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Article

Development of Triploid Seedless Nagpur Mandarin (Citrus reticulata Blanco) through Endosperm Rescue

by
Vijayakumari Narukulla
1,*,
Yogesh Lahane
1,
Shashi Pandey
1,
Krutika Fiske
1,
Priya Uge
1,
Kartiki Kawale
1 and
Vasileios Ziogas
2,*
1
Tissue Culture Laboratory, ICAR-Central Citrus Research Institute (CCRI), Amravati Road, Nagpur 440010, Maharashtra, India
2
Institute of Olive Tree, Subtropical Plants and Viticulture, Hellenic Agricultural Organisation DIMITRA (ELGO-DIMITRA), 73134 Chania, Greece
*
Authors to whom correspondence should be addressed.
Agronomy 2023, 13(11), 2814; https://doi.org/10.3390/agronomy13112814
Submission received: 12 October 2023 / Revised: 7 November 2023 / Accepted: 9 November 2023 / Published: 14 November 2023
(This article belongs to the Special Issue Fruit Growing: Production Practices and Post-Harvest Management)
Browse Figures
Review Reports Versions Notes

Abstract

:
Nagpur mandarin is a popular table fruit across India and is exported to various countries. Only 1% of the total production is processed into various products. The development of seedless cultivars will boost agricultural incomes by enhancing the potential for export and processing. At present, only a couple of commercially seedless varieties are available, but these have yet to become popular. A research study was undertaken to quickly develop a high-quality seedless variety of Nagpur mandarin by combining the available technologies, viz., endosperm rescue, somatic embryogenesis, and mini-grafting, at CCRI, Nagpur. Complete plantlets of C. reticulata Blanco cv. Nagpur mandarin was successfully regenerated from hybrid endosperm tissue via somatic embryogenesis after attempting various permutations and combinations of media at various stages of regeneration, right from the primary callus to complete plantlet production. Maximum response (93.33%) and survival (91.67%) for primary callus induction were obtained in Murashige and Tucker (MT) + Malt Extract (ME) + Casein Hydrolysate (CH) (500 mg/L) +2,4-dichlorophenoxyacetic acid (2,4-D) (2 mg/L) medium. Maximum stimulation for embryogenesis and morphogenesis occurred in 2MT + CH (500 mg/L) + adenine sulfate (ad.s) (2 mg/L) + Benzyl Adenine (BA) (0.25 mg/L). The highest response (95.84%) for shoot differentiation occurred in MT + adenine sulphate (2 mg/L) + Gibberellic Acid (GA3) (1 mg/L) + BA (1 mg/L) (94.85%). The surviving plantlets were tested for ploidy status through flow Cytometry, chromosomal counting/cytogenetic technique, leaf morphology, stomatal characteristics, and the appearance of prominent thorns. In initial evaluation trials, the fruits of the triploid field-planted trees were found to be commercially seedless. These results demonstrated the recovery of stable triploids from the hybrid endosperm via somatic embryogenesis, which is the first of its kind in the field of Citrus triploid breeding in India.

1. Introduction

Nagpur mandarin, popularly known as Nagpur Santra across India, is the most important commercial fruit crop in Central India and is grown over an area of 252,000 ha with a total production of 3,200,000 MT [1]. Fruit is mainly consumed as table fruit, and less than 1% of the total production is processed. Nagpur mandarin is currently exported to neighboring countries such as Bangladesh, Nepal, Bhutan, the United Arab Emirates, etc. [2]. The Indian mandarins ripen earlier and are available in the market for export when there are no fruits in the European market. The Maharashtra State Agricultural Marketing Board is exploring and promoting the export of Nagpur mandarin to newer markets through promotional schemes.
Seed number determines the degree of acceptance by fresh fruit consumers and the processing industries. The fruits of Nagpur Santra normally contain about 12 or more seeds per fruit. Seed is the chief source of the bitter principle, viz., limonin; hence, seedless cultivars are preferred by the processing industry. The popularity of many otherwise high-quality cultivars, viz., Duncan grapefruit, Dancy tangerine, Tangors, and Tangelos, was reduced by the high seed number within the fruits.
The development of seedless cultivars with acceptable fruit quality has been an important breeding/horticulture objective. Therefore, horticulturists and breeders employed different approaches, viz., (i) Selection of natural seedless bud mutants, (ii) Mutation Breeding (iii) Ploidy manipulation (through colchicine), followed by inter-ploid hybridization (2n × 4n: 4n × 2n), embryo rescue/culture [3,4,5], (iv) Somatic hybridization via protoplast fusion, and (v) Development of Transgenic plants to reduce the seed content in fruit crops. Triploid plants were reported from another culture [6,7]. The frequency of naturally occurring triploids with desirable fruit quality in citrus is low. Mutation breeding through irradiation can enhance the frequency of seedless/triploid mutants, but the selection of desirable seedless mutants is a time-consuming process.
The reported causes of seedlessness in citrus were male sterility, female sterility, incompatibility, parthenocarpy, and early embryo abortion [8,9]. The unique reproductive biology of Citrus (incompatibility, high degree of apomixes) and long gestation period pose problems for the implementation of conventional breeding methods [10,11,12]. The researchers combined both conventional breeding and advanced biotechnological techniques to overcome these limitations and develop seedless citrus cultivars. The development of techniques for the successful rescue of triploid endosperm and plantlet regeneration and the inherent seedless character of triploids have opened new opportunities for seedless cultivar development in various fruit crops. Many Citrus breeding programs around the world are focused on the development of tetraploids through inter-ploid crosses to generate seedless triploids [3]. In citrus, ‘Beibai’ Pummelo triploid plants were obtained by endosperm culture. It has demonstrated the possibility of obtaining endosperm plants in other Citrus species [13].
Endosperm culture is used in Citrus spp., viz., Citrus sinensis (L.) Osbeck, the sweet orange; Citrus maxima (Burm.), the Pummelo and Citrus reticulata Blanco cv. Kinnow mandarin, to produce triploid plants. In China, triploid plants of Citrus sinensis (L.) Osbeck [14] were regenerated from endosperm callus. In 1990, Professor Gmitter’s group produced triploid plants through the endosperm culture of Citrus grandis at the Citrus Research Center, University of Florida, USA. These authors induced callus from the endosperm and regenerated plantlets. They confirmed the triploid status of regenerated plants by using the chromosome count method. Various attempts have also been made to produce seedless Kinnow mandarins via irradiation and endosperm culture (for the production of triploid plants) in Pakistan, but success has been limited [15].
The basic chromosome number in the genus Citrus and other members of the subfamily Aurantioideae is x = 9. Almost all wild and cultivated forms of Citrus are diploid and comprise small-sized chromosomes [16]. The mitotic index is mostly low in root tips. High-grade metaphase preparation requires a proper, high-resolution metaphase spread. Cytogenetic studies such as chromosome counting in various in vitro and in vivo ploidy experiments and in-situ hybridization of higher ploidy species help in cultivar improvement. The currently available and routinely used squash preparation methodology is unable to obtain good-quality chromosome spreads. The enzymatic digestion method for leaf chromosome preparation is a reliable technique to solve drawbacks in conventional squash preparation methodology [17]. Emerging shoot tissue is a reliable source of active metaphase chromosomes in any plant species. However, ploidy level analysis in Citrus by cytogenetic methods, whether from root tips or from emerging leaf tissue, is a slow and inadequate process when large populations of plants have to be analyzed; however, detecting polyploid seems to be convenient by flow cytometry, which is an easier and much faster method that was adopted in confirmation of the ploidy level in many citrus improvement projects [6,18,19,20,21,22].
Stomata length and frequency have often been used as morphological markers for identifying ploidy levels in many plant species [23,24,25,26]. In general, stomata and epidermal cell frequency per unit leaf area decreased, while stomata guard cell length improved with an increase in ploidy level [26]. Stomatal frequency and size were previously studied in several Citrus species [27].
In Nagpur mandarin, the selection of naturally occurring less seedy mutants resulted in the development of Mudkhed seedless and Nagpur seedless varieties. However, these cultivars have not become popular so far. In a long-term evaluation trial conducted from 1988 to 2002, Nagpur seedless (NRCC 4) and Mudkhed seedless cvs proved to be commercially seedless with 4.11 and 2.63 seeds per fruit, respectively. It is observed that in the Nagpur seedless cultivar, about 50% of fruits were totally seedless, 25% of fruits contained one to two seeds, and 25% of fruits contained two to three seeds [28].
In view of the need for the development of seedless triploid cultivars of Nagpur mandarin to improve farm incomes by harnessing the export potential and enhancing the processing potential, a research project was taken up at CCRI, Nagpur, by combining endosperm rescue, somatic embryogenesis, and mini-grafting techniques.
The aim of this research project was to “produce triploid plants of Nagpur mandarin having desirable horticultural traits and seedless fruits” through endosperm rescue, somatic embryogenesis, and organogenesis, as the development of triploids in Citrus through conventional breeding is a cumbersome and time-consuming process. This paper describes the outcomes of the research undertaken.

