http://dx.doi: 10.11594/jtls.13.03.08
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Research Article
Effect of Adenine Sulphate on In Vitro Micropropagation of Acacia holosericea A.
Cunn. ex G. Don Leaflets and Leaf Rachis Explants
Anupama Shukla 1, Anita Narang 1*, Surinder Kaur 2
1 Department of Botany, Acharya Narendra Dev College, University of Delhi, New Delhi-110019, India
2 Department of Botany, S.G.T.B. Khalsa College, University of Delhi, Delhi-110007, India
Article history:
Submission September 2022 Revised December 2022 Accepted December 2022
ABSTRACT
In this study, different explant types (leaflets and leaf rachis) excised from 15-day- old seedlings of Acacia holosericea A. Cunn ex G. Don differentiated into multiple shoots when grown on B5 medium supplemented with various growth regulators.
Results revealed that the shoots differentiated directly and indirectly in all the cyto- kinins [Benzyl adenine (BA); Kinetin (Kn), 2-isopentenyladenine (2iP); Zeatin (ZN)] adjuvanted media. The ZN-supplemented B5 medium supported maximum multiple-shoot differentiation (3.2 ± 0.8 shoots per explant). However, with the ad- juvant (AdS) present in the same ZN-supplemented B5 medium, the explants pro- duced a higher number of shoots (7.0 ± 3.8 per explant). Likewise, the best leaflet regeneration medium was observed for B5medium supplemented with 2.5 mg/L ZN and 200 mg/L of AdS. The B5 medium with the lower amount of ZN (1.5 mg/L) and 50 mg/L of AdS produced a maximum number of shoots (6.3 ± 3.8 shoots per explant) in the leaf rachis explants. Nearly 100% of the in vitro B5 basal medium- grown shoots developed roots directly at their base, consequently leading to their successful transfer to the soil.
Keywords: Acacia holosericea, Fabaceous tree, Leaflets, Leaf rachis, Micropropa- gation
*Corresponding author:
E-mail: [email protected]
Introduction
Acacia holosericea (soapbush wattle) is a shrub or a small multi-stemmed tree characterized by a large, dense crown native to Australia. The tree is grown as an ornamental, valued for its silver foliage, long, yellow flower spikes, and twisted seedpods, as well as a windbreaker and soil stabi- lizer. Since it can thrive on a large variety of soils and tolerate drought, frost, and salinity, the tree is used in land restoration projects [1, 2, 3]. Oil ex- tracted from the seeds is used in manufacturing paints, surface coatings and soaps, while oil ex- tracted from the aril is an ingredient in cosmetics and toiletries. Meanwhile, the residue from the ex- traction process forms a protein-rich meal. Upon realising the plant's importance, A. holosericea was introduced in India for afforestation programs [4]. However, scientific investigations on the mor- phogenic potential of A. holosericea remained sparse, except for a few reports [5,6]. Use of
correct concentrations of plant growth regulators in specific media during micropropagation of se- lected plant species is important to determine their success in forming whole plants [6, 7]. In this per- spective, the study aimed to develop a reproduci- ble protocol for in vitro micropropagation of this plant species using different explants, cytokinins, and adjuvants.
Material and Methods
Seed treatment for aseptic purpose
Initially, the A. holosericea seeds were scari- fied by soaking in concentrated sulphuric acid (98%,v/v) for 5-35 mins to identify the best treat- ment duration. The seeds were then surface-steri- lized with freshly prepared chlorine water for 45 min, followed by washing thrice with sterilised water. The chlorine water was prepared by bub- bling chlorine in 500 ml of distilled water for 15-
20 min. For this, Chlorine was obtained by adding 30 ml of concentrated hydrochloric acid (LR) to 4 g of potassium permanganate [8]. The seeds were then implanted aseptically onto semi-solid Knop’s medium [9]. The seedlings grown on the Knop’s medium were used for preparing explants for the experiments conducted in the present study.
Medium preparation for growth of A.
holosericea leaflet and leaf rachis explants The leaves comprising leaflets and leaf rachis were excised from 15-day-old in vitro raised seed- lings and cultured on Gamborg B5 basal media [10] in the absence or presence of growth regula- tors such as cytokinins [Benzyl adenine (BA); Ki- netin (Kn), 2-isopentenyladenine (2iP); Zeatin (ZN)] and/or auxins [Indole acetic acid (IAA), In- dole butyric acid (IBA), Naphthalene acetic acid (NAA)] of HiMedia brand. All growth regulators were used at 0.5-3 mg/L concentrations. Experi- ments were also conducted parallelly by supple- menting cytokinins and adjuvants (AdS; 50- 250mg/L). AdS was used because it acts as a pre- cursor for natural cytokinin synthesis or enhances natural cytokinin biosynthesis. The B5 medium containing 3% sucrose and 0.8% agar was ad- justed to pH 5.8 using 1N NaOH or 1N HCl before autoclaving at 121oC with 1.06 kg/cm2 pressure for 15-20 min. Cultures were maintained at 25 ± 2ºC and 55 ± 10% relative humidity under white fluorescent light (40μ cE.m-2.s-1), emitted by 40W Crompton incandescent tubes programmed for 16 hr photoperiod.
