5. CLONAL PROPAGATION OF BOSWELLIA SERRATA ROXB.

Transcription

1 5. CLONAL PROPAGATION OF BOSWELLIA SERRATA ROXB. Clonal propagation through tissue culture offers an alternative to vegetative practices used in the part and has the potential to provide high multiplication rates of uniform genotypes, resulting in short term gains and providing an alternative for species which are difficult to root (Gupta et al., 1993). Boswellia serrarta Roxb. (Burseraceae), commonly named Salai guggul or india olibanum is an important medicinal tree grows in the hilly areas of India (Kapoor, 1990; Shah et al., 2008). Currently Boswellia serrata is categorised an endangered species (Sharma 1983; Purohit et al., 1995; Ghorpade et al., 2010). The oleo-gum resin, obtained by incision made on trunk of the tree is effective for the treatment of inflammatory disorder such as arthritis and in cardiovascular diseases (Ammon, 2006). The pharmaceutical properties of oleo-gum resin is well documented (Gyathri et al., 2007; Kruger et al., 2009) has been characterized as a rich source of Boswellic acids. The rate of natural propagation of Boswellia serrata is too slow where older trees get propagated through root suckers. However the root sucker technique has not been exploited commercially. Poor fruit setting (2.6 to 10%) under open pollination, inadequate production of viable seeds and scanty seed germination (10-20%) restricts the distribution and therefore limits the available ability of natural source (Purohit et al., 1995 ; Ghorpade et al., 2010). This has resulted in declining the natural stand and production of medicinally important gum-resin as well as active metabolites of boswellic acid. Tissue culture technique is one of the efficient tools to achieve the goals of conservation of rare, endangered and threatened plants (Ahire et al., 2011). 77

2 Till date there is no report on tissue culture studies on Boswellia serrata. In the present investigation in vitro responses was studied and result obtained are presented in this chapter. 5.1 RESEARCH METHODOLOGY MICROPROPAGATION Axillary Bud Break and axillary Shoot Proliferation (a) COLLECTION OF EXPLANT Explants in the form of nodal segments ( cm) were collected from healthy, disease free mature field grown plant of Boswellia serrata. Morphological tall, high fruit bearing superior trees were identified from Ranakpur ( and E) and near Sundha mata temple, Jhalore Rajasthan ( N and E). These trees were marked and explants were collected year round from these marked trees. (b) SURFACE STERILIZATION AND INOCULATION OF EXPLANTS Nodal segments measuring 3.0 to 4.0 cm with axillary buds were first surface-swabbed with 70% ethanol soaked cotton swab and then washed with Tween 80 in gently agitating manner for 5 minutes followed by rinsing with distilled water. Pre sterilization treatments were given to reduce the contamination, where nodal segments were treated with a mixture of aqueous solution of fungicide bavistin (0.2%) and bactericide streptomycin (0.1%) for 10 minutes. For surface sterilization, nodal segments were treated with mercuric chloride (w/v). The surface sterilized nodal segments were rinsed 3-4 times with sterilized distilled water thoroughly in order to remove the traces of sterilant in Laminar Air Flow. The sterilizing agents were tested for different concentrations and different time period. The surface sterilized axillary buds were inoculated on semi-solid MS medium supplemented with cytokinin (BAP/TDZ) were tested either alone or in combination with auxin 78

3 NAA. The ph of the medium was adjusted to 5.8 prior to autoclaving at C for 20 minutes. Cultures were maintained at 28 ± 2 0 C temperature with 16 hours illumination with a photon flux density of 2500 lux from white fluorescent tubes. Effect of season Influence of season of explants collection on in vitro axillary bud break and axillary shoot proliferation was investigated. One calendar year was divided in four seasons viz. March to May, June to August, September to November and December to Februrary. Nodal explants were collected throughout the year and were inoculated on MS medium supplemented with BAP (13.31µM) and NAA (0.54 µm)+ Additives (50 mg/l ascorbic acid+ 30 mg/l citric acid + PVP 100mg/l). Effect of Plant growth regulators In order to study the effect of cytokinin on axillary bud break and axillary shoot proliferation, cytokinins viz. BAP and TDZ was incorporated in full strength semisolid MS medium at different concentrations 6 to 16µM, whereas TDZ was incorporated at lower concentrations ranging from 0.2 to 2 µm in MS medium. IN VITRO MULTIPLICATION OF SHOOTS Various experiment were conducted for in vitro shoot multiplication. The proliferated axillary shoots were excised from the explant and cultured on fresh MS medium. Effect of Plant growth regulators Effect of cytokinin viz. BAP and TDZ were studied on multiplication of in vitro raised shoots. These cytokinins were incorporated in full strength MS 79

