Culture Of Zygotic Embryos Research Articles

Expression profiling of cell cycle regulatory genes E2F and CDKA during early stages of zygotic embryo culture in coconut (Cocos nucifera L.)

Expression profiling of cell cycle regulatory genes E2F and CDKA during early stages of zygotic embryo culture in coconut (Cocos nucifera L.)

Comparison of tissue culture-induced variation in triticale regenerants obtained by androgenesis and somatic embryogenesis

Triticale is becoming an increasingly important livestock crop production. This is evidenced by increasing triticale-producing areas and by improved yields. In addition, meeting the increasing demand for cereals involves the introduction of high-yielding and stress-resistant varieties into breeding. In vitro culture techniques can accelerate the development of new varieties. Therefore, it seems extremely important to develop efficient plant regeneration methods through in vitro cultures and to understand the mechanisms involved in gaining regenerants. Obtaining regenerants of triticale through somatic embryogenesis and androgenesis may lead to tissue culture-induced variation. In the present study, we compared regenerants obtained in both regeneration systems (anther and immature zygotic embryo cultures), considering the level of genetic and epigenetic changes observed in different DNA sequence contexts for methylated cytosine (CG, CHG, CHH). The changes concerning the DNA sequence (so-called sequence variation) and the changes concerning the DNA methylation patterns, i.e., the removal of methylated cytosine (DNA demethylation) and the introduction of methylation to cytosine (de novo DNA methylation), were analyzed. We observed that regenerants derived via somatic embryogenesis and androgenesis differ notably for demethylation in the symmetrical CG sequence context and de novo methylation in the asymmetrical CHH context. These changes may be related to the reprogramming of microspore development from gametophytic to sporophytic and lack of such process in zygotic embryos.

Agronomic Traits and Genetic Fidelity of Four Cocoa Clones Derived from Somatic Embryogenesis Culture

Morphological and genetic characterization of MCBC1, PBC230, KKM22 and KKM4 cocoa clones derived from staminode and immature zygotic embryo culture were compared with those conventionally grafted. Somatic embryogenesis culture successfully produced true-to-type progenies of elite cocoa clones of MCBC1, PBC230, KKM22 and KKM4 (from staminode culture). Phenotype variations (p < 0.05) were observed only in KKM4 clone from immature zygotic embryo culture which exhibited lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight. The genetic stability of the cultured clones was tested using fragment analysis with 12 SSR primers to validate these results. Eleven of these SSR primers detected mutations only in the allelic profiles of KKM4 clone from immature zygotic embryo. These results validated those variations in KKM4 clones of immature zygotic embryo culture were due to interactions between genotypic and explant types. Unfortunately, these variations were negative attributes to cocoa productivity. Thus, it is suggested that successful production of true-to-type KKM4 cocoa clone should consider other means of propagation including modification of the culture conditions.
 HIGHLIGHTS
 
 It is crucial to evaluate and confirm the quality of the immature zygotic and staminode regenerated cocoa plant before using the culture technique for commercial production
 The quality can be detected in both phenotype and genotype by comparing the regenerated cocoa plant with those regenerated from the conventional asexual propagation method of grafting
 Phenotype and genotype differences were only observed in KKM4 clone regenerated from immature zygotic embryo culture and thus validated that this clone exhibited variation
 KKM4 clone from immature zygotic embryo culture which has lower quantities in the fresh pod weight, number of flat beans per pod, seed length, seed width and individual seed weight confirmed the negative effect of using immature zygotic embryo explant for KKM4 clone propagation. It is suggested to modify the culture technique
 
 GRAPHICAL ABSTRACT

Enhanced production of pinosylvin stilbene with aging of Pinus strobus callus and nematicidal activity of callus extracts against pinewood nematodes