2. Materials and Methods

2.1. Materials

A few high-yielding trees of C. reticulata Blanco cv. Nagpur mandarin, having desirable fruit quality parameters, were selected in the experimental farm block of CCRI, Nagpur, as an explant source. The objective was to induce seedless character in the fruits of these otherwise desirable plants. Around 3000 flowers of Nagpur mandarin were tagged during the ‘ambia bahar’ flowering season (February) during anthesis. The tagged open-pollinated immature fruits were harvested 75–85 days after anthesis and brought to the lab. Fruits were sterilized using Sodium hypochlorite 1% (w/v) for 10 min followed by washing 3 times with autoclaved distilled water. Fruits were dissected aseptically to excise seeds. The immature seeds were then wiped and dissected to excise the endosperm. The endosperms were carefully excised. The excised endosperm with no or minimal injury was used to initiate cultures. A single endosperm was cultured in each test tube. The experiment consisted of five treatments with four replications, and in each replication, 10 test tubes were inoculated with the endosperm.

2.2. Callus Induction

Murashige and Tucker (MT) medium with amendments was used to induce primary callus from endosperm. Response of endosperm for primary callus induction and embryogenesis was tested in five different types of media formulations (treatments), viz., (i) Murashige and Tucker (MT) + casein hydrolysate (CH) 500 mg/L +2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/L; (ii) Murashige and Tucker (MT) + Malt extract (ME) 500 mg/L; (iii) Murahige and Tucker (MT) +Casein Hydrolysate (CH) 500 mg/L +2,4-dichlorophenoxyaceticacid (2,4-D) 2 mg/L + Benzyl Adenine (BA) 5 mg/L; (iv) Murashige and Skoog (MS) + Malt Extract (ME) 500 mg/L; and (v) Murashige and Tucker (MT) + Malt Extract (ME) + Casein Hydrolysate (CH) 500 mg/L +2,4-D 2 mg/L. Media were gelled with 0.7% (w/v) agar, pH 5.8, and autoclaved for 20 min. Cultures were grown in darkness at 26 ± 1 °C.
The observations were recorded on the percentage of endosperm responses to callus and embryogenesis induction and the days taken for the initiation of primary callus. Proliferated somatic embryos arising from endosperm-derived primary callus were isolated and cultured in 25 mm × 150 mm tubes containing 25 mL of Murashige and Tucker (MT) [29] medium supplemented with Benzyl Adenine (0.25 mg/L) and Murashige and Skoog (MS) [30] medium supplemented with Casein hydrolysate (CH).

2.3. Effect of Media on Morphogenesis of Endosperm Calli

The effect of different media on the morphogenesis of endosperm calli was studied. The primary calli induced was transferred to different media, viz., fresh basal Murashige and Skoog (MS) medium + malt extract (ME) + casein hydrolysate (CH), Murashige and Tucker, and Murashige and Tucker media supplemented with lower Benzyl Adenine (BA) concentrations, Gibberellic Acid (GA3), and Adenine Sulphate for increasing proliferation, stimulation for embryogenesis, and further morphogenesis. Observations were recorded on the percentage of callus induction and the number of globular, heart-shaped, torpiodo, and cotyledonary embryoids.

2.4. Effect of Phytohormones on In Vitro Plantlet Regeneration

To improve the efficiency of shoot and root regeneration, the embryoids from endosperm-derived embryogenic callus were isolated and subcultured on six media formulations containing growth regulators, viz. gibberellic acid (GA3), Indole-3-butyric acid (IBA), adenine sulphate, and Benzyl Adenine (BA). Elongated, strong shoots from in vitro endosperm cultures were mini-grafted onto five-month-old vigorous, rough lemon rootstock to facilitate ease of transfer to the screen house and overcome the challenges of transfer to the external environment.

2.5. Ploidy Analysis

The ploidy of regenerated plants was analysed using a flow cytometer (PartecGmbh, Munster, Germany). The ploidy was presented in the form of a histogram of integral fluorescence, with the peaks depicting the ploidy level of the respective sample. In the cells labeled with fluorescent stain (4′,6-diamidino-2-phenylindoledihydrochloride), individual cells or particles get illuminated by the excitation light, and the fluorescent light intensity, which is proportional to DNA content, is measured and analysed to depict the respective number of chromosomes and estimate the ploidy level of the samples. The run sample was analysed in a UV-LED with light emission at 365 nm, adjusted to fluorescence optical detection to gain as per the control sample of plants. More than 5000 nuclei were assessed in each sample. Histograms were constructed using CyView software (serial no. 140500541, Partec Gmbh, Meckenheim, Germany).

2.6. Chromosome Counting

The protoplast-dropping method developed for the preparation of chromosomes [17], with some modifications, resolved the problem of spreading and visualization of chromosomes in citrus crops. Citrus chromosomes are small in size, i.e., 2–4 µm [16], and the mitotic index is mostly low. This protocol improved the quality of chromosome spreads, unlike the routinely used traditional squash preparation methodology.

