Interaction Effects of 6-Benzylaminopurine (BAP) and Indole-3-Butyric Acid (IBA) on Ex Vitro Propagation of Sugarcane

Ex vitro propagation of micropropagated sugarcane plantlets of three selected sugarcane genotypes was carried out with the objective of evaluating their propagation responses to the interaction effects of BAP and IBA. Accordingly, six levels of IBA (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg L-1) and eight levels of BAP (0.0, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 and 2.25 mg L-1) with three sugarcane genotypes, each replicated three times were tested. The treatments were arranged in a factorial completely randomized design. Data on the number of tillers per shoot, average shoot length (cm) and number of active leaves per shoot were collected twice every 30th day after 30 days of treatment application. Analysis of variance revealed that the interaction effects of BAP, IBA and the sugarcane genotypes was very highly significant (P<0.0001) on the number of tillers per shoot, average shoot length and number of active leaves per shoot. The optimum number of tillers per shoot (5.67), average shoot length (40.77 cm) and number of active leaves per shoot (6.97) were obtained at 0.1 mg/l IBA+0.75 mg/l BAP in C132-81. In C86-56, the optimum number of tillers per shoot (4.50) was obtained at 0.1 mg/l IBA+1.5 mg/l BAP while the maximum average shoot length (48.33 cm) and maximum number of active leaves per shoot (7.67) were obtained at 0.2 mg/l IBA+0.75 mg/l BAP. In SP70-1284, the optimum number of tillers per shoot (6.43) was obtained at 0.3 mg/l+1 mg/l BAP with 16.05 cm average shoot length and 4.87 active leaves per shoot. Thus, it can be deduced that production of an average of 5.5 plantlets per shoot within a month can be possible.


Introduction
Sugarcane is a perennial grass which produces seed under suitable conditions, but for commercial production, it is propagated from stalk cuttings. Propagation through stalk cuttings is the traditional method of sugarcane propagation in which stalk cuttings containing one or more buds, termed sets are used for commercial planting [1]. The Ethiopian Sugar Industry used this traditional method of propagation alone for the last 50 years till 2012. However, under the new plan of the Industry to expand the existing farms and establishment of vast sugarcane plantation projects with many high crushing capacity sugar factories, the tradition propagation method alone was seen to have various draw backs. Among these, availability of enough amount of quality disease free planting material within short time, transport of bulky unclean seed cane from existing farms to the remote project farms, the low rate of propagation(usually 1:10), lack of methods for fast commercialization of improved and adapted varieties, obsolation of productive commercial varieties due to disease, lack of alternative techniques for rejuvenation and disease cleansing of the old contaminated sugarcane varieties were the major limitations identified as challenges to the vast expansion and new development plans [2,3]. With a view to minimize the challenges, microproagation technology was adopted and implemented to supplement the tradition method of sugarcane propagation in all sugar estates and projects. Microproagation Technology is a technique through which group of genetically identical plants all derived from a selected individual multiply vegetatively and rapidly by aseptic culture of meristematic regions under defined nutritional and controlled environmental conditions in vitro [4]. Nowadays, unlike the traditional propagation method, it is the only practical means of achieving rapid and large scale production of disease free quality planting materials in sugarcane [5][6][7] and alternative approach for fast multiplication of a genotype in its original form. It is very effective in entire disease cleansing, rejuvenation and subsequent mass propagation of well adapted and promising varieties facing gradual deterioration in yield, quality and vigor due to accumulation of pathogens during prolonged vegetative cultivation and hence sustains the productive potential of sugarcane crops for a longer period [8,9]. Furthermore, micropropagated sugarcane plants were reported to give superior in cane and sugar yield as compared to their donors under similar agronomic management systems [10][11][12][13][14]. However, the Ethiopian Sugar Corporation is procuring the micropropagated sugarcane plantlets from different organizations where the cost of procurement is high (about US$ 0.205 per plantlet). The erratic supply, long distance transport of the delicate plantlets followed by reduced survival, the increasing demand of micropropagated plants with the subsequent procurement cost increment were found to be the major limitations for the profitability and sustainability of the micropropagation technology. Therefore, this experiment was carried out with the objective to evaluate the effects of 6-benzylaminopurine (BAP) in vivo propagation of tissue culture raised sugarcane plantlets of three sugarcane genotypes (C132-81; C86-56 and SP70-1284) with a view to complement microproagation technology and to ensure continuous supply, cut down the cost of plantlets procurement and propagation of sufficient amount of quality planting materials at the farm gate nursery within short period of time.

Materials and Methods
The study was carried out at Metahara sugarcane plantation, located at Eastern part of the country, at about 200 km away from Addis Ababa, Ethiopia. Metahara Sugar estate is situated at 80 53 ' N latitude and 390 52 ' E longitudes at an altitude of 950 m above sea with a semiarid climatic condition. The experimental materials were in vitro propagated sugarcane genotypes of C132-81, C86-56 and SP70-1284. The primary acclimatized plantlets of these sugarcane varieties were delivered with intact coco-peat from Mekelle Technology Institute Tissue Culture Laboratory and directly planted in white polyethylene bag (8cm diameter with 10 cm height) filled with mixture of Luvisol, sand and compost in the ratio of 8:2:1. and eight levels of BAP (0.0, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 and 2.25 mg L -1 ) resulting in 144 treatment combination arrangements each replicated three times. Each plot contains 30 pots (one plantlet a pot) and data on the number of tillers per shoot, shoot length and number of active leaves per shoot were collected from ten randomly selected plantlets at 30 th and 60 th days of treatment application. Then the average data were subjected to analysis of variance using SAS software version 9.2 while separation of significant means' was done using REGWQ (Ryan-Einot-Gabriel-Welsch) Multiple Range Test.

