Thesuccess of plant tissue culture largely depends on the type of nutrient medium,including its chemical composition and physical form (Murashige, 1974).
Various typesof nutrient media have been reported forbanana shoot tip culture viz., B5 media (Gamborg et al., 1968), N6 media (Chuet al., 1975), Linsmaier and Skoog (LS) (Linsmaier and Skoog, 1975) media etc.but most of the investigators used modified Murashige and Skoog (MS) media(Brown et al., 1995). The microbialcontaminants are normally invade the medium right from the beginning and theexposure time varies with the volume of the liquid to be sterilized.
Inthe present study, the high grade chemicals were used (Hi-Media, India) forstock solution preparation and the growth regulators were of Sigma made (SigmaResearch Laboratory, America) that are comparatively free from impurities. Stocksolutions of major and minor salts, vitamins and plant growth regulators wereprepared in double distilled water at required concentrations and kept in refrigeratorsat 4oC until subsequent use. The stock solutions areexamined periodically for possible precipitation or contaminants. In thepresent investigation MS (Murashige and Skoog, 1962) nutrient medium, was used tostandardize the banana micropropagation protocol.
TheMS nutrient media was prepared by adding appropriate quantity of nutrients andgrow regulators into a one liter standard flask containing double distilledwater. The carbon source, 3% sucrose wasadded and dissolved in the media. The pH of the medium was adjusted between 5.6– 5.8 by adding 0.1 N NaOH or 0.1 N HCl using electronic digital pH meter. Themedia was polymerized with 0.
8 % bacteriological grade Agar – Agar (Hi-Media,Mumbai) on hot plate, well whirled for proper mixing and homogenized. Afterhomogenization about 40 ml of media was dispensed into culture bottles, thesebottles were closed with ebonite screw caps and autoclaved at 15 lb/in2 (1200C)for 15 minutes, care must be taken not prolong the autoclaving beyond times asit may bring about degradation of sugars, vitamins and growth regulators andeven of agar – agar. The media is stored for about six days after sterilizationto observe the presence of contamination by microorganisms. 3.2.Directorganogenesis: 3.2.1.
Material collection and maintenance The healthy four weekold sword suckers from Musa paradisiaca Monthan cv. Karibale weighing about onekilogram uniformly sized were identified from elite plants of superiorqualities such as bunch weight of above 40 kg’s, more than ten number of handsin a bunch, more than eighteen number of fingers in second hand from top andlength of peduncle more than 20 to 24 inches was selected and cut from thepseudostem 15 cm above the base level, weighing 500 to 1500 g were collected fromthe banana fields of a progressive farmer and used as starting material formicropropagation.Thesesuckers were transported to the Department of Biotechnology and Bioinformatics,Kuvempu University, Shankaraghatta and arranged on sand bed under shade andwatered regularly. Periodically fungicides/ bactericides are applied to thesesuckers till they are taken for initiation process. The disinfection of suckerswas carried out thoroughly washed with tap water, soaked in a solution of 0.
2%bavistin and 0.1% streptocyclin for overnight. The corm portion was trimmed toa width of 1 inch and height about 2 inches, and sterilized using 0.
001 Mteepol solution for an hour and washed 3-4 times using sterile distilled water toremove traces of teepol. 3.2.
2.Surface sterilization: The trimmed explants after removing a whorl of leaf weresurface sterilized in laminar air flow chamber with 70% ethanol for six min and0.1% mercuric chloride solution (with few drops of tween 20) for ten minutes. Rinsethe explants 3-4 times repeatedly with sterile distilled water for 5 minutes toremove the traces of sterilant. The process was repeated for once again and thecorm portion was sterilized with 1 % sodium hypochlorite (with few drops of tween20) for 15 min. and rinsed 3-4times repeatedly with sterile distilled water for5 minutes. Then the cut portion of the sucker was treated with antibioticsolution containing Cifotaxime 0.1% and Gentamicin 0.
05 % for 5 min. Aftersurface sterilization suckers were treated with ascorbic acid (100mg/l) for 10min to avoid tissue blackening due to phenolic exudation.3.
2.3.Initiation of organogenesis:Thesurface sterilized explants were aseptically inoculated on MS (Murashige , 1962) basal medium supplemented with 160mg/l Adenine sulfate (ADS), 0.8% Agar agar, 3 % sucrose along with the growth regulator BAP (6 –Benzyl aminopurine) at the range of 1.0 – 10.0 mg/l.
