Somatic Plantlets Research Articles

Promoting the application of Pinus thunbergii Parl. to enhance the growth and survival rates of post-germination somatic plantlets

ObjectiveThere is a growing need for nematode resistant Pinaceae species plantlets to cope with the global scale degradation of coniferous forests, due to the prevalence of pine wilt disease. One of the bottlenecks that limits the commercialization of Pinaceae species plantlets is regeneration following their transfer from controlled sterile environments to the field while maintaining high survival rates.MethodsThe growth factors of somatic plantlets (SPs), such as sucrose, media, culture substrate, brassinolide and spectrum were investigated to promote the application of somatic nematode-resistant P. thunbergii plants in afforestation.ResultsThe 1/2 WPM liquid medium, culture substrate (perlite and vermiculite =1:1), and carbohydrate (20 g/L sucrose) were effective in stimulating the growth of rooted SPs. While for unrooted SPs, 1 ug/L of brassinolide enhanced plantlet growth and rooting. And blue light (B) significantly promoted the longitudinal growth of shoots, while red light (R) was beneficial for root growth during the laboratory domestication stage. High quality SPs were obtained at a R/B ratio of 8:2. Following this acclimatization protocol, the P. thunbergii SPs could be directly transplanted to the field with a higher survival rate (85.20 %) in a forcing house.Conclusionthis acclimatization protocol extremely improved the survival rate of P. thunbergii SPs. Moreover, this work will contribute to enhancing the possibilities for somatic plant afforestation with Pinus species.

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets.

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

Somatic Embryogenesis and Indirect In Vitro Plant Regeneration in Amorphophallus konjac K. Koch by One-Step Seedling Formation

Konjac (Amorphophallus konjac K. Koch) is a well-known tuberous vegetable belonging to the important medicinal family Araceae, and the plant grows from an underground tuber. Here, we used a “one-step seedling regeneration” tissue culture system to improve the plantlet regeneration efficiency of konjac using young leaves as an explant source. In the current study, we used several sterilization methods for tuber sterilization. Moreover, various plant growth regulator combinations were applied to achieve efficient somatic embryogenesis and plantlet regeneration. Our results showed that the optimal tuber sterilization was method C (75% alcohol for 15 s + 0.1% HgCl2 for 15 min + washing by double-sterilized water three times). Three types of embryogenic calli were induced on full-strength Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L of 6-benzylaminopurine (6-BA), 0.5 mg/L of naphthaleneacetic acid (NAA), 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 g/L of sucrose. Of the three types of embryogenic calli, only type Ⅲ further regenerated plantlets, with a callus induction rate of 55.73% and a seedling induction rate of 92.73%. This suggests that the addition of the above hormones gives the optimal callus induction. The proliferation rate achieved was 38% on the MS basal medium containing 1.0 mg/L of 6-BA, 1.0 mg/L of indolebutyric acid (IBA), 0.2 mg/L of kinetin (KT), and 50 g/L of sucrose. The one-step seedling formation achieved in MS medium contained 2.0 mg/L of 6-BA, 0.5 mg/L of NAA, 0.1 mg/L of gibberellic acid (GA3), and 30 g/L of sucrose, and the number of regenerated shoots per explants was 6 ± 2. Therefore, we establish a one-step seedling regeneration system through indirect plant regeneration, which shortens the time for konjac in vitro regeneration, significantly increased the micropropagation efficiency, and decreased the cost of the konjac tissue culture.

