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(A constituent Institute of MANIPAL UNIVERSITY)
B. Tech Biotechnology (7th Semester)
REG NO. 150924014
(A constituent Institute of MANIPAL UNIVERSITY)
Date: 21/09/2018
This is to certify that this seminar report entitled “Effect of silver nitrate on in vitro shoot growth of coffee” has been completed by K Vijay Nayak Roll No. 6, B. Tech Biotechnology (7th Semester), in partial fulfilment of the award of the degree in Bachelor of Technology in Biotechnology of Manipal Institute of Technology, Manipal University during the 7th Semester, July-December, 2018

Signature of Guide
Signature of the Student
Name of the Student
Due credit must be given to those whose guidance one uses to complete a project like this. I thank Dr. Praveen Kumar of the Department of Biotechnology at Manipal Institute of Technology, Manipal, for guiding me through the course of this project, on how to frame this report and how to conduct this seminar.

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I also thank Dr.Narasimhan, Mr.Muthukumaran and Mr.Rajesh for giving me clear and concise directions on preparing for this seminar.

I would like to thank Dr. Bharath Raja Guru, Head of the Department of Biotechnology at Manipal Institute of Technology, Manipal for giving us this opportunity to show case our research and presentation skills, as this is an essential aspect of being in the scientific community.

Date: 21/09/2018 K Vijay Nayak

S.No. Title Page No.

1. Abstract 5
2. Introduction 6
3. About silver nitrate 7
Materials and methods 8
5. Results and discussion 9
6 References 11
Different concentrations of silver nitrate had a greatest effects on the various growth parameters.’Cafestol (caf) and kahweol (kah) are the two diterpenes are the uniquely associated with the unsaponified lipid fraction of coffee brewsand are reported to be responsible for an increase in serum cholesterol and tShoots of Arabica and Robusta coffee showed increased growth when incubated on MS medium containing silver nitrate and indole acetic acid and benzyladenine and riglycerides levels.’ some of the plant growth regulators (PGRs) indole-3-acetic acid (IAA), N6benzyladenine (BA), and thidiazuron (TDZ);some of the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some of the PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA). These used to determine the variation of caf and kahin in somatic embryos of Robusta coffee (Coffea canephora, CxR variety).

Keywords: Plant growth regulators ,Inhibitors, coffee, shoot explant, plantlets, silver nitrate, diterpenes.

Arabica coffee plant Robusta coffe

Traditional breeding of coffee is difficult because it will take long time before seeds are set. Plants are regenerated through induction of somatic embryos and their culture methodology for plant propagation has been reported. Also variations in such plants cannot be ruled out. The plants produced from seed-derived zygotic embryo cultures in vitro but the initial growth of the plantlets is very slow it will takes 3–5 months to reach 2–3 cm . Silver nitrate will inhibits the ethylene activity because it will promotes the growth in various systems . This project will concerns the influence of silver nitrate on the coffee shoot tips grown in vitro. We know Coffee is a popular non-alcoholic beverage that is brewed from ground beans of Arabica coffee and Coffea canephora var. Robusta coffee are the two commercially important Coffea species cultivated in coffee-growing countries. In southern India, coffee is mainly grown in the Mullayanagiri areas of Chickamagalur, Kodagu, and Hassan the state of Karnataka and also in some regions of Kerala, Tamil Nadu, Andhra Pradesh, and Orissa.Coffee is a complicated blend of phytochemicals, together with a significant quantity of caffeine, caffeic acid, chlorogenic acids, and protocatechui acid. Among these caffeine and chlorogenic acids are the most considered components of the coffee with regard to their profiles and physiological activities. Other main components present in the coffee are the diterpenes cafestol, kahweol, and 16-O-methyl cafestol these are unique to coffee. For genetic manipulation of coffee, it is necessary to first develop an efficient in vitro culture system to obtain competent explants for plant transformation and regeneration.Among invitro culture techniques, somatic embryogenesis is most widely used for clonal propagation and genetic modifications in higher plants. Traditionally the growth regulators,ethylene,andpolyamine inhibitors will influences endogenous pools of caffeine in in vitro cultures of Coffea spp.

Silver nitrate is a very powerful inhibitor of ethylene action and it is widely used for plant tissue culture. silver nitrate (AgNO3) is used to regulate the plant growth and developments. It is a simple hydrocarbon (C2H4), but has maximum effects on growth, cellular differentiation, fruit ripening and senescence in plants even at concentrations as low as 0.01 µl?L?1 or 10?6 % v/v. The important properties of silver nitrates such as easy available, soluble in water, specificity and stability make it very practical for various applications in exploiting plant growth regulation and morphogenesis in vivo and in vitro. Ethylene is a gaseous plants hormones are complicated in many aspects of plant life cycle such as seed germination, root hair development, root nodulation, flower senescence, abscission, and fruit ripening.

