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*Corresponding author’s e-mail: jdmanishkapoor@yahoo.com1Department 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 cloningRupinder 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

ABSTRACTProtocol 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 spindleleaf (Shoot tip), used as explant source, were cultured on different media compositions based on Murashige and Skoogsalts. Cultured explants exhibited swelling followed by direct shoot regeneration on media containing NAA, in all the threevarieties. The highest frequency of shoot regeneration (87.58%) occurred on MS medium supplemented with NAA (5.0mg/L) and Kinetin (0.5 mg/L) in variety, CoJ83. Medium devoid of NAA and supplemented with only kinetin did notinduce direct shoot regeneration in any of the varieties thus tried. Subsequently profuse rooting of shoots was observed onthe same medium and complete plantlets were recovered within 6 weeks. The sugarcane plantlets were acclimatized ingreenhouse. 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 asingle step method without callus interphase for direct plant regeneration, which can be used commercially for rapid masscloning of elite germplasm of sugarcane.

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

INTRODUCTIONSugarcane (Saccharum officinarum. L.), an

important cash crop of the world, is the major source of sugarand a perennial monocot plant. It is the member of the familyGramineae that belongs to genus Saccharum in which invitro propagation protocol need to be standardized forcommercial viability. Sugarcane is among the top 10 foodcrops, cultivated on a large scale in tropical and sub-tropicalregions, grown for sugar and for other industrial products,viz. furfural, dextran and alcohol. In India, the sugar industryplays a vital role towards socio-economic development inthe rural areas, by mobilizing rural resources and generatinghigher income and employment opportunities. India is theworld’s second largest producer of sugar and biggestconsumer of sugar. In India, sugarcane plays a pivotal rolein national economy by contributing 1.9 per cent to GDP.The crop is being cultivated on 5.15 million hectareproducing 355 million ton with productivity of 70 ton perhectare, thus contributing more than 20% of global cane sugarproduction and sharing about 41.11% of Asian SugarProduction (Murthy, 2010). India stands next to Brazil (5.77million 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 sugarcaneproductivity, India (70 ton) stands tenth, the first beingColombia (92.29 ton).

Being a tropical crop, it requires hot and humidweather for its flowering and seldom flowers in the NorthIndian states. Varieties of sugarcane are highly heterogeneousand generally propagated vegetatively by setts (stem cuttingwhich are nodal sections of sugarcane with 2 or 3 nodes)(Tiel et al., 2006). There is a systemic buildup of infectionsby systemic diseases particularly viruses, generation aftergeneration, which ultimately lead to reduced vigor, are aserious problem. Lack of suitable multiplication procedures,for the mass multiplication of the elite germplasm, results invarietal decline (Kaur and Kapoor, 2016). The yield potentialof sugarcane varieties is deteriorating, due to susceptibilityto diseases, insects, admixture and changing edaphic andclimatic environment (Gill et al., 2006). Moreover, the lackof rapid multiplication procedures has long been a seriousproblem in sugarcane breeding programs, as it takes 10-15years of work to complete a selection cycle. In Punjab, redrot caused by Colletotricum falcatum is a serious diseaseand almost all the commercial varieties are susceptible to avariable extent (Lal and Singh, 1994).

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Micropropagation (propagation through apicalmeristem) is an established, as well as a popular mean ofclonal propagation, is the most viable and successful methodfor production of pathogen free stock material. The mainadvantage of micropropagation is the rapid multiplicationof new varieties, improved plant health and its usefulness ingermplasm storage. It is the best method for propagation asit produces plants phenotypically similar (true to type) tothe mother plant and gives much more rapid multiplicationrate. Shaw (1990) also reported that, micropropagation wasbeing used in some sugarcane industries, for the developmentof disease free clones, mostly to facilitate their safe andspeedy movement through quarantine. Lal et al., (1996)demonstrated micropropagation system by exhibiting a invitro production potential of 75600 shoot from a single shootapex explant in a period of about 5.5 months. Gosal et al.,(1998) had reported that micro-propagated plants, grown inthe field had up to 44.96% more canes per plot and up to22.9% greater cane yield per plot than plants conventionallypropagated from three-budded setts. In order to ensurehighest possible yield and quality of field crops the diseasefree stock can be extended to the growers by using thistechnique (Khan et al., 2008; Khan et al., 2009).

