Effects of Phytohormone and Regulators on Shoot Tip and Nodal Explants for In Vitro Shoot and Root Clonal Propagation of Vitex negundo L

Medicinal plants are one of the most vital natural resources, but many of them are currently endangered due to habitat loss. Consequently, it is critical to emphasize the importance of using micropropagation techniques for mass propagation of plantlets on a commercial scale, in addition to germplasm conservation and distribution. Nodal explants and shoot tips were expunged from 15 days of the explant by aseptic seedlings, an effective, quick, and better in vitro plant regeneration procedure for Vitex negundo L. has been developed. The recent study was considered to develop an in vitro procedure for the regeneration of V. negundo L., a traditional medicinal plant. Nodal segments and shoot tips were cultivated on MS medium enhanced with numerous plant growth regulators. For multiple shoots and root regeneration, various cytokinins were examined. 6-benzylaminopurin (BAP), kinetin (Kin), and 1H-indole-3-butanoic acid (IBA) were all tested as a supplement to Murashige and Skoog (MS) medium including auxin phytohormone, such as Indole acetic acid (IAA) and 1naphthaleneacetic acid (NAA). The furthermost effective surface sterilization treatment for explants of V. negundo has been found 0.1% HgCl2 for 8 minutes. In all treatments, multiple shoots were collected from shoot tips and nodal segments. In MS media added with 2.0mg/l BAP, the most shoots were seen in V. negundo. Furthermore, V. negundo regeneration shoots rooted effectively in half MS containing 1.0 mg/l IBA. Finally, proliferated plantlets were effectively adapted in soil, where they grew normally without morphological anomalies and had a survival rate of 92 percent.

aseptic clonal propagation over traditional methods is the ability to develop a large numeral of plants from a single plant in a short amount of time and space (Zhi-Qiang et al., 2020).
Micropropagation has been used to propagate a variety of plant species, including several therapeutic plants (Lyndsey et al., 1986;Murashige, 1977). The regeneration of therapeutic plants such as Catharanthus roseus, Cinchona ledgeriana, and Digitalis spp., as well as Rehmannia gluthinosa, Rauvolfia serpentina, and Isoplexis canariensis from the shoot and stem meristems, has shown encouraging results. (Paek et al., 1995;Perez et al., 2002;Salma et al., 2008). The high quality of such medicinal plants could be capable to fulfill both the desires of our pharma companies and our basic needs (Wenle et al., 2020). V. negundo (Linn.) is a well-known traditional medicinal plant with a wide range of pharmacological properties (Yo-gendra et al., 2020). The plant includes a wealth of phytochemicals or secondary metabolites from root to fruit, giving it an unmatched range of medical applications (Neha et al., 2021). The current study deals with the comparative performances of a variety of cytokinins including IBA, BAP, and Kinwere all tested singly or in numerous mixtures to MS medium with also the various combination of NAA, and IAA on the in vitro revival of root and shoot by using shoot tips and nodal segmentsof V. negundo by successful restoration plants into field environments.

Explants preparation and inoculation -
The explant source was V. negundo, which was found from a commercial field in Gulshan in the Dhaka district, Bangladesh. The extraneous elements of the gathered materials, such as mature shoots and leaves, were eliminated. The remaining shoot segments were split into nodal segments of a manageable length (3-4 cm) and kept in a separate conical flask. The com-ponents were first rinsed under running water, then put in a conical flask filled with purified water and a few droplets of "Tween 20" and washed for 10 minutes while shaking constantly. To remove all residues of the foregoing substances, the second washing was done with an advanced change of distilled water. For surface sterilization, the selected explants were rinsed with 70% ethyl alcohol followed by washing with DW. To maintain appropriate time for contamination-free culture, surfaces were disinfected with 0.1% HgCl 2 solution for varied times (6, 8, and 10 minutes). After that, they were washed in sterile DW used for 10 minutes, resulting in 4-5 changes. It was detected that sterilizing shoot tips for 8 minutes with 0.1% HgCl 2 was beneficial. Explants were properly inoculated in culture glass tubes with agar gelled nutritional media supplied with various hormone doses. During inocul-ation, extra caution was needed to confirm that the explants penetrated but did not drop into the medium.

Nutrient
Media and culture condition -For shoot regeneration and multiplication, along with root induction, Murashige and Skoog, (1962) media added with numerous quantities and mixtures of growth regulators was utilized. The pH generally adjusted to 5.0-6.0 of the medium before autoclaving. As a solidifying or gelling agent, agar was usually used in the generation of semisolid (or solid) tissue culture medium. 7 gm (0.7%) agar was added to the present culture medium. Autoclaving at 1.05 kg/cm2 pressure used for 20 minutes at 121°C disinfected the culture vessels. Inoculated culture containers were situated in a growth chamber, which provided a unique cultural environment. In the growing chamber, the vessels were arranged on the shelves of a cabinet. All cultures were maintained in a growth chamber with 40-watt white fluorescent tubes installed 30-40 cm away from the culture shelves. The cultures were kept at 26°C for 16 hours with a light power of 1500 lux (about) emitted from fluorescent tubes. Each day, the reactivity to the culture vessels was measured.

