Department of Biotechnology, College of Dry Land Agriculture and Natural Resources, Mekelle University, Mekelle, Ethiopia
Received date: May 04, 2017; Accepted date: May 12, 2017; Published date: May 19, 2017
Citation: Hailu M (2017) In Vitro Propagation of Selected Sugarcane (Saccharum officinarum L.) Varieties (C 86-56 and C 90-501) through Apical Meristem. Adv Crop Sci Tech 5:282. doi: 10.4172/2329-8863.1000282
Copyright: © 2017 Hailu M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Sugarcane (Saccharum officinarum L.) is monocotyledonous crop plant that mostly propagates through conventional methods. However, conventional propagation lacks rapid multiplication procedures to commercialize newly released varieties within a short period of time. Hence, the objective of this work was to optimize in vitro micro propagation protocol for two sugarcane varieties (C 86-56 and C 90-501) through apical meristem explants. The two varieties were cultured on MS medium supplemented with different concentrations of growth regulators on shoot initiation, multiplication, rooting and acclimatization stages. Analysis of variance (ANOVA) revealed that the two varieties showed statistically significant difference in their response to the various hormonal treatments with regard to the parameters measured. For initiation stage vars. C 86-56 and C 90-501 performed best on 1.0 mg/l and 1.5 mg/l of BAP, respectively. On the other hand, multiplication stage was best in MS media enriched with 1.5 mg/l BAP+1.0 mg/l NAA and 1.0 mg/l BAP+1.0 NAA as manifested in terms of mean number of shoots and mean shoot length for vars. C 90-56 and C 90-501, respectively. With regard to root induction, best rooting response in terms of mean root number and mean root length was achieved best in 1/2 MS media enriched with 1.5 mg/l NAA+0.5 mg/l BAP and 1.0 mg/l NAA+0.5 mg/l BAP for vars. C 86-56 and C 90-501, respectively. Survival rate during acclimatization was best on coco peat media for both varieties C 86-56 and C 90-501 survived 90% and 70%, respectively. Lastly, factors causing low acclimatization, tissue dying, contamination and phenol exudation in the study should be further investigated.
Acclimatization; Apical meristem; In vitro; Growth medium; Rooting
Sugarcane (Saccharum officinarum L.) is a monocotyledonous crop plant that belongs to the family of Poaceae. It is a clonally propagated crop from which multiple annual cuttings of stalks are typically obtained from each planting. This crop is especially vulnerable to diseases and propagation from cuttings facilitates the spread of pathogens and may results in epidemics. Sugarcane stalks can be infected by various pathogens without exhibiting any symptoms, and therefore there is a high risk of disease transfer during exchange and transport of sugarcane cuttings. Its growth is closely related to temperature . Sugarcane, being a vegetatively propagated crop, has a low seed multiplication rate, which means one hectare of seed cane suffices six to eight hectare of commercial plantation. Lack of flowering potential and multiplication procedures has long been a serious problem in sugarcane breeding programs .
However, Propagation of sugarcane conventionally gives low availability of adequate quantity, highly susceptible to disease. On the other hand, availability of adequate amount of quality and disease free planting materials within a short time is the major limiting factor to attain large scale sugarcane production using the conventional method of propagation and the yield of the existing few and old commercial sugar cane varieties is declining and some productive sugarcane varieties also obsolete due to lack of alternative technologies for disease cleansing and rejuvenation. Besides, it is also difficult to understand the different types of sugarcane varieties in response to resistance to different contaminants during traditional propagation. Moreover, due to shortage of sufficient quantity planting material, commercialization of introduced and adapted improved varieties of sugarcane takes several years using the conventional route of planting material multiplication.
