Ploidy analysis among Citrus mutants using leaf meristematic tissue

A promising method for preparing metaphase spread for counting the number of chromosomes from the emerging shoot tissue is described in this report. In the present study, we adopted enzymatic digestion of shoot tips to analyse the chromosome number. The chromosomes in metaphase stage of cell division are highly condensed and easy to count in routine cytological technique. Even the morphological features like position of centromere can be seen in metaphase. In prophase it may not be clear as the chromosomes are getting ready for cell division. In enzymatic digestion even the prophase chromosomes are visible, which can be counted. Hence enzymatic digestion technique is more efficient in citrus as compared to acid digestion method as the citrus crop is a perennial crop with small-sized chromosomes. Furthermore, the sample collection in the field was easy and actively growing vegetative flush was available throughout the year. This technique was attempted in the tissue culture lab of ICAR- CCRI in various in vito and in vivo ploidy induction experiments in Citrus sinensis Osbeck (Sweet orange cv. mosambi), C. reticulata Blanco (Nagpur mandarin) and C. jambhiri Lush (Rough lemon), for confirmation of diploidy (2n=2x=18), triploidy (2n=3x=27), tetraploid (2n=4x=36), hexaploid (2n=6x=54)

Induction of Polyploidy in Citrus Rootstocks through <i>In Vitro</i> Colchicine Treatment of Seed-Derived Explants

Polyploidy, frequently observed in citrus species, aids in achieving better adaptation to environmental stresses. In this context, the current work aims to develop stable tetraploids in citrus rootstock cultivars, viz., Rough lemon, Rangpur lime and Alemow, through in vitro colchicine treatments. Seed-derived explants were obtained by culturing sterile seeds on MS basal media. Seedlings with a size of 5–8 mm (hypocotyl) were exposed to colchicine treatment. After treatment, the surviving seedlings were minigrafted onto six-month-old rootstock for better growth. Colchicine concentrations of 0.1%, 0.2%, 0.3% and control for durations of 16 or 24 h were tested with respect to the induction of polyploidisation. Treatment with 0.1% colchicine for 24 h resulted in high rates of mutation for polyploidisation and showed the highest tetraploid induction percentage (18.3%) in all the rootstock cultivars. High colchicine concentration and long exposure time decreased the survival of the observed seedlings. Flow cytometry and cytological methods were used for confirmation of autotetraploidy in the analysed samples. The surviving seedlings were identified on the basis of morphological and cytological variables, such as leaf area and stomata size, which significantly increased with increasing ploidy level. The proposed method was found to be an effective way to induce the polyploidy in Rangpur lime, Rough lemon and Alemow rootstocks

Development of Triploid Seedless Nagpur Mandarin (<i>Citrus reticulata</i> Blanco) through Endosperm Rescue

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