EFFECT OF THIODIAZURON ON MULTIPLE SHOOT INDUCTION OF DIPLOID COTTON (G. ARBOREUM CV.PA255)

About Authors:
Nitin Deorao Rewatkar
Department of Biotechnology, Kamla Nehru College,
Sakkardara Square, Nagpur
nitinrewatkar@gmail.com

Abstract
The effect of TDZ on regeneration studies of cotton G.arboreum cv PA255 was performed for in-vitro culture studies, healthy and disease free seeds, after surface sterilization were inoculated on seed germination media. Seeds germination efficiency was observed to be 73.38%. Shoot tips and cotynode explants aseptically isolated from in Vitro germinated seedlings of 7 and 14 days old were inoculated on MS basal salt supplemented with 30 gm/L glucose, 10 mg/L thiamine, 100 mg/L Inositol with four different concentration of TDZ. The proliferation of 7 and 14 days old shoot tip was found to 100% in media combination with 0.08 TDZ and 0.02 TDZ respectively. Cotynode explants of 7 days old was 100% responded in 0.02 TDZ and 0.08 TDZ, 14 days old cotynode shows highest 85% explants responded in 0.02 TDZ media combination but multiple shoots observed higher in 0.05 TDZ medium.TDZ induces callus formation and inhibit root formation, TDZ shows good responsein very small concentration otherwise increase concentration more than 1 mg/l it shows toxic effect on explants and completely dried. Browning and subsequent death of the cultured explants are major problems in TDZ supplemented media. Calculate the amount of phenol excreted from shoot tip and cotynode during in vitro regeneration of cotton cv. PA255. In the media combination 0.1mg /L TDZ evaluate  highest phenol secreted, shoot tip secrete average 0.014mg and cotynode secrete 0.022mg phenol by per explants.

Reference Id: PHARMATUTOR-ART-1414

Abbreviation : TDZ- Thiodiazuron,

Introduction
The field of plant biotechnology is concerned with developing ways to improve the production and productivity of plants in order to meal the world’s needs for food, fiber and fuel. As our population grows, our needs also grow. But land under cultivation remains the same. Therefore with the help of Plant tissue culture technique can be improved for better production. Tissue culture in combination of genetic engineering has helped to produce large number of transgenic plant for biotic stress resistance and improve quality        Cotton is known as a white gold because it is important cash crop of global significance which is cultivated in tropical & subtropical region of more than 70 countries the world over. The major producers of cotton are USA, China, India, Pakistan, Uzbekistan, Brazil, Mexican and Turkey. These countries contribute about 85% to the global cotton production. 111.

Through the use of biotechnology, desirable genetic traits can be transferred from one organism to another by transfer of DNA. Many more plants with the desirable DNA can be regenerated from small pieces of the transformed plant tissue. Due to the economic importance of cotton through genetic engineering, a great deal of effort has been made to develop systems to recover fertile transgenic cotton plants (Aragaro et al, 2005). Thus there is considerable interest in the development of tissue culture and gene transfer protocol in this species. Cotton as such is relatively difficult for in- vitro culture. Initially efforts were made to transform cotton using Agrobacterium tumefaciens and first transgenic cotton was developed (Firoza bady et al, 1987), and (perlak et al 1987). The transgenic cotton developed by these workers were later commercialized in USA and rest of this countries cultivar showing regeneration of cotton by somatic embryogenesis are few and mostly related  to coker genotypes. 22

There have been reports that genotypes other than coker have also been tried to investigate by somatic embryogenesis (Zhao et al 1996) .Though most of the genotypes showed low frequency of somatic embryogenesis, the embryos are either abnormal or not mature prosperity, therefore low rate of germination of genotypes these was genotype specific and even seedlings specific (Trolinder and xhixian 1989) 33

Direct shoot regeneration is another approach for plant regeneration Gould et al(1991), (Nandeshwar 1995)  Nandeshwar et al (2002) have reported regeneration by shoot tip explants and explain the possibility of using this protocol in genetic transformation Agrawal et al (1997) used cotyledonary node and shoot tip are regenerated cotton plants by direct shoot organogenesis or multiple shoot induction .It has been observed that incidence of genetic mutation and somaclonal variations are low as compare to plants regenerated by somatic embriyogenesis (Gould et al 1991, 1997), Nandeshwar et al,2002) 44

One of the most important factors for transformation in plants is the establishment of an efficient regeneration protocol of cells and/or tissues. In the specific case of cotton, most of the genotypes of commercial interest present problems of plant regeneration through callus or cells in suspension (Bayley et al., 1992). It has been considered a recalcitrant species to in vitro proliferation (Gupta et al., 1997). Therefore, improvement of methods of Regeneration in vitro is necessary to make viable research on transformation and regeneration of this species.55

Thiodiazuron (TDZ) is one of the various kinds of substituted urea that has been investigated for induction of multiple shoots in plants. Although it has been initially developed as an herbicide, its use as a growth regulator in woody species in vitro is considered one of the most positive innovations in tissue culture during the last few years (Huetteman and Preece, 1993). There are several reports about the positive effects of TDZ in the proliferation of auxiliary shoots. Huettman and Preece (1993) reported results by several authors from various species, in which positive effects of TDZ to the culture medium have been observed. Bhagwat et al. (1996) obtained multiple shoots of cassava from nodal explants by exposition to TDZ with concentrations ranging from 0.11 to 0.22 μM in liquid medium for 6 to 8 days. 66

Browning and subsequent death of the cultured explants are major problems for many tissue culture non hard-adaptive species and usually dependant on the phenolic compounds and the quantity of total phenols many studies, using shoot tip as explants source indicated various problem such as phenol exudation, media discoloration, rooting deficiencies and explants browning and death. 77

The phenol is synthesized by the plants and in many tissue culture studies, excreted and the oxidized phenol effect in vitro proliferation negatively. In addition, amount of phenols can be more or less in different stages of organogenesis due to metabolic actions.

Therefore determination of phenols and calculating, the amount of phenols may be another research area for many tissue culture studies. In this study, phenol amount of  shoot tip and cotynode leached in media were evaluating during in vitro of regeneration of cotton PA255 Singleton-Rossi method based folin-ciocalteu reagent was used for determination of total phenol concentration of the media.

Methods

In-vitro seed germination
Seed sterilization
Seeds  of good quality were selected and taken in a conical flask and washed with tap water for five min .To this was then added autoclaved DD water with few drops of liquid detergent agitated for few min followed by 3-4 washes with DD water. Seeds were then transferred in the flask containing Bavistin solution (1% w/v) and kept on orbital shaker  for 20 min .this was again washed with autoclaved DDW  3-4 time till the trace of bavistin is completely removed . The seeds were then treated with HgCl2 solution (0.1%) for 7 min, washed 4-5 times and inoculated in the flask containing of half MS at the rate of 15-20 seeds /flask.