To solve the multitude problems of the conventional propagation method the sugar industry utilizes the advantage of micro propagation technology, which is characterized by rapid multiplication to obtain disease free sugarcane varieties. The nutritional requirement for in vitro propagation protocol of sugarcane should be according to genotype and explants used ; varieties (genotypes) of the same species respond differently to media , besides rapid clonal propagation of sugarcane planting materials depends on the genotype and the plant growth regulators combinations used and needs to develop plant growth regulators combinations for each genotype. Similarly, plant growth regulators requirements for in vitro propagation responses vary from cultivar to cultivar in sugarcane . The nutritional requirement for every sugarcane variety is specific and exact . In addition, an efficient protocol is needed for any new variety or clone to get rapid shoot initiation, shoot multiplication, root induction and elongation . So it is recommended that an efficient protocol is needed for every new variety or clone of sugarcane to get rapid callus induction, shoot initiation, shoot multiplication and root induction and elongation . Therefore, this study was carried out to develop or optimize in vitro protocol for mass propagation of two sugarcane varieties (C 86-56 and C 90-501) through apical meristem.
Objective of the study
The general objective of this study was to develop optimization protocol for in vitro regeneration of two sugarcane varieties namely C 86-56 and C 90-501 from shoot apical meristem explants.
Specific objectives were
• To determine appropriate concentrations of BAP hormone on initiation culture of apical meristem.To determine appropriate concentration of BAP and NAA hormones for shoot induction and multiplication.To determine appropriate concentration of NAA and BAP hormones for root growth. To evaluate survival rate of plantlets under greenhouse condition.
Description of the study area
The study was conducted at Tigray Biotechnology center; plant tissue culture laboratory which is located at Mekelle town, specifically at Ellala near to Tigray Agricultural Research Center that is located at latitude of 13°29’N, longitude of 39°28’E and altitude of 2076 meters above sea level.
Plant material and explant preparation
Mother plants of the two varieties namely C 86-56 and C 90-501 that were used as a source of explants were raised from stem cuttings (setts) obtained from Welkayt sugar factory. According to Dereje  those two Cuban varieties were imported in 2006 and passed through agronomic performance evaluation. They were among the selected ones to be commercialized (Figure 1). Before planting, the setts were treated with hot water at 50°C for 2 hours. Explant preparation were made following the method employed by Belay . The actively growing shoot tips with apical mersitem were collected from three months old mother plants to serve as explants. Shoot tips were cut from mother plants at the base with some nodes. After trimming of the leaves, the shoot tips were taken to the laboratory for surface sterilization and explant preparation. Trimmed shoot tips were washed thoroughly under running tap water, outer leaf sheath were removed and cut into about 10 cm length. Thereafter, the shoot tips further washed three times each for 15 minutes with tap water containing liquid soap solution and three drops of Tween-20. Then, explants were taken to laminar airflow chamber, immersed in 0.3% (w/v) Kocide solution for 30 minutes followed by three times washing each for five minutes with sterile distilled water. The shoot tips were then be rinsed in 70% alcohol for one minute and washed with sterile distilled water three times each for five minutes. Finally, the explants were treated with 10% (v/v) sodium hypochlorite solution (4% active chlorine) for 20 minutes (Figure 2). After discarding the sodium hypochlorite solution, the explants were washed with sterile distilled water three times each for five minutes and the surface sterilized explants were excised and sized to 2.5 cm long for culturing.
Culture media preparation
Full strength Murashige and Skoog (MS) basal medium Murashige and Skoog  were used as a culture medium. MS basal medium consisted of 30 g/l sucrose for initiation of apical meristem and shoot initiation, whereas 60 g/l sucrose for rooting. The pH of the medium was adjusted to 5.8 using 1 N KOH and 1 N HCl before being gelled with 5.0 g/l agar and autoclaved at 121°C, 15 psi for 20 minutes (Figure 3). While molten, the medium (40 ml) was dispensed into glass culture jar for culturing and stored under aseptic condition at +4°C until use for shoot initiation.
Initiation of apical meristem
For shoot initiation, the sterilized shoot tips were aseptically transferred to MS-medium prepared as indicated above with supplementation of PGR (BAP) at a concentration of 0.5, 1.0, 1.5 and 2.0 mg/l. MS medium without PGR were used as control. The treatments of initiation for both varieties were as follows
T1=MS+30 g/l sucrose+5.0 g/l agar+0.0 mg/l BAP
T2=MS+30 g/l sucrose+5.0 g/l agar+0.5 mg/l BAP
T3=MS+30 g/l sucrose+5.0 g/l agar+1.0 mg/l BAP
T4=MS+30 g/l sucrose+5.0 g/l agar+1.5 mg/l BAP
T5=MS+30 g/l sucrose+5.0 g/l agar+2.0 mg/l BAP
The explants were maintained in dark for 8 hour and light for 16 hour duration. The experiment was laid out in a completely randomized design (CRD) with three replicates.
