Influencing in vitro clonal propagation of Chonemorpha fragrans(moon)Alston by culture media strength,plant growth regulators,carbon source and photo periodic incubation

Tasiu Isah·Shahid Umar

Abstract Chonemorpha fragrans is an endangered medicinal woody climber, regarded among alternative plant sources of camptothecin. Camptothecin is a monoterpene indole anti-cancer alkaloid with annual trade value of over three billion U.S.dollars in the recent,and is used in the production of its analog drugs approved for the chemotherapy of cancer of varied types.Effects of plant growth regulators,culture media strength and photoperiodic duration on the micropropagation efficiency of C.fragrans from nodal segment explants were studied on Murashige and Skoog (MS) medium amended with Thidiazuron(TDZ),Benzylaminopurine(BAP)or Kinetin(Kin).Thidiazuron was more efficient over BAP and Kin when half basal MS medium was used over full or quarter strength. Results of carbon source experiment showed sucrose as the most effective over glucose,fructose,and maltose in the clonal production.Studies on the photoperiodic incubation duration showed 12 h as the best light period and sub or supra-optimal resulted in the production of abnormal and albino micro shoots.Experimental results on the evaluation of physiological,biochemical parameters showed the role of pigment molecules and antioxidant systems in the production of albino micro shoots.

Keywords Micropropagation·Micro shoot·Plantlets·Chonemorpha fragrans·Plant physiology·Albino shoot·Basal callus·PGRs(plant growth regulators)·Antioxidants

Introduction

Chonemorpha fragrans Syn.C.grandiflora is an endangered medicinal plant of the family Apocynaceae and produces many phytochemicals,including chonemorphine,taraxasterol and b-sitosterol ascribed bioactivities shown by extracts isolated from the species(Rastogi and Mehrotra 1993;Khan et al.2005).The woody climber is regarded an alternative natural source of Camptothecin(CPT),the third most important cytotoxic quinoline alkaloid from which many analog drugs used in the chemotherapy of cancer were developed(Kulkarni et al.2010;Kai et al.2015;Isah 2016).Annual international trade value of the alkaloid was in the recently reported to have exceeded three billion U.S.dollars but, the supply relies upon plant sources(Sankar 2010).Seeds can be used to achieve clonal propagation of C.fragrans.However,slow growth and poor germination capacity are an impediment to efficient and timely production of its clones to meet the phytochemical needs of pharmaceutical industry(Kulkarni et al.2010;Ali et al.2016).Low rooting efficiency hampers its vegetative propagation by stem cuttings.Therefore,in vitro clonal propagation offers an alternative strategy for rapid clonal multiplication with application in efficient production of the clones(Madke et al.2014;Lalita et al.2016)for isolation of the anticancer alkaloid CPT to meet demand of the pharmaceutical industry.

In vitro growth of plants is affected by many factors that include explant type and condition,the culture media and strength,carbon source and concentration amended,and photoperiodic incubation(Perveen et al.2011;Wang and Yao 2017;Bhadrawale et al.2017).Adjusting physiological,nutritional,metabolism and genomic expression systems constitutes the essential needs for an efficient in vitro performance,and to achieve growth and clonal production in the in vitro cultures(Isah 2015).Explant condition determined by the stage of development and physiology have some impact on the in vitro clonal propagation.Added carbon sources into the culture media provide energy that plays a role in osmotic potential maintenance of cultures(Isah 2015).The type and concentration of an added carbon source influences in vitro performance,based on parameters evaluated in the in vitro cultures(Lipavska and Konradova 2004). Culture media strength, Plant Growth Regulators(PGRs)supplemented and photoperiodic duration also influence micropropagation efficiency of in vitro cultivated plants,and ability to overcome recurrent morpho-physiological anomalies in regenerates is determined by the degree of adjustments plantlets can achieve in the cultures(Madke et al.2014;Isah 2015).Environmetal condition of the in vitro cultures incubation that includes temperature, light, medium strength, composition and conditions play some roles in micropropagation efficiency and frequency of albino shoot formation.Genetic factors are also ascribed important and determinant role on albinism occurrence in shoot cultures,and in number of plants,it had been proved to be a recessive trait governed by many loci(Kumari et al.2009).Keeping this into consideration,this study evaluates the effect of explant conditioning,carbon source amended in the culture media,plant growth regulators and photoperiodic incubation on the in vitro clonal propagation of C.fragrans.Biochemical,physiological differences between the leaves of albino and normal micro shoots obtained in the study were also evaluated.

Materials and methods

Seed collection,cultures establishment and explants pre-treatment

Authenticated mature seed samples of C.fragrans were procured from Indian Institute of Horticultural Research(IIHT)Bangalore,India during March 2013.The seeds were immersed in water or Thidiazuron (TDZ)(1.0 mg L-1)solution for 1-2 h to soften seed coat and influence in vitro seed germination while control seeds were not given any treatment before kept in cetrimide solution for 20 min.Seeds were subjected to a jet of running tap water for 30 min and later surface sterilized under laminar airflow hood chamber using 70%ethanol for about 5 min.This was followed by rinses with autoclaved distilled water 2-3 times.Seeds were further treated with freshly prepared 0.1% mercuric chloride solution for 2-3 min followed by rinses with sterile distilled water 2-3 times and allowed to partially dry up to avoid possible cultures contamination.Surface sterilized seeds were given two early seedling recovery treatments that involved slanted cut of the seed coat to expose the radicular end of the seed embryo explants to the culture media or aseptic excision of seed embryo axes or on the alternative,inoculation of the surface sterilized whole seeds(control).Nodal explants were excised from 4-5 weeks old axenic seedlings for the in vitro studies.The excised explants were cultured or subjected to pre-treatment with autoclaved TDZ(1.0 mg L-1)solution by dipping for few seconds.This was followed by a blot of the adherent solution using autoclaved Whatman filter paper before their inoculation on solidified culture media.

