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Translating Applied Biology for Future Sustainability

Hatten Hotel Melaka, Malaysia 29 June – 1 July 2018

Editorial board: Nazlina Ibrahim Muhamad Hafiz Che Othman Shevin Rezal Feroz Fareed Sairi Mohd. Afiq Senen

Organised by: The Malaysian Society of Applied Biology http://msabsimposium.blogspot.my/

Hak Cipta Terpelihara. Tiada bahagian daripada terbitan ini boleh diterbitkan semula, disimpan untuk pengeluaran atau ditukarkan ke dalam sebarang alat juapun, sama ada dengan cara elektronik, mekanikal, cetakan, rakaman dan sebagainya tanpa kebenaran bertulis daripada Penerbit terlebih dahulu. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the Publisher. Hakcipta/Copyright© Diterbitkan oleh/Published by: Persatuan Biologi Gunaan Malaysia/Malaysian Society of Applied Biology Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. Perpustakaan Negara Malaysia

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Symposium of Malaysian Society of Applied Biology (15th : 2018 : Melaka, Malaysia) MSAB 2018 THE 15th SYMPOSIUM OF MALAYSIAN SOCIETY OF APPLIED BIOLOGY: Proceedings / Editorial board: Nazlina Ibrahim, Muhamad Hafiz Che Othman, Shevin Rezal Feroz, Fareed Sairi, Mohd. Afiq Senen ; Organised by The Malaysian Society of Applied Biology. ISBN 978-983-44335-5-0 1. Biology--Research--Malaysia--Congresses 2. Microbiology--Research--Malaysia--Congresses 3. Ecosystem Management--Research--Malaysia--Congresses 4. Government publications—Malaysia. I. Nazlina Ibrahim. II. Muhamad Hafiz Che Othman. III. Shevin Rezal Feroz. IV. Fareed Sairi. V. Mohd. Afiq Senen. VI. Persatuan Biologi Gunaan Malaysia, organizer. VII. Title. 570.72

The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka Article ID 2018026

GROWTH PERFORMANCE AND CHEMICAL PROPERTIES OF ELITE Labisia pumila PRODUCED BY TISSUE CULTURE AND CUTTING METHODS NORHAYATI SAFFIE*, FARAH FAZWA MD ARIFF, SYAFIQAH NABILAH SAMSUL BAHARI and SITI SUHAILA ABD RAHMAN. Forestry Biotechnology Division, Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia *E-mail: [email protected] ABSTRACT Plant growth performance is strongly affected by environmental conditions. The changes in weather and soil conditions from time to time may cause the variability on the plants morphology and also to its chemical contents. Labisia pumila was selected for this study as it is highly demanded by the industry and being selected as one of the 18 species under National Key Economic Area (NKEA). It also has several phytochemicals that make it an effective towards into anti-cancer, antiinflammatory, anti-microbial and anti-obesity. One elite planting materials that have performed well all over the locations from the previous study was selected and massproduced using tissue culture and leaf cutting techniques. This is one of the effort to sustain the production of planting materials of L. pumila in the future. Thus, in this study, L. pumila plants produced from both techniques were compared by planting them at three different locations; two FRIM’s Sub-stations (SPF Mata Ayer and SPF Maran) and, FRIM Kepong. The purpose of the study is to identify suitable production technique that could provide good growth performance and contain high chemical content before any large scale production could be conducted. Evaluation on the growth performances and chemical content (Total Phenolic content, TPC) were made at 12 months after planting. Analysis of Variance (ANOVA) showed there is no significant difference between the two techniques. Results also showed that the plant growth and TPC at Mata Ayer, Perlis was slightly higher as compared to other two locations. Keywords: Labisia pumila, mass produce, growth performance, total phenolic content INTRODUCTION Labisia pumila from Primulaceae family is an herbaceous under shrub plant that native to Malaysia. This herb is locally known as Kacip Fatimah (Fatimah’s betel cutter). Malays women often use it as well as in other Southeast Asia regions for


