TISSUE CULTURE CHAPTER VII

Transcription

1 CHAPTER VII TISSUE CULTURE INTRODUCTION Plant cell and tissue culture has become a major tool in the study of in plant science. It is used globally for the conservation of plants. Conservation of critically endangered plants has been achieved through tissue culture (Gupta et al. 1997; Verma and Kant 1996; Hogue et al. 2000; Nichol et al & Palai et al. 2000). Other two reports are genus Abutilon indicum in in vitro study (Jyoti, et al & Nataraja and Patil, 1984.) However, there is no report on in vitro regeneration of Abutilon ranadei Woodr. & Stapf. During the present investigations, the aim was to establish an efficient protocol for regenerating large number of plantlets in vitro from the explants derived callus cultures. The growth of the in vitro plantlets depends mainly on the media utilized and consequently, on the prepared solutions. The Murashige and Skoogs (1962) and Murashige (1974), basal medium is used a lot in Abutilon plantlet production in laboratories; its concentration of salt and vitamins are adequate for the normal growth of plantlets in vitro conditions. Presently, the basal media are easily obtained in their commercial presentation; however, the use of stock solutions makes possible the availability of culture media for many years, and reduces the costs of plantlet production. Additionally, the research in tissue culture requires the utilization of growth regulators, which must be appropriately prepared and preserved for their maximum effect. On the other hand, in spite of sterile conditions and good management, the presence of bacteria in the medium is possible; the use of antibiotics could be helpful in the temporary maintenance of the plantlets. It is necessary to establish proper method of sterilization, to avoid the abuse of antibiotics, which damage the 144

2 genetic stability of the plant and alter the resistance levels of the bacteria. Efforts will be made to establish protocol to prepare salt, hormones, antibiotics, and other stock solutions used to prepare culture media. MATERIAL AND METHODS Sterilization of Equipments and Glasswares All operations for in vitro culture were carried out inside a laminar air flow cabinet under aseptic conditions using sterilized plant materials, equipments, glass materials and chemicals. A horizontal laminar flow cabinet (Envirco Corporation, Foster City, California, USA) with HEPA filter was used. The hood surface was wiped clean with paper towel soaked in 70 % ethanol (Figure 3.2) and sterilized by germicidal ultraviolet light for 10 min prior to use. All surgical instruments, glassware and other accessories were sterilized in autoclave at 121 ºC with 15 psi for 30 min and then dried in oven. Surgical instruments like scalpel, forceps, and scissors were sterilized by dipping in 100 % ethyl alcohol and flaming prior to use. Surface sterilization of Explants The young healthy explants of Abutilon ranadei Woodr. & Stapf. were collected from Western Ghats of Maharashtra and planted in green house of botanical garden of easy availability of explants(table-1). The potential explants (the starting tissue originated from the donor plant) consist mostly of root, shoot (internodes and nodal segments), leaf (lamina segments with ribs), petiole, anther, hypocotyl fragments or cotyledons. Generally, younger, more rapidly growing tissue or tissue in early developmental stage are the most effective. Therefore, the initial quality of the explants will determine the success of the conservation procedure. The criteria for a good quality explants are: normal, true to type donor plant, vigorous and disease free. Plant fragments are initiated into axenic culture from various sterilization procedures depending of the tissue used. 145

3 As a common rule, fragile tissues (meristem, immature embryos, cotyledons, hypocotyls) requires less exposure to sterilizing agents than seeds or lignified organs. A successful sterilization is achieved when the explants were fully decontaminated and remains viable. An alternative for obtaining uncontaminated explants is to obtain explants from seedlings, which are aseptically grown from surface-sterilized seeds. An overview of successful sterilization for in vitro culture of A. ranadei is shown in Table 21. Table -21: Sterilization strategies developed of Abutilon ranadei. Sr. No. Explant type Sterilizing agent Concentratio n Time (min) (%) 1 Root Mercuric Chloride 2 Internode Mercuric s Chloride 3 Node Mercuric Chloride 4 Petiole Mercuric Chloride 5 Leaf Mercuric Chloride 6 Anther Mercuric Chloride They were washed first under running tap water (15 20 min) to remove surface adhered particles and then with 5% liquid hand wash dettol for 5 min followed by 70% ethanol for 5 Min. The material was 146

4 rinsed in distilled water (three four times) and transferred to Laminar air flow cabinet. The explants were then surface sterilized by 0.1% (w/v) HgCl 2 for (Table-21). Finally, the explants were washed in sterile distilled water for three five times to remove the residual HgCl 2 and then cut into appropriate sizes for inoculation on to the sterile medium. The callus induction medium composed of MS containing 3% (w/v) sucrose, 2% (w/v) clarigel with different concentrations of NAA alone or in combination with BAP and Kn for callus induction. The calli were transferred to the fresh medium for further proliferation and maintenance. The well developed calli were selected and sub-cultured on regeneration media. MS was supplemented with different concentrations of Kn and BAP alone or in combinations with NAA for shoot regeneration. Individual regenerated shoots were excised and used for rooting. Root induction was carried out on full strength of MS supplemented with NAA, IBA and IAA at different concentrations. Medium without plant growth regulators was used as a control. The ph of the medium was adjusted to 5.8 before autoclaving for 15 min at 121ºC. All the cultures were incubated at 25 ± 2ºC with a 16 hr photoperiod (40 µe/cm 2 /min/sec) provided by cool white fluorescent tubes. Well developed rooted shoots were removed from the culture vessels, washed gently under running tap water and planted in pots containing 50 % soil and 50 % cocopith (1:1). The plantlets were kept in the greenhouse for acclimation (two three weeks) before their subsequent transfer to the field. Humidity was maintained by sprinkling water regularly (Jasrai et al. 1999). Plants were gradually exposed to the normal conditions and finally transferred to the Departmental Botanical Garden of Dr. Babasaheb Ambedkar Marathwada University, Aurangabad. 147