In vitro multiplication of sugarcane has receivedconsiderable research attention and micro-propagation iscurrently the only realistic means of achieving rapid, large-scale production of disease-free quality planting material asseed canes of newly developed varieties in order to speedup the breeding and commercialization process in sugarcane(Feldmenn et al., 1994; Lal and Krishna, 1994). The presentresearch work was undertaken by keeping in view theimportance of tissue culture technology in sugarcaneimprovement and establishment of efficient protocols formass scale propagation of healthy, disease free and premiumquality planting material through micropropagation toenhance 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 AgriculturalUniversity Sugarcane Research Station, Ludhiana, weresurface sterilized (0.1 % HgCl2 for 10 minute). Nodal portionof spindle was discarded and the outer two or three whorlsof leaves were removed using a sharp scalpel blade.Innermost leaf whorls were cut obliquely (0.5-1.0 cm) inorder to achieve callus initiation, and were used as an explantfor culturing. Callus cultures were established by culturingthe spindle segments on MS medium (Murashige and Skoog,1962) supplemented with various combinations andconcentrations of naphthalene acetic acid (NAA), kinetin(Kin) and benzyl amino purine (BAP). Various mediacompositions 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 beforeshifting to illumination of 5000 lux white fluorescent light,with 16/8 hour (light/dark) photoperiod regimes.Regenerated plants from young leaves were sub-cultured forfurther multiplication at fortnight intervals. The observationswere recorded for length of shoots, number of shoots andnumber of albino plants.

Subsequently profuse rooting of shoots wasobserved on the same medium after five weeks and completeplantlets were recovered within 6 weeks, without callusinterphase. Whole plant (both shoots and roots) wereregenerated without transferring the plantlets to rootingmedium. Plantlets with well-developed root system wereremoved from the test tubes and thoroughly washed underrunning tap water. Plants were then hardened by keepingthem on cotton soaked in half strength MS medium (withoutsugar and vitamins), in open test tubes, under strongfluorescent light (5000 lux) for about 15 days for elongationof roots and for hardening of plants at 28oC. Medium waschanged daily to avoid contamination. The hardened in vitroproduced rooted plants were transferred to normal field soil,in polythene bags containing field soil, coco peat andfarmyard manure (1:1:1) in the greenhouse.

In all the above experiments, each culture tubereceived a single explant. Each replicate contained 20cultures and one set of experiment is made up of 3 replicates(60 leaf sections were cultured for one set of experiment foreach of the sugarcane varieties. Data was subjected tostatistical analysis ANOVA (analysis of variance, p<0.05)as per Factorial Completely Randomized Block Design tosee the level of significance and least significant difference(LSD) for media, variety and for variety media interactionwas calculated. Data presented in the tables were arcsinetransformed before being analysed for significance (Gomezand Gomez, 1984).RESULTS AND DISCUSSION:Direct regeneration: The healthy spindle leaves, excisedand cultured aseptically, on different concentrations andcombinations of auxin and cytokinin. Cultures wereincubated in diffused light for first fortnight and then shiftedto high light intensity in medium MS1 to MS7, exhibitedswelling and unwhorling during the first week of incubationin all the three varieties (Figure 1a). Whereas, the explantscultured on media MS8 and MS9 supplemented with onlyKin (0.5 and 1.0 mg/L), respectively, remained quiescentand started turning brown after 4 weeks of incubation. Duringthe third week of incubation, leaf segments cultured on auxinand cytokinin supplemented media i.e., MS1 to MS7 exhibiteddirect shoot regeneration from their cut ends (Figure 1a, band c). It was remarkable to note that maximum number ofshoots that regenerated from one leaf segment was 15–20 in

