The experiment was carried out in the Department of Crop Science and Technology, and Plant Breeding and Gene Engineering Laboratory under Rajshahi University and in the field of Shyampur, Katakhali, Rajshahi, Bangladesh during the period from December, 2010 to March, 2012. Two exotic potato varieties Diamant and Cardinal and two indigenous varieties Shilbilati and Lalpakri were used in the investigation collected from A. H. Z. BIOTEK SEEDS Ltd. nursery, Namo Bhadra, Rajshahi.

The plant growth regulators (PGR) such as indole-3-butyric acid (IBA) under auxin group; 6-benzyl aminopurin (BA) and 6-Furfuryl aminopurin (KIN) under cytokinin group; and Gibberellic acid-3 (GA₃) were used in this study. Generally 0.1% HgCl₂ was used as surface sterilizing agent, tween-20 (polyoxyethelene soribitan monolaurate) and savlon (Antiseptic, ACI Pharma, Bangladesh) were used as surfactant and detergent. MS medium was used for all kinds of in vitro experiments.

Different plant growth regulators (PGR) viz. auxin (IBA), cytokinin (BA and KIN), and gibberellic acid at different concentrations were used in two dimensions for making different media combinations. For the primary establishment of meristems there involved four treatment combinations composed of KIN and GA₃ at their various concentrations viz. 0.1 mg/L KIN + 0.1 mg/L GA₃, 0.1 mg/L KIN + 0.5 mg/L GA₃, 0.5 mg/L KIN + 0.1 mg/L GA₃ and 0.5 mg/L KIN + 0.5 mg/L GA₃. After the establishment of primary meristems they were subcultured on to MS semisolid basal medium supplemented with IBA, BA, KIN and gibberellic acid in different combinations at various levels of concentrations formulated by four media treatments viz. 0.5 mg/L BA+ 1.0 mg/L IBA; 0.1 mg/L KIN + 0.1 mg/L GA₃; 0.5 mg/L BA + 0.5 mg/L GA₃ and 0.5 mg/L KIN + 0.5 mg/L GA₃ were used to identify the suitable media compositions for shoot proliferation.

MS medium was used for all in vitro experiments which includes meristem culture, micropropagation of meristem derived plantlets, induction of microtuber etc. Sucrose was added at the rate of 30 g/L. According to requirements different growth regulators were supplemented to different media separately. Agar was added at the rate of 8 - 10 g/L for the preparation of semi-solid medium. For primary meristem culture MS liquid medium and bridges were used in liquid to support the meristem and primary culture medium.

Shoot tips of 25 - 30 day old potato plants of Diamant, Cardinal, Shilbilati and Lalpakri grown in the A.H.Z BIOTECH SEEDS Ltd. nursery were used as sources of meristems. The shoot tips were excised with the help of sharp blade and collected in a reagent bottle containing distilled water with few drops of savlon and few drops of tween-20 and quickly brought into the laboratory. Then the explants were washed for 2 - 3 times with gradual change of sterile distilled water. The plant materials were transferred to a 250 mL sterilized conical flask. Surface sterilization was carried out by dipping the plant materials into 0.1% HgCl₂ solution with gentle shaking for 2 - 8 minutes followed by 3 - 5 times washing with sterile distilled water in front of running laminar air flow cabinet.

Shoot tips of surface sterilized plant samples of four potato varieties were laid on the sterile tiles using sterile forceps. Immediately after dissection a single excised meristem was carefully placed on the “M” shaped filter paper bridge within the culture tubes containing liquid MS medium (Figure 1). The neck of the tube was flamed with spirit lamp and then plugged. The inoculated culture tubes were incubated in a growth chamber providing a special culture environment. The cultures were maintained at 22˚C ± 2˚C with light intensity varied from 200 -

3000 lux. Generally the photoperiod was maintained giving 16 hours light and 8 hours dark. The culture tubes were checked daily to note the morphogenic response of cultured meristem.

Three to four weeks after primary culture, the explants those showed morphogenic response were removed aseptically from the culture tubes and transferred into tubes containing agar gelled MS medium supplemented with different growth regulators (Figure 2). After 30 - 35 day of culture initiation shoots were removed aseptically from the culture tubes and placed on a sterile tile and cut into convenient (basal end of the shoot) size and again transferred into test tubes containing different plant growth regulators supplemented in semisolid MS media.

