{'fraction_non_alphanumeric': 0.035982420802050906, 'fraction_numerical': 0.02426295550265519, 'mean_word_length': 4.6890625, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Influence of Plant Growth Regulators on Micropropagation of Gwarpatha [Aloe vera (L.) Burm.]

Desert medicinal plant Gwarpatha (Aloe vera L. syn. Aloe barbadensis Miller) belongs to family Asphadelaceae (Souza and Lorenzi 2005). It is an ancient, semi tropical medicinal plant indigenous to Africa, Madagascar and Arabia and introduced plant in India. This genus has more than four hundred species of flowering succulent plants. Aloe vera is coarse looking evergreen perennial plant, grows up to 80-100 cm in height, with a strong fibrous root and at large stem supporting a rosette of narrow lanceolate leaves. The leaves are whitish green on both sides and bear spiny teeth on the margins. The International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 3 (2020) Journal homepage: http://www.ijcmas.com

yellow to orange drooping flowers produced in an inflorescence of 90-110 cm tall, each flower is pendulous, with yellow tubular corolla 2-3 cm and hermaphrodite. It has valuable medicinal properties and is commercially used in pharmaceuticals, cosmetics, food industries, as nutraceuticals and for many herbal preparations. There are about more than 40 Aloe based formulations being marketed in the global market. In India, tissue culture research began nearly six decades ago with the first report on production of test tube fertilization. Plant cell and tissue culture is defined as the capability to regenerate and propagate plants from single cells, tissues and organs under sterile and controlled environmental conditions (Murashige and Skoog, 1962). Tissue culture methods have also been employed to study the basic aspects of plant growth, metabolism, differentiation and morphogenesis and provide ideal opportunity to manipulate these processes. Advancement in tissue cultural methodology led many recalcitrant plants amenable to in vitro regeneration and to the development of haploids, somatic hybrids and pathogen free plants (Gupta et al., 2014).
In nature (in vivo), Aloe vera is propagated through lateral buds which are slow, very expensive and low income practice (Meyer and Staden, 1991). Sexual reproduction by seeds due to male sterility in Aloe vera is almost ineffective and vegetative propagation through lateral shoots only possible during growing seasons (Keijzer and Cresti, 1987). Thus large scale plantation of Aloe vera through in vivo system of plantlet multiplication is insufficient to meet the requirements of farmers and pharmaceutical industries demand (Bhandari et al., 2010). There is a need to develop suitable and alternative method for large scale propagation for rapid plant multiplication to meet the demand of farmers and pharmaceutical industries (Abrie and Staden, 2001). Therefore, the present study aimed to develop an alternative protocol for rapid and high frequency in vitro propagation of Aloe vera, looking through its increasing demand for pharmaceutical industry at global level.

Materials and Methods
The present investigation was carried out at Tissue Culture Laboratory of Department of Plant Breeding and Genetics, S.K.N. Agriculture University, Jobner, Rajasthan, India during 2018-19.

Plant growth regulators
Different concentrations of plant growth regulators were incorporated singly and in combinations in the MS medium for direct shoot proliferation from auxiliary explants.

Induction of roots
Explants were inoculated for induction of roots in the medium supplemented with different concentrations of plant growth regulators added alone or in combinations.
1. Plant growth regulators incorporated singly in the medium NAA/IBA: 0.5, 1.0, 1.5 and 2.0 mg/l. 2. Plant growth regulators incorporated in combinations: NAA: (0.5, 1.0, 1.5 and 2.0 mg/l) + IBA: (0.5 and 2.0 mg/l) MS basal medium without supplementation of any growth regulator was used as a control in all the experiments. All the cultures were maintained in an air conditioned culture room at a temperature of 25 ± 2 0 C under fluorescent light in a 14:10 hours' photoperiod.

Acclimatization of plantlets
After 40-50 days of culture on rooting media, the plantlets were shifted to polythene bags/earthen pots for their hardening prior to final transfer to soil in natural conditions. After removing media, these were dipped in 1% w/v solution of Bavistin to prevent any fungal infection to newly developed plants. After Bavistin treatment the plantlets were carefully planted in polythene bags/earthen pots containing 1:1 mixture of soil and vermicompost. Then the plants were shifted to shade house with indirect sunlight. After 50-60 days, shade house plants were finally transferred to the field.

Observations recorded
Observations on period of initiation of shoot, number of shoots per explant, morphogenetic response (Percent), shoots length (cm), number of days taken for root induction, number of roots, root length (cm), mean days taken for callus initiation, colour and texture of the callus are recorded following standard procedures.

Statistical analysis
Each experiment was conducted in completely randomized design and data were analyzed for means and standard error accordingly as described by Snedecor and Cochran (1972). Standard error was calculated only after value transformation for the characters where response was less than 100 per cent. The value for each replication was transformed by square root transformation as follows Where, Y= original value Tests of significance were done according to Duncan's Multiple Range Test (DMRT) for different traits (Gomez and Gomez, 1984).

