Middle East Journal of Agriculture Volume : 06 | Issue : 02 |April-June | 2017 Research Pages:424-432 ISSN 2077-4605

In vitro Propagation and Molecular Analysis of Pokeweed ( Americana)

Alaa M. El-Minisy 1, Adel El-Sawy 1, Hattem M. El-Shabrawi1, Mohamed H. Soliman2, Salah El-Assal2 and Heba A. Mahfouze3

1Plant Biotechnology Department, National Research Center, Dokki, Cairo, Egypt. 2Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt. 3Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, 12622, Egypt Received: 20 March 2017 / Accepted: 30 April 2017 / Publication date: 02 May 2017

ABSTRACT

The aim of this study was to establish a micropropagation protocol for pokeweed .Stem cutting explants were cultured on MS medium supplemented with different concentration of BAP (1,2,3 and 4 mg/L) combined with NAA (0.2 mg/L) for shoot formation. The combination of BAP (2mg/L) with NAA (0.2 mg/L) was recorded as optimum concentration yielding 2.6 shoots/explant .Gelling agents (agar, gelrite and agar + charcoal) have been investigated for root formation. Half strength of MS supplemented with 0.4 mg/L NAA was the best for root formation. An efficient in vitro regeneration protocol from culture has been established successfully for . The RAPD method was utilized to evaluate the genetic stability of pokeweed resulted from tissue culture. Eight primers used were able to amplify the DNA from all the plant materials. The amplified products of the RAPD profiles ranged from 100 to 530bp. A total of 167 bands were observed for two plant materials, using the eight primers. In conclusion, comparison of the bands with the mother plant revealed the monomorphic nature and true-to type clones.Genetic stability of the in vitro grown analyzed by RAPD markers ,indicated no somaclonal variation among the in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetically effects.

Key words: pokeweed, in vitro propagation, RAPD.

Introduction

Phytolacca Americana, commonly known as “pokeweed,” is an herbaceous growing up to 2 meters in height and belonging to the family . It is native to and has significant toxicity (Owen, 1988). Pokeweed reproduced only by seeds .The are perfect, radially symmetric, white or green (Owen, 1988) . The seeds have a long viability and can germinate after many years in the soil. All parts of the plant are toxic and pose risks to human and mammalian health (Owen. 1988 and Dasgupta, 2011). Although the plant is toxic, it is used as a traditional herbal medicine that can treat a wide range of maladies including mumps, arthritis and various skin conditions. The from the Phytolacca are one of the best sources for obtaining ribosome- inactivating proteins (RIPs). Pokeweed antiviral protein (PAP) is a ribosome inactivating protein produced by the plant Phytolacca Americana (Irvin, 1975 and Irvin and Uckun, 1992) also considered as anti-pathogenic protein toxins and broadly distributed throughout the kingdom of plants and fungi (Olsnes, 2004 and Domashevskiy and Goss, 2015). Also, pokeweed is used as an ornamental in horticulture, mainly for their attractive berries (Olsnes, 2004 and Domashevskiy and Goss, 2015).The plant and its tissue culture have been investigated as a source of saponins, betaline, mutagine and antivirual protein. Optimal conditions for germination of Pokeweed seeds have been investigated: alternating temperatures, light and nitrate are three factors of importance to obtain appreciable seed germination; soaking seeds in concentrated H2SO4 prior to sowing increases rates of germination (Edwards et al.,

Corresponding Author: Alaa M. El-Minisy, Plant Biotechnology Department, National Research Center, Dokki, Cairo, Egypt. E-mail:[email protected] 424 Middle East J. Agric. Res., 6(2): 424-432, 2017 ISSN: 2077-4605

1988). Micropropagation technique is essential to achieve rapid mass multiplication and distribution of improved plant as well as disease free plantlets of this most important plant. No micropropagation protocol was developed including molecular studies for pokeweed. The purpose of this study was to develop a micropropagation protocol for pokeweed using stem cutting and investigate the genetic stability of the micropropagated plants using RAPD analysis.

Materials and Methods

This investigation was carried out at the Department of Plant biotechnology, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Giza, Egypt.

Source of plant material:

Pokeweed seeds (phytolacca Americana) were obtained from the Leibniz institute of plant science and crop plant research (LPK), Gastersleben, Germany.

1. Micropropagation

Pokeweed micropropagation was performed using the following four stages procedures.

1.1. Establishment stage:

A. Sterilization of seeds:

Pokeweed seeds were treated with concentrated sulfuric acid for 10 min. then running water and sterilized distilled water. Seeds were sterilized by immersion in Clorox 10% (commercial bleach) for 10 min, subsequently washed six times in sterile distilled water, three minutes each.