4 medium supplemented with different concentration and combination of cytokinin and auxins. Effect of liquid and semisolid medium To compare the effect of liquid and semisolid medium on in vitro shoot multiplication, MS medium supplemented with BAP (8.88µM)+ NAA (0.54 µm) and additives (50 mg/l ascorbic acid+ 30 mg/l citric acid+ PVP 100mg/l); gelled with 0.7% agar (semisolid) and without agar (liquid) was tried. Propagules of four shoots each were cultured MACROPROPAGATION Macropropagation The conventional methods used for vegetative propagation of plants are also referred as macropropagation. Macropropagation is preferred over micropropagation of tree species for its (a) cost effectiveness (b) low requirement of skilled manpower and (c) simple infrastructure requirements. Source and preparation of cutting The cutting were collected from selected plus tree natural growing at sundha mata (Jhalore) and Ranakpur. Cuttings were harvested using sterile pruning scissors in morning time. After harvesting, these cutting were screened for desired diameter cm for semi hard wood cuttings then cuttings were kept in ice box (for prevention from damage) for transportation to laboratory site. After preparation cuttings were given a prophylactic treatment against fungal attack using aqueous solution of 0.1% Bavistin (Carbendazim 50% WP, Systemic fungicide, BASF India Limited, Bombay) for 20 minutes, subsequently washed with distilled water. After fungicidal treatment they were subjected to various auxin treatments Hartmann et al., (1990). 80

5 The discovery of natural plant rooting hormone (Thimann and Went, 1934) for propagation, it has been intuitive to apply these substances to the basal end of cuttings to produce new roots. Five concentration of aqueous rooting solutions i.e. control, 250, 500, 750 and 1000ppm (parts per million) of IBA, IAA and NAA, was prepared in 1N NaOH: autoclaved distilled water (v/v). Each group of cuttings were given hormone treatment by the Basal Long Methods (Kroin, 1992, 2011ab) by dipping 2/3 portion of basal ends of cutting for 15 minutes. Untreated cutting was considered as control and was taken for each rooting media. After auxin treatment, the cuttings were sown in root trainer (500cc) containing rooting media (sand and soil). The 1/3 basal cut portion was inserted in different rooting media. Placing the cuttings in rooting medium is called as "sticking". The medium has to be damp and the surface even. In the present investigation stem cuttings were collected from mature trees and a minimum of 45 cuttings have been used for each treatment. These cuttings were raised in root trainers and kept in mist polyhouse for root induction. Effect of different auxins Effect of different auxins IBA, IAA & NAA, their concentration were studied for rooting of stem cutting. Rooting response was recorded in terms of rooting percentage, average number of roots produced and average root length. Auxin is one of the major endogenous hormones known to be intimately involved in the process of adventitious rooting (Wiesman et al., 1988) and the physiological stages of rooting are correlated with changes in endogenous auxin concentrations (Heloir et al., 1996). High endogenous auxin concentration is normally associated with a high rooting rate at the beginning of the rooting process (Blazkova et al., 1997; Caboni et al., 1997). Auxins have 81

6 been shown to be effective inducers of adventitious roots in many woody species (De Klerk et al., 1999) and are usually synthesized in the stem tip and tender leaves of aerial parts of plants and then transported to the action site (Ljung et al., 2001). When applying exogenous auxin on cuttings, the endogenous auxin concentration reaches a peak after wounding (Gaspar et al., 1996; Gatineau et al., 1997) coinciding with the initiation of the rooting process. The widely used sources of growth hormones for rooting of cuttings are the IBA (indole-3-butyric acid), IAA (indole-3-acetic acid) and NAA (αnaphthalene acetic acid) and commercialized root promoters (root-growing powders). IAA was the first to be used to stimulate rooting of cuttings (Cooper, 1935) and soon after another (IBA) auxin which also promoted rooting. IBA was discovered and was considered even more effective (Zimmerman and Wilcoxon 1935). The application of exogenous plant growth regulators, as the auxins group, can increase the success of adventitious rooting in vegetative propagules collected from selected genotypes of Eucalyptus indole-3-butyric acid (IBA) is the widely used (Wendling et al., 2000; Wendling and Xavier 2005; Almeida et al., 2007; Schwambach et al., 2008; Brondani et al., 2010; Wendling et al., 2010). The objective of the present study is to investigate the effect of different auxin and their concentrations on root induction in stem cuttings of Boswellia serrata. Growth conditions The cuttings were kept under intermittent mist (misting flow for 60 sec in 30 min of interval on a root trainer) under relative humidity (60-80%) and temperature (25-30 C/15-20 C day/night). The cuttings were regularly watered and treated with 0.1% Bavistin to avoid desiccation damage and attack of pathogens at every 15 d interval. The rooting experiment was run for 60 d. Then rooted cuttings were transferred to polythene bags (16 x 28 cm) containing soil + FYM (field yard manure) (5:1) and kept in poly house for 15 to 20 d. The polythene bags were moved daily in order to minimize misting 82

