Review on In Vitro Propagation of Sugarcane to Advance the Value of Tissue culture

Sugar cane is an economically important Agra agro industrial crop and propagated conventionally by sets, however, low propagation rates, its time demand, huge land requirement and potential transmission of pathogens through seed cane from generation to generation are the major limitations of conventional propagation. The ultimate objective of this review is the effect of propagation method for increasing yield by reducing varietal deterioration. In vitro propagation is the best alternative to overcome such limitations to produce disease free and sufficient amount of planting material. Micro propagation is however a very sensitive technique, which requires aseptic condition in each stages (0-4) namely mother plant establishment, initiation and establishment of aseptic culture, multiplication, rooting, and acclimatization. This can be concluded that knowledge is needed regarding sensitivity of work for contamination and the economical importance of tissue culture to support the conventional propagation. This will allow the sugar industry estates to produce sufficient planting material within short period of time and cost effective.


Introduction
Sugarcane is a perennial grass family crop that normally reproduces through sexual and asexual modes. It reproduces asexually by three or two buds stem cutting called sets, and innovative approaches of in vitro propagation by taking parts of it such as shoot tip, apical merited, axillary shoot, bud and leaf. It also reproduces through seed propagation via flower (fuzz), which is used for breeding purposes. Sugarcane varieties are developed at sugarcane breeding institutions of the world through screening of large number seedlings raised from fuzz (true seeds) obtained from through breeding. Currently in Ethiopia, the breeding program involves only the import of fuzz and selection of exotic lines. However, most of the time the flower of sugarcane is not viable, and the reason behind this might be highly variable. This variation source is not required to maintain continuity of the variety to be stable for commercial propagation rather than using for the variety development.

Conventional propagation
Sugarcane is propagated commercially by vegetative method, which involves the planting of the stem cuttings of premature cane about 8 to 12 months old grown with special care are recommended for seed cane [1]. The seed cane that is used as planting material may be either whole stalks or stalks` cut up in shorter segments called sets [2]. The growth of sugarcane has different phases: emergency, tillering, stalk growth, and maturation. The germination is a critical event in the plant life to assure a good harvest. It is initially dependent on the set nutrients and water till developing its own root system for three weeks under proper conditions, though, the initial growth of sugarcane is influenced by several internal and external factors such as set age, cultivar, sets nutrients, temperature, soil aeration; sets position on the stalk and humidity [1].
A cane sett is the main conventionally propagation system for sugarcane growing countries in the world, including Africa. In some instances, the buds scooped out of the cane using a bud-chipping machine or knives are used for raising the seed nursery [3]. These seed canes involve a three bud system after treating with hot water or aerated steam therapy to kill pathogens and pests harboring the seed pieces and treating the sets with a fungicide. The treated sets planted in seed nurseries are used to raise primary seed which is used as planting material to grow a foundation seed and the foundation seed in turn is used to raise the certified seed nurseries. Higher seed rate of 75,000 two-bud sets per hectare is needed for raising breeder's seed to compensate for germination loss due to heat therapy [3].
In addition, one bud shoot produces four to five shoots in a year [4] and 1ha of seed cane is only sufficient to plant 10 ha commercial fields (1:10) in 7-10 months [5]. Thus, The newly released varieties take 6-10 years to produce enough quantity of initial seed material for the required vast area [6,7], while its seed propagation rate ranging from 1:7 to 1:10 [8,9]. However, the seed accumulates diseases and pests during several cycles of field production. In general, nonavailability of quality and true to type planting material of newly released varieties is a major constraint in their quick adoption for commercial use, and improving sugarcane productivity. Even well adopted commercial varieties cultivation required availability of quality seed to ensure better cane yield, sugar yield, pathogens and pest-free crop [10]. Further, traditional method of cultivation using three-budded set requires large quantity of seed, which is costly, time consuming and land demanding [11].
Therefore, development of tissue culture technology for rapid multiplication of disease-free planting material has been an important step towards sufficient, true to type and quality seed production in sugarcane. Australia, India and the Philippines in the Asia-Pacific region have already applied this technology for commercial seed production and the benefits have become evident through rapid propagation and distribution of elite varieties and increased sugarcane production [3]. The experiences of these countries would be of considerable benefit to all those who are in the process of adopting micropropagation for their seed production programs like Ethiopia and others.

