The micropropation is an important biotechnological tool for obtaining and maintaining mother vine plants with high quality plant health. The objective was to evaluate the establishment and multiplication in vitro and ex vitro acclimatization of grape genotypes with potential for Southern Brazil. Vine nodal segments were cultured in five culture medium formulations without adding growth regulators. It was evaluated the number of leaves and roots, length of roots and shoots, replication rate, relative chlorophyll index, percentage of regenerated and rooted plants, dry biomass of shoot, root and total plants grown in vitro and after acclimatization. In vitro propagation of IAC 571-6 rootstock and cv. Poloskei Muskotaly through nodal segments provided high rates of regeneration and rooting. High survival rates were obtained in the acclimatization of IAC 571-6 and P ?l ?skei Muskotaly. Considering all the variables, the culture medium Roubelakis showed the best growth rates and development for shoots and roots, and in vitro multiplication rate for IAC 571-6 and Poloskei Muskotaly grape varieties.
Viniculture is a very important activity in Santa Catarina, including the primary and secondary sectors in the production chain through production and commercialization of in natura grapes and the processing of raw material for the preparation of juice, wine and jams. The scenery in Santa Catarina has been changing in the last years, and traditional regions in grapes production have showed a decrease of the area under vines and a reduction in harvesting. Since the 1990s it is observed the decrease in vineyards productivity and the increase of plants mortality. Pathogenic fungi causing vascular wilt (Fusarium oxysporum f. sp. herbemontis) and root rot (Cylindrocarpon sp., Armillaria mellea, Rosellinia necatrix), ground-pearl (Eurhizococcus brasiliensis) and viruses are the main problems for the viticulture in the south of Brazil [
Varietal resistance is one of the most indicated strategies to overcome those problems. Recently, Dalbó et al. [
Other matters related to plant health in the South of Brazil are fungi diseases on shoots of the vine, especially downy mildew (Plasmopara vitícola) [
The establishment of “Poloskei Muscotaly” cultivar aiming white wine production as well in natura fruit consumption has been advocating in Santa Catarina, due to its productive performance (above 20 t∙ha−1) and good tolerance to downy mildew and powdery mildew (Uncinula necator) [
Phytosanitary conditions of mother plants, shoots and/or rootstock are directly linked to the capacity of the plant material in expressing all its genetic potential. The use of infected seedlings from other countries or other states in combination with the propagating method promotes the spread of diseases, mainly viruses [
This study had as objective to evaluate the establishment and in vitro multiplication of vine genotypes with potential in the south of Brazil, in different culture medium and ex vitro acclimatization.
Experiments were performed in Biotechnology Laboratory of EPAGRI-Empresa de Pesquisa Agropecuária e Extensão Rural de Santa Catarina (Company of Agriculture Research and Rural Extension of Santa Catarina), Experimental Station, in Lages/SC.
Mother plants from IAC 571-6 rootstock (Vitis caribaea × Pirovano 57) and from Poloskei Muscotaly canopy cultivar (Zalagyöngye × (Gloria Hungariae × Afuz ali)) were kept in greenhouse for providing explants.
Independent experiments were performed for each vine cultivar by a completely randomized design, with five treatments (saline formula) and thirty repetitions for each treatment in the evaluations during in vitro phase, and ten repetitions during acclimatization phase.
It was tested saline formulas by Galzy [
2-cm-nodal segments, with a single bud were used as explants source for in vitro cultivation. Under aseptic conditions, nodal segments were superficially sterilized, embedded in 70% alcohol (v/v) for 15 seconds and rinsed two times in sterile water; after, they were immersed in 80% sodium hypochlorite solution (v/v) and 0.1% Tween 20 (v/v) during 15 minutes and rinsed three times in sterile water. Explants were transferred into the test tubes (110 mm × 23 mm) with 10 mL of different culture media.
