Callus induction and Organogenesis in Sugarcane ( Saccharum officinarum L.) var 93v297

. An efficient protocol for induction of callus and regeneration of a sugar cane var 93v297 has been developed and reported here. Callus induction from immature young leaf explants derived from 2-3-month-old plants was achieved on Murashige and Skoog’s (MS) medium supplemented with different auxins viz, 2,4-D, NAA and IAA. Among different auxins, 2, 4-D at 3.5mg/l + 0.5mg/l BAP was found favourable in inducing callus. Addition of coconut milk and BAP further enhanced the growth of callus maximum being on MS medium supplemented with 0.5mg/l BAP (3602.33±0.88mg). Calli were further evaluated for regeneration. MS medium supplemented with 1.0 mg/l BAP was found suitable where 100% calli regenerated with maximum number of multiple shoots per callus mass (41.40±0.89). Highest number of root emergence (28.33±1.16) and maximum root length (3.40±0.67cm) was achieved on MS medium supplemented with 3mgl/l NAA. The in vitro grown plants were transferred to polycups containing a mixture of sterilized sand, soil and cocopeet (1:1:1) for hardening. The hardened plants were transferred to green-house conditions where they survived with 90% frequency.


INTRODUCTION
Sugarcane (Saccharum officinarum L.) is known in India from time immemorial. It belongs to the family of grasses (Poaceae). It occupies a distinct position as an agro-industrial crop of India, covering around 5.06 million hectares area (over 3% of the total cultivated area) with an annual production of 341.20 million tons and annual yield of 6.74lakh Hg/Ha (FAOSTAT, 2014), contributing about 9.5% to the gross value of the agricultural production in the country. Sugarcane accounts for nearly 70% of the worlds' sugar and is an economically important cash crop in tropical and sub-tropical climatic regions (Chatenet et al., 2001). Apart from use in sugar production, it is gaining importance for production of ethanol and some by-products like molasses, stock feed, alcoholic drinks, bagasses and cane wax. Sugarcane is a monoculture, clonally propagated crop and is prone to variety of pests and diseases and propagation from cuttings facilitates the spread of the pathogens and may result in epidemics (Schenck and Lehrer, 2000). Due to its importance globally, constant efforts are being made world over for its improvement, through tissue culture techniques (Heinz and Mee, 1969;Ahloowalia and Meretzki, 1983;Liu, 1993). Barba and Nickel (1969) who first independently demonstrated that plantlets could be developed from sugarcane callus cultures, and from the published results, it is evident that every part of the sugarcane plant is capable of producing callus (Liu, 1993). Callus induction is a very important for inducing genetic modifications in this crop (Matsuoka et al., 2001). Immature leaf rolls (Rao and Jabeen 2013; Soares et al., 2014), apical meristem (Kaur and Gosal, 2009;Ramgareeb et al., 2010) and the young leaves (Chengalrayan and Gallo-Meagher 2001) are capable of producing morphogenic callus to any appreciable level. Callus based regeneration will be very important as in vitro mutations and somaclonal variants could be induced and subsequently used for crop improvement. Keeping in view of the importance of callus induction and callus mediated regeneration, the present investigations is carried out for optimizing a complete plant tissue culture protocol (callus induction, regeneration and acclimatization).

MATERIALS AND METHODS
The plant material sugarcane var 93v297 was obtained from Vyuru Agriculture Research Station, Andhra Pradesh and from fields nearby Kalaburagi (India). Young leaf sheath of 2-3months-old plants were used as explants. The explants were thoroughly washed under running tap water to remove surface adhered particles and then washed with 1% (v/v) soap solution and Bavistin for 10 minute and further washed with distilled water till froth was completely removed, then they were surface sterilized for 3 min in 0.1% (w/v) mercuric chloride, rinsed with deionised water thrice to remove traces of mercuric chloride. Further the outer 2-3 layers of the explants were removed and inner leaves were cut into pieces measuring approximately 1cm and were then aseptically inoculated on Murashige and Skoog's (1963) medium supplemented with 3% sucrose and different types of auxins viz., 2, 4-dichlorophenoxyaceticacid (2, 4-D), α-naphthaleneacetic acid (NAA), and Indole acetic acid (IAA) at various concentrations and medium was gelled with 0.8% agar (w/v). pH of the medium was adjusted to 5.7 prior to autoclaving at a pressure of 15psi at 121 ºC for 15 minutes, then dispensed into pre-sterilized culture tubes and the cultures were maintained under cool-white florescent light at 60µM -2 S -1 (16h light/8h dark) at 26±1 ºC for four weeks. A complete randomized design with 30 explants per culture was maintained and all the experiments were repeated thrice. Relative growth rate of callus was determined after 4-weeks of culture. For regeneration, approximately 250±10 mg callus were placed on MS medium supplemented with 3% sucrose containing MS salts with or without cytokinins.

