Chapter 2 Indirect Organogenesis and histological analysis of organogenic and non-organogenic calli obtained from in vitro

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1 Chapter 2 Indirect Organogenesis and histological analysis of organogenic and non-organogenic calli obtained from in vitro cultures of Justicia adhatoda L ABSTRACT Leaf, axillary bud and root tip explants of J. adhatoda were employed for calli production and indirect organogenesis on Murashige-Skoog media supplemented with different combinations of indole 3- acetic acid (IAA), α- naphthyl acetic acid (NAA), indole 3- butyric acid (IBA), 6- benzylaminopurine (BA) and kinetin (Kn). Combination of 6 mg L -1 IAA and 6 mg L -1 Kn gave best callusing of leaf explants. Combinations of 3 mg L -1 IBA, 3 mg L -1 BA and 3 mg L -1 IBA, 6 mg L -1 BA gave maximum callus response with axillary bud and root tip explants respectively. Multiple shoot induction occurred per callus within six weeks on medium containing 6 mg L -1 BA and 4 mg L -1 Kn. High frequency rooting was recorded on Murashige-Skoog medium with 6 mg L -1 of IBA and NAA. A histological study of calli of in vitro propagation was carried out. The phenotypic differences of callus cultures derived from J. adhatoda L. were evaluated based on their morphology and ultrastructure. The organogenic and nonorganogenic calli are the result of hormonal variation in the medium. In nonorganogenic callus, cells redifferentiated into xylem elements forming clusters of nest like structures. In organogenic callus, the undifferentiated cells of callus were found to differentiate into vascular nodules called meristemoids, which then develop into xylem elements, especially tracheids. On culturing on the shooting medium, these nodules differentiated into shoot apical meristem. This adventitious origin provided chances for variability. Key words: indirect organogenesis, histological study, J. adhatoda L., multiple shoot, tracheids, meristemoids, xylem elements.

2 34 Chapter INTRODUCTION With an ever-increasing global inclination towards herbal medicine, there is not only an obligatory demand for a huge raw material of medicinal plants, but also of right stage when the active principles are available in optimum quantities at the requisite time for standardization of herbal preparations. Ideally, the herbal plants should be grown under uniform environmental conditions and the planting material must have the same genetic make- up as of the selected high-yielding clones, which is possible when they are cloned through an in vitro strategy, i.e. micropropagation, at least in cases where conventional vegetative propagation methods are insufficient or wanting to achieve the goal (Chaturvedi et al., 2007). A number of medicinally important plant species have been successfully propagated on a mass scale with the use of in vitro techniques. In vitro propagation helps in production of a very large number of plants from a tiny explant (Majumder et al., 2011). According to Shanmugapriya and Sivakumar (2011) tissue culture has been successfully used for the commercial production of pathogen-free plants to conserve the germplasm of rare and endangered species. Morphological and histological studies of callus induction of plant are important for increasing the incidence of callus production (Feng et al., 2007; Tan et al., 2009; Yan et al., 2012). In the present study, the morphology and histology of the callus produced from the leaf, axillary bud and root tip were systematically studied. The differences in morphological and histological characteristics between the green compact and white or brown soft, friable calli were also studied.

3 Indirect Organogenesis and histological analysis of REVIEW OF LITERATURE Unlimited exploitation of the natural resource for medicine is causing dwindling of the existing plant population. Large scale cultivation of the plants is the only remedy for ensuring future availability of medicinal plants. A major problem faced when we go for large scale cultivation is the scarcity of the planting materials. Hence there is a necessity for developing an in vitro culture technique for regeneration of the plants, which yields large number planting materials at all seasons (Viji and Parvatham, 2011). Largescale plant tissue culture offers a controlled supply of biochemicals independent of plant availability and more consistent product quality (Nalawade and Tsay, 2004). Plant regeneration from in vitro culture has been possible via organogenesis and somatic embryogenesis. Plant tissue culture offers many unconventional techniques for crop improvement. Callus induction and plant regeneration is one method of plant propagation useful for experimental work. Callus is a disorganized mass of undifferentiated tissue comprised of actively dividing cells. The cells of callus dedifferentiate and thus regain their meristematic properties, including rapid proliferation (Alatzas et al., 2008). Due to these meristematic properties, callus cells are totipotent, or capable of undergoing organogenesis, where they may potentially differentiate into any plant part, including roots, shoots, flowers or stems (Razdan, 2003). Callus is generally induced by a wound response with auxins and cytokinins which are plant growth regulators or PGRs. Auxins are involved in cellular division, cell elongation and callus induction. Cytokinins are also associated with cellular division and cell expansion and are widely used in

