In vitro plant regeneration from juvenile shoots of




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IN VITRO PLANT REGENERATION FROM JUVENILE SHOOTS OF Nelumbo nucifera Gaertn. ssp. nucifera (Chinese/Asian Lotus), A WATERPLANT SPECIES AS AN ALTERNATIVE FOOD SUPPLY
N. Mahmad and R.M. Taha
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia, Email: fara_aid@siswa.um.edu.my

Phone:+603-79677121, Fax:+603-79674178


Abstract

The lotus, Nelumbo nucifera Gaertn. ssp. Nucifera, is an important aquatic economic plant, not only as an ornamental flower but also as a source of edible and herbal medicine with strong bioactive ingredients such as alkaloids, flavonoids, antioxidants, antisteroids, antipyretic, anticancer, antiviral and anti obesity properties. An in vitro protocol is described for rapid and large scale propagation of Nelumbo nucifera Gaertn. ssp. nucifera (Nelumbonaceae), also known as sacred lotus. Juvenile shoots (2-weeks-old) were excised and cultured on solid Murashige and Skoog (1962) medium for axillary shoot proliferation. After 2 weeks of inoculation, one shoot elongated from each explant. Multiple shoots induction was achieved with efficient shoot elongation and repeated subculture of rootstock segments resulted in 4-5 shoots per explant after 8 weeks. The best growth was observed on solid MS medium supplemented with the combination of 0.5 mg/l NAA and 1.5 mg/l BAP. Rooting was obtained by growing shoots on solid MS without growth regulators (MS basal medium) within 4 weeks.


Keywords: lotus, plant regeneration, multiple shoots, 6-benzyl aminopurine (BAP), α-naphtaleneacetic acid (NAA)

Introduction

Aquatic ecosystems such as rivers, lakes, ponds and wetlands, are expected to be the most severely affected by global warming phenomenon such as UVB radiation, decreasing water quality, salinisation, elevated temperature and water level as they occupy the lowest areas in the landscape (Brock et al., 2005; Goodman et al., 2010; Hart et al., 2003; James et al., 2003; Walker et al., 2002). Despite being recognised as areas of ecological complexity and conservation importance (Davis et al., 2006), aquatic systems continue to be among the world’s most threatened ecosystems (Zedler and Kercher, 2005). The threats will lead the aquatic biota become increasingly stressed, resulting in reduced growth and reproduction (Sim et al., 2006) and ultimately death (Kefford et al., 2007; Nielsen et al., 2003), leading to a decline in species richness (Hart et al., 1990). Lotus one of most important aquatic plants and has the critical role in ecological systems of wetlands, lakes and ponds. The growth of lotus is also affected by water level and its fluctuation. The deepest water level recorded is about 2-3 m for wild lotus (Wang and Zhang, 2005). Recently lotus has been included in the list of endangered species in China (Dong and Zheng, 2005) and America (Sayre, 2004).


Nelumbo nucifera

The genus Nelumbo, commonly known as lotuses, was previously included in the family Nymphaeaceae. However, based on recent molecular phylogenetic analyses (Barkman et al., 2000; Lee and Wen, 2004), many researchers now place the genus in a separate family, Nelumbonaceae (Kubo et al., 2009). The Nelumbonaceae also known as the lotus family is a small family of perennial, aquatic emergent angiosperms which traditionarily consists of the two species Nelumbo nucifera Gaertn. (Indian or sacred lotus) and Nelumbo lutea (Willd.) Pers. (American lotus or water chinquapin). The former is distributed in Asia and North Australia and the latter is found in North and South America (Borsch and Barthlott, 1996 Han et al., 2007;). Although these two species have distinctive characters, such as petal color and leaf size, they also share many similar characteristics and can easily be hybridized. Hundreds of Nelumbo cultivars exist, mostly in Asia (Zou et al., 1997). The family is characterized by simple, peltate leaves which lack stipules and are borne on the surface of the water. Lotus is a typical hydrophyte that has been cultivated to produce edible tubers in the wetlands of eastern Asian countries. This species has a large, round leaf that expands horizontally at the top of its petiole. The diameter and area of the leaf exceed 0.5 m and 0.25 m2, respectively, in a full growing period (Takagi et al., 2006). There is a common red-flowered form where petals have darker red lines and a very rare form where the petals are dark red at the apices; less common are a white form and an intermediate white-red form. Flowers with less than 25 petals are usually known as singled-flowered, those with 25-50 petals are known as intermediate and those with over 50 petals sre called double-flowered (La-ongsri et al., 2008). The lotus is an important aquatic economic plant, not only as a dainty and ornamental flower but also as a source of herbal medicine with strong bioactive ingredients such as alkaloids, flavonoids, antioxidants, antisteroids, antipyretic, anticancerous, antiviral and anti obesity properties (Mukherjee et al., 1997; Sinha et al., 2000; Qian, 2002; Sridhar and Bhat, 2007). Lotus usually propagated vegetatively through rhizome division or tuber production but normally with low propagation rate growth (Shou et al., 2008). It also can be multiplied through seed but for quick and better germination the seeds need to be scarified by rubbing the outer hard seed coat gently on the sand paper at both ends and finally immersing in water to initiate germination. Scarified seeds will germinate after 3-4 days while normal seeds take 10-15 days to germinate. If the hard coating stays intact, the seed will remain viable for centuries and if placed in water it may take a few years for the seed to sprout (Hartman et al., 1990).



