Handling culture medium composition for optimizing plant cell suspension culture in shake flasks

Douglas-fir is a conifer species of major economic importance worldwide, including Western Europe and New Zealand. Herein we describe some characterization and significant refinement of somatic embryogenesis in Douglas-fir, with focus on maturation. The most typical structures observed in the embryonal masses were large polyembryogenic centres (up to 800–1500 µm) with a broad meristem, creating a compact cell “package” with suspensor cells. Singulated somatic embryos composed of both a embryonal head (300–400 µm) and long, tightly arranged suspensor were also frequent. Embryo development was enhanced following embryonal mass dispersion on filter paper discs at low density (50–100 mg fresh mass). Moreover, increasing gellan gum concentration in maturation medium (up to 10 g L^?1) improved both the quantity and quality of cotyledonary somatic embryos (SEs), which were subsequently able to germinate and develop into plantlets at high frequency. Embryogenic yield was highly variable among the seven embryogenic lines tested (27–1544 SE g^?1 fresh mass). Interestingly secondary somatic embryogenesis could be induced from cotyledonary SEs of both low- and highly-productive lines with some useful practical outcomes: secondary lines from low-performance lines (30–478 SE g^?1 fresh mass) displayed significantly higher embryogenic yield (148–1343 SE g^?1 fresh mass). In our best conditions, the total protein content in cotyledonary SEs increased significantly with maturation duration (up to 150 µg mg^?1 fresh mass after 7 weeks) but remained below that of mature zygotic embryos (300 µg mg^?1). The protein pattern was similar in both somatic and zygotic embryos, with major storage proteins identified as 7S-vicilin- and legumin-like proteins.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Physiological and morphological characteristics during development of pedunculate oak (Quercus robur L.) zygotic embryos

The developmental stages of oak zygotic embryos (ZEs) are characterized here according to morphological and physiological features. Seeds were harvested from June to September in 1-week intervals. Excised embryos were classified into four stages of development by using growth parameters. For physiological characterization, endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), l-proline, starch content and water status were determined. The expression of the oak legumin storage protein gene was tested in immature cotyledonary ZEs before and after ABA treatment. The ABA levels of the embryos showed a significant peak during the intermediate stage of maturation (stage III) and then decreased again at the end of the late maturation phase (stage IV). Concomitant with ABA, the moisture content declined with the maximum embryo size. High IAA levels were found at the beginning of embryo enlargement as exponential growth occurred (stage II) but decreased during further development. Starch accumulated gradually in the course of maturation, whereas significant values were found in stage IV ZEs near shedding. Proline, on fresh weight basis, was high during stages I and II. Osmotic potential increased when, by rapid dry matter accumulation, stage II ZEs reached their maximum size during early intermediate development. Expression of precocious germination was higher on hormone-free medium, in particular, among stage II and stage III ZEs. Variations in phytohormone levels in combination with changes in tissue water status seem to be important factors for oak ZE development.     

Photoautotrophic culture of Coffea arabusta somatic embryos: photosynthetic ability and growth of different stage embryos

Coffea arabusta somatic embryos were cultured and development of stomata, rate of CO2 fixation or production, chlorophyll content and chlorophyll fluorescence were studied in embryos at different stages of development. Cotyledonary and germinated embryos have photosynthetic capacity, although pretreatment at a high photosynthetic photon flux (PPF) (100 micromol m(-2) s(-1)) for 14 d increased photosynthetic ability. Except in a very small number of cases, stomata did not develop fully in precotyledonary stage embryos and were absent in torpedo stage embryos. Low chlorophyll content (90-130 microg g(-1) fresh mass) was noted in torpedo and precotyledonary stage embryos compared with cotyledonary and germinated embryos (300-500 microg g(-1) fresh mass). Due to the absence of stomata and low chlorophyll content in the torpedo and precotyledonary stage embryos, the photosynthetic rate was low and, in some cases, CO2 production was observed. These data suggest that the cotyledonary stage is the earliest stage that can be cultured photoautotrophically to ensure plantlet development. When grown photoautotrophically (in a sugar-free medium with CO2 enrichment in the culture headspace and high photosynthetic photon flux), torpedo and precotyledonary stage embryos lost 20-25% of their initial dry mass after 60 d of culture. However, in cotyledonary and germinated embryos, the dry mass of each embryo increased by 10 and 50%, respectively. By using a porous supporting material, growth (especially root growth) was increased in cotyledonary stage embryos. In addition, photoautotrophic conditions, high PPF (100-150 micromol m(-2) s(-1)) and increased CO2 concentration (1100 micromol mol(-1)) were found to be necessary for the development of plantlets from cotyledonary stage embryos.     