In vitro propagation
Conventional propagation of sugarcane suffered from low propagation rates, expensive labour, time consuming and potential transmission of pathogens through seed cane from generation to generation, which limits the efficiency of this method [12]. This long time taken of propagation causes a major bottleneck in commercial propagation and breeding programmers [13]. Thus, the growing demand of newly released varieties could not be fulfilled by only conventional propagation methods. Therefore, application of plant tissue culture techniques provides an alternative method for multiplication and improvement of sugarcane [14]. Plant tissue culture offers the best methodology through micro propagation of sugarcane for quality and phytosanitary planting material at a faster rate in a shorter period. Tissue culture can increase the propagation potential by 20-35 times [15]. About 18, 520 plants, produced from a single shoot through micro propagation, were required as compared to 8.8 tons of cane seed in conventional methods for planting in one hectare. Thus, multiplication ratio was 100-150 times using tissue culture plants as compared to 11-12 using conventional cane sets, leading to drastic reduction in seed cane requirement [16]. Kuar & Sandhu [17] showed the shoot multiplication rates were ranged from 4 to 25 fold in CoPb 91 and CoJ 83 cultivars, respectively and the complete plantlets were produced in 157 days with 97% survival rate. The fidelity of this protocol for agri-business industry was tested by producing approximately 0.1 million saleable HTM sugarcane plantlets in a small-scale (150 m2) tissue culture unit.
In addition, plants can be disease indexed and healthy material multiplied in half time compared to the conventional route [18]. Hence, methods of more efficient regeneration protocol for propagation have been developed through micro propagation [3]. Currently, it is the only realistic means of achieving rapid, largescale production of disease-free seed canes of newly developed varieties in order to speed up the breeding and commercialization process in sugarcane. Further, it is an important tool for the production of thousands of genetically uniform and safe plantlets, and its usefulness in germplasm storage [4]. Khan et al. [19] stated that in vitro propagation produce millions of plantlets from single shoot tip within a short period in contrast to conventional method where one bud produces, 4-5 shoots. Lee [20] and Lal et al. [21] produced around 10,000 identical plantlets in about 3-4 months and 75600 shots from a single shoot apex explants in a period of about 5.5 months.
Hendre et al. [22] and Biradar et al. [5] estimated that it is possible to produce some 260,000 shoots in four months and 2x10 8 plantlets in a year over 4-5 weeks sub culturing cycles from single shoot tips of seedcane respectively. Moreover, the micro propagated plants (CV. Co 83) grown in the field had up to 44.96% more canes/plots and up to 22.9% greater cane yield/plot than plants conventionally propagated from three-budded sets [23]. Ben sheikh et al. [24] also stated that gross yield obtained from in vitro multiplication derived plantlets seedcase source becomes over than their source plants.

Stock/mother plant establishment
A pre-propagation stage requires proper maintenance of the mother plants in the greenhouse under disease and insect free conditions with minimal dust. Collection of plant materials for in vitro propagation should be treated using appropriate pretreatment via, fungicides and bactericides to minimize contamination in the in vitro cultures [25]. To enhance the probability of success, the mother plant should be grown under optimal conditions in the greenhouse to maintain the crop physiology normal besides minimizing contamination in vitro [26].
Explants taken from field plants have problem of microbial contamination, as total sterilization of these explants is generally difficult. Moreover, physiological status of source plant also influences the response of explants [27]. As a result, the explants are then taken from greenhouse grown stock plants give rise to better results for in vitro propagation study as the load of contaminants is minimal compared to the ones grown in field conditions [28]. There are huge variations regarding in vitro culture response of explants excised from plants grown in field condition depending on weather conditions during the year, hence, the best results obtained from explants excised from in vitro grown seedlings [27]. About 85-95% shoots tip cultures reached successfully to the shoot proliferation.

