13 Analele ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi Tomul LVII, fasc. 2, s. II a. Biologie vegetală, 2011 INFLUENCE OF GENOTYPE AND CULTIVATION CONDITIONS ON VITROPLANTLETS EVOLUTION OF SOLANUM TUBEROSUM L. LOCAL VARIETIES IUSTINA BRÎNDUŞA CIOBANU * , DANA CONSTANTINOVICI ** , L. CREŢU *** Abstract: The local potato varieties from Gene Bank Suceava are maintained in a field collection and also by slow growth of in vitro culture. Slow growth method allows limited development of plantlets and the extension of time between two subcultures. This paper presents a part of the results about the influence of genotype, inhibitors from the media and conditions from the conservation room (temperature and light reduction), on potato plantlets preserved in vitro for different periods. Three grows inhibitors (daminozide, mannitol and sorbitol) and five local potato varieties of were included in the experiments. Several morphological features: number, length and branching of shoots, rooting, number of viable nodes, number and size microtubers and the viability rate of the shoots, were evaluated. Although there were differences in the growth type of potato varieties the main effect was the reduction of plantlets height, by shortening of internodes and leaves size. The leaf blade became very short (1,5 – 2 mm), many of them growing on the surface of some branches having a hypertrophic and translucent aspect. Could be noted a tendency to generate, also, well-defined microtubers bearing microbranches. The results on the response of plantlets developed on storage media recommended the medium C24, with 40 g / l sorbitol, as the best in terms of in vitro conservation of local potato varieties. Keywords: potato, inhibitors of growth, slow growth, biometry Introduction Potato (Solanum tuberosum L.) is a major crop worldwide, providing at least 12 essential vitamins, minerals, proteins, carbohydrates and iron [2, 8, 10]. Starting from the need to preserve a longer time a valuable biologic material, in terms of agronomic qualities, and avoid the excess of material resulting from the multiplication, were found effective methods for in vitro conservation of potato. An appropriate storage method for germplasm is the conservation of material under slow growth [1]. The principle of storage by slow growth allows the safe use of in vitro culture without frequent subculturing disadvantages. Cultures can be observed while growing up and can be returned to normal subculture for multiplication [11].Conservation by slow growth techniques have been developed for medium-term storage of crop plants [3, 9]. The various methods used to achieve these techniques include: use of growth inhibitors, a minimal growth medium, osmotic active substances, reduction of O 2 concentration, type and size of culture vessels, low temperature, light intensity or a combination of several methods [7]. Combination of osmotic active substances with lowering of temperatures and light intensity is the most effective way to extend the period of subculture on potato [4]. This method of in vitro conservation is complementary to other forms of storage and particularly useful for local potato varieties. * The Technical College ,,Samuil Isopescu” Suceava, Romania, [email protected]** Gene Bank Suceava, Romania *** University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania
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Analele ştiinţifice ale Universităţii „Al. I. Cuza” IaşiTomul LVII, fasc. 2, s. II a. Biologie vegetală, 2011
INFLUENCE OF GENOTYPE AND CULTIVATION CONDITIONS ONVITROPLANTLETS EVOLUTION OF SOLANUM TUBEROSUM L.
LOCAL VARIETIES
IUSTINA BRÎNDUŞA CIOBANU*, DANA CONSTANTINOVICI**, L. CREŢU***
Abstract: The local potato varieties from Gene Bank Suceava are maintained in a field collection andalso by slow growth of in vitro culture. Slow growth method allows limited development of plantlets and theextension of time between two subcultures. This paper presents a part of the results about the influence ofgenotype, inhibitors from the media and conditions from the conservation room (temperature and light reduction),on potato plantlets preserved in vitro for different periods. Three grows inhibitors (daminozide, mannitol andsorbitol) and five local potato varieties of were included in the experiments. Several morphological features:number, length and branching of shoots, rooting, number of viable nodes, number and size microtubers and theviability rate of the shoots, were evaluated. Although there were differences in the growth type of potato varietiesthe main effect was the reduction of plantlets height, by shortening of internodes and leaves size. The leaf bladebecame very short (1,5 – 2 mm), many of them growing on the surface of some branches having a hypertrophicand translucent aspect. Could be noted a tendency to generate, also, well-defined microtubers bearingmicrobranches. The results on the response of plantlets developed on storage media recommended the mediumC24, with 40 g / l sorbitol, as the best in terms of in vitro conservation of local potato varieties.