Enhancement of somatic embryogenesis and plant regeneration in Japanese larch (<Emphasis Type="Italic">Larix leptolepis</Emphasis>)

Different nitrogen sources, abscisic acid (ABA), gellan gum at various concentrations, and osmotica were evaluated for their effects on maturation of somatic embryo (SE) in Japanese larch (Larix leptolepis). Different concentrations of l-glutamine or casein hydrolysate (CH) in the medium were also compared. The highest number of matured embryos was obtained with ½ Litvay (LM) medium supplemented with 1.71 mM l-glutamine and 250 mg l^?1 CH. In terms of osmoticum effect, the highest number of cotyledonary SEs was produced in medium containing 0.2 M maltose. As for the effects of ABA and gellan gum concentration, the highest number of cotyledonary SEs was achieved on a medium containing 60 μM ABA and 0.8% gellan gum. In addition, the best plantlet conversion frequency (35.5%) was obtained with SEs derived from the treatment with 60 μM ABA and 0.8% gellan gum.     

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant,Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.     

Polyethylene glycol and maltose enhance somatic embryo maturation in loblolly pine (Pinus taeda L.)

Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine.     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos

The present study was conducted to understand the role of sucrose in the medium on the maturation of black spruce and white spruce somatic embryos. A maturation medium containing 6% sucrose, which hydrolyzed into glucose and fructose, gave significantly more embryos than a medium containing 3.16% of each glucose and fructose. Preventing the complete sucrose hydrolysis by a daily transfer of the tissues onto fresh medium significantly decreased the yield of somatic embryos compared to when sucrose was allowed to complete its hydrolysis. This reduction was not due to the manipulation of the tissues during the transfer, since a daily in situ transfer did not affect embryo production. To verify if the better embryo production observed on a medium containing 6% sucrose was due to the increasing osmotic pressure of the medium, this increasing osmotic pressure was simulated with a sequence of media containing different concentrations of glucose and fructose. Unexpectedly and for both species, this simulation did not improve somatic embryo production, which stayed similar to the one obtained on constant osmotic pressure. To understand these results, embryos produced on the different treatments were analyzed in terms of sucrose, glucose, fructose and starch levels and protein contents. The embryo carbohydrate content was independent from the carbohydrate used in the maturation medium. However, embryos matured on 6% sucrose allowed to hydrolyze during the maturation period contained significantly more soluble and insoluble proteins than embryos matured on any other treatment. Furthermore, embryos with a higher protein content also exhibited a higher epicotyl appearance frequency. The role of sucrose as a regulatory factor during the maturation of spruce somatic embryos is discussed.     

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.     

Development of white spruce somatic embryos: I. Storage product deposition

Summary The growth and development of white spruce somatic embryos was followed from the filamentous immature to the mature cotyledonary embryo stage. Histochemical examination of the various stages of embryo development showed that lipids, proteins, and polysaccharides were produced to varying degrees during the process. During early stages (1 to 2 wk on ABA), mostly polysaccharide was produced, whereas during later stages, polysaccharides, lipids, and protein accumulated. Electron microscopy indicated that lipid deposition in somatic embryos started during the first week after transfer to ABA-containing medium. Deposition of the storage products began at the basal end of the embryonal mass and within the proximal zone of the suspensors. Accumulation continued to the peripheral regions and then inward toward the cortex of the developing embryo. In all cases, polysaccharide accumulated first, followed by lipid and lastly, protein. Quantitatively, cotyledonary stage somatic embryos had less lipid and protein and more starch when compared to zygotic embryos at the same developmental stage. Total protein profiles elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the majority of proteins were similar in zygotic and somatic embryos. Prominent protein bands were found at 30, 20, 19.5, 15, 14.4, 12, and 10 Kd. However, protein bands at 40, 15, and 12 Kd in total protein from somatic embryos were either absent or highly underexpressed.     