Root initiation and multiplication -
For root induction from micro-cutting of in vitro regenerated shoots of V. negundo, different proportions of NAA (0.1,0.5, 1 and 2 mg/l) and IBA (0.1,0.5, 1, and 2 mg/l) in half-strength MS medium and MS 0 medium were used.

Acclimatization and transfer of plantlets to soil -
The regenerated explants were regarded appropriate for soil transfer after establishing a considerable root system. The growing plants were carefully excised from the culture jars. The roots of the plantlets were carefully rinsed under running tap water to eliminate any agar that had stuck to the root zone. They were immediately placed in small polythene bags with a 1:1:1 mixture of sand, compost, and soil after being washed. The plantlets in pots were enclosed with polythene bags to prevent sudden deformation. Interior sides of this bag were sprayed with water each 24 hours to keep high humidity around the growing plants. The plantlets were gradually vulnerable to the outside normal environment by perforating the polythene envelopes, which were then removed after seven days. By this point, the plantlets have embedded them in the soil. They were eventually relocated to a test field.

Statistical analysis -
All of the tests were repeated three times, with each treatment receiving an average of 25 replicates. SPSS ver. 16 (SPSS Inc., Chicago, USA) were used to perform statistical calculations of the data. DMRT (Duncan's multiple range test) was used to determine the significance of variations between means at P ≤ 0.05. The results of the three experiments are reported as means and standard deviations. Data were collected using the following parameters and the methods for data collection are given below:

i) For shoot induction a) Percentage of explants induced shoots
After the needed days of culture, data on various parameters from various treatments of shoot proliferation were recorded. The subsequent method was used to compute the percentages of explants that developed adventitious shoots.

b) Shoots number per plantlet
After the required days of culture, the shoot numbers of each explant were calculated. The subsequent method was used to calculate the mean number of adventitious shoots for each explant.
Mean number of shoot = Total number of shoot/Quantity of regenerated explants c) Length of lengthiest shoot For each explant, the length of the lengthiest shoot was measured on a cm-scale. The subsequent calculation was used to decide the average length of the shoot.
The average length of shoot = Total length of Shoot/number of shoot

ii) For root induction a) Percentage of explants induced roots
The frequency of shoots induced to develop roots was calculated by using the following formula:

b) Number of roots per shoot
After the required days of culture, the number of roots per shoot was calculated. Using the similar technique as for the number of shoots per explants, the mean numbers of roots and SE per shoot were computed.

c) Mean length of the root
For each shoot, the average length of the longest root was measured in centimeters. As previously stated, the average length of the root was computed.

Standardization of explant (Vitex negundo L.) surface sterilization
After cutting into suitable size the nodal segments and shoot tips of V. negundo were washed with the continuous flowing of tap water for 20m and then treated with antiseptic (4-5 drops liquid soap along with 2-3 drops of Tween-20) and then again washed with ddH 2 O. HgCl 2 was utilized as a surface sterilant for the plant used in this investigation. After one week of sterilization and inoculation it was detected that, when the experimented plant parts were treated with 0.1% HgCl 2 for 8 minutes, 95% of V. negundo L. explants were found contamination-free and healthy also. Ex-plants treated for a longer time (8-10 minutes), causes no contamination but partial or complete tissue killing was observed (S-1).

Shoot Multiplication through Shoot Tip
Culture -Shoot tips approximately 1-2 cm in size were obtained from field-grown plants and cultivated in MS medium with various doses and mixtures of plant growth regulators (cytokinins and auxins) to determine the best culture media for the multiplication of shoot (Shahen et al., 2019).

Effects of diverse concentrations of BAP and Kinetin on shoot initiation
Shoot tips were cultivated on MS media added with different doses of BAP, Kinetin (0.5, 1.0, 2.0, 3.0 mg/l) in this experiment. After 20-25 days, the cultured explants began to develop. In the instance of BAP, the proportion of cultures that responded to shoot formation at various levels ranging from 33.3 to 73.3%. When explants were cultivated in media addedby 3.0 mg/l BAP, the greatest percentage of shoot induction was observed (Fig 1A-1C, 1F). Conversely, the response of shoot formation to different levels of Kinetin ranged from 33.3 to 73.3%, with the greatest response observed at 1.0 mg/l concentration ( Table 1).  (Fig 1D-1F, 1G). Besides, the medium addedby 0.5 mg/l Kinetin showed the lowest response (S-2).

