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volume 44 issue 5 (october 2024) : 885-891,   Doi: 10.18805/ag.DF-621

Thidiazuron Role in In vitro Flowering of Murraya paniculata and Fortunella hindsii via Embryogenic Protoplast and Hypocotyl Segments Cultures

H
Hasan Basri Jumin1,*
1Garduate School in Agriculture Islamic University of Riau Jl Kaharuddin Nasution 113 Pekanbaru 28284, Indonesia.
Cite article:- Jumin Basri Hasan (2024). Thidiazuron Role in In vitro Flowering of Murraya paniculata and Fortunella hindsii via Embryogenic Protoplast and Hypocotyl Segments Cultures . Agricultural Science Digest. 44(5): 885-891. doi: 10.18805/ag.DF-621.

ABSTRACT

Background: Procedures to regenerate protoplast into plants of citrus relatives is important to establish the genetic improvement for sweet oranges by using somatic hybridization and transgenic. Protoplasts are a good material to genetically manipulate and to become gene transfer. This study aims to characterize the regeneration sequence of Fortunella hindsii and Murraya paniculata protoplasts via somatic embryogenesis into flowering plants in vitro. This study also describes the sequence of flowering in vitro originated from hypocotyl segments of plantlets of two species.

Methods: Protoplast sources that were isolated from callus of Fortunella hindsii and Murraya paniculata. Embryogenic callus was put in Erlenmeyer and mixed together with 0.4% macerozyme, 0.2% cellulose and 0.1% driselase and pH was adjusted to 6.0. Two drops of purified protoplasts were cultured with media supplemented with 2.0% glucose and 0.0, 0.001, 0.01, 0.1 or 1.0. 10.0 mg/l thidiazuron (TDZ) and thicken with 0.9% gelrite. 

Result: The media supplemented with 0.001 mg/l TDZ yielded the maximum plating efficiency, while 0.001 mg/l TDZ produced the highest percentage of shoot formation, approximately 80%. After being cultured on the same TDZ concentration for 14 days, the protoplasts that survived developed cell walls. Fortunella hindsii and Murraya paniculata underwent somatic embryogenesis to grow into plantlets. Thidiazuron has demonstrated efficacy in forming flower buds that grow normally. Murraya paniculata and Fortunella hindsii shoots that emerged from hypocotyl segmments flowered in vitro on half-strength MT media containing 0.001 to 0.01 mg/l TDZ and 2-3% sucrose after two months of culture and they eventually went on to flower.

INTRODUCTION

Thidiazuron (TDZ) encourages flowers in low concentration and there could be increased plant metabolism and reserve plants. Thidiazuron may produce high quality bud floral and develop to normal flowers (Olah et al., 2003). Thidiazuron with suitable  concentration  have been  increase the  axillary bud and are possible to increase frequency flowering in vitro. Addition of TDZ to the culture, triggered the adventitious buds to develop into  early flowering in vitro. Thidiazuron also belongs to the group cytokinin synthetic as well as the benzylaminopurine. Thidiazuron have a role in stimulating endogenous cytokinin and they  play a role as a cytokinin oxidase inhibitor that is an enzyme (Parveen and Shahzad, 2010).
       
Fortunella hindsii is one of citrus relatives that are potentially to use as an genetic resources for quality improvement and make the resistance traits of citrus, because the fruiting time is relatively short (Fu et al., 2003; Chen, 2012; Jumin and Nito, 1996a; Jumin and Nito, 1996b).  As a citrus relative’s member, they are used as a garnish while they also could be consumed as a food substitute to the citrus. Murraya paniculata potentially to be used as rooting sources for citrus  resistance to nematodes, water flooding, saline soil and substitution a genetics improvement (Swingle and Reece.1967; Sykes. 1988; Raveh. 2012; Jumin. 2013). Apparently the both varieties are related to citrus which are the easiest to use as sources as stem grafting and establish somatic hybridization with sweet orange (Citrus sinensis). Citrus sinensis is one kind of sweet orange that is in the highest demand in the market mainly in Indonesia and south east Asian countries.
       
Procedures to regenerate protoplast into plants of citrus relatives is important to establish the genetic improvement for sweet oranges by using somatic hybridization and transgenic plants. Protoplasts are a good material to genetically manipulate and to become gene transfer (Ahmed et al., 2021; Thakur et al., 2022; Chen et al., 2023). Plants regenerated from seedlings take a long time to reach flowering. Fortunella hindsii as rooting to make the citrus become dwarfed for garnish purpose. Plant grafting in the citrus could be used as a way to shorten the early flowering plants. Shoot resulted from regenerated protoplasts as alternatively as stems to be used for grafting of citrus.    
       
These technologies may be utilized as an additional method for producing wide hybridizations via protoplast fusion in order to improve the rootstock. This study aims to characterize the regeneration sequence of Fortunella hindsii and Murraya paniculata protoplasts via somatic embryogenesis into flowering plants in vitro. This study also describes the sequence of flowering in vitro originated from hypocotyl segment of plantlets of two species.

