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In Vitro direct plant regeneration using shoot tip ... · PDF file single step method without callus interphase for direct plant regeneration, which can be used commercially for rapid

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    *Corresponding author’s e-mail: 1Department of Biotechnology, DAV College, Amritsar-143001, Punjab, India.

    In Vitro direct plant regeneration using shoot tip explants in sugarcane (Saccharum officinarum L.) for rapid mass cloning Rupinder Kaur1 and Manish Kapoor*

    Department of Botany, Punjabi University, Patiala-147 001, Punjab, India. Received: 14-03-2017 Accepted: 29-05-2017 DOI: 10.18805/asd.v37i2.7981

    ABSTRACT Protocol for mass propagation, through direct plant regeneration in three commercial cultivars of sugarcane (CoJ64, CoJ83 & CoJ86) was standardized. Cultures were established from the young leaf segments (1.0 - 1.5 cm), excised from spindle leaf (Shoot tip), used as explant source, were cultured on different media compositions based on Murashige and Skoog salts. Cultured explants exhibited swelling followed by direct shoot regeneration on media containing NAA, in all the three varieties. The highest frequency of shoot regeneration (87.58%) occurred on MS medium supplemented with NAA (5.0 mg/L) and Kinetin (0.5 mg/L) in variety, CoJ83. Medium devoid of NAA and supplemented with only kinetin did not induce direct shoot regeneration in any of the varieties thus tried. Subsequently profuse rooting of shoots was observed on the same medium and complete plantlets were recovered within 6 weeks. The sugarcane plantlets were acclimatized in greenhouse. The plantlets were hardened and transferred to soil, which exhibited good survival ranging from 85-90%. Tissue culture derived field-grown plants were normal and exhibited faster growth and better tillers. This protocol is a single step method without callus interphase for direct plant regeneration, which can be used commercially for rapid mass cloning of elite germplasm of sugarcane.

    Key words: Direct plant regeneration, Micropropagation, Organogenesis, Rapid mass cloning, Saccharum officinarum L., Shoot tip culture.

    INTRODUCTION Sugarcane (Saccharum officinarum. L.), an

    important cash crop of the world, is the major source of sugar and a perennial monocot plant. It is the member of the family Gramineae that belongs to genus Saccharum in which in vitro propagation protocol need to be standardized for commercial viability. Sugarcane is among the top 10 food crops, cultivated on a large scale in tropical and sub-tropical regions, grown for sugar and for other industrial products, viz. furfural, dextran and alcohol. In India, the sugar industry plays a vital role towards socio-economic development in the rural areas, by mobilizing rural resources and generating higher income and employment opportunities. India is the world’s second largest producer of sugar and biggest consumer of sugar. In India, sugarcane plays a pivotal role in national economy by contributing 1.9 per cent to GDP. The crop is being cultivated on 5.15 million hectare producing 355 million ton with productivity of 70 ton per hectare, thus contributing more than 20% of global cane sugar production and sharing about 41.11% of Asian Sugar Production (Murthy, 2010). India stands next to Brazil (5.77 million hectare) in area and production 232.32 million ton (India) next to Brazil (420.12 million ton), in sugarcane

    production. However, in terms of per hectare of sugarcane productivity, India (70 ton) stands tenth, the first being Colombia (92.29 ton).

    Being a tropical crop, it requires hot and humid weather for its flowering and seldom flowers in the North Indian states. Varieties of sugarcane are highly heterogeneous and generally propagated vegetatively by setts (stem cutting which are nodal sections of sugarcane with 2 or 3 nodes) (Tiel et al., 2006). There is a systemic buildup of infections by systemic diseases particularly viruses, generation after generation, which ultimately lead to reduced vigor, are a serious problem. Lack of suitable multiplication procedures, for the mass multiplication of the elite germplasm, results in varietal decline (Kaur and Kapoor, 2016). The yield potential of sugarcane varieties is deteriorating, due to susceptibility to diseases, insects, admixture and changing edaphic and climatic environment (Gill et al., 2006). Moreover, the lack of rapid multiplication procedures has long been a serious problem in sugarcane breeding programs, as it takes 10-15 years of work to complete a selection cycle. In Punjab, red rot caused by Colletotricum falcatum is a serious disease and almost all the commercial varieties are susceptible to a variable extent (Lal and Singh, 1994).

