Applicable Rapid Propagation Protocol Through Organogenesis of Alocasia Cuprea for Plant Development Using Micro-Bulblet Explant

. Alocasia of Indonesian endemic has significant market potential. However, the scarcity of qualified seedlings hampered their commercialization. The purpose of this study was to obtain the best sterilization technique and media for in vitro propagation of Alocasia cuprea through the organogenesis pathway using microbulblet explants. Randomized complete block designs with five to ten replicates were applied in the experiments. Application of 5 and 10% clorox for 5 and 10 minutes resulted in 40.6% aseptic explants, 10% browning explants, and 90% explant growth potential. The best shoot initiation was achieved on the MS added 4.54 µM thidiazuron and 2.22 µM BAP. This medium had an 85% explant initiation, 4.2 shoots per explant, and 6.5 days initiation time. The best shoot proliferation was discovered on MS medium with 4.44 µM BAP, which produced 10.5 initial shoots with a shoot height of 1.3 cm and a clump diameter of 1.3 cm. The best plantlet regeneration was determined on ½ MS medium with 6.5 cm plantlet height, 3.5 leaves total, 3.8 cm leaf length, 2.6 cm leaf width, and 3.8 roots. This propagation method will be applicable to the propagation of qualified seedlings for commercial purposes and potentially used for other endemic Alocasia.


Introduction
Alocasia is a group of ornamental plants with beautiful leaves that belong to the Araceae family, the Aroideae sub-family, and the Colocasieae tribe [1,2].Alocasia of Southeast Asian endemic found in Indonesian forests, about 50 species found in Kalimantan, Sulawesi and Papua [3].One of the spesies of Alocasia endemic to Indonesia that is currently popular and in great demand in the market is Alocasia cuprea or more popularly known as 'Keladi Tengkorak', which is often found in the forests of Kalimantan (Borneo).The attractiveness of this alocasia lies in the specific, unique and exotic shape with texture and pattern of the fungicide Score 250 EC, and insecticide (Decis and Curacron, local insecticide product in Indonesia) of 0.15% each were applied once a week .Daily routine irrigation or irrigation adapted to the needs of the plant media.From donor plants that were mature (older than a year), micro-bulblet explants (0.5-1.0 cm in size) were taken.

Research stages.
The protocol was prepared starting from: (1) sterilization of explant, (2) shoot initiation, (2) shoot proliferation, and (3) plantlet regeneration, enlargement, and rooting.In detail, the stages of preparing the protocol are described as follows:

Sterilization of Alocasia cuprea micro-bulblet explant using different methods.
Alocasia cuprea micro-bulblets (0.5-1.0 cm in size) were harvested from donor plants and sterilized in the following stages: (1) cleaned of soil residues, washed with tap water for 30 minutes, and peeled until white micro-bulblet, (2) soaked in detergent solution for 30-45 minutes, followed by soaking in 1-2% bactericidal-fungicide for 30-45 minutes while shaking on a shaker with a speed of 80-90 rpm, (3) sterilized in a laminar with 2 methods of sterilization, namely: S1 (1 and 2% HgCl 2 ) and S2 (5 and 10% clorox) @ for 5 and 10 minutes, washed with sterile water 3 times @ 5 minutes, and air dried on a dry sterile tissue.Micro-bulblets were grown MS medium [15] and maintained for 4 weeks to see the treatment response and initial selection of explants.The initial response of micro-bulblet after being sterilized and cultured began to be observed 2 weeks after explant culture, beginning with the appearance of contaminants, browning of explants, and growth potential of explants.

Shoot proliferation from micro-bulblet explant on different media
The shoot that resulted from the micro-bulblet initiation shoot stage provided during 1-2 months after initiation culture, was subcultured on various proliferation treatment media.Before the shoots were subcultured on to the treatment medium, were exfoliated and given external wounds to stimulate the emergence of lateral shoots surrounding the main shoots.At this point, 4 treatment media compositions for proliferation were tested, namely a combination of MS media and BAP (0.00, 1.33, 2.22, and 4.44 µM).Sucrose, gelrite, pH and culture incubation applied was similar to previous experiment.Subcultures were repeated every month until the third subculture.The number of shoots per clump, shoot height (cm), and clump diameter (cm) were all measured.Until the 12 th week, observations were made every month.