5 Each experiment was repeated more than two times. Data were recorded on the percentage of response, number of shoots per explants, number of roots and root length per shoot. Means and standard errors were estimated for each treatment. Culture Room The explants were incubated in a culture room where the temperature was maintained at ºC, humidity at 85 % and either under continuous dark or under a photoperiod of 16 h light (25 µmol s-1m-1) and 8 h dark. Preparation of Media The media formulation described as by Murashige and Skoog (1962) referred as MS medium was selected as the optimal culture medium. For MS medium preparation four different stocks used are major salts (20 X), minor salts (200 X), iron (200 X) and organic nutrients except sucrose (200 X). Quantities for each stock were mentioned according to table 3.1. For the preparation of stock solution, each chemical is separately dissolved in distilled water to avoid precipitation. After preparation, they are stored in freeze until use (not more than one month).one liter MS medium was prepared by taking 50 ml of major salts, 5ml of minor salts, 5 ml of iron stock and 5 ml of organic nutrient stock. After addition of each stock in known volume of water, required quantity of sugar was added. The final volume was made up with doubled distilled water. After mixing, the ph (5.8) of the medium is adjusted using 0.1 N NaOH and 0.1 N HCL. 1% gelling agent i.e. agar was added in the medium. After addition of agar, the medium was heated in a water bath for homogenization of agar. The medium was then autoclaved at C at 15 kpa for 20 minutes. Separate stock solutions was prepared for each growth regulator by dissolving minimal quantity in appropriate solution and making up the 148

6 final volume with doubled distilled water. Auxins like 2, 4- Dichlorophenoxy acetic acid (2,4-D) Indole-3-acetic acid (IAA), 3- Indole butyaric acid (IBA), Napthaleneacetic acid (NAA) were dissolved in 0.1 N KOH. Cytokinins like Kinetin, 6-Benzyladenine (BA or BAP) were dissolved in 0.1 N HCL. GA was dissolved in water. For each growth regulator 1mg/ml stock was prepared. Stocks were stored in glass bottles under refrigeration not more than one month. According to need stock solutions of required quantity were added in the medium by filter sterilization with micro-filters of pore size 0.22 µm. After addition of growth hormones in the autoclaved medium, it was distributed in sterilized culture tubes (25 X 150 mm). Mouth of the culture vessels were closed with non absorbent cotton to avoid microbial contaminant, but allowing free gas exchange. After solidifying, medium was used for different experiments. The formulation and composition of MS medium is as follows. Sr. No Constituent Amount added In gm/lit Stock A 1 NH 4 NO KNO CaCL 2.2H KH 2 PO H 3 BO MnSO 4.H 2 O ZnSO 4.7H 2 O KI NaMoO

7 Dissolve them in 200 ml of distilled water. Keep the solution in a conveniently labeled vial at 4CC. 2. Weigh 5 mg of the following reagents: CuSO 4.5H 2 O CoCl 2.6H 2 O Dissolve them in 10 ml of distilled water. To 1 ml of the previous solution add 200 ml of distilled water. Keep the solution in a conveniently labeled vial at 400. B. Stock b: MgSO4 Weigh 3.7 p of MgSO 4.7H2O in 100 ml of distilled water. Keep the solution in a conveniently labeled vial at 4 C. C. Stock c: 1. Weigh 0.75g of Na 2 EDTA. Dissolve while hot in 20 ml of distilled water. Let the solution cool. 2. Weigh 0.55 g of FeSO 4.7H 2 O Dissolve it in 20 ml of distilled water 3. Mix both solutions and fill up to 100 ml by adding distilled water Keep the solution in a dark, conveniently labeled vial at 4 C. D. Stock d: Vitamins: Sr. No Constituent Amount 1 Thyamine HCI 20mg 2 Glycine 100mg 3 Nicotinic acid 25 mg 4 Pyridoxine HCI 25 mg Dissolve them in 500 ml of distilled water stir well. Dispense the solution in 20 ml vials and keep at 0CC. MS Basal solution For 1 liter of basal medium mix: 150

8 100 ml of Stock Solution A 10 ml of Stock Solution B 5 ml of Stock Solution C 10 ml of Stock Solution D 100 mg of lnositol Make up to 1 liter with distilled water Stocks A+B+C+D Preparation of solution V (Vitamins) This solution was previously called MSA. Preparation of hormone stock solution Gibberellic acid (GA3): Stock solution of gibberellic acid: 1,000 ppm 1. Weigh 0. 2 g of gibberellic acid and dissolve well with some alcohol drops. Add 200 ml of distilled water 2. Keep in a conveniently labeled vial at 0 C. The gibberellic acid may be sterilized together with the culture medium: however, the loss of some activity is also possible. One ml of concentrate solution (1,000 ppm) contains 1 my of gibberellic acid. Naftalenacetic acid (NAA) 1 Stock solution of NAA 1,000 ppm Weigh 0.2 g of NAA and dissolve well with some NaOH 1N drops. 2. Add 200 ml of distilled water. Keep it in a conveniently labeled vial at 0 0 C. One ml of stock solution (1,000 ppm) contains 1 mg of NAA. Indoleacetic acid (IAA): Stock solution of IAA: 1,000 ppm 1. Weigh 0.2 mg of IAA and dissolve well with some alcohol drops. Add 200 ml of distilled water. 2. Keep it in a conveniently labeled vial at 0 C. Sterilization by filtration is recommended. One ml stock solution (1,000 ppm) contains 1 mg of IAA. Indoleacetic acid (IBA) Stock solution of IBA: 1,000 ppm 151