    Agric. Sci. Digest., 37(2) 2017: 94-99 Print ISSN:0253-150X / Online ISSN:0976-0547

  • Volume 37 Issue 2 June 2017 95

    Micropropagation (propagation through apical meristem) is an established, as well as a popular mean of clonal propagation, is the most viable and successful method for production of pathogen free stock material. The main advantage of micropropagation is the rapid multiplication of new varieties, improved plant health and its usefulness in germplasm storage. It is the best method for propagation as it produces plants phenotypically similar (true to type) to the mother plant and gives much more rapid multiplication rate. Shaw (1990) also reported that, micropropagation was being used in some sugarcane industries, for the development of disease free clones, mostly to facilitate their safe and speedy movement through quarantine. Lal et al., (1996) demonstrated micropropagation system by exhibiting a in vitro production potential of 75600 shoot from a single shoot apex explant in a period of about 5.5 months. Gosal et al., (1998) had reported that micro-propagated plants, grown in the field had up to 44.96% more canes per plot and up to 22.9% greater cane yield per plot than plants conventionally propagated from three-budded setts. In order to ensure highest possible yield and quality of field crops the disease free stock can be extended to the growers by using this technique (Khan et al., 2008; Khan et al., 2009).

    In vitro multiplication of sugarcane has received considerable research attention and micro-propagation is currently the only realistic means of achieving rapid, large- scale production of disease-free quality planting material as seed canes of newly developed varieties in order to speed up the breeding and commercialization process in sugarcane (Feldmenn et al., 1994; Lal and Krishna, 1994). The present research work was undertaken by keeping in view the importance of tissue culture technology in sugarcane improvement and establishment of efficient protocols for mass scale propagation of healthy, disease free and premium quality planting material through micropropagation to enhance the yield and recovery per hectares of sugarcane. MATERIALS AND METHODS:

    Healthy spindles (1.5 cm diameter and 4-5 cm long) of three commercially important sugarcane cultivars (CoJ64, CoJ83 & CoJ86) procured from Punjab Agricultural University Sugarcane Research Station, Ludhiana, were surface sterilized (0.1 % HgCl2 for 10 minute). Nodal portion of spindle was discarded and the outer two or three whorls of leaves were removed using a sharp scalpel blade. Innermost leaf whorls were cut obliquely (0.5-1.0 cm) in order to achieve callus initiation, and were used as an explant for culturing. Callus cultures were established by culturing the spindle segments on MS medium (Murashige and Skoog, 1962) supplemented with various combinations and concentrations of naphthalene acetic acid (NAA), kinetin (Kin) and benzyl amino purine (BAP). Various media compositions MS1-MS9 were used for direct plant

    regeneration.The cultures were incubated at 25 ± 2ºC at 70- 80% humidity and diffused light for first 15 days before shifting to illumination of 5000 lux white fluorescent light, with 16/8 hour (light/dark) photoperiod regimes. Regenerated plants from young leaves were sub-cultured for further multiplication at fortnight intervals. The observations were recorded for length of shoots, number of shoots and number of albino plants.

    Subsequently profuse rooting of shoots was observed on the same medium after five weeks and complete plantlets were recovered within 6 weeks, without callus interphase. Whole plant (both shoots and roots) were regenerated without transferring the plantlets to rooting medium. Plantlets with well-developed root system were removed from the test tubes and thoroughly washed under running tap water. Plants were then hardened by keeping them on cotton soaked in half strength MS medium (without sugar and vitamins), in open test tubes, under strong fluorescent light (5000 lux) for about 15 days for elongation of roots and for hardening of plants at 28oC. Medium was changed daily to avoid contamination. The hardened in vitro produced rooted plants were transferred to normal field soil, in polythene bags containing field soil, coco peat and farmyard manure (1:1:1) in the greenhouse.

    In all the above experiments, each culture tube received a single explant. Each replicate contained 20 cultures and one set of experiment is made up of 3 replicates (60 leaf sections were cultured for one set of experiment for each of the sugarcane varieties. Data was subjected to statistical analysis ANOVA (analysis of variance, p


    Treatments Media composition Sugarcane varieties CoJ64 CoJ83 CoJ86

    MS1 MS + NAA (5.0mg/L) + Kin (0.5mg/L) 85.49 (67.59)* 87.58 (69.35)* 79.44 (63.02)* MS2 MS + NAA (5.0mg/L) + Kin (1.0mg/L) 84.30 (66.64)* 78.20 (62.15)* 77.29 (61.52)* MS3 MS + NAA (10.0mg/L) + Kin (0.5mg/L) 82.50 (65.25)* 79.45 (63.03)* 77.01 (61.32)* MS4 MS + NAA (10.0mg/L) + Kin (1.0mg/L) 80.20 (63.56)* 77.14 (61.42)* 74.97 (59.96)* MS5 MS + NAA (5.0mg/L) + Kin (1.

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