2.7. Stomatal Morphology

Stomata analysis was carried out using molds produced through the application of transparent nail paint. A thin layer was applied to the abaxial surface of the developed leaves of triploid plants. The dried nail polish layer on the leaves was removed with the help of transparent sticky tape and transferred to a glass slide. Analysis was performed with the Leica phase contrast microscope (40 and 100× magnification) equipped with a video camera connected to a computer. Stomata frequency and stomata length per 500 µm area were observed.

2.8. Statistical Analysis

The triploidy induction via endosperm rescue and somatic embryogenesis experiment was conducted using a Completely Randomized Block (CRD) design with 4 replications having 10 explants for each replication. Stomata morphometry (stomata length and density) was also analyzed using the student’s t-test analysis.

3. Results

The aim of this research project was to “produce triploid plants of Nagpur mandarin having desirable horticultural traits and seedless fruits” through endosperm rescue, somatic embryogenesis, and organogenesis. The development of triploids in Citrus through conventional breeding is a cumbersome and time-consuming process.
Endosperms were found to be in a suitable stage of development for culture initiation when fruits were harvested 75–85 days post-anthesis. Up to 75–85 days post-anthesis, endosperms, which were elastic to a soft texture, were found to be more responsive. Excising the endosperm was difficult when fruits and seeds were older with thicker peels and seed coats [31].
Five media formulations were used to induce and support the proliferation of primary callus from endosperm. MT and MS media were used in all phases of the regeneration protocol and amended with Malt extract and BAP as needed.

3.1. Primary Callus Induction

Endosperms (without the embryos) excised from immature seeds were cultured in five different media compositions and kept in the dark at 26 ± 1 °C (Table 1). Callus initiation from cultured endosperm occurred 25–35 days after inoculation. The primary endosperm callus was generally creamish and compact but not hard and unorganized (Figure 1). When the media dried, the cultures were shifted to the same medium for proliferation of callus and incubated in dark conditions, further extended for one more month. A total of 200 endosperms were cultured on five media compositions (mentioned in para 2.2 above @ 40 endosperms per treatment) to find out the best media for callus induction (Table 1).
Response and survival of endosperm for primary callus induction were tested in various media, and maximum response (93.33%) and survival (91.67%) were obtained in MT + ME + CH (500 mg/L) +2,4-D (2 mg/L) medium. The analysis of the data indicated that the response of Nagpur mandarin endosperm to callus initiation were significantly higher (50.94%) in MT + ME + CH (500 mg/L) +2,4-D (2 mg/L), followed by MT + ME + CH (500 mg/L) +2,4-D (2 mg/L) + Kinetin (5 mg/L) (46.9%). The callus initiation response in different media varied between 26.56% and 50.94% (Table 1).

3.2. Influence of Media on Morphogenesis of Endosperm Calli

One month after dark incubation, all the proliferating callus cultures were sub-cultured on five media formulations (Table 2). The transition of the callus from amorphous form to greenish embryogenic form occurred (Figure 2) when cultures were shifted to five different media, but maximum stimulation for embryogenesis and morphogenesis (Figure 3) happened in 2MT+ CH (500 mg/L) + ad.s (2 mg/L)+ BA (0.25 mg/L). Maximum cotyledonary embryoid induction was observed in 2MT medium with adenine sulphate and BA. The next best result for stimulation of endosperm towards morphogenesis of cotyledonary embryoids was found in the same medium composition: MT + CH (500 mg/L), +GA3 (1 mg/L), and BA (0.25 mg/L) without Adenine sulphate. Almost all the media supported the callus to embryogenesis proliferation; sustained and maximum proliferation was obtained in 2MT + CH (500 mg/L) ad.s (2 mg/L) + BA (0.25 mg/L). On the embryogenesis stimulation medium, the creamishcalli turned greenish in 22 to 30 days, and morphogenesis occurred in various kinds of embryoids. Approximately 2MT + CH (500 mg/L) + ad.s (2 mg/L) + BA (0.25 mg/L) induced the highest percentage of cotyledonary embryoids, followed by MT + CH (500 mg/L) + GA3 (1 mg/L) + BA (0.25 mg/L). The best callus induction stimulation to embryogenesis and later morphogenesis to embroids occurred when the medium was supplemented with CH (500 mg/L) in all the stages. The best response to embryogenesis and morphogenesis occurred in 2MT + CH (500 mg/L) + ad.s (2 mg/L) + BA (0.25 mg/L), followed by 2MT + CH (500 mg/L) + BA (1 mg/L).
Endosperm primary callus from one surviving endosperm culture was distributed to three test tubes, so likewise, from all the treatments, endosperm-derived embryogenic calli were further cultured in 240 test tubes.
The regenerating potential of the proliferating embryogenic callus was observed for a further year by subculturing.

3.3. Influence of Phytohormones on In Vitro Shoot and Root Regeneration

From each proliferating embryogenic callus, two to three well-developed cotyledonary embroids were harvested and transferred to the shooting and rooting media. Regeneration was induced from cotyledonary embroids of endosperm calli when sub-cultured on MT and MS media supplemented with adenine Sulphate (10 mg/L) and GA3 (1 mg/L). The highest response (95.84%) for shoot differentiation occurred in MT + adenine sulphate (2 mg/L) + GA3 (1 mg/L) + BA(1 mg/L) (94.85%), followed by MT + GA3 (1 mg/L) (87.5%). Even the highest number of shoots and the maximum length of the shoot were observed in the same medium (Figure 4). On the regeneration media, green cotyledonary embryoids showed shoot bud differentiation from 11 to 28 days after culturing. The shoot bud attained a length of 1.5 to 3.5 cm in 60–75 days, depending on the medium used. MS media supplemented with BA (2 mg/L), IBA (1 mg/L), and NAA (1 mg/L) showed early root induction (8.54 days) (Figure 5).
Treatment effects were found to be statistically significant for all the parameters studied, right from the induction of shoots and roots (Table 3).
Actively growing strong shoots of three months’ age and 3 cm length were successfully mini-grafted on 5-month-old rough lemon rootstocks raised in protected greenhouse conditions.