Long-term subculture affects rooting competence via changes in the hormones and protein profiles in Cedrela fissilis Vell. (Meliaceae) shoots

Long-term subculture plays an essential role in the large-scale multiplication and production of somatic plantlets. We investigated the effects of long-term subculture on in vitro shoot development and ex vitro rooting associated with changes in the hormones and protein profiles in Cedrela fissilis. The number of subcultures of shoots induced a decrease in the ex vitro rooting response. The reduction in adventitious root (AR) formation was associated with decreases in the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), 12-oxo-phytodienoic acid (OPDA), putrescine (Put), and spermine and increases in jasmonic acid (JA), jasmonoyl-isoleucine, trans-cinnamic acid, and salicylic acid contents in shoots at the fourth subculture compared to the first. The ornithine decarboxylase enzyme preferentially functions in the Put biosynthesis pathway, and it was related to the highest AR formation in shoots at the first subculture. Down-accumulation of the auxin-binding protein ABP19a in shoots from the fourth subculture compared to the first subculture was related to a decrease in both the IAA content and AR formation. In addition, down-accumulation of glucose-6-phosphate isomerase; glutamine synthetase leaf isozyme, chloroplastic; 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; l-ascorbate peroxidase, cytosolic; monodehydroascorbate reductase; and 2-Cys peroxiredoxin BAS1-like, chloroplastic and up-accumulation of caffeoyl-CoA O-methyltransferase 1 and isoforms of peroxidase 4 proteins in shoots from the fourth relative to the first subculture were associated with a reduction in AR formation. These results showed that the understanding of hormonal and molecular mechanisms related to the potential of AR formation in shoots under successive subcultures is relevant to improving large-scale plantlet production in C. fissilis.

Hybrid Pine (Pinus attenuata × Pinus radiata) Somatic Embryogenesis: What Do You Prefer, Mother or Nurse?

Development of hybrid pines of Pinus radiata D. Don for commercial forestry presents an opportunity to diversify the current resource of plant material. Climate change and different land uses pose challenges, making alternative species necessary to guarantee wood and non-wood products in the future. Pinus radiata var. cedrosensis × Pinus attenuata hybrid possesses different attributes, such as tolerance to drought conditions, better growth and resistance to snow damage at higher altitudes, and more importantly, different wood quality characteristics. Embryogenic cell lines were successfully initiated reciprocal hybrids using as initial explants megagametophytes, excised zygotic embryos and excised zygotic embryos plus nurse culture. However, the questions raised were: does the initiation environment affect the conversion to somatic plantlets months later? Does the mother tree or the cross have an effect on the conversion to somatic plantlets? In the present work we analysed the maturation rate, number of somatic embryos, germination rate, and the ex-vitro growth in cell lines derived from different initiation treatments, mother tree species, and crosses. Differences were not observed for in vitro parameters such as maturation and germination. However, significant differences were observed due to the mother tree species in relation with the ex-vitro growth rates observed, being higher those in which P. radiata acted as a mother. Moreover, embryogenic cell lines from these hybrids were stored at −80 °C and regenerated after one and five years.

Tolerance of Douglas Fir Somatic Plantlets to Aluminum Stress: Biological, Cytological, and Mineral Studies.

Aluminum (Al) is well known as a potent inhibitor of plant growth and development. It is notably present in soils in the soluble and bioavailable form Al3+ when the soil pH drops below 5. This situation is frequent, especially in softwood forests when litter decomposition is slow. In the present work, we studied the effects of Al3+ on the growth and development of Douglas fir plantlets. Somatic plantlets, regenerated via somatic embryogenesis, were grown in vitro on media supplemented with different concentrations of aluminum chloride (AlCl3): 0 µM, 200 µM, 500 µM. and 1 mM. We show that a concentration of 500 µM AlCl3 in medium significantly reduced root elongation (−21.8%), as well as stem growth (−14.6%). Also, a 25% reduction in dry mass of the plantlets was observed in presence of a concentration of 200 µM of AlCl3. Histological analysis of root tissues revealed significant damage, especially in conducting vessels. In addition, mineral cation content of plantlets was disturbed under Al exposure. More particularly, the Mg and K contents of needles and the Ca content of stems and needles were significantly reduced in presence of a concentration of 500 µM AlCl3 in the culture medium (−35.6%, −33.5%, −24%, and −34% respectively). However, all these damages appeared at relatively high Al concentrations when compared with other herbaceous species. This study shed light on the ability of Douglas fir in vitro plantlets to cope with the acid-driven toxicity of Al.