Agricultural Research & Technology: Open Access Journal
Apical/axillary meristem culture Plant tissue cultures are initiated from tiny pieces, called explants, taken from any part of a plant. Practically all parts of a plant have been used successfully as sources of explants. Plant segments used in tissue culture as explants are shoot tip, apical meristem, axillary bud, root tip, leaf, flower, ovule, cotyledon and hypocotyls. In sugarcane, shoot tip from 1-2 cm [24,[29][30][31], apical meristem from 1-6mm [32][33][34][35], leaf roll disk [4,[36][37][38]. These explants form direct and indirect organs, embryos, though, shoot tips and meristems give successful results for direct shoot regeneration [39]. Apical meristem is a small group of cells that develops to shoot, and communicates signals to the rest of the plant [40]. Apical Meristem tips are perhaps the most popular source of explants to tissue cultures [7,41,42]. Because the apical meristem is the origin of the shoot, it has four functions; initiating new organs and tissues, communicating signals to the rest of the plant, and maintaining itself as a formative region [43].
It is the most distal to outer portion of the shoot and comprises two groups of cells: the initial/source cells and the cells that are progenitors for tissues and lateral organs or it is a region just proximal to the meristem where lateral organ primordial are formed. Moreover, in vitro propagation through apical and axillary meristem shoot is the most common technique in India and Australia for commercial mass production [3]. This is due to the cells of apical and axillary meristems that are uniformly diploid and least susceptible to genotype changes, ensures genetic stability of the clones [44]. Eight sugarcane clones plantlet derived using meristem culture method were phenotypic ally uniform and 4mm size of meristem was the most suitable for establishment of culture while meristems were treated with a solution of ascorbic acid (100mg/l)+citric acid (150mg/l) for 10-15 minutes, phenolics could be controlled [45].

Size of explants
The sizes of explants were determined by the purpose of the experiment and the efficiency of regenerating multiple shoots. Virus free in vitro derived plantlets were successfully regenerated from 0.07 to 2 mm meristem size [42,46,47]. Apical meristem and shoot tip having 0.03 to 2cm size [29,33,35 48,49] used for genetically uniform and potential in vitro propagation across the world. However, the culture of small meristems exhibits lower rates of survival and regeneration during shoot initiation than larger blocks of meristem. This happens probably because meristems having larger sizes provide more amounts of readily available nutrients that require for initiation of shoot primordial than those of smaller sizes [49]. They obtained higher regeneration potential at 4 mm size (60%) than in smaller ones at size of 2 and 3 mm (40%) during initiation.
Ali et al. [48] also obtained high regeneration of apical meristems at 4mm (100%) for two genotypes. Ali et al. [33] obtained 100% survival with 90% regeneration potential at 3mm size within 12 days, and the time for shoot formation was increased by decreasing the size of the meristem. However, when the explants became extremely large, contaminations are highly serious [49]. In contrary, successful results from highly small explants are reported in previous works. Parmessur et al. [46] reported Larger meristems (>1mm) are likely to be dying, whereas smaller ones (<0.3mm) are unlikely to develop into plantlets, and the success resides in the ability to isolate the meristematic with one or two leaf primordial. Jahangir et al. [42] reported successfully regenerates of apical meristem at 0.07mm length size for disease free and rapid mass production of sugarcane cultivars. Generally, the efficient genetically stable in vitro propagation to produce potential multiple shoots, 4-6mm size shoot apical meristem was the best in sugar cane [35].

Explants surface sterilization and shoot establishments
Even though it is not a crucial stage of micro propagation, shoot initiation and establishment is the most difficult step due to regeneration potential and contamination especially grass family crops like sugarcane and other woody trees. Contamination in tissue culture can originate from two sources, both on the surface and in the tissues of explants or through faulty procedures in the laboratory. Establishing of tissue culture depends on the explants used, surface and entophytic microorganisms. In maritime culture, most organisms would be eliminated due to its small size, whereas in large explants (leaf, stem etc), most if not all microorganisms in the tissues may be carried over [26]. To avoid contamination, the explant has to be washed and cleaned up prior to surface sterilization by using liquid soap, commercial detergent, kocide (antifungal), tween 20 or 80 etc with tap water.
Although different sterilization agents such as Ca(OCl)2, H2O2, NaOCl, HgCl2 and ethanol can be used for surface sterilization, ethanol, NaOCl and HgCl2 are the most common frequently used agents. Tiwari et al. [50] studied four sterilization agents (EtOH, NaOCl, HgCl2 and H2O2) in combination and alone using leaf sheath exploits of two fields grown sugarcane varieties. The authors showed that the use of only one sterilizing agent is not successful, and HgCl2 (0.1%) for 5min and Et OH (90%) for 10min was the best along with prior washing and surface sterilization with tween 20 and bavistin. Benisheikh et al. [24] used 70% ethanol for 30 second to one-minute using shoot tip explants, followed by 0.1% HgCl2 for another five minutes. However, HgCl2 is highly carcinogenic, and toxic to the plant cells entering through the xylem during sterilization. Hence, other safer alternative sterilization methods are developed to avoid this risk through replacing HgCl2 by NaOCl and ethanol [47]. Cheong et al. [51] used 70% ethanol for surface sterilization of apical meristem and auxiliary bud. Khan et al. [4] reported 50% Clorox (Berekina, 5.25% active chlorine) for 30 minutes then put in 70% ethanol for 45 minutes. Mekonnen et al. [52] reported surface sterilization with 25% Berekina (5% active chlorine) for 25min exposure time is optimal for sugarcane shoot tip decontamination, and this treatment could replace 0.1% mercury chloride for 10 minutes. Generally, shoot regeneration in several tropical plant species has been studied and found satisfactory efficiency (82-93%) for maize, citrus species, Brassica spp. and Agricultural Research & Technology: Open Access Journal winged been except sugarcane (53%) which was thought to be due to the presence of surface hairs on the leaves and stalks [53].

