MATERIALS AND METHODS

Materials & Methods

MATERIALS AND METHODS Plant Material In the preliminary experiments, 30 varieties of grain Sorghum viz., IS 15, IS 28, IS 720, IS 745, IS 8887, IS 3040, IS 3949, IS 81, IS 4100, IS 8191, IS 6711, IS 7155, IS 14357, IS 18804, IS 18855, IS 18856, IS 18945, IS 18852, IS 23217, IS 2746, IS 11167, IS 23279, IS 23334, IS 23253, IS 18852, IS 27703, IS 23193, IS 23237, IS 23279 and IS 23193 were screened. The concerned seed material was obtained from ICRISAT (International Crop Research Institute for Semi- Arid Tropics), Patancheru, Hyderabad, A.P, India. All the varieties were maintained through self pollination. Cross pollination was prevented by covering the panicles with brown paper bags. All varieties were grown in the experimental farm, Department of Botany, Andhra University, Visakhapatnam. For callus initiation, healthy immature embryos, mature embryos, immature inflorescences and shoot meristems were used as explants. Out of the 30 Sorghum varieties screened, only six varieties i.e., IS 3040, IS 3949, IS 81, IS 8191, IS 2746, IS 8887 were observed to be efficient for callus induction and high frequency of regeneration. Hence, further experiments were carried out with these six varieties only. Chemicals and Glassware All the chemicals used in the present experiments were of excelar grade from Qualigens Chemical Company (India), Hi-Media chemical Company (India) and Sigma Chemical Company (USA). Culturing was carried out in culture tubes of different sizes (25 200 mm, 25 150 mm, and 18 180 mm), and in Petri dishes (100 15 mm) and baby jars (500 ml) of Borosil Company. These glassware were cleaned with liquid 28

detergent (Labolene) followed by thorough washing with tap water. The cleaned glassware were rinsed with double distilled water and dried in a hot air oven at 100ºC. The conical flasks and culture tubes were plugged with non-absorbent cotton wrapped in cotton gauze. The baby jars were capped with polypropylene closures. Preparation of medium for callus induction Fresh stock solutions were prepared (twice in a month separately) for both micro and macro nutrients, vitamins, amino acids and plant growth hormones. All stock solutions were stored in the refrigerator at 5ºC. Required volume of stock solutions were pipetted out and carbohydrate source was added before making of the final volume of the medium with double distilled water. For callus initiation, the basal MS medium was supplemented with different auxins and cytokinins viz., 2,4-D, 2,4,5-T, IBA, IAA, NAA, KN and ZN in different concentrations and combinations from 0.5 mg/l to 3.0 mg/l for all experiments. The ph of the medium was adjusted to 5.8 using 1N HCl and 1N NaOH. For the preparation of semi solid medium 0.8% agar was used as the gelling agent and the medium was boiled for proper mixing. The supplemented medium was dispensed into culture tubes, Petri dishes and baby jars as per the requirement. The culture tubes were closed with non absorbent cotton, Petri dishes with Parafilm and the baby jar bottles were capped with polypropylene closures. The medium was sterilized in autoclave at 120 ºC and 15 lb / in 2 pressure for 15 minutes. 29

Preparation of hormonal stocks (1 mg/ml) 2, 4-D 2, 4-D (50 mg) was dissolved well with few drops of 1N NaOH; the volume was made up to 50 ml, and stored at 4ºC. Kinetin Kinetin (50 mg) was dissolved well with few drops of 1 N HCl; the volume was made up to 50 ml, and stored at 4ºC. BAP BAP (50 mg) was dissolved well with few drops of 1 N HCl; the volume was made up to 50 ml, and stored at 4ºC. IBA IBA (50 mg) was dissolved well with few drops of 1N NaOH; the volume was made up to 50 ml and stored at 4ºC. IAA IAA (50 mg) was dissolved well with few drops of 1 N NaOH; the volume was made up to 50 ml, and stored at 4ºC after filer sterilization. 30

Preparation of Regents HCl (1N) The concentrated HCl (36.5 38% v/v; 8.6 ml) was added to 91.4 ml of distilled water in a beaker to prepare 100 ml of 1N HCl NaOH (1N) NaOH (4g) was added to 50 ml of distilled water, and the volume was made up to 100 ml with distilled water to prepare 100 ml of 1 N NaOH. Developmental stages of explants Immature embryo: Field grown immature seeds of Sorghum, containing immature embryos were harvested from main spikes of the caryopses of 14-17 days after anthesis. The explants were washed thoroughly under running tap water, followed by treatment with 70% alcohol for 5 minutes and subsequently washed three times with sterile double distilled water. The panicles harvested were kept moist at 4 C until immature embryos were excised. Mature embryo: Self pollinated mature seeds were collected from the experimental farm and stored in a low temperature (5 C). Fifty seeds of each variety were wrapped in cheesecloth and initially rinsed with 70% ethanol for 3 minutes. They were then rinsed with sodium hypochlorite solution (5% activated chlorine) for 20 min followed by 3 rinses with sterile water. The wrapped seeds were kept in a Petri dish and incubated overnight at 25±2 C. 31