Shoot induction and regeneration
For shoot initiation, MS basal medium supplemented with BAP in a concentration of 1.0, 1.5, 2.0 and 2.5 mg/l combined with 0.5 mg/l of NAA was used. MS basal medium without PGR, i.e., BAP and NAA were used as a control. Details of the treatments for both varieties were as follows:
T1=MS+30 g/l sucrose+5.0 g/l agar+0.0 mg/l BAP+0.0 mg/l NAA
T2=MS+30 g/l sucrose+5.0 g/l agar+0.5 mg/l BAP+1.0 mg/l NAA
T3=MS+30 g/l sucrose+5.0 g/l agar+1.0 mg/l BAP+1.0 mg/l NAA
T4=MS+30 g/l sucrose+5.0 g/l agar+1.5 mg/l BAP+1.0 mg/l NAA
T5=MS+30 g/l sucrose+5.0 g/l agar+2.0 mg/l BAP+1.0 mg/l NAA
Then after, cultures were maintained in a growth room at a temperature of 25 ± 2°C under 16/8 hours light/dark photoperiod adjusted with fluorescent light having 2500 lux light intensity (Figure 4). The incubation chamber had relative humidity of 75-80%. Shoots were allowed to grow 2 to 4 cm and then transferred to rooting media. The experiment was laid out in a completely randomized design (CRD) with a two factors treatment combinations each with three replicates (Figure 5).
Well grown 3-5 cm long shoots were aseptically transferred to 1/2 strength MS basal medium containing 0.0, 0.5, 1.0, 1.5, and 2.0 mg/l of NAA. The treatments of rooting for both varieties were as follows:
T1=1/2 MS+30 g/l sucrose+5.0 g/l agar+0.0 mg/l NAA+0.0 mg/l BAP
T2=1/2 MS+30 g/l sucrose+5.0 g/l agar+0.5 mg/l NAA+0.5 mg/l BAP
T3=1/2 MS+30 g/l sucrose+5.0 g/l agar+1.0 mg/l NAA+0.5 mg/l BAP
T4=1/2 MS+30 g/l sucrose+5.0 g/l agar+1.5 mg/l NAA+0.5 mg/l BAP
T5=1/2 MS+30 g/l sucrose+5.0 g/l agar+2.0 mg/l NAA+0.5 mg/l BAP
All the cultures were incubated at 25 ± 2°C under 16/8 hours light/ dark photoperiod adjusted with fluorescent light having 2500 lux light intensity (Figure 6). The relative humidity of the growth chamber was 75-80%. The experiment was laid out in a completely randomized design (CRD) with NAA factor treatment each with three replicates.
After four weeks of culture in a rooting media, well rooted in vitro plantlets were taken out gently from each PGR treatment bottle and washed under tap water to remove traces of agar that prevent the absorption of nutrients from the acclimatization culture substrates by roots. After this, plantlets were transferred to polystyrene tray that contains three different substrates, namely, coco peat, that is a multipurpose growing medium made up of coconut husk; garden soil: sand: compost in the ratio of 1:1:1 and garden soil and cow dung in the ratio of 1:1. Then, the polystyrene tray was arranged in completely randomized design in computerized green house with relative humidity (RH) gradually reduced from 90 to 60% and temperature of 30 to 31°C for two weeks for primary acclimatization (Figure 7). All the plantlets that survived the primary acclimatization were put in a direct sunlight and nursery shades were provided with adequate amount of water for secondary acclimatization and their performance were monitored for three weeks. Finally, plantlets that survived secondary acclimatization were transplanted to open field.
The following in vitro plant growth variables were recorded from this experiment and served as sources of quantitative data.
