The present invention is drawn to the production of fertile transformed wheat plants. The method involved subjecting wheat tissues to high velocity microprojectile bombardment, selecting for transformed cells, and regenerating stably transformed fertile plants from the transformed cells.

Methods for stable transformation of wheat

Chickpea (Cicer arietinum L), in its reproductive stage, is sensitive to heat stress (32/20C or higher as day/ nighttemperatures)withconsequentsubstantiallossofpotentialyieldsathightemperaturesThephysiologicalmechanisms associated with reproductive failures have not been established: they constitute the basis of this study Here, we initially screened a large core-collection of chickpea against heat stress and identified two heat-tolerant (ICC15614, ICCV 92944) andtwoheat-sensitive(ICC10685,ICC5912)genotypesThesefourgenotypesweresownduringthenormaltimeofsowing (November-March)andalsolate(February-April)toexposethemtoheatstressduringreproductivestage(>32/20C)The genotypes were assessed for damage by heat stress to the leaves and reproductive organs using various indicators of stress injury and reproductive function In the heat-stressed plants, phenology accelerated as days to flowering and podding, and biomassdecreasedsignificantlyThesignificantreductioninpodset(%)wasassociatedwithreducedpollenviability,pollen load, pollen germination (in vivo and in vitro) and stigma receptivity in all four genotypes Heat stress inhibited pollen function more in the sensitive genotypes than in the tolerant ones, and consequently showed significantly less pod set Heat stress significantly reduced stomatal conductance, leaf water content, chlorophyll, membrane integrity and photochemical efficiency with a larger effect on heat-sensitive genotypes Rubisco (carbon-fixing enzyme) along with sucrose phosphate synthase(SPS)andsucrosesynthase(SS)(sucrose-synthesisingenzymes)decreasedsignificantlyinleavesduetoheatstress leading to reduced sucrose content Invertase, a sucrose-cleaving enzyme, was also inhibited along with SPS and SS The inhibition of these enzymes was significantly greater in the heat-sensitive genotypes Concurrently, the anthers of these genotypes had significantly less SPS and SS activity and thus, sucrose content As a result, pollen had considerably lower sucrose levels, resulting in reduced pollen function, impaired fertilisation and poor pod set in heat-sensitive genotypes

Heat-stress-induced reproductive failures in chickpea (Cicer arietinum) are associated with impaired sucrose metabolism in leaves and anthers

The rising temperatures (>35°C) are proving detrimental to summer-sown mungbean genotypes that experience inhibition of vegetative and reproductive growth. In the present study, the mungbean plants growing hydroponically at varying temperatures of 30/20°C (control), 35/25, 40/30, and 45/35°C (as day/night 12 h/12 h) with (50 μM) or without ascorbic acid (ASC) were investigated for effects on growth, membrane damage, chlorophyll loss, leaf water status, components of oxidative stress, and antioxidants. The ASC-treated plants showed significant improvement in germination and seedling growth especially at 40/30 and 45/35°C. The damage to membranes, loss of water, decrease in cellular respiration, and chlorophyll were significantly prevented by ASC treatment to plants growing at these temperatures. The oxidative stress measured as malondialdehyde and hydrogen peroxide content was observed to be significantly lower at high temperatures with ASC application. The activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased at 40/30°C but decreased at 45/35°C in the absence of ASC while with its application, the activities of these enzymes were appreciably resorted. Among all the antioxidants, the endogenous ASC content decreased to the greatest extent at 45/35°C grown plants indicating its vital role in affecting the response of mungbean to heat stress. Exogenously applied ASC raised its endogenous content along with that of glutathione and proline at 45/35°C. The findings indicated that heat stress-induced inhibition in growth and chlorosis was associated with decrease in leaf water status and elevation of oxidative stress, which could partly be prevented by exogenous application of ASC. Its role in imparting protection against heat stress is discussed.

Heat-stress induced inhibition in growth and chlorosis in mungbean (Phaseolus aureus Roxb.) is partly mitigated by ascorbic acid application and is related to reduction in oxidative stress

Chickpea is a heat sensitive crop hence its potential yield is considerably reduced under high temperatures exceeding 35 °C. In the present study, we evaluated the efficacy of proline in countering the damage caused by heat stress to growth and to enzymes of carbon and antioxidative metabolism in chickpea. The chickpea seeds were raised without (control) and with proline (10 μM) at temperatures of 30/25 °C, 35/30 °C, 40/35 °C and 45/40 °C as day/ night (12 h/12 h) in a growth chamber. The shoot and root length at 40/35 °C decreased by 46 and 37 %, respectively over control while at 45/40 °C, a decrease of 63 and 47 %, respectively over control was observed. In the plants growing in the presence of 10 μM proline at 40/35 °C and 45/40 °C, the shoot length showed improvement of 32 and 53 %, respectively over untreated plants, while the root growth was improved by 22 and 26 %, respectively. The stress injury (as membrane damage) increased with elevation of temperatures while cellular respiration, chlorophyll content and relative leaf water content reduced as the temperature increased to 45/40 °C. The endogenous proline was elevated to 46 μmol g−1 dw at 40/35 °C but declined to 19 μmol g−1 dw in plants growing at 45/40 °C that was associated with considerable inhibition of growth at this temperature. The oxidative damage measured as malondialdehyde and hydrogen peroxide content increased manifolds in heat stressed plants coupled with inhibition in the activities of enzymatic (superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase) and levels of non-enzymatic (ascorbic acid, glutathione, proline) antioxidants. The enzymes associated with carbon fixation (RUBISCO), sucrose synthesis (sucrose phosphate synthase) and sucrose hydrolysis (invertase) were strongly inhibited at 45/40 °C. The plants growing in the presence of proline accumulated proline up to 63 μmol g−1 dw and showed less injury to membranes, had improved content of chlorophyll and water, especially at 45/40 °C. Additionally, the oxidative injury was significantly reduced coupled with elevated levels of enzymatic and non-enzymatic antioxidants. A significant improvement was also noticed in the activities of enzymes of carbon metabolism in proline-treated plants. We report here that proline imparts partial heat tolerance to chickpea’s growth by reducing the cellular injury and protection of some vital enzymes related to carbon and oxidative metabolism and exogenous application of proline appears to have a countering effect against elevated high temperatures on chickpea.