9 1. Weigh 0.2 mg of IBA and dissolve well with some alcohol drops. Add 200 ml of distilled water. 2. Keep it in a conveniently labeled vial at 0 C. Sterilization by filtration is recommended. One ml stock solution (1,000 ppm) contains 1 mg of IBA. 2, 4-D (Dichlrophenoxiaceti acid Stock solution of 2,4-D: 1,000 ppm 1. Weigh 0.2 g of 2,4-D and dissolve well with some alcohol drops. Add 200 ml of distilled water. 2. Keep in a vial conveniently labeled at 0 C. 2,4-0 may be sterilized together with the culture medium; however, a loss of its activity is also possible. One ml of the stock solution (1 000 ppm) contains 1 mg of 2, 4-0. Benzylaminopurine (BAP] Stock solution of BAP: 1,000 ppm 1. Weigh 0. 2 g BAP and dissolve well with some drops of NaOH 1N. Add 200 ml distilled water. 2. Keep in a conveniently labeled vial at 0 C. BAP may be sterilized together with the culture medium; however, the loss of some activity isalso possible. One ml of stock solution (1,000 ppm) contains 1 mg of BAP Kinetine (KIN) Stock solution of KIN: 1,000 ppm 1. Weigh 0.2 g KIN and dissolve well with some drops of NaOH 1N. Add 200 ml of distilled water. 2. Keep in a conveniently labeled vial at 0 C. KIN may be sterilized together with the culture medium; however, the loss of its activity is also possible. One ml of the stock solution (1,000 ppm) contains 1 mg of KIN. 152

10 Preparation of antibiotics Rifampicin (Rimactan 300) 1. Cut small squares of filter paper (1 0 mm x 1 0 mm). 2. Place them in a petri dish and sterilize them. 3. (In a flow chamber) Place the squares carefully on sterilized petri dishes, slightly separated one from the other. 4. Dissolve a capsule of Pimactan (300 mg) in 1 50 ml of distilled water. Sterilize with filters of 0.22 µm. 5. Place 3 drops of the antibiotic solution, approximately 0.09 ml, on each square. 6. Let the antibiotic dry in the flow chamber Keep all the squares in petri dishes, covered and sealed with parafilm. 7. Keep the temperature at 4 C, until the petri-dishes are ready to be used. When ready to use: with forceps, take a square containing antibiotic by one side, and introduce it in a tube. Press over the medium close to the place where the node will be planted. The antibiotic will diffuse and cover the planted area including the node. Sodic cefotaxim (claforan) 1 Cut small squares of filter paper (5 mm x 5 mm). 2. Place them in a petri dish and sterilize them. 3. (In a flow chamber) Place the square very carefully with a forceps, on the surface of sterilized petri dishes, slightly separated from each other. 4. Prepare an antibiotic solution, dissolving 1 g of Claforan in 25 ml of sterile distilled water Sterilize with 0.22 pm filters. 5. (In a flow chamber) Place a drop of approximately 0.03 ml on each square. 6. Let the antibiotic dry in the flow chamber. Keep all the squares in petri dishes, covered and sealed with parafilm. 7. Keep the temperature at 4 C, until the petri dishes are ready to be used. 153

11 When ready to use: with forceps, take a square with antibiotic by one side and put it into a tube. Press over the medium close to the place where the node will be planted. The antibiotic will diffuse and cover the planted area including the node. Preparation of Calcium hypochlorite 1. Weigh 50 g of calcium hypochlorite Dissolve it In ml of distilled water (5%). 2. Shake it for 3 to 4 hours and let it rest B to 8 hours, or over night. 3. Filtrate the solution by using a filter paper and maintain it hermetically closed in a flask in a safe place. 4. Use 50 ml of solution and add 50 ml of distilled water Preparation of solutions for ph adjustment Solution to bring down the ph - Hydrochloric acid (HCI) 1. N 1. Pour 91.4 ml of distilled water into a beaker (Use a mask and gloves to protect you from the acid vapors). 2 With a pipette take out 8.8 ml of hydrochloric acid (commercial concentrate, %). 3. Homogenize and keep in a broad-mouth vial, closed and at room temperature. Solution to bring up the ph Potassium hydroxide (KOH) I N 1. Place 50 ml of distilled water in a beaker 2. Add 5.6 g of KUH and dissolve well. 3. Bring to 1 00 ml with distilled water Keep it in a closed broad-mouth vial at room temperature. Uses According to ph of the medium, add the solutions drop by drop until the required ph is reached. 154