Percent of initiated culture: Percent of culture formed from the apical meristem explants after three weeks.
Average number of days to shoot emergence: Number of days needed by the explants to induce shoots from the first day of culturing in the shooting.
Mean number of shoots: Is the average number of dissectible shoots regenerated and induced from each cultured explants in each type of treatment.
Average number of days to root emergence: Number of days needed by the shoots to induce roots from the first day of culturing in the rooting media.
Mean number of roots: Is the average number of dissectible roots regenerated from each cultured shoot in each type of treatment.
Mean length of shoot: Is the average length of Shoots developed from the base of the shoot to the shoot apex before transferring to rooting media from each cultured explants. Lengths of the shoot were measured using sterilized ruler.
Mean length of root: Is the average length of roots developed from each cultured explants. Length was measured on the 30th day of transferring the shoot to the rooting media. Root length measurement was taken from the point that the root attached to the shoot to root tip.
Survival rate: Is the competence or the ability of the in vitro derived plantlets to endure in the in vivo condition for acclimatization. Data on this parameter was taken after one month after root transferred to poly house. Accordingly, the survival rate was calculated after three weeks as the ratio of plantlets survived to the total number of plantlets transferred to the poly house and expressed by percentage.
Data were analyzed on the effect of treatments using SAS version 9.1 and means were compared using fisher’s Least Significant Difference (LSD) test at p<0.0.
Percent of initiated culture
Initiation culture from the apical meristem explants was observed within two weeks after inoculation of the explants on MS medium containing five different concentrations of BAP (0.0, 0.5, 1.0, 1.5, and 2.0 mg/l). The results showed that shoot apical meristem culture initiation or establishment responses in the sugarcane varieties was dependent on the effect of sugarcane varieties (genotype) and BAP (Figure 8). Among the different of BAP tested, sugarcane variety C 86-56 gave the highest initiation culture responses (73.33%) on MS medium containing 0.5 mg/l BAP while C 90-501 gave highest initiation culture responses (76.667%) on MS medium supplemented with 1.5 mg/l BAP as shown in Table 1. This indicated that initiation response in these two varieties is different with respect to the amount of BAP to be used. In line with this, Variation of initiation culture response to different concentration of hormones with variety of sugarcane was reported by Dereje ; Tilahun . Control showed no response for initiation in which all explants cultured on control (0.0 mg/l BAP) dried out after explanation.
|Treatments||C 86-56||C 90-501|
|T1(0.0 mg/l BAP)||0.000d||0.000d|
|T2(0.5 mg/l BAP)||73.33a||66.667ab|
|T3(1.0 mg/l BAP)||60.00ab||60.000c|
|T4(1.5 mg/l BAP)||53.33bc||76.667a|
|T5(2.0 mg/l BAP)||43.33c||50.000c|
Means followed by the same letter are not significantly different at 5% significance level, CV=Coefficient of variance, LSD=Least significant different.
Table 1: Effect of different concentrations of BAP on the percent of initiated culture from apical meristem explants of C 86-56 and C 90-501 sugar cane varieties.
Shoot regeneration and multiplication
Number of days to shoot emergence: Shoot initiation was observed in PGRs treated cultures at all concentrations. However, no shoot initiation was observed in PGRs free (control) treatments. Previously, Dereje  reported that shoot-tip (apical meristem) explants of sugarcane variety C86-12 cultured on hormone free MS medium had slow shooting response. The fact that no shooting response/ delayed shooting response observed in PGRs free medium show that the available endogenous hormones may not be sufficient to induce shooting (Figure 9). The result also showed that the number of days to shoot emergence was found to be influenced by different concentrations of BAP combined with constant concentration of NAA. Relatively, C 86-56 variety had faster response at all PGRs concentrations than C 90-501 variety in terms of shoot emergency (Table 2). In both varieties, shoot emergence was found to be earlier at lower concentration, and number of days to shoot emergence increased with increasing PGRs concentrations (Table 2). This finding was in line with that of Sowal  who reported the effectiveness of low concentration of BAP to result in rapid shoot multiplication.
|Sugarcane varieties||Hormones (mg/l)||No of days to shoot mergence||No of shoots per expt.||Shoot length(cm)|
Means followed by the same letter within a column are not significantly different at 5% significance level, No=Number, Expt. =Explant, Wt. =Weight, CV=Coefficient of variance, LSD=Least significant different.
