Effect of Bulbils Position on Leaf Branches to Plant Growth Responses and Corms Quality of Amorphophallus muelleri Blume

Bulbil (corm leaf) is one material source of vegetative propagation in Amorphophallus muelleri Blume. Based on the position in branches there are two types of bulbil, middle and edge bulbils, which are different in shape and size. It has been reported that bulbil size affected not only the growth response of seedlings and plants but also the quality of the produced corms. Therefore, the objectives of this experiment were to study the effect of bulbils origin on A. muelleri Blume (Porang) growth and the quality of harvested corms. The quality of corms was assessed based on glucomannan and (CaOx) content. Bulbils from the middle and edge of branches were grown in polybag ( 5 cm) containing compost:fertilizer (1:1) mixed media under 40% shade. Each bulbil origin was repeated 8 times. The results showed that growth responses of plant from middle bulbil were significantly better than that from edge bulbil. However, at the harvesting time, the weight and diameter of corms derived from both types of bulbils were not significantly different. The glucomannan and calcium oxalate (CaOx) content of harvested corms grown from middle bulbil tends to be higher than that from edge bulbil.


INTRODUCTION 
Porang, Amorphophallus muelleri Blume (A. muelleri) is one members of the Araceae that produce corms which is actually an underground stem.It is native to Indonesia [1] especially in teak forests in East Java and in undisturbed areas such as riversides and bamboo forests in Central and West Java [2].It can grow in lowlands areas up to 1000 m above sea level, with temperatures between 25-35°C and rainfall between 300-500 mm per month during the period of growth [3].The plant will grow better under shade.Environment with shade of 50-60% has high production of corms [1].Amorphophallus muelleri has unique, highly dissected, umbrella-shaped leaf blade which is supported by pesudo-stem or petiole [4,5].The dissected leaf blade has some branches (leaflets) which are supported by petiolule.
Amorphophallus muelleri can be regenerated through generative and vegetative propagation [1,3].Bulbils or corms leaf are organ source of vegetative propagation of A. muelleri besides corms.The bulbils of A. muelleri are classified as epiphyllar bulbil because they were produced on the surface of branching points of the leaf [4].Based on the position on leaf branches there are two types of bulbils: middle and edge bulbils which are produced on leaf [6] and leaflet branching points, respectively.Spatial distribution of bulbils on leaf contributed to variations in shape, weight and size the bulbils [3].Middle bulbils have brown color and round shape while edge bulbils have oval shape and smaller than the former.Edge bulbils on the primary leaflet are bigger than that on secondary and tertiary leaflet.Propagation of A. muelleri from bulbils is more promising than from seeds.Plants derived from bulbils grew faster than those derived from seeds.At the third stage of growth period plants derived from bulbil were higher ( 180 cm) than those derived from seeds ( 140 cm) [7].Considerable degree of seed heterogeneity in size, weight and quality has been widely reported [8,9,10].The effects of bulbil size on the plant growth and corms quality of A. muelleri derived from different bulbil size have also been reported [7,11,12].Different sizes of bulbil showed no difference in the ability to grow but it has effect on plant growth responses [11].Large bulbils generate higher plants than small bulbils but the diameter of stem and leaves and the number of shoots were not significantly different [12].It has also been reported that bulbil size determined the amount of the stage of vegetative growth and dormancy [7].Different sizes of bulbil that were grown in medium containing various doses of lime produced corms with different level of glucomannan content [11].Position-dependent effects have been widely studied on seed or fruit [13,14,15].The position of a seed or fruit on plant can affect its morphology, mass and dormancy/germination [16,17,18].However, regarding to corms studies still focused on the placement position on propagation media or planting area [19] rather than the corms position on the maternal plant.The duration of Liatris daughter corms' growth to maturity and their growth rate vary with their position on the mother corm [20].
Currently market demand for the A. muelleri chip corms is quite high.Amorphophallus muelleri corms have been widely used as raw materials in food, cosmetics and pharmaceutical industry because they contained valuable source of glucomannan, a soluble, non-cellulosic polysaccharide [21].Chips derived from A. muelleri corms are highly exported to Japan, Hongkong and China and other countries.Therefore, today in Indonesia A. muelleri has been cultivated in several methods of propagation to produce high quality of corms to meet the increasing market demand.
Considering some previous reports which showed that bulbil size affects the growth response, therefore the spatial distribution of A. muelleri bulbil on leaf and leaflet branching is necessary to be examined both in physiological and morphological responses.Therefore, the objectives of the present study were to examine plant growth response and corms quality grown from middle and edge bulbils produced on leaf branches of A. muelleri.