MATERIALS AND METHODS

Plant materials
 
Plant Materials using protoplasts and seedling hypocotyl segments. Protoplasts were isolated from embryogenic calluses of Murraya paniculata and Fortunella hindsii. Seedling hypocotyl segments internode of cultured seeds of both species. The experiments were conducted in Biotechnology and Genetic Universitas Islam Riau, Jl Kaharuddin Nasution 113 Pekanbaru 28284 Indonesia during 2020 to 2024.
 
Protoplast sources
 
Protoplast sources that were isolated from callus of Fortunella hindsii and Murraya paniculata by using (Jumin and Nito. 1996abc) procedures. Formation callus emerged from plantlets in the culture after 3 mounts sub-culturing on MT media. Media prepared to induce embryogenic callus with 5% glucosa and addition of plant 0.01 mg/l kinetin, 600 mg/l malt extract and growing under 52.9 mmol.m-2.s-1 light intensity and a photoperiod of 16 h at 25°C.
 
Isolation of protoplasts
 
Embryogenic callus was put in Erlenmeyer and mixed together with 0.4% macerozyme, 0.2% cellulose and 0.1% driselase and pH was adjusted to 6.0. After overnight incubation on the gyratory shaker, the culturing of enzymes and callus were separated by reciprocal shaker at 25 rpm, for 5 min and 3 times.
 
Protoplast cultures
 
Two drops of purified protoplasts were cultured with media supplemented with 2.0% glucose and 0.0, 0.001, 0.01, 0.1, 1.0. or 10.0  mg/l thidiazuron (TDZ) and thickened with 0.9% gelrite. The culturing protoplasts were maintained for 40 days at 25°C temperature, 16 h photoperiod at 18.7 mmol.m2.s-1 light intensity. Furthermore, protoplasts developed into embryogenic callus and were maintained in the growth chamber at 25°C with 54 mmol.m2/s light intensity. Plating efficiency was counted as the percentage of living cells which become cells clustered in the media during 70 days following (Jumin and Nito, 1996c) procedures. The cell wall recoveries of the protoplasts was checked by fluorescein diacetate staining under an inverted microscope beginning from 10 days of culture.

Embryo induction and development
 
Sub-culturing of living cells has been subcultures 2 times  every 30 days beginning from 30 days of living cell cultures till  90 days using media MT media supplemented with 2.0% glucose, 0.0, 0.001, 0.01, 0.1, 1.0 and 10.0 mg/l thidiazuron. (TDZ) solidified with 0.2% gelrite. After somatic embryos emerged, about 2 mm in length, they were carefully transferred into media consisting of 4% sucrose with 0.001 mg/l TDZ and 0.3% gelrite, 6.0 pH at 25°C maintained for 80 days.
 
In vitro flowering
 
Embryogenic callus developed into globular embryos and then grew into cotyledon embryo-like structures after about 50 days of culture. Torpedo somatic embryos like-structure grow into plantlets with two or more leaves and transferred into fresh media with the same treatments. The percentage of flowering was checked beginning from 15 days till 70 days of culturing in the media at room temperatures 25°C with 54 mmol.m-2s-1 light intensity and 16 days photoperiod.
 
Hypocotyl sources
 
Hypocotyl segments of cultured seedlings of Murraya paniculata and Fortunella hindsii were cut to 5 segments 5 mm  in long and they were cultured into half-strength MT media consisting either 0.0, 0.001, 0.01, 0.1, 1.0 and 10.0 mg/l TDZ  and either 2.0%, 3% and 4% sucrose and distributed to  25 × 150 mm glass tubes.

RESULTS AND DISCUSSION

Fresh protoplasts 30 minutes post isolation a diameter of 20-30 mm was cultured in half strength MT medium (Fig 3A). About 80% of the protoplasts were successful in recovering the cell wall and growth normally (Table 1, 2, Fig 3B) Cells grow into colonies 70 days after cultures and develop into globular somatic embryos (Table 3). Media with 0.001 mg/l TDZ significantly triggered cells to develop cotyledon somatic embryos-like structure and grow to torpedo shaped embryos like-structures about 50 days after cultures (Table 4). Torpedo shaped somatic embryos like structures  grow into plantlets.
 

Table 1: Effect of TDZ and sucrose on plating efficiency (%) Murraya paniculata.


 

Table 2: Effect of TDZ and sucrose on plating efficiency (%) Fortunella hindsii.


 

Table 3: Effect of TDZ and sucrose on development of protoplast- derived globular somatic embryos (%) Fortunella hindsii 40 days after culture (30 globular somatic embryos tested for each treatment).


 

Table 4: Effect of TDZ and sucrose on shoot formation of Murraya paniculata 50 days after torpedo shaped embryos like- structure culture (20 torpedo shaped embryos like-structure tested for each treatment).