Acclimatization of plantlet
Plantlets with strong roots that have 3-4 leaves and a height 5-6 cm were hardened for ± 1 month outside the culture incubation room that settled by (temperature 25-27 o C with humidity 50-60%).The plantlets are washed under tap water, soaked in a 1-2% bactericidal-fungicide 15 minutes, air-dried, and planted in a compote of 100 plantlets on a plastic tray containing roasted husks medium and covered in plastic, and incubated on a shelf in a greenhouse at a light intensity of 35-50%, temperature of 23-25 °C with 70-80% humidity.

Design of Experiment and Analysis of Data.
A randomized complete block design (RCBD) with five to ten replicates were applied in the experiments with 20 units per experiment.Data were analyzed using analysis of variance (ANOVA) with SPSS version 26 [16].Differences between means were further tested using the Least Significant Difference (LSD) with a 95% confidence level.

Results
Based on periodic observations at each stage of the research, some important information at each stage of the research was obtained regarding the in vitro organogenesis process of Alocasia cuprea.In this study, 28-40.6%aseptic micro-bulblet explants were obtained with a browning level of 10-28%, and a growth potential of 78-90%.Initiation of shoots begins with swelling and the appearance of callus around the micro-bulblet and then ruptures into shoots (organogenesis) occurring in the range of 6.5-25.0days with 54-85% initiation of explants producing 1.4-4.2shoots per explant.The shoots that were successfully initiated were then subcultured on proliferation treatment media.The proliferation response was demonstrated shown by the emergence of new shoots around the primary shoots, with a fairly variable range of 1.7-10.5 shoots per explant, shoot height of 0.6-1.5 cm, and clump diameter of 0.4-1.3cm.The proliferation response was demonstrated by the emergence of new shoots around the primary shoots.The proliferated shoots will then regenerate into plantlets ready for acclimatization.The process of enlargement and rooting of plantlets was carried out on several media that were tested resulting in variations in response with plant height 5.3-6.4cm, number of leaves 1.9-3.5 per plantlet, leaf length 2.8-3.8cm, leaf width 1.8-2.6 cm, and roots number 2.6-4.4 cm.The treated plantlets grew successfully in the greenhouse, with more than 90% success (Figure 1).

Sterilization of Alocasia cuprea micro-bulblet explant using different methods
This study was successful in obtaining 28-40.6%aseptic explants with 10-28% browning of the explants but the growth potential of the explants was quite high, namely 72-90%.The success of sterilization in method S2 (5 and 10% Clorox for 5 and 10 minutes) was better than method S1 (1 and 2% HgCl 2 for 5 minutes and 10 minutes) resulting in 40.6% sterile explants, 10% browning explants, and 90% explant growth potential (Figure 2).In the S1 treatment, generally, bacteria, presumably because the concentration of HgCl 2 was too high and or the application time was too long, causing the explants to die.Whereas in S2 treatment the contaminants that appear are generally fungus, it is suspected that the dose and or duration of application is not optimal.According to the findings of this study, the sterilization method should be retested by modifying the pre-sterilization combination, the type and concentration of the sterilant, and the time of application.

Responses of different media (combination of MS and TDZ-BAP concentrations) on shoot initiation from micro-bulblet of Alocasia cuprea
The growth response of the micro-bulblets Alocasia cuprea cultured was different in each initiation media (Table 1).Shoot initiation was observed at 6.5-25.0days after culture with 54-85% initiation explant, 1.4-4.2initial shoots per explant, and all explants morphogenesis to form shoots directly (organogenesis).The best shoot initiation was produced on the MS medium fortified by 4.54 µM TDZ and 2.22 µM BAP.This medium had an 85% initiation explant, 4.2 shoots per explant, and 6.5 days initiation time (Table 1; Fig C1-2).The same letter in the same column following means are not different based on LSD, p=0.05.Abbreviations: EC (embriogenic calluses); SE (somatic embryos); S (shoots).