Cultures were maintained in the test room at 25˚C ± 2˚C during the fourth days in the dark, and after for a photoperiod of 16 hours of light per∙day?1 and luminous intensity of 50 μmol∙m?2∙s?1. Sub-crops were done each 45 days, segmenting sprouts in nodal segments of 1 cm length with a single bud. After the second sub-crop, in vitro cultures were maintained in growth for 60 days and were evaluated for number of leaves and roots, length of the bigger root and the aerial part, replication rate, chlorophyll index, dry matter from aerial parts and roots after drying in the oven at 60˚ for 48 hours, percentage of regenerated and rooted plants.
Replication rate was obtained by counting explants number which are originated when the material is replicated. For indirect measures of chlorophyll (chlorophyll index) in SPAD value, readings from adaxial face of the leaf located on the medium part of each bud, selecting completely expanded leaves through Handheld Chlorophyll Gauge SPAD-502 (Soil Plant Analysis Development, Konica Minolta®, Japan). Readings values performed with the gauge were calculated based on the quantity of transmitted light by the leaf on two wavelengths; the light that has passed through the leaf reaches a receptor which converts light on analog signals, and those ones on digital signals that are used to calculate SPAD values [
Ex vitro acclimatization was performed with the third sub-cultures after 60 days in in vitro growing. Roots were washed in water and pruned (2 cm length); aerial part was maintained with 2 or 3 basal leaves. Buds were transferred to honey-combed trays with 72 cells (100 mL) containing sterilized substrate at 121˚C for 1 hour with Dystroferric Red Nitosol, sand and commercial substrate Tecnomax® (1:1:1, v/v/v). Trays were packed into plastic boxes, covered with glass and put in an acclimatization room during 60 days. Analyzed variables were the same as the experiments performed in vitro, exception to replication rate.
Statistical models were considered according to the nature of the variable response. For the variables Roots Number, Leaves Number and Replication Rate, it was used Poisson’s distribution. Model prepositions were verified using Kolmogorov-Smirnov’s Tests for normality of residuals, and Bartlett’s test for variance homogeneity.
In order to verify the model adjustment, it was used normal plot with simulated envelopes for deviance residual [
In experiment 1, independent of the saline formula of each nutritive environment, in vitro cultures of Poloskei Mukotaly cultivar showed 100% of regeneration and rooting. Higher rooting rates were described by Biasi et al. [
Greater leaves quantity was found when Poloskei Muskotaly cultivar was cultivated in environments with Roubelakis’ and Galzi’s formula in relation to Zlenko’s one (
Roubelakis’ formulation promoted greater number of roots than C2D and DSD1 ones which induced higher mass accumulation of dry material; however, the culture environment did not affect the length of the longest root; the values were from 8.5 cm to 10.4 cm. When comparing MS [
The longest aerial parts were found when Poloskei Muskotaly was cultivated in the middle of Roubelakis, with aerial part formation of 6.4 cm, not significantly differing from Galzy and DSD1 formulations with 5.5 cm and 5 cm, respectively (
Replication rate reflects the quantity of seedlings from a single explant. As a reflection of “number of leaves” and “stem length” variables, the higher replication rate of Poloskei Muskolaly cultures was in Roubelakis’ formulation, generating around 6.2 new plants, that do not differ significantly from cultures with Galzy, C2D and DSD1 formulations which, when replied, generated 5.6, 5.1 and 5.6 new plants, respectively (
In relation to chlorophyll index, values of SPAD-502 reading showed culture medium formulas influenced chlorophyll rate (