When the generated shoots were 2 - 8 cm in height with 4 - 8 leaves they were removed aseptically from the culture tubes (Figure 3) and placed on a sterilized Petridish, cut from the basal of the shoots using sharp scalpel. Then each base of the shoot was inoculated on freshly prepared semisolid MS medium supplemented with different combinations and concentrations of plant growth regulators for further shoot multiplication and root induction.

The use of serological methods for rapid detection of plant viruses in vitro grown meristem-derived plantlets is popularly known as serodiagnosis [11] [12] [13]. Among the various serological methods so far developed, the double antibody sandwich enzyme-linked immuno-sorbent assay (DAS-ELISA) is extensively applied for its various merits over the other methods are reported by Clark and Bar-Joseph [14]. Different samples representing each symptom were tested

by DAS-ELISA.

After 4 week of incubation when the plantlets attained a height of 7 - 9 cm micropropagation was started. The plantlets of four potato varieties identified as disease-free (checked by ELISA) were used for massive micropropagation.

Nodal segments about 5 - 6 mm long was isolated from disease free plantlets having auxiliary bud (Figure 4). Isolated nodes were transferred into a 250 mL flasks or test tubes containing agar-solidified medium. Node cuttings were incubated at 22˚C ± 2˚C with a 16 hours photoperiod. The developed plantlets were subculture as required after every 5 - 6 weeks (Figure 4).

Well-rooted plantlets from meristem about 4 - 5 cm in height were ready for transplanting into soil. The plantlets grown inside the flask or test tubes were brought out from the growth chamber. After deplugging the culture tubes were kept in the room temperature for 4 - 5 days to bring them in contact to normal temperature. After 3 - 5 days of hardening the plantlets were taken out very carefully from the test tubes. The roots attached to the agar were gently and carefully washed out under running tap water.

After proper acclimatization of in vitro produced virus free plantlets of four potato varieties obtained from the meristem derived plantlets were planted on December, 2011 in the experimental field of Shyampur, Rajshahi covered with insect proof nylon net. The experiment was conducted following complete randomized design with three replications of each variety. The unit plots were 100 × 40 cm in size which accommodated 30 plantlets at a spacing of 20 × 10 cm.

The plantlets were watered immediately after transplanting and the beds were provided with shades from 10 am to 3 pm until the establishment of the plantlets into soil. The soil was prepared with cow dung and course sand (1:1 v/v), Urea, TSP and MP were applied at the rate of 75, 60 and 75 kg/ha. The entire does of TSP and MP and 50% of Urea were applied at the time of land preparation. The rest 50% of Urea was applied as top dressing into two equal installments at 35 and 50 days after planting. The intercultural operations such as earthing up, weeding and mulching were done when needed. Fungicides and insecticides were applied to protect the crops. The experimental plots were irrigated frequently to maintain optimum soil moisture and also to keep the soil cool. The crop was harvested at maturity in the month of March, 2012 from the field within 90 - 100 days of its plantation.

Data from in vitro grown meristem-derived plantlets on percentage of regenerated plantlet, virus free plantlet, explants responded, and days to shoot initiation, number of shoots/explants and shoot length were recorded. Number of shoots/explants and shoot lengths were calculated after 28 days of culture. Percentage of explants responded was calculated using the following formula.

% ofexplantsresponded = Number   of   explants   responded Total   number   of   explants   cultured × 1 00

Data for field-grown plants on different characters were noted from the randomly selected 10 plants of each variety. The plantlets that survived in the field were expressed in percentage according to variety. Days to maturity was recorded when the leaves just began to die. Plant height, number of branches, leaves, tubers and tuber weight per plant, and percentage of tuber grades were recorded at the time of harvest. Weight of tubers was taken with a weighing balance. The harvested tubers were expressed in percentage and categorized in the following grades: 1) Grade A (<20 mm), 2) Grade B (20 - 30 mm), 3) Grade C (30 - 40 mm), and 4) Grade D (>40 mm).

