Development of Shoot Cultures from Leaf Explant of Portulaca quadrifida

Portulaca quadrifida (Portulacaceae) is an annual succulent herb having medicinal value and is consumed as a vegetable or salads in India. In the present study, leaf explants were inoculated on Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and combinations of N-benzyladenine (6-BA) and kinetin (KIN) individually and in combination with 1naphtalene acetic acid (NAA). Rapid regeneration was observed in medium fortified with combinations of 6-BA (8 μM) and NAA (1 μM) which formed 19.40 ± 0.64 shoots with 100% response. Variation in sucrose concentrations (4-6%) was tried but it failed to increase the shoot number. When the optimized medium was fortified with different carbon sources viz. dextrose, glucose and maltose, they could not evoked better response and sucrose proved to be more effective for regeneration. Rooting of in vitro shoots was achieved in 1⁄2MS + sucrose (1%) + indole-3-butyric acid (IBA, 2 μM).


Introduction
Portulaca quadrifida L. commonly called 'looni' which belongs to the family Portulacaceae is a small diffused, succulent, annual herb found throughout the tropical parts of India.It emits a mild pleasant smell and is used in salads or consumed as a cooked vegetable.In India, boiled leaves of the plant are mixed with sorghum or pearl millet flour in preparing a kind of bread.The plant has medicinal properties and is used to cure asthma, cough, urinary discharge, inflammation, ulcers, abdominal complaints, erysipelas and hemorrhoids (Kirthikar and Basu, 2001;Mulla and Swamy, 2010).It also has neuropharmacological effects and antifungal activities against Aspergillus fumigates and Candida albicans (Hoffman et al., 2004;Syed et al., 2010).Due to its nutritional and medicinal importance it was taken up for regeneration studies.
Leaves are commonly used explant for efficient shoot regeneration in many important medicinal plants (Joshi and Padhya, 2010;Joshi et al., 2010;Pathak and Joshi, 2017) and is also suitable for genetic transformation and protoplast fusion (Kumar et al., 2010;Xu et al., 2013).However the regeneration of plant under in vitro conditions depends on many factors, like genotype, explant, medium composition, growth regulators, gelling agent, light intensity and quality, photoperiod, temperature and vessels (Reed, 1999).The cultures developed under in vitro conditions need external carbon sources, which help in proliferation and differentiation of shoots (Kozai, 1991;Gurel and Gulsen, 1998;Nowak et al., 2004).
The aim of the present study was to optimize the medium for shoot regeneration fortified with a suitable carbon source using leaf explants of P. quadrifida.

Plant material and surface sterilization
Young healthy twigs of Portulaca quadrifida L. were collected from the Botanical Garden of the M.S.University of Baroda.Pieces of stem containing two to three nodes were kept in running tap water for 1 h, and then washed with labolene (Fisher Scientific, India) for 5 min.Surface sterilization was done with bavistin solution (0.01%) followed by HgCl2 (Merck, India) solution (0.1%) for 3 min each.Entire leaves were excised and placed abaxially on medium.
Culture medium for shoot regeneration Murashige and Skoog (MS, 1962), medium fortified with sucrose (SRL, Mumbai, India) (3%) served as a control for the study.This medium was supplemented with 6-BA and KIN (SRL, Mumbai, India) individually (5 and 10 µM) and in the range of 0.5-10 µM with NAA (0.5 and 1 µM) (SRL, Mumbai, India).The pH of the medium was adjusted at 5.80 using NaOH/HCl (1 N) and agar (SRL, Mumbai, India) (0.8%) was used as the solidifying agent.Media sterilization was done in autoclave at 121 ºC (15 psi) regeneration of 19.40 ± 0.64 shoots was observed in medium fortified with 6-BA (8 µM) + NAA (0.5 µM) with 100% response within eight weeks (Table 1).Further increase in 6-BA concentration to 10 µM with NAA (0.5 µM) induced same morphogenic response, but number of shoots decreased to 16.70 ± 0.53.Increasing the NAA concentration to 1 µM with same 6-BA concentrations (0.5-10 µM) resulted in similar response and higher concentration of 6-BA (8 and 10 µM) formed nodular callus which further differentiated 15.10 ± 0.81 and 13.30 ± 0.53 shoots respectively (Table 1).Three week old nodular callus was fixed in FAA and the anatomy showed large number of meristemoids.These were round structures with isodimetric cells containing dense cytoplasm and prominent nucleus which were darkly stained and the differentiating shoot buds as leaf primordia (Fig. 1F).When 6-BA was replaced by KIN in combination with NAA, it induced callus which differentiated roots.This callus ceased to proliferate and even after transferring to a fresh new medium, it only increased the length and number of roots till eight week.
There are mainly two type of organogenesis i.e. direct and indirect, and the ratio of cytokinin to auxin in the medium is a key factor behind this (Skoog and Miller, 1957).In the present study, leaf explants of P. quadrifida differentiated callus with different morphology in presence of 6-BA and NAA.Similar results are reported for Suaeda nudiflora (Singh et al., 2004), Gomphrena globosa (Jack et al., 2005) and Hemidesmus indicus (Pathak and Joshi, 2017) where BAP and NAA influenced callus development along with variation in its morphology.In P. quadrifida lower concentrations of 6-BA was able to induce only callus whereas in higher concentration differentiated shoots.Similarly high cytokinin level was responsible for organogenesis from leaf explant whereas lower level formed only callus in leaf explant of Hemidesmus indicus (Pathak and Joshi, 2017).Optimum regeneration was achieved in MS medium fortified with 6-BA and NAA, and in leaf explants of Astragalus adsurgens (Luo and Jia, 1998), Beta vulgaris (Yildiz et al., 2007) and Astragalus nezaketae (Erisen et al., 2010) maximum shoots regenerated in medium having 6-BA + NAA.When KIN was supplemented in the medium, it suppressed callus formation, as well as shoots buds, and instead regenerated roots in all the combinations.Similarly in Hemidesmus indicus (Pathak and Joshi, 2017) the combination of KIN with NAA evoked less callus formation as compared to combinations of 6-BA with NAA.However contrary result was observed in leaf explants of Suaeda nudiflora (Singh et al., 2004) where medium fortified with Kn and NAA produced multiple shoots.Bhojwani and Razdan (1996) reported that the ratio between both the types of PGRs regulates the type of organ differentiation in vitro.In P. quadrifida leaf explants adopted an indirect mode of organogenesis which can become a source of somaclonal variants which enrich the genetic diversity (Patel et al., 2014).