Effects of various levels of BAP with Kinetin
In vitro developed shoots were individually cultured by MS medium enhanced with three concentrations and mixtures of BAP with Kinetin. Among the three concentrations highest (80%) and the lowest (53.3%) percentage of multiple shooting were observed in the medium added with 0.5 mg/l BAP + 1.0 mg/l Kinetin and 1.0 mg/l BAP + 0.5 mg/l Kinetin. After 90 days of cultivation, data was obtained. Furthermore, the cultured plantlets in the mixture comprising 0.5 mg/l of BAP + 1.0 mg/l of Kinetin had the largest average size of shoots (3.2 cm) (S-2).  Table 2).  Fig 3A-3B).

Effects of cytokinins with auxins on the regenerating of multiple shoots
Again, the most successful compositions for multiple shoots induction were observed to be 3.0 mg/l Kinetin +0.1 mg/l NAA, with averages of 87% culture reacted to shoot formation, including shoot per explant of 5.8 and the average size of shoot 1.94 cm long in asimilar media. Subsequently 90 days, the culture data were collected ( Table 2).
The highest percentage of culture reacted 86.6% in a medium having 3.0 mg/l Kinetin +0.1 mg/l IAA, with an average quantity of shoots per explant of 5.5 in the similar media. After 90 days of culture, all data were collected (Fig 3C-3D) (S-3).

Shoot Multiplication through Nodal Segment
Culture -For the multiplication of shoots, nodal segments of the field-grown plants were cultured on MS medium addedby various concentrations of BAP, Kinetin alone, or in combination with NAA and IAA.    (Fig 2A-2B, 2G) ( Table 3).    (Fig 2C, and  2H).

Effects of various BAP and Kinetin concentrations -
The medium including 0.5 mg/l BAP +1.0 mg/1 Kinetin had the highest percentage of culture responses (86.6%) of the three BAP+ Kinetin combinations (0.5+1.0, 1.0+0.5, and 1.0+1.5). In a medium having 1.0mg/l BAP+1.5 mg/1 Kinetin, however, the lowest (60%) percentage of culture was responded to. Every combination resulted in many shoots. S-4 presents the results.

2D) (S-5).
With varied concentrations and mixtures of BAP and NAA, the explants cultured in a medium having 3.0 mg/l BAP + 0.1 mg/l NAA responded with the highest percentage (93.3%) of culture. Furthermore, when the explants were grown in a medium including 2.0 mg/l BAP + 0.1 mg/l IAA, the largest proportion (86.6 percent) of culture responded. (See Table 4) (Fig 4A). Additionally, the medium added with two compositions, 1.0 mg/l Kinetin + 0.5 mg/l NAA and 3.0 mg/l Kinetin + 0.1 mg/l NAA, had the highest percentage of culture response (90%). However, in a medium containing 3.0 mg/l Kinetin + 0.1 mg/l NAA, the maximum average shoots count per explant was 7.2, and the maximum average size of the shoot was 4.3 cm. Furthermore, the medium added with 2.0 mg/l Kinetin+ 0.1 mg/l IAA (Table 4) yielded the highest percentage of culture response (80%) (Fig 2D, 4B) (S-5). From the overall experiments, it is observed that BAP is the best active hormone for multiple shoot initiation and growth in V. negundo and the suitable concentration was 2.0 mg/l BAP.

Root induction -
For root stimulation from micro-cutting of in vitro proliferate shoots of V. negundo, several concentrations of NAA (0.1,0.5, 1 and 2 mg/l) and IBA (0.1,0.5, 1 and 2 mg/l) in MS medium including half-strength and MS 0 media were used ( Table 6). MS 0 medium was employed for root initiation in this experiment. Induction of root is relatively low in such medium. In diverse NAA and IBA concentrations containing media ranging from 0.1 to 2.0 mg/l, about 93.3% of shoots promote rooting. In a medium involving 0.5 mg/l NAA, the best response of root stimulation in each culture was detected. In a medium including 1.0 mg/l NAA, the root's average length was the longest. In IBA, nearly identical results were observed. The best number of root/culture saverage was found 13 in a medium with 0.5 mg/l IBA, while the longest average size of the root was 2.0 cm in a medium with 1.0-2.0 mg/l IBA (Table 6, Fig 2E).

Establishment of regenerated plantlets to
field environments -After sufficient root development, the rooted shoots were planted in small poly stacks comprising garden soil, sand, and compost in a 1:1:1 ratio. To maintain the bags moistly, they were covered by plastic film and progressively acclimatized to outdoor conditions. Plantlets resulting from a variation of in vitro cultures were successfully transplanted into the soil. In open-air soil pots, about 92% of plants survived (Fig 2F).