Keywords: potato, inhibitors of growth, slow growth, biometry
Introduction
Potato (Solanum tuberosum L.) is a major crop worldwide, providing at least 12essential vitamins, minerals, proteins, carbohydrates and iron [2, 8, 10].
Starting from the need to preserve a longer time a valuable biologic material, interms of agronomic qualities, and avoid the excess of material resulting from themultiplication, were found effective methods for in vitro conservation of potato. Anappropriate storage method for germplasm is the conservation of material under slowgrowth [1]. The principle of storage by slow growth allows the safe use of in vitro culturewithout frequent subculturing disadvantages. Cultures can be observed while growing upand can be returned to normal subculture for multiplication [11].Conservation by slowgrowth techniques have been developed for medium-term storage of crop plants [3, 9]. Thevarious methods used to achieve these techniques include: use of growth inhibitors, aminimal growth medium, osmotic active substances, reduction of O2 concentration, typeand size of culture vessels, low temperature, light intensity or a combination of severalmethods [7].
Combination of osmotic active substances with lowering of temperatures and lightintensity is the most effective way to extend the period of subculture on potato [4]. Thismethod of in vitro conservation is complementary to other forms of storage and particularlyuseful for local potato varieties.
* The Technical College ,,Samuil Isopescu” Suceava, Romania, [email protected]** Gene Bank Suceava, Romania*** University of Agricultural Sciences and Veterinary Medicine, Iaşi, Romania
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Materials and methods
Experiments were conducted at Suceava Genebank with five local potato varietiescultivated in vitro by slow growth. The development of microcuttings on three conservationmedia and, also, on a multiplication medium, as control, for a period of 12 months, in orderto assess the role daminozide (as grows inhibitor) and the influence of mannitol and sorbitol(as osmotic agents) on medium-term potato conservation was studied.
The culture media used for conservation were based on Murashige-Skoogformulation (1962) in ½ dilutions, with low concentrations of growth regulators, sucrose 2-3%, with the addition of daminozide, mannitol and sorbitol, resulting three variants ofculture media. Control samples were maintained in the same ambient conditions, like theconservation samples, on MS medium [6] supplemented with 40 g/l sucrose and 6 mg/ldaminozide (Tab. I). Knowing the positive effect of kinetin on the tolerance of plantlets tolow temperatures and long periods of subculture [5], in order to help the control samples toovercome the storage conditions, the amount of this growth regulator in the medium was 50times higher. Thereby was favored the growth and development of potato plantlets duringthe experiment.
Table I. Variants of cultured media used for in vitro preservation local potato genotypes
Culture medium variant Basal culture medium and hormonal balance Other components
The research was initiated using microcuttings with 1-2 nodes taken from localpotato populations SVGB 14376 (genotype 1) SVGB 15079 (genotype 2), SVGB 15102(genotype 3), SVGB 15140 (genotype 4) and 15446 SVGB (genotype 5), maintained atSuceava Genebank on in vitro collection.
The pH of culture media was adjusted to 5.7 with 0.1N NaOH before autoclavingand was solidified medium with 0,75% agar. Sterilization of media was done byautoclaving at 1210C for 20 minutes. Culture vessels used were the glass jars of 170 ml(control medium) and 120 ml (for storage media), with wide opening, containing 20 mlculture medium. For each media variant, including control, respectively for each localvariety of potato were inoculated twenty microcuttings, respecting their polarity. In each jarwere placed two microcuttings. After placing the inoculums, the jars were covered withdouble polyethylene film and introduced in the growing room under the light of 2000-2500lx, with a photoperiod of 16 hours per day, at a temperature of 20 - 220C. The moisture inthe growth chamber was 60-70%.
After 4 weeks, the samples were transferred to storage room and kept at 6-120C,under the alternation of light (photoperiod 10/24 hours), in white fluorescent illuminationhaving an intensity of about 1000 lx.
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After 7 and 12 months, starting from initiation of the experiment, some biometricscharacteristics like the number, length and branching shoots, rooting, number of viablenodes and the percentage of viable shoots from the four culture media were studied.
Results and discussions
The data obtained from biometric studies performed on the plantlets grown on thethree storage media, and those grown on control medium, reveals that the three growthinhibitors (daminozide, mannitol and sorbitol) determined great response variability interms of analyzed characters.