Seeds of Coffea arabica variety Hemavathy and C. canephora variety 274 from fresh adult fruits were stained overnight and surface-fumigated with 1% Sodium hypochlorite(v/v) for 20 min and washed three times with sterile distilled water. A second sterilization was with 0.1% Mercury chloride solution for 5 min and washed three times with sterile distilled water. Embryos were separated under aseptic conditions and they are inoculated onto the following media. For arabica, half-strength MS (Murashige and Skoog) macro and micro salts were supplemented with 2% sucrose, 200 mg/l inositol, 1 mg/l thiamine HCl, 2 mg/l cysteine HCl, 0.5 mg/lcalcium pantothenate, 1 mg/l niacin, 1 mg/l adenine, 0.8 mg/l pyridoxine HCl, 3 mg/l glycine. For robusta, full-strength MS medium supplemented with 1 mg/l absiscic acid and 3% sucrose were used. After 30 days the robusta embryos that grown were further cultured on medium without of ABA. Media were set to pH 5.6 and gelled with 0.8% agar. The cultures were incubated in the dark at 25ºC for 2 months. When the shoots that cultured had tips 1–1.5 cm long, they were inoculated directly onto 40 ml growth medium comprising MS components supplemented with 2 mg/l 6-benzyladenine (BA) and 0.5 mg/l indole acetic acid (IAA) and 3% sucrose. Some of the explants were used as controls and cultured continuously for 45 days. The other explants were inoculated after 3 days onto the same growth medium strengthened with 5–40 µM silver nitrate. Ten explants were used in each treatment and the experiment was repeated twice. The cultures were incubated at 25ºC with 16:8 h photoperiod at a light intensity of 2500 lux for 45 days. The shoot length, number of nodes, number of leaves and leaf area were noted down, and the total chlorophyll content was determined by the method of Spectrophotometry. The Significant differences were tested using one way ANOVA and the results were analysed using Tukey’s Multiple Comparison softwer. After these measurements the shoots were cultured on the growth medium for a period of 45 days, so that they were well grown. They were then transferred to a rooting medium of MS salts, 1 mg/l thiamine HCl, 75 mg/l inositol, 1 mg/l pyridoxine HCl, 1 mg/l niacin, 1 mg/l calcium pantothenate, 0.3 mg/l BA, 0.2 mg/l IAA, 2% sucrose, 0.6% agar (pH 5.6) and incubated for 6 weeks at 25ºC. The rooted plantlets were then cultivated in a mixture of 6:2:1 soil, sand and farmyard manure, strengthened under controlled temperature and humidity for 2 months and then transferred to the field.

Silver nitrate amplified the growth of both types of coffee. At 40 µM it nearly doubled the growth of arabica shoots .whereas leaf number and area were more with 10 µM and the chlorophyll content was higher with 20 µM. In robusta,20 µM silver nitrate gave the greatest shoot length and most leaves. But 10 µM induced more leaf area and chlorophyll. The seedling-based shoot tips were not open to silver nitrate for the first 72 h because it seemed likely that ethylene may be required for growth during this period. They found that 20 mg/l silver nitrate is required to get arabica shoots 3 cm long only after 70–80 days on a medium containing 1 mg/l BA. In the present study, much lower levels of silver nitrate with IAA- and BA-containing medium gave long shoots with more nodes and leaves in only 45 days. The effect of silver nitrate on in vitro shoot growth of robusta coffee has not been described previously. After rooting, 60–70% of plants remain hardening in a greenhouse and all grew well in the field.

Lopes CR, Monaco LC (1979) Chemotaxonomic studies of some species of the genus Coffea. J of Plant Crops 7:6–14
Mazzafera P, Crozier A, Magalhães AC (1991) Caffeine metabolism in Coffea arabica and other species of coffee. Phytochem 30:13913–3916
Skirvin RM, Norton M, McPheeters KD (1993) Somaclonal variation: has it proved useful for plant improvement? Acta Hortic 336:333– 340
Sridevi V, Giridhar P (2008) Recent trends in coffee biotechnology towards quality improvement. Ind J Bot Res 4:5–12
Evans JM and Batty NP (1994) Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture. Plant Cell Reports 13: 676–678.
Ganesh SD and Sreenath HL (1996) Silver nitrate enhanced shoot development in cultured apical shoot buds of Coffea arabica cv. Cauvery (S.4347). Journal of Plantation Crops 24: 577–580.

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