12 In Vitro Culture Techniques Micropropagation Studies in Abutilon ranadii : Micropropagation technology is being widely utilized commercially in the ornamentals industry and in other plant production organization. This propagation method was widely used after the discovery of plant growth regulators, auxins and cytokinins. The discovery of auxin (IAA) and cytokinin (kinetin) created the great opportunities for in vitro propagation of higher plants (Pierik, 1997). There are five basic stages for successful Micropropagation of plantlets. The first stage, the preparative stage or stated as phase zero, involved the correct pretreatment of the starting plant material so as to ensure they are disease free as far as possible. The second phase is the establishment of clean starting tissue for aseptic growth and development. It involves a sterilization protocol for producing aseptic tissues. These aseptic tissues will be used for the next stage of shoot multiplication which can be carried out in a number of ways. Generally plant growth regulators are used for shoot multiplication. The shoots obtained in phase two will be used for root induction at the third phase either in vitro or in vivo. Finally, at phase four, the in vitro planlets are acclimatized for better survival when transferred to greenhouse conditions or to the soil (Pierik, 1997). Plant Growth Regulators The most usual groups of plant growth regulators (PGR) used in tissue culture research are the auxins and cytokinins. The amount of PGR in the culture medium was critical in controlling the growth and morphogenesis of the plant tissues (Skoog and Miller, 1957). Generally a high concentration of auxin and a low concentration of cytokinin supplemented into in the medium could promote cell proliferation with the formation of callus. On the other hand, low auxin and high cytokinin concentration in the medium resulted in the induction of shoot morphogenesis. Auxin alone or with the presence of a 155

13 very low concentration of cytokinin was important in the induction of root primordia (Pierik, 1997). There are a number of naturally occurring auxins, however, most of these are not generally available for routine use. Because of their stability, synthetic auxins are extensively employed. The most commonly used are 2,4- Dichlorophenoxyacetic acid (2,4-D), 1- napthaleneacetic acid (NAA) and indole- 3-butyric acid (IBA). In some chemical compounds which are not strictly auxins, such as dicamba (3,6- dichloro-o-anisic acid) or picloram (4-amino-3,5,6- trichloropyridine-2- carboxylic acid), have been used as auxin to substitute IBA. Both of these compounds are herbicides when used at higher concentration (Davies, 1987). They are found to occur naturally in many plants including olive and tobacco (Epstein et al., 1989). In many instances, addition of any one of these auxins to a basal medium may be enough to initiate and sustain callus growth. However since there may be different sites of action or target molecules, it can be helpful to use more than one auxin simultaneously or achieving the correct balance of the auxin and cytokinin especially when the tissue is recalcitrant (George and Sherrington, 1984). According to Murthy et al. (1998), recalcitrance could be mitigated by the application of other potent synthetic plant growth regulators such as thidiazuron (N-phenyl-n-1,2,3,-thidiazol-5- ylurea). Tissue culture of monocotyledons, particularly cereal grains and palms, had been achieved in some cases through the use of rather high levels of synthetic auxins like 2,4- D. High levels of auxin could act as herbicides but cell proliferation in the absence of exogenous cytokinin was frequently achieved, Morphogenesis such as the formation of somatic embryos or adventitious organs from callus tissues was observed when the auxin was removed or lowered in the culture medium (Krikorian et al., 1987). Cytokinins of adenine derivatives are characterized by the ability to induce cell division in tissue cultures usually 156

14 in the presence of auxin. The most common type of cytokinin found in plants is zeatin. Cytokinin also occurs as ribosides and ribotides. In tissue culture and crown gall culture, cytokinins promote shoot initiation. Lee and Chan (2004) reported that multiple shoots could be produced from the nodal segments of Orthosiphon Staminous using MS mg/l BA. In moss, cytokinins induce bud formation. Kinetin, the prototype molecule for the synthetic adenyl cytokinins and zeatin which is about 10 times more potent and generally considered the prototype of the naturally occurring cytokinins, are widely used in tissue culture. Dihydrozeatin, also naturally occurring, is not widely used compared to kinetin or zeatin (N6- triangle2-isopentenyl adenine) (Davies, 1987), Benson (2000) reported that TDZ (1-phenyl-3-(1,2,3-thia-diazol-5-yl)urea) could display both auxin- and cytokinintype activities and this was most likely due to it having both phenyl and thidiazol groups. Adenine was occasionally added to tissue culture media and acted as a weak cytokinin by promoting shoot formation (Beyl, 2002). Gibberelic acid (GA3), the endproduct of GA metabolisme in G. fujikuroi, has been commercially available for many years. Its application to dwarf or rossette plants, dormant buds, or dormant seeds can result in dramatic and diverse effects on growth. GA3 can also stimulate the production of numerous enzymes notably alpha-amylase in germinating cereal grains. For fruit setting and growth, this can be induced by exogenous applications in some fruit (e.g., grapes). GA3 can also induce maleness of dioecious flowers (Metzger, 1987). In tissue culture, GA3 was used for inflorescence proliferation to bypass juvenility and maintain the adult phase as most of the perennial plants usually passed through a long juvenile phase of vegetative development before flowering. Lin et al. (2004) reported that ginseng buds were cultured on B5 medium supplemented with 1 mg/l BA 157

15 and 1 mg/l gibberellic acid to develop new inflorescences for somatic embryogenesis. The regenerated plantlets from the embryogenic callus were found to have a juvenile phase and grew normally. Ohlsson & Berglund, (2001) found that giberellic acid could enhance anthocyanin content in the cell culture of periwinkle. This indicated that GA3 could also enhance the metabolic activity within pathway that lead to stress related secondary metabolites and anthocyanin biosynthesis. Effect of different hormones on shoot initiation 1. Effect of cytokinins on shoot initiation For observing effect of cytokinins on the shoot initiation different cytokines like Kinetin, BAP alone and Kinetin, BAP combine were used in MS medium. For each treatment various concentrations are used and for each concentration 10 culture tubes were prepared. All concentrations are in mg/l. All cultures were incubated in a culture room at 25 0 C and a 16 hrs light- 8 hrs. dark period. Observations were recorded after 15 days. Observations were compared with basal medium culture. 2. Effect of auxin on shoot initiation In order to study the combine effect of kinetin and auxins on shoot initiation, equal concentrations of kinetin along with auxins were added in the basal MS medium. Concentrations of kinetin were selected on the basis of result of experiment one. Auxins used are 2,4-D, IAA,IBA, NAA. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C.Observations were recorded after 15days. Observations were compared with basal medium culture. 3. Effect of combination of BAP and auxins on shoot initiation To observe the effect of BAP and different auxins in combination the following experiment was design. In this experiment BAP along with auxins, in equal concentration was used in basal MS medium. Concentrations of BAP were selected on the basis of result of experiment one. Auxins used are 158