B. Seed germination:

MS salts (Murashige & Skoog, 1962) supplemented with vitamins, 3% sucrose and 0.2% gelrite (germination medium). The pH was adjusted to 5.6 – 5.8 then autoclaved at 121º C and 1.5 lb for 20 min. Seeds were cultured in 300 ml glass jars containing 40 ml germinated medium for germination. Jars were incubated at 25 ± 2º C under a 16/8 h light/dark photoperiodic regime (1000 lux). One month later, seedlings were obtained and used as source materials for multiplication experiments.

1.2. Multiplication stage:

Stem cuttings were implanted on MS medium supplemented with vitamins (Murashige& Skoog, 1962), 3% sucrose,0.2% gelrite and different concentration of BAP(1,2,3 and 4 mg/L) and NAA(0.2 mg/L), Shoot length and shoot number were recorded after a month from culture.

1.3. Rooting stage:

Shootlets-derived in vitro from multiplication stage (2-3 cm) were individually excised and placed on half strength of MS medium contained vitamins, supplemented with 0.04 mg/L NAA and different types of gelling agent (i.e. gelrite 2 gm/L, agar 8 gm/L and agar 8 gm/L + charcoal 2gm/L). Cultures were incubated at 25±2º C under a 16/8 h light/dark photoperiodic regime (1000 lux), root length (cm) and number of roots per shootlet were recorded after one month.

1.4. Acclimatization: Plantlets resulting from the rooting stage were washed from medium residues then immerged in fungicide and transplanted into pots contained a mix of peat, perlite and sand (1:1:1). Pots were covered with plastic bags and kept in a growth room at 25± 2º C.

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One week later, the plastic bags were removed gradually. They were totally removed after 3-4 weeks before transplanting outdoors .Transplanted pots were kept in a growth room till new leaves grew and moved outdoor for planting in the open field till flowers and seeds were formed.

2. Molecular analysis

Genomic DNA isolation:

Genomic DNA was isolated from seeds using ZR plant/Seed DNA MiniPrep™ (D6020). Samples were placed in a sterile mortar containing liquid nitrogen and thoroughly crushed with a sterile pestle. The powdered leaves were subjected to DNA extraction.

RAPD-PCR analysis:

The PCR reactions were carried out in 20 µl volumes containing 100 ng of genomic DNA , 1.5 µl of each primer , 10 µl of Master mix,. A 100 bp DNA ladder (Fisher) was used as a standard with molecular sizes of 1000, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp. Eight random oligonucleotide (10 mer) primers (Operon technologies Inc., Alameda, California) were designed for use in RAPD analysis. The primers are as follow:

Table 1: RAPD primers code and their sequences. Primer code sequence OP-A01 CAGGCCCTTC OP-A02 TGCCGAGCTG OP-A05 AGGGGTCTTG OP-A11 CAATCGCCGT OP-A18 AGGTGACCGT OP-B15 GGAGGGTGTT OP-B18 CCACAGCAGT OP-C01 TTCGAGCCAG

PCR was performed using BIO-RAD T100™ thermal cycler. PCR conditions included 95 ºC /3 min (1 cycle) , 95 ºC / 30 sec, 36 ºC / 30 sec, 72 ºC /1min ( 34 cycle ) and 72 ºC/10 min (1 cycle ), then held at 4ºC. The amplification products of PCR were size-separated by gel electrophoresis in 1.5% agarose gel with 1 x TBE buffer using submarine gel electrophoresis apparatus and stained with0.5 µg/mL ethidium bromide and visualized with Gel documentation and photographed.

Data analysis

RAPD-PCR-amplified fragments were scored as present (1) or absent (0). Only clear and major bands were scored (Collard, Mackill, 2009). Pairwise comparisons between materials, based on the proportion of shared bands produced by the primers used, were calculated manually.

Results and Discussion

1. Micropropagation

1.1. Establishment stage:

To obtain high rate of seed germination, Seeds of pokeweed were treated with concentrated sulfuric acid for 10 min. (Edwards et al., 1988) before sterilization with 10 % Clorox for 10 min. which gave the best result with no contamination. Sterilized treated seeds were cultured on MS basal medium without growth regulators. Germination began after 7 days and reached its maximum at four weeks. After month, seedling 5 cm in height has been obtained and used as a source of explant. Regarding to seed germination the phenomenon of autotoxicity may be more widely spread that

426 Middle East J. Agric. Res., 6(2): 424-432, 2017 ISSN: 2077-4605 currently suspected. This study confirmed the optimum condition for laboratory germination of seeds by soaking seeds in acid solution (concentrated H2SO4) to increase the rate of germination, as mentioned by Edwards et al. (1988).(Fig.1.A).