Effect of sucrose concentration on shoot regeneration
Optimum shoot regeneration was achieved in MS medium supplemented with sucrose (3%) and 6-BA (8 µM) along with NAA (0.5 µM).The effect of higher sucrose concentrations (4-6%) on regeneration was also evaluated.

Effect of carbon source variation on shoot regeneration
As sucrose at 3% concentration was considered as control, it was replaced by other carbon sources like dextose, maltose and glucose at 3% concentration and their effect on shoot regeneration was evaluated.

Rooting stage
Eight weeks old shoots (> 3 cm) were cultured into ½MS liquid medium supplemented with sucrose (1%) and IBA (2 µM).Leaves from lower nodes of the shoots were excised and they were transferred to tubes containing a filter paper bridge, with the arms of bridge dipping in liquid medium (20 ml).Single shoot was inserted in the center of the bridge and the last 2-3 nodes were dipped in the medium.The tubes were kept in the culture room and lower portion of the tube was covered with aluminium foil till the root induction started.

Statistical analysis
All experiments were carried out in ten replicates, mean and standard error for each combination was calculated.The means were analyzed using ANOVA (α = 0.05) and significant means were further analyzed by Dunnett's test using Graph Pad Prism 6.01.

Results and Discussion
Establishment of shoot cultures from leaf explant Entire leaves of P. quadrifida when inoculated on basal MS medium as well as on medium with 6-BA and KIN individually, resulted in only swelling and failed to induce any morphogenic response.Synergistic combinations of 6-BA and NAA when tried, the explants swelled and friable callus differentiated in all the combinations within a week.Lower concentrations of 6-BA (0.5, 1, 2 and 4 µM) with NAA (0.5-1 µM) failed to regenerate shoots, whereas indirect organogenesis was observed in higher concentrations i.e. 8 and 10 µM of 6-BA (Table 1).In medium fortified with 6-BA (8 µM) and NAA (0.5 µM) callus formation was observed at cut end which proliferated, turned nodular with simultaneous appearance of pink protuberances at the end of second week (Fig. 1A).They formed shoot buds by the end of fourth week (Fig. 1B) and after subculture they proliferated along with elongation into shoots during sixth week (Fig. 1C).This proliferation and elongation of shoots continued till eight week and at the end healthy shoots were observed (Fig. 1D).Optimum