DISCUSSION:
For the vast majority of the world's inhabitants, medicinal plants constitute a crucial source of lifesaving medications. There are quite high opportunities for the production of plant-based medicine from in vitro regenerated medicinal plants (Radha et al., 2011). This may be accom-plished using a wide range of techniques, including micropropagation. In the field of forestry and flori-culture, micropropagation is commonly employed. Plant species that are endangered or unusual can also benefit from micropropagation. Tween 20 and 0.1% mercuric chloride (HgCl 2 ) solution were utilized as surface sterilizing agents for various periods in the current research, surface sterilization using HgCl 2 has come to light in several studies (Borthakur et al., 2000;Minakshi and Singh, 2017). Treatment of V. negundo explants with 0.1 % HgCl 2 solution for 8 minutes resulted in contamination-free culture with excellent survival, following the findings. The majority of authors reported that many medicinal plants were highly responded in the MS medium, e.g. Calotropis gigaritea (Roy and De, 1990), Asclepias curassavica (Pramanik and Datta, 1986), Solanum torvum (Jaiswal and Narayan, 1985), Rauvolfia serpentina (Roy et al., 1994), Cephaelisi pecacuanha (Jha and Jha, 1989), Adhatoda vasica (Raageeva and Shahnawaz, 2011), Azadirachta indica (Homen et al., 2017), and so many as well as other medicinal herbs. 2 mg/l BAP was combined with 0.5 mg/l NAA in this report, which is appropriate for the nodal segment and shoot tip culture from a field-grown plant. The best result was 2 mg/l BAP. When a medium containing 2 mg/l BAP was used, the largest amount of shoots per explant on average was observed from both nodal segments and shoot tips. In Chrysanthemum, (Waseem et al., 2011 andYesmin et al., 2014) achieved a more or less identical result. Kiran and Trevor, (1990) found that BAP (2.0 mg/l) had a better impact on shoot multiplication. 2.2 mg/l BAP was discovered to be the most efficient concentration for shoot tip culture in pear plants (Lane et al., 1979). While the basal MS media, BAP @2.5mg/l, and NAA @0.5mg/l demonstrated the top response to regeneration via callus induction, with a typical of 5.1 shoot buds per culture in 4-5 weeks (Suvalaxmi et al., 2017).
The highest percentages of shoot propagation were achieved in mediums added with 9.0 M BAP and 0. were rooted and grown into complete plantlets. Root and shoot development derived from shoots are particularly critical for tissue culture-derived shoot establishment (Sayeed et al., 2015).
To find the optimal root induction strategy, many tests had been completed using a half-strength MS medium added with different amounts of nutrients, such as auxins (NAA, IBA). Farhana et al. (2008) unclosed that the medium with 1.0 mg/l IBA produced the most roots. However, Jawahar et al. (2008) investigated that the regenerated shoots were discovered to be effective when rooted on MS media added with 0.5 mg/l IBA to induce root from in vitro grown shoots of V. negundo.
Half-strength MS medium added with 0.5 mg/l IBA was used to grow rooting shoots (Steephen et al., 2010). Nevertheless, in this inquiry IBA was used alone for rooting, it surpassed other auxins (NAA). In both half-strength, MS mediums with 1.0 mg/l IBA, the optimal concentration for root development and proliferation was obtained. Dhir et al. (1998) also utilized the same strategy for acclimatization as this investigation. In vitro clonal cultivation, germplasm collection, and dissemination also may assist V. negundo, for further research. The new study indicated that acclimatized plants survived at a rate of 92%, which is greater than the previous result (Jawahar et al., 2008).

CONCLUSION:
This work generated an in vitro protocol for the proliferation of shoots, root induction, and effective adaptation of the medicinal herb V. negundo L. For greater frequency shoot multiplication, HgCl 2 was utilized to surface sterilize the explants. After treatment in 0.1% HgCl 2 for 8 minutes, over 90% of explants were found to be free of surface contaminants with minimal tissue injury. To investigate clonal propagative effectiveness, explants were cultivated on MS medium treated with various concentrations and mixtures of cytokines, auxin, and 2,4-D, auxin. The greatest response was seen with V. nigundo at 2.0 mg/l BAP. Moreover, regenerated shoot tips are induced to root in halfstrength MS media supplemented with various doses of NAA and IAA for rooting. A half MS with 2.0 mg/l IAA yielded enough healthy roots. When the seedlings were transplanted to the soil, more than 92% of them had effectively acclimatized. This method could be used for both commercial plantlet bulk propagation, germplasm preservation, and distribution. Optimization might be taken in the future procedure for greater outcomes.

ACKNOWLEDGMENT:
The authors are very much thankful to the Department of Biotechnology and Genetic Engineering, Islamic University Kushtia-7003, Bangladesh and Atomic Energy Commission, Savar for research facilities and other logistic supports.

CONFLICTS OF INTEREST:
The authors declared there is no conflict of interest.