Proliferation of initial shoots Alocasia cuprea from micro-bulblet explant on different media composition (combination of MS and BAP concentration)
The combination of MS and BAP concentrations in media affected the proliferation of initial shoots from micro-bulblet explant of Alocasia cuprea.MS medium augmented with 4.44 µM BAP was the best medium for shoot proliferation, regenerating 10.5 initial shoots with a shoot height of 1.3 cm and a clump diameter of 1.3 cm.Another best shoots proliferation was produced by MS added with2.22 µM BAP which produced 6.7 initial shoots with a shoot height of 1.3 cm and a clump diameter of 0.7 cm (Table 2; Fig D1-2).Application of MS with BAP below 2.22 µM had very low proliferation (1.7-3.0 initial shoots per explant), even on MS without BAP no shoots appeared but induced growth of roots (Table 2 ; Figure E1).The same letter in the same column following means are not different based on LSD, p=0.05.

Enlargement and rooting of Alocasia cuprea plantlets on different media (combination of basic media with activated charcoal)
The best plantlet regeneration such as enlargement and rooting of plantlets were carried out on MS medium without activated charcoal (AC), producing plantlets with a height of 6.5 cm, 3.5 leaves total, 3.8 cm leaf length, 2.6 cm leaf width, and 3.8 roots.The second best medium was ½ MS with 0.1% AC which produced the highest roots number, namely 4.4.
The other treatments Hyponex with and without 0.1% AC gave lower yields with the lowest yields recorded on Hyponex without AC (Table 3).The same letter in the same column following means are not different based on LSD, p=0.05.

Discussion
This research had succeeded in developing an in vitro mass propagation protocol of Alocasia cuprea from micro bulblet explants.The protocol begins with the selection of explant sterilization methods and media for the initiation, proliferation, and regeneration of plantlets that are ready for acclimatization.
Sterilization is a critical initial stage and the key to getting aseptic explants ready for initiation culture.As a result, an appropriate sterilization method is required to obtain sufficient quantities of aseptic explants, particularly for donor plants and explant types that are in short supply.The application of 20% Clorox ® twice on the rhizome buds of Alocasia longiloba 'Watsoniana' and several spesias of Alocasia , yielded about 50% aseptic explants [10;12].The low number of aseptic explants obtained (<50%) is not only due to the ineffectiveness of the method, but also condition of the donor plants in the green house and the type of explants.Micro bulbs grow at the bottom around the roots of the planting medium, so the condition and cleanliness of the media is very decisive and the risk of contamination is higher compared to other explants such as shoots and leaves.
The stages of in vitro organogenesis of Alocasia cuprea starting from shoot initiation to producing quality plantlets (uniform, healthy, and vigorous) are a long series that are influenced by many factors after aseptic explants are obtained.Other studies have reported in vitro organogenesis of several alocasia species using various explants and media compositions, with varying results.MS medium added with 49.2 µM 2-iP for A. micholitziana 'Green Velvet' and 44.4 µM BA for A. amazonica and A. cuculata was successfully multiplied and regenerated shoots directly on axillary shoots cut from tubers within 6-8 weeks [8].Furthermore, on A. amazonica and A. cuculata, the highest shoot proliferation was achieved in MS medium containing 22.2 BA with 4.6 and 5 shoots per culture, respectively.MS media with low concentrations hormone was used for shoot rooting.Similar results were also on A. amazonica using shoot explants in the MS medium augmented with 2.27 μM TDZ produced an shoots per explant [9].The best multiple shoots of A. longiloba 'Watsoniana' were induced from the aseptic bud explants on MS containing 8.8 µM BA and 2.46 µM IBA, and produced 3-4 buds per explant.Roots induction on micro shoots occurred when cultured on MS media supplemented with 0.5 mg L -1 BA [10].
The optimum medium for the proliferation of A. macrorrhiza from mature seeds explant was MS added 26.64 µM BA, 6.97 µM kinetin, and 0.49 µM IBA with proliferation coefficient more than 6.0, the optimum medium for seedling strengthening and rooting was ½ MS containing 0.1% AC and 2.0 mg L -1 PP333 with strong seedling rate up to 82.50% and rooting rate up to 100%, and the optimum transplanting medium was peat soil combined with vermiculite(3:1) with survival rate up to 93.33% [12].MS medium containing 8.88 µM BA stimulated 5.2-5.3shoots per explant, while roots were formed only on MS medium with low concentrations of BA and without BA [12].The highest shoot length (7.26 cm) and maximum number of shoots per explant (18) A. longiloba from seed explants were recorded at 13.32 µM BAP and for in vitro rooting 2.46 µM IBA [13].High indirect organogenesis from young leaf explants of A. amazonica with 11.6 shoots per callus clump and 10.1 cm in length was proved on MS medium with 6.66 µM BAP and 2.69 µM NAA.An average of 22.20 ± 0.87 roots per shoot was recorded in ½ MS medium added with 14.76 µM IBA, 11.42 µM IAA and 10.74 µM NAA and 90% survivability of plantlets were noted in acclimatization stage [14].
The growth and development of plants in vitro is dominantly affected by the media , growth rate, elongation, and quality of morphogenesis [17;18].In general, MS media with the addition of TDZ and BAP individually or together were used for shoot initiation and proliferation on Alocasia.The application of TDZ and BAP in media is also used in the in vitro propagation of various types of plants, including ornamental plants [19][20][21][22][23][24].On medium of MS TDZ-BAP free, shoot initiation and proliferation did not occur, nor did roots grow.Root formation did not occur at high concentrations of BA, proving that high concentrations of cytokinins generally inhibit root formation in plants [25].The presence of TDZ and BAP in the culture medium is thought to influence A. cuprea high organogenesis potential.The combination of TDZ and BA promotes cell division, callus formation, and growth [26].TDZ acts as a cytokinin oxidase inhibitor, preventing the degradation of adenine-type cytokinins and translocating BA to injured explant tissue parts.Increasing the accumulation of BA in these cells causes cell division to become more active [27][28][29].TDZ plays an important role in modulating endogenous hormones resulting in hormonal balance and the ease with which culture can be initiated.TDZ also influences cell membranes, energy levels, nutrient absorption, and nutrient assimilation, all of which contribute to increased cell responses during morphogenesis [30].In general, BA plays a role in regulating cell division and expansion, proliferation and differentiation of shoots, and inhibits root formation [31].
After 4 weeks of acclimatization, the A. cuprea plantlet grew normal and healthy (Fig. 1B) with more than 90% survivability.The regenerated plants had similar performances to their mother plants morphologically.The established procedure provided a foundation for future in vitro experiments in Alocasia.This propagation method will be applicable to the commercial propagation of qualified seedlings and may be used for other endemic Alocasia.