Stress caused by the addition of inhibitor in the culture media and reduction oftemperature in the storage room, influenced the number of shoots/plantlet, the shootsheight, and the response varied according to genotype, media variant, and was influencedby the storage period, too.
The five potato genotypes analyzed showed different capacities of in vitroformation of shoots and the extension of conservation period was accompanied by thereduction of viable shoots numbers to the plantlets grown on media M14 and C22 and theincrease of this number on media C23 and C24 (Fig. 1). Decreased number of viableshoots/plantlet was more pronounced on the control medium (M14).
After 7 months of plantlets maintenance in conservation conditions, the highestaverage number of shoots/plantlets was obtained from genotypes 1 and 3 (with an averageof 12,7), followed by genotypes 5 and 4 (average of 10,2 and 10,0 viable shoots/plantlets);the lowest number was recorded in genotype 2, with an average of 7,5 viableshoots/plantlet.
After 12 months of storage in conditions of slow growth the highest averagenumber of shoots/plantlet was obtained from genotype 1 (with an average of 16,5),followed by genotype 3 (13,2), genotype 5 (11,2) and genotype 4 (10,7). The genotype 2has the fewest number of viable shoots/plantlet (average 6,0), also, after 12 months ofconservation.
The culture medium influenced, also, the number of shoots formed. Thus, after 7months of conservation the highest average number of shoots/plantlet was obtained oncontrol medium (M14), followed by C24 medium. Medium variant C22 and C23 gave the mostreduced results regarding this character. The average recorded values were much lower thanthe control medium, with up to 71,4% and 73,3% in genotypes 2 and 4 from the C22medium, and with up to 72,7% and 68,4% in genotype 3 and 5 on the C23 medium. Theextension of the storage period to 12 months, decreased the number of viableshoots/plantlet on M14 and C22 media and, generally, increased the number ofshoots/plantlet maintained on the media C23 and C24; the highest average number ofshoots/plantlet has been obtained on the C24 medium with sorbitol.
Regarding the interaction genotype x culture medium the number of viableshoots/plantlet can be seen that the highest values of this character are recorded, after 7months of storage, to the plantlets of genotype 3, from the control medium (M14), with anaverage number of 22,0 shoots/plantlet, and after 12 months of conservation, to theplantlets of genotype 1, on the medium with sorbitol (C24), with an average of 31,0 viableshoots/plantlet.
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By extending the storage period from of 7 to 12 months, the average length ofplantlets decreased, more or less, in all varieties and all variants of media analyzed (Fig. 2).A stronger inhibition of shoot elongation could be observed on the media C22 and C23, forall genotypes studied. Thus, compared to the control, there has been an average reduction ofshoot height with 2,88 cm after 7 months and 3,70 cm after 12 months, to medium C22 andwith 3,38 cm after 7 months and 2,36 cm after 12 months, to medium C23. In the case ofmedium C24 the average height of shoots reduction was of 2,56 cm after 7 months and 1,32cm after 12 months of in vitro conservation.
Regarding control medium (M14), the extension of the storage period from 7 to 12months caused a reduction in height of shoots, with an average of 1,96 cm, but allowed theincrease of viable shoots/plantlet to SVGB 14376 and SVGB 15102 varieties. The shootson the medium M14 were frailer, with longer internodes compared to plantlets from thethree storage media.
The number of viable nodes is very important for the regeneration phase of thebiological material, after phase of conservation. The average number of nodes/plantlet wasinfluenced by genotype and the type of inhibitor of growth.
Of the five genotypes studied, plantlets of genotype 1 had the bigger number ofviable nodes (with an average of 34,7 nodes/plantlet, after 7 months and 55,2 nodes/plantletafter 12 months of storage), followed by plantlets of genotype 3 (with an average of 29,6nodes/plantlet, after 7 months and 37,1 nodes/plantlet after 12 months storage) (Fig. 3). Thehighest average number of viable nodes/plantlet was recorded for plantlets maintained onthe control medium (M14) after 7 months and on the medium with sorbitol (C24) after 12months of maintenance in vitro (Figs. 4a, 4b).