The experiment was carried out using Murashige and Skoog(1962)medium(MS)containing carbon sources(as specified in the results)and solidified with 0.8%Agar(Agar Agar Microbiology Mumbai,India).Media pH was adjusted to 5.6-5.8 using 1N NaOH or HCl before autoclaved at 121°C for 21 min.The cultures were maintained under 12 h day/light period provided by cool fluorescent tubes(Phillips India)having photon flux density of 40 W,50 μmol m-2s-1(except otherwise clarified in set experimental results)and culture room condition of 25±2°C temperatures with a relative humidity of 50-70%.For the variable experimental light durations,cultures were maintained under 12,18,16,8,3 h light/dark or complete 24 h light or dark duration incubation.

Evaluation of seed germination

Seed germination capacity was evaluated according to the earlier description(Paul 1972).It was defined as the percentage germinability of seed sample(number)used for which germination was recorded after 5,10 and 15 days by the inter-seminal growth that led to the emergence of one live embryo and expressed using the formula:

Evaluation of chlorophyll,carotenoids and cellular osmolytes contents

Total chlorophyll contents, chlorophylls a and b, carotenoids content in the leaves of albino and normal shoot cultures were evaluated as reported by Arnon(1949)and their concentration calculated according to the description given by Wellburn(1994).Contents of glycine betaine in the leaves were measured by the periodic calorimetric method described by Grieve and Grattan (1983) and modified by Lokhande et al.(2010).Proline contents were estimated according to the procedure described by Bates et al. (1973) while total soluble sugars (TSS) using anthrone method(Watanabe et al.2000)as modified by Lokhande et al.(2010).

Lipid peroxidation

Lipid peroxidation in the leaves of albino shoots over the normal ones was measured in terms of Malondialdehyde(MDA)contents as described previously(Heath and Packer 1968)and slightly modified by Lokhande et al.(2011).Contents of generated hydrogen peroxide in the leave of the shoots were evaluated by the method described by Alexieva et al.(2001).Briefly,500 mg of leaves samples(each)were homogenized in 10 ml trichloroacetic acid(0.5%w/v)in an ice bath.It was followed by centrifugation at 10,000 rpm for 10 min at 4°C.Hydrogen peroxide(H2O2) contents determination were performed using 0.5 ml of the supernatant by mixing with 0.5 ml of potassium phosphate buffer(KPO4)(100 mM,pH 7.0)and 1 ml freshly prepared potassium iodide(1 M),and the reaction mixture incubated in the dark condition for about 1 h.The absorbance of this mixture was measured at 390 nm after the incubation.The amount of the H2O2accumulated by the leaf tissues of normal and albino shoots was calculated from a standard curve that was prepared using known concentration of H2O2.

Antioxidant enzymes activities

Normal and albino fresh leaves samples were harvested from the shoot cultures(500 mg)and homogenized in 5 ml of ice-cold sodium phosphate buffer(50 mM,pH 7.0)that contained EDTA(0.1 mM)and polyvinylpyrrolidone(1%w/v)using chilled mortar and pestle.The homogenates were filtered using single-layered cheese cloth.It was followed by centrifugation at 10,000 rpm for 20 min at 4°C.An appropriate aliquot of the obtained supernatant was used as crude enzyme(s)for the antioxidant enzyme activities assay.Soluble protein contents in the enzyme extract were determined according to reported procedure by Lowry et al.(1951),by the use of bovine serum albumin as standard.Activities of Superoxide Dismutase(SOD)were assayed according to reported procedure by Becana et al.(1986)through monitoring the inhibition of photochemical reduction of Nitro Blue Tetrazolium(NBT).The 1 ml reaction mixture contained phosphate buffer(50 mM,pH 7.0)and EDTA(0.1 mM)to which oxygen-generating system that contained methionine (14.3 mM), NBT(82.5 μM)and riboflavin(2.2 μM)were in situ added freshly.Reactions were initiated by the addition of 25 ml of crude enzyme and the entire system kept about 30 cm below light source(six 15 W fluorescent tube light)for 30 min.The reactions were terminated by switching offthe tube light.For the light blank,all reactants without the enzyme extracts were incubated in the light while reactants along with 25 μl enzyme extract were incubated in dark condition for the dark blank.Reductions in NBT were measured through monitoring change in the absorbance at 560 nm.SOD activities were expressed as μKat of SOD activity mg-1protein. Catalase (CAT) activities were measured by the decomposition of H2O2as described by Cakmak and Marschner(1992)with slight modifications.The activity was measured in 1 ml reaction mixture that contained phosphate buffer(50 mM,pH 7.0)and H2O2(300 mM).The reaction was initiated by the addition of 50 μl enzyme extract and determined as a result of H2O2decomposition by monitoring decrease in its absorbance at 240 nm(e=36 mM-1cm-1)for 2 min at an interval of 15 s.Slope of the obtained readings between time intervals was considered as DA and enzyme activity expressed as μKat of CAT activity mg-1protein.Ascorbate Peroxidase(APX)activity was determined as reported by Nakano and Asada (1981). The reaction mixture (1 ml) contained phosphate buffer(50 mM,pH 7.0),ascorbate(0.5 mM)and H2O2(0.1 mM).Reaction was started by the addition of 50 μl crude enzyme.Ascorbate oxidation was monitored for 1 min through measurement of the decrease in absorbance at 290 nm at every 15 s(e=2.8 mM-1cm-1).The enzyme activity was expressed as μKat of the APX activity mg-1protein. Guaiacol Peroxidase (GPX) assay was assayed according to the Hemeda and Klein(1990)procedure.The 1 ml reaction mixture contained phosphate buffer(50 mM,pH 7.0),guaiacol,H2O2(200 mM)and 10 μl enzyme extract.The reaction was started by the addition of H2O2(200 mM).Increase in the absorbance due to the oxidation of guaiacol(e=26.6 mM-1cm-1)was monitored at 470 nm. The enzyme activity was expressed as μKat mg-1proteins.