The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka

numerous purpose especially in promoting women’s health (Wan Ezumi, 2009). Traditionally, they claim this herb can fasten and ease the delivery of baby. A lot of researches have been done on L. pumila recently and they found it have important bioactive compounds with high biological activity that make it effective against various illnesses (Karimi et al., 2013). Several phytochemicals in L. pumila that contribute to this effectiveness are phenolics, flavonoids, carotenoids, ascorbic acids, saponin, alkenyl compounds and benzoquinone derivatives (Chua, 2011; Karimi et al., 2011; Karimi & Jaafar, 2011). This is the reason for this species to have highest antioxidant properties especially in leaves part compared to others such as stem and root (Karimi et al., 2011). Other potential pharmacological activities that have been identified are anti-cancer, anti-inflammatory, anti-microbial and anti-obesity (Norhanisah et al., 2013). All these benefits and potential have make L. pumila getting high demand and keep incresing year by year. However, the demands may not be fullfiled as the raw materials are insufficient. It is reported that 83% of medicinal plant raw materials were harvested from natural forest and only 17% were cultivated (Rohana et al. 2017). Besides, excessive collection from harvesters, over exploitation and logging activities in the forest also contribute to this number. As a result, Malaysia herbal industries need to import the raw materials from overseas to meet the demands. This alternative causes the adulteration that will reduce the quality of the herbal raw materials. Researchers from Forest Research Institute Malaysia (FRIM) have make some effort by mass produce this species using two techniques which are tissue culture and leaf cuttings. The plants sourced from the two techniques were then planted at three different locations in order to evaluate their growth performance and chemical contents. Output from the study can provide some guidelines on the growth requirement of the species for commercial plantation in the future. MATERIALS AND METHODS Preparation of planting materials A total of 180 plants produced from tissue culture and cuttings were prepared for each location. Tissue culture plants were prepared using Temporary Immersion System (TIS). Whereas, for cuttings plants, leaves of L. pumila were cut into approximately 30 cm2 of size and dip into rooting hormone. The media used was 100% river sand. Acclimatization at nursery

Plantlets produced from tissue culture were acclimatized for a period of one month in acclimatization chamber. Rooted cuttings plants were acclimatized under 50% of shade at nursery. The acclimatization process is to ensure that sufficient numbers of plants survive and grow vigorously when transferred to the field.


The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka

Preparation of plots Two locations in FRIM’s Sub-Stations and FRIM, Kepong were selected as experimental plots. The land was cleared from debris and weeds. The soils were ploughed to improve soil structure and provide necessary soil conditions which can enhance the successful of tissue culture and cuttings plants at field conditions. At each location, 90 plants from tissue culture and 90 plants from cuttings plants were planted in randomized complete block design (RCBD) arrangement. The plants were shaded with 70% of shade and irrigated by sprinkler system. Evaluation of plant growth Data of plant growth such as plant height (cm), leaf number, leaf length (cm), leaf width (cm) and collar diameter (mm) were measured at 12 months after planted at three locations. The data were analysed using one-way analysis of variance (ANOVA). Data collected was subjected to analysis of variance (ANOVA) and the means value were compared by Tukey’s HSD test at p ≤ 0.05 using the IBM SPSS Statistics version 22. Quantification of Total Phenolic Content (TPC) A total of 10 g of fresh healthy leaves were harvested from tissue culture and cuttings plants from the three locations. Total phenolic content analysis was determined by using Follin-Ciocaltue Reagent as being described by Singleton & Rossi (1965) with slight modifications (Vimala et al. 2003). Samples were expressed as gallic acid equivalents (GAE-TPC mg/gm of crude extract) based on gallic acid standard curve. RESULT AND DISCUSSION Evaluation of growth performances Table 1 indicated that cuttings plants of elite L. pumila showed better growth performance compared to tissue culture plants at 12 months after planting. However, for plant height, tissue culture plants produced higher values compared to the cuttings plants. In terms of value, location from FRIM Kepong gave the highest (7.52 cm  0.22) value for plant height, followed by SPF Maran (7.03 cm  0.35) and SPF Mata Ayer (5.87 cm  0.22). The similar results have also been observed in the study by Singh et al. (2013), which in-vitro turmeric plants was significantly taller than the conventional plant. Height is one of the indicators to show the vigorousness’ of plants (Sheela et al., 2001). For herbal plant such as L. pumila, the important plant part that required by most herbal industry is the leaves which can contribute to the high number of biomass. Thus, in this study, it showed that cutting plants gave higher values in terms of leaf number, leaf length and leaf width after planted at all locations (SPF Mata Ayer, SPF Maran and FRIM Kepong). Leaf number was recorded higher at FRIM Kepong (7.71  0.28) followed by SPF Mata Ayer (5.00  0.17). However in SPF Maran, tissue