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Proline induces heat tolerance in chickpea (Cicer arietinum L.) plants by protecting vital enzymes of carbon and antioxidative metabolism

The mechanisms affecting the heat sensitivity of chickpea are largely unknown. Heat-tolerant (ICCV07110, ICCV92944) and heat-sensitive (ICC14183, ICC5912) chickpea genotypes were sown in February in the soil-filled pots. At the time of flowering, these were subjected to varying day/night temperatures of 30/20, 35/25, 40/30 and 45/35°C in the growth chambers (12 h light/12 h dark; light intensity, 250 μmol m−2 s−1, 80% relative humidity). The pollen viability, pollen germination, tube growth, pollen load and stigma receptivity decreased with increases in temperatures to 45/35°C. The heat-tolerant genotypes experienced significantly less damage to pollen and stigma function. Membrane integrity, chlorophyll content, photochemical efficiency and cellular oxidizing ability were inhibited by the increase in temperature, with greater impacts on the sensitive genotypes. Oxidative injury as lipid peroxidation and hydrogen peroxide content was significantly greater in sensitive genotypes at 40/30 and 45/35°C. Enzym...

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Effect of varying high temperatures during reproductive growth on reproductive function, oxidative stress and seed yield in chickpea genotypes differing in heat sensitivity

A friable and fast-growing type of callus was isolated from a long term shoot-competent cell culture of wheat. The suspension cultures established from this callus consisted of small, densely cytoplasmic cells which divided more rapidly but with a lower plant regeneration frequency than the original culture. A high yield of protoplasts was released from suspension cells (2 to 3×107 protoplasts per ml packed cell volume) when treated with enzyme mixtures. The isolated protoplasts divided at a relatively high frequency (20% to 50%) in both liquid and agarose-solidified KM8p medium. Up to 0.21% of the dividing protoplasts continued to divide and form micro-calli. Sixty-eight plants were regenerated from micro-calli, and among the 30 plants which were transplanted to the greenhouse, 3 have survived.

Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.).

Hexaploid wheat plants were easily regenerated from young embryo-derived callus for twelve genotypes tested. After a 2.5 years culture period, however, most of the callus cells lost their ability to regenerate into shoots, but not into roots.A novel approach was used to regenerate shoots from the long-term suspension cultured cells. In general, instead of selecting embryogenic callus as source material, this approach requires the inoculation of unselected callus into liquid medium followed by removing the free floating cell portion, selecting out non-root forming cell clumps from the root forming primary suspension culture, and growing the putative shoot-competent clumps in liquid medium with reduced auxin concentrations. We have successfully established shoot-competent wheat suspension cultures for cv. 'Mustang'. High (>80%) frequencies of plant regeneration were observed from plating of 2.5 year suspension cultures. The suspension cultures established by this approach have been utilized to select for heat tolerant variants and will be an ideal source material for protoplast culture and transformation studies. This approach can also be applied to other cereal crops which form roots easily but are unstable in maintaining long term regenerable cultures and which are not easily adaptable to suspension culture.

A novel approach for efficient plant regeneration from long-term suspension culture of wheat.

The objectives of this study were to compare thermotolerance in whole plants vs. suspension cell cultures of winter wheat, and to evaluate the synthesis of heat shock proteins in relation to genotypic differences in thermotolerance in suspension cells. Whole plant genetic differences in the development of heat tolerance were identified for three wheat genotypes (ND 7532, KS 75210 and TAM 101). Suspension cell cultures of these genotypes were used to evaluatein vitro response to heat stress. Viability tests by triphenyl tetrazolium chloride (TTC) and by fluorescein diacetate (FD) were utilized to determine the relationship of cellular response to heat stress (37°C/24 h, 50°C/1h). KS 75210 and ND 7532 are relatively heat susceptible. TAM 101 is heat tolerant. Both tests at the cellular level were similar to the whole plant response. Thus, cellular selection for enhancing heat tolerance seems feasible. Heat shock protein (HSP) synthesis of two genotypes, ND 7532 and TAM 101 were determined for suspension cultured cells. In suspension cultures, HSPs of molecular weight 16 and 17 kD were found to be synthesized at higher levels in the heat tolerant genotype (TAM 101) than the susceptible genotype (ND 7532), both at 34° and 37°C treatments for 2 hours and 5 hours. HSP 22 kD was synthesized more at 34°C for TAM 101 than ND 7532, but not at 37°C; whereas, HSP 33 kD was synthesized at 37°C at similar abundance for both genotypes, but not at 34°C.

Wen Chung Wang

Papers

Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.).

A novel approach for efficient plant regeneration from long-term suspension culture of wheat.

Thermal stress evaluation of suspension cell cultures in winter wheat.