7 variation. After this, polythene bags containing rooted mini-cutting were transferred to Agro shade house for hardening. In Agro-shade house, plants were manually irrigated by tap water once in a day. After days of hardening, plants were planted in field and were manually irrigated by tap water once in a week. Analysis of rooting The percentage of rooting was recorded by observing all mini-cuttings of each replication. Roots from five propagules of each replication were randomly collected to study rooting percentage, number of roots and root length (cm). 5.2 Results Micropropagation Axillary Bud Break and axillary Shoot Proliferation Disinfection of nodal segments Nodal segment containing axillary buds were surface disinfected with 0.1 % and 0.2% HgCl 2 for 5-10 min. It was observed that, pretreatment of nodal segments with 0.2% bavistin prior to mercuric chloride treatment improved the surface disinfection of the axillary buds. Best result of 83.33% viable aseptic explants was obtained when a pretreatment of 0.2% bavistin and bactericide streptomycin (0.1%) for 10 minutes was used. Followed by surface disinfection with 0.1% HgCl 2 for 7 min was given to nodal segments (Table-1). It was observed that 0.2% HgCl 2 gave increase percentage contamination free explants, but the treatment was stronger as compared to 0.1% HgCl 2, causing injury to the explants, more phenolic exudation and ultimate death of nodal segments. Hence to obtain healthy and viable nodle segments, disinfection with 0.1% HgCl 2 for 7 min was adopted. Leaching out of phenolics from the cultured nodal segments was the problem for axillary bud break. The cultured nodal segment produced 83

8 phenolics which caused browning of nodal segments as well as the surrounding medium. To avoid browning problem and subsequent death of explants, incorporation of adsorbents PVP 100mg/l, antioxidant (50 mg/l ascorbic acid, 30 mg/l citric acid ) or rapid subculturing (in every three days) helped to overcome the effect of phenolic exudation. Seasonal effect of explant collection Explants collected in different seasons of the year showed variation in percentage of axillary bud break response. It was found that nodal segments collected during September to November showed maximum bud break response (57.88%) on MS medium supplemented with BAP (8.88µM)+ NAA (0.54 µm) and additives (50 mg/l ascorbic acid+ 30 mg/l citric acid+ PVP 100mg/l) (Table-30). Nodal segments collected in other months produced reduced axillary bud break response either due to contamination of explants, phenolic exudation or due to the presence of dormant meristem during cold season. Nodal segments collected during February to April gave 37.78% bud break and during the rainy season (June to Agust) 22.22% bud break response was obtained. Explants collected during rainy season were more susceptible to contamination %bud break was obtained from nodal segments collected during winter season (December to February).Thus results obtained showed that February to April was the appropriate time for nodal explants collection and axillary bud break response. Effect of phytohormones Two cytokinins (BAP and TDZ) were tested individually at different concentration for axillary bud induction. Result exhibited that bud induction strongly influenced by the presence of cytokinin, as on cytokinin free medium. 84

9 MS medium supplemented with BAP (8.88µM) and additives (50 mg/l ascorbic acid+ 30 mg/l citric acid+ PVP 100mg/l) gave highest bud break induction of 37.78% with an average shoot length (Table 31). Percentage response was influenced by the concentration of BAP in the meedium. At low concentration 4.44µM BAP in MS medium, bud break response decreased and was 11.11%, at increase concentration of BAP in the medium, also resulted in decreased percentage response of 17.77% at 17.76µM BAP supplemented MS medium. Nodal segments cultured on TDZ supplemented MS medium did not enhance the axillary bud break response. Maximum response of axillary bud break was only 20.00% and the average number of axillary shoots proliferated was only 1.44 (Table-32; Plate32). At increased concentration of TDZ in MS medium it resulted in decreased bud break response, as at 1.82µM TDZ only 17.78% explants showed bud induction. Effect of cytokinin-auxin interaction Addition of small doses of auxin (NAA) along with BAP increased the axillary bud induction with some callusing from the cut end of nodal segment. The bud break response of 57.78% was obtained on MS medium supplemented with 8.88µM BAP µm NAA+ additives (50 mg/l ascorbic acid+ 50 mg/l adenine sulphate + 30 mg/l citric acid+ PVP 100mg/l). Any increase in concentration of auxin NAA ( µm) in same composition of MS medium resulted in high callusing from the basal cut end of the explants. These proliferated development of axillary shoots were excised from the mother explants and sub-cultured on MS medium for the establishment of in vitro shoot cultures (Table-33; Plate 31). 85

