Comprehensive Report Submitted to The Principal,

Mahatma Gandhi MissionCollege of Agricultural Biotechnology, Gandheli, Aurangabad (Affiliated to Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani)

(ISO 9001:2008 Certified)

Bachelor of ScienceIn

Agricultural Biotechnology

Training organizationCollege of Agricultural Biotechnology, Gandheli, Aurangabad

Submitted by, Sonone Manjiri Vijay

(Reg. No. MGMCABT/2011/70)Semester-VIII (New), HOT 481-Hands on Training

Duration: 18.12.2014 to 30.04.2015.

“Induction of callus in pomegranate (Punica granatum) cv. Bhagwa through cotyledonary explant by using BA”

Sr. No. CONTENTS

PART-1: PROJECT REPORT

1. INTRODUCTION

2. MATERIAL AND METHODS

3. RESULT OF THE PROGRAMME

4. OUTCOME OF THE PROGRAMME

5 SUMMERY OF THE PROGRAMME

PART-2: INDUSTRIAL VISIT REPORT

1. INDUSTRIAL VISIT

2. LITERATURE COLLECTED

• Pomegranate, one of the oldest favorite table fruits of tropical and sub-tropical regions of the world.

• Name pomegranate derives from Latin pomum “apple” and “granatus” (grainy), or seeded apple.• Family punicaceae.• Native to Iran, including Afghanistan and Southern Pakistan's Baluchistan region to the Himalayas in north.• Commercially cultivated varieties : Mridula, Bhagwa, Ruby, Arakta, Ganesh, Super Bhagwa Jalore Seedless, and G 137. • Nutritional value per 100gm :

INTRODUCTION

Punica granantum L.

Energy 72 Kcal

Protein 1.0 gm.

Carbohydrate 16.6 gm.

Vitamin c 7 mg.

Iron 0.7mg

calcium 13 mg.

Cultivation and area of pomegranate production

• Maharashtra is considered as pomegranate basket in India contributing about 54.8 per cent of pomegranate production i. e. 492 MT and 6 lakh tones productivity followed by Karnataka, Gujarat, Andhra Pradesh, Madhya Pradesh and Tamil Nadu.

• In Maharashtra commercial cultivation is done in Sholapur, Sangli, Nasik, Ahmednagar, Pune, Dhule, Aurangabad, Satara, Osmanabad and Latur districts.

Source:-Anonymous, 2012. www.apeda.gov.in/pomegranate

AREA (thousand hect)

PRODUCTION PROUCTIVITYMT/ha

EXPORT(Lakh tone)

World 892 1190 7

India 112.2 745 MT 6.3 7.45

Maharashtra 78 408 MT 5.2 5.5

• Conventionally, pomegranate is propagated vegetativly by the rooting of hardwood and softwood cuttings, but about 1 yr. is required before the rooted cuttings can be transplanted to the field • Propagation by seed is not preferred because of the resulting variability in tree and fruit characters.• Production and yield of quality of pomegranate is affected by bacterial blight

disease (oily spot bacterial blight/ Telya). Cercospora fruit spot, alternaria fruit spot.

• It observed that there are 60-80% losses in pomegranate production in India (Kumar, 2009).

• Genetic improvement of fruit trees is general by conventional breeding methods. This method is a slow and have long life cycle.

Problems in pomegranate

Oily spot bacterial blight

• Callus is undifferentiated, swollen cell mass forming under the influence of elevated plant hormone levels.• Actively growing callus is initiated on culture media with an even physiological balance of cytokinin and auxin.• The most successful explants are young tissues of one or a few cell types

having pith cells.• Explants with high mitotic activity are good for callus initiation.• The WPM medium with addition of BA and NAA was used as treatments to

investigate callus induction of Punica granatum L.

• Transgenic plants are then regenerated from transformed callus.

• To produce true to type offspring.• For in-vitro selection of soma-clonal

variants.• To produce large number of plants.• For mutant variety preparation like

specific toxin reistant via soma-clonal variation. Also for secondary metabolite production.

 

Need of tissue culture

• To establish the in-vitro protocol for callus induction of pomegranate using BA and NAA.

• To find out the optimum concentration of BA for callus induction in pomegranate.