3.4. Triploid Induction Ratio in Nagpur Mandarin (Citrus Reticulata Blanco)

In Citrus reticulata (Blanco) cv. Nagpur mandarin, Callus was induced from cellular endosperm excised 11–12 weeks post-anthesis. A series of media formulations were used to induce primary callus from the endosperm. The best media out of the five tested media combinations was MT + ME + CH (500 mg/L) +2,4,-D (2 mg/L). Induction of somatic embryogenesis and morphogenesis from the primary callus was found to be maximum in (2MT + CH (500 mg/L) + Adenine sulphate (2 mg/L) + BA (0.25 mg/L) out of 5 tested media, which led to the differentiation of viable embroids. Maximum shoot bud and root differentiation occurred in MT along with adenine sulphate (2 mg/L) + GA3 (4 mg/L) + BA (4 mg/L) (Figure 6). Putative in vitro triploid shoots developed were mini-grafted on 5-month-old screen house-grown potted rough lemon rootstock in the year 2016 to transfer triploid regenerants from the lab to the external environment (Figure 7). The triploid plants developed from the experimental cultures in successive years were also transferred to the field on 5-month-old rough lemon rootstocks raised in protected greenhouse conditions. The confirmed triploid plants were field-planted on the experimental farm in 2019.
The summary of this study is presented in Table 4. Out of 90 numbers of tender seeds, 40 endosperm were rescued, and from these putative in vitro triploid shoots were developed and mini-grafted with a 54% survival rate (10 out of 18 pot-transferred grafts survived). (Table 4). The confirmed triploid plants were field-planted on the experimental farm in 2019. The initial field evaluation results revealed a lower seed content of Nagpur mandarin triploid fruits, and the same was documented and presented, which further indicates the triploid nature of the endosperm-derived Nagpur mandarin trees in the field (Figure 7). Further, triploid plants displayed dark green leaves (Figure 8).
Out of the 10 successfully pot-transferred putative triploids, 3 plants were certified or validated as triploids by flow Cytometry/cytogenetic and morphological studies. The ratio of tender seed to stable triploids was (30:1). The ratio of rescued endosperm to stable triploids worked out to be 13:1.
There is a potential for developing one confirmed triploid out of the 15 tagged and harvested immature fruitlets, 3 months old post-anthesis. So, the regeneration ratio of confirmed triploids from the tagged harvested fruitlets worked out to be 1:5, which is equal to 20%. From the excised immature endosperm of Nagpur mandarin, 20% of the regenerated plants were confirmed as triploids with 27 chromosomes (2n = 3x = 27), compared to 18 chromosomes in diploids (2n = 2x = 18) by flow Cytometry and chromosome counting, indicating the authenticity of the triploid nature and culture conditions followed in this study.
In the present study, even though somatic embryogenesis and shoots and roots were developed from cotyledonary embryos, it was not possible to produce plants with complete shoot and root systems for successful lab-to-lab transfers; vigorous and putative triploid shoots were mini-grafted to recover actively growing plants. Ten individual plants were recovered from 18 mini-grafts. The lab-to-land-transferred triploid plants showed varied vigor and vegetative morphology.
Triploid plants showed variation regarding important morphological traits such as leaf morphology, stomatal characteristics, and the appearance of prominent thorns as compared to diploid plants.
This research has demonstrated the feasibility of recovering triploid hybrid plants via in vitro embryogenesis of the endosperm-derived callus of Nagpur mandarin. An enhancement in the rate of plant regeneration efficiency is necessary before this method can be commonly used for breeding and citrus improvement studies.

3.5. Characterization of Triploid Plants by Flow Cytometry

Ploidy analysis was carried out using a flow cytometer. Flow Cytometry works by estimating the volume and florescence of isolated nuclei. The ploidy was presented in the form of a histogram of integral fluorescence, with the peaks depicting the ploidy level of the respective sample. Nuclear DNA histograms were constructed using CyView software, which determined the peak position and relative ploidy level of the tested samples (diploids and triploids).
The results confirmed the triploid nature of the endosperm-derived and lab-to-land-transferred Nagpur mandarin plants (Figure 9).

3.6. Triploid Characterization by Cytology

The chromosome number count was carried out using enzyme digestion and the protoplast drop technique to confirm the results obtained (Figure 9) by flow cytometry. The control plants were 2n = 2x = 18 (Figure 10a), and the triploid plants were 2n = 3x = 27 (Figure 10b).

3.7. Stomatal Analysis

The results of the morphometric analysis of the stomata (guard cells) indicated significant differences between diploid and triploid plants in terms of the number of guard cells and the length of the stomata. Triploid plants had lower numbers of guard cells per 500 µm/sq2 leaf area compared to diploid leaf samples and around a 33.33% reduction in the number of guard cells. The average lengths of stomata in diploid and triploid samples were 38.56 µm and 54.39 µm, respectively, and the observed stomata length in triploid samples was 41.05% more extended when compared to the diploid sample. The student’s t-test results, with a significance level of p > 0.01, indicated that the differences observed between diploid and triploid samples were statistically significant. There is a significant difference in the mean length of the stomata between the two samples, i.e., diploid and triploid, which further indicates that the higher the ploidy level, the higher the length of the stomata (Figure 11) (Table 5).