Explants were sub-cultured on a fresh medium every 25-30 days, and data were recorded at 10- 15d intervals, with the final data scored after 60 d of culturing. The average number of shoots per re- sponding explant was represented as a mean value
± standard deviation (mean ± S.D). The percent- age of responding explants was subjected to a Chi- square test, considering variance at a 5 % level.
The in- vitro-reared shoots were excised and then reared on B5 medium in the absence or presence of 1-3 mg/L of various auxins (IAA, IBA, NAA) for root induction. A minimum of 30 replicates were grown for each treatment, and all experi- ments were repeated twice.
Results and Discussion Differentiation of shoots
Before inoculating the leaflets, a scalpel was used to make slight incisions on the ventral surface
of the explants. Generally, the cut ends turned brown within a week of culturing on the basal me- dia, followed by the formation of small, compact, green, and friable greenish-white structures at the margins and cut ends of the explants. The B5me- dium turned out to be the best in supporting shoot morphogenesis in leaflets and leaf rachis, as pre- viously seen in the cotyledon explants [6].
In the present study, within 20-25 days of in- cubation on a ZN (0.5-3mg/L) supplemented me- dium, the leaflet explants enlarged and developed friable to compact green nodular calluses at their cut ends and margins. Shoot buds were induced in both types of explants only after 10-15 days, either directly or via callus. For the indirect mode, leaf- lets were seen to split longitudinally into two halves and nodular green structures formed along their entire margins (Figure 1A). The callus nod- ules were differentiated into shoot buds after 7-10 days (Figure 1B). A maximum of 16% of the ex- plants formed an average of 3.2 ± 0.8 shoots per explant, when grown on a B5 medium supple- mented with 2.5 mg/L ZN. In fact, certain explants produced shoots accompanied by small roots at their bases, forming a complete plantlet.
The number of shoots per explant also in- creased (7.0 ± 3.8 per explant) when the B5 basal medium was adjuvanted with AdS (200 mg/L).
The shoots developed both directly on the ex- plant’s surface or via callus, though the latter was initially friable and white before turning translu- cent and compact. Distinct nodular green struc- tures appeared in callus masses after 15-20 days of inoculation, which later differentiated into multi- ple shoot buds. For maximum caulogenesis, the B5medium containing 2.5 mg/L ZN and 200 mg/L AdS was adjudicated as the optimum (Table 1).
The study observed that even small masses of cal- lus, if separately cultured, formed multiple shoots on this medium. Moreover, some literature has shown that adenine, in various forms, could en- hance the growth of the isolated meristem tips and promote the proliferation of axillary shoots in shoot cultures. The compound also facilitates the formation of adventitious shoots directly from the explants or indirectly via callus [11, 12, 13].
Therefore, using adenine as an AdS could stimu- late cell growth and shoot multiplication, in which the compound acts as an organic nitrogen source and/or a precursor for natural cytokinin synthesis [14, 15, 16, 17].
The leaf rachis explants also showed multiple
shoot formations directly from the explant’s sur- face on the ZN (0.5-3 mg/L) supplemented me- dium. A maximum of 3.0 ± 1.5 shoots per explant were observed in the 1.5 mg/L ZN medium. Sup- plementation with AdS to this regeneration me- dium (B5 ± 1.5 mg/L ZN) further enhanced the number of multiple shoot regeneration. Similar re- sponses have been reported earlier by different re- searchers [18,19,20].The best response (6.3 ± 3.8 shoots per explant) was obtained on a medium
containing 50 mg/L AdS (Table 2). In addition to direct caulogenesis, shoot regeneration also oc- curred from the nodular green calluses that formed on the surface of the explant (Figure 2A, B).