Objective

• Experimental site: Plant Biotechnology MGM College of Agricultural Biotechnology, Gandheli Aurangabad.• Experimental details: The WPM basal medium was supplemented with BA at variable conc. (8, 9, 10, 11, 12 and 13 µM/L) and NAA (5.5 µM/L) is constant for the induction of callus in pomegranate (Punica granatum L. cv. Bhagwa) through cotyledonary explant. 

MATERIAL AND METHOD

Statistical design

Sr.

No.

Symbol

(T)

Concentrations(µM/L)

1. T1 BA (8.0)

2. T2 BA (9.0)

3. T3 BA (10.0)

4. T4 BA (11.0)

5. T5 BA (12.0)

6. T6 BA (13.0)

• Statistical Design : Completely Randomized Design (CRD)• No. of treatments : 06• Total no. of replications : 03• Treatment details : Concentrations of BA and constant NAA.

Experimental details

Plant material :seeds collected from the ripe fruit

kept under running tap water for an hour and washed with laboline

0.2% bavistin for 5 minutes followed by three washings with

distilled water.

15% sodium hypochlorite and tween20 for 20 minutes followed by three washings with distilled water.

Keep the seeds in water for 48 hours

70% ethanol for 30 seconds and rinsed

thoroughly with distilled water.

Excise the embryo and keep the seeds for germination in half

strength MS media

• WPM Media preparation:Woody Plant Medium (WPM) was used

for the experiments containing CaCl2, Vitamin and sucrose. The pH 5.6-5.8will be adjusted by 1N NaOH or 1N HCl and volume will be made up by distilled water. Then added agar, antifungal and antimicrobial supplement. After that the media was uniformly mixed with magnetic stirrer. The media was autoclaved at 121ºC for 20 min.• Zygotic embryo germination: The half strength MS (Murashige and Skoog 1962) basal medium was supplemented with 3% sucrose and 0.8% agar. The pH of all media adjusted to 5.6-5.8 by using either 0.1N NaOH or 0.1N HCl with the help of a digital pH meter. The volume was finally adjusted and required amount of agar, streptomycin and antimicrobial solution was added into the medium before autoclaving. The cultures was incubated at 25 °C (±2 °C) under a dark for one week in Zygotic embryo germination stage (Sharon and Sinha, 2011).

Media preparation and explant preparation

• Radical and primary shoot of 20 daysold seedling was removed and cotyledonary node will be inserted into slant tube containing 20 ml WPM media.• WPM medium was supplementedwith different concentrations of BA ranging from 8.0 μM to 13.0 μM will be used in combination with 5.5 μM NAA for the induction of callus from cotyledon explant.

Inoculation

• INCUBATION:Cultures was maintained at 25°C (±2°C) under 2500 Lux light density

with photoperiod of 16 hour provided by cool white fluorescent and 70% relative humidity.

1. No of days for callus initiation form cotyledonary explant was expressed as mean number of days. 2. Fresh weight of callus expressed as mean weight of callus induced. For fresh weight the callus was transferred to pre-weighed foil boat (W1) and quickly take the weight (W2). Fresh weight = W1 – W2.3. Dry weight of callus was done by drying the callus along the pre-weighted foil in an oven for 35oc till a constant will be obtained. Dry weight percentage = (weight of foil with dried callus – weight of foil) / (weight of foil with fresh callus – weight of foil).4. Diameter of callus expressed as mean diameter.

Biometric observations

RESULT OF THE PROGRAMMME

Sr. No.

Treatment (T) Days required for callus initiation

Diameter of callus (mm)

Fresh weight of callus (40 DAI) (gm)

Dry weight of callus (mg)

    A B C D

1 T1 18.33 2.66 0.16 16.1

2 T2 14.33 6.33 0.50 50.463 T3 15.66 4.33 0.31 32.10

4 T4 19.33 3.33 0.21 20.96

5 T5 22.66 2.33 0.11 11.7

6 T6 25.33 0.66 0.02 2.5

Mean 19.27 3.27 1.33 22.30

S.E.± 0.52 0.33 0.01 1.90

CD at 1 % 2.27 1.44 0.08 8.25

Biometric observations of punica granatum for callus initiation.