4. Discussion

Fresh fruit consumers and the processing industries prefer seedless Citrus fruits. Horticulturists/breeders had attempted various techniques for the development of seedless cultivars of the economically important Citrus species, despite the breeding barriers like long gestation periods, vegetative propagation, apomixes, nucellar embryony, and embryo abortion.
The conventional approaches, such as the selection of natural bud mutants and mutation breeding, had yielded limited success. Interploid hybridization has extensively been used, but it is cumbersome and time-consuming [32].
The endosperm of most angiosperms, including Citrus, is a unique tissue in its hybrid origin, development, and ploidy level [33]. The development of protocols for the regeneration of shoots/complete plantlets from triploid endosperm tissue facilitated the development of triploid seedless cultivars by overcoming barriers such as nucellar embryony and embryo abortion.
Even though endosperm culture was reported in more than 64 species, complete plantlet regeneration was reported only in 32 species. Confirmed triploid plants were regenerated and reported from only 15 species. Hence, regeneration from endosperm tissue was often time-consuming and technically challenging [34].
Triploid plants were successfully produced via somatic embryogenesis and organogenesis in vitro in Pummelo and Actinidia [35,36]. The regenerated endosperm-derived plants of Citrus were validated as triploids (2n = 3x = 27). The successful plants regenerated from endosperm culture were very few. Successful regeneration of complete plants via somatic embryogenesis and organogenesis and stable triploid citrus plants were reported in Ridge pineapple and Dweel-tanger [13,37].
No studies were reported in India on the development of field-transferred seedless triploids in Citrus species in general and particularly in Nagpur mandarin (Citrus reticulata Blanco), which is the most popular. This is the first reported research project undertaken in India to develop seedless triploid Nagpur mandarin plants through endosperm culture.
The flowers were tagged during anthesis, and the flowering period of each individual tree lasted for 7–10 days only. This observation indicates the need for advanced planning in implementation. The studies established that the endosperms rescued from fruitlets harvested 75–85 days after anthesis had better responses to somatic embryogenesis and organogenesis.
The efficient rescue of endosperm without injury from immature seeds required manual dexterity, patience, and diligence, factors that were also reported previously [13]. It was found that excising the endosperm from the mature seeds was difficult when fruits and seeds were older with thicker fruit peels and seed coats. Tender, elastic cellular endosperm with a liquid-to-soft texture was found to be more responsive [13,31,38]. Nucellar/zygotic embryos were observed while excising the endosperm from the fruits beyond the age of 14 weeks post-anthesis; hence, the endosperm needs to be rescued up to 12 weeks. Similar observations were reported [13].
In the present study series of media, formulations were used to induce callus via somatic embryogenesis and complete plantlet regeneration from the rescued hybrid endosperm of Citrus reticulata Blanco cv. Nagpur mandarin. The efficiency of callus regeneration from immature endosperm depends on the type of culture, medium, sampling time, and culture conditions. In this study, maximum callus induction was obtained in MT [29], supplemented with rich sources of organic nitrogen such as malt extract and Casein, and 2,4-D as an auxin source. Similar observations were reported by other groups [13,14,39,40,41].
In Nagpur mandarin, endosperm cultures survived and proliferated better in the dark, as callus grows better in low-light conditions [13,42]. The further response of endosperm was low to primary callus induction both in MT and MS basal media with ME (500 mg/L). All proliferated calli, one month after dark incubation, were transferred to light in five different media combinations for the induction of somatic embryogenesis and morphogenesis. Exogenous application of BA and adenine sulphate resulted in stimulation of the endosperm and an increase in morphogenesis. BA was the most effective cytokinin for the induction of morphogenesis in Nagpur mandarin endosperm cultures [13,31,34,43,44].
Five media formations were tested for their ability to induce the primary callus, support callus growth, and continue embryonic development leading to plantlet production. The best response for somatic embryogenesis and morphogenesis from primary callus induction occurred in 2 MT with CH (500 mg/L), BA (0.25 mg/L), and adenine sulphate (2 mg/L). Similar findings were also reported by other workers for embryogenesis and cotyledonary embryoid production [13]. Complete plantlet regeneration was achieved in Nagpur mandarin. In Citrus species, successful plantlet regeneration from immature seeds and endosperm culture was reported [13,14,37]. Detailed reviews of the scientific progress made regarding the regeneration of triploid plants from endosperm culture as a powerful tool for fruit breeding programs were reported [34,45].
In the work of Gmitter et al. [13], it was observed that shoots of triploid endosperm origin possess a large number of multicellular glands and appear fused morphologically. Successful transfer of such plants (with morphological aberrations in the regenerated shoots) from the lab to a green greenhouse and their acclimatization in soil (or) transfer by mini-grafting technique was a major challenge [46], but this was achieved with reasonable success. The ploidy status was analyzed, and the triploid status (2n = 3x = 27) of the transferred plants was confirmed.
In general, stomata density per unit leaf area diminished while stomata guard cell length improved with an increase in ploidy. Triploid plants possessed broader, thicker, and darker green leaves as compared to diploid plants, as evidenced by more leaf length and leaf width. Further flow cytometry analysis and chromosomal counting using leaf meristematic tissue through enzymatic digestion of shoot tips confirmed the ploidy levels of field-transferred triploids. A positive relationship was found between the ploidy level and stomata guard cell length, whereas a negative relationship was observed between the stomata density and ploidy level [22,25,47].
The ratio of successfully surviving stable triploid plantlets from endosperm rescue worked out as 13:1 (7.69%). It means that out of 13 rescued endosperms, one stable triploid plant was regenerated successfully. Variation in ploidy level is one of the common outcomes of endosperm culture [34]. Varied ploidy levels were also reported in Apple [48], Papaya [49], and Kiwi fruit [50].
The ratios of (i) fruitlets to seeds, (ii) seeds to endosperms, (iii) endosperms to putative plants, and (iv) putative plants to confirmed triploid plants were worked out to facilitate researchers in deciding this work targets based on the required number of triploid plants.
In the present study, a considerable variation in the morphological characteristics of leaves, density, and size of stomata and the thorny nature of the trees were observed, indicating the triploid nature of the field-transferred plants. Further fruit analysis indicated less than four seeds (few seeds), which further demonstrated the triploid nature of the plant produced through the endosperm rescue, which is the ultimate goal of this study. Modern analytical methods (flow) Cytometry and cytogenetic confirmation/chromosomal counting) were used in this research for confirmation of the ploidy [22,47].

5. Conclusions

This study had successfully demonstrated the feasibility of developing triploid seedless plants of Nagpur mandarin using plant tissue culture techniques, viz., endosperm rescue, somatic embryogenesis, regeneration, and mini-grafting at CCRI Nagpur. The present work resulted in the production of three confirmed stable triploid plants of Nagpur mandarin. The fruits of these triploid plants were found to be commercially seedless in initial evaluation trials. These plants can be further developed as a triploid seedless clone.

Author Contributions

Conceptualisation, methodology, writing—original draft preparation, visualisation, V.N.; data curation, formal analysis, Y.L., S.P., K.F., P.U. and K.K.; review and editing, V.N. and V.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ICAR-CCRI, and project no. CCRI/2.7. Vijayakumari N. is thankful to the ICAR for financial help in the execution of this research work.

Institutional Review Board Statement

Not applicable for studies not involving humans or animals.

Informed Consent Statement

Not applicable; it does not require any ethical statements as it does not involve either humans or animals.

Data Availability Statement

Data are available with the lead author and the PI of the project.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of this study, in the collection, analysis, or interpretation of data, in the writing of this manuscript, or in the decision to publish this results.