Retention of morphogenic potential
The study found that the leaflets retained their morphogenic potential for up to six months, akin to those from cotyledonary explants [6]. A similar response was also reported in cotyledonary node Figure 1. Morphogenic potential of leaflets of A. holosericea on shoot regeneration medium (B5+2.5 mg/L
ZN+200 mg/L AdS) (A) Nodular green structures developing on the surface of explants X 7(B) Mul- tiple shoot buds developing from the callus X 5.2 (C) Shoot regenerating from explants after 6 months X 4.3 (D) Shoots developing from callus pieces after 6 months of subculture. X 3.3.
explants of Acacia nilotica [8]. In this study, ex- plants grown on B5medium supplemented with 2.5 mg/L ZN and 200 mg/L AdS produced multi- ple shoot buds that formed either directly or indi- rectly via callus. Also, explants regularly sub-
cultured at 25-30 days intervals on a fresh medium of the same composition continued to regenerate multiple shoot buds (Figure 1C; arrows). Even small masses of callus (0.5-1 mm width), when isolated and sub-cultured, gave rise to multiple Table 1. The morphogenic response of A. holosericea leaflets on B5 medium supplemented with 2.5 mg/L ZN
and AdS. The following data were recorded after 60 days of inoculation AdS(mg/L) Number of ex-
plants
Explants forming callus(%)
Explants forming shoots(%)
Average number of shoots/explant
B5 basal medium 42 21 0 0
ZN + 0 42 100 16 3.2 ± 0.8
50 37 100 11 1.2 ± 0.4
100 42 100 17 5.0 ± 3.9
150 42 100 15 1.0 ± 0.0
200 42 100 33 7.0 ± 3.8
250 42 100 5 2.0 ± 1.0
Figure 2. The morphology response of leaf rachis explants on B5 medium supplemented with 1.5 mg/L ZN and 50 mg/L AdS. A cluster of shoots develops directly from the (A) surface X 6 and (B) from the green nodular callus. X 5.5.
Table 2. Morphogenic response of leaf rachis of A. holosericea on B5 mediumsupplemented with 1.5 mg/L ZN and AdS (adenine sulfate) after 60 days of culture
AdS(mg/L) Number of ex-
plants Explants forming
callus (%) Explants forming
shoots (%) Average number of shoots/explant
B5 basal medium 52 50 12 1.0 ± 0.0
Zeatin + 0 52 100 54 3.0 ± 1.5
25 52 100 38 2.4 ± 1.3
50 52 100 54 6.3 ± 3.8
100 52 88 23 1.5 ± 0.7
150 52 69 73 2.7 ± 1.6
200 52 58 15 1.2 ± 0.4
250 52 100 15 2.2 ± 1.0
shoots (Figure 1D). It is worth highlighting here that shoot buds differentiated from 5-6 months old calluses successfully developed into long shoots.
Rooting and transfer of plantlets to soil
The excised in vitro raised shoots (0.5-2 cm long) cultured on B5basal medium produced long, healthy roots at the basal cut ends, with almost 100
% rooting in all explants, after 10-15 days of incu- bation. After 30-40 days of incubation on the B5 basal medium, the plantlets were transferred to small pots containing garden soil and sand (1:1), incubated in a culture room and covered with plas- tic bags to maintain high humidity. The bags were removed periodically to reduce the humidity and to aid the plants’ acclimatization to the soil. Rapid defoliation was noted in some plantlets after soil transplantation (Figure 3), likely due to inadequate hardening processes before transfer. Other factors contributing to defoliation include water loss and desiccation caused by cuticular and stomatal tran- spiration, as well as altered mesophyll structure, poor photosynthetic activity, malfunctioning sto- mata, and a marked decrease in cuticular waxes [21,22]. In some plants, diluting the basal medium before transplanting to soil or using soil, vermicu- lite, and peat mix in various proportions reportedly helps harden the plants [23,24].
Conclusion
The present study shows successful micro- propagation of leaflets and leaf rachis explants of A. holosericea. The explants retained their poten- tial to regenerate multiple shoots even after 6 months. The B5 medium supplemented with zea- tin and adenine sulphate was ideal for explant re- generation. While a lower amount of zeatin (1.5mg/L) and 50mg/L AdS resulted in maximum shoot production in rachis explants, a higher level of zeatin (2.5 mg/L) along with 200 mg/L AdS was found to be the best regeneration medium for leaflets explants. A hundred percentage (100%) of the in vitro raised shoots developed roots directly at their base on B5 basal medium which led to the successful transfer of the in vitro-raised A.
holosericea plantlets to the soil. The present study, which evaluates the efficiency of media in the in vitro production of A. holosericea plant through leaflets and leaf rachis explants, is the first report of its kind. This will make significant contribu- tions to the literature.
Acknowledgment
The authors gratefully acknowledge the Coun- cil for Scientific and Industrial Research for the fi- nancial assistance. We are also thankful to the Principals of Acharya Narendra Dev College and SGTB Khalsa college for their constant support.
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