Experimental design and statistical analysis

The experiment was arranged in a randomized complete block design(where applicable)and carried out thrice.Collected data for each set of the separate experiments were analyzed using SPSS ver 21(U.S.A.).Significant difference between treatments was assessed by Analysis of variance(ANOVA)followed by Tukey's range test and obtained results expressed as mean±standard error of the replicated experiments.

Results and discussion

In vitro studies of C.fragrans

Seed germination

In the seeds of higher plants,seed germination involves emergence of a seedling from encased embryo,and is influenced by internal and external conditions(Peter et al.2005).It is the reactivation of metabolic machinery that leads to the utilization of seed-stored food reserves,followed by radicle and plumule emergence for establishment of complete seedling. Success of the germination is determined by the number of germinated seeds into seedling at a given moment.To study the effect of pre-germination treatments on seedling recovery from the obtained seeds,surface sterilized seeds were subjected to treatment in water or TDZ(1.0 mg L-1)solution and control.This resulted in differential germination capacity after 5,10 and 15 days incubation on half basal or full strength MS medium(Table 1).Maximum germination percentage was shown by seeds subjected to treatment with TDZ over the water with 98%germination to 79%after 15 days and compared to the 29%in control seed cultures,suggesting efficiency of water and TDZ treatments in promoting in vitro seed germination capacity. The germination capacity,defined as the emergence of embryonal radicle,was better when half strength MS medium was used(98%to 93%)over the full strength(79%to 72%),implying role of media nutritional strength on influencing seed germination in C.fragrans(Table 1).Although treatment with water alone promoted germination of the seeds over the control,it was not up to that with the TDZ-pretreated seeds(Table 1).Thidiazuron has been described as an efficient PGR that promotes clonal propagation of woody plants(Huetteman and Preece 1993).It effects efficient germination of seeds by promoting bud break,induction and stimulation of seed sprouting ability,cotyledon growth and development, the formation of trichomes and stomatal appearance on floral parts(Guo et al.2011).It enhanced germination in lettuce(Baskakov et al.1981)and neem(Murthy and Saxena 1997),and was substitute to the requirement of chilling pulse for seed germination in Pyrus species(Lin et al.1994).Thidiazuron enhanced seed germination in Striga asiatica(Babiker et al.1992)and all the tested cultivars of olive oil through inhibition of ethylene production(Rinaldi 2000).At lower concentration,it promoted seed germination in Digitalis purpurea by alleviating stress factors but,higher concentration inhibited the physiological process(Patil et al.2012).

Plant growth regulators and media strength

Formation and proliferation of in vitro cultures needs exogenous application of cytokinins for their role in cell division promotion and expansion through regulating gene expression and activities of enzymes involved in the process by stimulation initiation of meristem formation with influence on shoot differentiation and multiplication,the axillary buds formation and other in vitro morphogenic pathways(Mok and Mok 2001).They can initiate production of shoot primordial from explants at high efficiency when amended in the culture media.In the second experiment,effects of plant growth regulators(PGRs)and media strength on the in vitro clonal multiplication of C.fragrans from in vitro-derived nodal segment explants were studied.The PGRs type,concentration and cultivation media strength were found to influence the number and percentage of micro shoots produced by the cultured aseptic nodal segment explants after 21 days incubation(Figs.1d-f,2A-C).For the PGRs types tested,TDZ was more efficient in producing the shoots followed by Benzylamino Purine(BAP)and Kinetin(Kin),in decreasing order by response.However,most of the TDZ-induced micro shoots showed stunted growth while those induced with the BAP were more healthy-looking and vigorous in growth,although all the in vitro cultures of C.fragrans showed slow growth.Cultivation of the stunted TDZ-induced micro shoots on BAP or Kin-added media favored their elongation(Fig.1e,f).Thidiazuron was more efficient in inducing maximum number and percentage of micro shoots per nodal explant.For the concentration effect of the PGRs,0.5,1.0,2.0,3.0,4.0 and 5.0 mg L-1were tested.Among them,best formation of the shoots was observed with TDZ at 4.0 mg L-1concentration where an average of 6.48±0.15 shoots/explant was produced at 100% efficiency. Least shoot formation response was observed with Kin concentration of 0.5 mg L-1for which an average of 1.0±0.12 shoot/explant was produced at 29%efficiency.However,there were slight,insignificant differences on the shoot formation response between given lower concentration of the PGRs tested.Significant differences were observed between higher and lower PGRs treatment concentrations, suggesting their differential concentration effect in influencing shoot formation response in C.fragrans(Figs.1d,e,2A,B).Thidiazuron enhanced in vitro clonal propagation of strawberry and blueberry cultivars(Cappelletti et al.2016)and nodal explants of Stemona hutanguriana(Prathanturarug et al.2012).It was more efficient than other cytokinins tested in producing micro shoots of black gram during in vitro clonal propagation(Srilatha et al.2014).However,BAP was more efficient for the in vitro clonal multiplication of Ceropegia bulbosa from nodal segment explants(Goyal and Bhadauria 2006).

