Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Intergeneric somatic hybrids of rice [Oryza sativa L. (+) Porteresia coarctata (Roxb.) Tateoka]

Somatic hybrid plants were obtained following the electrofusion of rice (Oryza sativa L. cv ’Taipei 309’, 2n = 2x = 24) cell suspension–derived protoplasts with non-dividing leaf protoplasts of Porteresia coarctata (2n = 4x = 48), a saline-tolerant wild species. Fusion-treated protoplasts were plated on the surface of cellulose nitrate filter membranes, overlaying Lolium multiflorum nurse cells. The nurse cells were embedded in KPR medium containing 0.5 mg l⁻¹ 2,4–dichlorophenoxyacetic acid and semi-solidified with SeaPlaque agarose. Putative somatic hybrid cell colonies were selected on the basis of their growth, whereby faster growing colonies were transferred preferentially to MS-based medium with 2.0 mg l⁻¹ kinetin, 0.5 mg l⁻¹α-naphthaleneacetic acid, 30 g l⁻¹ sucrose and 4.0 g l⁻¹ SeaKem agarose to induce shoot regeneration. One hundred and nineteen regenerated plants were micropropagated clonally on MS-based medium containing 2.0 mg l⁻¹ 6–benzylaminopurine, 50 g l⁻¹ sucrose and 4.0 g l⁻¹ SeaKem agarose, prior to DNA extraction of plant samples. Putative somatic hybrids were initially identified by RAPD analysis, and 8 plant lines were selected for further investigation by flow cytometric ploidy determination and cytology. Plants of one line had an allohexaploid chromosome complement (2n = 6x = 72) and, following examination of its vegetative clones by GISH, were confirmed as somatic hybrids containing full chromosome complements of both O. sativa and P. coarctata. Received: 27 July 1998 / Accepted: 19 December 1998     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

Poorly formed chloroplasts are barriers to successful interspecific hybridization in chickpea following in vitro embryo rescue

Albinism is viewed as a major experimental bottleneck during wide hybridization in several species; the phenomenon is also widely reported in androgenesis and doubled haploid cultures. In this study, when chickpea (Cicer arietinum) was crossed with distant wild relatives, C. judaicum and C. pinnatifidum, the only hybrid embryos that survived were those rescued using in vitro techniques. Fourteen to 21-day-old embryos were incubated in ML6 medium with 90 g l⁻¹ sucrose, 1 mg l⁻¹ zeatin and 0.25 mg l⁻¹ indole acetic acid. Germinating embryos were dissected from the ovule and transferred to fresh medium without growth regulators. Later, shoots were micropropagated on solid MS medium, supplemented with B5 vitamins, 30 g l⁻¹ sucrose, 0.1 mg l⁻¹ 6-benzyladenine and 0.01 mg l⁻¹ naphthaleneacetic acid. Although some post-hybridization barriers were overcome by rescue in vitro, regenerated hybrid plantlets failed to thrive in culture and few survived transfer to soil. Here we report findings from characterization of this valuable breeding material, as a step towards a better understanding of albinism in chickpea wide hybrids and other plant tissue cultures. Following proliferation, hybrids were phenotyped as green, pale green and albino. Genotype affected pod set, regeneration and albinism. Plastid ultrastructure studies using transmission electron microscopy revealed that thylakoid membranes were well-formed in green hybrids but not in albinos. Spectrophotometric analysis of chlorophyll a, chlorophyll b and total carotenoids revealed that chlorophyll content was three to fourfold lower in albino compared to green hybrids; moreover green hybrids had two to threefold lower total chlorophyll content than in vitro-grown plantlets of their parents. In contrast, carotenoids were higher in some albino phenotypes and lower in others; however overall differences in carotenoids were less marked between all genotypes compared to chlorophyll pigments. Genetic variability between different wide crosses provides an opportunity to select certain chickpea parents and wild species which give rise to more frequent green hybrid regenerants. In future, only these hybrids will be maintained and multiplied for transfer to the glasshouse in our program.     

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer)

Summary Hairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l⁻¹ (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l⁻¹) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g⁻¹, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Gentiana dinarica</Emphasis> Beck.

Gentiana dinarica Beck, rare and endangered species of Balkan Dinaric alps, was in vitro propagated (micropropagated) from axillary buds of plants collected at Mt. Tara, Serbia. G. dinarica preferred MS to WPM medium, with optimal shoot multiplication on MS medium with 3% sucrose, 1.0 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was not clearly separated from shoot multiplication since BA did not completely inhibit root initiation. Spontaneous rooting on plant growth regulator-free medium occurred in some 30% of shoot explants. Rooting was stimulated mostly by decreased mineral salt nutrition and a medium with 0.5 MS salts, 2% sucrose and 0.5–1.0 mg l⁻¹ IBA was considered to be optimal for rooting. Rooted plantlets were successfully acclimated and further cultured in peat-based substrate.     