Effect of sucrose concentration on shoot regeneration
Sucrose is known to be a potent source of carbohydrate, which affects the regenerative potency of the explant.Hence optimized medium [6-BA (8 µM) and NAA (0.5 µM)] was fortified with different concentrations of sucrose (4-6%) and its effect on shoot regeneration was observed.Increasing the level of sucrose to 4% in medium evoked an early response and within three week callus started to differentiate shoot buds.An increase in shoot buds was observed however at the end of eight weeks 17.56 ± 0.58 shoots were observed in 100% cultures (Table 2).Increase in sucrose concentration to 5% and 6% induced the leaf explants to differentiate shoot buds, but number of shoots and % response was less in comparison to 3% sucrose.A total of 12.34 ± 0.84 number of shoots were formed in medium fortified with 5% sucrose (75% response) and 9.12 Table 1.Effect of BA (0.5-10 µM) with NAA (0.5-1 µM) on shoot regeneration from leaf explants of P. quadrifida (eight weeks)  ± 0.23 in presence of 6% sucrose (50 % response) (Table 2).
Generally sucrose has been used as a major carbon source which helps in maintaining the osmotic potential of culture and permits the absorption of mineral nutrients present in the medium.This is prerequisite for optimal proliferation, however it may vary from culture to culture (Nowak et al., 2004;Siwach et al., 2011).In shoot cultures of Paederia foetida (Amin et al., 2003) and Elaecarpus robustus (Rahman et al., 2004), sucrose at 3% concentration evoked optimum growth and development of shoots.Gubiš et al. (2005) also tried different concentrations of sucrose (1-3%) in tomato and observed that sucrose at 3% concentration regenerated maximum shoots.Naik et al. (2010) reported that lower sucrose (2% and 3%) were better for shoot regeneration whereas further increase in sucrose concentration adversely affected shoot regeneration in Bacopa monnieri.This may be due to the growth suppression by osmotic stress of high concentration of sucrose, as sugars are perceived by cells as chemical signals and their high concentrations in vitro acting as stressing agents (Steinitz, 1999;da Silva, 2004).Charriere and Hahne (1998) observed the direct influence of sucrose concentration on uptake of 6-BAP into the sunflower explants, and it modified the endogenous auxins and cytokinin level which in turn triggered organogenic or embryogenic response.Nowak et al. (2004) reported that the sucrose in the medium, not only acts as a carbon source but also as an osmotic, which was also supported by findings of Karim et al. (2007) in Araria elata.

Effect of carbon source variation on shoot regeneration
Sucrose (3%) regenerated optimum shoots and hence different carbon sources were used to evaluate their effect on shoot regeneration.When dextrose was supplemented in the medium, it evoked similar response as sucrose but resulted in inducing less number of shoots and was able to regenerate only 15.38 ± 0.82 shoots within eight weeks (Table 3).Presence of maltose in the medium also showed a poor response and only 11.70 ± 0.28 vitrified shoots were formed.Whereas the explants when inoculated on medium fortified with glucose as a carbon source it failed to induce any type of morphogenic response (Table 3).
Organic nutrition mostly in the form of sugars is the source of carbon and energy for growth of explants in culture media and its type as well as quantity plays a significant role in organogenesis.Earlier studies have reported that the sucrose is one of the important factors which controls induction and growth of shoots in vitro (Gurel and Gulsen, 1998;Gibson, 2000).Although sucrose has been the carbohydrate of choice in the majority of work on regeneration in different species, sometimes it is ineffective as the response is genotype dependent (Thompson et al., 1987;Cuenca et al., 2000).Results of variation in carbon source have depicted that sucrose is superior in comparison to other carbon sources as it differentiates healthy shoots from leaf explants.Gubis et al. (2005) used sucrose, glucose and maltose in the medium for tomato regeneration and observed that sucrose regenerated optimum shoots.The shoots regenerated in maltose supplemented medium showed vitrification and similar report by Bahmani et al. (2009) for apple in which the concentration of carbon source affected regeneration frequency and resulted in hyperhydricity of shoots in all the carbon sources.Cuenca and Vieitez (2000) reported that medium fortified with fructose showed vitrification symptoms in shoot cultures of Fagus species.Pasqualetto et al. (1988) has also reported that concentrations of carbon source and gelling agent influenced the vitrification in apple cultivars in vitro.Regeneration frequency and number of shoots varied between the carbon sources and this may be due to the complex interplay of sugar and hormone signalling in plant growth and development (Leon and Sheen, 2003).

Rooting stage
Generally in vitro rooting is affected by salts of the medium and reduced strength of MS medium (half or quarter strength) has been proved to be beneficial for rooting (Murashige, 1979;Skirvin and Chu, 1979;Garland and Stoltz, 1981;Zimmerman and Broome, 1981).In vitro shoots were excised and transferred to ½MS liquid medium supplemented with sucrose (1%) and IBA (2 µM).Within a week of inoculation, there was increase in shoot length.During second week, root formation was observed from the nodes and its length increased till four weeks (Fig. 1F).IBA has been used for rooting of many medicinal plants like Bacopa monnieri (Joshi et al., 2010) and Hemidesmus indicus (Pathak and Joshi, 2017).Similar reports were documented for Schinopsis balansae (Pedro et al., 2003), Bambusa balcooa (Das and Pal, 2005) and Pappea capensis (Mng'omba et al., 2007) where rooting was achieved in half strength MS medium supplemented with IBA.

Table 2 .
Effect of sucrose variation on shoot regeneration from P. quadrifida leaf explant (eight weeks) Means (n = 10) were subjected to ANOVA and significant means (*) were analyzed using Dunnett's test in comparison to control i.e. medium with 3% sucrose concentration (p < 0.05). a

Table 3 .
Effect of different carbon source on shoot regeneration from P. quadrifida leaf explant (eight weeks) a Means (n = 10) were subjected to ANOVA and significant means (*) were analyzed using Dunnett's test in comparison to control i.e. medium having sucrose (p < 0.05).