In vitro Poloskei Muskotaly cultures showed similar accumulation of dry mass among the treatments, with total biomass production in the interval from 39.92 mg to 50.82 mg, without statistical difference among culture medium formulations (
In relation to the allocation of in vitro supplies, Poloskei Muskotaly showed around 80% of dry mass accumulation in the aerial part, without significant difference among the culture medium (
The largest root dry mass accumulation of Poloskei Muskotaly was obtained when cultivating in Roubelakis formulation, with 8.89 mg of dry roots, superior to DSD1 (6.17 mg) and Galzy (5.61 mg) formulas and without differing significantly from C2D and ZL with an accumulation around 8.00 mg of dry roots. Although not the best formula for root dry mass variable, Poloskei Muskotaly cultivated with DSD1 is between the limits of 2.7 mg and 7.6 mg of dry root as defined by Borghezan et al. [
For ex vitro acclimatization, Poloskei Muskotaly cultivar showed, after 60 days, 100% of survival independently of the culture medium used on in vitro propagation phase. In relation to the effects of acclimatization environment on survival rate of Jales’ vine rootstock, Biasi et al. [
After acclimatization, it was possible to detect effects of culture medium formulas on Poloskei Muskotaly cultivar plants (
Longer aerial parts of acclimatized Poloskei Muskolaly cultivar are found when being previously cultivated in Roubelakis, ZL, C2D and DSD1formulas, in an interval of 8.20 cm to 10.60 cm; however, Galzy substract was significantly inferior to Roubelakis and ZL formulas (
IRC of acclimatized plants was lower than in vitro readings and it was not determined effects of culture medium in this phase, possibly because plants were submitted to the same nutritional conditions unlike in vitro phase (
After 60 days of acclimatization, the accumulation of dry biomass was bigger for Poloskei Muskotaly plants
Culture Medium | Number | Length (cm) | |||||
---|---|---|---|---|---|---|---|
Leaves | Root | Longer Root | Aerial Part | TR | IRC | ||
Roubelakis | 7.5 a | 3.1 a | 10.1 a | 6.4 a | 6.2 a | 27.4 a | |
ZL | 5.3 b | 2.6 ab | 10.4 a | 4.7 b | 4.7 b | 24.2 b | |
Galzy | 7.2 a | 1.9 ab | 8.5 a | 5.5 ab | 5.6 ab | 21.7 bc | |
C2D | 6.1 ab | 1.8 bc | 9.1 a | 4.7 b | 5.1 ab | 27.1 a | |
DSD1 | 6.54 ab | 1.9 bc | 9.6 a | 5.8 ab | 5.5 ab | 21.2 c | |
C.V. (%) | 18.63 | 70.1 | 33.99 | 29.65 | 29.12 | 6.71 | |
Culture Medium | Dry Mass (mg) | R (%) | E (%) | ||||
Roots | Aerial Part | Total Biomass | |||||
Roubelakis | 8.9 a | 39.6 a | 49.2 a | 100.0 | 100.0 | ||
ZL | 8.0 ab | 40.8 a | 50.8 a | 100.0 | 100.0 | ||
Galzy | 5.6 b | 34.3 a | 39.9 a | 100.0 | 100.0 | ||
C2D | 8.0 ab | 35.8 a | 44.4 | 100.0 | 100.0 | ||
DSD1 | 6.2 b | 34.0 a | 40.2 a | 100.0 | 100.0 | ||
C.V. (%) | 33.71 | 38.13 | 20.79 | -- | -- | ||
Averages followed by the same letter in the column do not differ among them by Tukey test (p < 0.005).
Culture Medium | Number | Length (cm) | IRC | ||
---|---|---|---|---|---|
Leaves | Root | Longer Root | Aerial Part | ||
Roubelakis | 7.3 a | 4.0 a | 26.4 ab | 10.6 a | 17.8 a |
ZL | 8.7 a | 3.5 a | 29.3 a | 10.6 a | 17.7 a |
Galzy | 4.8 b | 2.8 a | 13.1 b | 7.8 b | 17.9 a |
C2D | 6.2 a | 4.4 a | 25.2 ab | 8.2 ab | 17.8 a |
DSD1 | 5.5 ab | 3.7 a | 19.0 ab | 8.4 ab | 17.9 a |
C.V. (%) | 34.57 | 32.98 | 35.57 | 15.38 | 12.78 |
Culture Medium | Dry Mass (mg) | TS (%) | |||
Roots | Aerial Part | Total Biomass | |||
Roubelakis | 65.7 a | 210.1 a | 275.8 a | 100.0 | |
ZL | 82.8 a | 226.5 a | 309.3 a | 100.0 | |
Galzy | 26.5 b | 124.7 b | 151.2 b | 100.0 | |
C2D | 72.7 a | 220.2 a | 293.5 a | 100.0 | |
DSD1 | 37.2 b | 142.6 b | 179.8 b | 100.0 | |
C.V. (%) | 33.60 | 21.18 | 23.70 | -- |
Averages followed by the same letter in the column do not differ among them by Tukey test (p < 0.005).