Induction and growth characteristics of embryogenic avocado cultures

Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ myo-inositol, 30 g l⁻¹ sucrose, 0.41 μM picloram and 8 g l⁻¹ TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l⁻¹ sucrose, 4 mg l⁻¹ thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sucrose enhances phosphoenolpyruvate carboxylase activity of in vitro solanum tuberosum L. under non-limiting nitrogen conditions

Summary The effect of sucrose on in vitro potato (ev. Kennebec) metabolism was evaluated. Plants were grown in three different media: Murashige and Skoog basal medium containing high nitrogen concentration with 0 or 20 g l⁻¹ sucrose; or modified medium containing reduced nitrogen amount and 20 g l⁻¹ sucrose. Plants fed with 20 g l⁻¹ sucrose and high N exhibited higher phosphoenolpyruvate carboxylase (PEPC) and pyruvate kinase activities and high PEPC protein concentration at 7, 20 and 33 d of culture compared to those grown with 20 g l⁻¹ sucrose and low N, or with 0 g l⁻¹ sucrose and high nitrogen (control). The highest accumulation of starch and sucrose was found in plants grown with sucrose and low nitrogen. This accumulation occurred concomitantly with a reduced enzyme activity resulting from a low utilization of α-ketoglutarate by nitrogen assimilation, when plants were grown with reduced nitrogen. Our investigations on tricarboxylic acid cycle activity showed that sucrose led to the reduction of organic acid amounts in both leaves and roots when high nitrogen was supplied to plants. This was probably due to the intense exit of α-ketoglutarate, which was confirmed by measurements of cytosolic isocitrate dehydrogenase activity. The low leaf glutamine/glutamate ratio observed in plants grown with 20 g l⁻¹ sucrose and high nitrogen compared to their counterparts cultivated with low nitrogen might be due to glutamine conversion into proteins when nitrogen assimilation was intense. These results demonstrate that sucrose enhanced PEPC activity by increasing protein synthesis. They also suggest that sucrose metabolism is involved in the replenishment of the tricarboxylic acid cycle by providing carbon skeletons required to sustain phosphoenolpyruvate utilization during high nitrate assimilation.     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Somatic embryogenesis from leaf callus of mature plants of the gymnospermCeratozamia mexicana var. robusta (Miq.) dyer (Cycadales)

Summary Embryogenic callus was induced from explanted pinnae of newly emerged leaves of mature plants ofCeratozamia mexicana var. Robusta (Gymnospermae, Cycadales) on a modified B5 formulation with 1 mg·liter⁻¹ kinetin and 1 mg·liter⁻¹ 2,4-dichlorophenoxyacetic acid. Proembryos developed on induction medium, but they were more numerous after subculture onto phytohormone-free medium, which also enabled suspensors to elongate. For nearly 1.5 yr after explanting, subsequent development of somatic embryos was not observed as suspensors dedifferentiated to form embryogenic callus on phytohormone-free medium. After this time, cotyledonary somatic embryos developed at the distal end of the suspensors. Somatic embryos have germinated on phytohormone-free medium. This is the first report of regeneration by somatic embryogenesis of a gymnosperm species from a mature tree. This technique has great potential for preservation of the highly endangered cycads.     

Rapid clonal propagation of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn., an endangered medicinal plant

An efficient protocol was developed for in vitro clonal propagation of Curculigo orchioides Gaertn. through apical meristem culture. Multiple shoots were induced from apical meristems grown on Murashige and Skoog (MS) basal medium supplemented with 1.5 mg l⁻¹ 6-benzyladenine (BA), 100 mg l⁻¹ adenine sulfate (Ads) and 3% sucrose. Inclusion of indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) in the culture medium improved the formation of multiple shoots. The highest frequency of multiplication was obtained on MS medium supplemented with 1.5 mg l⁻¹ BA, 100 mg l⁻¹ Ads, 0.25 mg l⁻¹ IBA and 3% sucrose. Rooting was achieved upon transferring the micro-shoots to half-strength MS medium containing 0.25 mg l⁻¹ IBA and 2% sucrose. Micropropagtated plantlets were hardened in the greenhouse and successfully established in soil.     