Pinosylvin stilbenes are phenolic compounds mainly occurring in the Pinaceae family. We previously reported that the accumulation of two pinosylvin stilbene compounds, dihydropinosylvin methyl ether (DPME) and pinosylvin monomethyl ether (PME), in Pinus strobus trees was highly enhanced by infection with pine wood nematodes (PWNs: Bursaphelenchus xylophilus), and these two compounds showed strong nematicidal activity against PWNs. In this work, we established a system of pinosylvin stilbene (DPME and PME) production via the in vitro culture of P. strobus calli, and we examined the nematicidal activity of callus extracts. Calli were induced from the culture of mature zygotic embryos of P. strobus. Optimized growth of calli was obtained in 1/2 Litvay medium with 1.0 mg/L 2,4-D and 0.5 mg/L BA. DPME and PME accumulation did not occur in nonaged (one-month-old) calli but increased greatly with prolonged callus culture. The concentrations of DPME and PME in three-month-old dark-brown calli were 6.4 mg/g DW and 0.28 mg/g DW, respectively. The effect of methyl jasmonate treatment on the accumulation of DPME and PME was evaluated in cell suspension culture of P. strobus. However, the treatment appeared to show slight increase of DPME accumulation compared to callus browning. A test solution prepared from crude ethanol extracts from aged calli (three months old) containing 120 µg/ml DPME and 5.16 µg/ml PME treated with PWNs resulted in 100% immobilization of the adult PWNs and 66.7% immobilization of the juvenile PWNs within 24 h. However, nonaged callus extracts did not show any nematicidal activity against juvenile PWNs and showed less than 20% nematicidal activity against adult PWNs. These results indicate that pinosylvin stilbenes can be effectively produced by prolonged culture of P. strobus calli, can be isolated using simple ethanolic extraction, and are applicable as beneficial eco-friendly compounds with nematicidal activity against PWNs.

Overcoming germination barriers through in vitro culture of mature zygotic embryos of grapevine cultivars

Grape seeds have physical and physiological dormancy. Conventional germination methods are not very efficient. The in vitro culture of seeds and of mature zygotic embryos can be an alternative for overcoming germination barriers. The objective was to develop methodologies that increase seed germination and reduce the time to obtain seedlings. The following assays were developed: (1) conventional ex vitro germination, in a 2 × 3 factorial arrangement, of seeds with or without stratification for 90 days, from cultivars 'Niágara Rosada', 'Itália' and 'Red Globe'; (2) in vitro seed germination, in a 3 × 3 factorial arrangement, of the three cultivars and three explants (intact seeds, seeds sectioned in the micropyle region and seeds with a cross-section); and (3) in vitro germination of mature zygotic embryos, in a 4 × 2 factorial arrangement represented by four concentrations of mineral salts (MS, ½MS, WPM and ½WPM) and two cultivars ('Niágara Rosada' and 'Itália'). The conventional ex vitro germination assay resulted in a low seed germination for the three cultivars. The two types of section associated with in vitro culture provided an increase in seed germination. However, the highest germination rate (over 90%) in the shortest time (17 days) was achieved by the embryos grown in vitro. This study defines an efficient methodology to obtain high germination in grape seeds— over 85%—with uniformity and in a short time, using mature zygotic embryos grown in ½WPM culture medium. This methodology can accelerate grapevine breeding programs. Index terms: Vitis vinifera; Vitis labrusca; dormancy; seed scarification.

Standardization of zygotic embryo culture from Nerium oleander L. and comparative analysis of biosynthesized cardiac glycosides within in vitro and acclimatized plants

The primary result of our experiment revealed that the germination percentage of N. oleander mature seeds is only 30%. From this observation, the concept of protocol standardization for zygotic embryo culture of this plant was originated. Zygotic embryo culture was proved an efficient in vitro multiplication system of N. oleander. The maximum germination percentage (96%) of zygotic embryos was observed on ¼ MS medium with 15 gm/L sucrose, whereas the best growth medium was optimized as ½ B5 with same sucrose concentration. The second part of this study was aimed to find out the cardiac glycoside accumulation pattern in both in vitro and acclimatized plants. For this purpose, one-month-old in vitro plantlets and acclimatized plants were subjected to LC-MS analysis and 09 cardiac glycosides were detected and quantified in both the systems. Most of the cardiac glycosides including odoroside A (32.71 mg/gm DW), odoroside H (4.69 mg/gm DW) and oleandrin (0.52 mg/gm DW) were found to be accumulated at maximum level within in vitro plantlets. CG 840b (1.89 mg/gm DW) is the only cardiac glycoside, which was maximally accumulated in acclimatized plants. From this study, it can be concluded that, zygotic embryo culture is a better choice for in vitro multiplication of N. oleander when compared to matured seeds and in vitro grown plantlets of this species favor cardiac glycosides biosynthesis in comparison to acclimatized plants. Therefore, all future research on the enrichment of cardiac glycosides from this plant may be conducted on zygotic embryos derived in vitro grown plantlets or cultures.