10 IN VITRO SHOOT MULTIPLICATION Effect of plant growth regulators The proliferated in vitro axillary shoots were excised from mother explants after second subculture (8weeks) and these axillary shoots were subcultured on semisolid MS medium supplemented with cytokinin (BAP and TDZ,) & in combination of NAA for the multiplication of in vitro shoots. It was observed that proliferated shoots were not able to multiply in semisolid and liquid MS medium supplemented with cytokinin alone and in combination with NAA. limited success was obtained towards in vitro shoot multiplication in the present case. The axillary shoots when transferred on MS medium supplemented with different concentration of BAP (2.22µM to 8.88 µm) alone survived for 8 weeks (two subculture cycle) with development of 1-2 new in vitro shoots. However, beyond this the leaves turned brown and start falling on the medium followed by stem necrosis leading to death of in vitro shoots. A number of hormonal combination were tried in combination with BAP in MS medium (BAP+ Kn; BAP+IAA; BAP + Zeatin; BAP + NAA etc) to overcome the problem of leaf fall and necrosis in general, so as to proceed with in vitro multiplication of shoot. All attempt failed, except in BAP+ NAA supplemented MS medium in which 3-4 new in vitro shoot developed and culture s survived for 3-4 months (Plate 33). Kn alone (4.6, 9.29, µm) in MS medium did not helped in multiplication of in vitro shoots. The axillary shoot transferred on this media failed to grow and multiply and later died. TDZ supplemented MS medium (0.04, 0.23 and 0.45µM) helped limited in vitro shoot multiplication. However in vitro multiplied shoot did not grow further remained green for next 3-4 subculture cycles and later died (Plate 34). 86

11 All attempts made for in vitro shoot multiplication such as use of different source of energy (carbohydrate) in MS medium, addition of different types of amino acids like cystine, proline, L-arginine, L- asparagine in MS medium, addition of antioxidants, silver nitrate, adenine sulphate and use of different set of physical condition (light & temperature) could not induce in vitro shoot multiplication which evidently means that culture need a different set of Physico-chemical requirement. Macropropagation Clonal propagation technique plays an important role in conservation and multiplication and also large scale production of an endangered forest tree species. This technique is most valuable for true-to-type of plant production. There are several method present for vegetative propagation of trees. But, rooting of stem cutting is the most convenient, less expensive and in many cases it has already been proved to be successful for mass multiplication of selected trees. Auxin have been shown to promote the rooting of shoot cuttings of many woody plants. (Nanda 1968; Hartmann and Kester 1983; Pal 1988). The cuttings were collected from selected mature tree of year age, naturally growing near Sundha mata temple and Ranakpur (Rajasthan) during October-November. Untreated cutting was considered as control (cuttings were dipped in distilled water serves as control) and was taken for each rooting media. The Basal Long Soak Methods were used for treating cuttings (Kroin 1992; 2011ab). After auxin treatment, the cuttings were sown in root trainer (500cc-conical cells) containing rooting media (sand and soil). The one third basal cut portion was inserted indifferent rooting media. Placing the cuttings in rooting medium is called as sticking. The medium was made damp with autoclaved water and the surface was made even. Holes were punched into the medium to allow the insertion of the cuttings 87

12 without damaging the cambium or removing the rooting hormone. After the cuttings were stuck, the rooting medium was pressed slightly around them. Experiments were repeated thrice and data represents the mean of three experiments. Each treatment consisted of minimum 15 cuttings. In present finding, among all the auxins IBA, IAA and NAA used, IBA showed better result. Stem cutting of Boswellia serrata were treated with 500ppm IBA for 15min which gave better result in combination with sand: soil (1:1) rooting media. The data (Table-34) revealed that cutting planted in sand:soil rooting media and 500 ppm IBA gives significantly improved result in terms of rooting percent (60%), number of roots (6.11) and root length 6.10cm per rooted cutting. At increased concentration of IBA, it resulted decreased rooting percentage, as at 1000ppm 28.88% stem cutting showed rooting (Plate 37). Rooted stem cutting were transferred to polybags containing sand:soil: FYM (1:1:1), after 2-3 months transferred in the field (Plate 39,40). When stem cuttings were treated with different concentration of IAA & NAA, the cuttings showed small amount of rooting % and sprouting, and the cutting survived for nearly 10 day. (Plate 38) 88