Adventitious shoot proliferation
The multiplications of shoots are a crucial stage in the propagation of any species for commercial exploitation and the most rapid rates are required. The most common additives to standard media are cytokines usually as BAP, BA and Kinetin alone or with combination of low amount axing like NAA, IBA and IAA. Typically, the same medium and environmental conditions are used for both shoot initiation and multiplication [54]. The rate of shoot multiplication mainly depends on a number of factors. These are type and combination of plant growth regulators, explants type, culture medium composition, and genotype. Jagadeesh et al. [55] showed that high ratio of cytokinin and axing was essential and better for production of adventitious shoots rather than cytokinin alone in sugarcane. The authors found the highest multiple shoot on MS medium with 3mg/l BAP+2mg/l IAA+2mg/l Kin.
Bhor and Mungse [56] obtained the maximum number of shoots (9.8 in Co-86032 and 8.1 in CoM-88121) on MS medium+1.0mg/I BAP+0.5mg/l NAA. Koy and Kabir [57] obtained the maximum of 17.2 shoots and 7.2 shoot length on MS +1.5mg/l BA with 0.5mg/l NAA in Isd32 genotype. Abdu et al. [58] reported the highest number, length and vigor of shoots in all the genotypes on MS media containing 1.0 and 1.5 mg/L BA with 0.2mg/L NAA. Gopitha et al. [59] also achieved best regeneration of shoots on MS medium fortified with BAP 1.0mg/L and IBA 0.5mg/L. A maximum of 24 and 29 shoots per bud for cv. Mex 68-P23 and cv. MY 55-14 respectively in six weeks on 2mg/l Kin with 1mg/l NAA were reported [60]. Mamun et al. [61] obtained best shoots for Isd-28 and Isd-29 on MS medium fortified with 1.5mg/l BA and 0.5mg/l NAA. Best multiplication on 0.5mg/l BAP with 0.5mg/l NAA and 15% CW was also obtained [62]. In addition, Yadav et al. [63] reported best response of multiplication on MS medium with BAP, Kin and NAA (0.5mg/l each). Sahoo et al. [64] obtained multiple shoots from meristems on MS medium with 1.0mg/l BA, 0.5 mg/l Kin and 0.25mg/l NAA.