A. Number of days required for callus initiation

T1 T2 T3 T4 T5 T60

5

10

15

20

25

30

18.33

14.33 15.66

19.33

22.6625.33

Treatment of BA

No

of d

ays f

or c

allu

s ini

tatio

n

• Plate 1. callus induced in T2.• Mean number of days - 19.27

Plate 1

T1 T2 T3 T4 T5 T60

1

2

3

4

5

6

7

2.66

6.33

4.33

3.33

2.33

0.66

Treatment of BA

N

o of

day

s for

cal

lus i

nita

tion

B. Diameter of callus induced

• Plate 2. Diameter of callus in T2• Mean diameter of callus induced- 3.27 mm.

Plate 2.

C. Fresh weight of callusPlate 3.

Plate 3. Maximum fresh weight of callus in T2 0.50 gm.Mean fresh weight of callus 1.33 gm.

T1 T2 T3 T4 T5 T60

0.1

0.2

0.3

0.4

0.5

0.6

0.16

0.5

0.31

0.21

0.11

0.02

Treatment of BA

Fres

h w

eigh

t of c

allu

s in

gm.

D. Dry weight of callus

Plate 4.

Plate 4. treatment T2 with max dry weight 50.46 mgMean dry weight :- 22.30 mg.

T1 T2 T3 T4 T5 T60

10

20

30

40

50

60

16.1

50.46

32.1

20.96

11.7

2.5

Treatment of BA

Dry

wei

ght o

f cal

lus i

n m

g.

• The callus initiation was found superior in concentration of BA 9.0

µML-1 in combination with NAA 5.5 µML-1 in 14 days (DAI).• The concentration of BA 9.0 µML-1 in combination with NAA 5.5

µML-1 was found the best hormonal concentration to yield highest fresh weight callus 0.50 gm, dry weight 50.46 mg and diameter 6.33 mm produced in Punica granatum L cv. Bhagwa.

• The Concentration exceeds or below than BA 9.0µML-1 and NAA 5.5 µML-1 shows inhibitory effect on growth rate of callus.

 

OUTCOME OF THE PROGRAMME

For experimentation young, healthy, disease free pomegranate were selected. Seeds were surface sterilized .The sterilized seeds were transferred on Whatman filter paper No.1 wetted with the solution prepared by adding4-5 drops of antifungal and antimicrobial supplement in sterile distilled water for 10-12 days at 25ºC in a dark condition for germination.

The standard semisolid WPM media was prepared and autoclaved at 121ºC for 20 min. After autoclaving the WPM medium was supplemented with different concentrations of BA (8, 9, 10, 11, 12, 13 µM/L and NAA 5.5 µM/L) by using syringe filter.

SUMMERY OF THE PROGRAMME

Twenty day old seedling explant were placed in slant tube having 15 ml WPM. The radical and the primary shoots were cut and cotyledonary nodes.

Callus cultures was maintained at 2500 Lux light density with photoperiod of 16 hour provided by cool white fluorescent, at 25°C (±2°C) and 70% relative humidity and sub-cultured on the fresh WPM medium after 4 weeks. The biometric observations were recorded weekly.

Number of days for callus initiation form cotyledonary explant was expressed as mean number of days per explant, diameter of callus expressed as mean diameter in mm, fresh weight of callus expressed as mean weight of callus induced and dry weight percent

Al-obied, R.S., Shaheen, M.A., Saad, F.A. and Said, A. E. 1990. In-vitro culture and propagation of Punica granatum L. Arid Land Agriculture Science. 1:67-75.

Anonymous, 2013.http:// www.national horticulture board, India. Anonymous, 2014. wikipedia.org/wiki/pomegranate. Anonymous, 2012. www.apeda.gov.in/pomegranate. Bhojwani, S.S. and Razdan, M.K. 1983. Plant Tissue Culture: Callus Theory and Practice. Else-vier Science

Publishers BV, The Netherlands, pp. 502. Bonyanpour, A. and Khosh, K.M. 2013. Callus induction and plant regeneration in Punica grantum L. Nana from

leaf explants. Journal of Central European Agriculture. 14(3): 928-936. Bottino, P. J. 1981. Methods in plant tissue culture. Kemtec Educational Corp., Kensington, Maryland.9(22): 60-72. Kumar, R.S., Jahagridar, M.R., Yenjerappa, S.T. and Patil, H. B. 2009. Epidemiology and management of bacterial

blight of pomegranate caused by Xanthomanas axonopodi Punicae. Acta. Horticulture. 818:291-296.

LITERATURE CITED

Kanwar, K., Jommy, J., and Deepika, R. 2010. Comparison of in vitro regeneration pathways in Punica granatum L. Plant Cell Tissue Organ Culture. 100:199–207.