4 36 Chapter 2 conjunction with auxins to induce callus. Cytokinins have been found to promote shoot regeneration in higher concentrations presumably through the change in the auxin-to-cytokinin ratio. A wide array of factors can influence callus growth and morphology, including plant genotype, nutrient medium composition, the explant material used and abiotic factors such as light and temperature. Callus may be globular or friable and both may exhibit variable coloration. The PGRs auxins and cytokinins are required for both callus induction and organogenesis. Furthermore, the ratio of auxin to cytokinin concentrations determines if callus, shoots or roots will be induced. Successful callus induction in other plant systems has been achieved with mature explants materials such as cotyledons, internodes and petiole. However, it is believed that the best results are derived from immature tissues showing meristematic properties, due to their increased culture survival rates, growth and totipotency in vitro. These tissues include meristems, leaves, roots, shoots and inflorescences (Carter et al., 2011). Callus culture and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies (Catapan et al., 2002). Somatic embryogenesis generally occurs through two different pathways, i.e. direct and indirect organogenesis. Direct organogenesis occurs directly from the explants and indirect organogenesis, indirectly following callus formation from explants (Baskaran and Jayabalan, 2010). Indirect plant regeneration can be employed as an alternative means for genetic upgrading, and its application largely depends on the reliable plant regeneration system. A good regenerating system may be suited for transformation where the production of transformants by direct organogenesis is desired (Thomas and Sreejesh, 2004).

5 Indirect Organogenesis and histological analysis of 37 Production of regenerated plant through indirect organogenesis is one possible way to contribute to genetic improvement, because there are some advantages of shoot regeneration from callus over direct shoot regeneration. A callus phase is commonly included in tissue culture protocols with the objectives of generating variability to introduce new desirable traits and generating transgenic plants to introduce traits such as pest resistance in crops. Moreover, callus production is also a necessary step for obtaining protoplasts used in protoplast fusion, a useful tool in genetic improvement of vegetatively propagated plants for introducing useful genes or producing new crops (Yan et al., 2009). Genetic transformation is an important and effective technique to improve the yield and quality of crops. It is a prerequisite for transgenic studies to establish a highly effective callus induction and regeneration system (Ruan et al., 2009). Formation of vascular nodules in callus cultures may represent or be associated with an early stage of the development of shoot meristems (Sujatha et al., 2003). They reported that the nodules containing xylem elements in callus of Pelargonium developed into shoots when moved to an auxin free medium. Such nodules that protrude out from the callus and leaf buttresses, later on developed into distinct shoot or leaf primordium. It is also reported that callus differentiation begins when peripheral meristematic activity is replaced or supplemented by the formation of centres for cell division deeper in the tissue SPECIFIC OBJECTIVES: The present investigation aimed at the development of an indirect micropropagation for large scale regeneration of plantlets from explants of

6 38 Chapter 2 mature plants with a view to cloning high alkaloid containing genotypes. A histological study of different types of calli was also done. The main specific objectives of this study are, 1. To develop a protocol for indirect micropropagation from the various explants of J. adhatoda L. 1a) To study the effects of hormones, auxins and cytokinins in callogenesis. 2. Histological analysis of organogenic and non-organogenic calli obtained from in vitro cultures of J. adhatoda L MATERIALS AND METHODS INDIRECT ORGANOGENESIS Plant Material As mentioned in chapter one (Refer 1.6.1) Inoculation and Incubation As mentioned in chapter one (Refer ) Subculture Sub culturing of the cultures were done after every 30 days, using fresh medium and same culture conditions. The frequency of callus formation was recorded as percent of the explants forming callus. After four weeks, the callus formed was sub cultured on MS medium containing varying concentrations of BA and Kn (Central Drug House (P) LTD, India) for multiple shoot induction. Excised multiple shoots were separated after six weeks and transferred to MS medium containing different concentrations of IAA, IBA and NAA (Central Drug House (P) LTD, India)