Results and Discussion
Analysis of variance revealed that the interaction of Indole-3butyric acid (IBA), 6-benzylaminopurine (BAP) and sugarcane genotype have a very highly significant (BAP * IBA * Genotype=p<0.001) effect on all the response variables tested: numbers of leaves per shoot, average shoot length and number of leaves per shoot in all the sugarcane genotypes tested: C132-81, C86-56 and SP70-1284 ( Table 1).  Comparison of the sugarcane genotypes revealed that all the three sugarcane genotypes showed marked variation in all the responses tested: number of tillers per shoot, average shoot length and number of leaves per shoot ( Table 2). Regardless of the other treatment, comparison of the sugarcane genotypes shows us SP70-1284 produced the highest number of tillers per shoot C86-56 gave the highest average shoot length (37.28 cm) and maximum number of leaves (6.36) per shoot ( Table 2). In sugarcane genotype C132-81, the lowest number of tillers per shoot (1.63) was found on 0 mg/l IBS+0 mg/l BAP (control treatment) with 27.27 cm average shoot length and 4.00 leaves per shoot. However, increasing the concentration of IBA from 0 mg/l to 0.1 mg/l and BAP from 0 mg/l to 0.75 mg/l, increased the number of tillers per shoot from 1.63 to 5.67, average shoot length from 27.72 cm to 40.77 cm and number of leaves from 4.00 to 6.97. The maximum number of tillers per shoot (5.67), highest average shoot length (40.77 cm) and number of leaves per shoot (6.97) was obtained at 0.1mg/l IBA and 0.75 mg/l BAP in sugarcane genotype C132-81 (Table 3). Holding the concentration of BAP at 0.75 mg/l and increasing the concentration of IBA beyond 0.1 mg/l up to 0.5 mg/l showed a declining trend in number of tillers per shoot, average shoot length (cm) and number of leaves per shoot.  Similarly, at 1 mg/l BAP, increasing the concentration of IBA from 0.1 up to 0.3 mg/l showed an increasing trend in the number of tillers per shoot and number of leaves per shoot while further increase revealed a decreasing effect in both response variables (Table 3) both have no any biological and economic advantage (Table 3). In sugarcane genotype C86-56, the lowest number of tillers per shoot (1.50) and average shoot length (22.67 cm) was found on the control treatment (0 mg/l IBA and BAP) while the lowest number of leaves per shoot (4.10) was obtained at 0.5 mg/l IBA and 2.25 mg/l BAP (Table 3). In C86-56, increase in the number of tillers per shoot from 1.50 to 2.67; average shoot length from 22.67 cm to 34.00 cm and number of leaves per shoot from 4.70 to 7.00 was obtained as a result of 0.1 mg/l IBA+0.75 mg/l BAP (Table 3) (Table 3). The rate of sugarcane propagules multiplication depends upon auxin -cytokinine balance of culture medium [11]. A low concentration of auxin is often beneficial in conjunction with higher levels of cytokinine during shoot multiplication and exogenous auxin does not promote auxiliary shoot proliferation; however, their presence in culture medium may improve the culture growth. Although cytokinines are known to stimulate cell division, but does not induce DNA synthesis. Nevertheless, the presence of auxin promotes DNA synthesis. Hence, the presence of auxin together with Cytokinine stimulates cell division and control morphogenesis thereby influences shoot multiplication [2,3].

Conclusion
The traditional method of sugarcane planting material propagation has many limitations while microproagation technology is efficient to solve all the limitations except it is costly to produce or procure the micropropagated plantlets. Hence, ex vitro propagation technology (IVPT) was developed to complement tissue culture technology, but reproducibility of the IVPT protocols is dependent on the genotype, environmental weather conditions, soil mixture used, plant growth regulators and interaction of the factors used. Therefore, the aim of this study was to optimize in vivo propagation protocol for three recently released sugar genotypes: C132-81, C86-56 and SP70-1284. The result proved that ex vitro propagation of the sugarcane genotypes tested is highly dependent on the interaction effects of IBA, BAP and the sugarcane genotypes. Treatment combination containing 1 mg/l BAP and 1.5 mg/l gave optimum propagation responses. Sugarcane genotype C132-81 4 tillers per shoot with 30.67 cm average shoot length and 6 active leaves per shoot while C86-56 produced 3.53 and 4.67 tillers per shoot, 29.33 cm average shoot length and 7.0 and 6.33 active leaves per shoot per month, respectively. Thus, the current finding will help minimize the current challenges of sugarcane production by rapidly availing adequate amount of quality planting material of sugarcane while reducing the cost of plantlets procurement and hence the cost of sugar production.
In sugarcane genotype C132-81, the optimum number of tillers per shoot (5.67), highest average shoot length (40.77 cm) and number of active leaves per shoot (6.97) was obtained at 0.1 mg/l IBA and 0.75 mg/l BAP while in C86 -156), the optimum number of tillers per shoot (4.50) was obtained at 0.1 mg/l IBA+1.5 mg/l BAP while the optimum average shoot length (48.33 cm) and maximum number of leaves per shoot (7.67) was obtained at 0.2 mg/l IBA+0.75 mg/l BAP. However, in SP70-1284, the optimum number of tillers per shoot (6.43) was obtained at 0.3 mg/l IBA+1.0 mg/l BAP with 16.05 cm average shoot length and 4.87 leaves per shoot. Thus, from this result, it can be deduced that this result can be used to produce sufficient quantity (5 plantlets per shoot within a month) of quality planting materials and therefore can guarantee sustainable supply of planting materials with the lowest possible cost.