Adjusting culture media strength is an essential strategy for efficient high frequency in vitro clonal propagation of plants(Cardoso and Ono 2011),and salt strength composition of MS media provides nutrition that promotes cellular metabolic processes that support morphogenesis by overcoming toxicity effect(Fadel et al.2010).For the influence of media strength on the production of micro shoots, maximum efficiency was observed with half strength over quarter and full strength MS medium,possibly due to influence of the differences in water potential and availability of nutrients in the culture media that affected cellular metabolic physiological processes with resultant influence on growth and development of induced micro shoot.However,there was no significant difference in the number and percentage of micro shoot production efficiency with most of the PGRs concentrations tested between the use of quarter basal and full strength MS medium(Fig.2A,B).Many of the explants cultures on full strength MS medium showed basal swelling accompanied with callus formation before shoot induction response,and was more pronounced in media amended with the BAP.The induced calli were compact,nodular and greenish with slow growth(Fig.1f).It was earlier reported that basal callus produced along with micro shoot could become a sink to synthesized endogenous hormones,leading to the reduction in shoot formation response(Moyo et al.2011).In vitro clonal propagation of Typhonium flagelliforme was affected by media strength and culture system through growth parameters that were influenced by nutrients uptake(Rezali et al.2017).Half strength MS medium was more efficient in micropropagation of Mentha spicata over full and quarter strength media(Fadel et al.2010).

Thidiazuron pre-treatment of nodal explants

Fig.1 a C.fragrans seed germination on half basal MS medium,b seed embryo axes excised from the surface sterilized mature seeds,c 3-week-old aseptic seedling obtained from the mature seed embryonic axes cultured on half basal MS medium,d,e cultured nodal segment explant showing formation of healthy shoots under 12 h photoperiodic incubation,f basal callusing on the induced micro shoots from nodal segment explants grown on media added with Thidiazuron(1.0 mg L-1)and incubated under 3 h photoperiod,g formation of albino micro shoot among cultures incubated under 18 h light period,h rooting of the healthy micro shoot on medium added with indole butyric acid(1.0 mg L-1),i aclimatization of the regenerated plantlets.a(bar 1.0 cm),b(bar 0.6 cm),d and h(bar 1.45 cm),c,eg and i(bar 1.89 cm)

Fig.2 Results of micro propagation studies showing A influence of PGRs types,concentration and media strength on shoot formation(numbers)in C.fragrans,B percentage of the micro shoots formed,C effect of TDZ pulsing on number of the induced micro shoots,D effect of carbon sources on the number of shoots formation,E effect of carbon source on percentage shoot formation, and F influence of photoperiodic incubation on micro shoot formation

Fig.2 continued

Because the aim is to maximize shoot formation in the in vitro cultures of C. fragrans and that TDZ showed more efficiency in the previous experiments, treatment of the nodal explant with the PGR pulse before cultivation on solidified culture media were tested. Nodal explants were treated with autoclaved liquid TDZ (1.0 mg L-1) for few seconds, and the adherent liquid blotted out with autoclaved What man filter paper before cultured on solid media. The pulse treatment promoted early and higher micro shoots production efficiency over culture of the explants without the TDZ pulse treatment (Figs. 1d-f; 2A-C). Maximum formation of the shoots were observed on solid media added with 4.0 mg L-1where an average of 6.93±0.17 micro shoots/explant were produced and least on 0.5 mg L-1added media with 2.89±0.11 average micro shoots/explant after 21 days culture,suggesting the enhancing effect of TDZ pre-treatment pulse in producing the micro shoots.Similarly,half strength MS medium cultivation was more efficient in producing the shoots over quarter and full strength,further confirming the influence of media strength on micro shoot formation in C.fragrans,even with the TDZ pre-treatment pulse(Figs.1d,e,2C).Thidiazuron pulsing promoted growth and shoot development in peach palm but,with restriction in the development of other morphogenetic process(Graner et al.2013),similar to the observation made in this study.It showed carry over enhancing effect on shoot multiplication and proliferation in the light and dark-incubated cycles of banana cultivars in vitro cultures through influencing endogenous hormone levels.The cultivars showed differential shoot proliferation rates between dark condition and 8 or 16 h light incubation conditions(Makara et al.2010).