7 Indirect Organogenesis and histological analysis of 39 for root induction. The rooted plantlets were removed from culture tubes and transferred to conical flask containing MS medium for two weeks. Then the rooted plantlets were hardened Statistical Analysis Fifty tubes each were inoculated for each hormone concentration and each explants and this was repeated three times and average callus response was calculated as a mean of three replicates. Data were expressed as mean±se for three replicates each for each hormone combination. Statistical analysis was done by ANOVA using the statistical package INSTAT and means were compared by Tukey-Kramer Multiple Comparisons Test HISTOLOGICAL EXAMINATION Samples of calli were prepared for histological examination after 15 days in culture. The calli at different stages of growth after initiation (15, 30, 45, 60, 90 and 120 days) were selected. The samples were fixed in FAA 50 [formalin-acetic acid-70% ethanol (5:5:90)] (E. Merck (India) Limited), dehydrated in a graded ethanol series (70%, 90% and 100%, three changes in each concentrations for 30 minutes), xylene (E. Merck (India) Limited), (three changes, each for 30 minutes) and embedded in paraffin wax (Central Drug House (P) LTD, India) (melting point: C). Serial sections of 10µm thickness were obtained with a rotary microtome (Reichert Jung, 2050 Super cut, Heidelberg, Germany). Sections were stretched on glass slides previously treated with 100 mg ml -1 poly-l-lysine (Sigma Aldrich, India), exposed to xylene-ethanol series to remove paraffin and stained with 0.1% safranin (Central Drug House (P) LTD, India). They were then mounted in DPX (dibutyl phthalate xylene/ distrene polystrene xylene) (Sigma Aldrich, India) and observed under light microscope (Olympus).

8 40 Chapter RESULT INDIRECT ORGANOGENESIS In the present study micropropagation of the plant was attempted by using different explants Callogenesis In the present work the explants started callusing within two weeks and callus induction was obtained from leaf, axillary bud and root tip with different levels of IBA, NAA, IAA, BA and Kn. Different explants responded differently to various plant growth regulator combinations (Table 2.1, 2.2 and 2.3). In J. adhatoda, leaf explants responded well to callusing. The nature of callus developed was different when auxins in combination with cytokinins, were added to the medium. The texture and colour of the callus depend on the source of origin of cells and the growth regulators in the medium. In the present study the growth regulator combination in the medium as well as the type of the explants influenced the mass, the colour and the texture of callus. The leaf explants showed creamish white callus in the medium containing IBA or IAA alone (Plate 2.1). With BA and Kn alone callus produced was loose creamish brown (Plate 2.2). But the same explants showed green colored compact callus when added with a combination of IAA, NAA and IBA with BA and Kn (Plate 2.3). The callus developed from axillary bud was brown in colour, irrespective of the hormone concentration. Friable white callus was formed from root tip explants with IBA, NAA, BA and Kn.

9 Indirect Organogenesis and histological analysis of 41 Plate 2.1 Friable creamish white callus from leaf explant Plate 2.2 Loose creamish brown callus from axillary bud

10 42 Chapter 2 Plate 2.3 Green compact callus from leaf explants Callusing of leaf explants was at its best when the medium had been supplemented with 6 mg L -1 IAA and 6 mg L -1 Kn. Significantly higher percentage callusing was also observed at this concentration (Table 2.1). Axillary bud explants showed significantly higher callus response at the hormone concentration of 3 mg L -1 IBA and 3 mg L -1 BA (Table 2.2).

11 Indirect Organogenesis and histological analysis of 43 When root tip explants were used for callusing significantly higher callus response with respect to average days for callusing and average weight of callus was found when 3 mg L -1 IBA and 6 mg L -1 BA were added to basal MS medium (Table 2.3). Table 2.1. Effect of auxins and cytokinins on 50 leaf explants on MS medium after 30 days Sl.No. MS medium+ Phytohormones IBA IAA KN BA % of callus induction Average callus response (50 explants in three replicates) Average wt. of callus in mg/ 30 days Average days required for callusing Nature of callus ± ± ±0.57 Creamish white ± ± ±0.57,,,, ± ± ±1.00,,,, ± ± ±1.1,,,, ± ± ±1.00 Loose Creamish white ± ± ±1.00,,,, ± ±8.0 24±1.00,,,, ± ± ±1.52,,,, ± ± ±1.53 Green compact ± ± ±1.52,,,, ± ± ±1.15,,,, ± ± ±1.15,,,, ± ± ±1.00,,,, ± ± ±1.52,,,, ± ± ±0.57,,,, ± ± ±1.52,,,, ± ± ±2.00,,,, ± ± ±1.00,,,, ± ± ±1.52,,,, ± ± ±1.52,,,, ± ± ±0.57,,,, ± ± ±1.53,,,, ± ± ±1.52,,,, ± ± ±1.53,,,, (*P <0.0001) CD value 3.76 CD value 16.5 CD value 2.45