cultivated in vitro in Roubelakis, ZL and C2D formulas in relation to DSD1 and Galzy. This result shows that conditions of nutrients availability from culture medium in in vitro micropropagation phase have influence on plants growing in the posterior phase of ex vitro acclimatization, during heterotrophic and autotrophic conditions.
In respect of biomass allocation in different acclimatized plant organs, the greatest accumulation occurred on the aerial part and the best results for dry material from “aerial part” and “roots” variables followed the same order of total biomass (
In experiment 2, in vitro cultures from IAC 571-6 rootstock showed greater growth of aerial parts and roots in Roubelakis and ZL culture medium. This is reflected on dry biomass accumulation; likewise, replication rate and leaves and roots number was superior in Roubelakis medium, followed by ZL (
For “leaves number” variable, the greatest leaves quantity in IAC 571-6rootstock is found when it is cultivated in Roubelakis formula with a formation of 10.6 leaves per explant (
Concerning the number of roots for IAC 571-6 rootstock, Roubelakis showed greater average with formation of 2.05 principal roots per plant, which did not differ from ZL formula (
Longer length of roots from IAC 571-6 rootstock was observed when cultivating in Roubelakis and ZL mediums, differing significantly from the others (
The best development of vine aerial part cultivars in Roubelakis formula was observed by Roubelakis-Ange- lakis and Zivanovitc [
In relation to IRC, values for SPAD-502 readings revealed formulas from culture medium have influenced chlorophyll index (
For total biomass production of IAC 571-6 rootstock, the greatest dry mass accumulation was reached when rootstock was cultivated in Roubelakis and ZL formulas forming 69.8 mg and 57.2 mg of dry mass (
In relation to in vitro biomass allocation, similarly to Poloskei Muskotaly cultivar, IAC 571-6 rootstock showed higher dry mass accumulation from the aerial part, representing in average 76% of the total (
In vitro cultures of IAC 571-6 rootstocks expressed high potential of regeneration and rooting, whatever the culture medium used (
Culture Medium | Number | Length (cm) | |||||
---|---|---|---|---|---|---|---|
Leaves | Root | Longer Root | Aerial Part | TR | IRC | ||
Roubelakis | 10.6 a | 2.1 a | 13.5 a | 7.4 a | 9.5 a | 22.6 abc | |
ZL | 7.1 b | 1.8 ab | 14.9 a | 6.5 a | 6.6 b | 22.0 bc | |
DSD1 | 5.8 b | 0.7 c | 8.9 b | 3.9 b | 4.54 c | 24.2 ab | |
C2D | 6.3 b | 1.2 bc | 7.6 b | 4.5 b | 5.3 c | 25.7 a | |
Galzy | 6.2 b | 1.2 bc | 7.7 b | 4.3 b | 4.9 c | 20.4 c | |
C.V. (%) | 12.94 | 23.21 | 39.5 | 28.15 | 14.37 | 16.82 | |
Culture Medium | Dry Mass (mg) | R (%) | E (%) | ||||
Roots | Aerial Part | Total Biomass | |||||
Roubelakis | 18.4 a | 54.5 a | 69.8 | 100.0 | 100.0 | ||
ZL | 11.3 b | 45.9 a | 57.2 a | 100.0 | 100.0 | ||
DSD1 | 7.6 b | 26.7 b | 31.9 b | 100.0 | 100.0 | ||
C2D | 3.2 c | 25.0 b | 28.2 b | 100.0 | 100.0 | ||
Galzy | 4.5 c | 22.3 b | 26.8 b | 85.0 | 100.0 | ||
C.V. (%) | 28.75 | 32.41 | 33.77 | -- | -- | ||
Averages followed by the same letter in the column do not differ among them by Tukey test (p < 0.005).