Optimizing culture conditions for high frequency somatic embryogenesis and plantlet conversion in Daucus carota L

The present investigation was carried out to establish a simple and efficient in vitro method for rapid propagation of Daucus carota L for different applications. Root, stem, leaf and seed explants were tested to induce callus on Gamborg medium supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) for cell suspension culture establishment. The leaf, stem, seed explants induced calluses were more embryogenic and viable when compared to callus derived from tap root explants. Plant regeneration potential of embryogenic callus from tap root showed very slow response producing only fewer shoots, whereas the response of other explants derived calluses like stem, leaves and seeds exhibited rapid multiple shoot formation within three weeks’ time. Tap root derived suspension culture showed more clumps with less viable embryogenic cells, in contrast to the stem and seed derived suspension cultures, where cells were more embryogenic and proliferative. Explant, concentration of carbohydrates, 2,4-D and amino acids were tested for embryo induction response and it was found that 3% sucrose, 1.0 mg/L 2,4-D and amino acid alanine had great influence on embryo induction. The somatic embryos were subjected to desiccation prior to plating on the regeneration medium. It was found that 1 h desiccation increased the regeneration percentage of the embryos and the conversion frequency of embryos to plants decreased on increased desiccation time. These optimized conditions will be very useful for studies involving genetic transformation, cell cycle, cell proliferation, synthetic seed production and other physiological studies of D. carota species.

<b>Effects of glutathione supplementation and carbon source during somatic embryogenesis of <i>Acca sellowiana</i> (O.Berg) Burret (Myrtaceae)

The present study intended to investigate the effects of different glutathione (GSH) levels (0, 0.1, 0.5 and 1 mM) on the somatic embryogenesis (SE) induction of Acca sellowiana . Besides, we evaluated the effect of different carbon sources (sucrose and maltose) on the somatic embryos conversion. GSH-supplemented treatments resulted in improved SE induction rates (~70%) as compared to the control GSH-free (~35%) after 50 days of culture. The total number of somatic embryos obtained did not differ between treatments, but significant differences were observed for the embryonic stages after 80 days of culture. After 80 days of culture, 0.5 and 1 mM GSH-supplemented treatments showed the largest amount of torpedo-staged somatic embryos. In contrast, treatments supplemented with 0 and 0.1 mM GSH showed equal amounts of somatic embryos at all embryonic stages. These results indicate that GSH accelerates the SE induction process and increases the synchrony of the somatic embryo formation of A. sellowiana . The use of maltose for the somatic embryos conversion, as compared to sucrose, did not influence the conversion rate of normal chlorophyllous somatic embryos, but increased the formation of normal achlorophyllous somatic plantlets. This finding can be attributed to the rapid hydrolysis of sucrose, contributing to an enhanced chlorophyll synthesis.

Rooting of Pinus radiata somatic embryos: factors involved in the success of the process

In vitro conditions of the culture media, plant growth regulators and culture containers may cause anatomical and physiological changes that have negative effects on rooting and ex vitro acclimatization of somatic plantlets. The control of these factors could contribute to the improvement of somatic embryogenesis systems in conifers, especially in pines. The influence of macronutrient concentrations, explant type and culture containers in Pinus radiata D. Don in vitro somatic embryo rooting were analyzed. The highest rooting percentage was observed using half-strength macronutrient concentrations, complete micronutrients and vitamins of Quoirin and Lepoivre medium. Although the use of glass culture vessels was the best to increase the efficiency of the somatic embryogenesis process in terms of rooting, the use of ventilated containers resulted in a significant increase in the percentage of plants able to be planted in the field.

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of Carica papaya.

Efficient protocols for somatic embryogenesis of papaya (Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C. papaya. To study the effects of PEG on somatic embryogenesis of C. papaya, we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG(control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C. papaya somaticembryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.