       
The relationship between shoot formation and flowering in vitro showed as interdependent in linear equation with y = 0.7542x+11.608 R² = 0.9029 (Fig 1) for Fortunella hindsii  and  y = 1.426x-12.541 R² = 0.9602 for Murraya panicuata (Fig 2). Meaning that an increase of shoot formation followed by an increase of flowering and they are connected to each other as a linear pattern. As a result there’s an improvement in the somatic embryos and that is also an increasing percentage of flowering of the plantlets Fortunella hindsii and Murraya paniculata (Fig 4).
 

Fig 1: Relationship between shoot formation and flowering in vitro of Fortunella hindsii.


 

Fig 2: Relationship between shoot formation and flowering in vitro of Murraya paniculata.


       
Murraya paniculata look more responsive to TDZ and sucrose than Fortunella hindsii. However both species have been sensitive to low concentration of TDZ. Concentration 0.001 mg/l TDZ is the best concentration to trigger protoplasts to recover the cell walls. The same response that the low concentration of TDZ is also significantly recovered cells grow into globular somatic embryos like-structure and to form cotyledon embryos like-structures. The concentration of  0.001 mg/l TDZ  significantly triggered cells to develop cotyledon somatic embryos-like structure and grow to  torpedo shaped embryos like-structures about 50 days after cotyledon somatic embryo like-structure cultures (Table 3). Torpedo embryos like-structures  grow into plantlets (Ralker et al., 2008;, Neumann et al., 2020).
       
Murashige and tucker media supplemented with cytokinin (TDZ) after the hypocotyl segments of Fortunella hindsii and Murraya paniculata grew to make the plantlets with more leaves. The number of leaves in each plantlet is averaged 2 to 4 leaves.  
       
Media supplemented with cytokines supported the tissues to grow and become  new tissues (Murashige and Tucker, 1969; Abubakar and Pudake, 2019; Fonseka and Aluthgamage, 2021).
       
At concentrations ranging from 0.001 to 0.10 mg/l TDZ, in vitro shoots of Murraya paniculata and Fortunella hindsii exhibited a favorable reaction to generate plantlets. With 80% of the hypocotyl segments developing into shoots and 75% beginning blossoms. TDZ at 0.01 mg/l shown to be the most effective to develope in flower in vitro (Fig 3C). A study by Srinivasan and Mullin (1978) indicated that endogenous cytokinin in ascending xylem sap is triggered by exogenous TDZ, hereafter  stimulating flower buds and then causing the plant to initiate flower development.
 

Fig 3: (Left). Fresh protoplasts 30 minutes after isolation (bar 30 mm).


               
Murraya  paniculata protoplast and hypocotyl seedling near  apical meristems using exogenous cytokinin to generate flowers in vitro. Moreover,  Fortunella  hindsii and Murraya paniculata hypocotyl segments initiated the mitotic cycle, which typically occurs prior to flowering. Bernier et al., (1977) discovered that cytokinin (TDZ) given to citrus relatives’ protoplasts and plantlets may cause in vitro flowering. When Fortunella  hindsii and Murraya  paniculata plantlets generated from protoplasts developed in to flower bud in media supplemented with 0.001 mg/l TDZ and 2.0% sucrose and then flowering frequency rise. Media containing 2.0% sucrose and 0.00 mg/l TDZ added to media, the hypocotyl segment produced the best flower blooming (Fig 1, 2). According to Jumin and Ahmad (2000), when plantlets were cultured in MT media with 0.01 mg/l cytokinin (BA) and 5% sucrose, Fortunella hindsii and Murraya paniculata floral buds grew into regular flowers. If there are high quantity of cytokinin, plantlets are sufficient to develop flower buds (Jumin and Ahmad (2000); Scorza and Janick, 1980; Sing, 2023).  Most flowers were normal and complex and appeared in the leaf axils. In Suitable concentration of cytokinin flowers fromed in vitro are common in citrus relatives when media, photoperiod, temperature, light intensity and other factors are supported  (Jumin and Nito. 1996ab; Jumin, 2016; Jayaraman and Ramachandran. 2024). 
       
Murraya paniculata protoplast and hypocotyl seedling apical meristems using exogenous cytokinin to generate flowers in vitro. Moreover, Fortunella hindsii hypocotyl segment initiated the mitotic cycle, which typically occurs prior to flowering. Bernier et al., (1977), Jumin and Nito (1996ab) discovered that cytokinin (TDZ) given to citrus relatives’ protoplasts and plantlets may cause in vitro flowering. when plantlets were cultured in medium with 0.01 mg/l cytokinin (BA) and 5% sucrose, floral buds in Fortunella  hindsii and Murraya  paniculata grew into regular flowers. 
       
Flower formation occurred when Fortunella hindsii and Murraya paniculata plantlets derived from protoplasts cultured in media supplemented with 0.001 mg/l TDZ and 2.0% sucrose. The optimal flowering originating from hypocotyl segments occurred from media  with 0.00 mg/l TDZ and 2.0% sucrose. This is Jumin and Mukhtar (2000) mentioned that, In Fortunella hindsii and Murraya paniculata floral buds developed into complete and normal flowers when plantlets cultured in media at 0.01 mg/l cytokinin (BA), 5% sucrose. Plantlets are proper to form floral buds if 0.01 mg/l BA (cytokinin) and 2.0% sucrose mixed to media (Scorza and Janick, 1980; Jumin, 2016).
       