Table 2: Different shoot parameters measured for apical meristem explants treated with different concentrations of BAP combined with 1.0 mg/l of NAA. Values are mean ± SE, n=3.
Mean number of shoots: Number of shoot/explant was significantly higher in PGRs treated explants than PGRs free cultured explants. Number of shoot /explants was also significantly varied between PGRs treatments with highest number (10.667) counted at 1.5 mg/l of BAP combined with 1.0 mg/l of NAA for variety C 86-56 and 9.333 shoot/ explants for variety C 90-501 at 1.0 mg/l of BAP combined with 1.0 mg/l of NAA (Table 2). Previously, Tarique  reported that 1.0 mg/l BAP+0.5 mg/l NAA and 1.0 mg/l BAP+0.5 mg/l IBA showed the best result for induction and multiplication of shoots for sugar cane varieties of B52-298 and NCO-334, respectively (Figure 10). This shows that different varieties of sugar cane respond differently to different types and concentrations of PGRs, suggesting unique optimization for better performance. Moreover, Genotype specific response to number of shoot regeneration was reported by Gandonou  and Behara .
Shoot number /explant appeared to increase with increasing concentration of BAP up to 2.0 mg/l. It has been reported that a high level of cytokinin in combination with a low auxin level was essential for the differentiation of adventitious shoots in sugarcane Belay . However, it was observed that mean number of shoots per explants was found to decline with further increase in the concentration of BAP beyond optimum (2.0 mg/l BAP) for both varieties. This finding agrees with Khalafalla  who reported that BAP at the concentration of 5.0 mg/l gives low number of shoot per explants and concluded that shoot number decreases as BAP concentration increases beyond optimum. Increasing trend in shoot number per explants up to optimum level is due to the fact that cytokinin (BAP) stimulates protein synthesis and participates in cell cycle control in a cell division George .
Effect of growth regulators on shoot length: Average shoot length was significantly higher in PGRs treated explants than explants culture on PGRs free media. Shoot length also showed significant difference between PGRs treatments. Shoots cultured on MS media containing 1.5 mg/l BAP and 1.0 mg/l NAA showed significantly higher mean shoot length (8.533) compared to all other treatments for var. C 86-56. For var. C 90-501 highest mean shoot length (5.633) measured was on MS medium containing 1.0 mg/l BAP and 1.0 mg/l NAA (Table 2). Similar to this result, Dereje  reported maximum shoot length 8.4 ± 0.008 for Cuban sugar cane variety C 90-501 when cultured on BAP (1.5 mg/l)+kin (0.5 mg/l). Behera  also reported that maximum shoot length of 6.2 ± 0.37 and 4.0 ± 0.61 under BAP (2.0 mg/l)+IBA (0.5 mg/l) and BAP (2.0 mg/l)+IBA (1.0 mg/l) for two sugarcane varieties namely B52-298 and NCO-334, respectively (Figure 11). On the other hand, it was observed that shoot length was found to decline with the increase in the concentration of BAP beyond optimum (2.0 mg/l BAP). This findings agrees with that of Bhatia who explained that increasing the concentration of the PGRs over optimum supplements may lead to negative effects on the morphology of the in vitro shoots.
Effect of growth regulator (NAA) on root induction
Number of days to root emergence: Root initiation was observed in PGRs treated shoots at all concentrations. However, no root initiation was observed in PGRs free (control) treatment. Similar result has been reported by Rashid. The number of days to root emergence was also found to be influenced by different concentrations of NAA. The two varieties showed variation in responding to the different concentrations of NAA in terms of time taken for root emergence. Variety C 86-56 took 13.0 and 18.33 days for root emergence at 0.5 mg/l NAA and 2.0 mg/l containing MS medium, respectively. Whereas C 90-501 formed root earlier (13.67 days) at the highest (2.0 mg/l) of NAA and root formation was delayed at the lowest (1.0 mg/l) of NAA.