Effect of plant growth regulators on shoot proliferation
BAP was used as cytokinin for shoot bud proliferation in the present investigation. The medium devoid of growth regulators failed to initiate bud break or callus formation from any of the explant. BAP when incorporated singly in the basal MS medium, induced multiple shoot buds at all the levels (Table 1), however the most profuse shoot bud breaks (12.8) and shoot length (8.9 cm) were obtained at 4.0 mg/l BAP ( Fig. 1) followed by 6.0 mg/l BAP (11.2). Slight to medium loose/semi compact callus was also induced at the base of auxiliary explants incubated at different levels of BAP (1.5-2.5 mg/l). Lower (0.5-1.0 mg /l) and higher (3.0-6.0) levels of BAP inhibited callus differentiation. These results were in close agreement with other reports of micropropagation of Aloe vera (Ahmed et al., 2007 andDwivedi et al., 2014). Eufrocinio and Malasa (2005) in A. barbadensis reported best multiple shoot bud induction at medium containing 1.0 mg/l BAP. However, these results were contrary to the results of present investigation with the level of BAP might be due to differences in species and explants used in the particular study. Callusing was also observed at the base of auxiliary explant in present investigation which was similar to the observations of Kawai et al., 2006 andYadav, 2008 in Aloe arborescens and Aloe vera, respectively. Auxin (BAP) combined with cytokinin (NAA) enhanced the shoot bud multiplication considerably (Table 2). Highest shoot proliferation (12.6) was observed at medium supplemented BAP (4.5 mg/l) with NAA (0.6 mg/l) (Fig. 2). These were at par with the level of BAP (4.0 mg/l) where maximum shoot proliferation was observed upon supplementation in the medium alone. Callus formation was completely inhibited at the base of explants in the medium supplemented with combination of BAP and NAA.
These results are in accordance with the results obtained by Khanam et al., (2014). They reported that a perfect combination of auxin and cytokinin is needed for optimum shoot induction. MS basal medium in combination with 4.0 mg/l BAP and 0.2 mg/l NAA was found to be the best on which explants began to show emergence of shoot buds within one week. Similar observations were also found by Nayankantha et al., (2010) and Kiran et al., (2017), who reported synergetic effect of BAP and NAA in shoot bud break.

Effect of plant growth regulators on root induction
Root induction was observed at all the levels of IBA and NAA added either singly or in combination with 100 per cent frequency (Table 3). Maximum number of root induction (6.6) was obtained in the medium supplemented with 1.5 mg/l IBA with shoot length (7.64 cm) followed by (5.4) 1.0 mg/l IBA (Fig. 3). In IBA supplemented media, roots were thin and long, whereas thick roots were induced in medium supplemented with NAA (Fig. 4).
In combination, maximum number of roots (5.6) was observed at 0.5 mg/l IBA with 2.0 mg/l NAA with longest root length (8.3 cm) followed by 0.5 mg/l IBA + 1.5 mg/l NAA (Fig. 5). In comparison to NAA, IBA produced higher number of roots at all the levels except at 2.0 mg/l IBA. There was significant difference among the levels of IBA and NAA for number of root induction per explant. Results of present investigation were similar to the reports of Abrie and Staden (2001) in Aloe polyphylla with respect to type of plant growth regulator (IBA), they observed maximum root on medium supplemented with 0.5 mg/l IBA.
However, in the present study maximum root induction was ovserved at 1.5 mg/l IBA. This might be due to difference in the species under study. Hashemabadi and Kaviani (2008) found the best root induction at MS medium supplemented with 1.0 mg/l IBA with 1.0 mg/l NAA. Khanam et al., (2014) reported profuse root induction after one week of culture at 2.0 mg/l IBA and 1.0 mg/l NAA. However, these levels were not tested in the present investigation.

Acclimatization of plantlets
After 40-50 days of culture of in vitro developed shoots on most responsive rooting media were shifted to pots filled with mixture of 1:1 ratio of soil and vermicompost for their hardening prior to final transfer to soil, showed good percentage of survival (80 per cent) in culture room (Fig. 6). In shade house plants also showed 74 per cent survival rate (Fig. 7).
The growth and elongation of plants were less in culture room whereas, in shade house growth of plants was better and they also started to elongation and thickening of leaves in shade house. Perusal of literature on micropropagation of Aloe also indicated that rooting and hardening had never been encountered as a problem in Aloe's tissue culture. Various researchers have reported successfully in vitro rooting and hardening of Aloe vera (Kalimuthu et al., 2010 andRathore et al., 2011) and corroborate present study.     In the present investigation we did not investigate all the factors which in one way or other way block or stimulate micropropagation potential in Aloe vera, thus in the light of these factors we feel the prolonged and intensive in vitro efforts are needed to exploit the micropropagation in Aloe vera.
However, results of the present study may be recommended to develop protocols for mass multiplication of Aloe vera to meet the demand of farmers and pharmaceutical industries.