1.2. Multiplication stage:

Stem cuttings were transferred to MS basal media with different concentrations (1, 2, 3 and 4 mg/L) of BAP combined with 0.2 mg/L of NAA. The dose of cytokinin (BAP) is known to be critical for the induction of multiple shoots. Among the BAP concentrations, BAP at 2 mg/L was found to be more effective in producing a greater number of shoots / explant.The optimal culture for induction of clustered shoots were NAA (0.2 mg/L) + BAP (2 mg/L) after four weeks (Fig.1-B).It was observed that callus like structures formed at the base of the shoot during shoot multiplication. Our findings are shown in Table (2) that the stem cuttings respond after two weeks and the response included number of shoots / explant and the shoot height. After being culture on multiplication medium for 4 weeks the number of shoots resulted and its height were varied at different BAP concentration. Among the interaction of the different BAP concentration on the growth of the explant (Table 2) revealed that BAP at 2 mg/L gave the higher number of shoots/explant (2.6 ± 0.17 and 2.6 ± 0.26) than 3-4 mg/L of BAP. Regarding to the shoot height, the same trend was obtained and gave shoots higher (2.9cm ± 0.17 and 2.9 cm ± 0.1) than the other concentration (3or 4 mg/L). Growth and morphogenesis in vitro are regulated by the interaction and balance between the growth regulators supplied within a medium and the growth substances induced endogenously (Hartmann et al., 2002). Benzyl amino purine (BAP) is the best cytokinin growth regulator used for shoot induction in plant tissue culture (Singh and Sing, 2007; Sugla et al., 2007). In the respect of Zou L et al., (2008) mentioned that the optimal culture medium for callus induction was MS+NAA (0.2 mg/L) +BAP (2 mg /L),but GAO Li-chen et al. (2010) suggested that BAP (1 mg/L), GA3 (1 mg/L), LH (300 mg/L), Sugar (30 g/L), Agar (9 g/L) gave best result for shoot multiplication and and Wu H et al., 2013 (2013) observed that the optimal explant for callus induction and bud differentiation were stem sections. Finally, our findings indicated that the protocol of mass propagation of pokeweed via tissue culture are promoting, alternative for normal propagation and solving the seed germination problem.

Table 2: Effect of BAP concentration on the multiplication rate and shoot length per explant of pokeweed BAP concentration (mg/L) Height (cm) No. of shoots /explant 1 2.9±0.17 2.6±0.17 2 2.9±0.1 2.6±0.53 3 1.2±0.17 2.2±0.17 4 2.1±0.1 2.0±0

1.3. Rooting stage

Root formation was observed after 1-2 week and the optimum root number and root length are recorded after one month (Table-3). Gelling agent is one of the medium component that affects the rooting culture response.Variation in rooting response is observed with the different gelling agent. This investigation described the effect of three gelling agent on in vitro derived shoots cultures of pokeweed .the results shown in Table (3), indicated that the shootlet produced higher number of roots in case of gelrite than the other agents used (24 ± 3.6), but it was (7.3 ± 5.7) and (5 ± 1) in medium contained agar and agar plus charcoal, respectively. However the root length is approximately are equal in both of gelrite and agar (1 cm ± 0.1). In case of agar and charcoal it was shorter (0.6 ± 0.17).It can be concluded that the best response of rooting observed at gelrite medium (Table 3) (Fig.1- C). Unfortunately, the gelling agent is a major component that could significantly affect the performance of tissue culture media; thus, its inclusion must consider both quantitative and qualitative requirements.

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The search for an economical agar substitute with essentially the same gelling characteristics, particularly thermal reversibility, has led to a bacterial gellan gum, gelrite, now purchasable from retail suppliers by diverse trade names (e.g., Phytagel). It has become the preferred gelling agent of many plant tissue culture media because of its high purity and consistent quality, with one grade satisfying a variety of needs and substantially smaller quantities producing gels of hardness comparable to agar. Nevertheless, in spite of the advantages, gelrite has not supplanted agar as the universally employed gelling agent. This is partly because many potential users remain unaware of its existence. Some choose not to use it because gels of desired firmness have not been attainable by following the manufacturer's instructions. The recommended rate of 0.2% has resulted in softer or harder gels, depending on the basal nutrient formulation. And there are those that have been discouraged by reports that gelrite causes or enhances vitrification, or hyperhydricity, of regenerating shoots (Pasqualetto et al., 1986; Zimmerman and Cobb, 1989). Our studies are in harmony with that obtained by both of Zou L et al., 2008 and Wu H, 2013, showed that the optimal culture medium for rooting was half strength of MS with NAA (0.4 mg/L). An addition of an auxin, mainly IAA, IBA and NAA to the medium or free growth regulators was essential to induce rooting in the regenerated shoots (Hartmann et al., 2002). Daffalla et al. (2011) reported that roots may require a less concentration of auxin to grow, but root growth is strongly inhibited by its higher level because at this level, auxin induces the production of ethylene, a root growth inhibitor.