References

  1. Available online: https://agricoop.gov.in/en/StatHortEst (accessed on 27 July 2023).
  2. Available online: https://agriexchange.apeda.gov.in/indexp/Product_description.aspx?hscode=08051000 (accessed on 8 November 2023).
  3. Soost, R.K.; Cameron, J.W. Tree and fruit characters of Citrus triploids from tetraploid by diploid crosses. Hilgardia 1969, 39, 569–579. [Google Scholar] [CrossRef]
  4. Soost, R.K.; Cameron, J.W. ‘Oroblanco’, a triploid pummelo-grapefruit hybrid. HortScience 1980, 15, 667–669. [Google Scholar] [CrossRef]
  5. Soost, R.K.; Cameron, J.W. ‘Melogold’, a triploid pummelo-grapefruit hybrid. HortScience 1985, 20, 1134–1135. [Google Scholar] [CrossRef]
  6. Germanà, M.A.; Chiancone, B.; Lain, O.; Testolin, R. Anther culture in Citrus clementina: A way to regenerate tri-haploids. Aust. J. Agric. Res. 2005, 56, 839–845. [Google Scholar] [CrossRef]
  7. Sunderland, N.; Collins, G.B.; Dunwell, J.M. The role of nuclear fusion in pollen embryogenesis of Datura innoxia Mill. Planta 1974, 117, 227–241. [Google Scholar] [CrossRef]
  8. Liang, C.; Liu, M.; Jin, Z. Research progress on plant seeds abortion. Mol. Plant Breed. 2005, 1, 117–122. [Google Scholar]
  9. Gambetta, G.; Gravina, A.; Fasiolo, C.; Fornero, C.; Galiger, S.; Inzaurralde, C.; Rey, F. Self-incompatibility, parthenocarpy and reduction of seed presence in ‘afourer’ Mandarin. Sci. Hortic. 2013, 164, 183–188. [Google Scholar] [CrossRef]
  10. Grosser, J.W.; Ollitrault, P.; Olivares, F.O. Somatic hybridization in citrus: An effective tool to facilitate variety improvement. Vitr. Cell. Dev. Biol. 2000, 36, 434–449. [Google Scholar] [CrossRef]
  11. Korkmaz, N.; Aşkin, M.A.; Altunlu, H.; Polat, M.; Okatan, V.; Kahramanoğlu, İ. The effects of melatonin application on thedrought stress of different citrus rootstocks. Turk. J. Agric. For. 2022, 46, 585–600. [Google Scholar] [CrossRef]
  12. Keul, A.B.; Farkas, A.; Carpa, R.; Dobrota, C.; Iordache, D. Development of smart fruit crops by genome editing. Turk. J. Agric. For. 2022, 46, 129–140. [Google Scholar] [CrossRef]
  13. Gmitter, F.G.; Ling, X.B.; Deng, X.X. Induction of triploid Citrus plants from endosperm calli in vitro. Theor. Appl. Genet. 1990, 80, 785–790. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, D.; Chang, C.J. Triploid citrus plantlet from endosperm culture. Sci. Sin. 1978, 21, 822–827. (In Chinese) [Google Scholar]
  15. Jaskani, M.J.; Khan, I.A.; Husnain, S. Morphological description of citrus colchiploids. Proc. Intl. Soc. Citricult. 1996, 1, 130–132. [Google Scholar]
  16. Krug, C.A. Chromosome number in the subfamily Aurantioideae with special reference to the genus Citrus. Bot. Gazzette 1943, 104, 602–611. [Google Scholar] [CrossRef]
  17. Kesara, A.J. Preparation of chromosome from plant leaf meristems for karyotype analysis and in situ hybridization. Methods Cell Sci. 2003, 25, 91–95. [Google Scholar]
  18. Vahdati, K.; Jariteh, M.; Niknam, V.; Mirmasoumi, M.; Ebrahimzadeh, H. Somatic embryogenesis and embryo maturation in Persian walnut. Acta Hortic. 2006, 705, 199–205. [Google Scholar] [CrossRef]
  19. Alzea, P.; Juarez, J.; Cuenca, J.; Ollitrault, P.; Navarro, L. Recovery of citrus triploid hybrids by embryo rescue and flow cytometry from 2x × 2x sexual hybridisation and its application to extensive breeding programs. Plant Cell Rep. 2010, 29, 1023–1034. [Google Scholar] [CrossRef]
  20. Grouh, M.S.H.; Vahdati, K.; Lotfi, M.; Hassani, D.; Pirvali, B.N. Production of haploids in Persian walnut through parthenogenesis induced by gamma-irradiated pollen. J. Am. Soc. Hortic. Sci. 2011, 136, 198–204. [Google Scholar] [CrossRef]
  21. Hassanzadeh, K.H.; Rastegar, S.; Golein, B.; Golmohammadi, M.; Aboutalebi, J.A. Genetic Variation of Lime (Citrus sp.) Accessions Using Flow Cytometry Technique, Morphological Characteristics and Molecular Markers. Int. J. Hortic. Sci. Technol. 2018, 5, 199–208. [Google Scholar]
  22. Narukulla, V.; Lahane, Y.; Fiske, K.; Pandey, S.; Ziogas, V. Induction of Polyploidy in Citrus Rootstocks through In Vitro Colchicine Treatment of Seed-Derived Explants. Agronomy 2023, 13, 1442. [Google Scholar] [CrossRef]
  23. Mishra, M.K. Stomatal characteristics at different ploidy level in Coffea L. Ann. Bot. 1999, 80, 689–692. [Google Scholar] [CrossRef]
  24. Beck, S.L.; Dunlop, R.; Fossey, A. Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Bot. J. Linn. Soc. 2003, 141, 177–181. [Google Scholar] [CrossRef]
  25. Yuan, S.X.; Liu, Y.M.; Fang, Z.Y.; Yang, L.M.; Zhuang, M.; Zhang, Y.Y.; Sun, P.T. Study on the relationship between the ploidy level of microspore-derived plants and the number of chloroplasts in stomatal guard cells in Brassica oleracea. Agric. Sci. China 2009, 8, 939–946. [Google Scholar] [CrossRef]
  26. Ye, Y.M.; Tong, J.; Shi, X.P.; Yuan, W.; Li, G.R. Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.). Sci. Hortic. 2010, 124, 95–101. [Google Scholar] [CrossRef]
  27. Jaskani, M.J.; Khan, M.M.; Khan, I.A. Growth, morphology and fruit comparison of diploid and tetraploid Kinnow mandarin. Pak. J. Agric. Sci. 2002, 39, 126–128. [Google Scholar]
  28. Available online: https://timesofindia.indiatimes.com/city/nagpur/seedless-variety-of-nagpur-mandarin-released/articleshow/49706271.cms (accessed on 8 November 2023).
  29. Murashige, T.; Tucker, D.P.H. Growth factor requirements of citrus tissue culture. Proc. First Int. Citrus Symp. 1969, 3, 1155–1161. [Google Scholar]
  30. Murashige, T.; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473–497. [Google Scholar] [CrossRef]
  31. Rakhi, C.; Maharaj, K.R.; Sant, S.B. An efficient protocol for the production of triploid plants from endosperm callus of Neem, Azadirachta indica A. Juss. J. Plant Physiol. 2003, 160, 557–564. [Google Scholar]
  32. Aleza, P.; Juarez, J.; Ollitrault, P.; Navarro, L. Production of tetraploid plants of non apomictic citrus genotypes. Plant Cell Rep. 2009, 28, 1837–1846. [Google Scholar] [CrossRef]
  33. Frost, H.B.; Soost, R.K. Seed production: Development of gametes and embryos. Citrus Ind. 1968, 2, 290–324. [Google Scholar]
  34. Wang, X.; Cheng, Z.M.; Zhi, S.X.F. Breeding Triploid Plants: A Review. Czech. J. Gen. Plant Breed. 2016, 52, 41–54. [Google Scholar] [CrossRef]
  35. Gui, Y.L.; Gu, S.R.; Xu, T.Y. studies on morphological differentiation of endosperm plantlets of Chinese gooseberry in vitro. Acta Bot. Sinica 1982, 24, 216–221, (In Chinese with English summary). [Google Scholar]