Reproductive Characteristics of the Selected Cocoa (Theobroma cacao L.) Clones after Regenerated from the Somatic Embryogenesis Culture

This study was conducted to evaluate the reproductive characteristics of 4 elite cocoa clones (MCBC1, PBC230, KKM22 and KKM4) propagated via somatic embryogenesis culture. From the findings, all clones have similar reproductive characteristics with clones from conventional grafted. However, only KKM4 clone from immature zygotic embryo culture produced the shortest staminode to style distance of 1.83 mm. This consequently influenced flower stability by reducing the efficiency of pollination by insects. It was found that this clone also has the highest number of flowers drop after anthesis (5 flowers) and lowest production of cherelle (5 cherelles). Further observation revealed that floral development from first bud visible (BBCH51) to flower anthesis (BBCH68) of all clones took around 31 days. These cocoa flowers which remained receptive soon after anthesis at 10 am (day-31) until the next day (day-32) suggesting 2 days’ period of receptivity.
 HIGHLIGHTS
 
 It is crucial to assess the presence of off-type characteristics in the reproductive organ structure such as the distance between staminode to style, period of reproductive cycle and stigmatic receptivity of cocoa clones regenerated from somatic embryogenesis
 The converging and parallel type of staminode to style distances are the ideal flower spatial arrangements for the optimal pollination in cocoa plant compared to splay type
 Only KKM4 clone propagated from immature zygotic embryo culture showed variation in the distance between staminode to style distance and this caused pollination failure by insect which then consequently caused minimum cherelle production
 All regenerated cocoa clones observed with typical period of the reproductive cycle and stigmatic receptivity
 
 GRAPHICAL ABSTRACT

Agronomic Performance and Genetic Fidelity of the Selected Elite Cocoa Clones Derived from Somatic Embryogenesis Culture

This study was conducted to compare the agronomic performance of four elite cocoa clones (MCBC1, KKM22, KKM4 and PBC230) regenerated from staminode and immature zygotic embryo culture with conventional grafted cocoa clones. From the results, it was found that the KKM4 clone propagated from immature zygotic embryo culture exhibited variations in the fresh pod weight (339.6 g), fresh individual seed weight (4.13 g) and number of flat beans per pod (4 beans) compared with the rest of the regenerated clones. The genetic stability of the somatic embryogenesis cultured clones and the donor clones was then tested using fragment analysis with five SSR primers, i.e. mTcCIR7, mTcCIR18, mTcCIR22, mTcCIR33 and mTcCIR40. Four of these primers identified variations in the allele size and allele addition in KKM4 clone from immature zygotic embryo. Molecular analysis validated that the difference in agronomic performance of the KKM4 clone from immature zygotic embryo culture was due to genetic mutation created during the immature zygotic embryo culture process.

Zygotic embryo culture is an efficient way to optimize in vitro growth in Panax ginseng

