Interaction Effects of 6-Benzylaminopurine and Kinetin on In vitro Shoot Multiplication of Two Sugarcane (Saccharum officinarum L.) Genotypes

Sugarcane (Saccharium officinarum L.) belongs to the Saccharium genus of the Andropogoneae tribe of the Poaceae (Gramineae/grass) family with an octaploid 2n=8x=80 number of chromosomes [1]. It is a tall perennial tropical grass that tillers at the base to produce unbranched stems, 3-4 m or more in height with a thickness of approximately 5 cm in diameter. It is one of the most efficient photosynthesizing plant or converter of solar energy to sugar stored in the internode [2]. The commercially cultivated crops of sugarcane have two geographic centers of origin in New Guinea and Northern India [3]. Although the major industries are found in Brazil, China and India, the crop is also commercially produced in many other countries, including South Africa [4]. But there is no well documented reference on how, where and when sugarcane was introduced to Ethiopia, although some records claim its introduction during the early 18^th century [5].

Sugarcane accounts approximately 75% of the world’s sugar and it is economically important cash crop in tropical and sub-tropical regions of many countries [6]. Its properties such as efficient photosynthesis and biomass production make it an excellent target for industrial processing, valuable alternative for animal feed and for the production of by-product such as ethanol from molasses [7]. In Ethiopia, this crop is grown as an important cash and industrial crop among many crops and it has an immense importance for the development of the economy of the country. It is used for the production of white and brown sugar and by-products like molasses, bagasse and press mud (filter cake) which have been used for different purposes in a daily life and there is no by-product thrown as non- useful matter. Furthermore, production of sugar in Ethiopia has created employment opportunities and foreign currency.

Day by a day increasing use of sugar and its relevant by-products has created a challenging situation for sugar producing countries, researchers and growers [8]. In Ethiopia the annual yield of sugar from three factories was nearly around 300,000 tons while the annual domestic demand is close to 450,000 tons [9] and the deficit was covered by importing from abroad. In addition, the yield per hectare of this crop is the lowest all over the world [8]. Considering the availability of abundant water resource coupled with a vast fertile land favorable for sugarcane cultivation, suitable agro-ecological conditions, cheap labor and huge domestic and foreign demand for sugar and for its by-products [9], this yield is very minimal in the country. Hence, by considering these opportunities the government has planned to establish ten sugar factories on 370,000 ha of plantation area.

Yield potential of sugarcane varieties is deteriorating day by day due to segregation, susceptibility to diseases, insects, admixture, and changes in edaphic and climatic factors [10]. Improvement of sugarcane varieties is very difficult, because it is not an ideal candidate crop for conventional plant breeding, since its flowering is not-synchronized, it has low sexual seed viability and it is a perennial crop [11]. Hence, its improvement takes up to ten years from initial crosses to final agronomic assessments [12,3]. But also the lack of rapid multiplication procedure has long been a serious problem in sugarcane conventional breeding programs as it takes 10-15 years of work to complete a selection cycle [13,8]. Commercially, sugarcane is propagated from stem cutting with each cutting or set having two or three buds [14,15]. Vegetative propagation by cutting is a very low rate of propagation which is about 1:6 to 1:8 [15] and 1:7 to 1:10[16,17]. In addition to low rate of propagation on an open field, it favors pathogens to keep on accumulating generation after generation which reduces the yield and quality of sugarcane [14,18]. For instance Ratoon stunting disease is a common disease in sugarcane and conventionally it is treated with hot water that could be ineffective or could damage the set [19]. Hence, availability of quality planting material of newly released varieties is a major constraint in their adoption and commercialization within a short period of time. The time spent for conventional multiplication is considered as a serious economic problem, mainly in view of the higher yields that would be obtained by planting the new variety earlier on a large commercial scale, therefore efficient propagation systems are mandatory for mass multiplication.

Tissue culture of sugarcane has got a considerable research attention because of its economic importance as a cash crop. Plant multiplication or regeneration via tissue culture is a viable alternative for improving the quality and quantity of sugarcane [20]. Plant tissue culture (Micro propagation) holds immense potential for mass multiplication, subsequent rejuvenation and quality production of sugarcane [21]. By in vitro propagation, it is possible to produce some 260,000 shoots in four months [22] and 2x108 plantlets within 4-5 weeks [14] from single shoot tip of sugarcane. It is demonstrated that micro propagated system exhibited a potential in vitro production of 75600 shoots from a single shoot apex explant in a period of about 5.5 months [23]. It is reported that around 2500 seedlings could be generated from one bud in a 12 week period on MS medium supplemented with 1.5 mgL^-1of BAP and GA₃[24]. Rapid micro propagation is also achieved [25] by producing 78408 plantlets in three months on MS media supplemented with BAP (0.2 mgL^-1) and Kinetin (0.1 mgL^-1) and he conclude that by using tissue culture it would be possible to commercialize a new variety within 1-2 years. In Ethiopia, there is no sugar breeding facility and new varieties have be imported by sett and propagated for commercialization by cutting so far. Hence, this experiment was launched to optimize a protocol for in vitro mass propagation of newly introduced genotypes to supplement the conventional vegetative propagation.

