Strategies for overcoming genotypic limitations of in vitro regeneration and determination of genetic components of variability of plant regeneration traits in sorghum

Development of suitable strategy to overcome genotypic limitations of in vitro regeneration in sorghum would help utilize high yielding but poor tissue culture responsive genotypes in genetic manipulation programmes. A factorial experiment was conducted with two explants (immature embryos and inflorescences), eight genotypes (five Sorghum sudanense and three Sorghum bicolor genotypes), three levels of 2,4-D (1 mg l⁻¹, 3 mg l⁻¹, and 5 mg l⁻¹), and two levels of kinetin (0.0 mg l⁻¹ and 0.5 mg l⁻¹). The induced callus was transferred to the regeneration media with factorial combinations of IAA (1.0 mg l⁻¹ and 2.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹ and 1.0 mg l⁻¹). S. sudanense regenerated at significantly higher frequency (38.91%) and produced shoots more intensely (2.2 shoots/callus) than S. bicolor (26.93%, 1.26 shoots/callus). Immature inflorescences regenerated at a much higher frequency (46.48%) and produced significantly more number of shoots (2.71 shoots/callus) than immature embryos (22.35%, 0.99 shoots/callus). Moreover, differences for plant regeneration between genotypes of the same species were minimal when using immature inflorescences. Increase in the 2,4-D concentration in callus induction media exhibited inhibitory effect on callus induction, growth, shoot induction and number of shoots/callus but inclusion of kinetin in callus induction media improved these responses. Use of immature inflorescence explant and inclusion of kinetin in callus induction media could overcome genotypic limitations of plant regeneration to a large extent. The extent of variability, heritability and expected genetic advance was more in plant regeneration traits than in callus induction traits. This indicated that the variability in respect of these attributes in the genotypes may be due to the additive gene action and selection of genotypes for these characters would be rewarding.     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

The influence of NO3 - and NH4 + nutrition on the carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) plants

The carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) grown hydroponically at a constant pH on either 4 mM or 12 mM NO₃ ^- or NH₄ ⁺ nutrition were investigated using either ¹⁴C or ¹⁵N techniques. Greater allocation of ¹⁴C to amino-N fractions occurred at the expense of allocation of ¹⁴C to carbohydrate fractions in NH₄ ⁺-compared to NO₃ ^--fed plants. The [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were 1.5-fold and 2.0-fold greater in shoots and roots respectively of 12 mM NO₃ ^--compared to 12 mM NH₄ ⁺-fed wheat. In both 4 mM and 12 mM N-fed maize the [¹⁴C]carbohydrate:[¹⁴C]amino-N ratios were approximately 1.7-fold and 2.0-fold greater in shoots and roots respectively of NO₃ ^--compared to NH₄ ⁺-fed plants. Similar results were observed in roots of wheat and maize grown in split-root culture with one root-half in NO₃ ^--and the other in NH₄ ⁺-containing nutrient media. Thus the allocation of carbon to the amino-N fractions occurred at the expense of carbohydrate fractions, particularly within the root. Allocation of ¹⁴N and ¹⁵N within separate sets of plants confirmed that NH₄ ^--fed plants accumulated more amino-N compounds than NO₃ ^--fed plants. Wheat roots supplied with ¹⁵NH₄ ⁺ for 8 h were found to accumulate ¹⁵NH₄ ⁺ (8.5 g ¹⁵N g^-1 h^-1) whereas in maize roots very little ¹⁵NH₄ ⁺ accumulated (1.5 g ¹⁵N g^-1 h^-1)It is proposed that the observed accumulation of ¹⁵NH₄ ⁺ in wheat roots in these experiments is the result of limited availability of carbon within the roots of the wheat plants for the detoxification of NH₄ ⁺, in contrast to the situation in maize. Higher photosynthetic capacity and lower shoot: root ratios of the C₄ maize plants ensure greater carbon availability to the root than in the C₃ wheat plants. These differences in carbon and nitrogen partitioning between NO₃ ^--and NH₄ ⁺-fed wheat and maize could be responsible for different responses of wheat and maize root growth to NO₃ ^- and NH₄ ⁺ nutrition.     