The pH of the media was adjusted to 5.8and thirty replicates were used for each of the concentrations. 3.2.4.Growth condition: Thecultures were maintained in incubation room with controlled temperature, lightand humidity conditions. The cultures were incubated for fourweeks at 25±2ºC, with 16 h photoperiod and 40% relative humidity illuminated bycool, white fluorescent lamps (40 Watts). After shoot elongation the cultureswere taken for multiplication stage.
3.2.5. Massmultiplication of propagules The regenerated shoots were vertically marked intosmaller pieces (usually 2 to 4) and transferred on to the MS mediumsupplemented with 4 to 6 mg/l BAP, 0.1 to 1.2 mg/l Thidiazuron (TDZ) and 0.1 to0.
9 ml/l coconut water for the induction of multiple shoot buds. The cultureswere incubated for 4 to 5 weeks. The multiple shoots formed were transferred tonew organogenic media supplemented with the same concentration of growthregulators. To minimize tissue blackening due to phenol exudation, meristematictissues were transferred to the similar fresh medium frequently every 10 to 12days for about one month by removing the blackish tissues using sterilizedscalpel during the initial phase.
During subculture,the cultures were checked for bacterial and fungal contamination which mayappear within 15 days of incubation. At each subculture, the multiple shoots weretransferred to the fresh media supplemented with the same concentration ofgrowth regulators. The clumps of shoot propagules developed on multiplicationmedia were transferred to shoot elongation media supplemented with 5 mg/l BAP. Atthis stage the regenerants were maintained for 2 -3 weeks and transferred on tothe rooting media. 3.2.6.
RhizogenesisAt the end ofmultiple shoot generation cycles, individual shootlets attained the height of4-5 cm were carefully isolated from the shoot clump and aseptically transferredto rooting media composed of MS basal nutrients augmented with 0.5 – 1.5 mg/lIBA, 0.5 – 1.5 mg/l IAA, 0.
5 – 1.5 mg/l NAA and 0.2% activated charcoal toinduce root formation. The observations were made on the development pattern ofplantlets after four weeks of incubation and the data were recorded. 3.3.
Indirect organogenesis: 3.3.1. Plant material and disinfection: The leaves of Musa paradisiaca Monthan cv. Karibale were obtained from plantsgrowing at farmyards of Shivamogga District, Karnataka, India.
These weretreated with 5% teepol (w/v) for 5 min, and washed three times with sterile distilledwater. This was followed by surface sterilization with 0.1% (w/v) mercuricchloride (HgCl2) for 5 min followed by washing with sterile double distilledwater inside the laminar airflow chamber to remove traces of HgCl2. 3.3.2.
Organogenic callus induction: The leaf segments (1.0–1.5 cm) were excisedaseptically and transferred onto the callus induction MS media augmented with3% (w/v) sucrose, 0.8% (w/v) agar (PGRs), pH 5.8 and the plant growthregulators 2, 4-dichlorophenoxyacetic acid (2, 4-D) (2 – 4mg/l) and Benzylamino purine (BAP) (0.0 – 0.
9 mg/l). For shoot organogenesis four weeks oldleaf derived calli were transferred onto the regeneration media amended withdifferent concentrations BAP (2 – 4 mg/l) and Thiadiazuran (TDZ) (0.4 – 0.6mg/l).
All the cultures were maintained in a growth room with a 16 hphotoperiod (cool, white fluorescent light – 3000 lux light intensity) and thetemperature was maintained at 25 ± 2ºC, with 50 – 80% relative humidity. Thepercent explants forming callus, the number of regenerated shoots per unitcallus and shoot lengths were recorded after 8 weeks of incubation. The percentinduction was calculated using the following equation Frequency = No. ofexplants showing response X100 Total no. of explants In vitro differentiatedshoots measuring 3.
0 – 4.0 cm in length were excised and cultured on rootingmedium augmented with IBA (0.25-1.0 mg/l). The data collected in this study weresubjected to the statistical analysis by using ezANOVA tool (0.98 versions).
3.3.3. Hardening Once the plantlets arefully developed they are carefully acclimatized to the green house and later toleast protected field conditions. During hardening, the plantlets undergophysiological adaptation to changing external factors like water, temperature,relative humidity and nutrient supply. Hardening ofplantlets were carried out in two stepsi) PrimaryHardening The plantlets from culture bottles areshifted from the laboratory to a room at ambient temperature and kept aside forfour days so that relative humidity was less promoting the formation of bettercuticular wax.