16 2,4-D, IAA, IBA, NAA with BAP. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C.Observations were recorded after 15 days. Observations were compared with basal medium. 4. Effect of kinetin and auxins on shoot initiation For observing effect of high ratio of kinetin and auxin, various concentrations of kinetin along with 2, 4-D, IAA, IBA, NAA were added in the MS medium. Concentrations of kinetin were selected on the basis of result of experiment one. Same concentrations were used for remaining auxins. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C.Observations were recorded after 16 days. Observations were compared with basal medium culture. 5. Effect of cytokinins in combination with auxins on multiplication of shoots After fifteen days of shoot initiation, shoots were transferred on multiplication medium. In order to study the effect of BAP along with auxins (2,4-D, IAA, IBA, NAA) on shoot multiplication, various concentrations of BAP and auxins were added in the basal MS medium. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C.Observations like number of multiple shoots and shoot length were recorded after 16 days. Effect of different hormones on Rhizogenesis: 1. Effect of auxins on Rhizogenesis: In vitro multiplied shoots separated and transferred on the MS medium having various levels of auxins for rooting. Auxins employed during this experiment were IAA, NAA, IBA and 2,4-D. All auxins were used separately. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C. Observations were taken after 10 days, which includes length of initiated roots and nature of root. 159

17 2. Effect of IBA and other auxins on Rhizogenesis: In vitro regenerated shoots were separated and transferred on the MS medium having various IBA concentrations along with other auxin (IAA, NAA and 2,4-D) concentrations. According to the previous records (2.0 mg/l) of IBA was used along with other auxins for induction of rooting. After inoculation cultures were incubated under 16 hrs light- 8 hrs. dark period and at 25 0 C. Observations were taken after 10 days, which includes length of initiated roots and nature of root. 3. Effect of IBA and other cytokinins on root initiation In vitro regenerated shoots were separated and transferred on the MS medium having various IBA concentrations along with other cytokinin (Kinetin and BA) concentrations. According to the previous results, rooting was achieved with IBA (2.0 mg/l) along with cytokinins. After inoculation cultures were incubated under 16 hrs light- 8 hrs.dark period and at 25 0 C.Observations were taken after 10 days, which includes length of initiated roots and nature of root. Hardening of In vitro regenerated plants: Rooted plants were taken out for hardening. For hardening different proportions of Sphagnum moss and coco pit were used. Before taking in vitro regenerated plants for hardening cotton plugs were loosen to adjust moisture conditions. After two days plants were removed from culture tube and transferred to sphagnum moss and coco peat mixture in a proportion (2:0, 2:1, 1:1, 1:2. 0:2). Results for hardening were observed after twentyfive days. Survival percentage was calculated in each proportion. Healthy plants were shifted to glass house and these plants were subjected to biochemical analysis. Transferred plants were covered with cloth dome to maintain the moisture. 160

18 Callus initiation and organogenesis using various explants A) Callus initiation a) Preparation of explants for callus initiation The young healthy explants of Abutilon were collected from Western Ghats of Maharashtra (Torana fort District Pune). These healthy plants were maintained in garden. From young and healthy Abutilon plants 2-3 cm long apical shoots were cut with sharp scalpel. Explants were washed under running tap water for 20 minutes. After washing excess of the leaves from shoot tip were removed with scalpel. All the sterile materials were used for inoculation like glassware, forceps, scalpel etc. Explants was carried out in laminar air flow and treated with 0.1 % mercuric chloride for 2 minutes followed by treatment with 70% ethanol followed by two washing with sterile distilled water. These explants were trimmed with sterile scalpel to avoid blocking of nutrient passage from medium to explants. Before inoculation shoots were cut into transverse sections with sterile, sharp scalpel. Then sections were inoculated in the suitable MS medium. Cultures then incubated in a culture room at 25 0 C and in dark period. Observations were taken at a regular interval for callus initiation and contamination. If contamination was observed then cultures were removed from growth room. After ten days cultures were observed for callus initiation. If callus initiation was observed, cultures were shifted in light condition (16 hrs light- 8 hrs.dark) for multiplication. Later on callus mass were used for various experiments. b) Effect of different hormones on callus initiation 1. Effect of auxin on callus initiation Effect of different auxins on callus initiation was observed by using different auxins like IAA, IBA and 2,4-D. These auxins were used separately in the basal MS medium and for each concentration five replicates were 161