The experiment was conducted at Plant Tissue Culture Laboratory of Jimma University College of Agriculture and Veterinary Medicine (JUCAVM), Ethiopia. Two sugarcane genotypes (C86-12 and C86-56) were used for the study. They were imported from Cuba in 2006 and passed through agronomic performance evaluation. They were among the selected ones to be commercialized. The sets of these genotypes were prepared and treated with hot water. The setts were taken to JUCAVM green house and planted. After two to three months of growing, shoot tip explants were taken from the sugarcane plants. The explants were prepared according to [14] procedures. The surrounding leaf sheaths of sugarcane tops were carefully removed one by one until the inner white sheaths were exposed. The explants were sized to about 10 cm length by cutting off at the two ends, locating the growing point somewhere near to top. They were washed under running tap water and liquid detergents. They were socked in fungicide solution (0.3% kocid) for 30 minutes under laminar flow cabinet containing three drops of tween-20. After the kocid was properly washed off from the explants, they were rinsed three times with distilled water and disinfected with 70% ethanol for one minute. The ethanol was poured off and the explants were rinsed again with sterile distilled water. Disinfection of explants was done with 0.1% of HgCl₂ for 10 minutes [26] followed by 3-4 washing with sterile distilled water. The required amounts of all stock solutions of MS [27] media, 30 gL^-1 [28] sucrose and combinations of different concentrations of BAP and Kin were mixed in a beaker and the pH was adjusted to be 5.8. This was followed by addition of 0.8% agar for solidifying the media. Then, it was heated to melt the agar and 30 ml media was dispensed in to culture jars. Finally, it was autoclaved at temperature of 121°C for 20 minutes with 15 psi of pressure.

Initiated explants were cultured under laminar flow hood aseptically and transferred to the growth room at which growth conditions were adjusted to be 16 hours of photoperiod with 25 μmolm⁻²s⁻¹ photosynthetic photon flux intensity and 26 ± 2°C of temperature. The experiment was laid down in factorial treatment combination in complete randomized design with two factor factorial treatment combination arrangements. Each of treatment was replicated three times. Data on number of shoots per explant, number of leaves per shoot and shoot length were collected after 30 days of culturing. Finally data were subjected to two-way analysis of variance (ANOVA) using SAS statistical software version 9.2 (SAS Inc., 2008) and treatments’ means were separated by using REGWQ (Ryan-Einot-Gabreil-Welsch Multiple range test) mean separation method.

Analysis of variance revealed that the interaction among BAP and Kin combinations and genotypes was highly significant (p=0.001) on number of shoots per explant, number of leaves per shoot and shoot length. MS medium without PGRs did not result in shoot multiplication on both genotypes (Table 1). However, increasing the concentration of kinetin from 0.0 mgL^-l to 1 mgL^-l without BAP increased the mean number of shoots per explant from 0.0 ± 0.0 to 10.2 ± 0.445 and 0.0 ± 0.0 to 13.6 ± 0.548 for C86-12 and C86-56 respectively. Similarly, increasing the concentration of BAP from 0.0 mgL^-l to 2.0 mgL^-l for C86-12 and 0.0 mgL^-l to 1.0 mgL^-l for C86-56 without Kin showed a significant increase in the mean number of shoots per explant from 0.0 ± 0.0 to 17.8 ± 0.447 and 0.00 ± 0.00 to 14.0 ± 0.707 respectively. This showed that addition of exogenous PGRs is a must to have shoot multiplication. Moreover, the increasing trend in shoot number per explant is due to the fact that cytokinin (BAP and Kin) stimulate protein synthesis and participate in cell cycle control in a cell division [29]. If cytokines are used for shoot culture media, they can overcome apical dominance and release lateral buds from dormancy and enhance shoot multiplication [29].