Despite many studies on tissue culture of Panax ginseng, the propagation of cultivars is still dependent on seeds due to low survival rate of plants produced in vitro. Healthy in vitro grown plants guarantee high survival rates after they are transferred to soil, but limited information exists on the optimum medium for the in vitro growth of P. ginseng. In this study, zygotic embryo culture was used to develop an optimum medium suitable for in vitro cultivation of P. ginseng. After 4–6 months of culture, the aged aerial parts turned yellow, and the in vitro grown roots (IGRs) had thickened taproots with dormant rhizomes. The seedling growth was evaluated using various concentrations of sucrose and basal media at various strengths. Of the tested media, 2% sucrose and 1/3 strength SH medium showed superior results. Subsequently, IGRs were transferred to soil and evaluated for their morpho-anatomical characteristics. Furthermore, their plants were analyzed for genetic fidelity using molecular markers. IGRs showed outstanding morphological differences compared with conventionally grown 1-year-old ginseng roots; however, there was no difference in the sprouting and survival rate after soil transfer. 2-year-old IGRs deprived from the IGRs have intermediate properties between 1-year-old and 2-year-old root. Unlike the 2-year-old roots, the morphology of 2-year-old IGRs had shorter primary roots with several lateral roots. Despite the morpho-anatomical differences, molecular marker analysis also confirmed that the IGRs had genetic fidelity.

SEED DORMANCY BREAKING OF AN ENDANGERED MEDICINAL TREE SPECIES (TAXUS BACCATA L.) USING EMBRYO CULTURE

Natural regeneration of Taxus baccata L. is constrained due to the depth of seed dormancy requirements (often taking two or more years) and low seed germination. Further, the conventional method of vegetative propagation by cuttings is associated with difficulties in rooting. Hence, for the first time, this study describes an efficient and reproducible in vitro protocol for breaking the dormancy of seeds from the endangered forest tree T. baccata L. via zygotic embryo culture. Embryos isolated from 100% sterile seeds were cultured on DCR medium that contains sucrose (30 g/l), agar (8 g/l), and activated charcoal (5 g/l), fortified with different concentrations of Plant Growth Regulators (PGRs), and held at a temperature of 25 ± 2 ºC in a growth room. The results revealed that the in vitro embryo germination percentage was mostly affected by gibberellic acid (GA3) and thidiazuron (TDZ). Among the nine treatments, the treatments with 0.5 mg/l TDZ and 1 mg/l GA3 showed the highest germination (100%), while the other treatments all increased the germination percentages significantly compared to the control (37.5%). The 1/2 DCR medium with the addition of 0.1 mg/l indole-3-butyric acid (IBA) resulted in the highest rooting ratio (94%). However, the greatest root and hypocotyl elongation (59.37 ± 3.77 and 62.75 ± 4.43 mm, respectively) occurred when seedlings were cultured on 1/2 DCR medium containing 0.5 mg/l BA. Plantlets were transplanted into plastic pots containing an autoclaved garden soil, sand, and vermiculite mixture (1:1:1) and held at a temperature of 25 ± 2 ºC in a growth room for 4 weeks before being transplanted into the greenhouse. These results indicated that the protocol developed during the current study will be useful to overcome seed dormancy and for multiplication and conservation of the species T. baccata L.

Seed storage behavior of Musa balbisiana Colla, a wild progenitor of bananas and plantains - Implications for ex situ germplasm conservation

Musa balbisiana (BB) and M. acuminata (AA) are the two wild diploid progenitors of the cultivated, triploid, tetraploids bananas and plantains. Collection and safe conservation of crop wild relatives (CWR) has gained tremendous importance, as they are the reservoir of genes and traits required to combat climate change effects and the emerging biotic and abiotic stresses. The purpose of this study was to investigate seed storage behavior of M. balbisiana for devising and applying suitable strategies to conserve CWR of banana/plantain germplasm in gene banks, both for short-and long-term. Seeds (from mature fruits) of three accessions of M. balbisiana from Field Gene Bank of ICAR-NBPGR, New Delhi (EC653579) and natural populations from ICAR-NBPGR, Thrissur, Kerala (IC630992) and Mizoram University, Mizoram (IC633382) were augmented and desiccated to various moisture contents (MC at 5,10,15, 20 and 25 % on fresh weight basis). Fresh and desiccated seeds were stored at three temperatures (25, -20 and -196 °C) to assess freezing sensitivity. Whole seeds as well as excised zygotic embryos were cryopreserved at the optimized MC using simple air dehydration technique. For recovery, data on in vitro regeneration of zygotic embryos was recorded and analyzed statistically to determine optimal explant and conservation regime. Whole seeds in general were difficult to germinate whereas in vitro regeneration of zygotic embryos yielded high results (95 ± 5 %). Seeds were found to be desiccation tolerant (viable up to 5 % MC) and desiccated seeds (5–10 %) were insensitive to ultra-low temperature (-20 and -196 °C), without significant loss of viability and germination potential. High moisture content (15, 20 and 25 %) in seeds led to low regeneration potential (≤50 %) due to cold injury of cells at ultra-low temperatures (-20 and -196 °C). No significant differences were observed in regeneration of fresh and cryopreserved desiccated (5–10 % MC) excised embryos (90 ± 10 %). Our studies confirm that M. balbisiana seeds are ‘orthodox’ in storage behavior, and can be easily conserved for short-term at 25 °C, medium-term at -20 °C and long-term at -196 °C with 5–10 % MC. However, unlike other typical orthodox seeds, regeneration of conserved seeds is only feasible through zygotic embryo culture under in vitro conditions, as desiccated whole seeds fail to germinate using standard protocols. For cryobanking, whole seed or excised zygotic embryo cryopreservation is a feasible option for germplasm conservation of M. balbisiana.