Immature inflorescence: It is very difficult to identify immature inflorescence before the emergence of the flag leaf. The immature inflorescence (size ranging from 1-4 cm in length) was taken, before the emergence of the last leaf; the uppermost internode was collected and washed with tap water for one hour and surface sterilized by swabbing with 70% ethanol. Shoot meristem: Mature seeds were washed with running tap water and then soaked in 70% ethanol for 5 minutes and finally kept for germination on wet filter papers arranged in Petri dishes for 3-4 days in the dark at 26±2ºC. Shoot tips were isolated from aseptically germinated seedlings and top portion ( 5 mm) was cut. Sterilization agents Various sterilization agents were used, the most commonly used compound being Sodium hypochlorite (NaOCl), either as a chemical grade reagent or as a commercial household bleach formation. Other sterilization agents used included calcium hypochlorite (CaCl 2 ), mercuric chloride (HgCl 2 ), ethanol (70%), and hydrogen peroxide. Surface sterilization and inoculation of the explants Immature embryo Immature embryos were isolated at 14-17 days after pollination and were washed with mild detergent (teepol 0.1%). Explants of the size ranging from 0.5 2.0 mm in length were isolated from the main spikes under dissecting microscope. Again they were washed with 0.4% sodium hypochlorite, then rinsed 5 times with sterilized distilled water and blotted on sterile papers. Approximately 50 such immature 32

embryos were aseptically inoculated. They were placed with their axes in contact with the MS medium in a single Petri dish and their scutellum facing upwards. Mature embryo Fifty mature seeds of each variety were wrapped in cheesecloth and initially rinsed with 70% ethanol for 3 minutes. They were then rinsed with sodium hypochlorite solution (5% activated chlorine) for 20 min followed by 3 rinses with sterile water. The wrapped seeds were kept in a Petri dish and incubated overnight at 25±2 C. For callus induction, mature embryos were aseptically moved with a scalpel from the imbibed seeds by using a dissecting microscope, embryos were placed furrow downwards (embryo side exposed) on callus induction medium in sterile glass tubes containing 25-30 ml of solid agar medium. Immature inflorescence Immature inflorescence enclosed in the boot leaf were collected from field grown plants, swabbed with 70% ethanol after removing outer whorl of leaves. Small panicles (1-4 cm) were aseptically cut open and were surface sterilized in 70% ethanol for a minute and 3% sodium hypochlorite for 10 minutes and then rinsed twice with sterilized distilled water. It was cut into small pieces (0.5 cm or less) and then 10-12 explants were placed on the MS medium with their bases touching the medium and kept at 25 ± 2ºC in dark. 33

Shoot meristem Mature seeds were washed with running tap water and then soaked in 70% ethanol for 5 minutes; surface sterilized in 4% sodium hypochlorite for 20-30 minutes and rinsed thrice with sterile distilled water. The seeds were finally kept for germination on wet filter papers arranged in Petri dishes for 3-4 days in the dark at 26±2ºC. Shoot tips were isolated from aseptically germinated seedlings and top portion ( 5 mm) was cut. Two methods were used for isolation of shoot apical meristems from shoot tips. In the first method, outer whorl of leaves were removed, the central core of the leaves was isolated and plated in contact with the medium. In the second method, a vertical slit was made from top till the joint of mesocotyl. This gave rise to a Y-shaped shoot tip. It was plated on the medium so as to make the Y-junction came indirect contact with the medium. Approximately 50 such shoot meristems were inoculated on MS medium. In vitro characters Total callus Quantity The quantity of embryogenic callus and non embryogenic callus together was measured as the total callus quantity. Callus volume was measured 3 weeks after inoculation. For measuring the callus volume, a thin transparent polythene graph paper drawn in millimeters was used. The area occupied by the callus was measured by counting the number of squares on the graph paper. 34