Effect of carbon source

Plant growth in the in vitro condition is affected by quality and concentration of amended sugar in the culture media and photosynthetic efficiency of chlorophyll plays critical role in growth of plants(Afreen 2005;Isah 2015).However,excess sugar in the culture media affects growth through downregulating photosynthetic efficiency, and may serve as chemical signal or stress agents(Kaul and Sabharwal 1971;Koch 1996;Rahman and Alsadon 2007;Yaseen et al.2013).Because plant cell carbon metabolism efficiency from the culture media varies with effect on performance through in vitro growth and development,in this study,different carbon sources were tested using the most efficient PGR in promoting shoot formation in C.fragrans(TDZ at 4.0 mg L-1).Various carbon sources also exerts influence on growth and morphogenesis of the in vitro cultures by virtue of the role they play in nutrition through osmotic potential and energy supply with influence on cell division and morphogenesis through influencing biosynthetic pathways involved in cellular processes for developmental program in the in vitro cultures(Yaseen et al.2013).In this study,effects of glucose,sucrose,fructose,and maltose in producing micro shoots were investigated at 2,3,4 and 5 mg L-1concentrations added to the solid culture medium(Fig.2D,E).Maximum shoot formation was observed with sucrose in all of the concentrations tested.It was the best at 4 mg L-1,producing an average of 6.72±0.14 micro shoots/explant at 99.4%efficiency after 21 days culture on solid medium amended with the selected TDZ concentration.Least shoot formation response was observed with maltose at 2 mg L-1where an average of 3.98±0.18 micro shoots/explant was formed at 68.4%efficiency.Sucrose is regarded major transported sugar in phloem,and its morphogenetic potential in plants in vitro cultures can be manipulated by concentration added into the culture media.Although it proved better in the overall performance in this study,other carbon sources were also effective but,to a lesser extent than the sucrose.The ease in availability of sucrose in the culture media facilitates in vitro growth and development by impacting adjustment of cellular osmolarity,and it was explained that sucrose is broken down into glucose and fructose during autoclaving through the activity of invertase to promote efficient utilization by in vitro cultures of higher plants(Pierik 1987;Abou Rayya et al.2011).It is also regarded most important carbon source for plant tissue cultures,been the most common carbohydrate that is found in phloem sap involved in the control of various plant developmental processes(Gibson 2000).However,glucose is utilized earlier for the trigger of morphogenesis coordinated by energy supply and other metabolic activities in cells.Both carbon sources at 5.0 mg L-1showed formation of micro shoot/explant at lower numbers and efficiency accompanied with induced solid compact growth that could be ascribed to osmotic stress,implying that the concentration is inhibitory to the formation of micro shoot,more particularly with the sucrose(Fig.2D,E).This suggests that sucrose at 4 mg L-1using half strength MS medium is the best for the in vitro clonal propagation of C.fragrans from aseptic nodal segment explants.Sucrose initial concentration affects growth and biomass production in the in vitro cultures,and its higher levels can retard development of the cultured cells through causing cell cycle termination when there is limitation on nutrients(Wu et al.2006).In this study,low and a higher concentration of the sucrose in half strength MS medium affected in vitro growth through the number and percentage of the formed shoots per explant after 21 days incubation(Fig.2D,E).Similar findings were reported during in vitro clonal propagation of Phalaenopsis and Dendrobium hybrid(Gnasekaran et al.2010;Nambiar et al.2012;Sudipta et al.2013).Inhibitory effect of high carbon sources supplemented into the culture media observed in this study could be attributed to the inhibition of photosynthesis that plays a role in the in vitro performance of micropropagated plants.High carbon sources supplementation in the culture medium inhibited rubisco activity in the leaves of Tobacco plants with effect on photosynthetic systems during micropropagation, and sucrose at 4%was the best concentration for the optimal in vitro performance(Roh and Choi 2004).Yildiz et al.(2007) observed tissue necrosis and decrease in shoot formation response during in vitro regeneration of sugar beet lines at higher sucrose concentration while in vitro cultivated pears showed detrimental effects due to declined osmotic potential(Jain and Babbar 2003).In Ginkgo biloba and Tomato,medium supplemented with over 4%carbon source showed lower growth when MS medium was used(Schnapp and Preece 1986;Park et al.2004).Explants cultured on solid medium that was not amendment with carbon source showed very poor response to the in vitro morphogenesis,indicating the critical role played by sugars in the in vitro growth of C.fragrans in vitro cultures.