Rooting
The success of in vitro propagation relies on efficient rooting in regenerated shoot and their subsequent acclimatization. Once the sufficient numbers of shoots have been generated, portion of explants that contains one or more shoots could be transferred to a medium that contains higher concentration of axing, resulting in root formation. The initiation of roots is easily achieved in some species by reducing the cytokinin level [38] or on MS medium with or without the addition of extra root promoting axing [11]. In sugarcane, axing especially IBA from 0.5-3 mg/l [4,54] and NAA from 0.5-7mg/l [38,41,65] alone or in combination are the most common used axing for rooting.
Most of researchers reported in vitro regenerated roots of sugarcane shoots on MS basal medium fortified with auxins [47,64,65]. Khan et al. [4] reported vigorous root development on MS medium containing 6% table sucrose +1mg/l IBA among the combinations used. However, the MS medium without growth regulators promoted rooting in more than 90% of two cultivars after 30 days of culture [11,66]. In addition to the presence and absence of growth regulators, rooting was greatly dependent on the strength of MS medium in various plant species. Jagadeesh et al. [55] reported that half MS media were more responsive than full MS medium for rooting of sugarcane. This resulted in 77.78% of root inducing shoots from 14.3 days of shoots inoculated on ½MS+6 mg/l NAA medium. Sahoo et al. [64] reported that rooting of shoots was achieved on half MS basal medium with 2mg/l NAA plus 6% sucrose. Tiwari et al. [49] obtained 100% rooted shoots on ½ MS medium supplemented with 50g/l sucrose and 5.0mg/l NAA at pH 6.0 within two weeks. Rooting (85-92%) was induced by transferring shoots on 1/2 MS medium supplemented with 2mg/l NAA and 1.0mg/l IBA [67].
Rooting was highly influenced by the different types and concentrations of axing used. Even if there also results reported on the IBA and IAA, NAA was the most efficient axing for root initiation of sugarcane in vitro propagation [4,54]. Jagadeesh et al. [55] reported NAA was better than IBA either alone or in combination with other hormones for rooting of sugarcane. In general, many researchers reported that 5 mg/l NAA was good for rooting [16,45,63,65], but more than 5mg/l NAA inhibits rooting [5]. In contrary, many researchers obtained best rooting at lower concentration of NAA from 0.5-3mg/l [38,64,68,69].

Acclimatization of in vitro regenerates
Acclimatization of in vitro propagated plantlets to the ex vitro environment is a critical step for successful propagation. It is ultimately depending on their ability to withstand the conditions transferring from in vitro to ex vitro because the in vitro environments are highly conducive than ex vitro environment. In sugarcane, successful acclimatization can be possible by taking in vitro shoots at two different stages of the plantlets. Either this is when in vitro regenerated plantlets have an optimum shoot/root ratio [42,55] or after optimum shoot formation but before rooting on in vitro medium what is called ex-vitro rooting. Furthermore, the acclimatization of in vitro regenerated shoots can be achieved efficiently if the plants are initially maintained with high humidity conditions. Tiwari et al. [49] reported that over 6000 rooted Agricultural Research & Technology: Open Access Journal shoots were transferred to greenhouse for hardening, of which 94% of the plantlets survived. Yadav et al. [63] reported that 90% survival rate was recorded in the greenhouse condition. Similarly, easily acclimatized sugarcane plantlets using soil as substrate and the initial plantlets cultured in mist chamber condition by adding fertilization weekly was demonstrated [11,70].
The media compositions with its ratio also play a vital role to increase survivality of plantlets. Ather et al. [71] reported that in vitro grown plantlets survived successfully with the rate of 96% after four weeks when farmyard manure was used with garden soil in the ratio of 1:4 as a potting mixture. Jagadeesh et al. [55] reported that the treatment combination of vermicompost: soil: sand (1:1:1) gave the highest survival percentage of 75%, followed by the treatment combination of press mud: soil: sand (1:1:1) which produced 50% survival. Dibax et al. [66] also reported that use of composed substrate of vermiculite + MS salts was effective for acclimatization. In addition, the type of media used such as liquid and semisolid are also a highly important detrimental factor. Snyman et al. [72] obtained approximately 18,000 plants/leaf roll by using temporary immersion in vitro culture in 12 weeks when compared with approximately 2000 plants/leaf roll produced on semi-solid medium. However, due to hyperhydricity, only ~34% of the plants produced in RITA® were survived in acclimatization.

Summary and Future Line
There is a high transmission and incidence of disease through stem cutting seedcane grown plants. This is particularly true in Ethiopia since yield losses can be increased due to disease under suitable environmental conditions in mineral soils. Using in vitro propagation derived plantlets decrease diseases transmission like smut, yellow leaf virus, Leaf scald, mosaic virus, ratoon stunting disease and others that spread through generation and sugar estates in the case of seedcane with subsequent cost reduction of disease control and increasing yield. In line with this, healthy seed cane supply is the lifeline in the productivity and profitability of sugarcane, hence for the sustainability of sugar industry; all the Ethiopian sugar estates/projects should be used tissue culture derived sugarcane planting materials.