Flower formation gradually supple above 0.01 mg/l TDZ The optimal concentration of TDZ for flowering in vitro in both  species has only 0.001 mg/l TDZ and 2.0% sucrose. The frequency of flowers declined gradually after 0.01 mg/l TDZ and 3.0% sucrose.  With the optimal TDZ concentration and sucrose, there were a mean of 2 buds formed from the inflorescence developed from hypocotyl segments Fortunella hindsii and Murraya paniculata resulting from the floral buds from hypocotyl segments or plantlet derived from protoplasts. The tobacco thin cell layer stem originated from explants that produced either flower at suitable media, while leaves emerged at unsuitable media (Cousson et al., 1981).  
       
Floral gradient  for plantlets formation and flower in vitro on the first flower was observed between  the hypocotyl segments  positions if taken from the base of the hypocotyl segments during 80 days of culture. The plantlets derived from segments of hypocotyl segment commence flowers just if they were pieced node from stem originating close to the apex and cultured to media with proper plant hormone (Jumin, 2016; Cousson et al., 1981).
       
Plantlets originating from hypocotyl segments at the peak exhibited the highest percentage of flower development and as the distance from the peak increased, flowering emerged at a steadily decreasing rate (Fig 4). These findings suggest that there is a "floral gradient" in the position of hypocotyl segment both young and aged tissue.  The largest percentage of in vitro blooms for Murraya paniculata and Fortunella hindsii is seen when the hypocotyl segment started near to the apex (Jumin and  Nito, 1996; Jumin and Nito, 1996b).
 

Fig 4: Percentage of Flowering in vitro on plantlet sources.


       
They have the ability to produce flowers that descend basipetally from the hypocotyl segments base of hypocotyl.  This relates to the finding that there is a gradient in the ability of flowers to regenerate, with the position of the shoots and flowers on the hypooctyl segments clearly influencing their production in vitro. According to Scorza (1982); Scorza and Janick (1980) and McDaniel et al., (1989) flower buds derived from hypocotyl segments developed into normal declining from the apical to the basal regions of hypocotyl segments. These findings suggested that endogenous gradients of growth substances in the stem may be the cause of this apex-to-base floral gradient.
       
The floral variation may result from changes in a cell’s ability to produce or respond to a floral stimulus, from a gradient of a floral promoter or inhibitor at the time of explant excision, or from a gradient that combines promoter and inhibitor   When trying to obtain de novo flowers in thin-cell-layer explants of a photoperiodically sensitive tobacco cultivar, the position of the branch internodes in the se on the intact plant is essential (Tran-Thanh-Van, 1973; Tran Thanh Van et al., (1974;  McDaniel et al., 1989; Wardell and Skoog, 1969; Lang, 1987). Bridgen and Veilleux,(1985);  Rajeevan and Lang 1987) and Altamura et al., (1989) mentioned that because there is insufficient capacity to generate the flowering factor (s) or for the meristems to respond to the flowering factor, the position of the hypocotyl segments is insufficient to commence flowering (Lang, 1965;  Hackett, 1985; Wardell and Skoog, 1969a). Tobacco branches have been shown to be able to generate flowers in vitro (Wardell and Skoog, 1969a; Wardell and, Skoog, 1969b), while young Passiflora suberose leaf discs (Scorza, 1982) are unable to produce flowers. According to the current findings, a significant portion of branch internodes could generate blooms. Based on Chang and Hsing (1980) reported that mature Panax ginseng root callus was used to produce embryos, which in turn produced flowers with viable pollen. This effective system developed into a model that could be used to examine how feeding affects the reproductive activities of plants.

CONCLUSION

Surviving  of isolated protoplasts in media supplemented by suitable concentration thidiazuron (0.001 mg/l) and  sucrose (2.0%) grow into  embryogenic callus, globular somatic embryos,  and then  grow into spherical heart shaped somatic embryos-like structure,  further then into plantlets.  Plantlets originated from the branch internode of  Murraya paniculata  and Fortunella hindsii  induced flowers in vitro under low concentration of thidiazuron and sucrose.
       
This efficient system from the protoplasts to somatic embryos and to plantlets in vitro of two species of have been potentially used as genetic manipulation to repair the genetics of these two species. 