Effect of photoperiodic incubation

Plants in vitro propagation are conventionally carried out under low photosynthetic photon flux of 30-80 μmol m-2s-1in comparison to the higher photoautotrophic condition of the ex vitro(Afreen 2005).It has been established that light influences quality of in vitro propagated plants through influences on the photosynthetic efficiency that co-commitantly influence tissue culture system under various cultivation conditions of the in vitro regenerated plants(Rodriguez et al.2012)through possible influence on endogenous hormones balance that results in changes in growth efficiency(Pinero et al.2014).Somaclonal variation,the appearance of phenotypic variants that may be in the form of shoot and foliar pigmentation in the in vitro cultures,defined as the percentage of regenerates that show the aberration or phenotypic changes in relations to parental plants is known to occur in plant cell cultures(Larkin and Scowcroft 1981).Formation of albino shoots characterized by whitish or cream-coloured leaves or micro shoots lacking chlorophyll is a somaclonal variation that could affect in vitro clonal production of plantlets.To test the effects of photoperiod on the micropropagation of C.fragrans,standard light duration of 12 h were tested against 3,8,16,18 and 24 h.Maximum formation of the shoots was observed with 12 h daily light/dark incubation duration wherein an average of 7.75±0.19 micro shoots was produced per nodal explant using half strength MS medium added with TDZ(4.0 mg L-1)and sucrose at 4%.Least response was recorded with the complete dark incubation, producing an average 3.12±0.17 micro shoots/explant.This was followed by complete light incubation with 4.03±0.11 and 3 h light incubation period that produced an average 4.62±0.14 micro shoots/nodal explant after 21 days culture.Complete light/dark incubation produced very short,unhealthy-looking shoots with yellow pigmentation.Light intensity duration has significant effect on physiological processes in the in vitro cultured plants with resultant impact on growth parameters(Moyo et al.2014).Cultured plants encounter certain physiological anomalies that may lead to unusual morphological features induced by inefficient photosynthesis influenced by light and gas exchange through the stomata of leaves and low chlorophyll content which could result in defects.Among the cultures incubated below 12 h or complete light photoperiod,formation of albino shoots bearing complete white shoot and leaves and some albinism spots on the normal shoots were observed.It occurred at higher frequency in cultures that were incubated under complete light condition while those in complete dark showed yellow leaves with white spots on the shoots(Fig.1g).The albino phenotype observed might have been caused by the photoperiodic light illumination essential for chlorophyll formation and possibly contributed in parts by the TDZ-pulse treatment or their interaction.The use of TDZ in the in vitro cultures of Bamboo had been linked to the occurrence of albinism(Liu et al.2007).Under these light conditions and durations, high frequency of albino shoot formation observed can also be explained by the lack of proplastids differentiation into chloroplast influenced by light illumination and due to the possible poor development of thyllakoid membrane.However,the degree of albino shoot formation between the cultures(in terms of numbers)was hard to assess due to its sporadic occurrence and difficulty in establishing sharply demarcated formation in relations with the photo period exposure duration.The albino shoots were not different from the normal ones in terms of micro shoot height and size of the leaves(Fig.1g).Under these conditions,the micro shoots showed excessive basal callusing,particularly with the cultures incubated in 18 and 16 h dark duration,and possibly resulted in shorter shoots having thin and less number of leaves observed in some of the cultures(Fig.1f).Light intensity and duration have significant effect on physiological processes in the in vitro cultured plants with resultant impact on growth parameters.Culture media and light intensity influenced albino shoot formation in anther culture-derived plantlets of rice varieties(Mohiuddin et al.2011).In this study,there was significant reduction in the number of induced shoots/nodal explant with decrease in the light period below 12 h.The length of the shoots also showed similar productivity trend(data not shown).Light exposure duration of C.fragrans in vitro cultures and frequency of albino shoot formation showed some association but,not with the culture media composition(Fig.2F).Photoperiodic duration was found to affect micropropagation parameters of multiple shoot formation and calli in Citrus assamensis using nodal explant cultures(Yaacob et al.2014).Longer or shorter light duration incubations influenced the occurrence of albinism in the in vitro cultures of rice varieties(Mohiuddin et al.2011).It has been reported in Wasabi plantlets that micro environment of culture vessels influence plantlets interaction with the culture medium,thus affecting physiological processes of photosynthesis,shoot and roots development and stomatal function essential for biomass production(Hoang et al.2017).Occurrence of albinism in the in vitro cultures of wheat varieties was ascribed to genetic factors and change in medium composition was effective in lowering its frequency in plantlets(Alikina et al.2016)

Rooting of the micro shoot

For successful in vitro clonal propagation of plants,rooting of the produced micro shoot is a major step towards survival and growth of regenerates during aclimatization.Medium strength and carbon source play a role in rhizogenesis efficiency of micro shoot.Osmotic stress induced by high sugar concentration and media strength is perceived by the induced roots,leading to abscisic acid signaling that decreases aerial tissues permeability with the resultantly reduced uptake of carbon source and repression of lateral roots formation(Yaseen et al.2013).In this study,in vitro regenerated shoots were rooted(Fig.1h)with indole butyric acid(1.0 mg L-1),based on an earlier report(Kulkarni and Malpathak 2006).The regenerated plants were acclimatized,first in potted mixture of soilrite/perlite(Fig.1i),followed by transfer to the field condition,and albino shoots showed low survival of 49.2%compared to the 89.6%higher shown by the normal ones.

Physiological,biochemical differences between the leaves of albino and normal micro shoots

Plants deficient in chlorophyll are referred to as albino and offers genetic material for gaining insight into molecular mechanism involved in chlorophyll biosynthesis, and development of chloroplast in higher plants under the in vitro culture condition(Jung et al.2003).The morphological anomaly may be induced by high exogenous applied PGRs that induces an elevation in the levels of endogenous hormones biosynthesis, prolonged in vitro culture or extended passage time,and is regarded a rare somaclonal variation that occurs at low frequency.To figure biochemical, physiological differences between normal and albino leaves of C. fragrans, associated parameters of antioxidant systems and photosynthesis that have an impact on the in vitro cultured plants growth and development were studied.