The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka

culture plants gave higher number of leaves (5.04  0.23) compared to cutting plants (4.37  0.20). According to Hazarika et al. (2006), leaves that develop through invitro generally lack well-developed epicuticular waxes and stomata may not close normally, poorly structured internal anatomy and may not be photosynthetically efficient. This showed that the development of leaves from tissue culture plants will take more time and effected by environmental manipulation. However, in other research such as turmeric in vitro (Singh et al., 2013), banana in vitro (Buah et al., 2000), they showed that tissue culture plants established quickly, grow vigorous and fast increase in the leaf length as well as in emergence of new shoots. Table 1: Plant growth performances of elite L. pumila produced from leaf cuttings and tissue culture at 12 months after planting at three different locations Plant growth Propagation Locations characteristics techniques SPF Mata SPF Maran, FRIM Ayer, Perlis Pahang Kepong, Selangor b b Plant height (cm) Cuttings 4.91  0.22 6.11  0.27 7.77  0.27a Tissue culture 5.87  0.22a 7.03  0.35a 7.52  0.22a Leaves no. Cuttings 5.00  0.17a 4.37  0.20b 7.71  0.28a Tissue culture 4.44  0.17b 5.04  0.23a 6.36  0.23b Leaf length (cm) Cuttings 13.96  0.29a 14.65  0.41a 17.43  0.32a Tissue culture 11.88  0.30b 14.99  1.21b 14.98  0.40b Leaf width (cm) Cuttings 5.99  0.12a 6.64  0.17a 7.37  0.12a Tissue culture 5.89  0.66a 6.24  0.19b 6.56  0.13b Collar diameter (mm) Cuttings 6.39  0.10a 5.74  0.28a 6.22  0.13a Tissue culture 5.45  0.12b 4.73  0.20b 5.51  0.14b Means followed by the same letter is not significantly difference at p < 0.05 In terms of collar diameter, cuttings plants also gave high values compared to tissue culture plants after planted at three locations. The range of values for collar diameter is 5.74 mm to 6.39 mm for cuttings plants whereas for tissue culture plants, the range is between 4.73 mm to 5.51 mm. Analysis of total phenolic content (TPC) Results 12 months after planting indicated that tissue culture plants produced higher values of total phenolic content compared to cuttings plants (Figure 1) except for SPF Maran, Pahang. Location SPF Mata Ayer gave the highest TPC (473.2 mg/100 g GAE), followed by SPF Maran (278.6 mg/100 g GAE) and FRIM Kepong (259.7 mg/100 g GAE). The significant differences for TPC may be attributed by different environmental conditions at the three tested locations (Goyali et al., 2013). Beside that, the production of secondary metabolites is much higher in shoot culture as the compounds are synthesized and/or stored in the aerial plant parts (Amoo et al.,


The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka

2012). Tissue culture plants also have getting more concentrations of plant growth regulators (PGRs) during culture, which may regulate and modify the concentration of secondary metabolites in plants differently. All this combination of factors may be contributed to these result of TPC. 600


Total Phenolic Content (mg/100 GAE)



a SPF Mata Ayer cuttings

b 300



SPF Mata Ayer Tissue culture SPF Maran cuttings SPF Maran Tissue culture



FRIM Kepong cuttings FRIM Kepong Tissue culture


0 cuttings

Tissue culture

SPF Mata Ayer


Tissue culture

SPF Maran


Tissue culture

FRIM Kepong

Locations and Types of Plant Propagation

Fig. 1. Total phenolic content of elite L. pumila produced from leaf cuttings and tissue culture at 12 months after planting at three different locations CONCLUSION The study demonstrated that cuttings plants are good in growth especially in leaves development (leaf number, leaf length and leaf width) when planted at three different locations with different environmental conditions. However, tissue culture plants seems more quickly growth and vigorous as the plants taller than cuttings plants. In addition, tissue culture plants produced more concentrated and maximum value of TPC than the cuttings plants. However, it is too early to conclude which is the best technique for future planting stock in the future. Further investigations are still required. ACKNOWLEDGEMENT The authors gratefully acknowledge the cooperation of Biousahawan Laboratory for their excellent in technical support and Staff in Plant Improvement Program for their assistance in this study. This study was financially supported under National Key Economic Area (NKEA) grants by the Ministry of Agriculture (MoA).


The 15th Symposium of Malaysian Society of Applied Biology, 29th June – 1st July 2018, Melaka

REFERENCES Amoo, S. O., Aremu, A. O., and Van Staden, J. 2012. In Vitro Plant Regeneration, Secondary Metabolite Production And Antioxidant Activity Of Micropropagated Aloe Arborescens Mill. Plant Cell, Tissue and Organ Culture, 111 (2012): 345-358. Buah, J. N., Kawamitsu, Y., Yonemori, S., and Murayama, S. 2000. Field Performance of In vivo-propagated and Sucker-derived Plants of Banana (Musa spp.), Plant Production Science, 3 (2) : 124-128. Goyali, J. C., Igamberdiev, A. U. and Debnath, S. C. 2013. Morphology, Phenolic Content and Antioxidant Capacity of Lowbush Blueberry (Vaccinium Angustifolium Ait.) Plants as Affected By In Vitro And Ex Vitro Propagation Methods. Can. Journal of Plant Science, 93: 1001-1008. Singh, T. D., Singh, C. H., Nongalleima, K., Moirangthem, S., and Devi, H. S. 2013.Analysis Of Growth, Yield Potential And Horticultural Performance of Conventional vs. Micropropagated Plants of Curcuma Longa var. Lakadon. African Journal of Biotechnology, 12(14): 1604-1608.