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Treatments Media composition Sugarcane varietiesCoJ64 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 (1.0mg/L) + BAP (1.0 mg/L) 79.80 (63.27)* 75.52 (60.33)* 72.62 (58.43)*MS6 MS + NAA (10.0mg/L) + Kin (1.0mg/L) + BAP (1.0 mg/L) 79.62 (63.15)* 73.75 (59.16)* 69.99 (56.76)*MS7 MS + NAA (1.0mg/L) + BAP (1.0 mg/L) 4.13 (11.62)* 7.37 (15.71)* 3.74 (11.03)*MS8 MS + Kin (0.5mg/L) 2.56 (8.99)* 2.91 (9.65)* 1.39 (5.95)*MS9 MS + Kin (1.0mg/L) 1.69 (6.99)* 1.86 (7.45)* 1.33 (5.60)*

Table-1: Per-cent shoot regeneration observed in sugarcane varieties on different media.

CD for variety means at 5% level of significance = 1.65 CV = 3.82CD for media means at 5% level of significance = 0.91CD for variety × media interaction at 5% level of significance = 2.86* Figures within parentheses denote arcsine transformed values

number, by culturing the spindle leaves on the auxin richmedium, without callus interphase whole plant (both shootsand roots) were regenerated without transferring the plantletsto rooting medium. This system for obtaining direct shootsfrom the cultured spindle leaves has advantage being a singlestep regeneration, which require less time and minimize thechances of somaclonal variations. This system can beexploited just like leaf disc method in dicots. All the threevarieties exhibited fairly good plantlet regeneration. Explantsof the three varieties cultured on MS medium supplementedwith NAA (5.0 mg/L) and Kin (0.5 mg/L) showed plantregeneration in the range of 78-87%. Variety CoJ64 exhibitedmaximum per cent plantlet regeneration i.e. 87.58%,followed by CoJ83 (85.49%) and CoJ86 (79.44%) (Table1). On further increase in the concentration of NAA from5.0 to 10.0 mg/L and Kinetin from 0.5 to 1.0 mg/L in themedia there was decrease in the per cent shoot regenerationin all the varieties. The increased level (10.0 mg/L) of NAAresulted in little callusing at the cut ends and thus reducedthe per cent shoot regeneration in all the varieties as a resultresponse in CoJ83 for shoot regeneration declined by14.00%. It is evident from Table 1, that CoJ83 show 79.45%and CoJ86 (77.01%) and CoJ64 (82.50%) shootregeneration. Thus addition of auxin (NAA) was necessaryfor induction of direct shoot regeneration but elevatedconcentration of NAA reduced the per cent plantregeneration. By using two concentrations of NAA (5 and10 mg/L) a comparison was made in all the three varieties indifferent parameters. It was observed that maximum numberof albino plants was regenerated in CoJ64. The Number ofshoots per plant was maximum in CoJ83 with maximumlength of shoots (Fibure 1c).

It is noticeable that on further increase in theconcentration of cytokinin and by the addition of anothercytokinin in the medium MS5 [(MS + NAA (5.0 mg/L) +Kin (1.0 mg/L) + BAP (1.0 mg/L))]; the per-cent shoot

regeneration declined in all the three varieties. When MSmedium supplemented with NAA (10 mg/L) and Kin (1.0mg/L), CoJ64 variety showed higher percentage of shootregeneration of 80.20% followed by CoJ83 (75.52) and74.97% in CoJ86. Addition of BAP (1.0 mg/L) along withKinetin and NAA in the medium i.e., MS5 and MS6 did notshow any synergistic effect in relation to direct shootregeneration. Statistical analysis of the date revealed thatthe varieties varied significantly from each other in responseto direct plant regeneration and the media were alsosignificantly different at 5% level of significance.Regenerated shoots in all the media tried were normal, greenand healthy.