MATERIALS AND METHODS Bulbil Germination
Middle and edge bulbils derived from the second growth period of plants were obtained from Oro-oro Waru Village, Saradan, Madiun District, in East Java, Indonesia.Eight bulbils of each type were germinated in plastic bags ( = 5 cm) containing  500 g fertilizer:compost (1:1) mixed media.The bulbils were planted 5 cm depth in the media and the plastic bag then placed in the experiment field of Biology Department, University of Brawijaya under 40% shading.After bulbil-derived seedling has reached a height of  20 cm then it was grown in larger polybag ( = 40 cm) containing @ 5 kg soil media.Plants were watered to about field capacity twice a week.

Plant Growth and Corms Harvesting
The measurement growths responses were started at three months after the coleoptile sheath merged on the soil surface, once a week for 10 weeks.The parameters of growth responses measured were plant height, stem girth and canopy width.Plant height was measured from petiole base on the soil surface to the top of leaf canopy.To measure canopy width, firstly the two range poles of the extreme edges of the canopy were marked.Subsequently, the distance between the two poles was measured with a measuring tape and recorded as the canopy width.The corms were harvested at  90 days after planting, and prepared for glucomannan and CaOx analysis.

Extraction and Analysis of Glucomannan Content of Fresh Harvested Corms
Fresh harvested corms were rinsed and the skin was peeled.Total 6 g of the rinsed flesh corms were mashed using blender to form pasty. Subsequently, glucomannan extraction and analysis were conducted based on the method of [22] with modification.Pasty of corm was put into beaker glass containing 200 mL of AlSO4 0.3% and then placed in an incubator at 55C for 15 min while stirred gently to maximize the process of glucomannan extraction.The extract yield was filtered with cotton cloth, diluted until the final volume reached 600 mL and then centrifuged at 1500 g for 15 min.Supernatant was added by isopropyl alcohol with ratio 1:1.The white precipitate formed was glucomannan.The precipitate glucomannan was oven-dried at 45°C overnight or until dry to obtain glucomannan yield.The weight of dry glucomannan relative to the weight of dried corm used for calculating glucomanan content.The weight of dried corms were obtained from 6 g of flesh corms that was rinsed, chopped and dried by oven at 45°C overnight or until dry.Dried glucomannan sample was weighed and glucomannan content per DW sample was calculated as: (precipitate DW/corms sample DW) x 100%.

Extraction and Analysis of Calcium Oxalate (CaOx) of Fresh Harvested Corms
Extraction and determination of CaOx content of A. muelleri corms were conducted by the method of Imouha and Kalu [23] with modification.Determination of CaOx consisted of three phases: digestion, oxalate precipitation and titration of permanganate.Corms were rinsed and then approximately ¼ parts of them was grated.The digestion phase was initiated by dissolving 10 g grated corms into 190 mL aquadest and then added by 10 mL of HCl 6N.The suspension was heated at 100C for 1 h then allowed to cool.

Effect of Bulbils to Plant Growth Responses and Corms Quality of A. muelleri Blume (Mastuti et al)
Aquadest was added into the suspension until reached volume 250 mL and it was filtered by fine cotton cloth.The second phase was initiated by adding six drops of methyl red into filtrate yield.The pH was adjusted by adding some drops of NH4OH until achieved pH 4-4.5.Subsequently filtrate was heated until 90C and then allowed to cool.Filtrate was filtered by whattman no. 1 to eliminate Fe.Filtrate was heated at 90 C, added by 10 mL of CaCl2 5% and homogenized by magnetic stirrer.Finally, the filtrate was centrifuged at 2500 rpm for 10 min.At the last phase pellet derived from previous centrifugation was put into 10 mL of H2SO4 20%.After added with aquadest until reached the volume of 100 mL the solution was heated until almost boiled.Subsequently, titration of this solution was conducted by standardized KMnO4 0.1 N until produced pink color for 1 min.The content of CaOx (g/FW corms/tubers) was calculated with Eq. 1.

Statistical analysis
Data were subjected to analysis of variance (ANOVA) and means were compared by Tukey test α 0.05 using the SPSS ver.17.