12 44 Chapter 2 Table2.2. Effect of auxins and cytokinins on 50 axillary bud explants on MS medium after 30 days Sl.No. MS medium + Phytohormones IBA NAA BA % of callus induction Average callus response (50 explants in three replicates) Average wt. of callus in mg/ 30 days Average days required for callusing Nature of callus ± ± ±.0.57 creamish brown ± ±6.2 24±1.0,,,, ± ± ±1.0,,,, ± ±6.1 20±1.0 brown,soft, friable ± ± ±0.57,,,, ± ± ±1.0,,,, ± ± ±1.52,,,, ± ± ±1.0,,,, ± ± ±1.0,,,, ± ± ±1.0,,,, ± ± ±1.0,,,, (*P <0.0001) CD value 4.42 CD value CD value 3.63

13 Indirect Organogenesis and histological analysis of 45 Table 2.3 Effect of auxins and cytokinins on 50 root tip explants on MS medium after 30 days Sl.No. MS medium + Phytohormones IBA NAA BA KN % of callus induction Average callus response (50 explants in three replicates) Average wt. of callus in mg/ 30 days Average days required for callusing Nature of callus ± ± ±1.15 Friable white ± ± ±2.08,,,, ± ± ±2.0,,,, ± ± ±1.0,,,, ± ± ±0.57,,,, ± ± ±1.15,,,, ± ± ±1.0,,,, ± ± ±1.15,,,, ± ± ±1.52,,,, ± ± ±1.0,,,, ± ± ±0.57,,,, ± ± ±1.15,,,, ± ± ±1.0,,,, ± ± ±0.57,,,, ± ± ±2.0,,,, ± ± ±1.52,,,, (*P<0.0001) CD value 5.6 CD value CD value 3.87

14 46 Chapter Multiple Shoot Induction When the callus obtained from leaf, axillary bud and root tip explants were transferred to shoot inducing medium significantly higher percentage shoot proliferation and number of total shoots per culture were observed when the medium was supplied with 6 mg L -1 BA and 4 mg L -1 Kn (Table 2.4), (Plate 2.4). Multiple shoots with green curly leaves appeared at this concentration. Irrespective of the source the callus obtained from leaf, axillary bud and root tip showed the same response. Plate 2.4-Multiple shoot induction

15 Indirect Organogenesis and histological analysis of 47 Table. 2.4 Influence of BA and Kn on multiple shoot induction from 25 callus cultures of J adhatoda L. after 30 days. Sl.No. Growth regulators(mg/l) BA Kn %Of callus showing shoot proliferation No.of total shoots /culture Average No. of leaves /shoot ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.94 (*P<0.0001) CD value 1.82 CD value 1.43

16 48 Chapter Root induction from callus Calli were rooted on MS medium supplemented with different concentrations of IBA and NAA. Statistically significant rooting was reported on MS medium with 6 mg L -1 IBA and 6 mg L -1 NAA (Figure.2.1). Roots formed in the medium were longer and white in colour (Plate 2.5). Plate 2.5 In vitro rooting on MS medium % of in vitro root induction 40 % IBA- 5IAA 6IBA- 6IAA 6IBA- 5NAA 6IBA- 5.5NAA 6IBA- 6NAA 6IAA- 6NAA concentration of growth regulators Figure2.1 Influence of IAA,IBA & NAA on in vitro root induction from 25 callus cultures of J.adhatoda L after 30 days

17 Indirect Organogenesis and histological analysis of 49 Table.2.5 Influence of IAA, IBA and NAA on in vitro root induction from 25 callus cultures of J. adhatoda L. after 30 days. Sl.No. Growth regulators(mg/l) IBA IAA NAA % of callus showing root induction No. of roots/ culture Average length of roots ± ±.54 Nature of root Pale, semifine, tuberous ± ±0.83,,,, ± ±0.70,,,, ± ±0.70,,,, ± ±1.30,,,, ± ±0.89,,,, ± ±1.30,,,, ± ±0.89,,,, ± ±0.84,,,, ± ±1.10,,,, ± ±0.83,,,, ± ±0.89,,,, ± ±1.30,,,, ± ±1.00,,,, ± ±0.71,,,, ± ±0.54,,,, ± ±0.84,,,, ± ±0.89,,,, ± ±1.10,,,, ± ±0.83,,,, ± ±1.00,,,, ± ±0.54,,,, ± ±0.71,,,, ± ±0.89,,,, ± ±0.83,,,, (*P<0.0001) CD value 1.21 CD value 1.34