Histological studies of cellular differentiation during somatic embryogenesis of coconut plumule-derived calli

<div><table cellspacing="0" cellpadding="0" align="center"><tbody><tr><td align="left" valign="top"><p>Since coconut is one of the most recalcitrant species to generate <em>in vitro</em>, it is necessary to study in detail about the cellular changes that occur during somatic embryogenesis to enhance our knowledge about this phenomenon. In the present study, coconut plumular tissues, the shoot meristem including leaf primordia, were used as explants for <em>in vitro </em>regeneration studies. Histological studies were carried out in different stages of plumule culture. No noticeable growth was observed in 15 days old cultures. After 30 days, meristematic cells could be identified. Abundance of meristematic cells, foremost to the development of callus structures, was observed after 45 days. After 75 days, globular friable calli were formed and histological studies revealed the presence of meristematic centers which eventually formed somatic embryos. The histological study of matured somatic embryos formed after 120 days of callus initiation showed a clear meristematic zone of parenchyma cells, surrounded by vascular bundles. Histological studies, carried out for certain abnormalities like compact calli, abnormal somatic embryoids with rudimentary shoots and multiplied roots, revealed the presence of intact cotyledonary leaves which seemed to inhibit the apical meristem development of somatic embryoids. The presence of vascular bundles in the early stages of callus formation might lead to the direct formation of meristemoids. These results could aid future studies leading to enhanced control of the somatic embryogenic process and greater efficiency of somatic embryo and plantlet formation in coconut.</p></td></tr></tbody></table></div>

Cryopreservation of somatic embryos of Alnus glutinosa (L.) Gaertn. and confirmation of ploidy stability by flow cytometry

Germplasm preservation plays an important role in current breeding programs. A simple vitrification procedure that allows for the reproducible cryopreservation of two alder embryogenic lines is presented for the first time. Somatic embryos clumps (1–2 mm) were precultured in hormone-free medium (Murashige and Skoog half-strength macronutrients, MS1/2) supplemented with 0.3 M sucrose for 3 days, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25 °C. Osmoprotected somatic embryos were dehydrated using plant vitrification solution 2 (PVS2). The effect of different PVS2 incubation times was evaluated, and 60 min at 0 °C was considered to be the optimum period. After changing the solution with fresh PVS2, the somatic embryos were directly immersed in liquid nitrogen. Following rapid thawing in a water bath at 40 °C for 2 min, the somatic embryos were transferred onto MS1/2 supplemented with 0.1 mg l−1 benzyladenine, 30 g l−1 sucrose and 6 g l−1 Vitro agar. The cultures were kept in the dark for 1 week prior to exposure to light (16 h/8 h light/dark cycle). The recovery rate of vitrified somatic embryos reached over 90 % in both embryogenic lines. Cryopreservation did not affect the plant regeneration potential of Alnus glutinosa through somatic embryogenesis. The ploidy stability of the regenerated material was assessed by flow cytometry. Analysis of DNA ploidy stability of the control, PVS2 treated, cryopreserved somatic embryos and plantlets showed no significant differences.

DEVELOPING REGENERATION SYSTEM FOR CRYOPRESERVATION IN SUGARCANE (SACCHARUM OFFICINARUM L.)

A regeneration system for cryopreservation of sugarcane (Saccharum officinarum L.), commercial cultivar ‘U-Thong 5’, has been established. Somatic embryogenesis has been done using immature inflorescence segments as explants. The highest induction percentage of the initial embryogenic callus was obtained in Murashige and Skoog media supplemented with 10-15 µM 2,4-dichlorophenoxy-acetic acid, 50 ml/L coconut water, 500 mg/L casein hydrolysate, and 6% (w/v) sucrose in dark condition. Embryogenic callus developed to somatic embryos and plantlets on MS media containing 50 ml/L coconut water, 0.5 gm/L orchid fertilizer (21-21-21) and 6% (w/v) sucrose. Shoots were able to root on these media. For cryopreservation, a vitrification protocol using different plant vitrification solutions was employed, PVS2, PVS3, and variants of PVS3 (various concentrations of glycerol and sucrose). Somatic embryos in the early stages have been precultured on MS media containing 50 ml/L coconut water, 2 µM abscisic acid, 0.3 M sucrose for 24 h. After 24 h, the somatic embryos have been cultured on the same media with 0.5 M for 24 h. Subsequently, the somatic embryos have been treated with loading solution comprising of 0.5 M sucrose and 2 M glycerol in MS media for 30 min. Then, somatic embryos were dehydrated with different types of PVSs for 30 min at 5°C prior to being immersed in liquid nitrogen for 7 days. After freezing in liquid nitrogen, regeneration percentages of the somatic embryos treated with PVS3 and variants of PVS3 were 34-42%. In contrast using PVS 2 during dehydration step, lower survival and regeneration percentages of the somatic embryos were obtained, when compared to those of PVS3.