Culture Medium | Number | Length (cm) | IRC | ||
---|---|---|---|---|---|
Leaves | Root | Longer Root | Aerial Part | ||
Roubelakis | 7.2 a | 2.2 a | 29.8 a | 11.1 a | 16.9 a |
ZL | 6.2 a | 1.8 a | 28.3 a | 11.4 a | 16.9 a |
DSD1 | 6.0 a | 1.7 a | 26.8 a | 9.7 a | 17.2 a |
C2D | 7.0 a | 1.6 a | 25.9 a | 11.1 a | 17.4 a |
Galzy | 6.7 a | 2.0 a | 25.5 a | 10.2 a | 17.0 a |
C.V. (%) | 2.17 | 46.77 | 22.60 | 12.50 | 19.35 |
Culture Medium | Dry Mass (mg) | TS (%) | |||
Roots | Aerial Part | Total Biomass | |||
Roubelakis | 62.4 a | 199.2 a | 261.6 a | 100.0 | |
ZL | 63.2 a | 196.9 a | 260.1 a | 100.0 | |
DSD1 | 50.7 a | 134.9 b | 185.6 b | 100.0 | |
C2D | 49.2 a | 154.9 ab | 204.1 ab | 85.7 | |
Galzy | 60.2 a | 173.3 ab | 233.5 ab | 100.0 | |
C.V. (%) | 30.42 | 18.76 | 20.68 | -- |
Averages followed by the same letter in the column do not differ among.
occurred the regeneration of 85% of inoculated nodal segments. However, all growing explants formed roots, so suggesting there is no need of growth regulator application which promotes rooting or specific phase for rooting. Similar results were found by Dzazio et al. [
At the end of acclimatization phase, IAC 571-6 vine rootstock showed high index of survival similar to that obtained in other researches [
Effects that culture medium carried out on in vitro development were not too expressive and evident in acclimatized plants (
Formulas of culture medium showed effects on morphological and physiological parameters of in vitro propagation and acclimatized vine cultivars. Multiplication rates and variant growth values of the cultivars demonstrated the importance of culture medium selection without growth regulators, since the addition of those ones to nutritive medium could not be favorable due to the induction of undesirable somaclonal mutations. Results in this research are in agreement with those found by Roubelakis-Angelakis and Zivanovitc [
In vitro IAC 571-6 rootstock and Poloskei Muskotaly cultivar propagation through nodal segments obtained from mother plants maintained in greenhouse has promoted high indexes of regeneration and rooting.
In vitro root formation of IAC 571-6 rootstock and Poloskei Muskotaly cultivar has occurred in culture medium without growth regulators, and it is not needed a specific phase for root formation.
Considering all the analyzed variables, saline formula Roubelakis has promoted better growing and development of aerial parts and roots as well the best in vitro multiplication of IAC 571-6 and Poloskei Muskotaly vine cultivars.
Thanks to the Coordination of Improvement of Higher Education (CAPES) for the scholarship award to the first author at the Graduate School. We also thanks to the Company of Agriculture Research and Rural Extension of Santa Catarina for providing the structures for execution of the work.
Jean CarlosBettoni,Murilo DallaCosta,João PetersonPereira Gardin,Aike AnnelieseKretzchmar,JulianaAparecida Souza, (2015) In Vitro Propagation of Grapevine Cultivars with Potential for South of Brazil. American Journal of Plant Sciences,06,1806-1815. doi: 10.4236/ajps.2015.611181