Temperature and GA3 on ROS and cytogenetic stability during in vitro cultivation of strelitzia zygotic embryos

ABSTRACT Tropical species may require higher temperatures as well as higher growth regulator concentrations for in vitro development. Since these conditions may affect plant metabolism, the objective of this study was to identify how different temperatures and gibberellin concentrations may affect the in vitro development of strelitzia embryos, analyzing the effect on ROS and cytogenetic stability. Zygotic embryos were cultivated on MS medium supplemented with 5, 10 and 20 µM GA3 under temperatures of 25 °C, 30/25 °C and 30 °C. After 60 days, higher embryonic germination rate (72%) and shoot length of plantlets (3.14 cm) were observed on medium containing 20 µM gibberellic acid (GA3). At this concentration, there was an increase in nitrate reductase activity with no change in the cytogenetic stability. The temperature influenced only shoot and root lengths, which were highest at 25 °C. At 30 °C, superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities increased compared with those at 25 °C. Thus, the addition of 20 µM GA3 to the culture medium and a temperature of 25 °C in the growth room should be used for zygotic embryo culture of strelitzia.

A review on regeneration potential and commercialization of Azadirachtaindica: A multifunctional tree species

“Azadirachta indica” commonly called as neem, nimtree or Indian lilac, is a tree representing plant family “Meliaceae”. It is commonly and natively found in tropical and semi-tropical regions. It has high industrial demand due to its immediate application as an eco-friendly, biodegradable pesticides and immediate availability. The only trading and commercialized way to produce neem is extraction of seeds from the plant, but the seed availability is very limited as the tree flowers once in a year. Also due to firm and harsh out-breeding nature of the plant, seeds are heterozygous, resulting in the production of inconsistent metabolite. But now a day the production of azadirachta has been influenced by various strategies which involve androgenic cultures, beta cell generation, callus cultures, leaf, ovary, zygotic embryo cultures or many other ways. A.indica has been now used as a supplement for drug industry, cosmetics, photochemical, pharmacological and as a therapeutic because of the high content of antioxidant. It is used for the treatment of skin disorders with the help of various commercial products. Azadirachta indica also possesses anticancer activity and as a wound healer. There are many products available in market that can be used for various purposes either it can be for human use or for agricultural or poultry use or as biopesticides, certified by the government. They do not have any side effects and only target the micro-organisms for which they are designed. During pandemic covid-19 also Azadirachta indica has been recommended for different purposes to minimize the infection rate. The present review is to highlight the importance of A.indica, commercialization and with special focus on regeneration protocol development to multiply and enhance the important phytochemicals within it.