18 50 Chapter Hardening and Establishment in Pots The plantlets were planted in poly cups containing sterilized mixture of sand and soil, irrigated and kept under fluorescent lights, covered with polythene bags (for maintaining humidity). 16/8h photoperiod and 25±2 0 C was maintained, for a week, and then transferred to field conditions. The hardened plants when transferred to field shown 90% survival. So this process can be adopted as an alternative to propagation through cutting. Plate In vitro propagated plantlets Histology Histological examinations of calli revealed indirect development of few shoots and no evidence of somatic embryogenesis was found. During the early stages of callus formation, the parenchyma cells of the mesophyll tissue near the vascular bundles produced an undifferentiated mass of cells which is called primary callus (Plate 2.7a). Primary callus underwent division and produced large parenchymatous cells as derivatives while the initials appeared as darkly stained clumps (Plate 2.7b). Narrow elongated cells formed procambium and it developed into distinct vascular elements, especially tracheids (Plate 2.7c). Vascular nodules were formed (Plate 2.7d). Meristemoid regions were seen which is characterized with densely stained small cells. On culturing on the shooting medium from vascular nodules small buds with the tunica corpus organization of a shoot apical meristem was developed (Plate 2.7e).

19 Indirect Organogenesis and histological analysis of 51 Plate 2.7a.Callus with undifferentiated cells Plate 2.7b. Callus with initials (Darkly stained) Plate 2.7c Procambium develops into distinct vascular elements Plate 2.7d Callus with vascular nodules Plate 2.7e Callus with shoot primordia

20 52 Chapter DISCUSSION It is well known that auxins and cytokinins are effective for callus and organ formation in tissue culture of many plants (Yakauwa and Harada, 1982). It was clear from the study that the leaf explants showed maximum callus response (Table 2.1). On increasing the concentrations of growth regulators gradual increase in percentage of cultures forming callus was noticed in all cases upto the optimum concentration (Faisal and Anis, 2003). The nature of callus developed was different when auxins in combination with cytokinins, were added to the medium. The texture and colour of the callus depend on the source of origin of cells and the growth regulators in the medium. In the present study the growth regulator combination in the medium as well as the type of the explants influenced the mass, the colour and the texture of callus (Plate 2.1, 2.2 and 2.3). The histological analysis of the regenerating calli clearly showed that the shoot buds had emerged from the peripheral nodular structures, which consisted of closely arranged and highly cytoplasmic cells (Plate2.7a-e). In some shoots the vascular supply was found to be continuous with the vasculature of the callus (Thomas and Puthur, 2004). The division and growth of callus cells continued for some time resulting in the enlargement of primary callus. According to Sujatha et al.,(2003) in a root or shoot apex, certain cells of the meristems undergo divisions in such a way that, one product of a division becomes a new body cell, called derivative and the other remains in the meristem, called initials. A similar pattern of meristematic activity was observed in J. adhatoda callus. Callus cultures contain vascular nodules which comprises vascular elements and parenchymatous cells. The organogenic and non-organogenic calli are

21 Indirect Organogenesis and histological analysis of 53 the result of hormonal variation in the medium. In non-organogenic callus, cells redifferentiated into xylem elements forming clusters of nest like structures. In organogenic callus, the undifferentiated cells of callus were found to differentiate into vascular nodules called meristemoids, which then develop into xylem elements, especially tracheids. On culturing in the shooting medium, these nodules differentiated into shoot apical meristem. The detailed histological analysis shows that the shoots regenerated from the leaf derived callus of J. adhatoda have no organized cellular connection with the original explant tissue, indicating an adventitious origin and hence, chances of genetic variability among the regenerants. White-friable calli and green-compact calli had similar histological structures. Shape and sizes of cells, which was composed of these tissues varied greatly. Compared with the loose morphology outside of calli, cells inside calli were compact relatively and developed some intercellular spaces. These two types of calli also showed similar features in ultra structure (Plate2.7a-e). Result of the histological study showed that cells composed of overgrown calli had similar morphology. Moreover a structure of a series of cell clusters could be observed.