Number of roots per shoots: Number of root/shoot was significantly higher in PGR treated shoot than PGR free cultured shoots. Number of roots was also significantly varied between PGR treatments with highest number (12.667) counted at 1.5 mg/l of NAA for variety C 86-56 and 9.0 for variety C 90-501 at ½ MS medium supplemented with 1.0 NAA (Table 3). This result can be complemented by a number of previous studies. For example, Behera  found highest number of roots per micro shoots (13.4 ± 1.5) on ½ MS medium supplemented 2.5 mg/l NAA for sugar cane varieties B52-298. The above result contradicts to Dereje  reported that maximum root/shoot (17.8) on ½ MS medium supplemented with 5.0 mg/l NAA for C 86-12 sugar cane variety.
|Sugarcane Varieties||Hormone(mg/l)||No. of days to root emergence||No. of roots per shoot||Root Length(cm)|
Means followed by the same letter within a column are not significantly different at 5% significance level. No=number, Wt. =weight, CV=Coefficient of variance, LSD=Least significant different.
Table 3: Effect of different concentrations of NAA combined with 0.5 mg/l BAP on rooting responses of C 86-56 and C 90-501 sugar cane varieties. Values are means and n=3.
Effect of growth hormones on root length: Root length was significantly (p<0.05) affected by different concentrations of NAA supplemented to ½ MS medium for both varieties (Table 3). Variety C86-56 produced maximum root length (5.667 cm) on half strength MS media containing 1.5 NAA and 0.5 mg/l BAP. But, variety C 90- 501 produced maximum root length (4.7667 cm) on half strength MS media containing 1.0 mg/l NAA and 0.5 mg/l BAP. In line with this, Belay  reported root length of 3.2 ± 0.25 cm when grown on ½ strength MS medium supplemented with 1.0 mg/l NAA alone for N14 sugarcane variety. Mangrio  obtained average root length of 2.5 cm on ½ MS media supplemented with 3.0 mg/l NAA for sugarcane Variety NCO-334. The effect of variations in the concentrations and combination of the same hormone in most of the cited literatures and in the present work is almost entirely due to variation in the varieties of sugarcane tested by different researchers. That is why it is of paramount importance to optimize genotype specific in vitro propagation protocols for every variety.
Survival rate in green house during acclimatization
In vitro induced shoots are very delicate and cannot resist sudden environmental changes that may damage the plantlets unless they are gradually adapted to the new environment. Thus, acclimatization is essential to enable the rooted plantlets to adapt the natural environment in ex vitro conditions at controlled temperature and humidity of greenhouse conditions. In the acclimatization stage of this experiment, a total of 150 and 90 well rooted plantlets for variety C 86-56 and C 90-501, respectively were transferred to greenhouse containing substrates namely, coco peat alone, that is a multipurpose growing medium made up of coconut husk; garden soil: sand: compost in the ration of 1:1:1 and garden soil and cow dung in the ration of 1:1. Then, the polystyrene tray was arranged in greenhouse with relative humidity (RH) gradually reducing from 90 to 60% and temperature of 30 to 31°C for two weeks for primary acclimatization (Figure 12). Generally the acclimatization phase of this experiment revealed that there was a difference in survival rate due to substrate nature and varietal difference. Both varieties had the highest survival value when grown on coco peat alone. On this media substrate, survival rate was 90% and 70% plantlet for C 86-56 and C 90-501 sugar cane varieties, respectively as shown (Table 4).
|Types of sugarcane varieties||Types of culture medium||Total No of plantlet transferred||Survived plantlets||Died plantlets||Percent of survived plantlets||Percent of died plantlets|
|C 86-56||Coco peat only||50||45||5||90%||10%|
|Garden soil:sand:compost (1:1:1)||50||36||14||72%||18%|
|Garden soil: cow dung(1:1)||50||28||22||56%||44%|
|C 90-501||Coco peat only||30||21||9||70%||30%|
|Garden soil:sand:compost (1:1:1)||30||18||12||60%||40%|
|Garden soil: cow dung(1:1)||30||16||14||53.33%||46.66%|
Table 4: Effect of different medium substrates on the survival of in vitroregenerated plantlets of the two varieties during acclimatization stage in green house.
