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.
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).
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.
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).
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.