ACKNOWLEDGEMENT

We thank to Rector Islamic University of Riau for Financial support to joins the ICABBBE 2024: 18. International Conference on Agricultural, Biotechnology, Biological and Biosystems Engineering, October 28-29, 2024 in Paris, France. The special thank to Director of  Post Graduate in Agronomy programm  for assist the administrative affair.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    Full Research Article

    volume 44 issue 5 (october 2024) : 885-891,   Doi: 10.18805/ag.DF-621

    Thidiazuron Role in In vitro Flowering of Murraya paniculata and Fortunella hindsii via Embryogenic Protoplast and Hypocotyl Segments Cultures

    H
    Hasan Basri Jumin1,*
    1Garduate School in Agriculture Islamic University of Riau Jl Kaharuddin Nasution 113 Pekanbaru 28284, Indonesia.
    Cite article:- Jumin Basri Hasan (2024). Thidiazuron Role in In vitro Flowering of Murraya paniculata and Fortunella hindsii via Embryogenic Protoplast and Hypocotyl Segments Cultures . Agricultural Science Digest. 44(5): 885-891. doi: 10.18805/ag.DF-621.

    ABSTRACT

    Background: Procedures to regenerate protoplast into plants of citrus relatives is important to establish the genetic improvement for sweet oranges by using somatic hybridization and transgenic. Protoplasts are a good material to genetically manipulate and to become gene transfer. This study aims to characterize the regeneration sequence of Fortunella hindsii and Murraya paniculata protoplasts via somatic embryogenesis into flowering plants in vitro. This study also describes the sequence of flowering in vitro originated from hypocotyl segments of plantlets of two species.

    Methods: Protoplast sources that were isolated from callus of Fortunella hindsii and Murraya paniculata. Embryogenic callus was put in Erlenmeyer and mixed together with 0.4% macerozyme, 0.2% cellulose and 0.1% driselase and pH was adjusted to 6.0. Two drops of purified protoplasts were cultured with media supplemented with 2.0% glucose and 0.0, 0.001, 0.01, 0.1 or 1.0. 10.0 mg/l thidiazuron (TDZ) and thicken with 0.9% gelrite. 

    Result: The media supplemented with 0.001 mg/l TDZ yielded the maximum plating efficiency, while 0.001 mg/l TDZ produced the highest percentage of shoot formation, approximately 80%. After being cultured on the same TDZ concentration for 14 days, the protoplasts that survived developed cell walls. Fortunella hindsii and Murraya paniculata underwent somatic embryogenesis to grow into plantlets. Thidiazuron has demonstrated efficacy in forming flower buds that grow normally. Murraya paniculata and Fortunella hindsii shoots that emerged from hypocotyl segmments flowered in vitro on half-strength MT media containing 0.001 to 0.01 mg/l TDZ and 2-3% sucrose after two months of culture and they eventually went on to flower.

    INTRODUCTION

    Thidiazuron (TDZ) encourages flowers in low concentration and there could be increased plant metabolism and reserve plants. Thidiazuron may produce high quality bud floral and develop to normal flowers (Olah et al., 2003). Thidiazuron with suitable  concentration  have been  increase the  axillary bud and are possible to increase frequency flowering in vitro. Addition of TDZ to the culture, triggered the adventitious buds to develop into  early flowering in vitro. Thidiazuron also belongs to the group cytokinin synthetic as well as the benzylaminopurine. Thidiazuron have a role in stimulating endogenous cytokinin and they  play a role as a cytokinin oxidase inhibitor that is an enzyme (Parveen and Shahzad, 2010).
           
    Fortunella hindsii is one of citrus relatives that are potentially to use as an genetic resources for quality improvement and make the resistance traits of citrus, because the fruiting time is relatively short (Fu et al., 2003; Chen, 2012; Jumin and Nito, 1996a; Jumin and Nito, 1996b).  As a citrus relative’s member, they are used as a garnish while they also could be consumed as a food substitute to the citrus. Murraya paniculata potentially to be used as rooting sources for citrus  resistance to nematodes, water flooding, saline soil and substitution a genetics improvement (Swingle and Reece.1967; Sykes. 1988; Raveh. 2012; Jumin. 2013). Apparently the both varieties are related to citrus which are the easiest to use as sources as stem grafting and establish somatic hybridization with sweet orange (Citrus sinensis). Citrus sinensis is one kind of sweet orange that is in the highest demand in the market mainly in Indonesia and south east Asian countries.
           
    Procedures to regenerate protoplast into plants of citrus relatives is important to establish the genetic improvement for sweet oranges by using somatic hybridization and transgenic plants. Protoplasts are a good material to genetically manipulate and to become gene transfer (Ahmed et al., 2021; Thakur et al., 2022; Chen et al., 2023). Plants regenerated from seedlings take a long time to reach flowering. Fortunella hindsii as rooting to make the citrus become dwarfed for garnish purpose. Plant grafting in the citrus could be used as a way to shorten the early flowering plants. Shoot resulted from regenerated protoplasts as alternatively as stems to be used for grafting of citrus.    
           
    These technologies may be utilized as an additional method for producing wide hybridizations via protoplast fusion in order to improve the rootstock. This study aims to characterize the regeneration sequence of Fortunella hindsii and Murraya paniculata protoplasts via somatic embryogenesis into flowering plants in vitro. This study also describes the sequence of flowering in vitro originated from hypocotyl segment of plantlets of two species.