19 prepared. Concentrations used are in high range After inoculation cultures were incubated culture room at 25 0 C and in dark period for ten days. Later on observations were recorded. Results were compared with basal MS medium. 2. Effect of cytokinins and gibberellin on callus initiation Different cytokinins like Kin, BA and gibberellins were used in various concentrations for observing effect on callus initiation. These hormones were used separately in the medium and for each concentration five replicates were prepared. Concentrations used are in high range i.e. 3, mg/l. After inoculation cultures were incubated culture room at 25 0 C and in dark period for ten days. 3. Effect of IAA and 2, 4-D on callus initiation On the basis of results of above mentioned experiments IAA and 2, 4-D was selected for further work. In this experiment low concentrations of IAA and 2,4-D were used. After inoculation cultures were incubated in culture room at 25 0 C and in dark period for ten days. Observation for initiation of callus was taken after 16 days of incubation. 4. Effect of low concentration of IAA and 2,4-D on callus initiation From results of above experiment further low range concentrations of IAA and 2, 4-D were tried in basal MS medium for callus initiation. After inoculation cultures were incubated in culture room at 25 0 C and in dark period for ten days. Observation for initiation of callus was recorded after 16 days of incubation. 5. Effect of 2, 4-D on regeneration of callus On the basis of results of above mentioned experiments 2, 4-D was selected for further work. In this experiment 2,4-D in concentration 0.1 mg/l was used in the basal MS medium. After inoculation cultures were incubated in culture room at 25 0 C and in dark period until callus initiation. After initiation of callus, it was transferred to a fresh medium containing various 162

20 concentrations of 2, 4-D to observed effect on regeneration. Culture tubes were shifted in light (16 hrs light- 8 hrs. dark) for multiplication. Observation for multiplication of callus i.e. weight of callus was recorded after 10 days of incubation. 6. Effect of auxins along with 2, 4-D on callus multiplication. Effect of other auxins along with 2,4-D on callus growth was observed in this experiment. For this auxins used are IAA, IBA, NAA in various concentrations along with 2,4-D. For induction of callus (5 mg) of 2-4 D was used. These cultures were maintained in light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C. Observations were recorded at regular interval of seven days. Callus growth was observed by taking biomass of grown callus. Remarks were mentioned on callus nature. Concentration of auxins giving good biomass were selected and used further to obtain callus. These cultures of calli were maintained by regular subculture and subjected for various biochemical analysis. 7. Effect of cytokinins and gibberellins along with 2, 4-D on callus Regeneration In this experiment, effect of 2,4-D along with cytokinins and gibberellins on callus multiplication were observed. Cytokinins used are Kn and BA in combination with 2,4-D. In each culture tube 5 mg callus was transferred and incubated at light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C. Callus mass was recorded at a regular interval. Remarks were noted on nature of callus. B) Organogenesis from Callus a) Effect of hormones on organogenesis 1. Effect of cytokinins on callus for shoot initiation. Callus obtained at optimum concentration of growth regulators were used for organogenesis. For shoot initiation various concentrations of cytokinins (Kinetin and BA) were used in the basal MS medium. After inoculation, 163

21 cultures were transferred in light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C. 2. Effect of BA and auxins on callus for shoot initiation From the above experiment the suitable concentration of BA was used along with other auxins. In this experiment, effect of high cytokinin and low auxin was observed on shoot initiation. Auxins used are IAA, IBA, NAA, 2-4-D Already initiated callus was used for experiment. After inoculation cultures were incubated at light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C. Results were documented for shoot initiation. 3. Effect of auxins on rooting of in vitro regenerated shoots Shoots obtained from above experiments were used for rooting. Rooting experiments were done by using various concentrations of auxins. In this experiment auxins used are IBA, IAA, NAA,2-4-D. After inoculation cultures were incubated at light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C.Observations were recorded after 15 days. 4. Effect of auxins and cytokinins on callus for root initiation According to the results obtained in the rooting experiment, auxins along with different cytokinins were tried for rooting. In the present experiment high concentration of auxin and low concentration of cytokinin were used. Cyokinins used are Kn and BA along with IBA and 2,4-D. Cultures were incubated at light conditions (16 hrs. light and 8 hrs. dark) and at 25 0 C.Observation were recorded for root initiation. 5. Effect of potting mixture on the hardening of plantlets through callus Root initiated plants later on used for hardening. For hardening different proportions of sphagnum moss and coco pit were used. Before taking plants for hardening cotton plugs were somewhat loosen, to adjust moisture conditions. After two days plants were removed from culture tube and transferred to sphagnum moss and coco pit mixture in a proportion (2:0, 2:1, 164

22 1:1, 1:2. 0:2). Transferred plants were covered with cloth dome to maintain the moisture. Moisture was reduced slowly. Results were observed for best hardening after ten days. Survival percentage was calculated in each proportion. After hardening at first stage plantlets were transferred to glasshouse. Afterwards plants were used for various biochemical analysis. RESULT AND DISCUSION Induction of Callus from root explants: Table -22. Effect of different concentrations of 2, 4-D or in combination with BAP or Kn in MS media for callus induction from root explants of A. ranadei. Growth regulators (mg/l) Response % Nature & color of Degree of callus formation Callus Hormone free MS - 2,4-D yellow green watery Yellow white green Yellow green Yellow green White green white green 2,4-D + BAP ,4-D + Kn watery Yellow green Yellow green White green white green yellow green Yellow white green Yellow green white green Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. In order to evaluate the effects of plant growth regulators (PGRs) on callus induction from root explants (Photo plate No. 15a), induction media based on MS medium containing different concentrations of 2,4-D, Kn and BAP were designed with orthogonal pattern. 165