  36. Wang, D.Y. In vitro culture of Citrus embryos. Chin. J. Bot. 1975, 17, 149–152. [Google Scholar]
  37. Mooney, P.; Watson, M.; Harty, A. Developing New Seedless Citrus Triploid Cultivars. Hort. Res. Publ. 2007, 1–13. [Google Scholar]
  38. Thomas, T.D.; Bhatnagar, A.K.; Bhojwani, S.S. Production of triploid plants of mulberry (Morus alba L) by endosperm culture. Plant Cell Rep. 2000, 19, 395–399. [Google Scholar] [CrossRef]
  39. Nakajima, T. Physiological studies of seed development, especially embryonic growth and endosperm development. Bull. Univ. Osaka Pref. Ser. B 1962, 13, 13–48. [Google Scholar]
  40. Rangswamy, N.S.; Rao, P.S. Experimental studies on Santalum album L. Establishemnt of tissue culture of endosperm. Phytomorphology 1963, 13, 450–454. [Google Scholar]
  41. Bhojwani, S.S.; Johri, B.M. Morphogenetic studies on cultured mature endosperm of Croton bonplandianum. New Phytol. 1971, 70, 761–766. [Google Scholar] [CrossRef]
  42. Thomas, T.D.; Rakhi, C. Endosperm culture: A novel method for triploid plant production. Plant Cell Tissue Organ Cult. 2008, 93, 1–14. [Google Scholar] [CrossRef]
  43. Otoni, W.C.; Paim, P.D.L.; Rocha, D.I.; Vieira, L.M.; Dias, L.L.C.; Silva, M.L.; Silva, C.V.; Lani, R.G.; Silva, L.C.; Tanaka, F.A. Organogenesis and somatic embryogenesis in passionfruit Passifloras sp. In Somatic Embryogenesis and Gene Expression; Aslam, J., Srivastava, O.S., Sharma, M.P., Eds.; Narosa Publishing House: New Delhi, India, 2013; pp. 1–17. [Google Scholar]
  44. Rocha, D.I.; Batista, D.S.; Faleiro, F.G.; Rogalski, M.; Ribeiro, L.M.; Mercadante-Simões, M.O.; Yockteng, R.; Silva, M.L.; Soares, W.S.; Pinheiro, M.V.M.; et al. Passiflora spp. Passionfruit. In Biotechnology of Fruit and Nut Crops (Agriculture), 2nd ed.; Litz, R.E., Pliego-Alfaro, F., Hormaza, J.I., Eds.; CABI: Boston, MA, USA, 2020; Volume 1, pp. 381–408. [Google Scholar]
  45. Hoshino, Y.; Miyashita, T.; Thomas, T.D. In vitro culture of endosperm and its application in plant breeding: Approaches to polyploidy breeding. Sci. Hortic. 2011, 130, 1–8. [Google Scholar] [CrossRef]
  46. ICAR Central Citrus Research Institute, Nagpur. 2018–2019. Available online: https://ccri.icar.gov.in/ccringp/PDF/AR2021.pdf (accessed on 8 November 2023).
  47. Wu, X.J.; Liu, S.Q.; Zhou, Y.K.; Qian, N.F.; Zhang, P.; Xie, H.Q.; Zhang, F.S.; Yan, Z.L. Induction of triploid apple plants from endosperm calli in vitro. Chin. Sci. 1978, 355–360. [Google Scholar]
  48. Sun, D.Q.; Lu, X.H.; Liang, G.L.; Guo, Q.G.; Mo, Y.W.; Xie, J.H. Production of triploid plants of papaya by endosperm culture. Plant Cell Tissue Organ Cult. 2011, 104, 23–29. [Google Scholar] [CrossRef]
  49. Huang, Z.X.; Huanpu, Y.L.; Xu, L.Y. Production of triploid plants from endosperm cultures of Actinidia chinensis Planchon. Chin. Sci. Bull. 1982, 27, 247–250. [Google Scholar]
  50. Diego, P.; Amr, M.; Benedetta, C.; Maria, A.G.; Patan, S.S.V.K. Ploidy levels in Citrus clementine affects leaf morphology, stomatal density and water content. Theor. Exp. Plant Physiol. 2013, 25, 283–290. [Google Scholar]
Agronomy 13 02814 g001
Figure 1. Induction of primary callus. Bar: 1 mm.
Figure 1. Induction of primary callus. Bar: 1 mm.
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Figure 2. Initiation of somatic embryogenesis begins at the primary induction callus. (a). Initiation of somatic embryogenesis, (b). Magnified view of somatic embryogenesis. Bars: 1 mm.
Figure 2. Initiation of somatic embryogenesis begins at the primary induction callus. (a). Initiation of somatic embryogenesis, (b). Magnified view of somatic embryogenesis. Bars: 1 mm.
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Figure 3. Differentiation of somatic embryos to morphogenesis. (a). The morphogenesis of somatic embryogenesis involves different stages. (b). Magnified view of cotyledonary embroids. (c). Rooting initiation in cotyledonary embroids. Bars: 1 mm.
Figure 3. Differentiation of somatic embryos to morphogenesis. (a). The morphogenesis of somatic embryogenesis involves different stages. (b). Magnified view of cotyledonary embroids. (c). Rooting initiation in cotyledonary embroids. Bars: 1 mm.
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Figure 4. Endosperm callus shows multiple shoot differentiation. Bar: 1 mm.
Figure 4. Endosperm callus shows multiple shoot differentiation. Bar: 1 mm.
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Figure 5. Endosperm-regenerated plants have fused stem-organ fasciation.
Figure 5. Endosperm-regenerated plants have fused stem-organ fasciation.
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Figure 6. Endosperm rescue and different stages of plantlet regeneration from callus induction via somatic embryogenesis and morphogenesis.
Figure 6. Endosperm rescue and different stages of plantlet regeneration from callus induction via somatic embryogenesis and morphogenesis.
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Figure 7. (a) Mini-grafting of endosperm rescued triploid shoots on rough lemon rootstock. Morphological characters of the C. reticulata cv. Nagpur mandarin triploid in the field. (b) Field-transfer triploid of Nagpur mandarin. (c) Mature triploid Nagpur mandarin tree in bearing. (d) Absence of thorns in diploid Nagpur mandarin. (e) Presence of the thorns in the field-transfer triploid of Nagpur mandarin. (f) Nagpur mandarin triploid fruit of the initial field trial. (g) Seedless character of endosperm derived Nagpur mandarin triploid.
Figure 7. (a) Mini-grafting of endosperm rescued triploid shoots on rough lemon rootstock. Morphological characters of the C. reticulata cv. Nagpur mandarin triploid in the field. (b) Field-transfer triploid of Nagpur mandarin. (c) Mature triploid Nagpur mandarin tree in bearing. (d) Absence of thorns in diploid Nagpur mandarin. (e) Presence of the thorns in the field-transfer triploid of Nagpur mandarin. (f) Nagpur mandarin triploid fruit of the initial field trial. (g) Seedless character of endosperm derived Nagpur mandarin triploid.
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Figure 8. Morphological characteristics of diploid and triploid leaves.
Figure 8. Morphological characteristics of diploid and triploid leaves.
