Indole -3- Butyric Acid Induces Plant Regeneration From Stem Cuttings Of Three Medicinal Plants

Field regeneration of three (3) medicinal plants Securidaca longepedunculata (violet tree), Ocimum gratissimum (scent leaf) and Pterocarpus mildebraedii by means of rooting of stem cuttings at different lengths of 10 -15 cm and 15 -20 cm were studied. The effect of the plant hormone Indole-3-Butyric acid (IBA) at 5g/l was tested on rooting, bud sprout and leaf formation of the rooted stem cuttings of the plant species as well as the effect of the length of the stem cuttings on their growth and survival rates. The results of the study revealed that stem cuttings of O. gratissimum treated with IBA and the control treatment sprouted within 5 8 days. The treated O. gratissimum stem cuttings produced a slightly higher number of buds (2.58 ± 0.86) when compared to the control (2.00 ± 0.89) with no significant difference at P ≤ 0.05. Stem cuttings of the treated P. mildbraedii cuttings sprouted within 8 -10 days with 1.40 ± 0.37 number of buds, with the control showing no sprouts. Consequently, IBA application produced more leaves (13.00 ± 5.58) and roots (1.33 ± 0.01) than the control. However, O. gratissimum stem cuttings had the highest leaf (13.08 ± 4.47) and root numbers (135.00 ± 13.45) followed by the control with values13.00 ± 5.58 for leaf development and 61.66 ± 2.34 for root number respectively. Irrespective of the treatments, none of the stem cuttings of S. longepedunculata sprouted. This study showed that exogenous application of IBA to stem cuttings of the tested plants, except S. Longepedunculata, improved the root number, number of leaves and number of buds.