23 Root segments were cultured on MS with various levels of growth regulators, namely 2, 4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explant were found to differ depending upon growth regulator supplements (Table-22). After one week of inoculation with 2, 4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 15a). Among different concentrations of auxins, 2.5 mg/l NAA alone was found to be most effective (85%) for callus induction (Photo plate No. 16). Callus induction gradually increased up to 2.5 mg/l 2, 4-D and then declined. Where a combination of 2, 4-D and Kn were applied, the highest callusing rate of 85% was observed for the root explant in the medium containing 2.5 mg/l 2, 4-D mg/l Kn. When different concentrations of 2, 4-D with BAP were tried, 2.5 mg/l 2, 4-D mg/l BAP produced 75% of callus. Several researchers observed that 2, 4-D was the best auxin for callus induction for root explants (Quan-Nan et al. 2010). In the present study 2, 4-D alone showed better effect for callus induction in A. ranadei. Similar results were also observed in root explants of Hevea brasiliensis Quan-Nan et al. (2010). Induction of Callus from node segment: Nodal segments were cultured on MS with various levels of growth regulators, namely 2,4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explants recorded were differing depending upon growth regulator supplements (Table -23). After one week of inoculation with 2,4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 15b). Among different concentrations of auxins empoyed, 2.5 mg/l 2, 4 D alone was found to be most effective (90%) for callus induction (Fig. 1B). 166

24 Callus induction gradually increased up to by adding 2.5 mg/l 2,4 D in medium and then declined. With 2,4 D and Kn the highest rate of callus formation 85% was recorded for the leaf as an explants in the medium containing 2.5 mg/l 2,4 D mg/l Kn. When different concentrations of 2,4 D with BAP were tried, 2.5 mg/l 2,4 D mg/l BAP produced 80% of callus. Table -23. Effect of different concentrations of 2, 4-D alone or in combination with BAP or Kn in MS for callus induction from node segment explants of A. ranadei. Growth regulators Response Nature & color of Callus Degree of callus (mg/l) (%) formation Hormone free MS 2, 4-D Friable yellow green Yellow white green friable Friable yellow White green yellow white green 2, 4-D + BAP Yellow white green friable Friable yellow yellow white green 2, 4-D + Kn Friable yellow green Yellow white green friable Friable yellow White green Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. 167

25 Several researchers have recorded that the 2,4 D was the best auxin combination for callus induction for monocot and dicot plants (Chee 1990, Malamug et al. 1991). In the present study 2,4 D alone shown better effect for callus induction in A. indicum. Similar results were also observed in leaf explants of sugarcane (Begum et al. 1995, Karim et al. 2002). Induction of Callus from petiole explants: Petiole segments were cultured on MS with various levels of growth regulators, namely 2,4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explant were found to differ depending upon growth regulator supplements (Table-24). After one week of inoculation with 2,4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 15c). Among different concentrations of auxins, 2.5 mg/l 2, 4 D alone was found to be most effective (85%) for callus induction (Photo plate No. 16). Callus induction gradually increased up to 2.5 mg/l 2, 4 D and then declined. Where a combination of 2,4 D and Kn were applied, the highest callusing rate of 85% was observed for the petiole explants in the medium containing 2.5 mg/l 2,4 D mg/l Kn. When different concentrations of 2, 4 D with BAP were tried, 2.5 mg/l 2,4 D mg/l BAP produced 80% of callus. Several researchers recorded that 2,4 D was the best auxin for callus induction for petiole explants (Sebastiana 2004). In the present study 2,4 D alone showed better effect for callus induction in A. ranadei. Similar results were also observed in petiole explants of Catalpa bungei (Lin Juan et al. 2010). 168

26 Table-24. Effect of different concentrations of 2, 4-D alone or in combination with BAP or Kn in MS for callus induction from petiole explants of A. ranadei. Growth regulators Response (%) Callus colour Degree of callus (mg/l) formation Hormone free MS 2,4 D Friable white green white Yellow white green friable White green yellow white green 2,4 D + BAP Friable yellow green Friable yellow White green white green 2,4 D + Kn white Yellow white green White green yellow Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. Induction of Callus from leaf explants: Leaf segments were cultured on MS with various levels of growth regulators, namely 2,4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explant were found to differ depending upon growth regulator supplements (Table-25). After one week of inoculation with 2,4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 169

27 15d). Among different concentrations of auxins, 2.5 mg/l 2, 4 D alone was found to be most effective (90%) for callus induction (Photo plate No. 16). Callus induction gradually increased up to 2.5 mg/l 2,4 D and then declined. Where a combination of 2,4 D and Kn were applied, the highest callusing rate of 85% was observed for the leaf explant in the medium containing 2.5 mg/l 2,4 D mg/l Kn. When Table 25. Effect of different concentrations of 2, 4-D alone or in combination with BAP or Kn in MS for callus induction from leaf explants of A. ranadei. Growth regulators (mg/l) Response (%) Callus colour Degree of callus formation Hormone free MS 2,4 D green Yellow green Yellow green green green 2,4 D + BAP green Yellow green Yellow green 2,4 D + Kn Yellow green green Yellow green Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. 170

28 different concentrations of 2,4 D with BAP were tried, 2.5 mg/l 2,4 D mg/l BAP produced 80% of callus. Several researchers observed that 2,4 D was the best auxin for callus induction for monocot and dicot plants (Chee 1990, Malamug et al. 1991). In the present study 2,4 D alone showed better effect for callus induction in A. ranadei. Similar results were also observed in leaf explants of Abutilon indicum and sugarcane (Jyoti 2009, Begum et al. 1995, Karim et al. 2002). Induction of Callus from Anther explants: The aim here is the production of haploid plants through the induction of androgenesis in the haploid cells of the immature pollen grain. Haploid plants are important for number of reasons. Because they possess single set of chromosomes, plant breeders interested in haploid plants because either spontaneous doubling of chromosome number (42) Survase et. al. (2012) or an application of the colchicine to double the chromosome number gives rise to homozygous diploid plants (Razdan, 2003). Immature anthers of Abutilon ranadei were cultured on the modified MS medium supplemented with 2,4 -D (2.0 mg/l), BAP and Kn ( mg/l) resulted in the induction of the callus. Single anther were cultured on MS with various levels of growth regulators, namely 2,4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explants were found to differ depending upon growth regulator supplements (Table-26). After one week of inoculation with 2,4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 15f). 171