Photosynthesis and non-enzymatic antioxidants

Photosynthesis and uptake of nutrients have substantial effects on the physiological process and could be suppressed,thereby enhance dark respiration that may affect metabolic processes in cells of cultivated plants,leading to inhibited growth and morpho-physiological anomalies(Isah 2015).Chlorophyll content of leaf in the in vitro grown plants could affect photosynthetic efficiency by reducing light capture and absorption through the photosynthetic machinery(Christensen et al.2008),leading to morphological anomalies such as the formation of albino shoots observed in this study.This might have resulted in the reduced efficiency in growth parameters of the in vitro cultured explants due to reduced efficiency in the in vitro performance observed with the albino shoots. Among factors likely affected is CO2utilization of photosystems that possibly resulted in humidity accumulation in the culture vessel during the photo period,thereby contributing to low micropropagation efficiency of plants under the studied biochemical parameters (Fig.3). CO2levels affected in vitro growth efficiency of poplars through an effect on various characteristics of the in vitro propagated plants(Richet et al.2012)as well as in Orchids cultivated under high levels of the CO2(Yoon et al.2009).

Chloroplast is an organelle that performs photosynthesis in the cells of plants through the‘‘housed''photosynthetic machinery having subunits,and mutation affects its structural units involved in the synthetic carbon activity.Chlorophyll and carotenoids participate in the capture or absorbance of light energy,conferring‘‘photo protection''to plants'photosystems against photooxidative damage by dissipating excess light energy(Horton et al.1996).Their deficiency or absence affects plant productivity through photoxidative damage(Mohanty et al.2005;Bode et al.2009).In this study,chlorophylls a and b and total chlorophyll contents of leaves in the albino shoot showed differential levels over that of the normal ones(Fig.3A).Leaves of the albino shoots accumulated relatively lower chlorophyll a contents with an average of 0.285±0.34 mg g-1FW compared to the higher 0.913±0.51 mg g-1FW accumulated by leaves of the normal micro shoots,representing 3.4-fold increase.For the chlorophyll b,the albino shoots accumulated higher with an average 0.316±0.15 mg g-1FW compared to the lower 0.184±0.24 mg g-1FW in the leaves of normal plantlets,representing 1.7-fold increase.However total chlorophyll content in the leaves of normal micro shoots was relatively higher with average of 1.246±0.69 mg g-1FW compared to the lower 0.389±0.43 mg g-1FW accumulated by the albino leaves,representing 3.2-fold increase(Fig.3A).

Carotenoids are lipid-soluble antioxidants regarded potential scavengers of reactive oxygen species(ROS)and lipid radicals that play a role in scavenging superoxide radicals during cellular oxidative stress(Hollander-Czytko et al.2005).They form part of light-harvesting complex that protects photosynthetic machinery from photo-oxidative damage(Pogson et al.2006).In this study,leaves of normal plantlets accumulated higher carotenoids with an average 5.17±0.16 mg g-1FW in comparison to the lower 2.15±0.42 mg g-1FW accumulated by the albino ones,representing a 2.4-fold increase(Fig.3A).This could be due to the higher photosynthetic efficiency in the former than the latter given its higher chlorophyll contents that represents high photosynthetic efficiency which is known to generate reactive oxygen species that needs to be scavenged to tolerable cellular levels.Lower pigment molecules in the leaves of albino shoots over the normal ones also indicated possible disruption of chlorophyll and carotenoids biosynthesis that resulted in low photosynthetic capacity in C.fragrans in vitro cultures.

Fig.3 Physiological,biochemical analysis between leaves of albino shoots and normal ones:A photosynthetic pigment molecules,B osmolytes molecules analysis,C antioxidant molecules analysis,D antioxidant enzymes assay

During tolerance to stressful growth conditions of the in vitro culture imposed by many factors such as photoperiodic incubation duration of cultures,plants synthesize and accumulate organic osmolytes such as soluble sugars, proline, glycine betaine and non-enzymatic antioxidants as H2O2. Accumulation of soluble sugars helps plants in osmotic adjustment for cells to maintain favorable water relations(Jin et al.2010).Proline,glycine betaine and total soluble sugars showed similar production trend with higher in the albino over the normal ones,indicating that C. fragrans accumulates the osmolytes during stressful condition and likely contributed to the formation of the albino shoots(Fig.3B).Total soluble sugars showed higher accumulation in the leaves of albino shoots with an average 18.59±0.56 mg g-1FW over the lower 10.17±0.14 mg g-1FW by the leaves of normal shoots.It is likely that the light and dark condition-induced stress promoted higher production of TSS contents in the leaves of albino shoots over the normal ones as indicated by the 1.83-fold higher accumulated in former over the latter.Proline can serve as storage sink to nitrogen and carbon or free-radical scavenger during in vitro stressful condition to plants(Chinnusamy et al.2005;Flors et al.2007)and its stabilizing role to subcellular structures,including membranes and proteins can serve as buffer to cellular redox potential when plants are growing under differential photo periods of the in vitro culture that likely cause morpho-physiological anomaly(Chinnusamy et al.2005;Kishore and Singh 2005).Proline contents were relatively higher in the leaves of albino shoots over the normal ones with an average 179±0.43 μg g-1FW in former to 112±0.48 μg g-1FW in the latter,representing a 1.6-fold increase(Fig.3B).Osmoprotectants such as glycine betaine can give stability to proteins quaternary structure and highly ordered state to membranes through alleviating osmotic stress in tissues.Their synthesis is dependent on energy production,thus,may affect growth of plants in the in vitro condition.Glycinebetaine production was similarly higher in the leaves of albino shoots with an average of 182.12±0.33 μg g-1FW accumulated over the lower in the normal ones that had 109.77±0.24 μg g-1FW, representing a 1.67-fold increase(Fig.3B).Accumulation of osmolytes and high antioxidant systems possibly played a major role in higher osmotic adjustment of C.fragrans albino shoot cultures during light/dark conditions incubation above or below 12 h/day photoperiods in comparison to the normal ones.