Embryogenic callus quantity Embryogenic callus was morphologically distinguishable from non embryogenic callus. The embryogenic callus was compact, organized and white to pale white in colour. The non embryogenic callus is soft, creamy in colour and unorganized. Based on the physiological nature, the amount of embryogenic callus area in the total callus was taken and expressed as embryogenic callus quantity. Measurement of callus growth rate Growth rate of callus was determined in terms of both fresh and dry weight. For determining the fresh weight, the callus was blotted on a sterile blotting paper immediately after removing from the medium, placed in a sterile vial (of known weight) and weighed using electric balance. The dry weight was determined after drying the callus at 60 ºC overnight. The increase in fresh and dry weights was calculated by subtracting the initial weight from the final weights (at the end of third, sixth, ninth and twelve weeks) Regeneration Medium Three week old calli were divided into small pieces and transferred to the regeneration medium supplemented with 0.5 3.0 mg/l of BAP, KN, ZN, GA 3 and TDZ either alone and in combination. For regeneration, the cultures were incubated in continuous white colour fluorescent light at an intensity of 25μ mol m -2 S -1 at 25 ± 2ºC and 16 hours photoperiod. All tissue culture experiments were performed under aseptic 35

conditions. All cultures were transferred to fresh medium every 21 days or earlier when necessary due to repeated secretion of phenolics. Rooting induction Regenerated shoots were separated from the cultures individually and used for root induction. The medium used for root induction was half strength MS medium supplemented with NAA in concentrations of 0.5, 1.0, 1.5 and 2.0 mg/l. Multiple Shoot induction For multiple shoot induction, three weeks old embryogenic calli were cut into small pieces and transferred onto multiple shoot induction medium. The MS medium was supplemented with 0.5 3.0 mg/l of BAP, KN, ZN, GA 3, IAA and TDZ in various combinations. In addition different concentrations of L-asparagine, L-proline and casein hydrolysate were added in the medium. The medium was dispensed in sterile Petri dishes, culture tubes and baby jars. After embryogenic callus transferred onto the multiple shoot induction medium, the cultures were incubated for a week at the temperature of 25 ± 2ºC in continuous darkness. After a week, the cultures were incubated in continuous cool white fluorescent light at an intensity of 25μ mol m -2 S -1. Direct somatic embryogenesis For direct somatic embryogenesis of Sorghum, fresh plant materials of immature embryo, mature embryo, immature inflorescence and shoot meristems were collected from the field and were washed several times with tap water before surface sterilization. 36

After surface sterilization the explants were inoculated on MS media supplemented with different plant growth regulators viz., 2, 4-D, 2,4,5-T, IBA, BAP, KN, GA 3, TDZ and ZN at various concentrations and combinations. In addition to the plant growth regulators, different concentrations of L-asparagine, L-proline, casein hydrolysate and AgNO 3 were added in the medium. After explants were inoculated onto the media, the cultures were incubated at the temperature of 25±2 C in light conditions at intensity of 25 μ mol m -2 S -1. Material preparation for scanning electron microscopic study The samples of calli at different stages of somatic embryogenesis for microscopic studies were transferred to vials and fixed in 3% Gluteraldehyde in 0.05 M phosphate buffer (ph 7.2) for 24 hours at 4 C and post fixed 2% aqueous osmium tetroxide in the same buffer for 2 hours. After the post fixation samples were dehydrated in series of graded alcohol and processed for critical point drying with Electron Microscopy Science CPD unit. Then dried samples were mounted over the stubs. Finally, a thin layer of gold metal was applied over the sample using an automated sputter coater (JEOL JFC-1600) for about 3 min. Then the samples were scanned in scanning electron microscope. Establishment of regeneration in the green house The plantlets regenerated through various in vitro techniques in this study were taken out from the culture medium and washed gently with double distilled water for removing of all traces of medium from the roots. The washed plantlets were then transferred to small plastic cups containing sterile soil. The plastic cups were covered 37

with sealed plastic vinyl bags to keep full humidity at 25 ± 2ºC in light conditions (Photon flux density = 25μ mol m -2 S -1 16 h). The plantlets were moistened with water. As the plants grew vigorous, the bags were poked with chopsticks to allow air into the bags until the plants become self-supported. The polythene bags were removed after 15-21 days. The plantlets were later transferred to larger pots containing sterile compost and soil (1:1 ratio) and kept under shade in the greenhouse for another two weeks before transferring to field. Fully established regenerates were later established in the field for further growth. Agrobacterium-mediated transformation Preparation of Agrobacterium culture LBA 4404 Agrobacterium tumefaciens strain harboring the plasmid pcambia 1305.1 with kanamycin and hygromycin resistance genes was streaked on LB solid medium having 50 μl/ml of kanamycin and 10 μl/ml of rifampicin. The plasmid has -glucuronidase (GUS) gene as reporter gene. That was grown for 2 days at 28 C to obtain single colonies. One such colony was picked and inoculated under aseptic conditions in 2 ml LB liquid culture medium having appropriate antibiotics, and was left for 24 h at 28 C in a shaking incubator at 200 rpm. The overnight liquid culture was diluted in 1:10 (v/v) ratio with MS liquid medium having 200 μm acetosyringone and the culture was grown with continuous shaking for another 24 h. The culture was grown under continuous shaking till the required density was obtained. If that was over grown, that was still diluted till the required OD was obtained. For infecting explants, a density of 0.5 1.0 OD units (A 600 ) were taken. Immature embryo, immature inflorescence and shoot meristem explants were selected for Agrobacterium infection. 38