RESULT AND DISCUSSION Growth Responses of Plants Derived From Different Types of Bulbils
Middle bulbils have bigger size than edge bulbils (Fig. 1).Both types of bulbils showed significantly different vegetative growth responses.Plants derived from middle bulbils grew faster than that from edge bulbils (Fig. 2A).Plant height derived from different bulbil types showed significantly differentt from the first week until the last week of observation (Fig. 2B).From week to week the plants derived from middle bulbils were taller than those derived from edge bulbils.At the last week observation the plant height derived from middle bulbils reached 60.0  4.7 cm while those from edge bulbils reached only 47.5  4.1 cm.But until the last week the average height of plants derived from each middle and edge bulbils was not significantly different from the first week observation.Stems girth of plants grown from middle bulbils (14  1.39 cm) were also significantly greater than those grown from edge bulbils (10  0.60 cm) (Fig. 3A).Canopy width from both types of bulbils was also significantly different.It derived from middle bulbils had longer diameters than that derived from edge bulbils (Fig. 3B).The de-

Effect of Bulbils to Plant Growth Responses and Corms Quality of A. muelleri Blume (Mastuti et al)
velopment of storage organ grown from middle and edge bulbils which was represented by weight and diameter of harvested corms were not significantly different.The weight of harvested corms of plant grown from middle and edge bulbils reached 73.3  37 g and 49.6  13.8 g, respectively.Diameter of harvested corms of plant grown from middle and edge bulbils reached 54.4  10.2 mm and 48.2  4.5 mm, respectively (Fig. 4A).Bulbil of A. muelleri is a small cormes duced on leaf branch.Bulbil is a sink containing storage resources translocated from photosynthetic organs.The size of bulbil indicated the amount of storage resources possessed.In this study the middle bulbils which have bigger size showed better primary and secondary growth responses than edge bulbils based on plant height, stem girth and canopy width.Middle bulbils of A. muelleri is apparently to contain greater numbers of photosynthate as food reserves so the process of photosynthesis occurs earlier and the vegetative organs formed faster than edge bulbils.Sumarwoto and Maryana [12] have reported that bigger size of A. muelleri bulbil produce higher plant than smaller ones.The same results were shown in other species.Bulb size influenced plant growth and development of Hyacin and Lily [24].Corms size affected the performance of Gladiolus grandiflorus [25,26] as well as stigma and corm yield of saffron (Crocus sativus L.) [19].Environment is predominant factor which determine size variation of seed produced as a source of generative reproduction [8].Withinplant variation can be determined by fruit position and seed position within a fruit or inflorescences.Bulbils of A. muelleri are vegetative reproduction organ which sized vary as a result of their position on the leaf branch of maternal plants.This maternal position effect of A. muelleri bulbils occur in a structurally ordered manner.Bulbils on more periphery branch have smaller size.Within-plant variation arising from position effects is inherited [8].Effect of seed position resulted in variations in size is always present in every generation.This shows that the inherited character which is more affected by position and the variation in size is due to the distribution of the position.

Corms Quality: Glucomannan and Calcium Oxalate (CaOx) Content
Corms quality both glucomannan or calcium oxalate content of corms harvested from plant grown from middle and edge bulbils were not significantly different.The average of glucomannan content was 48.63 and 37.78% DW corms harvested from plant grown from middle and Effect of Bulbils to Plant Growth Responses and Corms Quality of A. muelleri Blume (Mastuti et al) edge bulbil, respectively (Fig. 4B).In this study it is assumed that corms were not harvested at mature physiological age.Consequently, the content of glucomannan produced by the two plants derived from middle and edge bulbil was not significantly different.Glucomannan content was higher in mature corms than in young corms.Glucomannan within the developing corm changes throughout the growing season and was highest just before the foliage died off, prior to dormancy [27].Generally foliage died off 5-6 months after planting [3].
Calcium oxalate crystals were also obtained from corms of A. muelleri plants derived from bulbils.However there was no significant difference between two plant types in terms of its content.It is suspected that during corms development the formation of calcium oxalate crystals has not been maximized.There is an indirect relationship between the diameter and the density of calcium oxalate in the corms [28].

CONCLUSION
From this result it can be concluded that the position effect of A. muelleri's bulbils on the maternal leaf branch will influence the growth response.The content of glucomannan and CaOx have no significant difference in young corms.

2
Effect of Bulbils to Plant Growth Responses and Corms Quality of A. muelleri Blume(Mastuti et al) VKMnO4 is volume KMnO4 (mL), M KMnO4 is molarity KMnO4, ME is molar equivalent of KMnO4 in oxalic, DF is dilution factor, and mf is mass of fresh corms

Figure 3 .
Figure 3. Stem girth and canopy diameter of A. muelleri plants.(a) Stem girth and (b) Canopy width.Different letters following bars indicate significant differences at α = 0.05 of Anova test.Means  SD (n=8).

Figure 4 .
Figure 4. Quality of A. muelleri harvested corms planting from different types of bulbils at 90 days after planting: (a) Weight and diameter of harvested cormes and (b) Glucomannan and Calcium oxalate content.Means  SD (n=8).