STATISTICAL ANALYSIS
All the data was subjected to Tukey-Kramer multiple comparison test (One way ANOVA) to see the standard error (SE) and level of significance using Instat graphpad prism software made for windows (La Jolla, USA).

RESULTS AND DISCUSSION
Induction of Callus: Immature young leaf sheaths of sugarcane were used as starting material to generate callus on medium containing different types of auxins at various concentrations (Table 1). Callus induction was observed within ten days after inoculation of the explants on the medium. All the growth regulators used viz., 2, 4-D, NAA and IAA used at various concentrations (1.0-4.0mg/l) induced callus with varying frequencies. 2, 4-D at 3.5mg/l induced maximum amount of callus with 100% frequency followed by NAA at 1.0mg/l with 70% and IAA with 50% frequency at 1.0mg/l. Growth in terms of fresh weight of callus was 2740±0.94mg on 3.5mg/l 2, 4-D, followed by 1.0mg/l NAA which was 712±0.54mg (Plate: 1a, b, c) and the least was recorded on 1.0mg/l IAA supplemented medium (320±0.33mg), further increase in the concentrations of the auxins resulted in decreased growth rate of callus (Table-1). Supplementing coconut water at 5% to 2, 4-D containing medium further increased the growth of callus (3208±0.72mg). Highest growth of callus was observed on MS medium supplemented with 3.5mg/l 2, 4-D + 0.5 mg/l BAP (3602.33±0.88) (Table: 2 Organogenesis: Callus was evaluated for multiple shoot induction on MS medium supplemented with different cytokinins viz., BAP, Kn and TDZ. Approximately 100-150mg of fresh callus was sub cultured on medium containing cytokinins. It was noticed that lower concentrations of cytokinins induced higher number of multiple shoots from the callus with varying frequencies depending upon the type of growth regulator used. BAP (1.0mg/l) alone induced maximum multiple shoots (41.40±0.89) with 100% frequency, further increase in the concentrations of BAP resulted in decrease in the differentiation of shoots, however the frequency of shoot induction was not affected. Kn (1.0mg/l) and TDZ (0.2mg/l) were also found suitable for production of multiple shoots (29.12±0.33 and 18.91±2.03 respectively) but the frequency and number of multiple shoots inducted was lesser than shoots obtained on BAP supplemented medium (Plate: 2b, Table: 3). Similar reports were available on regeneration from callus using low concentrations of BAP ( ) are reported to be useful in root induction in sugarcane; however, rooting on medium devoid of growth regulators but only at high concentrations of sucrose (6-8%) has been reported (Meretzaki and Hiraki, 1980). Well rooted plants were transferred to the poly cups (Fig. 2d) containing cocopeet, soil and sand mixture (1:1:1). Plantlets were initially covered with plastic bags to maintain high humidity in the culture rooms. MS medium was added to the plantlets on daily basis during this period. After 15-20 days, plastic bags were removed and thus they were acclimatized to the normal temperature later brought to the green house with 90% success rate.

CONCLUSION
From the present investigation and results of the earlier literature it can be concluded that sugarcane mainly depends on the concentrations and combination of growth regulators irrespective of the variety. Callus induction requires auxins especially 2, 4-D and it is played beneficial role when it is supplemented with BAP and for regeneration through callus requires low concentrations of cytokinins (BAP) for efficient regeneration and induction maximum multiple shoots.