Direct somatic embryogenesis and plant regeneration from leaf sheath explants of mango ginger (Curcuma amada Roxb.)

An efficient and simple procedure was systematically developed for inducing direct somatic embryogenesis and plantlet regeneration from leaf sheath explants of Curcuma amada Roxb. A two-step culture system was used to induce somatic embryogenesis. The optimized procedure resulted in direct somatic embryogenesis from 93.3% explants after 3-wk culture. Leaf sheath explants were incubated for 2 wk on medium containing 2.24 μM 2,4-dichlorophenoxyacetic acid and 1.11 μM 6-benzyladenine to initiate direct somatic embryogenesis. Thereafter, these explants were transferred to a medium containing 9.10 μM thidiazuron and 1.33 μM α-naphthaleneacetic acid. Elongated somatic embryos obtained from these cultures germinated readily, and the optimal frequency of plantlet development (86.7%) was achieved when embryos were cultured in darkness on 1/2 strength Murashige and Skoog medium containing 1.44 μM gibberellic acid. Histological and scanning electron microscopic studies showed that the initial cell divisions that led to embryo formation occurred in epidermal and subepidermal cells, followed by the development of globular and elongated structures that appeared to be somatic embryos. The presence of a clear protoderm in the globular structures and procambial strands in the elongated structures confirmed that these structures were true somatic embryos. Plantlets derived from somatic embryos were acclimatized successfully to ex vitro conditions at a survival rate of 87.43% and developed with normal phenotypes.

Improved high-efficiency protocol for somatic embryogenesis in Peach Palm (Bactris gasipaes Kunth) using RITA® temporary immersion system

Bactris gasipaes Kunth (Arecaceae) is an Amazonian palm cultivated mainly for the production of fruits and heart of palm. Biotechnological tools based on somatic embryogenesis (SE) can be used for improvement and germplasm conservation of this species. The efficiency of different culture systems and culture medium composition were evaluated to optimize the efficiency of SE. The study also evaluated global DNA methylation levels, total protein and starch contents, and alcohol dehydrogenase (ADH) activity. The RITA® bioreactor showed enhanced multiplication of somatic embryos with increased protein, starch levels and ADH activity. Furthermore, a low DNA global methylation rate (27.52%) was observed, suggesting its relationship with the expression of proteins associated with the maturation of somatic embryos and their subsequent conversion to plantlets. Somatic embryo maturation was successfully achieved on Petri dishes and abscisic acid (ABA) supplementation in the culture medium. Chlorophyll pigmented somatic embryos were obtained in Petri dishes containing PGR-free culture medium, and RITA® provided the greatest number of plantlets to be acclimatized after reaching 6–7cm in length. In the acclimatization step, the best results were obtained with plantlets derived from RITA® after reaching 6–7cm in length. These new findings shed light on the importance of defining adequate culture systems for different steps of in vitro regenerative protocols and represent a breakthrough in the in vitro morphogenesis process of peach palm, resulting in a large number of somatic plantlets with enhanced physiological and morphological features.