Analysis of variance and subsequent significant test were done according to Gomez and Gomez, 1984. The mean values of all the parameters for four varieties were calculated and the analysis of variance was performed by “F” (variance ratio) test. The significance of differences among the treatment means were evaluated by least significant difference (LSD) test at 5% level of probability [15] using MSTAT-C programme.

Isolated meristems of four potato varieties Diamant, Cardinal, Shilbilati and Lalpakri were placed separately on “M” shaped filter paper bridge in culture tubes (125 × 25 mm) containing liquid MS medium supplemented with different concentrations and combinations of KIN and GA₃ to find out varietal performance of four potato varieties (Figure 1). Data on days to response, percentage of meristems responded and morphogenic responses were recorded after 21 days of culture and are presented in Table 1.

Days to response ranged from 4 - 9 for all varieties with all four combinations. Among four combinations 0.1 mg/L KIN + 0.5 mg/L GA₃ showed better effect on the meristem culture. Cardinal showed the best response within the shortest duration of time taken 4 - 5 days to grow in the media with 0.1 mg/L KIN + 0.5 mg/L GA₃ among four varieties.

Percentage of meristems responded to new growth ranged from 51% - 78% in all four combinations of KIN and GA₃ growth regulators with all potato varieties. The combination 0.1 mg/L KIN + 0.5 mg/L GA₃ exposed as the most appropriate for growth initiation for four potato varieties ranged from 56% - 78% and responding the highest percentage of meristems recorded in Diamant (78%) followed by Cardinal (73%) and Lalpakri (61%) where as the lowest was recorded in Shilbilati (56%). The meristems of four potato varieties produced shoots and roots in all treatment combinations of KIN and GA₃ except with the treatment

+ = Positive responses; − = Negative responses.

0.5 mg/L KIN + 0.5 mg/L GA₃ where Cardinal did not produce any root.

Among the meristem-derived plantlets, a serological DAS-ELISA test was carried out to detect virus before micropropagation. No positive colour was found in the microliter plates when the in vitro grown meristem-derived plantlets were used as samples. In contrast, colour appeared when field-grown plants were used as samples that proved in vitro grown meristem-derived plantlets were free from the viruses.

The nodal segments of the primary meristem-derived shoots developed from the isolated meristems of four potato varieties were cultured separately on to MS semisolid basal medium supplemented with BA, IBA, GA₃ and KIN in different combinations and concentrations. Data on days to shoot initiation, percentage of shoot formation, number of shoots/explant and shoot length (cm) were recorded after 4 weeks of culture and are presented in Table 2.

NS = Non significant.

Shoot multiplication from established meristems of four potato varieties were highly influenced by the four different combinations and concentrations of BA, IBA, GA₃ and KIN. The cultured explants of four potato varieties initiated shoots within very short days ranged from 4 - 13 days. Among four combinations and concentrations, all potato varieties showed shorter duration of shoot initiation taking 4 - 8 days in receiving the treatment 0.5 mg/L BA + 0.5 mg/L GA₃. Among the varieties Cardinal exposed better response with this combination that required 5 - 6 days for shoot initiation followed by Diamant which required 5 - 7 days for the same.

Percentage of shoot proliferation was ranged from 56% - 76% among four potato varieties. All varieties showed better performance regarding percentage of shoot formation with the treatment 0.5 mg/L BA + 0.5 mg/L GA₃ and ranged from 60% - 76% among four different combinations. The highest (76%) nodal segments induced shoot proliferation was observed in Cardinal with this combination of growth regulator.

Number of shoots/explant of four varieties ranged from 2.8 - 4.8. The highest number of shoots/explant was recorded 4.8 in Diamant among all potato varieties with the treatment of 0.5 mg/L BA + 0.5 mg/L GA₃. The length of shoots of four potato varieties ranged from 6.5 - 9.8 cm. The longest length (9.8 cm) of shoots was observed with the supplemented treatment medium of 0.5 mg/L BA + 0.5 mg/L GA₃.