Table 3: Rooting after one month Gelling agent No. of roots Length of roots (cm) Gelrite 24 0.9 Agar 7 1 Agar & charcoal 5 0.6

1.4. Acclimatization stage:

The best plantlets from rooting stage (after four weeks) were transplanted for acclimatization. Mix of peat, perlite and sand (1:1:1) were used and most plantlets were survived and after month they were ready to be transferred to soil (Fig.1-D).

2. RAPD analysis

In this study, seeds derived from in vitro plantlets and from mother plants puwere used as plant materials. The RAPD pattern of the seeds derived from in vitro plantlets was compared with the seeds of the mother plant to confirm the homogeneity of the in vitro plantlets. Genomic DNA of both materials were extracted and compared by RAPD-PCR, using random oligonucleotide primers. Eight primers (OP-A01, OP-A02, OP-A05, OP-A11, OP-A18, OP-B15, OP-B18 and OP-C01) successfully yielded 167 bands across the plant material tested. All primers showed high efficiency in generating successive monomorphic bands. Size ranged from 100-530 bp depending on the primer and plant material. Pair wise differences between the two materials detected and analyzed using computer, intensive bands were considered as present (1), while weak or absent bands were considered as absent (0).The use of RAPD marker, which amplify different regions of the genome, allows better chances for the identification of genetic variations in the samples (Martins et al. 2004). Table (4) and figure (2) show the reaction of eight primers (OP-A01, OP-A02, OP-A05, OP-A11, OP-A18, OP-B15, OP-B18 and OP-C01) with the two material and summarizes the relevant results of eight primers in pokeweed. Most of the obtained bands from in vitro derived seeds in the primers used are confirmed to be present in the fingerprints of ex vitro derived seeds and the percentage of polymorphism of 4.8 %.It means in all cases that the RAPD fingerprints produced with different primers were almost identical. The results of the present finding are in agreement with Castiglione et al. (1993), who found no RAPD fingerprint variation when different plants of the same clone of popular species. More importantly, it is generally considered to be an in vitro culture system with low risk of genetic instability (pierik,1991 ; Schoofs,1992), because the organized meristems are generally

428 Middle East J. Agric. Res., 6(2): 424-432, 2017 ISSN: 2077-4605 more resistant to genetic changes that might occur during cell division or differentiation under in vitro conditions (Shenoy and vasil, 1992).

Fig.1: Micropropagation protocol of Pokeweed. (A)Establishment stage (MS salts+ vitamins). (B)Multiplication stage (2 mg/L BAP +0.2 mg/L NAA). (C)Rooting stage (half strength of MS with NAA (0.4 mg/L). (D)Plantlet acclimatization and outdoor growth.

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Table 4: Primers with arbitrary sequence tested for their effectiveness in the RAPD –PCR analysis that produced polymorphic band of pokeweed. Primers Number of bands in the gel Size of polymorphic % of polymorphic Total Polymorphic bands(bp) OP-A01 16 0 -- 0 OP-A02 18 0 -- 0 OP-A05 15 1 280 6.7 OP-A11 15 1 160 6.7 OP-A18 32 2 200,300 6.3 OP-B15 28 0 -- 0 OP-B18 20 3 290,190,530 15 OP-C01 23 1 100 4.3 Total 167 8 4.58

Fig. 2: RAPD profiles using the ten primers [lane N.C negative control, lane E: Ex-vitro seeds; lane I: in-vitro seeds, M 1 kb marker (Fisher)].

Notwithstanding this consideration, there are numerous reports on the incidence of somaclonal variation among micropropagated plants. For example, reports have indicated the occurrence of somaclonal variation in micropropagated bananas and plantains (Shoofs, 1992) raised through meristem culture. Various kinds of chlorosis coupled with multiple apexing and dwarfing in strawberry (Martinelli, 1992) more jagged and pubescent leaves in grapevine (Grenan, 1992),spininess and albino strips in pineapple (Moore et al.,1992).Also, El-Sawy (2007) found that mass propagation via tissue cultures produced clones genetically similar to the mother plants, he also found that RAPD approach is convenient, fast and reproducible to detect the presence of genetic variation associated with tissue culture of strawberry.

Conclusion

RAPD analysis of the micropropagated pokeweed showed 95 % genetic similarity to the mother plant by applying eight random primers. Since the RAPD technique is considered as a powerful molecular technique .Hence, it can be concluded that the micropropagation protocol developed in this study is suitable for micropropagation and in the genetic transformation studies of pokeweed.

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