Regenerative callus of <Emphasis Type="Italic">Dianthus</Emphasis> ‘Telstar Scarlet’ showing mixoploidy produce diploid plants

Highly regenerative callus was isolated from the base of adventitious shoots on cotyledon explants of Dianthus hybrida Telstar Scarlet cultured on MS medium supplemented with 1 mg l⁻¹ TDZ and 0.1 mg l⁻¹ NAA. Flow cytometric analysis showed that cotyledon tissue is a mixture of diploid and tetraploid cells. Whereas the regenerative callus consisted of cells showing various ploidy levels of 2C to 16C, their regeneration ability was maintained as long as they were sub-cultured onto fresh media. More than 93% of regenerated shoots from the calluses were diploid. Only a few shoots were revealed as tetraploids and octoploids, suggesting that diploid cells had higher regeneration ability.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

High frequency androgenesis in indica × Basmati rice hybrids using liquid culture media

An improved procedure has been developed for high frequency androgenesis in indica × Basmati rice hybrids using a liquid culture medium. Anthers from fourteen genotypes comprising of indica × Basmati rice F₁ hybrids, F₂ plants and the parental rice cultivars, were floated in liquid RZM, N6M, and Heh5M media. Anther culture frequencies (percentage of anthers forming calluses) in most of the genotypes were significantly higher in RZM medium (16–75%) compared to those obtained in N6M (7–29%) and Heh5M (7–41%) media. Agarose (1.0% w/v)-solidified MSR1 medium containing 3.0% (w/v) maltose, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹α-naphthalene acetic acid (NAA) induced green shoot regeneration at high frequencies compared to the medium (MSR2) lacking BA. In all the genotypes, microspore calluses initiated in RZM medium regenerated green shoots with over tenfold higher frequencies compared to the calluses initiated in other two media. High plant regeneration frequencies (up to 270 green plants/1000 anthers) were obtained from microspore-derived calluses of some of the F₁ hybrids (Gobind × Basmati 370, Gobind × Taraori Basmati) and F₂ plants (Gobind × Basmati 370, Gobind × Taraori Basmati, HKR86-3 × Taraori Basmati) as compared to their actual parents. Cytological analysis of the root tips of the progeny seedlings of the microspore-derived plants revealed haploids at a frequency of about 50%; 22% of the microspore- derived plants had > 5% spikelet fertility and were diploid. Use of RZM liquid and MSR1 media, respectively for anther culture and plant regeneration resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice F₁ hybrids/F₂ plants which were comparable to those reported for japonica rice varieties/hybrids leading to the improved feasibility of using doubled haploids in genetic, breeding and mapping research with indica rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from different explants of neem

When different seedling explants, i.e. hypocotyl, epicotyl, cotyledonary node, root-shoot zone, cotyledon, leaves and roots from 7-day-old seedlings of neem were cultured on Murashige and Skoog's medium supplemented with 2 mg l⁻¹ benzyladenine and 0.1 mg l⁻¹indole-3-acetic acid, shoot buds were initiated from all the explants tested, with leaf explants producing the highest average number of shoots/explant. The regenerated shoots were further subcultured and later could be rooted on a medium supplemented with indole butyric acid (1 mg l⁻¹) and complete plants could be obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Uptake and transport of foliar applied zinc (65Zn) in bread and durum wheat cultivars differing in zinc efficiency

Using two bread wheat (Triticum aestivum) and two durum wheat (Triticum durum) cultivars differing in zinc (Zn) efficiency, uptake and translocation of foliar-applied ⁶⁵Zn were studied to characterize the role of Zn nutritional status of plants on the extent of phloem mobility of Zn and to determine the relationship between phloem mobility of Zn and Zn efficiency of the used wheat cultivars. Irrespective of leaf age and Zn nutritional status of plants, all cultivars showed similar Zn uptake rates with application of ⁶⁵ZnSO₄ to leaf strips in a short-term experiment. Also with supply of ⁶⁵ZnSO₄ by immersing the tip (3 cm) of the oldest leaf of intact plants, no differences in Zn uptake were observed among and within both wheat species. Further, Zn nutritional status did not affect total uptake of foliar applied Zn. However, Zn-deficient plants translocated more ⁶⁵Zn from the treated leaf to the roots and remainder parts of shoots. In Zn-deficient plants about 40% of the total absorbed ⁶⁵Zn was translocated from the treated leaf to the roots and remainder parts of shoots within 8 days while in Zn-sufficient plants the proportion of the translocated ⁶⁵Zn of the total absorbed ⁶⁵Zn was about 25%. Although differences in Zn efficiency existed between the cultivars did not affect the translocation and distribution of ⁶⁵Zn between roots and shoots. Bread wheats compared to durum wheats, tended to accumulate more ⁶⁵Zn in shoots and less ⁶⁵Zn in roots, particularly under Zn-deficient conditions. The results indicate that differences in expression of Zn efficiency between and within durum and bread wheats are not related to translocation or distribution of foliar-applied ⁶⁵Zn within plants. Differential compartementation of Zn at the cellular levels is discussed as a possible factor determining genotypic variation in Zn efficiency within wheat.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate

