studies are directed to overcome the difficulties with regeneration process . The source of somaclonal variation depends on genotype,explant type and culture conditions especially basal culture medium,concentration and combination of plant growth regulators etc. . Clear information about genetic mechanisms controlling organogenesis is still limited. Positive correlation between CDKA gene expression and the proliferation stage of pepper cotyledon tissue culture in vitro was proved . The authors concluded that CDKA expression seemed to be a useful molecular marker for the cell dedifferentiation phase. A better understanding of recalcitrant nature will provide an opportunity to identify the regulatory genes of pepper morphogenesis and process . Morphology characterization and random amplified polymorphic DNA analysis are used to evaluate the somaclonal variation in pepper [5,16]. In R0 generation variations in growth habit,stem and flower colour,pollen fertility,fruit morphology,productivity per plant etc. were observed . The studies of the inheritance of changes traits in the R1 generation were also conducted . Pepper lines with early flowering,increase of yield and yield components [18,19],different in fruits colour and shape ,quality traits and mineral content in the fruits  were developed on the base of somaclonal variation.
In vitro organogenesis and regeneration in pepper is affected by genotype,explants and concentration of basal medium. Cotyledons are more susceptible to regeneration than hypocotyls. Better regeneration was achieved in control medium. The best elongation of obtained shoots was registered in culture medium variant with 1 ½ MS0 supplimented with 0.2 mgL-1 AgNO3 and 0.3 mgL-1 GA3
Morphological differences in R0 were observed between in vitro regenerants and seed-derived plants. Somaclones in next R1 generation showed same favorable changes like increase of fruit weight,width and pericarp thickness. The obtained variability in both studied genotypes indicates the possibility of improvement of pepper through somaclonal variation.