The explants were immersed in Agrobacterium culture for 20 min following which, the explants were blot-dried with sterile filter paper and transferred onto the MS co-cultivation medium having 200 μm acetosyringone and were washed with MS liquid medium containing 400 mg/l cefotaxime to inhibit the bacterial growth, and were transferred onto culture medium supplemented with different concentrations and combinations of plant growth regulators and were cultured following standard protocol. For GUS staining, the Agrobacterium infected explants were treated with 5-Bromo-4-chloro-3-indolyl- -D-glucuronide (X-Gluc) stain after thoroughly washing with cefotaxime. One day after infection, GUS (reporter gene) histochemical analysis was performed to confirm the gene transfer into the plant tissue. Explants were stained in X-Gluc (the substrate solution for -glucuronidase) and the number of blue spots obtained, percentage of explants exhibiting the GUS expression was recorded. For this, the transformed explants from each Petri dish were dropped into X-Gluc solution and were incubated at 37 C for 24-48 hours. Agrobacterium transformation was confirmed by transient GUS expression in all the explants. Preparation of X-Gluc solution (following Jefferson et al., 1987) Reagents 1. Phosphate buffer (250 mm) A quantity of 2.4 gm of sodium hydrogen phosphate was dissolved in 100 ml of sterile distilled water to prepare 0.2 M monobasic salt solution. Similarly, 2.8 gm of sodium dihydrogen phosphate was dissolved in 100 ml of sterile distilled water 39

(0.2 M dibasic salt solution); 39 ml of monobasic salt solution was mixed with 61 ml of dibasic salt solution and the volume was made to 200 ml. 2. Phosphate buffer (50 mm) 10 ml of 250 mm buffer was diluted using 40 ml of sterile distilled water to obtain 50 mm buffer. 3. Potassium ferrocyanide (10 ml of 0.1 M) distilled water. A quantity of 330 mg of potassium ferrocyanide was dissolved in 10 ml of sterile 4. Potassium ferricyanide (10 ml of 0.1 M) distilled water. A quantity of 422 mg of potassium ferricyanide was dissolved in 10 ml of sterile Procedure: One mg of X-Gluc was weighed and dissolved in 1 ml of dimethyl sulfoxide (DMSO) and subsequent by 5 ml of phosphate buffer (50 mm), 1.0 ml each of 0.1 M potassium ferrocyanide and ferricyanide, 120 μl of triton X-100 were added and the volume was made up to 20 ml using 12.83 ml of sterile distilled water. Selection of putative transformants The GUS expressed calli were passed through two cycles of selection. First they were cultured on MS basal medium supplemented with 30 mg/l of hygromycin for two weeks and were then cultured on 50 mg/l of hygromycin. The surviving calli were then transferred to regeneration medium. Six weeks after selection, the surviving calli were 40

transferred on to MS regeneration medium supplemented with different concentrations of auxins and cytokinins devoid of selection agent. Then, the regenerants were transferred to rooting medium. Plantlets with well established shoots and roots were finally transferred to greenhouse. Statistical Data analysis using SPSS SPSS (Statistical Package for the Social Sciences) is data management and analysis product produced by SPSS. Among its features are modules for statistical data analysis, including descriptive statistics such as plots, frequencies, charts, and lists, as well as sophisticated inferential and multivariate statistical procedures like analysis of variance (ANOVA), factor analysis, cluster analysis, and categorical data analysis. Data on the effects of variety of hormone treatments at various concentrations and combinations and callus induction frequency, growth rate of fresh and dry weights, callus volume and shoot lengths were tested by using ANOVA. 41

Table-1. Composition of MS medium for one liter I. Macro nutrients NH 4 NO 3 1650 KNO 3 1900 MgSO 4.7H 2 O 370 KH 2 PO 4 170 CaCl 2 440 II Micro nutrients H 3 BO 3 620 MnSO 4. 4H 2 O 1000 ZnSO 4. 7H 2 O 860 CuSO 4. 5H 2 O 26 CoCl 2. 6H 2 O 26 Na 2 MoO 4. 2H 2 O 35 KI 85 III Iron Sources Na 2 EDTA 373 FeSO 4. 7H 2 O 278 IV Vitamins Myo-inositol 100 Pyridoxine HCl 1.0 Thiamine HCl 0.2 Nicotinic acid 1.0 Glycine 0.004 Sucrose Agar 30 gm 8 gm 42