Addition of the ethylene antagonist, silver nitrate (AgNO₃), into callus induction medium significantly enhanced embryogenic callus production (both induction frequency and callus growth) of field-collected male immature inflorescence cultures of buffalograss NE84-45-3 and 'Texoka'. No stimulatory effect of AgNO₃ was observed on embryogenic callus induction for female immature inflorescence culture of a female genotype `609' and `Texoka'. Calli initiated on AgNO₃-containing media had more shoot-regenerating calli than those initiated on AgNO₃-free media, when they were transferred to the regeneration media. Benzyladenine at 2.2 μM gave the best response for regeneration, regardless of the callus source. Although average number of shoots regenerated per callus was lower for calli initiated on AgNO₃-containing media, total number of shoots regenerated was higher. The stimulatory effect, however, was environment and genotype dependent. While the addition of AgNO₃ significantly stimulated embryogenic callus induction of NE84-45-3 immature inflorescences collected in Fall 1995 and May 1997, it only slightly increased the embryogenic callus induction frequencies in May 1996 when rainy conditions occurred. For male inflorescences of `Texoka' collected in early May, AgNO₃ significantly enhanced embryogenic callus production consistently over the two-year period (1996, 1997). Published as Journal Series No. 1351, Agricultural Research Division, University of Nebraska. This revised version was published online in June 2006 with corrections to the Cover Date.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

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.     

Influence of nitrogen form and NH4 +-N/:NO3 −-N ratios on adventitious shoot formation from pear (Pyrus communis) leaf explants in vitro

The effect of various basal salts media, containing different nitrogen levels on in vitro adventitious shoot regeneration from leaf explants of Louise Bonne Panachee and Seckel pear (Pyrus communis L.) were investigated. Among the different basal salt formulae tested, Nitsch (1969) gave significantly better regeneration in most of the experiments. Shoot regeneration was altered with different NH₄ ⁺-N/NO₃ ^–-N ratios. The best regeneration was obtained when NH₄ ⁺:NO₃ ^– was either 1:2 or 1:3 regardless of overall N concentration. In addition, these data show that NH₄ ⁺ was essential for adventitious shoot regeneration from pear leaf explants on White's (1943) medium.     

Shoot regeneration and the relationship between organogenic capacity and endogenous hormonal contents in pumpkin

To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after in vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0 mg l⁻¹). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5 mg l⁻¹ BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1 mg l⁻¹ BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1 mg l⁻¹ NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.     

High regenerative nature ofMentha arvensis internodes

Media and incubation conditions have been defined for highly efficient regeneration of shoots from internode explants of slow and fast growing cultivars ofMentha arvensis. Internodal segments excised from thein vitro raised shoots were inoculated on the MS medium supplemented with combinations of 5 concentrations of l-napthalene acetic acid (NAA) and 3 concentrations of 6-benzyl amino purine (BAP). The media containing 2 μg ml⁻¹ NAA, 10 Μg ml⁻¹ BAP and 1 μg ml⁻¹ NAA, 5 μg ml⁻¹ BAP proved best for shoot regeneration and growth responses on cv Himalaya and cv Kalka explants, respectively. In 12 weeks time, on average one explant of cv Himalaya produced about 200 shoots and that of cv Kalka produced about 180 shoots. The Himalaya explants required higher concentrations of NAA and BAP for high efficiency proliferation as compared to the Kalka explants. The experiments demonstrated that internodal tissue inMentha arvensis can be induced to obtain direct shoot regenerants with high efficiency. The analysis of the RAPD profiles of 100 regenerated plantlets each of cv Himalaya and Kalka showed more than 99.9% homogeneity in bands with respect to the parents.     

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm³ of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F₁, F₂ and F₃ generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.     

The effects of KNO3 concentration in callus induction medium for wheat anther culture

KNO₃ concentration was found to significantly affect the anther culture of wheat (Triticum aestivum L.). When KNO₃ was increased from 0 to 15 mM (in cultivar Jinghua 1) or from 10 to 15 mM (in cultivars 2531-10, Xiaoyan 759 and Norin 10), the callus induction frequency increased significantly. When KNO₃ was increased further above 20 mM, the callus induction frequency decreased significantly in all the tested cultivars. The subsequent frequency of green plantlet regeneration increased significantly, and the ratio of green to albino regenerants increased sharply when KNO₃ concentration increased. Further experiments found that the decrease of callus induction frequency in the medium with too much KNO₃ might be caused by NO₃ ^- ion alone, while the effect of KNO₃ on green plantlet regeneration might be caused by both K⁺ and NO₃ ^- ions, and that the effects of NO₃ ^- concentration were independent of NH₄ ⁺ concentration in the medium.