Conclusion
This study successfully obtained the complete protocols of in vitro organogenesis in Alocasia cuprea using plant micro-bulblet explant, beginning with sterilization, shoot initiation, and ending with acclimatization.The application of 5 and 10% clorox for 5 and 10 minutes was produced the best aseptic explants and the highest explant growth potential.MS media added by 4.54 µM TDZ and 2.22 µM BAP was optimal for shoot initiation, while MS medium with 4.44 µM BAP was appropriate for shoot proliferation.Nevertheles, ½ MS medium was the best for plantlet regeneration.Based on all of these accomplishments.This technology could be said to be reliable technology as a standard protocol for mass propagation of Alocasia cuprea and might be served as foundation for mass propagation in other Alocasia spp as endemic, mutant or hybrid.This technology also could be applicate to other purposes on breeding programme, identification of potential secondary metabolite candidates in Alocasia spp.and other deciduous ornamentals plant as well.Furthermore, this technology would be easy to deserve the seedling for ornamentals farmer, grower and deciduous ornamental industry in Indonesia and to aim increasing exports, thereby increasing national economic growth.This project is funded by a research collaboration between the Agriculture and Food Center (BRIN) 2023 and CV.Ranata Nursery.We also thank our colleague Dr. Drs.Budi Winarto, MSi for improving this manuscript.

Table 1 .
Comparison of initiation responses of Alocasia cuprea micro-bulblet explant on four media compositions(combination of MS and TDZ-BAP concentrations)

Table 2 .
Shoot proliferation responses of Alocasia cuprea on MS media with various concentrations of BAP (2 months)

Table 3 .
Enlargement and rooting of Alocasia cuprea plantlets on a combination of basic media with active charcoal