Introduction
From time immemorial, plants have been an important source of food, fibre and medicine (Nweze et al., 2004;Thangavel et al., 2014).
According to the World Health Organization (WHO), about 50% of people still rely primarily on traditional remedies such as herbs for their medicine and 65% of Indian populace domicile in rural areas use traditional form of medicine to meet their primary health needs (Alice et al., 2014). Of the medicinal plant species, about 10% are cultivated while the larger proportion (90%) are obtained from the wild. The domestic cultivation of medicinal plants is a well-accepted way of producing plant materials both for conservation and other relevant purposes (Tandon et al., 2009). However, harvesting from the wild poses serious challenges as was seen in cases of many plant species that became threatened and this led to the loss of genetic diversity and habitat destruction, disrupting biodiversity which is the store house of species richness (Afolayan and Adebola, 2004;Kuldeep et al., 2012). A lot of resources have been channelled into curbing the loss of biodiversity and various protocols have been successfully established.
Unfortunately, not much improvement has been noticed (Tripathi, 2008). Due to improved agronomic and medicinal traits, it has been reported that the development of regeneration methods is of major importance in medicinal plant biotechnology. Plant genetic resources are the major biological basis of world food security which, to a great extent, support every life on the planet earth. Hence, conservation of such a buffer is considered fundamental and provides priority in all sectors of global development (Tandon et al., 2009).
Plants are totipotent in nature, hence plant biotechnology takes advantage of this property of plant development to solve the issue of decreasing plant biodiversity (Slater et al., 2003).
When living organisms are injured or lose parts of their bodies, many are able to regenerate new tissues or organs to minimize the impact of the local damage (Ikeuchi et al., 2016), but the mode of this regeneration varies markedly among taxa (Birnbaum and Sánchez-Alvarado, 2008). The influence of plant growth regulators on plant regeneration has been reported in several medicinal plants by different researchers (Faridah et al., 2011, Sanoussi et al., 2012, Onyeulo et al., 2018. Regeneration is a widely conserved physiological response in both animals and plants (Pulianmackal et al., 2014) and has long been utilized for clonal propagation in the form of cutting and grafting (Melnyk and Meyerowitz, 2015). The regenerative capacity of plant cells can be enhanced when explants are soaked in solutions of plant hormones (Chupeau et al., 2013) with explants from juvenile plants regenerating shoots more effectively than those from the mature plants (Zhang et al., 2015).
Indole-3-Butyric Acid (IBA), a plant hormone in the "auxin" group is naturally abundant in potato tuber peels (Ludwig, 2000). It is applied exogenously and has been used successfully as a plant growth regulator promoting rooting (Hunt et al., 2011) and exerting different effects on plant growth and development, e.g. regulating responses of plants against biotic and abiotic stresses (Tognetti et al., 2010). Treating cuttings with auxins increases the percentage of rooting, root initiation, number of roots and uniformity of rooting (Elhaak et al., 2015).
Securidaca longipedunculata (Violet tree) of the family Polygalaceae is a medicinal herb indigenous to Africa (Alafe et al., 2014) (Aguiyi et al., 2000;Elizabeth et al., 2012). In Nigeria, it is found in the Savannah and coastal areas and it is called with various local names such as 'Efinrin' in Yoruba, 'Diadoyal' in Hausa and 'Nchuanwu' in Igbo (Owulade, 2004). It has been used in the treatment of various ailments including diabetes mellitus (Aguiyi et al., 2000), rheumatism, paralysis, epilepsy, high fever, sunstroke, influenza, gonorrhoea and mental illnesses (Soumen et al., 2012). It has antihyperglycemic effects (Simon and James, 2007), so it is transformed to herbal tea and used as anti-diabetic therapy (Mohammed et al., 2007). Pterocarpus mildbraedii of the family Fabaceae is a green leafy vegetable locally known as "Oha ojii" in eastern Nigeria (Rosemary et al, 2015). According to Alice et al., (2014) and Ngwuli et al., (2019), it is indigenous to Africa. Its leaves are used for food and medicinal purposes. It is endowed with antioxidant, hypocholesterolemic, chemo-protective and anti-bacterial properties (Rosemary et al, 2015).
Reports on the methods of regenerating S. longepedunculata Fresen using the stem cuttings are lacking. P. mildbraedii Harms and O. gratissimum L whose leaves serve as a source of food and medicine have been reported to be difficult to root using loamy soil, hence the need for the current study. This study therefore is necessary so as to provide an efficient and quick means of regenerating these plant species so as to curb the problem of extinction. Rooting of stem cuttings has proven to be the fastest method of producing large quantities of uniform seedlings, contributing significantly to the conservation of species, future biotechnological exploitation of its medicinal properties and food security, as well as making the species available for future research activities.

Plant collection and Soil composition
Three medicinal plants -S. longepedunculata, P. mildbraedii and O. gratissimum were used in this study. The branches of S. long epedunculata and P. mildbraedii were obtained from Nkalagu in Enugu State, Nigeria while that of O. gratissimum was obtained from the Botanical Garden of Plant Science and Biotechnology, University of Nigeria. The rooting medium was composed of Loamy soil (LS), Sawdust (SD) and Poultry manure (PM) in the ratio of 2:1:1, respectively, all placed in polythene bags and measured to the weight of 12 kg each. Indole-3-Butyric Acid (5g/l) (Sigma-Aldrich) was prepared according to the manufacturer's instruction.

Sample preparation
The branches of S. longepedunculata, P. mildbraedii and O. gratissimum were defoliated and cut at varying lengths of 10-15cm and 15 -20cm long, each bearing nodes. A total number of 52 stem-cuttings were used in this study consisting of 32 treated stem cuttings (10 cuttings of P. mildbraedii, 12 cuttings of O. gratissimum and 10 cuttings of S. longepedunculata) and 20 stem cuttings (5 cuttings of P. mildbraedii, 5 cuttings of O. gratissimum and 10 cuttings of S. longepedunculata.) served as control.

Sample Pre-treatment and Plant Procedure
The stem cuttings were treated with the rooting hormone IBA (5g/l) and soaked for 24hrs before planting in an erect position in the rooting medium according to the methods of Sanoussi et al., (2012). The experiment was carried out in a randomized complete block design (RCBD) and set up under a natural partial shade. This was mechanically irrigated daily and the resulting weeds handpicked while observations were made for signs of metabolic activities on the cuttings. The study was carried out during the rainy season (July to September, 2019) under a natural partial shade in the Botanical garden of the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka.