How to shorten a plant breeding program? A case study with ornamental peppers

Abstract Plant breeding of ornamental peppers (Capsicum spp.) can be supported by biotechnological tools. The objective of this study was to evaluate the efficiency of an in vitro culture of immature zygotic embryos (IZE) to reduce the breeding cycle of ornamental pepper (C. annuum) in comparison to the conventional system. Three ornamental pepper genotypes were used: UFPB 001, UFPB 004, and UFPB 099. Embryos at 30 days after selfing were inoculated in MS ½-strength culture medium, and at the same time, seeds were placed to germinate in a commercial substrate. Approximately 215 days are required from selfing until fruit ripening in the conventional system, whereas the IZE system requires an average of 153 days, a decrease of approximately 30% per selection cycle, corresponding to 496 days considering 8 selfing cycles. A decrease in time, labor, and inputs makes the IZE system a suitable tool for shortening the breeding program of ornamentals peppers.

In vitro culture of zygotic embryos and seeds of Caesalpinia ferrea Martius

ABSTRACT The aim of this study was to evaluate the in vitro germination of zygotic embryos and seeds of Caesalpinia ferrea Martius and the morphogenetic responses of the explants to different concentrations of growth regulators. Seeds and zygotic embryos were inoculated in MS culture medium and kept in a growth room at a temperature of 25 ± 2 ºC for 16 hours of photoperiod for 30 days. The seeds had a higher in vitro germination rate than the explants from zygotic embryos. However, zygotic embryos in MS medium supplemented with 0.9 mg L-1 BAP had the highest percentage of regeneration (50%), number of shoots (3.25), buds (2.85) and leaves (3.15), multiplication rate (27.75), and length of shoots (1.96 cm). The in vitro culture of zygotic embryos and seeds made possible the multiplication of a higher number of healthy seedlings. Thus, it can be used as an alternative technique for the propagation of this species.

Micropropagation in litchi: concept, constraints and recent advances – a review

Owing to the genetically diverse nature of seedlings produced, seed propagation in litchi is not advisable. Furthermore, the long juvenile phase of seed propagated plants accompanied with the tendency of irregular bearing makes this method of propagation uneconomical and inefficient. Conventionally litchi is propagated by vegetative means like air layering, which again are slow and inefficacious. Hence use of micropropagation for large scale production of litchi plants can prove to be a more potent alternative. Albeit, till date only marginal success could have been achieved in in vitro regeneration of litchi, various studies have shown that micropropagation indeed can be used in litchi with varied rate of success. In past few years, encouraging results have been reported in regenerating plantlets from callus culture derived from young leaf explants on MS media. Some studies have shown production of multiple shoots by using nodal explants of 3-4 year old litchi on woody plant medium. Embryo culture in litchi has reportedly been successful by utilizing immature embryos of different sizes and age in a range of different media and achieving multiplication through induction of adventitious buds from embryonic shoots. Multiple shoots have also been obtained by direct germination of litchi seeds in liquid MS media. Attempts involving induction of somatic embryogenesis in cultures of zygotic embryos of litchi and anther culture have also been reported. Like other woody perennials secretion of polyphenols is also a major constraint in litchi micropropagation. In order to combat this problem, antioxidants like ascorbic and citric acids, in the growth media have been used by some workers with varied degrees of efficacies. Another serious problem affecting the success of micropropagation in litchi is the fungal infection, to inhibit the growth of which use of Bavistin and elimination of organic nutrients from growth medium have been tried. In this communication, we aim to critically analyze the major issues that are restraining the use of micropropagation in litchi along with the recent advances made in this field and also to investigate its future implications.

PHYSIOLOGICAL DEVELOPMENT OF HELICONIAS DERIVED FROM IN VITRO CULTURE OF ZYGOTIC EMBRYOS AND CONVENTIONAL PROPAGATION

The aim of the current study is to compare the physiological development of Heliconia bihai (L.) L. Lobster Claw Two plants derived from in vitro culture of zygotic embryos and conventional propagation. Heliconias obtained from rhizomes and from in vitro multiplication were evaluated every 30 days for ten months under greenhouse conditions. The experimental design was completely randomized (CRD), with ten repetitions, and the 2x10 factorial arrangement consisted of two plant multiplication methods and ten evaluations performed at different times. The analyzed biometric parameters were plant height, number of leaves, number of tillers, leaf area, and color intensity in the bracts. The plants derived from in vitro culture showed significant differences in the development of the evaluated physiological parameters in comparison to the plants derived from rhizomes, and they also showed early flowering. Although the in vitro culture plants derived from zygotic embryos, no morphological changes were found in the vegetative and reproductive parts (inflorescence) of the plants or in the colorimetry. It shows that the in vitro culture of zygotic embryos may be used as a technique to produce seedlings on a large-scale, thus allowing the floriculture sector to grow in the region and all over the country.