Tissue culture of aquatic plants
During the past few years, there has been increasing interest in the use of aquatic vascular plants for the removal of pollutants from domestic and industrial sewage effluents (McDonald and Wolverton, 1980). Aquatic macrophytes can take up excessive nutrients and also play a crucial role in creating a favourable environment for a variety of chemical, biological and physical processes that contribute to the nutrient removal and degradation of organic compounds (Gumbricht, 1993; Chong et al, 2004). Of the several plants studied, lotus (N. nucifera) and water hyacinths (Eichhornia crassipes) are among the most commonly cited and appear to have the greatest potential for use in water pollution control and are known to accumulate nutrients (Hailer and Sutton, 1973; Ornes and Sutton, 1975 and Cornwell et al., 1977). Nelumbo is a good bioindicator of trophic changes, assimilation and removal of heavy metals such as chromium removal (Vajpayee et al., 1999) and also can absorb and accumulate Fe, Mn, Cu, Zn, Pb, and Ca (Sun et al., 1987). In order to produce large numbers of these potential aquatic plants for this type of uses, one has to rely on vegetative propagation or sexual reproduction. Unfortunately most aquatic plant species do not produce seeds and plants produced through asexual propagation are time consuming, labor intensive, expensive and not adequate to meet the demands of industry (Thullen and Eberts, 1995). Furthermore the harvesting of aquatic plants from their natural habitat will become a threat to the species richness (Lauzer, 2004).
Table 1.0: Wide range of aquatic plant species tissue culture.

Species

Explants

Media

Source

Nelumbo lutea

Excised embryos from immature flowers

Half strength MS + 100mg inositol, 0.4mg thiamine, 100mg/l GA3

Kane et al., 1988

Cryptocoryne lucen

Aerial plants

MS + 0.45mg BAP, 0.1mg NAA

Kane et al., 1990

Myriophyllum aquaticum

Nodal and internode segments

Liquid half MS + 8mg/l 2iP

Kane et al., 1991

Anubias barteri var. undulata

Lateral shoots

MS + 0.3mg/l BA,

0.01 thiadiazuron and 0.1 NAA



Huang et al., 1994

Trapa natans

Shoot tips and node

Nitsch's basal

liquid medium (NBL) + 10-6M BAP



Agrawal and Mohan Ram, 1995

Scirpus robustus

Seedling mesocotyl

MS + 1mg/l 2,4-D

Wang et al., 2004

Therefore tissue culture is a suitable method to mass propagate aquatic plant species and offers several advantages for industry. Among the advantages is good quality of planting materials which disease and virus free at a competitive price while conserving aquatic plants in their natural habitat. Large scale plant production also can be programmed and preservation of plant species in vitro is also possible (Yapabandara and Ranasinghe, 2006). In vitro propagation is a most efficient and cost effective method of propagating large number of planting materials. The plants produced by in vitro propagation are genetically uniform, vigorous and free from associations with other organisms and useful for the culture of aquatic plants where contaminating organisms can dominate other types of production systems (Alistock and Shafer, 2006). Many tissue cultured water plant species show a more bushy growth with more adventitious shoots, qualities that many will appreciate (Christensen, 1996). Tissue culture has been successfully employed for micro-propagation of a wide range of aquatic plants as stated in Table 1.0 but its application in lotus rarely reported possibly because its recalcitrance to regeneration in vitro (Zhao, 1999). So far, a protocol for flower lotus regeneration (Arunyanart, 1998; Arunyanart and Chaitrayagun, 2005) and in vitro multiplication of lotus through shoot proliferation from underground rhizomes (Shou et al., 2008) has been reported.


Materials and methods
Plant materials and tissue culture initiation
Nelumbo nucifera Gaertn. plant were obtained from natural lake, Chini Lake in Pahang, Malaysia. Two types of seeds were collected from intact plants including matured green seed and black seed. These seeds were initially washed with tap water and teepol. Then, seeds were sterilized with 100% sodium hypochlorite solution for 1 min and rinsed with distilled water for three times. In laminar flow, the seeds were dipped in 70% (v/v) ethanol for 1 minute and rinsed with steriled distilled water for three times. Seeds were cultured on solid basal germination medium composed of Murashige and Skoog (MS) (1962) salts and vitamins supplemented with 30g/L sucrose and 10 g/L agar. Green petiolar inside the seeds were cut into small pieces (3mm2) and cultured on MS media (Table 2.0) with 25 different combination hormones of NAA and BAP with 30 replicates for each treatment. Media were adjusted to pH 5.5 and sterilized by autoclaving (15 min, 121ºC) and 50 ml aliquots poured into pre-sterilised 290 ml plastic pottles (80 mm diameter x 60 mm high). All cultures were incubated in a growth room at 24ºC day and night temperature, with a 16-h photoperiod at 80-85 µmol m-2 s-1 under cool white fluorescent light. Every 4 weeks the in vitro plants were subcultured on the same media (Table 2.0).
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