From the two genotypes, C86-12 gave higher mean number of shoots per explant (33.8 ± 0.837) with 13.04 ± 0.089 mean number of leaves per shoot and mean shoot length of 8.4 ± 0.008 cm on MS medium supplemented with 1.5 mgL^-l of BAP and 0.5 mgL^-l of Kin (Table 1 and Figure 1A). With the same medium composition, C86-56 gave only 17.4 ± 0.548 mean number of shoots per explant with 8.4 ± 0.548 mean number of leaves per shoot and 3.22 ± 0.567 cm mean shoot length. In this genotype, the highest mean number of shoots per explant (25.6 ± 0.548) was obtained with mean number of leaves per shoot of 9.8 ± 0.447 and mean shoot length of 8.65 ± 0.724 cm on MS medium supplemented with 1.5 mgL^-l BAP and 1.0 mgL^-l Kin (Table 1 Figure 1B). However, the same medium in C86-12 resulted in 20.0 ± 0.707 mean number of shoots per explant; 6.3 ± 0.447 mean number of leaves per shoot and 4.27 ± 0.013 cm mean shoot length. For C86-12, as the concentration of kinetin increased from 0.0 mgL^-l to 0.5 mgL^-l keeping BAP at 1.5 mgL^-l, the mean numbers of shoots per explant, mean number leaves per shoot and mean shoot length showed a significant increase from 15.9 ± 0.224 to 33.8 ± 0.837, 8.0 ± 0.000 to 13.04 ± 0.089 and 3.82 ± 0.008 to 8.4 ± 0.008 respectively. However, for C86-56 only mean number of shoots per explant showed a significant increment from 12.2 ± 0.447 to 17.4 ± 0.548.

Table 1: Effects of 6-benzylaminopurine and kinetin on shoot multiplication

Figure 1: Shoot multiplication showing best results of
A: C86-12 genotype on MS medium containing 1.5 mgL^-1 BAP and 0.5 mgL^-1 Kin
B: C86-56 genotype on MS medium containing 1.5 mgL^-1 BAP and 1 mgL^-1 Kin.

The best result obtained in C86-12 is in agreement with the result reported by [30]. They reported that optimum multiplication from HSF-240 genotype exhibited 16.5 cm mean shoot length, 11 number of shoots per explant and 32 leaves per explant on medium supplemented with 1.5 mgL^-l BAP, 0.5 mgL^-l Kin and 30 gL^-1 sucrose after 30 days of culturing. Though they found higher number of leaves per shoot and shoot length, the present study is better in terms of mean shoot number per explant. This difference could be due to genotypic difference. The best results in both genotypes of the present study contradict with results reported in [31-33]. Best results were obtained from CO678 genotype on MS medium supplemented with 2 mgL^-l BAP+0.5 mgL^-1 Kin with 9.1 ± 0.1 mean number of shoots, 6.83 ± 0.12 mean shoot length and 5.67 ± 0.04 leaves per shoot. He also obtained 7.87 ± 1.06 mean number of shoots, 5.44 ± 0.19 mean number of leaves and 6.33 ± 0.21 mean shoot length on MS medium supplemented with 2 mgL^-l BAP+0.25 mgL^-l kin+30 gL^-1 sucrose from Co449 genotype but from both genotypes he reported much less number of shoot per explant than the result of this study[31]. Tilahun M (2011) [31] reported more number of shoots per explant (34 ± 1.54) than the current result but with less number of leaves per shoot (12 ± 0.17) and shoot length (6.95 ± 0.01 cm) on MS medium supplemented with 1 mgL^-l BAP+0.5 mgL^-l Kin+30 gL^-1 sucrose for B41-227 genotype. Comparable mean shoot number per explant (29.7 ± 1.0069) from BL-4 genotype on MS medium supplemented with 0.25 mgL^-l BAP and Kin each was reported [8]. These differences happened because it is an established fact that different genotypes may give different results on MS medium supplemented with different concentrations of plant growth regulator and combinations. Sharma [33] found 20 ± 0.15; 24 ± 0.22 mean number of shoots per explant and 7.0 ± 0.27; 7.4 ± 0.06 mean shoot length for CoJ 83 and CoS 8436 genotypes respectively after 21 days of culturing on MS medium supplemented with 1.0 mgL^-l BAP+1.5 mgL^-l Kin+30 gL^-1 sucrose . The difference is not only due to genotypic variation but also due to the number of days taken for culturing.

1. Therefore, the best results obtained on MS medium supplemented with1.5 mgL^-l BAP and 0.5 mgL^-l kin for B85-12 and 1.5 mgL^-l BAP and 1 mgL^-l kinetin for C86-56 showed genotypic difference in relation to concentrations of BAP and Kinetin combinations to be used for optimal shoot multiplication. It is because of the fact that different genotypes possess specific receptor proteins and differed in concentration for plant growth regulators [29]

The present results showed that MS medium fortified with 1.5 mgL^-l BAP and 0.5 mgL^-l of Kin for B86-12; and 1.5 mgL^-l of BAP and 1.0 mgL^-l of Kin for C86-56 were found to be the optimum media for shoot multiplication. Hence, by using these media combinations (protocol), these genotypes can be commercialized within a short period of time and supplement the conventional propagation which improves both the quality and quantity of the planting materials.

We would like to gratitude the Ethiopian sugar corporation for funding the research budget and, Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) for providing the Tissue Culture laboratory.