However, it was observed that genotypic traits of four varieties of potato were more or less same treated with all media formulations. The optimum response was exposed in the medium supplemented with the treatment 0.5 mg/L BA + 0.5 mg/L GA₃ for all potato varieties out of four media combinations. Diamant showed the highest response onto these PGR supplemented media among all varieties.

The data for field performance of in vitro produced meristem-derived plantlets were recorded on survival percentage of transplanted plantlets, days to tuber initiation (DTI), days to maturity (DM), plant height (PH), number of branches/plant (NB), number of leaves/plant (NL), number of tubers/plant (NT), tuber weight/plant (TW) and tuber grade (%), and the results are presented in Table 3 and Table 4. In vitro produced plantlets of all cultivars acclimated well in outdoor condition (Figure 4) and grew in the field normally up to maturity (Figure 5 and Figure 6).

The variety Cardinal showed the highest percentage (90%) of plantlets survived in the field was closely followed by Diamant (85%). The lowest survival rate was observed in the indigenous variety Lalpakri (62%). Between the two indigenous varieties the highest survival rate (70%) was found in Shilbilati (Table 3).

Each value indicates the average of three (3) replications with standard deviation in parentheses. DTI = Days to tuber initiation, DM = Days to maturity, PH = Plant height, NB = Number of branches/plant, NL = Number of leaves/plant, NT = Number of tubers/plant, TW = Tuber weight/plant. The mean values were calculated from three replications.

Each value indicates the average of three (3) replications with standard deviation in parentheses.

The number of days required for tuber initiation varied with different varieties (Table 3). Lalpakri took the maximum days (35.20) which was statistically different from other varieties. Cardinal needed the shortest period (29.0 days) for tuber initiation. The maximum number of days (105.42) needed for maturity of the plant was recorded in Lalpakri that significantly differed from the rest of the varieties (Table 3), and closely followed by Shilbilati (99.42). The minimum number of day was required for maturity of the plants in Diamant (90.20) followed by Cardinal (93.53) and both differed from each other significantly.

In terms of plant height, Diamant attained the longest height (67.50 cm) (Table 3) that took minimum number of days to maturity and its height was significantly different from the rest of the varieties. The shortest height was recorded in Lalpakri (49.50 cm) that closely followed by Shilbilati (50.50 cm). The highest number of branches/plant was observed in Shilbilati (7.48), which was significantly higher than the rest of the varieties (Table 3). The lowest number of branches/plant was observed in Lalpakri (3.45) which was closely followed by Diamant (3.99) and Cardinal (4.50). The number of leaves/plant varied significantly with the varieties (Table 3). The highest number of leaves/plant was recorded in Cardinal (240.30) that differed significantly from other varieties and the second highest number was in Diamant (207.50). The lowest number of leaves/plant was found in Lalpakri (151.30) followed by Shilbilati (170.50).

Shilbilati produced the highest number of tuber per plant (27.52) followed by Lalpakri (24.60) and Cardinal (21.50) while the lowest number was recorded in Diamant (19.32) (Table 3). The greatest tuber weight/plant was obtained from Cardinal (343.40 g), which was significantly higher than those of the rest varieties (Table 3). The second highest tuber weight/plant was found in Diamant (320.50 g), and the lowest was recorded in Lalpakri (102.30 g).

The results on tuber grade classified on the basis of different sizes are presented in Table 4. It was observed that the two high yielding exotic varieties Cardinal and Diamant always produced tubers more than 20 mm in size. Lalpakri produced the highest (76%) percentage of tubers of this grade.

Significantly higher percentage of tubers of 20 - 30 mm in size was recorded in the exotic variety Cardinal (79%) which was identically similar to Diamant (78%). In contrast, the indigenous varieties Shilbilati and Lalpakri produced the same grade (20 - 30 mm) of tubers those were significantly lower at 18% and 19% respectively. In the case of 30 - 40 mm size the highest percentage was observed in Diamant (17%) followed by Cardinal (15%) while the lowest percentage in this grade was observed in Lalpakri (5%).

The indigenous varieties Shilbilati and Lalpakri failed to produce tubers greater than 40 mm in size and only the high yielding varieties produced tubers of this grade. The highest percentage of tuber in this grade was observed in Cardinal (6%) followed by Diamant (5%) and these were identically similar to each other.