    MATERIALS AND METHODS

    Plant materials
     
    Plant Materials using protoplasts and seedling hypocotyl segments. Protoplasts were isolated from embryogenic calluses of Murraya paniculata and Fortunella hindsii. Seedling hypocotyl segments internode of cultured seeds of both species. The experiments were conducted in Biotechnology and Genetic Universitas Islam Riau, Jl Kaharuddin Nasution 113 Pekanbaru 28284 Indonesia during 2020 to 2024.
     
    Protoplast sources
     
    Protoplast sources that were isolated from callus of Fortunella hindsii and Murraya paniculata by using (Jumin and Nito. 1996abc) procedures. Formation callus emerged from plantlets in the culture after 3 mounts sub-culturing on MT media. Media prepared to induce embryogenic callus with 5% glucosa and addition of plant 0.01 mg/l kinetin, 600 mg/l malt extract and growing under 52.9 mmol.m-2.s-1 light intensity and a photoperiod of 16 h at 25°C.
     
    Isolation of protoplasts
     
    Embryogenic callus was put in Erlenmeyer and mixed together with 0.4% macerozyme, 0.2% cellulose and 0.1% driselase and pH was adjusted to 6.0. After overnight incubation on the gyratory shaker, the culturing of enzymes and callus were separated by reciprocal shaker at 25 rpm, for 5 min and 3 times.
     
    Protoplast cultures
     
    Two drops of purified protoplasts were cultured with media supplemented with 2.0% glucose and 0.0, 0.001, 0.01, 0.1, 1.0. or 10.0  mg/l thidiazuron (TDZ) and thickened with 0.9% gelrite. The culturing protoplasts were maintained for 40 days at 25°C temperature, 16 h photoperiod at 18.7 mmol.m2.s-1 light intensity. Furthermore, protoplasts developed into embryogenic callus and were maintained in the growth chamber at 25°C with 54 mmol.m2/s light intensity. Plating efficiency was counted as the percentage of living cells which become cells clustered in the media during 70 days following (Jumin and Nito, 1996c) procedures. The cell wall recoveries of the protoplasts was checked by fluorescein diacetate staining under an inverted microscope beginning from 10 days of culture.

    Embryo induction and development
     
    Sub-culturing of living cells has been subcultures 2 times  every 30 days beginning from 30 days of living cell cultures till  90 days using media MT media supplemented with 2.0% glucose, 0.0, 0.001, 0.01, 0.1, 1.0 and 10.0 mg/l thidiazuron. (TDZ) solidified with 0.2% gelrite. After somatic embryos emerged, about 2 mm in length, they were carefully transferred into media consisting of 4% sucrose with 0.001 mg/l TDZ and 0.3% gelrite, 6.0 pH at 25°C maintained for 80 days.
     
    In vitro flowering
     
    Embryogenic callus developed into globular embryos and then grew into cotyledon embryo-like structures after about 50 days of culture. Torpedo somatic embryos like-structure grow into plantlets with two or more leaves and transferred into fresh media with the same treatments. The percentage of flowering was checked beginning from 15 days till 70 days of culturing in the media at room temperatures 25°C with 54 mmol.m-2s-1 light intensity and 16 days photoperiod.
     
    Hypocotyl sources
     
    Hypocotyl segments of cultured seedlings of Murraya paniculata and Fortunella hindsii were cut to 5 segments 5 mm  in long and they were cultured into half-strength MT media consisting either 0.0, 0.001, 0.01, 0.1, 1.0 and 10.0 mg/l TDZ  and either 2.0%, 3% and 4% sucrose and distributed to  25 × 150 mm glass tubes.

    RESULTS AND DISCUSSION

    Fresh protoplasts 30 minutes post isolation a diameter of 20-30 mm was cultured in half strength MT medium (Fig 3A). About 80% of the protoplasts were successful in recovering the cell wall and growth normally (Table 1, 2, Fig 3B) Cells grow into colonies 70 days after cultures and develop into globular somatic embryos (Table 3). Media with 0.001 mg/l TDZ significantly triggered cells to develop cotyledon somatic embryos-like structure and grow to torpedo shaped embryos like-structures about 50 days after cultures (Table 4). Torpedo shaped somatic embryos like structures  grow into plantlets.
     

    Table 1: Effect of TDZ and sucrose on plating efficiency (%) Murraya paniculata.


     

    Table 2: Effect of TDZ and sucrose on plating efficiency (%) Fortunella hindsii.


     

    Table 3: Effect of TDZ and sucrose on development of protoplast- derived globular somatic embryos (%) Fortunella hindsii 40 days after culture (30 globular somatic embryos tested for each treatment).


     

    Table 4: Effect of TDZ and sucrose on shoot formation of Murraya paniculata 50 days after torpedo shaped embryos like- structure culture (20 torpedo shaped embryos like-structure tested for each treatment).