<b>Cultivation of immature <i>Capsicum</i> spp. embryos for incompatible-crossing embryo rescue

The in vitro culture of embryos is an important technique to enable the rescue of embryos from incompatible crosses. Studies analyzing the factors that affect the in vitro culture of zygotic embryos are scarce. This study evaluated the effect of the genotype (Capsicum baccatum and C. frutescens), the composition of the culture medium (MS and ½ MS medium with different concentrations of sucrose, IAA and GA3) and the stage of development (globular, cordiform, torpedo and cotyledonary) in the in vitro culture of immature embryos. Embryo germination was influenced primarily by the stage of development of the embryo and the composition of the culture medium. Regardless of the species, the most suitable culture medium for the germination of globular and cordiform embryos was ½ MS with 0.05 mg L-1 of GA3 and IAA and 40 g L-1 of sucrose. For torpedo and cotyledonary embryos, ½ MS culture medium with 20 g L-1 of sucrose without phytoregulators is recommended for germination. These results were better than those described in the literature for all development stages in the two species. The results in the present study will be useful for geneticists and genetic enhancers interested in applying germination techniques conducted in vitro for immature Capsicum embryos.

Physiological development of zygotic embryos of heliconias propagated in vitro and conventionally

ABSTRACT The aim of this study is to compare physiological development of Heliconia bihai cv. Lobster Claw Two plants derived from in vitro culture of zygotic embryos and conventional propagation. Heliconias obtained from rhizomes and from in vitro multiplication were evaluated every 30 days during ten months under greenhouse conditions. The experimental design was completely randomized, with ten repetitions, and the 2x10 factorial arrangement consisted of two plant multiplication methods and ten evaluations performed at different times. The analyzed biometric parameters were plant height, number of leaves, number of tillers, leaf area, and color intensity in the bracts. Plants derived from in vitro culture showed significant differences in the development of the evaluated physiological parameters in comparison to plants derived from rhizomes, and they also showed early flowering. Although the in vitro cultured plants were derived from zygotic embryos, no morphological changes were found in the vegetative and reproductive parts (inflorescence) of the plants or in the colorimetry. It shows that the in vitro cultures of zygotic embryos may be used as a technique to produce seedlings on a large-scale, thus allowing the floriculture sector to grow in the region and all over the country.

Coconut Tissue Culture: The Indian Initiatives, Experiences and Achievements

Coconut is one of the principal crops of India cultivated in over 35 districts mainly in the southern states. The productivity of the crop is declining in many of the traditionally cultivated regions owing to ageing plantations as well as biotic and abiotic stresses. These plantations are to be replanted with high yielding varieties/hybrids for which adequate quantity of quality planting material is not available. Even though tissue culture research was initiated in many laboratories in the country, the work was eventually phased out in most of the laboratories for want of a repeatable protocol. At ICAR-CPCRI, coconut tissue culture programs have been continuing for the past three decades. The attempts made include experimentation with different explants viz., immature inflorescence, plumular tissues, mature palm shoot meristem, ovary and anthers and different culture media supplemented with varying levels and types of hormones. Some of the successful protocols developed at the Institute include coconut zygotic embryo culture for collection and exchange of germplasm, cryopreservation and retrieval of zygotic embryos and pollen and plantlet regeneration from plumular tissues. Even though ICAR-CPCRI has succeeded in obtaining plantlets via direct organogenesis from inflorescence explants, the absence of friable calli formation from explants, the low rate of somatic embryo formation, large number of cultures turning to abnormal shoot development, non conversion of somatic embryos into plantlets, and formation of abnormal somatic embryos remain the major bottlenecks. Gene expression studies are being currently undertaken to decipher the molecular basis of in vitro recalcitrance.