This document outlines a research project on callus induction in sugarcane (Saccharum officinarum L.) cv CoM 0265 using 2,4-D. The objectives are to determine the optimal concentrations of 2,4-D and its interaction with kinetin for callus induction and the minimum number of days required. The experiment will use a factorial randomized block design with 12 treatments testing combinations of 2,4-D at 1.5, 2, 2.5, and 3 mg/L with or without kinetin at 0.1 or 0.2 mg/L. Nodal segments from sugarcane will be sterilized and cultured on MS medium with the hormone treatments. The effects on call

1. OUTLINE OF RESEARCH PROJECT WORK ON
“Callus induction in sugarcane (Saccharum officinarum L.)cv CoM 0265 by using
2,4 D”
Submitted to
The Principal
Mahatma Gandhi Mission
College of Agricultural Biotechnology, Aurangabad
(Affiliated to Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani)
(ISO 9001:2008 Certified)
Submitted by,
Name of student: Ingle Snehal Devidas. Reg. No.:2010/30
Academic Year: 2014-15 Session: Summer
Course No.: HOT 481 Degree: B.Sc. (Agri. Biotech.)
Course Title: Hands on Training, Semester: VIII (New)
Credits: 20 (0+20)
Research Project Guide
Dr. Ashwin B. Kshirsagar
Associate Professor
Department of Plant Biotechnology
MGM College of Agricultural Biotechnology, Aurangabad

2. CONTENT
Sr. No. TITLE Page. No.
1. Introduction 4
2. Objectives 6
3. Review of literature 7
4. Materials and method 10
5. Literature cited 13

3. Sr. No. Particulars
1. Introduction
2. History of Plant Quarantine
3. Types of Quarantine
4. List of Quarantine Diseases
5. Quarantine Labs
6. Laws & Regulation
7. Application
8. References

4. OBJECTIVES
The present investigation entitled “Callus induction in sugarcane (Saccharum officinarum L.)
CoM 0265 by using 2,4 D”
•To find out optimization concentrations of 2, 4- D and its interaction with kinetin for callus
induction of sugarcane.
•To determine the minimum number of days for callus induction.

5. MATERIALS AND METHODS
The details of materials to be used and methods will be adopted for conducting the present
investigation is described in this chapter under appropriate heads.
A. Experimental site:
The experiment will be conducted in Department of Plant Biotechnology MGM College of
Agriculture Biotechnology, Gandheli.
B. Experimental details:
The study will be conducted in the controlled laboratory conditions. The MS basal medium will
be used to study the synergistic effects of different combination of 2,4-D and KIN on callus
induction in Sugarcane.
C. Statistical Design : Factorial Randomized Block Design (FRBD)
No. of treatments : 12
Total no. of replication : 03
Treatment details : Concentrations of 2,4-D and KIN.

6. Sr. No Treatment (T) Concentrations (mg/L)
01 T01 MS+2,4-D(1.5)
02 T02 MS+2,4-D(2.0)
03 T03 MS+2,4-D(2.5)
04 T 04 MS+2,4-D(3.0)
05 T05 MS+2,4-D(1.5)+KIN(0.1)
06 T 06 MS+2,4-D(2.0)+KIN(0.1)
07 T07 MS+2,4-D(2.5)+KIN(0.1)
08 T08 MS+2,4-D(3.0)+KIN(0.1)
09 T09 MS+2,4-D(1.5)+KIN(0.2)
10 T10 MS+2,4-D(2.0)+KIN(0.2)
11 T11 MS+2,4-D(2.5)+KIN(0.2)
12 T12 MS+2,4-D(3.0)+KIN(0.2)

7. Other experimental details:
Plant material and surface sterilization:
The nodal segment used as explants of sugarcane (Saccharum officinarum L.) will be obtained
from the nearby farm at Gandheli.
The nodal segment of sugarcane will be cut into small segments and wash with running tap water
to remove dirt for 10 minutes. After these initial washing, the explants will be kept in an aqueous
solution of Bavistin (1%w/v) for 10minutes. Then explants will be washing with household
detergent (Laboline) with few drops of Tween-20 for 15 min followed by repeated rinsing with
distilled water for 10 min. After that explant will be transferred to laminar air flow for surface-
disinfect using 70% ethanol for 10-20 sec. and further sterilization with 0.1% mercuric chloride
(HgCl2) solution containing few drops of Tween-20 for 10 minutes and wash and rinse with
sterilized double distilled water (DDH2O) for 5 times. The explants will be trimmed before
transfer to the callus induction medium.
The cleaning of glassware for the growth of tissues in vitro by using chemical such as
hydrochloric acid (1N) for 2 hr or overnight and laboline and rinse thoroughly with tap water,
allow to dry in hot air oven at 140- 160ºC for 2 hr.
Preparation of Media:
The appropriate composition of the medium largely determines the success of the cultures. Plant
materials do vary in their nutritional requirements and therefore it is often necessary to modify
the medium to suit a particular tissue. Plant growth regulators: growth as well as differentiation
of tissues in vitro is controlled by various growth regulators.

8. Auxin (2,4-D) play a vital role in inducing callus and maintaining callus and suspension cells in
differentiated state usually used as sole auxin source (1-50µm) or in combination Cytokines are
often used in culture media for callus induction, growth of callus & cell suspension and induction
of morphogenesis (1-220µm) used in higher conc. (20-50µm) induce the rapid multiplication of
shoots , increased photosynthetic pigments, photosynthetic activity. It diminished the inhibitory
effect of pb on the chlorophyll content.