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Figure 9. Flow cytometry histograms represent diploid (a) and triploid (b) levels of Nagpur mandarin.
Figure 9. Flow cytometry histograms represent diploid (a) and triploid (b) levels of Nagpur mandarin.
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Figure 10. Processed metaphase cells of Citrus reticulata Blanco (cv. Nagpur mandarin) sample. (a) Diploid, (b) Triploid. The bar represents 2.5 μm.
Figure 10. Processed metaphase cells of Citrus reticulata Blanco (cv. Nagpur mandarin) sample. (a) Diploid, (b) Triploid. The bar represents 2.5 μm.
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Figure 11. Effect of ploidy level on leaf morphology and stomatal characteristics in Nagpur mandarin.
Figure 11. Effect of ploidy level on leaf morphology and stomatal characteristics in Nagpur mandarin.
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Table 1. Influence of media on the frequency of primary callus induction from endosperm.
Table 1. Influence of media on the frequency of primary callus induction from endosperm.
Sr No.MediaSurvival (%)Response (%)Days TakenCallus Initiation
1MT + ME (500 mg/L)81.67
(68.95) 		86
(62.97) 		(28.18) 26.56
(30.31)
2MT + ME + CH (500 mg/L) + BA(5 mg/L) + 2,4-D (2 mg/L)73.34
(63.95) 		50.83
(45.47) 		(29.54) 43.96
(41.28)
3MT + ME + CH (500 mg/L) +2,4-D (2 mg/L)91.67
(80) 		93.33
(81.20) 		(25.92) 50.94
(45.56)
4MS + ME (500 mg/L)65
(56.45) 		14.08
(22.0) 		(35.66) 28.83
(32.26)
5MS + ME + CH (500 mg/L) + Kinetin (5 mg/L) +2,4-D (2 mg/L)37.5
(37.50) 		8.51
(16.80) 		(27.05) 46.9
(43.12)
CD0.05%19.36 *15.51 *4.67 *10.59 *
0.01%26.20 **20.99 **6.33 **NS
Numbers in parentheses are arc sine-transformed means of four replicate batches. ** Highly significant (p < 0.01), * significant (p < 0.05).
Table 2. Influence of media on the morphogenesis of proliferating embryoids from endosperm calli.
Table 2. Influence of media on the morphogenesis of proliferating embryoids from endosperm calli.
MediaEmbryogenesis (Days)Embryogenic Callus (%)Differentiation of Embryoid
Globular
Shape		Heart ShapeTorpiodo
Shape		Cotyledonary
Shape
1MS + CH (500 mg/L)28.8833.31
(34.90)		32.98
(35.01)		16.13
(23.59)		26.41
(30.88)		24.95
(29.90)
2MT + CH (500 mg/L) + GA3 (1 mg/L) + BA (0.25 mg/L)26.6926.9
(30.82)		29.47
(34.35)		15.11
(22.82)		25.11
(30.03)		30.79
(33.88)
3MT + CH (500 mg/L) + BA (0.25 mg/L)25.7231.9
(33.97)		37.27
(37.57)		14.23
(22.10)		20.60
(26.95)		27.9
(31.92)
42MT + CH (500 mg/L) + BA (1 mg/L)23.0555.45
(48.24)		34.5
(35.93)		13.15
(21.38)		22.83
(28.33)		29.52
(32.87)
52MT + CH (500 mg/L) + Ad.S (2 mg/L) +
BA (0.25 mg/L)		22.6677.65
(64.05)		28.17
(32.00)		20.13
(26.63)		17.81
(25.27)		33.92
(35.56)
CD0.052.63 *11.22 *2.17 *2.50 *2.17 *5.77 *
0.013.57 **15.18 **3.01 **3.45 **3.01 **4.26 **
Numbers in parentheses are arc sine-transformed means of four replicate batches. ** Highly significant (p < 0.01), * significant (p < 0.05).
Table 3. Influence of media on complete plantlet regeneration from endosperm-derived embryoids.
Table 3. Influence of media on complete plantlet regeneration from endosperm-derived embryoids.
Sr.NoMediaResponse (%)Shoot Initiation
(Days)		Root Initiation (Days)Shoots (No)Roots (No)Length of Shoot (cm)Length of Root (cm)
1MT + Ad.S (2 mg/L) + GA3 (1 mg/L) + BA (1 mg/L)95.84
(85)		1110.793.954.173.483.54
2MT + GA3 (1 mg/L) + BA (1 mg/L)87.5
(75)		12.0413.683.293.252.843.12
3MT + IBA (2 mg/L)83.45
(70.07)		24.359.342.963.01.763.05
4MT + Ad.S (2 mg/L) + GA3 (1 mg/L) + IBA (0.5 mg/L)67.5
(50.88)		25.8711.511.912.701.682.79
5MS + IBA (2 mg/L) + BA
(0.5 mg/L)		60
(55.47		28.8511.511.831.831.551.79
6MS + BA (2 mg/L) + IBA (1 mg/L) + NAA (1 mg/L) 80
(70.07)		25.938.543.251.541.903.14
CD0.0517.52 *1.86 *2.26 *0.50 *0.63 *0.42 *0.53 *
0.0123.59 **2.51 **3.04 **0.37 **0.47 **0.31 **0.40 **
Numbers in parentheses are arc sine-transformed means of four replicate batches. ** Highly significant (p < 0.01), * significant (p < 0.05).
Table 4. Triploid induction ratio in Nagpur mandarin (Citrus reticulate Blanco).
Table 4. Triploid induction ratio in Nagpur mandarin (Citrus reticulate Blanco).
Stage of Triploid Plant DevelopmentSelected Best Media Total FruitsImmature SeedsTender Endosperm from an Immature Seed (No)Response of Endosperm for Callus Induction (%)Callus Induction (%)Induction of Somatic Embryogenesis (%)Morphogenesis- Cotyledonary Shape
(%)		Response to Plantlet Regeneration (%)Plants Generated (No)Putative Triploid
Plants (No)		Lab-to-Land Transfer by MinigraftingSuccess in Mini
Grafting		Confirm Triploidy Plants
Endosperm cultureMT + ME +
CH (500 mg/L) +2,4-D (2 mg/L)		159040--------------------
Primary callus inductionMT + ME +
CH (500 mg/L) +2,4-D (2 mg/L)		------93.3350.94----------------
Embryogenesis and morphogenesis2MT + CH (500 mg/L) + ad.s (2 mg/L) +
BA (0.25 mg/L)		----------77.6533.92------------
Complete plantlet regeneration MT + ad.s (2 mg/L)+ GA3 (1 mg/L) + BA (1 mg/L)--------------95.843318------
Transfer by minigrafting----------------------18103
Table 5. Morphometric analysis of stomata in triploid and diploid Nagpur mandarin.
Table 5. Morphometric analysis of stomata in triploid and diploid Nagpur mandarin.
Triploid StomataDiploid Stomata
Stomatal count1110
Avg. length of stomata54.3938.56
SD5.382.49
Variance28.956.24
T-Statistic8.494
T-table (0.05) *2.093
T-Table (0.01) **2.861
** Highly significant (p ≤ 0.01), * significant (p ≤ 0.05).
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Narukulla, V.; Lahane, Y.; Pandey, S.; Fiske, K.; Uge, P.; Kawale, K.; Ziogas, V. Development of Triploid Seedless Nagpur Mandarin (Citrus reticulata Blanco) through Endosperm Rescue. Agronomy 2023, 13, 2814. https://doi.org/10.3390/agronomy13112814

AMA Style

Narukulla V, Lahane Y, Pandey S, Fiske K, Uge P, Kawale K, Ziogas V. Development of Triploid Seedless Nagpur Mandarin (Citrus reticulata Blanco) through Endosperm Rescue. Agronomy. 2023; 13(11):2814. https://doi.org/10.3390/agronomy13112814

Chicago/Turabian Style

Narukulla, Vijayakumari, Yogesh Lahane, Shashi Pandey, Krutika Fiske, Priya Uge, Kartiki Kawale, and Vasileios Ziogas. 2023. "Development of Triploid Seedless Nagpur Mandarin (Citrus reticulata Blanco) through Endosperm Rescue" Agronomy 13, no. 11: 2814. https://doi.org/10.3390/agronomy13112814

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