High frequency embryogenesis in immature zygotic embryos of sunflower

In the present investigation, nutritional requirements for induction of a high frequency of well formed somatic embryos (SEs) from zygotic embryos (ZEs) of sunflower were assessed. Variables like genotype, embryo size (0.5–10 mm), sucrose concentration (30–240 g l⁻¹), carbohydrate source (sucrose, glucose, maltose), agar strength (0.2–1.0%), basal media (MS, Gamborg, Nitsch, White), photoperiod (light/dark) and temperature (20–36°C) were tested. All these variables except photoperiod had significant effect on the frequency of embryogenesis. Highest frequency of embryogenesis was facilitated by Gamborg basal salt media, 120–210 g l⁻¹ sucrose, 0.8–1.0% agar, smaller sized embryos (0.5–2 mm) and incubation temperature of 28–32°C. In addition to these, growth regulator combinations (2,4-D, 2,4-D+kinetin, BA+NAA) in varying concentrations were tried. Media supplemented with 2,4-D promoted direct embryogenesis, BA+NAA facilitated formation of single/multiple shoots while there was no response on 2,4-D+kinetin supplemented media. Zygotic embryos with well differentiated embryos were transferred to growth regulator free half strength MS medium for whole plantlet development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments

Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer‐insoluble 11S‐ globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer‐soluble 7S vicilin‐like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β‐mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H₂O g^?1 DM (mean ± sd ) than the mature non‐dried zygotic embryos; (1.16 ± 0.09 g H₂O g^?1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S‐vicilins) or below (11S‐globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.     

Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce

The ontogenetic course followed by somatic embryos of interior spruce is highly dependent on the media concentration of abscisic acid (ABA). Little or no organized development occurs in the absence of ABA and as the level of ABA is increased, a range of embryo types is produced. "Shooty embryo" structures predominate in many callus lines at low levels of ABA (1-10 μM), while 10-20 μM ABA promotes the formation of bipolar embryos that germinate precociously. When ABA is increased to 30-40 μM, precocious germination is inhibited and opaque cotyledonary embryos characteristic of their zygotic counterparts are formed which enter a period of quiescence. Only "mature" somatic embryos contain significant amounts of storage proteins and the level to which these proteins accumulate is dependent on the concentration of ABA. Indole-butyric acid (IBA) included with ABA increases the number of mature embryos. Root elongation, which was used as a measure of embryo quality, was never observed from shooty embryo Structures and was 2-3 fold higher in mature embryos compared to those that germinated precociously.     

Effects of carbohydrate source, polyethylene glycol and gellan gum concentration on embryonal-suspensor mass (ESM) proliferation and maturation of maritime pine somatic embryos

Summary The influence of carbon sources and polyethylene glycol combined with 0.45 and 0.9% (w/v) of gellan gum on the maturation of maritime pine somatic embryos was tested. The effect of the carbon source and polyethylene glycol varied widely between lines. One out of the five lines tested showed a striking response to polyethylene glycol (PEG) treatment; the addition of this osmoticum limited the embryonal-suspensor mass (ESM) proliferation while it enhanced the maturation rate. Conversely, the ESM proliferation was stimulated by PEG in the other lines without subsequent improvement of the maturation rate. The use of a high concentration of gellan gum (0.9%) improved the maturation of the five ESM lines. It was concluded that the most efficient culture medium to recover cotyledonary embryos from all lines is one supplemented with sucrose at 6% (w/v) and gellan gum at 0.9% (w/v) without PEG. The determining factor in the maturation of maritime pine somatic embryos is the genotype and/or the quality of ESM. The possible relationship between maturation performances and ESM morphology, particularly the suspensor organization, is discussed.     

Variations in storage protein and carbohydrate levels during development of avocado zygotic embryos

Variations in carbohydrates and proteins were monitored during avocado (Persea americana Mill.) zygotic embryo development and correlated with growth parameters in order to define specific markers characterizing distinct embryogenic phases. Hexose (glucose and fructose) levels were initially high and declined as embryo development advanced reaching the lowest levels in completely mature embryos. Sucrose and starch evolution showed an opposite trend with a progressive increase during embryo growth. The beginning of the maturation phase could be identified by a switch in the carbohydrate status from high hexose/sucrose ratio to low hexose/sucrose ratio. Storage protein accumulation began at early cotyledonary stages (7–8 mm), increasing significantly in the maturation phase where they represented 83% of total proteins. Mature embryos (38–40 mm) contained albumins, globulins and glutelins, albumins being the predominant and most heterogeneous fraction. Storage protein accumulation occurred in a sequential and specific way suggesting a possible role as indicators of embryo development. The complete maturation stage could be characterized by the synthesis and accumulation of a 49 kDa albumin.     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.