The plantlet having roots were isolated from the bottle andwashed thoroughly with water to remove the media contents. The roots weretrimmed to about 6 to 7 cm and the plantlets were dipped in 0.1% Bavistin to protectfrom fungal infections. Then the plantlets were placed in portrays containingcocopeat mixture along with growth nutrients. The trays were covered withpolythene and allowed to harden in green house, optimized at 27°C, 70% RH and15,000 lux2. After ten days, plants were transferred to secondary hardening process.ii) SecondaryHardening After primary hardening, the plantlets aretransferred from micropots to 18 x 15 cm polybags. Base substrate is generally themixture of sand, red soil and cow dung in 1:2:1 ratio.
Initially, in vitro plantlets are maintained in lowlight intensity under shade nets and 70% RH (Relative humidity). After 15days ofhardening the light intensity has been increased and 40% RH maintained. After 5to 6 weeks, the plants become ready for field planting having 3 to 5 welldeveloped leaves and a good mass of fibrous roots. During the process ofhardening, the stocks were evaluated for phenotypic variations at weeklyintervals. These could include vegetative deformities like dwarfism, leafvariegation, rosette foliage and leaf crinkiness. 3.3.4.
Manuring and plant protection innursery Plantletsshould be off two to three weeks old before any fertilizer is applied. In 1000ml of water, 0.5 g of NPK (19:19:19) was applied twice a week till three tofour weeks.
After 3-4 weeks, doubling the dosage of manure helps better growthof the plantlets in nursery and filed condition. Regular applications offungicide or bactericide avoid the risk of damage by pest and diseases.To protect the plant from the pathogens andother disease, 25 g of copper oxy chloride, 1 g streptocycline and 10 g ofbavistin was mixed in ten litres of water and sprayed regularly. 3.3.5. Field preparation and planting of invitro raised banana plants Well-developed plants were acclimatized ingreen house for about two months and transferred to the field to evaluate theagro-morphological variations along with vegetatively propagated Karibaleplants of the same age at the experimental farm.Cultivation practices such as, fertilizerapplication and irrigation followed conventional protocols (Chattopadhyay etal.
, 2001). Planting of tissue culture banana can be done throughout the yearas per the market demand except in severewinter and during heavy rains. The land was ploughed 2-4 times and levelledusing Ratovator to break the clump and bring the soil to a fine tilt.
Duringsoil preparation basal dose of FYM is added and thoroughly mixed into the soil.A pit size of 60cmx 60cm x 60cm was prepared and filled with 8kg of FYM, 200 gmof Neem cake and 20 gm of carbofuran. When the plants are 30 cm, polybag wasseparated from the plant without disturbing the root ball of the plant and thenplants are planted in the pits keeping the pseudo-stem 4cm below the ground level.3.
4. Evaluation of morphoagronomic charactersof the in vitro regenerants To evaluate the phenotypic variations, the in vitro raised plants after planting infarmyard were subjected to field trails at standing crop stage. For fieldevaluation, following yield parameters were recorded after two months ofbunching such as height of the plants (in ft), number of leaves, length of theleaves (in cm), width of the leaves (in cm), length of the bunch (in cm),weight of the bunch (in kg), number of hands in a bunch, number of fingers in ahand, length of the finger (in cm), girth of the finger (in cm) and girth ofthe pseudo stem (in cm) were recorded. All the data were analyzed by using ezANOVAsoftware (version – 0. 98). i)Plant height: The height of the plants was measured from base of the plant tothe tip of main shoot from five plants and mean plant height was calculated andexpressed in feet.
ii) Girth of the pseudostem: The girth of thepseudostem of the plants was measured at the base above two feet from theground level using the tape from five plants and expressed in cm. iii) Number of leaves per plant: The numberof leaves emerging from the main pseudostem of plants from five plants werecounted and the mean value was worked out. iv) Length of the leaves: The length from base of the leaf to the tipwas measured and expressed as length of the leaves and mean length of theleaves was expressed in cm.
v) Width of the leaves: The width of each oldleaves of the plants was recorded and mean value was worked out. vi) Length of the bunch: The length from baseof the bunch to the point where inflorescence ends was measured from fiveplants expressed as length of the bunch and mean value was expressed in cm. vii) Weight of the bunch: The weight of thebunch from five plants was recorded at the harvesting stage and the value wasexpressed in Kilograms.
viii) Number of hands in a bunch: The meanvalue of hands in a bunch from five plants were computed and recorded in thisparameter. ix) Number of fingers in a hand: From eachhand of a bunch the fingers were calculated and the mean value of fingers wascomputed. x) Length of the finger: In this parameterthe length of the finger from each bunch of five plants was calculated and themean value was expressed in cm.
xi) Girth of the finger: The girth of thefinger was calculated by using Vernier caliper and the data is recorded andexpressed in cm.