Besides using the higher concentrations of auxinfor spindle leaves regeneration a comparison was made withhigher concentrations of cytokinin. Cytokinins at very highlevel were not effective for the regeneration of spindle leaves.Cytokinins at very high concentrations tend to increase morechlorophyll in the tissue and also increase the compactnessof the tissue. Therefore at high level of cytokinin tissue mighthave got diverted toward the chlorophyll development ratherthan shoot regeneration. Table 1 reveals that on mediacontaining NAA (1 mg/L) and BAP (5 mg/L), per-cent shootregeneration ranges between 3-7%.

After five weeks of inoculation, profuse rootingoccurred from the base of shoots (Figure 1d). Variety, CoJ64exhibited profuse rooting in comparison to CoJ86 and CoJ83.Rooted shoot clumps when removed from the culture vesselsand transferred for hardening, continued to survive and didnot wilt. During hardening of plantlets developed secondaryand tertiary roots. The hardened plantlets transferred topolythene bags in the greenhouse (Figure 1f) were healthyand green with 95% survival of the hardened plants in thegreenhouse was recorded (Figure 1e and f).Hardening and transfer to soil: Tissue cultured regeneratedplants are very fragile and tender and most of these die if

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directly transferred to the field conditions because oftransplantation shock. Therefore, in order to improve theirsurvival in the field hardening is a very critical step.Regenerated plants both from the spindle leaves having welldeveloped root and shoot system were removed from testtubes and thoroughly washed under running tap water. Plantswere hardened by keeping in open test tube containing tapwater. Hardening of the plants was done for 15 days in thelaboratory conditions by changing the water daily. Thus, theprocess of hardening acclimatized the plantlets for theirsubsequent transfer to soil. The hardened plants were thentransferred to the normal field soil with farm yard manure inpolythene bags and were kept in the glass house. The plantstransferred to soil exhibited good survival ranging from 90-95%.

Using the novel tissue culture approach is beingsought for attaining rapid incremental improvement incommercial varieties. Earlier reports on sugarcane were forsomatic embryogenesis and transformation were based oncallus cultures, but excessive cell division during callus phaseleads to somaclonal variation which were not desirable duringcloning of elite germplasm of commercial varieties, thus the

direct plant regeneration protocol (Figure 2) developed formass clonal propagation of the three commercial varietiesof sugarcane, using young spindle leaf segments in thepresent study is of immense importance (Cheema andHussain, 2004; Ali et al., 2008; Biradar et al., 2009).Although direct plant regeneration methods are available indicotyledonous plants viz. tomato and potato (Webb et al.,1983; Asokan et al., 1984; Park et al., 1995), but similarsystem needs to be exploited in monocots, for commercialexploitation. Plant regeneration from callus cultures, is wellestablished in sugarcane (Heinz and Mee, 1969; Ahloowaliaand Meretzki, 1983; Islam et al., 1996; Kaur and Kapoor,2016), but it is advantageous only when obtaining somaclonalvariations is the aim (Kaur et al., 2002) and is highlyundesirable for in vitro, mass cloning of a newly developedvarieties, when the presence of even minimum callusinterphase poses the problem of genetic variability.Therefore, in the present study a relatively weak auxin NAAwas used instead of a strong auxin 2,4-D, that induces rapidcallusing. It was interesting to note that young leaf segmentscultured on MS medium supplemented with NAA (5.0 mg/L) and Kinetin (0.5 mg/L) did not exhibit any callusing and

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explant swelling was followed by direct shoot regeneration,which is an uncommon phenomenon in monocots. Theprotocol developed is efficient, highly reproducible and is a

single step method, without callus interphase; thus can becommercially used for quick mass propagation of elitegermplasm of sugarcane.

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