Lipid peroxidation as per evaluation of MDA content of leaves showed higher levels in the albino shoots over the normal ones with the production of 11.12±0.41 μM mg-1FW in former to the lower 7.23±0.19 μM mg-1FW in the latter,representing a 1.54-fold increase(Fig.3C).This indicates higher lipid peroxidation and membrane damage in former than the latter,possibly due to the excessive stress that the leaves were exposed to by deficiency in photooxidation defensive machinery of photosynthetic apparatus that possibly contributed to the formation of albino shoots(Fig.1g).It also suggests that extended or sub-optimal exposure to the light duration causes leaves bleaching and might have been contributed in part by the lack of chlorophyll,with a possible contribution by lipid peroxidation. Production of H2O2at higher levels is among evidence of oxidative stress to plant tissues and keeping levels of the generated H2O2,andto minimum in the cellular environment is essential for plant survival under stressful condition of the in vitro culture,more particular in plants that show morpho-physiological anomaly under the condition(Mallik et al.2011).H2O2content showed 1.53-fold higher levels in the leaves of albino shoots which accumulated 11.47±0.22 μM mg-1FW over the normal ones that had 7.51±0.13 μM mg-1FW in its leaves(Fig.3C),indicating higher accumulation of the compound possibly due to the higher ROS and hydroxyl radical species production through MDA and SODs activity in former than the latter.

Enzymatic antioxidant systems

Overcoming oxidative stress and damage caused by the ROS needs defensive antioxidant systems involving low molecular mass antioxidant enzymes.SOD that participates as the first line of defense against ROS showed higher activity in the leaves of albino shoots,producing 7.42±0.15 μKat mg-1protein compared to the lower 4.94±0.25 μKat mg-1proteinin the normal shoots(Fig.3D).This is1.5-fold higher in former than the latter,possibly due to the higher levels of ROS generated in the albino leaves than in the normal ones.High H2O2produced by the activity of the SOD due to high stress in the leaves of albino shoots is decomposed into less cellular toxic molecules by the activity of CAT,APX and GPX enzymatic machinery(Lokhande et al.2011).The CAT is mainly associated with the removal of H2O2in the micro bodies(Scandalios et al.1997)through decomposition of generated H2O2.In this study,CAT activity was higher in the leaves of albino shoots producing 4.21±0.73 μKat mg-1protein compared to the lower 2.85±0.16 μKat mg-1protein shown by the normal ones(Fig.3D),representing 1.48-fold increase in its activity in former than the latter,possibly contributing to the steady state levels of the H2O2(Sergio et al.2012).APX also involved in the dismutation of H2O2through ascorbate-glutathione cycle(Shigeoka et al.2002)and regarded most important substrate for the reduction of cellular accumulated H2O2through detoxification,showed higher activity in the leaves of albino micro shoots over the normal ones,with 1.52-fold higher levels through accumulation of 2.23±0.32 μKat mg-1protein in former to the lower 1.47±0.11 μKat mg-1protein in the latter(Fig.3D).GPX that has broad specificity with regards to an electron donor and participates in many physiological processes,showed lower activity in the leaves of normal plantlets, producing 1.93±0.16 μKat mg-1protein in comparison to higher 2.57±0.51 μKat mg-1protein demonstrated by the albino,which is 1.33-fold higher than in the latter(Fig.3D).

Conclusions

In conclusion,the results of this study revealed that photoperiod,PGRs amended in the culture media,carbon source type and concentration,culture media strength and explant pre-conditioning influence micropropagation efficiency of C.fragrans from nodal segment explants derived from axenic seedlings. The parameters are the shoot induction efficiency and morpho-physiological anomalies encountered.Photoperiodic incubation had dramatic effect on the physiology and morphology of C.fragrans cultivated in vitro from the nodal segment explants.Thus,can be explained by the changes in biochemical,metabolic pathways in response to cellular oxidative stress that possibly led to the formation of albino shoots in some of the in vitro cultures.Antioxidant systems showed elevated levels in the leaves of albino shoots over the normal ones,indicating higher cellular oxidative stress induced by the photoperiods of incubation that altered the biochemical parameters studied.The activities of antioxidant enzymes showed a similar trend to other biochemical parameters studied.SOD activity showed higher levels in the leaves of albino shoots over the normal ones while that of CAT,APX and GPX were lower in the levels but,relatively greater in the leaves of albino shoots,indicating their efficient role in scavenging ROS and reducing resultant accumulated H2O2in the leaf cells of the albino shoot cultures. Albinism affected metabolic processes and biosynthesis of associated pigment molecules with effect on physiology in Agave augustifolia plantlets due to epigenetic changes that influenced gene expression related to the phenotype produced(Duarte-Ake et al.2016;Us-Camas et al.2017).

AcknowledgementsAuthors thank Indian Institute of Horticultural Research for the permission and gift of Chonemorpha fragrans germplasm used in the study.We are grateful to Hamdard University New Delhi,India for support to this research work.Financial assistance provided to Tasiu Isah by the Department of Biotechnology,Government of India,New Delhi and the World Academy of Science(TWAS)for the Advancement of Science in the Developing World Trieste.Italy through DBT-TWAS Postgraduate Research Fellowship is also acknowledged.