13 Table-30 Effect of different treatments on surface disinfection of nodal segment collected from mature plant of Boswellia serrata. Data recorded after 4 weeks. Presterilization treatment Sterilization Time duration (min.) % Explants with contamination % of Aseptic cultures % HgCl % Bavistin % Streptomycin 0.2% HgCl Table-31 Effect of seasons on axillary bud proliferation of Boswellia serrata. Nodal segment were inoculated in MS medium Supplemented with 8.88µM BAP µm NAA + antioxidant. Data recorded after 4 weeks. Mean shoot Mean Shoot Seasons Response % number Length (cm) Mar-May ± 2.22 b 1.65 ± 0.12 b 1.89 ± 0.06 ab Jun-Aug ± 2.22 c 1.70 ± 0.15 ab 1.82 ± 0.08 ab Sept.- Nov ± 2.22 a 2.04 ± 0.08 ab 1.99 ± 0.05 a Dec.-Feb ± 2.22 c 1.60 ± 0.16 a 1.70 ± 0.08 b ** = P<0.01, *= P<0.05 Values with common superscript are not significantly different at P<0.05, Duncan s multiple range test. 89

14 Table-32: Effect of BAP on in vitro axillary bud induction of Boswellia serrata. MS medium supplemented with antioxidant. Data recorded after 4weeks. Mean shoot Mean Shoot BAP Response % number Length (cm) MS 0.00 ± 0.00 d 0.00 ± 0.00 c 0.00 ± 0.00 c ± 2.22 b 1.40 ± 0.24 ab 1.10 ± 0.06 b ± 2.22 a 1.82 ± 0.13 a 1.67 ± 0.07 a ± 2.22 b 1.42 ± 0.11 ab 1.31 ± 0.06 b ± 2.22 c 1.25 ± 0.16 ab 1.13 ± 0.05 b F= 44.09** F= 3.40** F=12.78** ** = P<0.01, *= P<0.05 Values with common superscript are not significantly different at P<0.05, Duncan s multiple range test. Table-33: Effect of TDZ concentration on in vitro axillary bud induction of Boswellia serrata MS medium supplemented with antioxidant. Data recorded after 4 weeks. Mean shoot Mean Shoot TDZ(µM) Response % number Length (cm) MS 0.00 ± 0.00 c 0.00 ± 0.00 c 0.00± 0.00 d ± 2.22 b 1.10± 0.10 b 1.13± 0.03 bc ± 0.00 a 1.44± 0.12 ab 1.25± 0.03 b ± 2.22 ab 1.79± 0.14 a 1.66± 0.07 a ± 2.22 b 1.38± 0.18 ab 1.05± 0.06 bc F= 34.50** F= 3.27** F=16.83** ** = P<0.01, *= P<0.05 Values with common superscript are not significantly different at P<0.05, Duncan s multiple range test. 90

15 Table-34: Effect of BAP interaction with NAA on in vitro axillary bud induction of Boswellia serrata. MS medium supplemented with antioxidant. Data recorded after 4weeks. Mean shoot Mean Shoot BAP+NAA(µM) Response % number Length (cm) MS 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00± 0.00 e ± 2.22 b 1.25 ± 0.16 b 1.26 ± 0.04 d ± 2.22 a 2.08 ± 0.08 c 1.98 ± 0.08 a ± 2.22 b 1.89 ± 0.12 a 1.47 ± 0.40 b ± 2.22 c 1.25 ± 0.16 b 1.18 ± 0.04 c F= ** F= 12.48** F=46.26** ** = P<0.01, *= P<0.05 Values with common superscript are not significantly different at P<0.05, Duncan s multiple range test. Table-35: Effect of IBA on rooting percentage, number of root, root length of Boswellia serrata stem cutting. IBA (ppm) Response % Mean root number Mean Root Length (cm) control 0.00 ± 0.00 e 0.00 ± 0.00 d 0.00 ± 0.00 e ± 2.22 d 1.25 ± 0.16 b 1.26 ± 0.04 d ± 2.22 a 2.08 ± 0.08 c 1.98 ± 0.08 a ± 2.22 b 1.89 ± 0.12 a 1.47 ± 0.40 b ± 2.22 c 1.25 ± 0.16 b 1.18 ± 0.04 c ** = P<0.01, *= P<0.05 F= ** F= 12.48** F=46.26** Values with common superscript are not significantly different at P<0.05, Duncan s multiple range test. 91