Sugae Wada

Improving in vitro mineral nutrition for diverse pear germplasm

Mineral nutrition in the media used for growth of in vitro plants is often difficult to optimize due to complex chemical interactions of required nutrients. The response of plant tissue to standard growth media varies widely due to the genetic diversity of the plant species studied. This study was designed as the initial step in determining the optimal mineral nutrient requirements for micropropagation of shoot tips from a collection of genetically diverse pear germplasm. Five mineral nutrient factors were defined from Murashige and Skoog salts: NH4NO3, KNO3, mesos (CaCl2·2H20–KH2PO4–MgSO4), micronutrients (B, Cu, Co, I, Mn, Mo, and Zn), and Fe-EDTA. Each factor was varied over a range of concentrations. Treatment combinations were selected using response surface methods. Five pears in three species (Pyrus communis ‘Horner 51,’ ‘Old Home × Farmingdale 87,’ ‘Winter Nelis,’ Pyrus dimorphophylla, and Pyrus ussuriensis ‘Hang Pa Li’) were grown on each treatment combination, responses were measured, and each response was analyzed by analysis of variance. The analyses resulted in the identification of the following factors with the single largest effects on plant response: shoot quality (mesos), leaf spotting/necrosis (mesos), leaf size (mesos), leaf color (mesos, NH4NO3, and KNO3), shoot number (NH4NO3 and Fe), nodes (NH4NO3 and KNO3), and shoot length (mesos and Fe). Factors with the largest effects (mesos and Fe) were similar among the genotypes. This approach was very successful for defining the appropriate types and concentrations of mineral nutrients for micropropagation of diverse pear genotypes.

Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl2, MgSO4, and KH2PO4) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl2 and MgSO4 with low KH2PO4; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl2 and moderate to low MgSO4 and KH2PO4 produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.

Mineral nutrition influences physiological responses of pear in vitro

Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoog mineral salt factors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface response experimental design. Pyrus communis ‘Old Home × Farmingdale 87,’ ‘Horner 51,’ and ‘Winter Nelis’; Pyrus dimorphophylla; and Pyrus ussuriensis ‘Hang Pa Li’ shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combinations. Analysis of shoot response to these treatments identified the factors that both contributed to physiological disorders and remedied them. Undesirable callus formation was common for pear shoots cultured on standard medium and decreased on formulations with increased NH4NO3, Fe, and mesos (CaCl2, KH2PO4, and MgSO4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH4NO3. Hooked and upwardly curled new leaves were seen in most genotypes and resulted from use of low mesos in P. communis and low nitrogen for ‘Hang Pa Li’ and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simultaneously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and moderate nitrogen compounds produced normal shoots without physiological disorders.

Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species

Inorganic nitrate (NO3−) and ammonium (NH4+) are the two major components in nitrogen (N) nutrition of typical tissue culture growth media, and the total amounts and ratios influence both shoot induction and differentiation. This study was designed to determine the optimal N requirements and interactions of NH4+ × NO3− to complete the optimization of a pear shoot culture medium. Pyrus communis ‘Horner 51’ and ‘OH × F 87’, P. cordata, P. pyrifolia ‘Sion Szu Mi’, and P. ussuriensis ‘Hang Pa Li’ from the pear germplasm collection of the US Department of Agriculture, National Clonal Germplasm Repository–Corvallis (NCGR) were evaluated. Response surface design was used to create and analyze treatment combinations of NH4+, K+, and NO3−. Cultures were evaluated for overall quality, shoot length, multiplication, leaf color and size, leaf spotting and necrosis, and callus production. Significant improvement was observed in multiplication and length for most genotypes. Reduced callus amounts were seen in two genotypes, and greener leaves were also seen in two genotypes. Each species had a distinct response, and the N form could be manipulated to produce longer shoots, more shoots, or less callus. For the best-quality shoots, both P. communis cultivars required high NO3− and low to moderate NH4+, P. cordata quality was best with high NO3− and NH4+, P. pyrifolia ‘Sion Szu Mi’ quality improved with moderate NO3− and high NH4+, and P. ussuriensis ‘Hang Pa Li’ required low NO3− and high NH4+. This study illustrates that optimizing the N components of a growth medium is very important when working with diverse plant germplasm.

Screening genetically diverse pear species for in vitro CaCl2, MgSO4 and KH2PO4 requirements

Conservation of important plant germplasm is often difficult due to the specific growth requirements of genetically diverse species including in vitro culture collections. Recently the mesos components (CaCl2, MgSO4, KH2PO4) of Murashige and Skoog medium were identified as one of the most influential groups of nutrients for five pear genotypes. To determine if this requirement also applied to a larger germplasm collection, 18 genotypes in six species were screened. Shoot quality, shoot length, leaf spots and leaf color were the most affected responses. Seven of nine Pyrus communis cultivars had improved shoot quality, five had significantly longer shoots, better leaf color and fewer leaf spots while two had more shoots. Two of the four Pyrus pyrifolia cultivars had improved shoot quality while three had better leaf color and fewer leaf spots. Pyrus calleryana ‘Capital’, Pyrus cordata and Pyrus ussuriensis ‘Harbin’ had longer shoots while Pyrus koehnei had less callus. P. ussuriensis ‘Hang Pa Li’ was the only genotype where shoot quality declined at high mesos concentrations. Quantitative ion analysis detected substantially higher concentrations of Ca, Mg and K, but significantly less Fe, in the shoots cultured on increased mesos compared to controls. This study confirms that increased mesos improved growth of P. communis and P. pyrifolia cultivars, but produced fewer significantly improved responses for four other species.

Micropropagation of pear (Pyrus sp.).

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 μM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.