Sugarcane, as a globally important industrial crop, mainly is a source of sugar, ethanol, and other important by products. Hence, due consideration to the use of advanced technologies for sugarcane production is mandatory to obtain the unfolded benefits tapped from the crop. On the other hand, in Ethiopia, sugar industry is increasing at an alarming rate and is expected to play a significant role in poverty reduction. Thus, multiplication of sufficient quality of seeding material is needed more than ever before. However, in sugarcane seeding material multiplication usually takes up to 10 years following conventional method; besides the method allows continuation of diseases over vegetative cycles, which leads to drastic yield and quality reduction. To get out of the situation, in vitro propagation that enables rapid and large scale production of disease free planting material as being exercised with different crops is a prerequisite.
Based on this fact plant regeneration protocol was optimized in this study through direct Organogenesis for two commercially important Cuban originated sugarcane varieties (C 86-56 and C 90- 501) using apical meristem explants. Accordingly, the information below was obtained. For initiation stage of apical meristem explants and more initiated culture var. C 86-56 best perform on 0.5 mg/l BAP mg/l, while var. C 90-501 best perform on 1.5 mg/l of BAP. Shoot parameters were also highly influenced by varieties and the type and combinations of various growth regulators. The effect of varieties and hormones combinations was highly significant (p ≤ 0.05) on average values of shoot parameters. The highest number of initiated explants cultures regenerated more number of shoots and optimum shoot length were observed in var. C 86-56 on full MS media supplemented with 1.5 mg/l BAP+1.0 mg/l NAA, whereas maximum percentage of initiated explants cultures regenerated more number of shoots and optimum shoot length were recorded on 1.0 mg/l BAP+1.0 mg/l NAA for C 90-501 variety. Roots regenerated 10 to 15 days after micro shoots were transferred to root induction media for both varieties. Highly significant (p ≤ 0.05) differences were observed among treatments and varieties were also highly significant (p ≤ 0.05). The highest percentage of shoots regenerated number of roots and root length were recorded for var. C 86-56 on half strength MS media containing 1.5 mg/l NAA combined with 0.5 mg/l BAP., whereas for var. C 90-501 maximum percentage of number of root and root length was recorded on half strength MS media containing 1.0 mg/l NAA combined with 0.5 mg/l BAP. As to the acclimatization response, relatively highest survivability percentages were recorded on coco peat media substrate as compared to the other medium substrates used. Besides from the two varieties C 86-56 survived higher percentage (90%) than C 90-501 (70%) on coco peat media.
Lastly, in this work, the results clearly indicated the importance of evaluating individual variety to optimize a given tissue culture protocol. In other words, genotypic specificity was highly reflected in all of the parameters tested. Genotypic specificity has been reviewed in the literature review part of this paper and many researchers have imposed the evaluation of individual variety to recommend a tissue culture protocol.
Based on the above findings, the following recommendations are made for further investigation of in vitro culture of the two varieties.
• Further studies are needed using other hormones such as Kn,2,4-D, IAA, IBA with different concentration and interaction effects for observing their ability to induce shoots and roots for the reproducibility of the protocol optimization through direct or in direct organogenesis.
• Factors causing contamination, low acclimatization, tissue dying, and phenol exudation in the study should be further investigated.
• It is necessary to study the performance and genetic stability of the in vitro regenerated seedlings after transplanting in the field necessary.
• To enhance the acclimatization rate of in vitro developed plantlets in the glasshouse, various methods have to be manipulated.
• To sum up, the present study has developed protocol optimization for in vitro micro propagation of new Cuban origin Saccharum officinarum L. varieties (C 86-56 and C 90-501) using apical meristem explants through direct organogenesis. Hence, it is beneficial to use this developed in vitro micro propagation protocol as a best road map to large scale propagation to generate large number of seedlings in short period of time.