It was found a direct correlation between the height of plantlets and the number ofnodes from the plantlets maintained on storage media for 12 months, but not in plantletsmaintained on control medium, where the length of internodes was much longer. Thiscorrelation was not found after 7 months of storage in vitro. The presence of daminozidedetermined the formation of biggest number of nodes/plantlet (an average of, 31,4) after 7months of storage; sorbitol and mannitol, with an average of 52,3 and respectively 34,8nodes/plantlet, influenced more this character after 12 months of conservation by slowgrowth.
The survival of shoots varied depending on genotype and culture medium after thetwo periods of in vitro preservation (Tab. II). After 7 months of in vitro conservation of thestudied genotypes, the rate of viability varied from 66,7% of the total shoots number, onC22 medium, up to 87,3% on C24 medium. Increasing the culture period on the samemedium to 12 months, led to a decrease of shoots survival with 24,9%, on the controlmedium (M14), with 21,6% on the medium with daminozide (C22), with 6,9% on mediumwith mannitol (C23) and 6,0%, on the medium with sorbitol (C24).
The highest survival rates of shoots were obtained on medium with sorbitol (C24)after 7 and, also, after 12 months of plantlets preservation under slow growth conditions.Among the genotypes used in the experiments, the plantlets of genotypes 1 and 5 recordedthe best evolution of shoots after 7 months, with a rate of viability of 93,07 and respectively95,50%; with 70,1% and 84,72% survival rates of shoots the same genotypes had the bestresults after 12 months of conservation (Figs. 5, 6). The plantlets of genotype 2 showed thelowest shoots evolution after both periods of conservation (60,9% after 7 months and48,1% after 12 months).
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Table II. Effect of culture media and genotype influence on survival of shoots after different periodsof storage in vitro
Viability rate shoots (%)Culture medium Genotype7 months 12 months
The experimental data showed the influence of growth inhibitors and physicalconservation conditions on the morphological characteristics of potato plantlets: length ofshoots was lower compared to the samples grown on the control medium (M14). Theplantlets, grown on M14 medium, recorded the highest growth in length of shoots, after 7and 12 preservation months, with a survival shoots rate of 84,3%, respectively 59,4%. After12 months of conservation, plantlets grown on the control medium became elongated andfragile, with pale leaf.
The conservation media inhibited progressively the growth of plantlets, producingshoots with very small leaves, having a dark green color and short internodes. The biggestinfluence in terms of a limited growth had the medium variant with 30 mg/l daminozide(C22) which reduced most the average length of shoots, but caused, also, the lowest survivalrate of plantlets after 12 months of conservation. The mannitol and the sorbitol added inculture medium (40 g/l), through the osmotic effect led to reduced shoot growth and had apositive effect for tolerance of plantlets at low temperatures and extended shelf-life.
The best medium option, from the standpoint of in vitro conservation on themedium term of local potato varieties under study, has proved to be variant C24, with 40 g/lsorbitol, where was recorded a survival rate of shoots of about 85,5% after 7 months and79,5% to 12 months of conservation. Among the five potato varieties analyzed, the bestdevelopment of shoots, after 12 months of maintenance in vitro by slow growth on the threestorage media used, had the variety SVGB 15446 (genotype 5), with a survival rate of
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shoots of 84,7%, and the lower evolution was recorded in the variety SVGB 15079(genotype 2), with 48,1% survival rate of shoots.
REFERENCES
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Explanation of the plates
Plate IFigure 1. Influence of genotype and culture medium on the number of viable shoots/plantlet at 7 and 12 months ofstorage under slow growth conditionsFigure 2. Influence of genotype and culture medium on the average length of viable shoots/plantlet at 7 and 12months of storage in conditions of slow growthPlate IIFigure 3. Influence of genotype and culture medium on the number of viable nodes/plantlet at 7 and 12 months ofstorage under slow growthFigures 4a, 4b. Plantlets developed after 12 months of conservation, on the culture medium with sorbitol (C24) tothe genotype SVGB-15140 (general imagine 4a and detail 4b)Figure 5. Plantlets developed after 7 months of conservation, on the culture medium with sorbitol (C24) to thegenotype 1 (SVGB-13476)Figure 6. Plantlets developed after 12 months of conservation, on the culture medium with sorbitol (C24) to thegenotype 1 (SVGB-13476)
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Iustina Brînduşa Ciobanu, Dana Constantinovici, L. Creţu PLATE I