Comparative proteomic analysis of off-type and normal phenotype somatic plantlets derived from somatic embryos of Feijoa (Acca sellowiana (O. Berg) Burret)

Morphological disorders in a relevant portion of emerged somatic embryos have been a limiting factor in the true-to-type plantlet formation in Acca sellowiana. In this sense, the present study undertook a comparison between normal phenotype and off-type somatic plantlets protein profiles by means of the 2-D DIGE proteomics approach. Off-type and normal phenotype somatic plantlets obtained at 10 and 20 days conversion were evaluated. Results indicated 12 exclusive spots between normal and off-type plantlets at 10 days conversion, and 17 exclusive spots at 20 days conversion. Also at 20 days conversion, 4 spots were differentially expressed, up- or down-regulated. Two proteins related to carbohydrate metabolism were only expressed in off-types at 10 days conversion, suggesting a more active respiratory pathway. A vicilin-like storage protein was only found in off-types at 20 days conversion, indicating that plantlets may present an abnormality in the mobilization of storage compounds, causing reduced vigor in the development of derived plantlets. The presence of heat shock proteins were only observed during formation of normal phenotype somatic plantlets, indicating that these proteins may be involved in normal morphogenesis of plantlets formed. These new findings shed light on possible genetic or epigenetic mechanisms governing A. sellowiana morphogenesis.

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.

Ploidy stability of somatic embryo-derived plants in two ecological keystone sedge species (Lepidosperma laterale and L. concavum, Cyperaceae)

We report on the development of a somatic embryogenesis system for Lepidosperma concavum R.Br. and L. laterale R.Br. and the determination of ploidy stability of plants derived from somatic embryos. These keystone Lepidosperma species cannot currently be returned to restoration sites because of propagation difficulties (i.e. seed dormancy, low seed fill and recalcitrance to vegetative propagation). Three explant types (in vitro-germinated seedlings, immature seed and immature inflorescences) were used for the assessment of callus production potential. Embryogenic callus was induced and multiplied on 1/2MS medium with 2,4-D either alone, or in combination with zeatin. Over 90% of seedling explants of L. laterale produced regenerative calli after 6 weeks and 53% of seedling explants of L. concavum produced calli after 16 weeks on media containing 2,4-D and zeatin. Inflorescence material appeared to be least responsive. High rates of conversion to plants were achieved on medium containing activated charcoal, followed by thidiazuron medium. Acclimatisation success of plants ranged from 86% to 95%. Acclimatised plants grew vigorously under standard nursery conditions. The DNA ploidy level of 486 somatic embryogenesis-derived plantlets was analysed by flow cytometry. Only one plant (=0.2% of all plantlets tested) was found mixoploid. All other plants showed a stable ploidy level and stable C-values within the species. There was a small but significant C-value difference between the two Lepidosperma species. Five variegated plants (=0.3%) were observed among a total of ~1600 plants acclimatised. The application of tissue culture techniques such as somatic embryogenesis brings large-scale production of Lepidosperma plants for revegetation and horticultural purposes closer to commercial feasibility.

EFFECT OF CRYOPRESERVATION ON GROWTH OF DIFFERENT ELITE EMBRYOGENIC CELL LINES OF NORWAY SPRUCE (PICEA ABIES (L.) KARST.)

Callus from seven different elite embryogenic cell lines of Norway spruce originated from the controlled crossing conducted by Skogforsk (Forest Research Institute of Sweden) have been cryopreserved in liquid nitrogen using the slow freezing method with sorbitol (0.2 and 0.4 M) and DMSO (5% v/v) as cryoprotectants. Cooloing of samples was performed in a programmable freezer Cryo Med 7452. The cryovials were inserted at +4°C and frozen at -0.3°C/min to -16°C. After 15 min at -16°C, to avoid rapid cooling and crystal formation in the cells, the cryovials were further cooled to -35°C at -0.3°C/min. At the end of the freezing program the cryovials were transferred and stored in a CBS V1500 liquid nitrogen storage unit at -196°C. After thawing and plating on proliferation medium, the recovered cells showed a short lag phase, after which they continued to grow. Lags in growth were also observed after the transfer to pre-maturation and maturation media. Somatic embryo maturation and plantlet regeneration occurred in all the tested embryogenic lines.