Optimization of Nutritional Factors for Exopolysaccharide Production by Submerged Cultivation of the Medicinal Mushroom Oudemansiella radicata

Summary Effects of nutritional factors on exopolysaccharide production by submerged cultivation of the medicinal mushroom Oudemansiella radicata were investigated in shake flasks. Sucrose and peptone were optimal carbon and nitrogen sources for cell growth and exopolysaccharide production. The exopolysaccharide production was increased with an increase in initial sucrose concentration within the range of 10–40 g l⁻¹ and initial peptone concentration within the range of 1–3 g l⁻¹. To enhance further exopolysaccharide production, the effect of carbon/nitrogen ratios was studied using central composite design (CCD) and response surface analysis. The maximum exopolysaccharide production of 2.67 ± 0.15 g l⁻¹ was achieved in medium with optimized carbon and nitrogen sources, i.e. 39.3 g sucrose l⁻¹ and 3.16 g peptone l⁻¹ in the same cultivation conditions. The information obtained is helpful for the hyperproduction of exopolysaccharide by submerged cultivation of O. radicata on a large scale.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

Regeneration of interspecific somatic hybrids between Helianthus annuus L. and Helianthus maximiliani (Schrader) via protoplast electrofusion

Helianthus maximiliani is one of the wild Helianthus species with the genes for resistance to many pathogens including Sclerotinia sclerotiorum. Unfortunately, a transfer of disease resistance genes from this species into the cultivated sunflower is limited by its poor crossability with the cultivated sunflower and sterility of interspecific hybrids. To overcome this problem, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clone of H. maximiliani were electrically fused with etiolated hypocotyl protoplasts of the cultivated sunflower inbred line PH-BC₁-91A. Fusion products were embedded in agarose droplets and subjected to different regeneration protocols. Developed microcalluses were released from the agarose and transferred into solid media. Shoot regeneration was achieved by culture of calluses on regeneration medium containing 2.2 mg l⁻¹ BAP and 0.01 mg l⁻¹ NAA after the treatment with a high concentration of 2,4 D for a limited period of time. A morphological and RAPD analysis confirmed a hybrid nature of the regenerated plants.     

The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (hybrid tea) cv. “First Prize”

Shoots of rose (hybrid tea) cv. “First Prize” were induced to flower in vitro on Murashige and Skoog (MS) medium containing various sucrose concentrations (15, 30 or 45 g l⁻¹) and different phytohormone combinations of different cytokinins [N⁶-benzyladenine (BA); thidiazuron (TDZ) and zeatin] with α-naphthaleneacetic acid (NAA). Results indicate that sucrose is the key factor in floral morphogenesis while cytokinin increases the flowering percentage and helps the normal development of floral buds. From the three cytokinins that were used, BA and zeatin were considered to be more suitable as inductive flowering agents than TDZ. Reduced inorganic and organic salt concentration in MS media had a positive effect on in vitro flowering. The morphology of shoots bearing floral buds varied with different cytokinin treatments. The highest percentage (45%) of flowering was obtained on MS medium supplemented with 3.0 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA and 30 g l⁻¹ sucrose.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. f. ex diels. axillary shoots in vitro

The effect of different concentrations and activities of cytokinins on the morphogenesis of regenerated Rhododendron forrestii Balf. f. ex Diels. shoots taken from nodal segments were tested. We evaluated zeatin, zeatin riboside, izopentyladenine, izopentyladenine riboside, kinetin, kinetin riboside, benzylaminopurine, benzylaminopurine riboside. The experimental results were evaluated by mathematical methods and regression analysis describing the effect of isoprenic and aromatic type of cytokinins. On the basis of this modelling, maximum axillary shoot production was attained with medium supplemented with 2.0 mg·l⁻¹ izopentyladenine riboside, 2.0 mg·l⁻¹ benzylaminopurine and 20 g·l⁻¹ sucrose. Minimal axillary shoots were produced with kinetin and kinetin riboside.     

Induction of somatic embryogenesis in Azadirachta indica

A modified culture protocol has been developed for the induction of somatic embryogenesis in Azadirachta indica (neem). Embryogenic calluses were initiated from cotyledons or hypocotyls using a Murashige and Skoog (MS) agar medium supplemented with 0.5 mg l⁻¹ α-napthaleneacetic acid (NAA), 1 mg l⁻¹ 6-benzylaminopurine (BA), 1 g l⁻¹ casein hydrolysate, and 50 g l⁻¹ sucrose. The calluses, when transferred to a liquid medium similar to the agar medium but with NAA replaced by 0.5 mg l⁻¹ indole-3-acetic acid (IAA), formed globular structures which further developed a rudimentary root, after 4 to 5 weeks incubation. Subsequently, these highly differentiated tissues when transferred into a hormone-free MS medium containing 1 g l⁻¹ casein hydrolysate and 50 g l⁻¹ sucrose, active embryo masses started to appear after 1 to 2 weeks. The embryo production was found to improve more than 2 fold by adding 0.2 mg l⁻¹ zeatin to the medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.