           
    The relationship between shoot formation and flowering in vitro showed as interdependent in linear equation with y = 0.7542x+11.608 R² = 0.9029 (Fig 1) for Fortunella hindsii  and  y = 1.426x-12.541 R² = 0.9602 for Murraya panicuata (Fig 2). Meaning that an increase of shoot formation followed by an increase of flowering and they are connected to each other as a linear pattern. As a result there’s an improvement in the somatic embryos and that is also an increasing percentage of flowering of the plantlets Fortunella hindsii and Murraya paniculata (Fig 4).
     

    Fig 1: Relationship between shoot formation and flowering in vitro of Fortunella hindsii.


     

    Fig 2: Relationship between shoot formation and flowering in vitro of Murraya paniculata.


           
    Murraya paniculata look more responsive to TDZ and sucrose than Fortunella hindsii. However both species have been sensitive to low concentration of TDZ. Concentration 0.001 mg/l TDZ is the best concentration to trigger protoplasts to recover the cell walls. The same response that the low concentration of TDZ is also significantly recovered cells grow into globular somatic embryos like-structure and to form cotyledon embryos like-structures. The concentration of  0.001 mg/l TDZ  significantly triggered cells to develop cotyledon somatic embryos-like structure and grow to  torpedo shaped embryos like-structures about 50 days after cotyledon somatic embryo like-structure cultures (Table 3). Torpedo embryos like-structures  grow into plantlets (Ralker et al., 2008;, Neumann et al., 2020).
           
    Murashige and tucker media supplemented with cytokinin (TDZ) after the hypocotyl segments of Fortunella hindsii and Murraya paniculata grew to make the plantlets with more leaves. The number of leaves in each plantlet is averaged 2 to 4 leaves.  
           
    Media supplemented with cytokines supported the tissues to grow and become  new tissues (Murashige and Tucker, 1969; Abubakar and Pudake, 2019; Fonseka and Aluthgamage, 2021).
           
    At concentrations ranging from 0.001 to 0.10 mg/l TDZ, in vitro shoots of Murraya paniculata and Fortunella hindsii exhibited a favorable reaction to generate plantlets. With 80% of the hypocotyl segments developing into shoots and 75% beginning blossoms. TDZ at 0.01 mg/l shown to be the most effective to develope in flower in vitro (Fig 3C). A study by Srinivasan and Mullin (1978) indicated that endogenous cytokinin in ascending xylem sap is triggered by exogenous TDZ, hereafter  stimulating flower buds and then causing the plant to initiate flower development.
     

    Fig 3: (Left). Fresh protoplasts 30 minutes after isolation (bar 30 mm).


                   
    Murraya  paniculata protoplast and hypocotyl seedling near  apical meristems using exogenous cytokinin to generate flowers in vitro. Moreover,  Fortunella  hindsii and Murraya paniculata hypocotyl segments initiated the mitotic cycle, which typically occurs prior to flowering. Bernier et al., (1977) discovered that cytokinin (TDZ) given to citrus relatives’ protoplasts and plantlets may cause in vitro flowering. When Fortunella  hindsii and Murraya  paniculata plantlets generated from protoplasts developed in to flower bud in media supplemented with 0.001 mg/l TDZ and 2.0% sucrose and then flowering frequency rise. Media containing 2.0% sucrose and 0.00 mg/l TDZ added to media, the hypocotyl segment produced the best flower blooming (Fig 1, 2). According to Jumin and Ahmad (2000), when plantlets were cultured in MT media with 0.01 mg/l cytokinin (BA) and 5% sucrose, Fortunella hindsii and Murraya paniculata floral buds grew into regular flowers. If there are high quantity of cytokinin, plantlets are sufficient to develop flower buds (Jumin and Ahmad (2000); Scorza and Janick, 1980; Sing, 2023).  Most flowers were normal and complex and appeared in the leaf axils. In Suitable concentration of cytokinin flowers fromed in vitro are common in citrus relatives when media, photoperiod, temperature, light intensity and other factors are supported  (Jumin and Nito. 1996ab; Jumin, 2016; Jayaraman and Ramachandran. 2024). 
           
    Murraya paniculata protoplast and hypocotyl seedling apical meristems using exogenous cytokinin to generate flowers in vitro. Moreover, Fortunella hindsii hypocotyl segment initiated the mitotic cycle, which typically occurs prior to flowering. Bernier et al., (1977), Jumin and Nito (1996ab) discovered that cytokinin (TDZ) given to citrus relatives’ protoplasts and plantlets may cause in vitro flowering. when plantlets were cultured in medium with 0.01 mg/l cytokinin (BA) and 5% sucrose, floral buds in Fortunella  hindsii and Murraya  paniculata grew into regular flowers. 
           