Observations and data collection
The parameters observed include Period of bud sprouting, number of sprouted buds per stem cutting, period of bud development into photosynthesising leaves, the number of leaves produced which were counted three weeks after planting at intervals of 7 days, leaf lengths and leaf diameters, root formation and effect of stem cutting length on seedling growth and survival. The resulting data were collected and documented.

Statistical analysis
Data collected were analyzed using one way Analysis of variance (ANOVA) at 95 percent probability levels (P≤0.05).

Results and Discussion
The results of the determination of periods of initial and final bud break varied among the stem cuttings of the 3 medicinal plants studied. The stem cuttings of O. gratissimum (both treatments and control) had the earliest period of bud sprout (5-8 days after planting) as seen in fig.1 and plate 1. This was followed by the treated stem cuttings of P. mildbraedii (8-10days after planting) as seen in fig. 1 and plate 2 with the control showing zero sprout. However, both the treated and the control stem cuttings of S. longepedunculata showed zero sprout throughout the study period. From fig.1  This is in agreement to an earlier report of Alagesaboopathi (2012) on Andrographis macrobotrys. Alagesaboopathi (2012) stated that the ability of the treated cuttings of A. macrobotrys to produce higher numbers of bud sprouts could be as a result of the combined effects of the applied rooting substances and inherent hormones in the plant. On the other hand, S. longepedunculata showed zero sprout. Table 1 shows the number of buds produced by the stem cuttings during the study period presented in their mean ± standard error values. These variations ranged from 0.00±0.00 to 2.58±0.86.
In   P. mildbraedii stem cuttings produced less number of leaves than O. gratissimum. The stem cuttings of P. mildbraedii (control) and that of S. longepedunculata produced no leaves. However, not all buds that sprouted in the stem cuttings of O. gratissimum and P. mildbraedii developed into leaves. The inability of these sprouted buds to develop into leaves is in agreement with the works of Nzekwe (2002) and Sani et al., (2016).
They revealed that not all buds that sprouted on the stem cuttings of Irvingia wombolu and Moringa oleifera persisted till leaf development.
Again, there is a significant difference between the treated and the control stem cuttings of P. mildbraedii. These variations ranged from 5.00 ± 1.58 to 9.75 ± 2.02 for observations made after 28 days of planting and 9.40 ± 3.05 to 13.08 ± 4.47 for observations made after 49 days of planting. 0.00 ± 0.00 0.00 ± 0.00 b 0.00 ± 0.00 b 0.00 ± 0.00 b Securidaca T1 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 T2 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 where cuttings of 15 -20 cm length sprouted earliest and had vigorous growth than cuttings of 5 and 10 cm length. The better growth result obtained in stem cuttings lengths of 15 -20 cm in the studied plants can be related to different factors such as level of endogenous auxins and other growth regulators which may be lower in small-sized cuttings thereby resulting to reduced growth percentage, the presence of more nodes on the stem cuttings with longer lengths resulted to more bud sprout and leaf formation (Hamilton et al, 2009). This however, implies that the length of the stem-cuttings contributes to the vegetative growth of the plant.
In determination of the number of roots per stem cutting (Table 3), the treated O. gratissimum had the highest number of roots followed by the control. The treated P. mildbraedii stem cuttings produced roots as well. This agrees with the previous reports of Tchoundjeu et al., 2002;Teklehaimanot et al., 2004;Sanoussi et al., 2012 who revealed that treating stem-cuttings by dipping in auxin solutions ranging from concentrations of 2.5 to 5 g/l or when sprinkled with rooting powder stimulate the rhizogenesis of Prunus africana, Osyris Lanceolatata and Vitex donania stem cuttings, respectively . This, however, showed that treating stem cuttings with a rooting hormone such as IBA is more adequate for obtaining best responses by the stem cuttings in terms of root production. The inability of P. mildbraedii (control) to produce roots can be