29 Table-26. Effect of different concentrations of 2, 4-D alone or in combination with BAP or Kn in MS for callus induction from Anther explants of A. ranadei. Growth regulators Response Callus colour Degree of callus (mg/l) (%) formation Hormone free MS 2,4 D yellow yellow yellow yellow 2,4 D + BAP yellow yellow 2,4 D + Kn yellow yellow Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. Among different concentrations of auxins, 2.5 mg/l 2,4 D alone was found to be most effective (70%) for callus induction of callus(photo plate No. 16). Callus induction gradually increased up to 2.5 mg/l 2,4 D concentration and then declined. Where a combination of 2,4 D and Kn were applied, the 172

30 highest rate of callus induction of 75% was observed for the leaf explants in the medium containing 2.5 mg/l 2,4 D mg/l Kn. When different concentrations of 2,4 D with BAP were tried, 2.5 mg/l 2,4 D mg/l BAP produced 70% of callus. Several researchers observed that 2,4 D was the best auxin for callus induction for monocot and dicot plants (Chee 1990, Malamug et al. 1991). In the present study 2,4 D alone showed better effect for callus induction in A. anther. Similar results were also observed in leaf explants of sugarcane (Begum et al. 1995, Karim et al. 2002). Induction of callus from floral bud explants: The flower buds at the uninucleate pollen-grains stage. On MS medium with supplemented 2, 4-D, of the explants showed callusing in 2 week. In response to 2, 4-D mg/l sepals and cut end of the pedicel showed proliferation in all the cultures within a week (Photo plate No. 15e). The callus was yellowish-white and friable; Floral buds were cultured on MS with various levels of growth regulators, namely 2,4-D alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) or in combination with BAP or Kn (0.25, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Morphogenic potentialities of the explants recorded were differing depending upon supply of growth regulator (Table-27). After one week of inoculation with 2,4-D (2.5 mg/l) the tissue started swelling (Photo plate No. 15e). Among different concentrations of auxins, 2.5 mg/l 2,4 D alone was found to be most effective (30%) for callus induction (Photo plate No. 16). Callus induction gradually increased up to 2.5 mg/l 2,4 D and then declined. 173

31 Table-27. Effect of different concentrations of 2,4-D alone or in combination with BAP or Kn in MS for callus induction from Anther explants of A. ranadei. Growth regulators (mg/l) Response (%) Callus colour Degree of callus formation Hormone free MS 2,4 D Yellowish-white and friable 2,4 D + BAP ,4 D + Kn Slight callusing. Considerable callusing and Profuse callusing. Data were taken after eight weeks of culture and each treatment consisted of 20 flasks. Where a combination of 2,4 D and Kn were applied, the highest callusing rate of 30% was observed for the leaf explant in the medium containing 2.5 mg/l 2,4 D mg/l Kn. When different concentrations of 2,4 D with BAP were tried, 2.5 mg/l 2,4 D mg/l BAP produced 30% of callus. Several researchers confirmed that 2,4 D was the best auxin concentration for callus induction for monocot and dicot plants (Chee 1990, Malamug et al. 1991). In the present study 2,4 D alone showed better effect for callus induction in A. anther. Similar results were also observed in floral buds 174

32 explants of Abutilon indicum (Natraja and Patil 1984). It failed to differentiate organs on subculture to the same medium. Callus induction was recorded from various explants viz. leaves, petiole, stem segment and root explants of Abutilon ranadeii on MS medium with different concentrations of 2, 4-D ( mgl-1) and BA (5.0, 2.5 and 1.0 mgl-1) (Table-28). Although the same in vitro conditions were provided to all explants, there was a marked difference between their callus induction days to callus size and type (Photo plate No. 15 and 16). The callus induction was found to be tissue-dependent. Experimental results show well developed callus among explants viz. leaves, petiole, stem segment and root. Callus was initiated in the seventh day from leaves explants, in the range of 7-10 days; in the fifth day from stem segment explants, the range was between 5-10 days; in the fifth day from petiole explants, the range was between 5-9 days. In addition, the callus morphology and growth state is different from four kinds of explants (leaves, petiole, stem segment and root). The callus induced from leaves explants were compact callus in structure, growing slowly. The callus induced from the stem segment was water soaked, easy browning. The callus induced from the leaf was loose and light green, fast growing. So, the callus induced using leaf as explants were the most appropriate. The callus induction was also found to be hormone dependent. There was a marked difference in their callus inducing capabilities (Table 28). In petioles, it was found that low concentration of 2, 4-D had significant effects on callus formation. The maximum callus induction capabilities were obtained at concentrations of 0.01 or 0.05mgl-1 NAA and 5.0, 2.5 or 1.0 mgl-1 BAP, with compact and pale callus (Photo plate No. 16) of 100% callus induction frequency. On the contrary, treatment with high concentration of 2, 4-D ( mgl-1) and 5.0, 2.5 or 1.0 mgl-1 BAP 175