    Flower formation occurred when Fortunella hindsii and Murraya paniculata plantlets derived from protoplasts cultured in media supplemented with 0.001 mg/l TDZ and 2.0% sucrose. The optimal flowering originating from hypocotyl segments occurred from media  with 0.00 mg/l TDZ and 2.0% sucrose. This is Jumin and Mukhtar (2000) mentioned that, In Fortunella hindsii and Murraya paniculata floral buds developed into complete and normal flowers when plantlets cultured in media at 0.01 mg/l cytokinin (BA), 5% sucrose. Plantlets are proper to form floral buds if 0.01 mg/l BA (cytokinin) and 2.0% sucrose mixed to media (Scorza and Janick, 1980; Jumin, 2016).
           
    Flower formation gradually supple above 0.01 mg/l TDZ The optimal concentration of TDZ for flowering in vitro in both  species has only 0.001 mg/l TDZ and 2.0% sucrose. The frequency of flowers declined gradually after 0.01 mg/l TDZ and 3.0% sucrose.  With the optimal TDZ concentration and sucrose, there were a mean of 2 buds formed from the inflorescence developed from hypocotyl segments Fortunella hindsii and Murraya paniculata resulting from the floral buds from hypocotyl segments or plantlet derived from protoplasts. The tobacco thin cell layer stem originated from explants that produced either flower at suitable media, while leaves emerged at unsuitable media (Cousson et al., 1981).  
           
    Floral gradient  for plantlets formation and flower in vitro on the first flower was observed between  the hypocotyl segments  positions if taken from the base of the hypocotyl segments during 80 days of culture. The plantlets derived from segments of hypocotyl segment commence flowers just if they were pieced node from stem originating close to the apex and cultured to media with proper plant hormone (Jumin, 2016; Cousson et al., 1981).
           
    Plantlets originating from hypocotyl segments at the peak exhibited the highest percentage of flower development and as the distance from the peak increased, flowering emerged at a steadily decreasing rate (Fig 4). These findings suggest that there is a "floral gradient" in the position of hypocotyl segment both young and aged tissue.  The largest percentage of in vitro blooms for Murraya paniculata and Fortunella hindsii is seen when the hypocotyl segment started near to the apex (Jumin and  Nito, 1996; Jumin and Nito, 1996b).
     

    Fig 4: Percentage of Flowering in vitro on plantlet sources.


           
    They have the ability to produce flowers that descend basipetally from the hypocotyl segments base of hypocotyl.  This relates to the finding that there is a gradient in the ability of flowers to regenerate, with the position of the shoots and flowers on the hypooctyl segments clearly influencing their production in vitro. According to Scorza (1982); Scorza and Janick (1980) and McDaniel et al., (1989) flower buds derived from hypocotyl segments developed into normal declining from the apical to the basal regions of hypocotyl segments. These findings suggested that endogenous gradients of growth substances in the stem may be the cause of this apex-to-base floral gradient.
           
    The floral variation may result from changes in a cell’s ability to produce or respond to a floral stimulus, from a gradient of a floral promoter or inhibitor at the time of explant excision, or from a gradient that combines promoter and inhibitor   When trying to obtain de novo flowers in thin-cell-layer explants of a photoperiodically sensitive tobacco cultivar, the position of the branch internodes in the se on the intact plant is essential (Tran-Thanh-Van, 1973; Tran Thanh Van et al., (1974;  McDaniel et al., 1989; Wardell and Skoog, 1969; Lang, 1987). Bridgen and Veilleux,(1985);  Rajeevan and Lang 1987) and Altamura et al., (1989) mentioned that because there is insufficient capacity to generate the flowering factor (s) or for the meristems to respond to the flowering factor, the position of the hypocotyl segments is insufficient to commence flowering (Lang, 1965;  Hackett, 1985; Wardell and Skoog, 1969a). Tobacco branches have been shown to be able to generate flowers in vitro (Wardell and Skoog, 1969a; Wardell and, Skoog, 1969b), while young Passiflora suberose leaf discs (Scorza, 1982) are unable to produce flowers. According to the current findings, a significant portion of branch internodes could generate blooms. Based on Chang and Hsing (1980) reported that mature Panax ginseng root callus was used to produce embryos, which in turn produced flowers with viable pollen. This effective system developed into a model that could be used to examine how feeding affects the reproductive activities of plants.

    CONCLUSION

    Surviving  of isolated protoplasts in media supplemented by suitable concentration thidiazuron (0.001 mg/l) and  sucrose (2.0%) grow into  embryogenic callus, globular somatic embryos,  and then  grow into spherical heart shaped somatic embryos-like structure,  further then into plantlets.  Plantlets originated from the branch internode of  Murraya paniculata  and Fortunella hindsii  induced flowers in vitro under low concentration of thidiazuron and sucrose.
           
    This efficient system from the protoplasts to somatic embryos and to plantlets in vitro of two species of have been potentially used as genetic manipulation to repair the genetics of these two species. 

    ACKNOWLEDGEMENT

    We thank to Rector Islamic University of Riau for Financial support to joins the ICABBBE 2024: 18. International Conference on Agricultural, Biotechnology, Biological and Biosystems Engineering, October 28-29, 2024 in Paris, France. The special thank to Director of  Post Graduate in Agronomy programm  for assist the administrative affair.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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