Lloyd, G.B., McCown, B.H. 1980. Commercially feasible micro-propagation of mountain laurel, Kalmia latifolia by use of shoot tip culture. Comb ProcInt Plant PropagSoc. 30:421–427.

Mineo, L. 1990. Plant tissue culture techniques. Conference of the Association for Biology Laboratory Education. 11: 151- 174.

Murashige, T. and Skoog, F. 1974. Plant propagation through tissue culture. Plant Physiology.22: 135- 165. Murkute, A.A., Patil, S., Patil, B.N., and Mayakumari. 2002. Micro-propagation in pomegranate callus induction

and differentiation. South Indian Hortic. 50 (1/3), 49– 55. Naik, S. K., Pattnaik, S. and Chand, P. K. 2000. High frequency axillary shoot proliferation and plant regeneration

from cotyledonary nodes of pomegranate (Punica granatum L.). Scientia Horticulture. 85:261-270. Panse, V. G. and Sukhatme, P. V. 1967. Statistical methods for agricultural workers. ICAR, New Delhi. 2:381. Raj, D. and Kanwar K. 2010. In-vitro regeneration of Punica granatum L. from different Satheesh, M. and Sridharan, T. B. 2014. Standardization of punica granatum explant and callus induction

through micro-propagation - indirect organogenesis. International Journal of Research in Engineering and Technology. 21(2)-116.

Sharon, M., Sinha, S. and Sharan, M. 2011. Somatic embryogenesis in different root segments of Punica granatum L. Annals of Biological Research. 2(5):104-112.

Singh, P., Patel, R. M. 2014. Factors influencing in vitro growth and shoot multiplication of pomegranate. An international quarterly journal of life sciences.9(3):1031-1035.

Soukhak, F., Khalighi, A. and Ghaemmaghami, S.A. 2011.Study of direct adventitious shoot regeneration in pomegranate (Punica granatum cv. Malassaveh) through cotyledonary explants. International Journal of Agricultural Science and Research. 2(3) 65-79.

Van., L.B. 2009. Plant tissue culture an overview. Plant Biotechnology Vietnam Open Course Ware University of science. 5(7):44-10.

Ultrabiotech modern labs like:- 1. Cell biology lab- operations like GMO and non-GMO plant

transformation done on okra, chilli etc. 2. Genomic lab- F1 production for disease resistant vegetable crop.

PCR was used to check hybrid purity and lots were compared to standard lots of marker 4 lots were used for a single crop to confirm result.

3. Molecular technology lab-To check genomic purity DNA extraction was performed in the lab and compared with standard primer sets to check purity upto 95% i.e pure hybrid. The Beckan counter method was used for fast DNA isolation for multiple samples at a time

PART-2: INDUSTRIAL VISIT REPORT

Biosafety lab 5. ISTA lab:-Sampling from the lot: Obtain a representative

sample, Purity and identification of other seeds, composition and identity of species - Separation and weighing of fractions. Germination: Determine the germination potential. Moisture content: Determine moisture content.

6. Seed treatment unit – It had seed polishing and treatment operations performed by machines. Labours sorted the seeds into different types and separated the best seeds for final packaging.

7. Seed packaging unit- the good quality seeds were packed into beejsheetal labeled bags having kalash seed logo.

8. Cold storage area:- Huge deep freezer could tonnes of seed saple before distribution.

9. Market distribution area:- from here the Kalash seed products were distriuted to farmers and ditributers.

 

We learned that biotech knowledge can make us aware of good pure-line varieties in 2-3 days rather than cultivating and waiting to grow it fully. Many mechanized new machines were also known in the industry like seed treatment, seed screening and packaging units. Our questions and queries were satisfied with the industry guide there. This was an unforgettable fully knowledge gaining experience.

LITERATURE CITED Anonymous. Beejo products profile. 2014. www.bejosheetalseeds.comAnonymous. Beejo sheetal info prolfie. 2014. www.bsbsfoundation.orgAnonymous. ICAR news bulletin prolfie. 2014.www.icar.org.inAnonymous. Kalashseeds products prolfie. 2014. www.kalashseeds.comAnonymous. Beejo products prolfie. 2014. www.mpkv.mah.nic.inAnonymous. Beejo products prolfie. 2014. www.mkv2.mah.nic.in

     

THANK YOU !